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Molecular Identification and Genetic Characterization of Cetacean Herpesviruses and Porpoise Morbillivirus

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MOLECULAR IDENTIFICATION AND GE NETIC CHARACTERIZATION OF CETACEAN HERPESVIRUSES AND PORPOISE MORBILLIVIRUS By KARA ANN SMOLAREK BENSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Kara Ann Smolarek Benson

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I dedicate this to my best friend and husb and, Brock, who has always believed in me.

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iv ACKNOWLEDGMENTS First and foremost I thank my mentor, Dr Carlos Romero, who once told me that love is fleeting but herpes is forever. He welcomed me into his lab with very little experience and I have learned so much from him over the past few years. Without his excellent guidance, this project would not have been possible. I thank my parents, Dave and Judy Smolare k, for their continual love and support. They taught me the importance of hard work and a great education, and always believed that I would be successful in life. I would like to thank Dr. Tom Barrett for the wonderful opportunity to study porpoise morbillivirus in his la boratory at the Institute for Animal Health in England, and Dr. Romero for making the trip possible. I especially thank Dr. Ashley Banyard for helping me accomplish all the objectives of th e project, and all th e wonderful people at the IAH for making a Yankee feel right at home in the UK. I thank Alexa Bracht and Rebecca Woodruff who have been with me in Dr. Romero’s lab since the beginning. Their con tinuous friendship and encouragement have kept me sane even in the most hectic of tim es. I thank the newest members of the lab, Shasta McClennahan and Rebecca Grant, for their input and support over the past few months. I also thank Linda Thomas and Mia Shandell for all their help. I would like to thank my committee memb ers, Dr. David Bloom, Dr. Donald Forrester and Dr. Charles Manire, for their guidance and expertise.

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v This project would not have been possi ble without the assistance of the many people and organizations that sent samples, especially Dr. Ruth Ewing, Dr. Charles Manire, Dr. Jeremiah Saliki and Dr. Forrest Townsend. Big thanks go to all of them! They keep this laboratory going. I would like to thank the computer guys at CSM for solving all my computer mishaps, and although they could not retrieve a ny of my lost thesis information when my computer crashed, I know th ey gave it their all. Last but not least, I thank my sister Ali, my Florid ian significant other Maura, and my husband Brock for always believing in me, even when I did not believe in myself. Finally, this work was supported by a gr ant from Harbor Branch Oceanographic Institution and Florida Fish and Wildlife Co mmission through the Marine Animal Health Program of the College of Veterinary Me dicine at the University of Florida.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 CETACEAN HERPESVIRUSES................................................................................1 Introduction................................................................................................................... 1 Materials and Methods...............................................................................................12 Sample Acquisition and DNA Extraction...........................................................12 Nested PCR Targeting the DNA Polymerase Gene............................................13 Cloning, Sequencing and Sequence Analysis.....................................................15 PCR Targeting the DNA Terminase Gene..........................................................16 Results........................................................................................................................ .17 Nested PCR Targeting the DNA Polymerase Gene............................................17 PCR Targeting the DNA Terminase Gene..........................................................38 Discussion...................................................................................................................40 2 PORPOISE MORBILLIVIRUS.................................................................................50 Introduction.................................................................................................................50 Materials and Methods...............................................................................................56 Analysis of PMV.................................................................................................56 Reverse transcription....................................................................................56 RACE and amplification of the terminal extragenic domains.....................57 Amplification of the complete PMV N gene...............................................58 Amplification of the C-terminus of the PMV N gene..................................60 Detection of N protein e xpression by Western blot.....................................60 Analysis of Stranded Cetacean Tissues for Morbillivirus...................................61 Sample acquisition, RNA extractio n and reverse transcription...................61 PCR targeting the P gene.............................................................................61 PCR targeting the -actin gene.....................................................................62 Results........................................................................................................................ .63

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vii Analysis of PMV.................................................................................................63 RACE and amplification of the terminal extragenic domains.....................63 Amplification of the complete PMV N gene...............................................65 Detection of protein e xpression by Western blot.........................................74 Analysis of Stranded Cetacean Tissues for Morbillivirus...................................74 Discussion...................................................................................................................74 APPENDIX A MARINE MAMMAL SAMPLES..............................................................................84 B CETACEAN TISSUE SAMPLES.............................................................................93 LIST OF REFERENCES...................................................................................................97 BIOGRAPHICAL SKETCH...........................................................................................106

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viii LIST OF TABLES Table page 1-1. Percent nucleotide identities of DNA polymerase gene fragments among members of the family Herpesviridae......................................................................31 1-2. Percent amino acid iden tities of DNA polymerase gene fragments among members of the family Herpesviridae......................................................................32 1-3. Percent amino acid similarities of DNA polymerase gene fragments among members of the family Herpesviridae......................................................................33 2-1. Percent nucleotide identities of morbillivirus N genes............................................67 2-2. Percent amino acid identitie s of morbillivirus N genes...........................................67 2-3. Percent amino acid similariti es of morbillivirus N genes........................................67 A-1. List of all samples tested for herp esvirus using the nested PCR approach..............85 B-1. List of all samples tested for morbilli virus nucleic acid using PCR targeting the phosphoprotein gene................................................................................................94

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ix LIST OF FIGURES Figure page 1-1. Schematic of the location of the primer s used in the nested PCR targeting the herpesvirus DNA polymerase gene..........................................................................14 1-2. Gel electrophoresis of herp esvirus nested PCR products.........................................17 1-3. Wart-like lesion on the genital sl it of a female dwarf sperm whale.........................18 1-4. Genital lesions of two captiv e Atlantic bottlenose dolphins....................................19 1-5. Blainville’s beaked whale penile lesion...................................................................20 1-6. Atlantic bottlenose dolphin dermatitis lesions.........................................................21 1-7. Epithelial cells from a skin lesion of a bottlenose dolphin......................................22 1-8. Atlantic bottlenose dolphin with lesions on the tongue and the penis.....................23 1-9. Gel electrophoresis of large DNA po lymerase fragments from cetacean gammaherpesviruses................................................................................................25 1-10. Multiple alignments of the nucleotid e sequences of the DNA polymerase gene fragments of cetacean herpesviruses........................................................................27 1-11. Multiple alignments of the deduced amino acid sequences of the DNA polymerase gene fragments ofcetacean herpesviruses.............................................30 1-12. Neighbor-Joining phylogenetic tree of th e deduced amino acid sequences of the herpesvirus DNA polymerase gene..........................................................................34 1-13. Neighbor-Joining phylogenetic trees of the nucleotide sequences of the DNA polymerase gene of cetacean herpesviruses.............................................................36 1-14. Neighbor-Joining phylogenetic trees of the deduced am ino acid sequences of the DNA polymerase gene of cetacean herpesviruses...................................................37 1-15. Comparison of the nucleotide sequences of the DNA terminase gene fragment of cetacean alphaherpesviruses................................................................................39

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x 1-16. Comparison of the deduced amino acid sequences of the DNA terminase gene fragment of cetacean alphaherpesviruses.................................................................39 2-1. Schematic of the primers used to sequence the full nucleocapsid gene of PMV.........................................................................................................................59 2-2. Gel electrophoresis of ta iled and untailed PCR products.........................................64 2-3. Nucleotide sequence comparison of the genomic promoter region of morbilliviruses..........................................................................................................65 2-4. Nucleotide sequence comparison of the antigenomic promoter region of morbilliviruses..........................................................................................................65 2-5. Gel electrophoresis of the overlapping PCR products used to obtain the full N gene sequence of PMV.............................................................................................66 2-6. Neighbor-Joining phylogenetic trees of the nucleotide sequences of the complete N gene of morbilliviruses.........................................................................................68 2-7. Neighbor-Joining phylogenetic trees of the deduced amino acid sequences from the complete N gene of morbilliviruses...................................................................69 2-8. Nucleotide comparison and consensus sequence for the full N gene of three morbilliviruses than infect marine mammals...........................................................70 2-9. Amino acid comparison and consensus sequen ce of the full nucleocapsid gene of morbilliviruses.....................................................................................................72 2-10. Expression of the N gene hypervariabl e C-terminus of DMV as detected by Western blot.............................................................................................................74

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xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MOLECULAR IDENTIFICATION AND GE NETIC CHARACTERIZATION OF CETACEAN HERPESVIRUSES AND PORPOISE MORBILLIVIRUS By Kara Ann Smolarek Benson August 2005 Chair: Carlos H. Romero Major Department: Veterinary Medicine Extracted DNA from tissues and lesions of captive and stranded cetaceans was analyzed for the presence of herpesvirus ge nomes by nested and direct polymerase chain reactions (PCR) using consensus primers. The targeted sequence corresponded to a region of the DNA polymerase gene containing multiple conserved amino acid motifs. Herpesvirus genomic DNA fragments were amp lified by nested PCR, from nine lesions out of 118 lesion samples from eight animals, while none of the stra nded cetacean tissue samples were positive for herpesvirus using th e same technique. Sequence analysis of the small DNA fragments indicated that alpha or gamma herpesvi ruses were present in the positive cetacean lesions. Alphaherpesvirus DNA was detected in skin lesions of two Atlantic bottlenose dolphins ( Tursiops truncatus ), while gammaherpesvirus DNA was amplified from genital lesions from three Atlantic bottlenose dol phins, one dwarf sperm whale ( Kogia sima ) and one Blainville’s beaked whale ( Mesoplodon densirostris ), as well as one oral lesion from an Atlantic bottlenose dolphin. Longer amplicons were

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xii obtained using direct PCR from the six geni tal gammaherpesviruses, but could not be amplified from lesions containing alphaherp esviruses. Upon sequencing and phylogenetic analysis of cetacean herpesviruses DNA polymer ase gene fragments, they were shown to be most similar to Varicell oviruses and Rhadinoviruses, re spectively. Furthermore, these analyses showed that the cetacean herpesvi ruses were genetically distinct from known herpesviruses of fish, marine turtles and pinnipeds. These findi ngs strongly indicate that these herpesviruses are cetacean specific and most likely coev olved with their hosts. The terminal extragenic domains and th e complete nucleocapsid (N) gene of porpoise morbillivirus (PMV) were sequen ced to better understand the phylogenetic relationship between PMV and dolphin morb illivirus (DMV). Furthermore, tissue samples from four species of cetaceans th at were involved in recent mass stranding events in the southeast U.S. were tested by PCR for the presence of morbillivirus nucleic acid using consensus primers that target the phosphoprotein (P) gene. High sequence homologies of the terminal extragenic dom ains and the complete N gene of PMV and DMV were suggestive of a common cetacean mo rbillivirus, with tw o strains: PMV and DMV. Phylogenetic analyses confirmed th at PMV and DMV were closely related and that the cetacean morbillivirus es represent an independent morbillivirus lineage, which likely evolved with their hosts. In agreemen t with previous studies, our results have indicated that the cetacean morbilliviruses are more closely related to rinderpest virus, peste-des-petits ruminant virus and measles vi rus than they are to canine distemper virus and phocine distemper virus of carnivores. None of the tissue samples from recently stranded cetaceans in waters of the US South east coasts were positive for morbillivirus by PCR.

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1 CHAPTER 1 CETACEAN HERPESVIRUSES Introduction Herpesviruses are enveloped viruses that vary in size from 120 to 200 nm in diameter. They have a unique tegument wh ich surrounds an icosahedral nucleocapsid that is approximately 100 nm in diameter. The genome is composed of a single, linear, double stranded DNA molecule ranging in si ze from 125 to 235 Kbp (Kilo base pairs). They encode between 70 (the smallest genome) and 200 (the largest genome) genes (Roizmann and Pellet, 2001). Herpesviruses ha ve two categories of genes: Those needed for viral replication (immediate early and early genes) and structural pr oteins (late genes). Some also produce a heterologous set of opti onal genes. Replication of new viral DNA and virus assembly occur in the nucleus. Herpesviruses obtain their envelope by budding through the inner layer of the nuclear envelope of the host cell and the integrity of the envelope appears to be essential for inf ectivity (Roizmann and Pellet, 2001). Landmarks of herpesvirus infection incl ude latency and reactivation. All known herpesviruses undergo persiste nt infection with periodic or continuous shedding of infectious virus. Viral reactivation is often caused by stress, intercurrent disease, trauma, hormonal irregularities, immunosuppression or waning immunity (Murphy et al., 1999). Eosinophilic intranuclear inclusi on bodies are characteristic of herpesvirus infection and are the result of late degene rative changes, as well as c ondensation and margination of chromatin. Some herpesvirus infections also produce syncytia resulting from the fusion of infected cells with neighboring infected or uninfected cells. In ge neral, herpesviruses

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2 have a narrow host range and transmission requires close cont act, usually between mucosal surfaces (Murphy et al., 1999). Herpesviruses are divided into three main subfamilies, Alpha-, Betaand Gammaherpesvirinae, which were originally ba sed on biological characteristics, but more recently, with gene content and sequence sim ilarity (Roizmann and Pellet, 2001). Most alphaherpesviruses grow rapidly, lyse infected cells and establish latent infections in sensory ganglia of the central nervous system Many produce localized infections of the skin and mucosas of the respiratory or ge nital tract, while general infection is characterized by necrosis of organs and tissu es. The latent genome of alphaherpesviruses is essentially silent except for the production of a latency-associated transcript (LAT). The subfamily Betaherpesvirinae comprises the cytomegaloviruses, which may remain latent in secretory glands, lymphoreticul ar tissues and kidneys. Betaherpesvirus replication is slow and cell lysis does not occur until several days after infection. Gammaherpesviruses are lymphotropic and beco me latent in lymphocytes. They often have a narrow host range and some can be linked to oncogenic transformation of lymphocytes. These viruses typically cause chronic infection lasting several months before clinical recovery. Gammaherpesvirus es replicate in lymphoblastoid cells, with different viruses being specific for either B or T lymphocytes. These viruses can also enter a lytic stage in which they can cause ce ll death without the production of virions. Herpesviruses are evolutionarily old and have been found in almost every species of bird and mammal that has been investig ated, as well as reptiles, amphibians and mollusks (Murphy et al., 1999). Molecular phylogen etic studies suggest that there are at least three distinct lineages of herpesvi ruses (mammalian/avian, fish/amphibian and

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3 invertebrate) that arose approximately 180 to 220 million years ago (McGeoch et al., 1995), and because each virus is unique, they are believed to have coevolved with their host species (Murphy et al., 1999). Herpesviruses infect a vari ety of aquatic vertebrates including teleost (bony) fish, chelonians (turtl es and tortoises), pi nnipeds (seals and sea lions) and cetaceans (dolphins, porpoises a nd whales) (Roizmann et al., 1992; KennedyStoskopf, 2001). The most studied fish herpesviruses in clude salmonid herpes virus-1 (SalHV-1), salmonid herpesvirus-2 (SalHV-2), channel catfish virus (CCV) and koi herpesvirus (KHV). Wolf et al. (1978) isolat ed SalHV-1 from rainbow trout ( Oncorhynchua mykiss ), and the virus was shown to indu ce syncytia and intranuclear inclusions when inoculated into other salmonid fish cell lines. Structura lly, the genome of SalHV-1 is characteristic of the Varicellovirus genus of the subfamily Alphaherpesvirinae. However, no genetic relationship was found between SalHV-1 and mammalian herpesviruses leading to the conclusion that this genome structure evol ved independently in both the mammalian and fish herpesvirus lineages (Davison, 1998). SalHV-2 was first isolated from landlocked masu salmon ( Oncorhynchus masou ) in Japan (Kimura et al., 1981). SalHV-2 is most closely related to SalHV-1, but is serologically distinct and has a wider host range. CCV was isolated during epizootics of an acute he morrhagic disease of high mortality in young channel catfish ( Ictalurus punctatus ) (Fijan et al., 1970) and was classified as a herpesvirus based upon the presence of intranuclear inclusions and syncytia as well as its virus particle morphology (Wolf and Darlin gton, 1971). Initially classified as an alphaherpesvirus based on virion morphol ogy, complete genome analysis found no relationship with mammalian herpesviruses; thus, CCV was placed in a new subfamily,

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4 Ictalurivirus (Davison, 1992). A herpesviru s was also isolated from adult koi ( Cyprinus carpio ), a strain of common carp that suffere d mass mortality during outbreaks in the mid-Atlantic region of the U.S. and in Israel (Hedrick et al., 2000). KHV was distinguished from other known herpesviruses of carp, namely carp pox ( Cyprinid herpesvirus ), by indirect fluores cent antibody tests. Herpesviruses infect many species of cheloni ans, including marine turtles, of which all seven species are either endangered or threatened. Herpesviruses have been associated with two diseases of mariculture-reared green turtles ( Chelonia mydas ) in the Cayman Islands. Lung-eye-trachea disease ( LETD) associated herpesvirus was isolated from juveniles with conjunctivitis, trachei tis and severe pneum onia (Jacobson et al., 1986). Gray-patch disease (GPD) was also conc luded to be of herpesvirus etiology based on virus particle morphology and the presence of intranuclear inclusi on bodies (Rebell et al., 1975). Fibropapillomatosis (FP) is a neoplastic disease that causes external fibropapillomas of the skin, eyes, oral cavity an d carapace, as well as internal fibromas of the visceral organs. Although the disease itself is usually not fatal, the large tumors can interfere with locomotion, vision, swallowing, and breathing, and can also affect organ function. FP was first described over 60 year s ago and has been rapidly increasing in prevalence worldwide. Un til recently, the etiology of FP was unknown, but multiple studies have suggested that a virus is involved in the pathog enesis of this disease. Papillomavirus was first suspected, but Sout hern blot hybridization of green turtle fibropapillomas failed to de tect any papillomavirus DNA (Jacobson et al., 1989). Multiple studies have now suggested that a herp esvirus (FPHV) is the etiologic agent that causes FP; however, definitive evidence of the involvement of papillomavirus, or any

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5 other agent, is not yet ava ilable. Polymerase chain re action (PCR) has demonstrated herpesvirus genomic DNA in fibropapillomas of four species of mari ne turtles including greens, loggerheads ( Caretta caretta ), olive ridleys ( Lepidochelys olivacea ) and Kemp’s ridleys ( Lepidochelys kempii ) (Quackenbush et al., 1998; Lac kovich et al., 1999; Lu et al., 2000; Yu et al., 2000; Quackenbush et al., 20 01; Yu et al., 2001; He rbst et al., 2004). In addition to the external tumors, various visc eral organs, such as kidney, heart, lung and brain, have also resulted in herpesvirus DNA amplification. An extensive study was performed to investigate the genomic vari ation of FPHV across seven geographic areas and three host species (Greenbl att et al., 2005). All variants tested showed greater than 96% nucleotide sequence conservation. Sequen ce variations correla ted with geographic location but not with species. Phylogenetic analysis of a 23 Kbp fragment suggested that FPHV was an alphaherpesvirus, but not a memb er of any of the currently established genera. Natural transmission is still larg ely unknown; however He rbst et al. (1995) demonstrated that FP could be experiment ally transmitted to young captive-reared green turtles using cell-free fibropapilloma extracts prepared from free-ranging turtles with spontaneous disease. Fibropapillomas developed in all recipients and were histologically indistinguishable from tumors of free-rang ing turtles. To date, no FPHV has been isolated from any species of marine turtle. In pinnipeds, three herpesviruses have been characterized to date, including phocid herpesvirus-1 (PhHV-1), phocid herpesviru s-2 (PhHV-2) and otarine herpesvirus-1 (OtHV-1). PhHV-1 was first seen in young harbor seals ( Phoca vitulina ) in captivity in The Netherlands and was associated with acu te pneumonia, focal hepatitis, general depression and high mortality (Osterhaus et al., 1985). Upon isolation, electron

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6 microscopy (EM) revealed herpesvirus-like pa rticles and intranucle ar inclusions. The virus was also shown to be antigenically related to canine herpesvirus (CHV) and feline herpesvirus (FHV), and was eventually classifi ed as a member of the Alphaherpesvirinae. A similar epizootic was seen in harbor seals being rehabilitated in California (King et al., 1998). Phylogenetic analysis of sequenced DNA fragments from the Pacific isolate showed greater than 95% ident ity in translated amino acids after comparisons with the Atlantic-European isolate (Har der et al., 1996). PhHV-1 has al so been associated with mass mortality of free-ranging harbor seals in northwestern Europe in 1988 (Frey et al., 1989). Goldstein et al. (2004) provided ev idence that PhHV-1 can be transmitted between harbor seals through di rect contact with oro-nasal secretions containing shedding virus. PhHV-2 has been isolated from lung tis sue of a juvenile cap tive California sea lion ( Zalophus californianus ) that was simultaneously infected with a retrovirus (KennedyStoskopf et al., 1986) and from leukocytes of a harbor seal from the German Wadden Sea (Lebich et al., 1994). At present, PhHV-2 has not been associated with clinical disease. Harder et al. (1996) performed antigenic and genetic analyses to further characterize PhHV-1 and -2. Herpesviruses were isolated from four harbor seals from Long Island as well as one harbor seal and one grey seal ( Halichoerus grypus ) from European waters. PCR was performed with partially degenerate primers based on conserved regions of the glycoprotein B and D genes of CHV, FHV and equine herpesvirus-1. Fragments amplified from PhHV-1 showed a high degree of homology with CHV and other members of the genus Varicellovirus No DNA amplification occurred with PhHV-2. However, based on sequence analysis of two EcoRI fragments, PhHV-2 was classified as a gammaherpesvirus, most closely related to equine herpesvirus-2. Serological studies

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7 have shown that herpesvirus infections occu r in at least 11 species of pinnipeds (for review, see Kennedy-Stoskopf, 2001) and antibodies have been reported in as many as 99% of subadult and adult harbor seals in No rth American populations (Goldstein et al., 2003a). OtHV-1 has been associated with urogenital carcinoma of California sea lions that stranded on the central California coast. Me tastatic carcinoma was found in sublumbar lymph nodes of ten sea lions, and upon histolog ical examination, intr aepithelial neoplasia was found in the genital tracts of all ten animals (Lipscomb et al., 2000). Eosinophilic intranuclear inclusions were seen in one anim al, herpesvirus-like pa rticles were seen in two others, and another was positive for Epstein-Barr virus (HHV-4) by immunohistochemical staining. Analysis of herpesvirus DNA sequences of the polymerase and terminase genes obtained by PCR allowed for the classification of this virus as a gammaherpesvirus in the genus Rhadinovirus Samples were also tested for papillomavirus by Southern bl ot and PCR; all samples we re negative by both methods. King et al. (2002) designed an OtHV-1 specific PCR and demons trated that the virus was present not only in tumors, but in brain a nd muscle tissue as we ll. Because viral DNA was evident in all tumors examined, it was hypothesized that OtHV-1 may be a factor in oncogenesis. Phylogenetic anal ysis of large fragments of the polymerase and terminase genes showed a clear grouping with othe r gammaherpesviruses; however OtHV-1 was distinct from PHV-2. Herpesvirus infections have been suspected to occur in three families of cetaceans, namely, Phocoenidae (porpoises), Monodontidae (belugas and narwhals) and Delphinidae (dolphins, killer whales, pilot whales and rela tives) (Van Bressem et al., 1999) for at least

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8 three decades. Although there are only a handf ul of documented cases, past reports have indicated that these viruses are important pat hogens that may be associated with localized infections of the skin and mucosas as well as systemic infections. Barr et al. (1989) described a focal necr otizing dermatitis in a young adult female beluga whale ( Delphinapterus leucas ) from the Churchill River, Manitoba, Canada. Pale, depressed lesions with irregular borders on the dorsal ridge a nd lateral side of the whale were first seen approximately 3.5 months after capture. Histologically, many eosinophilic intranuclear inclus ion bodies were seen in ep ithelial cells and herpesviruslike particles were demonstrated by transmission electron microscopy (TEM). The lesions were transient in nature, lasting appr oximately eight months, and appeared to be confined to the skin as no other health pr oblems were noticed in the animal. It was believed that the virus was either latent at capture or that it was transmitted from other beluga whales following capture. Once in captivity, the beluga wh ale also had contact with Atlantic bottlenose dolphins ( Tursiops truncatus ), California sea lions and harbor seals, all of which are sus ceptible to herpesvirus infec tions, although infections across species are rare events. A similar, yet mo re severe, viral dermatitis was reported in a free-ranging juvenile female beluga whale that stranded in the St. Lawrence Estuary, Qubec, Canada (Martineau et al., 1988). Lesions were found covering the body, were most apparent on the head and were described as pale, circular, depressed areas outlined by a narrow dark rim. Epith elial cells contained eosinophi lic intranuclear inclusion bodies shown by EM and viral particles simila r in morphology to those of herpesviruses were also seen. A second young female beluga from the same area was also reported as having widespread dermatitis, which was grossly and microscopically similar to the first.

PAGE 21

9 However, no viral particles were observed in the second animal sugges ting that lesions of the first animal may have been at an earli er stage of infection. Despite extensive dermatitis, it was determined that herpesviru s infection was not the cause of death of either whale. Beluga whales in the St. La wrence estuary tested for cetacean herpesvirus seroprevalence using bovine herpesvirus-1 (BHV-1) as the indicator in a serum neutralization test a nd a blocking enzyme-linked imm unosorbent assay (ELISA) were found to be 46 and 58 % antibody positive, respectively (Mikaelian et al., 1999). The presence of antibodies against BHV-1 was inte rpreted as previous infection of beluga whales with a closely related ceta cean alphaherpesvirus. Skin lesions associated with herpesvirus-lik e particles have been also reported in free-ranging sexually immature dusky dolphins ( Lagenorhynchus obscurus ) from Peruvian coastal waters (Van Bressem et al ., 1994). Skin lesions consisting of a few black points on the rostrum were noticed in three dusky dolphins, while a fourth had lesions dispersed all over its body. The ep ithelial cells of two dusky dolphins were shown by TEM to contain virus particles with morphological features similar to those of herpesviruses. No virus particles were det ected in the other two dolphins, as the lesions may have been in a convalescent stage. A lthough the clinical signs resembled those of alphaherpesvirus infection, antigenic or mol ecular characterization was not performed. The virus strain involved in these cases s howed a skin tropism and seemed to be only mildly pathogenic, as no other evidence of disease was observed. It should be noted that the dusky dolphin is a highly sociable species and can congregate in supergroups of 700 to 800 individuals, which may facilitate eas y direct transmission of this virus.

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10 Although the presence of herp esvirus or herpesvirus-like particles was not confirmed, skin lesions in a killer whale ( Orcinus orca ), a striped dolphin ( Stenella coeruleoalba ) and three harbor porpoises ( Phocoena phocoena ) were also reported to be the result of herpesvirus inf ection. Greenwood et al. (1974) reported a killer whale that developed a widespread rash of small ve sicles, which was described as closely resembling a varicella infection. Varicella zoster an alphaherpesvirus commonly known as chickenpox, is caused by human herpesvirus3. Baker (1992) surveyed skin lesions in wild cetaceans from British waters and reporte d possible herpesvirus infections in one striped dolphin and three harbor porpoises. The diagnosis of the four cases of herpesvirus infection was based on the simila rity of gross pathol ogy to the herpesvirus infections seen in beluga wh ales (Martineau et al., 1988; Ba rr et al., 1989). Histological examination of lesion samples revealed the presence of large eosi nophilic intranuclear inclusions in epithelial cells. A herpesvirus has been described also in association with genital lesions on the penile mucosa of an adult harbor porpoise found on the New Jersey coast (Lipscomb et al., 1996a). Histol ogically, epithelial ce lls contained many eosinophilic intranuclear inclus ion bodies and immunohistoche mical stainings for herpes simplex-1 and -2 were positive. More serious disease in th e form of herpesvirus ence phalitis was diagnosed in a juvenile female harbor porpoi se that stranded off the coast of Sweden (Kennedy et al., 1992). Skin lesions, which were morphologically similar to those of beluga whales (Barr et al., 1989; Martineau et al., 1988) were f ound on the head, thorax and abdomen of the animal, however, there was no histological or immunohistochemical evidence to support a herpesvirus etiology. Severa l tissues were examined for histopathologic lesions but

PAGE 23

11 acidophilic intranuclear inclusions were seen only in the cerebral cortex. Herpesviruslike particles were observed by EM in aff ected neurons and herpesviral antigen was detected by immunoperoxidase staining of the cerebral cortex. Immunologic crossreactivity between the presumed porpoise herp esvirus and antisera to pseudorabies virus, human herpesvirus-1 and bovine herpesvirus-1 i ndicated that the porpoise virus was most likely an alphaherpesvirus. The presence of encephalitis in this por poise indicates that fatal herpesviral disease in free-ranging cetaceans is possible. More recently, two novel alphaherpesviruses have been described in association with disseminated infection in Atlantic bot tlenose dolphins that stranded along the East coast of the U.S. (Blanchard et al., 2001). Two juvenile female bottlenose dolphins, one found on Hilton Head Island, South Carolina (case 1), the other on Prime Hook Beach, Delaware (case 2), presented with acute necrotizing lesions in multiple organ systems, which was the leading cause of death in both cases. Examination of various tissues by TEM revealed the presence of herpesvirus-lik e particles in the nuc leus and enveloped virions in the cytoplasm. PCR targeti ng the herpesvirus DNA polymerase and DNA terminase genes confirmed the diagnosis of al phaherpesvirus infection. Case 1 yielded a 189-bp product from lung tissue while case 2 yielded a 180-bp product from heart tissue of a highly conserved region of the DNA polymerase gene (GenBank accession numbers AF196646 and AF245443, respectively). A part ial terminase gene sequence of 375-bp was also obtained from case 1 (GenBa nk accession number AF196647). Phylogenetic analyses of these herpesvirus genome fragment s revealed that they were most closely related to each other and not very closely related to herpes viruses found in other marine animals, namely, harbor seals, sea lions and green sea turtles. Because the dolphins

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12 presented with symptoms characteristic of both herpesvirus and mo rbillivirus infection, such as syncytial cells, intranuclear in clusions and lymphoid depletion, reverse transcriptase PCR (RT-PCR) was also performed to detect possible morbillivirus; however, results were negative. This was the first report of disseminated herpesvirus infection in cetaceans as we ll as the first molecular ev idence of the existence of alphaherpesviruses in cetaceans. The objective of this research was to de velop a diagnostic assay for herpesviruses infecting tissues and lesions of captive and free-ranging cetaceans. A previously described PCR targeting highly conserved re gions of the herpesvirus DNA polymerase gene (VanDevanter et al., 1996) was selected as a starting point because it has been shown to amplify herpesvirus DNA in other aquatic animals. Utilizing this same technique, FPHV sequences were obtained from three species of marine turtles (Quackenbush et al., 1998), and a novel ga mmaherpesvirus was identified in Hawaiian monk seals ( Monachus schauinslandi ) (Goldstein et al ., 2003b). The results of the assays with cetacean samples were then used for the genetic identification and molecular characterization of these novel cetacean herpesviruses. Materials and Methods Sample Acquisition and DNA Extraction Lesion and tissue samples were obtained from stranded cetaceans from Florida, Georgia, North and South Carolina, Texas and Alaska waters. Scrapings or biopsy lesions were also collected from live cetacean s at wildlife parks and rehabilitation centers at various locations throughout Florida and Ca lifornia. In total, 118 lesion samples encompassing 12 cetacean species were analyzed for herpesvirus genomes; 87 from skin lesions and 31 from mucosal lesions. Sevent y-two tissue samples, mostly from lung and

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13 brain, were also tested from five cetacean species. In addition to cetacean samples, lesions from three species of marine turt les and one pinniped we re also tested. A comprehensive listing of all samples te sted is presented in Appendix A. Total DNA was extracted from all sample s using the DNeasy Tissue Kit (Qiagen Inc., Valencia, California, USA) according to the manufacturer’s protocol. Briefly, the sample was crushed in a microfuge tube w ith a sterile pestle and allowed to lyse overnight in lysis buffer at 55C. DNA wa s precipitated by the addition of absolute ethanol and applied to a spin column, washed a nd eluted in 200 l of elution buffer. The total DNA content and quality of the eluted DNA were determined using the Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). Nested PCR Targeting the DNA Polymerase Gene Herpesvirus DNA was amplified using publis hed degenerate primers designed to target a region of the DNA polymerase gene of herpesviruses that corresponds to highly conserved amino acid motifs (VanDevanter et al., 1996; Ehlers et al., 1999). These primers are known to direct the amplification of DNA polymerase gene fragments 215 to 235-bp in length for most herpesviruses and 315-bp for cytomegaloviruses. A nested PCR assay was performed with two forward a nd one reverse primers in the first reaction, and one forward and one reverse primer in the second reaction. Primer sequences for the first reaction were: DFA-5’GAY TTY GCI AGY YTI TAY CC -3' (forward), ILK-5’TCC TGG ACA AGC AGC ARI YSG CIM TI A A -3' (forward), KG1-5’GTC TTG CTC ACC AGI TCI ACI CCY TT -3' (reverse) Primers for the second reaction were: TGV-5’TGT AAC TCG GTG TAY GGI TTY AC I GGI GT -3'(forward), IYG-5’CAC AGA GTC CGT RTC ICC RTA IAT -3' (revers e). (See Figure 1-1.) Total DNA extracted from cell monolayers of Madin Dar by canine kidney (MDCK) cultures infected

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14 with canine herpesvirus (CHV) and from cell mo nolayers of tortoise heart (TH) cultures infected with the tortoise herpesvirus-1 (T HV-1) were used as positive templates for PCR. In the first PCR, approximately 500 ng of sample DNA was used as template. The first PCR mixture contained 400 nM of each primer, 100 M of each dNTP, 10 mM KCl, 10 mM (NH4)2 SO4, 20 mM Tris-HCl, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1 unit of Taq DNA polymerase (New England BioLabs Inc., Beverly, Massachusetts, USA). All PCRs were run in a PTC-100 Pr ogrammable Thermal Cycler (MJ Research, Inc., Waltham, Massachusetts, USA). Cycli ng conditions for the first and second PCR were: Initial incubation at 94C for 2 min fo llowed by 55 cycles at 94C for 20 sec, 46C for 30 sec, and 72C for 30 sec. A final exte nsion step at 72C for 10 min finished the cycling. In the second PCR, mixtures were identical to those in the first PCR but contained 400 nM of each of the second reaction primers and 2 l of the first PCR product as DNA template. Approximately 20 l from the second PCR were resolved by horizontal gel electrophoresis in 1.0% agarose containing ethidium bromide (0.5 ug/ml) and the DNA fragments were visualized by UV light transillumination and photographed using a gel documentation system. Amplified DNA fragments of the predicted size were purified using the MinElute PCR Purificati on Kit (Qiagen Inc., Va lencia, California, USA). Figure 1-1. Schematic of the locat ion of the primers used in the nested PCR targeting the herpesvirus DNA polymerase gene (VanDevanter et al., 1996). In order to obtain additional nucleotide sequence, samples that were positive for herpesvirus DNA by the nested PCR were used subsequently for the amplification of a

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15 larger fragment of the DNA polymerase ge ne, approximately 700-bp in length, using primers DFA and KG1. The PCR mixture co ntained approximately 500 ng of sample DNA, 400 nM of each primer, 100 M of each dNTP, 2 mM MgCl2, 1X Expand High Fidelity buffer and 3.3 units of Expand Hi gh Fidelity enzyme mix (Roche Applied Science, Indianapolis, Indiana, USA). Cy cling conditions were: Initial denaturing at 94C for 2 min followed by 40 cycles at 94C fo r 30 sec, variable annealing temperature for 30 sec and extension at 72C for 2 min. A final extension step at 72C for 10 min finished the cycling. The annealing temper ature varied between 42 and 47C depending on the species of DNA template being assa yed. If the amplification of the 700-bp fragment was unsuccessful, a third PCR was attempted with primers DFA and IYG to amplify approximately 500-bp of herpesvirus DNA. The Expand High Fidelity PCR system (Roche Applied Science, Indianapol is, Indiana, USA) was used in the PCR mixture and all the quantities were the same as above. Cycling conditions were: Initial denaturing at 94C for 1 min followed by 40 cycl es at 94C for 30 sec, variable annealing temperature for 30 sec, extension at 72C for 1 min and a final extension step at 72C for 10 min. As above, the annealing temperatur e varied between 42 and 47C depending on the species of DNA template being assayed. Amplified DNA fragments of the predicted size from both subsequent PCRs were purif ied using the MinElute PCR Purification Kit (Qiagen Inc., Valencia, Califor nia, USA) or excised afte r gel electrophoresis in 1.2% low-melting-point agarose and purified using the MinElute Gel Extraction Kit (Qiagen Inc., Valencia, California, USA). Cloning, Sequencing and Sequence Analysis Purified DNA fragments were cloned into the plasmid vector pCR2.1-TOPO T/A (Invitrogen, Carlsbad, California, USA). Two clones for each sample were selected for

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16 plasmid purification and sequenced in duplic ate from both ends using the CEQ 2000 XL (Beckman Coulter Inc., Fullerton, California, USA) sequencing instrument, following the manufacturer’s protoc ol. Briefly, the 20 l sequencing reaction contained approximately 100 fmol of total DNA, 2 l of either the forward or reverse M13 primer, 4 l DTCS Quick Start Master Mix, 1 l sequencing buffer and ultra pure water. Cycling conditions consisted of 50 cycles at 96 C for 20 sec, 50 C for 20 sec and 60 C for 4 min following an initial denaturation step at 96 C for 2 min. Exported chromatograms were manually reviewed using the Chromas 2.3 software (Technelysium Pty Ltd., Tewantin, Queensland, Australia). Genetic analyses were performed using the functions Seqed, Gap, Translate, Lineup, Pileup and Pretty of the University of Wisconsin Package Version 10.2, Genetics Computer Group (GCG ), Madison, Wisconsin, USA. Multiple sequence alignments and phylogenetic anal yses were performed using PAUP* 4.0 (Sinauer Associates, Sunderland, Massachus etts, USA). The BL AST function of the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) was used to iden tify herpesvirus sequences more closely related to those of the cetacean herpesviruses. PCR Targeting the DNA Terminase Gene Samples positive for herpesvirus polymera se gene DNA by the nested PCR were tested subsequently for cetacean herpesvi rus terminase DNA. Primers were designed from the terminase sequence (GenBank acces sion number AF196647) of an Atlantic bottlenose dolphin herpesvirus (Blanchard et al., 2001); 5’ACC AAC ACC GGC AAA GCT A -3’ (forward), 5’CAC GTA CAC GAA CAG TTC C -3’ (reverse). The PCR mixture contained approximately 500 ng of sample DNA, 400 M of each primer, 100

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17 mM of each dNTP, 10 mM KCl, 10 mM (NH4)2 SO4, 20 mM Tris-HCl, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1 unit of Taq DNA polymerase (New England BioLabs Inc., Beverly, Massachusetts, USA). Cycling conditions were: Initial incubation at 94C for 2 min followed by 40 cycles at 94C for 30 sec, 50C for 30 sec, and 72C for 30 sec. DNA fragments of the expected size were purified, cloned, sequenced and analyzed as previously described. Results Nested PCR Targeting the DNA Polymerase Gene The degenerate primer nested PCR appr oach, as previously described for the detection of herpesvirus genomic sequences of terrestrial mammals, was also successful in the amplification of DNA polymerase gene fragments from total DNA from cutaneous and mucosal lesions of cetaceans. Fragments of the herpesvirus DNA polymerase gene were amplified from nine lesion samples from three species of cetaceans, as well as 15 fibropapilloma samples from two species of turtles (Figure 1-2). Positive cetacean samples are described in detail below. Figure 1-2. Gel electrophoresis of herpesvirus nested PCR products; lane 1K311 bottlenose dolphin penile lesion, lane 2K263 bottlenose dolphin vaginal lesion, lane 3K310 bottlenose dolphin penile lesion, lane 4K308 bottlenose dolphin tongue lesion, lane 5K264 bottl enose dolphin penile lesion, lane 6K265 dwarf sperm whale vaginal lesion, lane 7K285 Blainville’s beaked whale penile lesion, lane 8K167 bottlenose dolphin skin lesion, lane 9K231 bottlenose dolphin skin lesion, lane 10green turtle fibropapilloma, lane 11Kemp’s ridley turtle fibropapilloma, lane 12negative control, lane 13positive control, CHV.

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18 An adult female dwarf sperm whale ( Kogia sima ) that had repeatedly stranded in Manatee County, Florida was euthanized in a deteriorating condition. Sample K265 was taken from a wart-like lesion located on the ge nital slit and tested pos itive for herpesvirus DNA. Several lymph nodes from this animal we re also tested, but were all negative by the nested PCR approach. Figure 1-3. Wart-like lesion (arrow) on the ge nital slit of a female dwarf sperm whale (K265). Photo provided by Dr. Nlio B. Barros. Two Atlantic bottlenose dol phins from a Florida wild life park presented with genital lesions. Sample K311 was a scraping of a plaque on the penile mucosa of an adult male (Figure 1-4A) and K263 was a scra ping of vaginal sores of an adult female (Figure 1-4B and C). Evidence of sexua l activity between these two animals was apparent as they have produced offspring.

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19 A C B Figure 1-4. Genital lesions of two captive Atla ntic bottlenose dolphins A) Penile lesions (K311) B) vaginal sores C) vaginal sores 10X. An adult male Blainville’s beaked whale ( Mesoplodon densirostris ) was found stranded on Kure Beach, North Carolina in January, 2004 with a papilloma-like penile lesion (K285) (Figure 1-5A). Penile epithelial cells contained 4 to 6 intranuclear inclusions shown by hematoxylin and eo sin (H&E) staining (Figure 1-5B).

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20 A B Figure 1-5. Blainville’s beaked whale (K285) A) Penile lesion B) H&E stain showing multiple cells that contain intranuclear inclusions (arrows). Bar = 20 m. Photos provided by Dr. Jeremiah T. Saliki. A stranded juvenile male Atlantic bottlenose dolphin was admitted to the Dolphin and Whale Hospital, Mote Marine Laboratory and Aquarium, Sarasota, Florida with mild dermatitis on the rostrum, head, dorsal fins, flanks, peduncle and flukes (Figure 1-6). Lesions were described as hundreds of 1 to 3 mm, spherical, raised, black papules and

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21 biopsies were taken 1 to 2 months post-admi ssion (K167). Lesions regressed slowly and were almost completely resolved by 3 mont hs post-admission. Hist ologically, epithelial cells contained intranuclear inclusions (Fi gure 1-7A) and herpesviru s-like virions were shown by TEM (Figure 1-7B). A B Figure 1-6. Atlantic bottlenose dolphin derm atitis lesions (K167) on the A) rostrum and B) skin. Photos provided by Dr. Charles A. Manire.

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22 A B Figure 1-7. Epithelial cells from a skin lesion of a bottlenos e dolphin (K167). A) Intranuclear inclusion bodies (arrows) seen w ith H&E staining. B) Transmission electron micrograph illust rating non-enveloped nucleocapsids (arrowheads) in the nucleus and an enve loped virion (arrow) in the cytoplasm adjacent to the nuclear membrane (nm). Uranyl acetate and lead citrate, bar = 250 nm. Images provided by Dr. Michael Kinsel.

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23 In June 2001, an adult male Atlantic bottlenose dolphin stranded offshore of Islamorada Key, Florida. Reddened ulceratio ns were widely appa rent on the tongue (K308) (Figure 1-8A) and plaque s were seen on the penile mucosa (K310) (Figure 1-8B). Total DNA extracted from the penile and oral lesions were both positive for herpesvirus by PCR. Amphophilic intranuclear inclusions an d syncytia were observed in a cluster of vessels in the lung; however DNA extracted from lung tissue yielde d negative results by nested PCR. This animal was severely debilitated and immunocompromised, and ultimately died of sepsis. A B Figure 1-8. Atlantic bottlenose dolphin with lesions on A) the tongue and B) the penis. Photos provided by Dr. Ruth Ewing. Sample K231 was obtained from a juvenile male Atlantic bottlenose dolphin from a wildlife park in Florida. The animal had multiple skin lesions on the right lateral side, which were described as 1mm black pinpoints that could be palpat ed. Histological changes were not suggestive of a herpesvirus infection.

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24 Sample K264 was obtained during necropsy fr om a penile lesion of an Atlantic bottlenose dolphin that stranded along the coast of Jacksonville, Florida. Sequencing of the DNA fragments amplified from cetacean lesions after the nested PCR approach demonstrated that they range d in size from 222 to 244 nucleotides in length, and translation of these nucleotid e sequences, resulted in DNA polymerase fragments that ranged in si ze from 73 to 80 amino acid residues. The subsequent PCR with primers DFA and KG1 amplified 731-bp am plicons that translated into proteins composed of 243 amino acid residues, from six animals (Figure 1-9). These primers were used also to amplify a 737-bp, 245 amino acid residue fragment from CHV. This sequence has been deposited in the GenB ank database under the accession number AY949827. Larger fragments from the remain ing samples that were positive for the small DNA polymerase fragment could not be obtained by any of the PCR approaches attempted. All cetacean herpesvirus sequen ces have been deposited in the GenBank database. Accession numbers for individual samples are: K285 Blainville’s beaked whale penile lesionAY949828, K265 dwarf sperm whale vaginal slit lesionAY949830, K311 bottlenose dolphin penile lesionAY 949831, K231 bottlenose dolphin skin lesionAY949832, K264 bottlenose dolphin penile le sionAY952776, K263 bottlenose dolphin vaginal lesionAY952777, K310 bottlenose dolphin penile lesionAY952778, K308 bottlenose dolphin tongue lesionAY952779, K167 bottlenose dolphin skin lesionAY757301. Multiple sequence alignments of the nucleotide and amino acid sequences derived from eight positive cetacean samples indicated that viruses that could be placed in two virus groups were present (Figures 1-10 and 1-11). The sequence from sample K308 was

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25 excluded from the alignment as it was identical to the sequence of a separate lesion (K310) from the same animal. Nucleotide and amino acid homologies with multiple known herpesviruses indicated that cetacean herpesviruses fell within the Alphaand Gammaherpesvirinae subfamilies (Tables 1-1, 12 and 1-3). Although genetic variability was observed within the alpha and gammaherpesvirus sequen ces, phylogenetic analyses clearly indicated that both al phaherpesviruses and gammaherp esviruses infect cetaceans (Figures 1-12, 1-13 and 1-14). Figure 1-9. Gel electrophores is of large DNA polymerase fragments from cetacean gammaherpesviruses; lane 1K263 bottl enose dolphin vaginal lesion, lane 2K311 bottlenose dolphin penile lesion, la ne 3K310 bottlenose dolphin penile lesion, lane 4K264 bottle nose dolphin penile lesi on, lane 5Blainville’s beaked whale penile lesion, lane 6K265 dwarf sperm whale vaginal lesion, lane 7negative control, lane 8positive control, CHV. The nested PCR approach using degenera te consensus primers has demonstrated that cutaneous lesions from cetaceans are associated with alphaherpesvirus infection while genital lesions, and in one case, an oral lesion, are associated with gammaherpesvirus infection. Alphaherpesvi rus genomic DNA was amplified from two cutaneous lesions from Atlantic bottle nose dolphins (K167 and K231). Bottlenose dolphin K231 appeared to be infected with th e same virus identified by Blanchard et al. (2001) that caused a disseminated infection in an Atlantic bottlenose dolphin from South Carolina in 1995 (GenBank accession number AF196646). Nucleotide and amino acid identities between the two dol phin alphaherpesviruses were 98.9 and 96.8%, respectively.

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26 Tissue samples for bottlenose dolphin K231 c ould not be tested for disseminated infection as the dolphin is still alive. Gammaherpesvirus genomic DNA was amplified from penile lesion samples from three A tlantic bottlenose dolphins (K311, K310 and K264) and one Blainville’s beaked whale (K285) as well as two vaginal lesions from an Atlantic bottlenose dolphin (K263) and a dwar f sperm whale (K265). In addition to the penile lesion (K310), one bottlenose dolphi n had ulcerations on the tongue (K308), and upon comparison of the sequences from both le sions, it was determined that the same virus was present in the oral and genital mu cosas. Of the cetacean tissue samples from stranded animals that were assayed none were positive for herpesvirus using the nested PCR approach. A pair of cetacean-specific primers wa s designed to amplify the small DNA polymerase gene fragment of both alpha a nd gammaherpesviruses, while a second pair was designed to amplify the large DNA polymerase gene fragment from the gammaherpesviruses. Unfortunately, neither set of primers were consis tently successful. Sequences obtained from the turtle fibr opapilloma samples were 237-bp in length and translated into polypeptides of 79 ami no acid residues. Upon comparison with FPHV sequences from the NCBI website, all amino acid homologies were greater than 96%, clearly indicating the presence of FPHV in our samples.

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27 1 50 10 0 BNDK311 .......... ..c....... .......... .......... .......... .......... .......... .......... .......... ......... BNDK263 ..t....... ..c....... .......... .......... .......... .......... .......... .......... .......... ......... BNDK310 ..a..c.... .cc....... .......... .......... ........c. .......... .......... .......... .......... ......... BNDK264 ..t....... ..t....... .......... ..g..t.... .c........ .......... .......... c......... .........c ......... BBWK285 .......... ..t....... .......... .......... .......... ...a.....t .....t..t. a......... .tt....... ...t....g DSWK265 .......... .ct....... .......... ..g....... .c.....t.. .........t ......c... a......t.. t.....t..c .at...ac. c BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ Consensus GACTTTGCTA GT-TGTACCC CAGCATCATT CAAGCCCACA ATCTGTGCTA CTCGACCCTC ATTCCCGACG GGGAGATGCA CCGGCACCCG ACGCTGCTC A 101 150 20 0 BNDK311 .......... .......... .......... .......... .......... .......... .......... .......... .......... ......... BNDK263 .......... .......... .......... .......... .......... .......... .......... .......... .......... ......... BNDK310 .......... .......... .......... .......... .......... .......... .......... .......... .......... ......... BNDK264 g......... .......... .......... .g........ ......c... .......... .c...c.... .......... ..a....... ....c.... BBWK285 ....g..... ...a.....c .....t.... .t.....t.. a.....c... ..a.....t. .g..t..... t..t...... .....t.... .a..c...t DSWK265 cc.aa..... ...c..a... .......... .g..t..g.. ...t.....t ..a..a.... .t........ t.....c..a .g...at... .t..g.... BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ Consensus AGGGCGACTT TGAGACGTTT CACATCAGTT CCGGGCCCGT GCACTTTGTG AAGAAGCACG TAACCTATTC CCTCCTATCC AAGCTGCTGG CGACTTGGC T 201 250 30 0 BNDK311 .......... .......... .......... .......... .......... .......... .......... .....a.... .......... ......... BNDK263 .......... .......... .......... .......... .......... .......... .......... .....a.... .......... ......... BNDK310 .......... .......... .......... .......... .......... .......... .......... .....a.... .......... ......... BNDK264 .......... .........a .......... .......... .......... ....c..g.. c...c..... .....g.... .......... ......... BBWK285 ...t.....t ........aa ....t..... ....g....t ..a.....t. .at.g..... ...a...... .....g.... ....t..t.. ......c.. DSWK265 ...g...... ........a. ....t...t. a..tgg...t ..c.....t. .a..c..... c..a.....t .....g.... .......... ...a..... BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~. BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~. Consensus GGCCAAGCGC AAGGCCATCC GGCGGGAGCT GAGCCAGTGC TCGGACCCCC AGCTTAAAAC TATCTTGGAC AAGCA-CAGC TCGCCATCAA GGTGACGTG T Figure 1-10. Multiple alignments of the nuc leotide sequences of the DNA polymerase gene fragments of cetacean herpesviruses. BNDbottlenose dolphin, BBWBl ainville’s beaked whale, DSWdwarf sp erm whale. K311penile lesion, K263vaginal lesion, K310penile lesion, K 264penile lesion, K285penile lesion, K265vaginal slit lesion, K167skin lesion, K231skin lesion.

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28 301 350 40 0 BNDK311 .......... .......... .......... .........t .......... .......... .......... .......... .......... ......... BNDK263 .......... .......... .......... .........t .......... .......... .......... .......... .......... ......... BNDK310 .......... .......... .......... .........t .......... .......... .......... .......... .......... ......... BNDK264 .....g.... .......... .........c .......... .......... .........c .....g.... .......... .......... ..g..g... BBWK285 ..t....... .t.....t.. a..t...... ..a......a .......... c..a...... .....t.... .a..c..... ....t..... ..a..g... DSWK265 .....c..t. .t..g..t.. t........a ..c..t..g. .a.....t.. a.....t... ..a..t..g. .ttc...a.. a..at..... ......c.c BNDK167 ...t.g.... ....g..... .......... ca......a. .......t.. .cc...c..g .c...c..a. .gac.atc.. .c..ga.... t.actc..c a BNDK231 ...t.g.... ....g..... .......... ca......g. ca........ .c....c..g .c...c..g. .gac.att.. tc..gat... ..gctgc.. a Consensus AACGCAGTGT ACGGCTTCAC GGGCGTGGCG TCGGGGCTCC TGCCGTGCCT GAAGATAGCA GAGACAGTCA CCCTGCAGGG GAGGCGCATG CTTGAAAGG T 401 450 50 0 BNDK311 ......g... .......... .......... .......... .......... .......... .......... .......... .......... ......... BNDK263 ......g... .......... .......... .......... .......... .......... .......... .......... .......... ......... BNDK310 ......g... .......... .......... .......... .......... .......... .......... .......... .......... ......... BNDK264 ....g..... .......... ..cg...g.. ...a...... .......g.. .......t.. .......... .......g.. .......... a...c.c.. BBWK285 .......... .......... ...g..t... .g.....t.. .........t ..a..ga.t. ..a.a.a... t..t.aa... t.t..a..t. aa.....t. DSWK265 .t..g..... ......a... .....t..tt ca.a...t.. a......... ....g...c. .ac...ata. gtg..a.... ..t..t.... .......c. BNDK167 .gcgcg.c.a .t.gc.ct.. tcgttggg.t a.gc..ga.c a.ctgg.gg. ..a.tttg.. gacg..tacg ..t.ccccgc t..c.tc..g cct.cagcg t BNDK231 ..cgcg.c.a .c.gc.c... gc.ctggg.g a.g.c.ga.c .cctggtgg. ..a.tttgac g.cg.....g ..g...cgc. ..tagcg..g cc.cccgcg c Consensus CAAAAAAGTT CATAGAGGCC ATAAACCACC GCAGGCTCGA GGAGCTCATC GGGCACGCGG TGGCCGGCGC CGACGGGAAT GCCGAGTTCA GGGTTGTGT A 501 550 60 0 BNDK311 t......... .....t.... .......... .........t .......... ........g. .......... .......... .......... ......... BNDK263 t......... .....t.... .......... .........t .......... ........g. .......... .......... .......... ......... BNDK310 t......... .....t.... .......... .........t .......... ........g. .......... .......... .a........ ......... BNDK264 c......... .....c.... .......g.. ......g..c .....g.... ....t...a. .......... .........g .......g.. ......c.. BBWK285 t..a.....a .....at... ....a..... t.....g..t ..a..g.ct. .t.....ta. a..t..t..t ..tt...... .t.....t.. .a....g.. t DSWK265 c..g.....t .....a.... ....t..... t..t..c..c ..a....... ....aa.ta. t.....t..t ..a....... .aa....... taac..ct. BNDK167 ctc.ctactc c..c.gggtg a.c.acggc. a.ac...ctc .gt~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ BNDK231 cac..tactc c....a.gt. a.c.acggc. a.ac...ctc .gt~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ Consensus -GGCGACACG GATTC-CTGT TTATCGAATG CCGGGGATATCGCTAGACT CCGTGTCG-A GTTCTGCGAC GCCCTGGCCT CCGTCACCAG CGGTACTCT G 601 650 70 0 BNDK311 .......... .......... .......... .......... .......... .......... ..a....... .......... ......c... ......... BNDK263 .......... .......... .......... .......... .......... .......... ..a....... .......... ......c... ......... BNDK310 .......... .......... .......... .......... .......... .......... ..a....... .......... ......c... ......... BNDK264 ...g...... ....c..... .......... .......... .......... ....c..... ..g....... ....c..... c.....t..a ......... BBWK285 ..t.a..... .......a.. ......a... ........c. .......... a........t ..g....... .......... t.....t..t ........t DSWK265 ..t.t...a. ........t. a.....t... .....a.... ....t..a.. ....cca... ..g....... .......a.. t..c..t.at ........t BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~ ~ Consensus TTCACGGACC CCATAAAGCT GGAGGCGGAA AAGACTTTTA AGTGCTTGCT CCTCTTGACC AA-AAGAGAT ACATTGGGAT ACTGTC-ACG GATAAAATC T Figure 1-10. Continued.

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29 701 731 BNDK311 .......g.. g......... .......... BNDK263 .......a.. .......... .......... BNDK310 .......a.. .......... .......... BNDK264 .......a.. .......... .......... BBWK285 .......g.. .......... .......... DSWK265 .......g.. .......... .......... BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~ BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~ Consensus TAATGAA-GG CGTCGACCTG GTGAGCAAGA C Figure 1-10. Continued.

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30 1 50 100 BNDK311 .......... .......... .......... .......... .......... .......... .......... .......... .......... ....... ... BNDK263 .......... .......... .......... .......... .......... .......... .......... .......... .......... ....... ... BNDK310 e......... ......h... .......... .......... .......... .......... .......... .......... .......... ....... ... BNDK264 .......... .......... ...a...... ...r...... .......... .....h.... .......... .......... .......... ....... ... BBWK285 .......... .......... ...e...l.. .......... .......... .......... .......... ........e. .......... ....... ... DSWK265 .......... .......... ..he.i.... n.tpe..d.. .......... .......... r......... ........g. .......... ....... ... BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ ~~~ BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ ~~~ Consensus DFASLYPSII QAHNLCYSTL IPDGEMHRHP TLLKGDFETF HISSGPVHFV KKHVTYSLLS KLLATWLAKR KAIRRELSQC SDPQLKTILD KQQLAIK VTC 101 150 200 BNDK311 .......... .......... .......... .....r.... .......... .......... .......... .......... .......... ....... ... BNDK263 .......... .......... .......... .....r.... .......... .......... .......... .......... .......... ....... ... BNDK310 .......... .......... .......... .....r.... .......... .......... .......... .......... .......... ....... ... BNDK264 .......... .......... .......... .....k.... ...dr.k... ....v..... .d........ .k.a...... .......... ....k.. ... BBWK285 .......... ...m...... ........c. .....k.... ...dy..... ...qt.td.. e.s....... .k........ .......... a...k.. ... DSWK265 .......... .......... ....s...c. .....k.... .....sk... ...r..pdtc e........d .......... .......... ...tn.. ... BNDK167 .s........ q......p.. a...ti..d. .lstrd.lhs r.atreq.aa df.d.y.spa pis.s.s..s i......... ~~~~~~~~~~ ~~~~~~~ ~~~ BNDK231 .s........ q..p...q.. a...ti..d. .l.trd.lh. h.atae..va df.dga.a.l las.p.p..s ih........ ~~~~~~~~~~ ~~~~~~~ ~~~ Consensus NAVYGFTGVA SGLLPCLKIA ETVTLQGRRM LERSK-FIEA –WINHRRLEE LIGHAVAGAD GNAP-A-PYE FRVVYGDTDS LFIECRGYSL DSVSEFC DAL 201 250 BNDK311 .......... .......... .......... .......... .......... BNDK263 .......... .......... .......... .......... .......... BNDK310 .......... .......... .......... .......... .......... BNDK264 .a.......a .......... .......... .......... .......... BBWK285 .....s...k .......... .......... .......... .......... DSWK265 .....n...m e......... ......p... .......n.. .......... BNDK167 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~ BNDK231 ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~ Consensus ASVTSGTLFT DPIKLEAEKT FKCLLLLTKK RYIGILSTDK ILMKGVDLVS K Figure 1-11. Multiple alignments of the deduced amino acid sequences of the DNA po lymerase gene fragments ofcetacean herpesviruses. BNDbottlenose dolphin, BBWBlainville’s beaked whale, DSWdwarf sperm whale. K311penile lesion, K263vaginal lesion, K310penile lesion, K264penile lesi on, K285penile lesion, K 265vaginal slit lesion, K167skin lesion, K231skin lesion.

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31Table 1-1. Percent nucleotide identities of DNA polymerase gene fragments among members of the family Herpesviridae. GenBank accession numbers for the virus sequences used are the same as in Figure 1-12. BBW K285 BND K264 BND K310 BND K311 BND K263 DSW K265 BND K167 BND K231 Human herpesvirus-8 62.9 66.3 66.1 66.1 66.2 64.0 51.2 50.2 Human herpesvirus-4 58.6 66.2 63.5 64.2 63.7 59.4 56.6 57.1 CA sea lion gammherpesvirus 60.8 59.4 60.2 60.4 60.1 60.2 47.4 44.8 Bovine herpesvirus-4 65.1 63.4 63.2 63.6 63.4 63.0 45.1 41.1 Ovine herpesvirus-2 64.6 67.8 66.1 66.3 66.1 63.7 49.6 53.3 Porcine lymphotropic herpesvirus-1 61.4 57.5 59.4 59.2 59.2 60.1 49.0 45.9 Equine herpesvirus-2 65.1 72.9 71.0 71.6 71.2 64.4 53.4 50.6 Beaked whale K285 100.0 80.8 82.8 83.3 83.2 76.3 40.6 54.1 Bottlenose dolphin K264 80.8 100.0 91.5 91.8 92.2 77.4 54.9 56.4 Bottlenose dolphin K263 83.2 92.2 99.3 99.6 100.0 78.4 57.8 58.1 Bottlenose dolphin K311 83.3 91.8 99.0 100.0 99.6 78.5 57.8 58.1 Bottlenose dolphin K310 82.8 91.5 100.0 99.0 99.3 78.4 57.8 58.1 Dwarf sperm whale K265 76.3 77.4 78.4 78.5 78.4 100.0 48.4 49.0 Dolphin alphaherpesvirus SC95 42.3 52.9 49.2 49.2 49.2 46.0 73.0 98.9 Dolphin alphaherpesvirus DE99 41.0 38.5 38.9 38.9 38.9 36.1 55.6 55.6 Bottlenose dolphin K167 40.6 54.9 57.8 57.8 57.8 48.4 100.0 78.8 Bottlenose dolphin K231 54.1 56.4 58.1 58.1 58.1 49.0 78.8 100.0 Human herpesvirus-1 52.4 59.3 56.3 56.7 56.6 53.4 71.6 71.6 Human herpesvirus-2 50.6 57.0 55.9 56.3 56.1 51.9 72.5 72.1 Green turtle FP herpesvirus 52.6 56.1 54.5 54.8 55.2 52.8 62.9 59.1 Bovine herpesvirus-1 52.6 58.9 58.5 58.6 58.8 53.4 69.8 69.0 Suid herpesvirus-1 52.7 61.6 59.5 59.7 59.6 57.2 61.5 75.0 Marek's disease virus 50.2 49.2 50.4 50.6 50.6 52.3 56.8 56.3 Canine herpesvirus 54.5 47.6 50.1 50.3 50.5 54.6 53.9 50.9 Phocine herpesvirus-1 40.7 35.7 37.7 38.0 37.7 38.1 55.1 55.1 Feline herpesvirus-1 53.1 51.2 51.2 51.4 51.4 53.9 60.3 57.8 Equine herpesvirus-1 53.8 55.5 55.6 55.9 55.5 53.7 72.9 68.1 Human herpesvirus-5 50.7 53.0 52.6 53.0 52.8 51.3 58.6 47.5 Murid herpesvirus-4 51.0 56.3 55.2 55.1 55.5 51.5 57.6 58.8 Porcine cytomegalovirus 53.9 54.8 53.0 53.4 53.5 53.1 54.1 56.2 Elephant herpesvirus-1 55.1 53.2 53.5 54.0 54.0 52.7 47.5 52.4 Koi herpesvirus 42.2 44.7 42.4 42.4 42.2 39.9 ND ND Channel catfish virus 37.2 41.4 39.2 37.7 39.0 39.6 42.4 41.9 Salmonid herpesvirus-1 34.9 36.6 39.5 39.3 39.3 38.2 ND ND

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32Table 1-2. Percent amino acid identities of DNA polymerase gene fragments among members of the family Herpesviridae. GenBank accession numbers for the virus sequences used are the same as in Figure 1-12. BBW K285 BND K264 BND K310 BND K311 BND K263 DSW K265 BND K167 BND K231 Human herpesvirus-8 70.7 70.7 69.8 70.7 70.7 70.2 53.5 58.0 Human herpesvirus-4 63.0 63.4 63.8 64.6 64.6 62.6 52.7 51.2 CA sea lion gammherpesvirus 64.4 63.6 62.0 63.2 63.2 62.0 42.9 46.0 Bovine herpesvirus-4 68.9 69.3 68.5 69.3 69.3 68.3 59.2 53.5 Ovine herpesvirus-2 68.1 66.8 66.8 67.6 67.6 67.6 45.3 46.7 Porcine lymphotropic herpesvirus-1 64.5 65.1 64.5 63.8 63.8 66.9 41.2 42.6 Equine herpesvirus-2 69.4 70.7 69.0 69.8 69.8 67.8 56.3 56.3 Beaked whale K285 100.0 90.9 91.4 92.2 92.2 86.4 45.1 47.9 Bottlenose dolphin K264 90.9 100.0 93.0 93.8 93.8 84.8 48.6 51.4 Bottlenose dolphin K263 92.2 93.8 99.2 100.0 100.0 87.7 50.0 52.8 Bottlenose dolphin K311 92.2 93.8 99.2 100.0 100.0 87.7 50.0 52.8 Bottlenose dolphin K310 91.4 93.0 100.0 99.2 99.2 86.8 50.0 52.8 Dwarf sperm whale K265 86.4 84.8 86.8 87.7 87.7 100.0 48.6 50.7 Dolphin alphaherpesvirus SC95 36.5 42.9 41.3 41.3 41.3 38.1 71.4 96.8 Dolphin alphaherpesvirus DE99 34.5 41.1 36.2 36.2 36.2 43.4 65.0 61.7 Bottlenose dolphin K167 45.1 48.6 50.0 50.0 50.0 48.6 100.0 77.2 Bottlenose dolphin K231 47.9 51.4 52.8 52.8 52.8 50.7 77.2 100.0 Human herpesvirus-1 50.4 51.9 51.3 51.7 51.7 50.6 72.0 68.0 Human herpesvirus-2 50.4 51.3 52.9 53.4 53.4 50.0 70.7 66.7 Green turtle FP herpesvirus 49.8 51.7 50.8 51.3 51.3 50.4 64.5 60.5 Bovine herpesvirus-1 50.4 50.6 53.8 54.2 54.2 52.5 68.4 67.1 Suid herpesvirus-1 50.4 51.7 51.7 52.1 52.1 52.1 73.6 69.4 Marek's disease virus 47.7 46.5 45.6 46.1 46.1 47.9 60.0 57.3 Canine herpesvirus 51.0 50.0 49.0 49.4 49.4 51.5 63.2 57.9 Phocine herpesvirus-1 48.8 49.7 48.5 49.1 49.1 49.7 65.3 61.3 Feline herpesvirus-1 51.7 50.8 50.8 51.3 51.3 52.1 68.4 64.5 Equine herpesvirus-1 51.0 52.3 50.6 51.0 51.0 50.8 76.0 70.7 Human herpesvirus-5 48.5 47.7 47.7 48.5 48.5 48.1 50.0 48.1 Murid herpesvirus-4 45.2 46.0 44.4 45.2 45.2 44.8 53.3 46.7 Porcine cytomegalovirus 46.7 46.9 45.2 46.1 46.1 46.9 51.4 45.9 Elephant herpesvirus-1 52.7 51.3 51.9 52.7 52.7 50.4 47.9 44.0 Koi herpesvirus 29.0 30.0 35.3 34.8 34.8 36.4 ND ND Channel catfish virus 27.2 25.3 23.3 23.7 23.7 24.9 20.8 21.1 Salmonid herpesvirus-1 27.8 25.9 25.9 25.9 25.9 30.8 ND ND

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33Table 1-3. Percent amino acid similarities of DNA polymerase gene fragments among member s of the family Herpesviridae. GenBank accession numbers for the virus sequences used are the same as in Figure 1-12. BBW K285 BND K264 BND K310 BND K311 BND K263 DSW K265 BND K167 BND K231 Human herpesvirus-8 78.9 77.7 78.9 78.9 78.9 79.3 59.2 60.9 Human herpesvirus-4 70.4 71.2 71.2 71.2 71.2 69.1 59.5 55.4 CA sea lion gammherpesvirus 70.6 70.4 69.9 69.9 69.9 69.3 50.8 54.0 Bovine herpesvirus-4 78.0 77.6 77.6 77.6 77.6 78.8 64.8 60.6 Ovine herpesvirus-2 75.5 73.4 74.3 74.3 74.3 74.7 50.7 57.3 Porcine lymphotropic herpesvirus-1 75.7 75.7 75.7 75.7 75.7 78.8 51.5 52.9 Equine herpesvirus-2 77.7 77.3 77.3 77.3 77.3 77.3 62.0 60.6 Beaked whale K285 100.0 93.4 94.2 94.2 94.2 90.1 57.7 56.3 Bottlenose dolphin K264 93.4 100.0 96.3 96.3 96.3 88.1 54.2 56.9 Bottlenose dolphin K263 94.2 96.3 100.0 100.0 100.0 90.5 55.6 59.7 Bottlenose dolphin K311 94.2 96.3 100.0 100.0 100.0 90.5 55.6 59.7 Bottlenose dolphin K310 94.2 96.3 100.0 100.0 100.0 90.5 55.6 59.7 Dwarf sperm whale K265 90.1 88.1 90.5 90.5 90.5 100.0 55.6 57.7 Dolphin alphaherpesvirus SC95 44.4 47.6 47.6 47.6 47.6 47.6 71.4 96.8 Dolphin alphaherpesvirus DE99 48.3 51.8 46.6 46.6 46.6 56.6 68.3 65.0 Bottlenose dolphin K167 57.7 54.2 55.6 55.6 55.6 55.6 100.0 77.2 Bottlenose dolphin K231 56.3 56.9 59.7 59.7 59.7 57.7 77.2 100.0 Human herpesvirus-1 61.3 60.7 60.4 60.8 60.8 60.7 78.7 74.7 Human herpesvirus-2 61.8 60.0 61.8 62.2 62.2 59.2 77.3 73.3 Green turtle FP herpesvirus 58.5 60.0 59.6 60.0 60.0 59.6 73.7 69.7 Bovine herpesvirus-1 60.0 58.9 61.3 61.2 61.2 60.0 73.7 72.4 Suid herpesvirus-1 61.0 61.0 61.0 61.4 61.4 61.9 77.8 73.6 Marek's disease virus 57.7 56.0 55.6 56.0 56.0 57.9 68.0 66.7 Canine herpesvirus 59.8 58.8 58.1 58.5 58.5 60.3 71.1 64.5 Phocine herpesvirus-1 56.5 58.1 56.9 56.9 56.9 58.8 68.0 65.3 Feline herpesvirus-1 60.0 57.9 57.5 57.9 57.9 58.8 72.4 68.4 Equine herpesvirus-1 61.1 60.3 59.8 60.2 60.2 60.4 77.3 74.7 Human herpesvirus-5 60.3 59.0 59.8 59.8 59.8 59.4 58.8 55.7 Murid herpesvirus-4 55.2 55.2 55.2 55.2 55.2 54.0 64.0 57.3 Porcine cytomegalovirus 56.7 56.0 55.6 55.6 55.6 56.4 59.5 54.1 Elephant herpesvirus-1 62.3 59.2 61.5 61.5 61.5 60.0 60.3 52.0 Koi herpesvirus 38.4 40.0 45.6 44.2 44.2 47.5 ND ND Channel catfish virus 37.1 36.9 34.7 34.7 34.7 35.5 30.6 26.3 Salmonid herpesvirus-1 31.5 29.6 29.6 29.6 29.6 38.5 ND ND

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Figure 1-12. Neighbor-Joining phylogenetic tr ee of the deduced amino acid sequences of the herpesvirus DNA polymerase gene. The tree was generated by Clustal X slow and accurate function us ing Gonnet 250 residue weight ta ble, gap penalty of 11, gap length penalty of 0.20, and 1000 bootstrap rep lications. The bar indicates a 0.1 dive rgence scale. The GenBank accession numbers of the DNA polymerase gene fragments used fo r the construction of this tree are: AF196646-Dolphin alphaherpesvirus SC95, AF245443-Dolphin alphaherpesvir us DE99, AF181249-Bovine herp esvirus-2, AB070848-Human herpesvirus-1, AY038367-Human herpesviru s-2, AF299109-Olive ridley turtle FP herpesvirus, AY646894-Kemp’s ridley turtle FP herpesvirus, AF239684-Green turtle FP herpesvi rus, AY646888-Loggerhead turtle FP herpesvirus, NC_001847Bovine herpesvirus-1, NC_006151-Suid herpesvirus-1, AF147806Marek’s disease virus, AY464052-Equine herpesvirus1, AF030027-Equine herpesvirus-4, NC _001348-Human herpesvirus-3, U92269-Phoc id herpesvirus-1, AJ224971-Feline herpesvirus-1, NC_006273-Human herpesvirus-5, AY186194-Rhesus cytomegalovirus, AF268040-Porcine cytomegalovirus, AY529146-Murid herpesvirus-4, NC_000898Human herpesvirus-6, AF005477-Human herpesvirus-8, AF250886-Gorilla rhadinovirus-1, AF290600Gorilla lymphocryptovirus-1, AJ 507799-Human herpesvirus-4, AF159033Macaca mulatta gammavirus, AF029302-Rhesus monkey rhadinovirus, AY270026-Baboon gammaherpesvirus, AF236050-CA sea lion gammaherpesvirus, NC_002665-Bovine herp esvirus-4, AF287948-Black rh inoceros herpesvirus, AF083424-Ateline herpesvirus-3, NC_001350-Saimiriine he rpesvirus-2, AF376034-Badger herpesvirus, NC_001650Equine herpesvirus-2, AF327831-Ovine herpesvirus2, NC_002531-Alcelaphine herpesvirus-1, AF118399-Porcine lymphotropic herpesvirus-1, AF118401-Porc ine lymphotropic herpesvirus-2, AF327830Bovine lymphotropic herpesvirus, AF322977-Elephant herpesvirus-1, AB047545Tortoise herpesvirus, AY572853-Ko i herpesvirus, NC_001493-Channel catfish virus, AF023673-Salmonid herpesvirus-1, AY949827-Ca nine herpesvirus. BNDbottlenose dolphin, BBWBlainville’s beaked whale, DWSdwarf sperm whale, K311penile lesion, K263vagina l lesion, K310penile lesion, K264penile lesion, K265vaginal lesion, K285pe nile lesion, K167skin lesion, K231skin lesion.

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35

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36 A B Figure 1-13. Neighbor-Joining phylogenetic tr ees of the nucleotide sequences of the DNA polymerase gene of cetacean herpesviruses. The tree was generated by Clustal X slow and accurate function using Gonnet 250 residue weight table, gap penalty of 15 and gap length pe nalty of 6.66. BNDbottlenose dolphin, BBWBlainville’s beaked whale, DWSdwarf sperm whale, K311penile lesion, K263vaginal lesion, K310pen ile lesion, K264pe nile lesion, K265vaginal lesion, K285penile lesion, K167skin lesion, K231skin lesion. A) Radial tree where the numbers repr esent the percent confidence of 100 bootstrap replications B) Phylogram with 0.1 divergence scale. 0.1 Divergence BNDK263 BNDK310 BNDK311 BNDK264 BBWK285 DSWK265 BNDK231 BNDK167 BBWK285 DSWK265 BNDK231 BNDK167 100.0 82.0 96.0 BNDK264 BNDK311 BNDK310 BNDK263 100.0

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37 A B Figure 1-14. Neighbor-Joining phylogenetic trees of the deduced amino acid sequences of the DNA polymerase gene of cetacean herpesviruses. The tree was generated by Clustal X slow and accurate func tion using Gonnet 250 residue weight table, gap penalty of 11 and gap le ngth penalty of 0.20. BNDbottlenose dolphin, BBWBlainville’s beaked whale, DWSdwarf sperm whale, K311penile lesion, K263vaginal lesion, K 310penile lesion, K264penile lesion, K265vaginal lesion, K285penile le sion, K167skin lesion, K231skin lesion. A) Radial tree where the number s represent the perc ent confidence of 100 bootstrap replications B) Phylogr am with 0.1 divergence scale. 0.1 Diver g ence BNDK263 BNDK311 BNDK310 BNDK264 BBWK285 DSWK265 BNDK231 BNDK167 BNDK264 BBWK285 DSWK265 BNDK231 BNDK167 100.0 99.0 100.0 BNDK310 BNDK263 90.0 BNDK311

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38 PCR Targeting the DNA Terminase Gene Negative results were obtained for the PCR targeting the terminase gene for five of the six samples tested, namely K167, K263, K265, K285 and K311. Only skin lesion sample K231 yielded a positive result. Upon sequencing, it was demonstrated that the sequence of K231 corresponded to an alphaherp esvirus that differed from the sequence obtained from a bottlenose dolphin from South Ca rolina that had died with disseminated herpesvirus infection (GenBank accession number AF196647) by two nucleotides at positions 208 and 294 (Figure 1-15). Th e deduced amino acid sequences of both terminase gene fragments were identical (Figure 1-16). The DNA terminase gene fragment obtained from a skin lesion of bottlenose dolphin K231 was deposited in the GenBank database under accession number AY949829.

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39 1 50 1 00 K231 .......... .......... .......... .......... .......... .......... .......... .......... .......... ........ .. AF196647 .......... .......... .......... .......... .......... .......... .......... .......... .......... ........ .. Consensus ACCAACACCG GCAAAGCTAG CACCAGCTTC CTCTTCAACC TCAAGTACTC CTCGGACGAC CTGCTCAATG TGGTCACGTA TATCTGCGAC GAGCACAT GG 101 150 2 00 K231 .......... .......... .......... .......... .......... .......... .......... .......... .......... ........ .. AF196647 .......... .......... .......... .......... .......... .......... .......... .......... .......... ........ .. Consensus ATCGCGTGCG CGTGCACACG AACGCCACGG CCTGTTCGTG CTACGTGCTG AACAAGCCGG TGTTCATCAC GATGGACGCG TCCATGCGAA ACACGGCC GA 201 250 3 00 K231 .......t.. .......... .......... .......... .......... .......... .......... .......... .......... ...c.... .. AF196647 .......c.. .......... .......... .......... .......... .......... .......... .......... .......... ...t.... .. Consensus GATGTTC-TG CCGAACTCGT TCATGCAGGA GATCATCGGC GGCGGCTCCG CGGACCCCGC GGCCGGCGGC GACGGCCCCG TGTTCACAAA GGC-GCGG CG 301 350 375 K231 .......... .......... .......... .......... .......... .......... .......... ..... AF196647 .......... .......... .......... .......... .......... .......... .......... ..... Consensus GACCAGTTTC TGCTCTACCG CCCCTCCACC ACCACGCGGC GAGGCGCGAT GGCCGAGGAA CTGTTCGTGT ACGTG Figure 1-15. Comparison of the nucleotide se quences of the 375-bp partial DNA terminas e gene fragment of an alphaherpesvirus amplified from a skin lesion of an Atlantic bottlenose dol phins (K231)skin lesion, AF196647lung, disseminated infection (Blanchard et al., 2001). 1 50 1 00 K231 .......... .......... .......... .......... .......... .......... .......... .......... .......... ........ .. AF196647 .......... .......... .......... .......... .......... .......... .......... .......... .......... ........ .. Consensus TNTGKASTSF LFNLKYSSDD LLNVVTYICD EHMDRVRVHT NATACSCYVL NKPVFITMDA SMRNTAEMFL PNSFMQEIIG GGSADPAAGG DGPVFTKA AA 101 125 K231 .......... .......... ..... AF196647 .......... .......... ..... Consensus DQFLLYRPST TTRRGAMAEE LFVYV Figure 1-16. Comparison of the deduced ami no acid sequences of the partial DNA term inase gene fragment from two Atlantic bottlenose dolphins. K231skin lesion, AF196647lung, disseminated infection (Blanchard et al., 2001).

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40 Discussion Applying PCR technology, we have been ab le to demonstrate the existence of both alpha and gamma herpesviruses in several species of cetaceans in which they cause, respectively, local or generalized cutaneous lesions, and localized genital and oral lesions. It is not known at this point whether the herp esvirus DNA present in these lesions corresponds to latent or to actively replicating virus, as in the few instances in which virus isolation was attempted, the re sults were unsuccessful. When accessible, central nervous and lymphoid tis sues were also tested in the form of tissue homogenates after a positive PCR result was obtained from a lesion; however, herpesvirus genomic DNA was not detected in any tissue tested, most likely i ndicating virus absence. Mesenteric, pulmonary and prescapular lym ph node samples were tested from dwarf sperm whale K265 in addition to the genita l lesion. A localized gammaherpesvirus infection was demonstrated in the lesion but not in the lymph nodes. Gammaherpesviruses are known to become latent in lymph tissue, thus, this infection was most likely active. Certain gammaherpesvir uses, such as human herpesvirus-8 (HHV-8), which causes Kaposi’s sarcoma in AIDS patie nts, and OtHV-1, have the potential for oncogenic transformation of host cells, but no ev idence of this was seen in any of the cetacean cases presented in this study. All cetacean tissue samples tested by nested PCR were negative for herpesvirus genomic DNA. Although no systemic infec tion was found during this study, Blanchard et al. (2001) recently used PCR to demonstrat e disseminated fatal herpesvirus infections in two Atlantic bottl enose dolphins. These viruses were unknown previously, thus, it was speculated that the natural host of such vi ruses could be a speci es outside the order Cetacea, and the cases described could represent atypical infections in an aberrant host.

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41 The herpesviruses found in the cetaceans of our study, as well as those found in the bottlenose dolphins described by Blanchard et al. (2001), appeared to be distinct from known herpesviruses of other marine animals, such as pinnipeds and marine turtles. Sequence homologies and phylogenetic anal yses demonstrated that the cetacean herpesviruses identified during this study we re more closely related to other cetacean herpesviruses of the respectiv e subfamily than to other known herpesviruses. Given the lower sequence homologies of cetacean herpesviruses with other mammalian herpesviruses, it seems unlikely that these vi ruses arose from a know n virus that spread into a new cetacean host. Our study suppor ts the hypothesis that the herpesviruses identified in various cetaceans are specific for the cetaceans examined and, most likely, coevolved with their hosts. The deduced amino acid sequences of the small DNA polymerase fragment of alphaherpesviruses from skin lesions from two Atlantic bottle nose dolphins (K231 and K167) shared 77.2% identity, while the two al phaherpesviruses identified by Blanchard et al. (2001) shared 65.0%. These findings s uggest that there may be several species of cetacean alphaherpesvirus. As with other herpes viruses, such as those associated with FP of marine turtles, the cetacean herpesviru ses could vary based on species and/or geographical location, but extensive epidemio logical studies are needed to make any definitive conclusions. Our findings, nevertheless, have shown that a stranded bottlenose dolphin from South Carolina (GenBank acces sion number AF196646) and a captive bottlenose dolphin in Florida (K231) were infe cted with the same virus. As further support, bottlenose dolphin K231 yielded the only positive PCR result for the terminase gene, similar to the bottlenose dolphin from S outh Carolina. If the terminase gene of

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42 cetacean alphaherpesviruses maintains as much conservancy/variation as the DNA polymerase gene, it is not surprising that th e primers used did not amplify sequences from any other sample. From these findi ngs it can be concluded that the same alphaherpesvirus can cause localized skin lesions as well as deadly disseminated infections. The six cetacean gammaherpesviruses identi fied in this study showed variations in deduced amino acid sequences of DNA pol ymerase gene fragments of 84.8 to 100%, which is much less than the sequence variation observed for the cetacean alphaherpesviruses (> 61.7%). The high degree of sequence homologies between species indicates that cetacean gammaherpesviruses ma y not be species specific, but rather family or order specific. For example, the four bottlenose dolphin sequences were more similar to each other than to the two whal e sequences. The amino acid identities of the DNA polymerase gene fragment of the gamm aherpesvirus from bottlenose dolphin K263 when compared with the ho mologous fragments from the other three bottlenose dolphins ranged from 93.8 to 100%, while the identities to beaked whale K285 and dwarf sperm whale K265 were 92.2 and 87.7%, respectivel y. This suggests that cetacean gammaherpesvirus variants could be correlated w ith the species they infect. On the other hand, bottlenose dolphins K263, K310 and K311 i nhabited the same general area in the Florida Keys, thus suggesting that ge ographic variants are also possible. Recent guidelines for differentiating herpes virus species include separation based on serologic and restriction endonuclease cleav age methods, as well as the occupation of different ecological niches (Roizmann et al., 1992). The cetacean herpesviruses identified in this study appear to occu py similar niches throughout both alphaand

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43 gammaherpesviruses. This observation along with the relatively comparable sequence homologies suggests that ther e are two subfamilies of cetacean herpesviruses; cetacean alphaherpesviruses and gammaherpesvirus, both with several strains encompassing either different viral strains or species. Based on phylogenetic analysis and multiple sequence homologies, it was determined that cetacean herpesviruses were most closely related to bovine herpesvirus1, suid herpesvirus-1 and equine herpesvirus-1 and -4 (genus Varicell ovirus) in the case of alphaherpesviruses, and to saimiriine he rpesvirus-2, ateline herpesvirus-3 and bovine herpesvirus-4 (genus Rhadinovirus) in the cas e of gammaherpesviruses. Similar analysis also revealed that cetacean herpesviruses are least similar to the fish herpesviruses KHV, SalHV-1 and CCV, with the majority of amino acid identities below 30%, thus supporting the theory of the existence of three distinct herpesvirus lineages (mammalian/avian, fish/amphibian and invertebrate). The cetacean-specific primers that were designed based on the small DNA polymerase gene fragment of both cetacean alphaherpesviruses and gammaherpesviruses were, essentially unsuccessful in PCR am plification, most likely due to the high variability among the target sequences. Furt hermore, cetacean-spec ific primers designed from the large DNA polymerase fragment seque nces of the gammaherpesviruses did not give consistent results, most likely due to the viral gene diversity among the species tested. As expected, these primers did not dr ive the amplification of the long fragment of the cetacean alphaherpesviruses. Cetacean-s pecific primers are essential not only to increase the specificity and sensitivity of the assay, but also to reduce the risk of error. The nested PCR, which has two rounds of reactions, is cumbersome and time consuming,

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44 and can lead to a greater possi bility for mistakes and cross-contamination. There is an urgent need for the availability of more cetacean herpesvirus seque nces to design better cetacean-specific herpesvirus primers. Herpesviruses are generally transmitted via direct contact of mucosal surfaces, and the gregarious nature of many cetacean species would favor this type of transmission. The cetacean alphaherpesviruses detected in skin lesions were probably transmitted through skin abrasions, which are similar to alphaherpesvirus infec tions of terrestrial mammals including humans, namely BHV-2, which causes mammillitis, and HHV-1, which produces both facial and oral lesions. Two captive dolphins from the same park, one male (K311) and one female (K263), were both diagnosed with genital infections from identical gammaherpesviruses (100% am ino acid identity). Sexual activity between these two animals was apparent as they have produced offspring, which strongly indicates that the cetacean gammaherpesviruses are sexually transmitted. This mode of transmission is unique, as very few gammaherpesvirus infections are transmitted through genital contact. One example is the Kaposi’s sarcoma-associated herpesvirus (HHV-8) in humans, and perhaps OtHV-1, given that it has a genital origin. In one case, identical gammaherpesvirus sequences were obtained fr om both a genital (K310) and oral (K308) lesion of a bottlenose dolphin. It is interesting that oral lesions were also present in this animal, as no other gammaherpesviruses have been shown to cause lesions of the oral mucosas. Although the possible modes of transm ission for herpesviruses among cetaceans are still largely unknown, vertical transmission and the incorporation of unidentified vectors cannot be ruled out. A recent study suggested that PhHV-1 may be vertically

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45 transmitted, as adult female harbor seals we re found to be shedding virus in vaginal secretions and premature newborn pups showed evidence of early inf ection (Goldstein et al., 2004). Tentative candidate vectors for the spread of FP among marine turtles have been identified recently. Greenblatt et al (2004) performed quantitative PCR on an assortment of parasites of Hawaiian green turtles and found that marine leeches ( Ozobranchus spp.) carried fibropapilloma-associat ed herpesviral loads sufficient to cause infection. There is evid ence that leeches may serve as mechanical vectors for other pathogenic agents, including hepa titis B virus in humans, seve ral parasites of fish and a blood protozoan of turtles. PCR has also b een used to detect FPHV sequences in the snout, gill and liver of a reef clean er fish, the saddleback wrasse ( Thalassoma duperrey ) (Lu and Yu, 2000). Toxic benthic dinoflagellates ( Prorocentrum spp.) have been suggested as yet another cofactor, since th ey have a worldwide distribution and are epiphytic on seagrasses that are norma lly consumed by green turtles. These dinoflagellates are known to produce a tumor promoter, okadaic acid (OA), which has been detected also in tissues of Hawaiian gr een turtles, suggesting a potential role of OA in the etiology of FP (La ndsberg et al., 1999). Herpesviruses, by virtue of being e nveloped viruses, are generally unstable outside the host, however, research has s hown that LETD-associated herpesvirus of marine turtles can remain infectious as l ong as 120 hours in natura l and artificial sea water at 23 C (Curry et al., 2000). It has been suggested that both FPHV and GPDassociated herpesvirus may also be able to survive for extended periods of time under harsh environmental conditions. The possibility remains that all marine herpesviruses, including those of cetaceans, may also be transmitted directly through sea water.

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46 Although the prevalence of cetacean herpes virus is not well documented, it is possible that false negative diagnoses may have been made in the past, thus, overlooking potential herpesvirus infections. The charac teristic papillomatous lesions of marine turtles with FP and the urogenital carcinoma of California sea lions were both originally thought to be caused by papillomaviruses. Ho wever, negative results for papillomavirus using Southern blot hybridization (Jacobs on et al., 1989) and PCR (Lipscomb et al., 2000) have detracted from these theories. Ot her likely misdiagnoses could include carp pox, a herpesvirus that infects farmed carp, whic h is also associated with papillomatous lesions (Buchanan and Richards, 1982) and cetacean genital lesions, which can be papillomaand wart-like in appearance. The mild dermatitis lesions of bottlenose dolphin K167 was initially thought to resemb le early poxvirus infection. Additional herpesviruses, cetacean or otherwise, may remain undiscovered due to a misdiagnosis of the etiological agent. Treatment for herpesvirus infections gene rally includes antivir al drugs, commonly acyclovir, ganciclovir and cidofovir, which block herpesvirus replication. Bottlenose dolphin K311 was treated with acyclovir for pe nile lesions without any improvement. Although acyclovir is often used to treat ge nital herpes in humans, the cause is an alphaherpesvirus, whereas gammaherpesviruses s eem to be the cause of genital lesions in cetaceans. The difference in host species c ould also be a factor on how effective the treatment will be. It is unknown how the trea tment in this case was administered, and no other accounts of treatment fo r herpesvirus infections in ce taceans have been reported. Mammalian herpesvirus disease can ofte n be linked with environmental stress; therefore, the correlation be tween skin disorders and various natural and anthropogenic

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47 factors is now being investigated for cet aceans. Environmental contaminants are commonly believed to influence the devel opment of disease in wild populations. Marine mammals, as long-lived top level predators, can accumulate high concentrations of chemicals in their tissues, which can then be passed to offspring through the milk. An extensive study was performed by Wilson et al. (1999) in which bottlenose dolphin populations were monitored in ten areas spa nning the globe. Skin lesion prevalence and severity was determined using photograph-identif ication of the dorsal fin and correlations were examined between multiple factors. Epidermal lesions were found in all ten populations in frequencies of 63 to 100%. High prevalence of epidermal disease was found to be related to low water temperature and low salinity. It was suggested that low salinity causes cellular damage to the epid ermis by disrupting the electrolyte balance, thus, weakening the ability to shield the anim al from infectious agents. Likewise, low water temperature may limit blood flow to th e skin and impede pathways for immune protection. Surprisingly, no si gnificant correlations were found between prevalence or severity of lesions and contaminants, namely organochlorine compounds and trace metals; however, only four populations were in cluded as the other six lacked baseline toxicological data. A similar study was performed in the Sado estuary, Portugal, where skin disorders of bottlenose dolphins were monitored over two time periods. Harzen and Brunnick (1997) concluded that 85% of the long term residents had skin disorders after the second study period, and most of the disorders appeared continuous and nonfatal. Lesion samples were not taken; theref ore, the etiological agents i nvolved could not be identified. However, skin disorders that matched the de scription of pox and herp es virus infections

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48 by Baker (1992) were noted. Periods of bloo m were also reported, in which the skin lesions were more dist inct. These bloom periods could be gin quickly and then enter into remission, but the reasons behind this remained unclear. One important factor that needs further research is that of habitat degrada tion. Areas where habitats are being destroyed, such as the Sado estuary, often experi ence excessive nutrient enrichment or euthrophication that can harm plant growth, wh ich in turn may lead to the damage of the entire food web. Contaminant-induced immune suppression can also occur. Anecdotal reports suggested that FP of ma rine turtles is most prevalen t in near-shore habitats, such as estuaries and lagoons that have been negatively impacted by human occupation (Herbst and Klein, 1995). Human activity has also directly added to the concentration of pathogens in the oceans, primarily though sewage discharge and storm water run-off. In Scotland, an alarming number of pathogens were identified in domestic sewage that was discharged into coastal waters. Specifical ly, viruses, including herpes, were found in concentrations of 10,000 to 10,000,000 per liter of raw sewage (Grillo et al., 2001). Natural environmental fluctuations ma y also play a role in herpesvirus prevalence. Under artificial culture conditions, stress, crowding and in some cases, increased temperature have been shown to enhance the risk of herpesvirus outbreaks among marine organisms, namely teleost fish, turtles, oysters and blue crabs (Buchanan and Richards, 1982). Similarly, thermal stress has been shown to exacerbate herpesvirus infection in hatchling green tu rtles (Herbst and Klein, 1995). It has been suggested that temperature variation causes a shift in ba lance between virus replication and the efficiency of host defense mechanisms.

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49 Much more research is needed on cetacean herpesvirus infections including; mode of transmission, species susceptibility, host defense mechanisms and identification of cofactors that aid in the sp read of these viruses. Th is information along with the phylogenetic comparisons made in the present study should lead to a better understanding of the epidemiology of cetacean herpesviruse s and may result in practical methods of control and prevention in cetaceans in cap tivity, those undergoing rehabilitation and possibly, in the not too distant future even those of wild populations.

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50 CHAPTER 2 PORPOISE MORBILLIVIRUS Introduction Morbilliviruses are members of the fa mily Paramyxoviridae; a family whose members have caused more morbidity and mort ality than any other group of diseases in history (Murphy et al., 1999). All paramyxoviru ses have genomes consisting of a single molecule of negative-sense, single-stranded RNA that is 15 to 16 Kbp (Kilo base pairs) in length. The pleomorphic (spherical or filame ntous) virions are enveloped, covered with large peplomers and contain a “herringbone-sha ped” helically symmetrical nucleocapsid. Glycoprotein peplomers aid in cell attach ment and pathogenesis of paramyxovirus infection. The two glycoproteins of mo rbilliviruses are the fusion (F) and the hemagglutinin (H) proteins, which, unlike that of other paramyxoviruses, lack neuraminidase activity. Morbilliviruses encode only six genes, which are located in the genome in the following order: 3’ N-PM-F-H-L 5’ (Griffin, 2001). The phosphoprotein (P) gene is spliced into three separate prot eins, namely, P, V and C. Replication is entirely cytoplasmic and newly synthesized negative-sense RNA associates with nucleocapsid (N) protein and tran scriptase to form nucleocapsids. As the virions mature, viral glycoproteins are incor porated into the host cell plasma membrane allowing the matrix (M) protein to associate with the host cell membrane. Mature virions are released by budding and thus, obtain their envelope from the host cell plasma membrane. Characteristics of morbill ivirus infection include the production of acidophilic intracytoplasmic and intranuclear inclusions as well as the formation of syncytia. In

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51 general, morbillivirus infection presents it self mainly as a respiratory disease of vertebrates, and most viruses have a relativ ely narrow host range (Murphy et al., 1999). Aerosol transmission is probably the mo st important route of infection for morbilliviruses, but indirect contact cannot be ruled out. Up until 1988, only four morbilliviruses were known, all of which infected terrestrial mammals, namely, human measles vi rus (MV), bovine rinderpest virus (RPV), peste-des-petits ruminant virus of goats and sheep (PPRV) and canine distemper virus (CDV). In the past 15 years, various epizoot ics in marine mammals have demonstrated the existence of three new morbilliviruses th at have become known as phocine distemper virus (PDV), porpoise morbillivirus (PMV) and dolphin morbillivirus (DMV). (For review see Kennedy, 1998 and Duigna n, 1999.) Between 1987 and 1988, it was estimated that more than half of the Atlantic bottlenose dolphin ( Tursiops truncatus ) population along the Atlantic coast of the U.S. died (Lipscomb et al., 1994). Initially thought to be caused by red tide toxins, retros pective studies showed that dolphins were infected with both PMV and DMV. At a pproximately the same time, a CDV strain, which is believed to have orig inated in terrestria l carnivores, killed thousands of Baikal seals ( Phoca siberica ) in Siberia (Barrett et al., 19 92). In the spring of 1988, over 18,000 harbor seals ( Phoca vitulina ) died in northwestern Europe (Mahy et al., 1988). Pup abortion was widespread and adult seals pr esented with CDV-like lesions, however, upon virus isolation it was determined that a ne w morbillivirus, PDV, was the cause of the mass-mortality. Grey seals ( Halichoerus grypus ) were also infected, but with relatively low mortality. Between 1990 and 1992, thousan ds of Mediterranean striped dolphins ( Stenella coeruleoalba ) died from a DMV outbreak that began in Spain and spread

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52 eastward to Greece and Turkey (Domingo et al., 1992). CDV was implicated as the cause of thousands of Caspian seal ( Phoca caspica ) deaths in the summer of 2000 in Kazakhstan, Azerbaijan and Turkmenistan (Kennedy et al., 2000). The origin of the virus was unknown, but there were local anec dotal reports of contact between Caspian seals and terrestrial carnivores. The la st major documented epizootic occurred in European harbor seals infected with PDV in 2002 (Harding et al., 2002). The death toll was comparable to that of the PDV outbreak in 1988, and sequence analysis of P gene fragments from seal tissues from 1988 and 2002 showed greater than 97% identity (Jensen et al., 2002). Smaller scale die-offs fr om PMV have also been reported in harbor porpoises ( Phocoena phocoena ) in Ireland (Visser et al., 1993) and Atlan tic bottlenose dolphins in the Gulf of Mexico (Lipscomb et al., 1996b). Serological studies have shown that numerous cetacean and pinniped species from every major ocean possess morbillivirus-neutralizing an tibodies. Antibodies agains t CDV were retrospectively detected in Crabeater seals ( Lobodon carcinophagus ) from Antarctica, suggesting that a morbillivirus epizootic may have occurred in 1955 (Bengtson et al., 1991). The list of seropositive species also includes walruses ( Odobenus rosmarus rosmarus ) from northwest Canada (Duignan et al., 1994), polar bears ( Ursus maritimus ) from Alaska and Russia (Follmann et al., 1996), and Florida manatees ( Trichechus manatus latirostris ) (Duignan et al., 1995b). Clinical signs and lesions of infected ma rine mammals are similar to those of CDV and are mainly seen in the lung, central ner vous system and lymphoid tissues. Affected tissues often present with acidophilic intranuc lear and intracytoplasmic inclusion bodies, and syncytia are commonly found in cetaceans, but not in pinnipeds. Signs commonly

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53 include bronchointerstitial pneumonia, respir atory distress, encepha litis, neurological disturbances, ulcerations of the skin and buccal mucosa, and abortion. Morbilliviruses induce immunosuppression; therefore, infected animals are much more susceptible to secondary infections by bacteria and opportunistic parasites. These same signs are seen in pinnipeds as well as cetaceans, but to a lesser extent. The social behavior of many marine mammals probably favor s horizontal transmission. An ti-CDV maternally-derived antibodies have been reported in immature grey seals (Carter et al., 1992). Possible reservoirs for marine mammal morbilliviruses include harp seals ( Phoca groenlandica ) and pilot whales ( Globicephala species). Both species ar e gregarious, migratory and numerous, which would facilitate lateral transm ission of these viruses. For example, harp seals from Greenland, with inapparent or subacute infections, were suspected of introducing PDV to northwestern Europe in 1988. In the western Atlantic, it was reported that 86% of stranded pilot whales had morbillivirus-neutralizing titers, suggesting that this whale species is in continuous contact with the virus and that immunity or natural resistance may have pr evented a serious outbreak from occurring (Duignan et al., 1995a). It is suspected that recent epizootics are the result of virus transfer to immunologically -nave populations. There is much debate over wher e to place these new marine mammal morbilliviruses in the paramyxovirus phyloge ny. Nucleotide sequence comparisons of PDV and other morbilliviruses indicate that PDV is most closely related to CDV, but should be regarded as a separate species. Cross reactivity studies of DMV and PMV with monoclonal antibodies against known morbilliv iruses revealed that the two cetacean viruses are more closely related antigenically to RPV and PPRV of ruminants than to

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54 distemper of carnivores (Visse r et al., 1993). Researcher s are still undecided as to whether PMV and DMV are separate viral spec ies, or simply different strains of a common cetacean morbillivirus (CMV). Previous attempts to establish the relatedness of DMV and PMV have concentrated on seque nce homologies of various genes. Polymerase chain reaction (PCR) utilizing universal morbillivirus P gene primers generated fragments 429-bp in length (Barrett et al., 1993). The amount of nucleotide variation between the P gene fragments of PMV and DMV was approximately 10%, which is similar to that observed between geogr aphically distinct isol ates of RPV. In a similar evaluation, the complete F proteins of PMV and DMV were compared and found to be 94% identical (Bolt et al., 1994). The results of these two studies indicate a close sequence relationship between DMV and PMV s uggesting that they should be considered two variants of CMV. Recently, van de Bild t et al. (2004) sequenced the hypervariable carboxyterminal (C-terminal) e nd of the N gene, and the complete H protein of both PMV and DMV. The divergence between the N gene C-terminal sequences of the two cetacean morbilliviruses was 18.3%. This wa s greater than 12.4% divergence between the two most distantly related strains of MV. The sequence divergence of the complete H gene gave similar results. PMV and DM V showed 15.5% divergence, while the MV strains only demonstrated 6.7%; thus, it was suggested that PMV and DMV be considered two different species of morbill ivirus. Based on results of Western blot analysis of the N protein and other differen ces in biological propert ies, Visser et al. (1993) concurred with the theory of two separate cetacean morb illivirus species. Areas of interest of the morbillivirus ge nome with relevance to this study include the N protein and the genome (leader) and antigenome (trailer) prom oter regions. All

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55 non-segmented, negative strand RNA viruses pro duce a nucleocapsid protein that serves to encapsidate viral RNA, thus, protecting it from cellular proteases as well as being involved in allowing the viral polymerase access to the RNA for transcription and replication. Multiple alignments of the N genes of MV, RPV, PPRV, CDV and PDV defined four regions within the N gene with varying degrees of homo logy (Diallo, et al., 1994). Region I (amino acid residues 1-222) wa s quite well conserved with identities between 75 and 83%, region II (residues 123144) had homologies below 40%, and region III (residues 145 to 420) was the mo st conserved with 85 to 90% homology. Region IV (residues 421 to the end) containe d the hypervariable C-terminus with less than 30% identity. The hyperv ariable region of MV, which is located outside of the nucleocapsid structure, has been shown to contain linear epitopes recognized by monoclonal antibodies, and has been used to separate MV into several different genotypes (Griffin, 2001). The bi ological significance of this area is uncertain, but this portion of the N gene may evolve rapidly, pos sibly indicating the lack of function or structural constraint. The N gene product is the most abundant vira l protein and one of the most variable in all morbilliviruses. Th e variation in sequence is impressive when compared to the high degree of conservation se en in other morbillivirus genes (van de Bildt et al., 2004). The N gene sequence diss imilarity between the two most distantly related MV strains is ~7%, and increases to 12.4% when the comparison is limited to the 456-bp of the C-terminal end. The N gene wa s chosen in this study, as it had been sequenced in full for all morbilliviruses, except PMV, and for the fact that the hypervariable 5’ end may possibly be used to distinguish infecti ons by PMV and DMV. Recent studies with morbilliviruses, as well as a number of other negative strand RNA

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56 viruses, have highlighted the importance of s hort untranslated regions at either end of the full length single stranded genome as playing a major role in virus pathogenesis. These regions, termed the genome and antigenome prom oters, generally take up less than 3% of the total genome length, but as promoters a nd regulators of both vi rus transcription and replication, they have been shown to play vi tal roles in viral pat hogenesis, although these events are not fully understood (Banya rd et al., 2003; Fujii et al., 2002). The objectives of the present study were to amplify and sequence the 3’ and 5’ extragenic regions, as well as the complete nucleocapsid gene of PMV in order to better understand its phylogenetic relationship to DMV and other morbilliviruses. The hypervariable C-terminal end of the N gene of the two cetacean morbilliviruses was expressed in order to aid in the distinction of PMV from DMV. Furthermore, cetacean tissues from recent mass stranding events were tested by PCR for the presence of morbillivirus nucleic acid in order to help de termine the cause of the mass strandings, as well as the possible presence of morbillivir us in the southeastern U.S throughout 2004. Materials and Methods Analysis of PMV Reverse transcription Porpoise morbillivirus (Belfast strain) RNA extracted from infected Vero cell cultures was kindly donated by Dr. Jeremiah T. Saliki from the Oklahoma State Animal Diagnostic Laboratory. The Belf ast strain of PMV was isolat ed from a harbor porpoise that stranded on the coast of Northern Ire land (Kennedy et al., 1988). Complementary DNA (cDNA) was prepared by reverse transcrip tion (RT) reactions us ing gene specific primers based on the sequence of the closely related DMV (GenBank accession number NC_005283). Briefly, 13.5 l of RNA and 2.7 M of primer were heated at 70C for 10

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57 min and snapped-cooled on ice. The final reactions contained 50 mM Tris-HCl at pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 333 M dNTP and 40 units RNase out. Reactions were incubated at 25C for 10 min followed by 2 min at 42C. Three hundred units of Superscript II (Inv itrogen, Carlsbad, California, USA) were added, and the reactions were incubated at 42C for 1 hr and inactivated at 70C for 15 min. Specific primers 8A and P2 were used to obtain c DNA for PCR targeting the complete N gene. Primers N2 and L were used to obtain cDNA to be used in the RACE technique for the amplification of the genome and antigenome promoter regions, respectively. Primer sequences were: 8A-5’ACC AGA CAA AGC TGG CTA GGG GT -3’ (forward), P25’GTC GGG TTG CAC CAC CTG TC -3’ (reverse), N2-5’CTG AAC TTG TTC TTC TGG ATT GAG TTC T -3’ (reverse), L5’TCG CGT CTG GAT CAG AGG -3’ (forward). RACE and amplification of the terminal extragenic domains The genome and antigenome promoter regions were obtained using the RACE (Rapid Amplification of cDNA Ends) techni que as described prev iously by Baron and Barrett (1995). Ten microlit ers of cDNA were added to 11.2 5 l ultra-pure water and 0.25 M NaOH, and incubated at 50C for 30 mi n. Acetic acid (0.3 M) was added and the mixture was purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences UK Limited, Little Chalfont, Buckinghamshire, UK). The tailing reactions contained 10 l cDNA, 25 mM Tris-HCl at pH 8.3, 37.5 mM KCl, 1.5 mM MgCl2, 5 mM DTT, 1 g BSA, 0.1 mM dC TP, and 19.8 units TdT (Promega UK Ltd., Southampton, UK). The mixture was h eated to 37C for 5 min followed by 65C for 10 min. The result of the RACE techni que produced run-off transcripts that were tailed with cytosine residues. The tailed cDNAs were subsequently used in a PCR

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58 containing 5 l of cDNA, 50 mM KCl, 10 mM Tris-HCl at pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl2, 100 M of each dNTP, 2.5 units Taq (Promega UK Ltd., Southampton, UK), 20 pM DMV specific internal primer and 25 pM of poly-Gn primer (5’GGG GGG GGG GAC CA -3’). The DMV sp ecific internal reverse prim ers used for leader and trailer amplification were: 5’TAC TTC AAC TAA TCT GAT GCT -3’, 5’TCG CGT CTG GAT CAG AGG -3’, respec tively. Cycling conditions were: Initial incubation at 95C for 2 min followed by 34 cycles at 95C for 45 sec, 55C for 45 sec, and 72C for 1 min 30 sec. A final extension step at 72C fo r 5 min finished the cycling. The RACE generated PCR products were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences UK Li mited, Little Chalfont, Buckinghamshire, UK) and sequenced in their entirety on both strands using the DMV specific primers described above. DNA sequencing was carri ed out using the Beckman automated CEQ8000 (Beckman Coulter Limited, Buckingh amshire, UK) sequencer as per the manufacturer’s instructions. Amplification of the complete PMV N gene Porpoise morbillivirus cDNA was used in two PCRs to obtain the full N gene. Primers 8A and N2 directed the amplification of appr oximately 1050-bp, while primers P2 and N1 directed the amplification of approximately 1650-bp with an overlap of approximately 230-bp (Figure 2-1). Prim er sequences were: 8A-5’ACC AGA CAA AGC TGG CTA GGG GT -3’ (forward), N15’ATA AAC CAA GGA TCG CTG AAA TGA T -3’ (forward), N2-5’CTG AAC TTG TTC TTC TGG ATT GAG TTC T -3’ (reverse), P2-5’GTC GGG TTG CAC CAC CTG TC -3’ (reverse). The PCR mixture contained 2 l of PMV cDNA, 400 nM of each primer, 100 M of each dNTP, 2 mM MgCl2, 1X Expand High Fidelity buffer and 3.3 units of Expand High Fidelity enzyme

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59 mix (Roche Applied Science, Indianapolis, Indiana, USA). Cycling conditions were: Initial incubation at 94C for 2 min followed by 40 cycles at 94C for 30 sec, 45C for 30 sec, and 72C for 3 min, followed by a fina l extension step at 72C for 10 min. Approximately 20 l of the PCR products were resolved by horizontal gel electrophoresis in 1.0% agarose containing et hidium bromide (0.5 ug/ml) and the DNA fragments were visualized by UV light transillumination a nd photographed using a gel documentation system. Figure 2-1. Schematic of the primers used to sequence the full nucleocapsid gene of PMV. Note that this figure is not drawn to scale. Amplified DNA fragments of the predicted size were purified using the MinElute PCR Purification Kit (Qiagen Inc., Valencia California, USA) and cloned into the plasmid vector pCR2.1-TOPO T/A (Invitroge n, Carlsbad, California, USA). Two clones for each sample were selected for plasmid pur ification and sequenced in duplicate from both ends using the CEQ 2000 XL (Beckman Co ulter Inc., Fullerton, California, USA) sequencing instrument following the manufacturer’s protocol as previously described in Chapter 1. Exported chromatograms were manually reviewed using the Chromas 2.3 software (Technelysium Pty Ltd., Tewantin, Queensl and, Australia). Genetic analyses and multiple sequence alignments were performed using the programs Seqed, Gap, Bestfit, Translate, Lineup, Pileup and Pretty of the University of Wisconsin Package Version 10.2, Genetics Computer Group (GCG), Madison, Wisconsin, USA. N gene P gene Leader 8A N1 N2 P2

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60 Phylogenetic analyses were performed using PAUP* 4.0 (Sinauer Associates, Sunderland, Massachusetts, USA) and the National Center for Biotechnology Information (NCBI) website was used to obtain sequences for all other morbilliviruses. Amplification of the C-terminus of the PMV N gene PCR was used to amplify the hypervariable C-terminus of the N gene of both PMV and DMV. The PCR procedure using Taq polymerase (Promega UK Ltd., Southampton, UK) was the same as above, and the primers used were: PMVDMVForward-3’AAC GAA TGG ATC CGG TCA AGA GAT GGT C AG GAG A -5’, PMVReverse-3’AAC CGT AAG CTT ATT AGC CGA GTA GGT C TT TGT CGT TAT -5’, DMVReverse-3’TTC GCC CAA GCT TAT CAG CCA AGT AGA TCT TTA TCA TTA T -5’. The PCR products were approximately 480-bp, whic h corresponded to amino acid residues 400 to 529 in the hypervariable C-terminus of the N gene of both viruses. The GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK) was used to purify multip le PCR products from each virus before digestion with BamHI and HindIII, and ligati on into the bacterial expression vector pQE30 (Qiagen Ltd., Crawley, West Sussex, UK). Th e vector-insert construct was used to transform competent M15 E. coli cells. Detection of N protein exp ression by Western blot Positive transformants, those that contai ned the 6xHis tagged morbillivirus protein, were expressed in M15 cells. Protein expres sion was induced by IPTG and detected by Western blot after Tris Tricine gel elect rophoresis, and protein transfer onto a nitrocellulose membrane. Primary and secondary antibodies used were HisTag monoclonal antibodies (EMD Biosciences, Inc., Novagen Brand, Madison, Wisconsin, USA) and peroxidase labeled anti-mous e immunoglobulin antibodies (Amersham

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61 Biosciences UK Ltd., Little Chalfont, Buck inghamshire, UK). ECL Plus Western Blotting Detection Reagent (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK) was then used to dete ct the truncated prot eins corresponding to the N-gene C-terminus. Analysis of Stranded Cetacean Tissues for Morbillivirus Sample acquisition, RNA extraction and reverse transcription Tissue samples were obtained from cetaceans that mass stranded on the coasts of Florida, Georgia, North and South Carolin a, and Texas throughout 2004. In total, 57 tissue samples, mostly from spleen, brain and lung, from four cetacean species were analyzed for morbillivirus genomes. A compre hensive listing of all samples tested is presented in Appendix B. Total cellular RNA was extracted from all samples using Trizol (Invitrogen, Carlsbad, California, USA). Briefly, chloro form was added to crushed tissue. After centrifugation, the top aqueous phase was collected and RNA was precipitated with propanol, washed with 70% ethanol, and eluted in 100 l of RNase-free water. The total RNA content and quality of the eluted R NA was determined using the Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biot ech, Piscataway, New Jersey, USA). Total RNA was used in RT reactions as previ ously described; however, 1.5 g of random hexamer primers were used as a substitute for DMV specific primers. PCR targeting the P gene PCR using universal morbillivirus primers designed to amplify a region of the P gene was used to detect genomic morbillivirus nucleic acid in tissues of recently stranded cetaceans. These primers are known to direct the amplification of a P gene fragment 429bp in length (Barrett et al., 1993). Prim er sequences were: 5’ATG TTT ATG ATC

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62 ACA GCG GT -3' (forward), 5’ATT GGG TTG CAC CAC TTG TC -3' (reverse). These primers had been thoroughly tested in our laboratories and shown to be very efficient at amplifying P gene fragme nts of DMV, PMV, MV, PDC and CDV (unpublished results). Total cDNA extracte d from monolayers of Vero cell cultures infected with the Edmonton strain of MV wa s used as a positive control for PCR. Five microliters of cDNA were used as amplification template, and the PCR mixture contained 400 nM of each primer, 100 M of each dNTP, 10 mM KCl, 10 mM (NH4)2 SO4, 20 mM Tris-HCl, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1 unit of Taq DNA polymerase (New England BioLabs Inc., Beverly, Massachusetts, USA). Cycling conditions were: Initial incubation at 94C for 30 sec followed by 40 cycles at 94C for 30 sec, 56C for 30 sec, and 72C for 1 min. A final extension step at 72C for 5 min finished the cyc ling. Approximately 20 l of the PCR products were resolved by horizontal gel electrophoresis in 1.0% agarose containing ethidium bromide (0.5 ug/ml) and the DNA fragments were visualized by UV light transillumination and photographed using a gel documentation system. PCR targeting the -actin gene To insure the integrity and amplification of the RNA, an internal control PCR was implemented on cDNA produced with random primers. Primers targeting the mammalian -actin housekeeping gene were used to amplify a 275-bp fragment from cetaceans. Primer sequences were: FP-5 ’GAG AAG CTG TGC TAC GTC GC -3’, RP5’CCA GAC AGC ACT GTG TTG GC -3’. The PCR mixture contained 5 l cDNA, 400 nM of each primer, 100 M of each dNTP, 10 mM KCl, 10 mM (NH4)2 SO4, 20 mM Tris-HCl, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1 unit of Taq DNA

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63 polymerase (New England BioLabs Inc., Beverly, Massachusetts, USA). Cycling conditions were 40 cycles at 94C for 30 sec, 60C for 30 sec, and 72C for 30 sec. Approximately 20 l of the PCR products were resolved by horizontal gel electrophoresis in 1.0% agarose containing et hidium bromide (0.5 ug/ml) and the DNA fragments were visualized by UV light transillumination a nd photographed using a gel documentation system. Results Analysis of PMV RACE and amplification of the terminal extragenic domains The poly-Gn primer amplifies 10 G bases to the 3’ end of the product, thus, if the RACE technique was successful, the tailed PCR product will be 10 nucleotides longer than the untailed PCR product (Figure 2-2). Upon sequencing the RACE generated PCR products, it was determined that the genome promoter region was 107-bp long, directly followed by the ATG start codon that begins th e N gene open reading frame. Similarly, the antigenome promoter region was 108-bp l ong and directly followed the L gene TAA stop codon. The leader and tr ailer sequences of PMV have been deposited into the GenBank database under accession numb ers AY949833 and AY949834, respectively. Nucleotide sequence comparisons between PMV and DMV showed 95.3% identity for the genome promoter and 80.6% identity for th e antigenome promoter. Comparisons of all morbillivirus genome and an tigenome promoter regions, w ith the exception of PDV, are shown in Figures 2-3 a nd 2-4, respectively.

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64 Figure 2-2. Gel electrophoresis of PCR products ; lane 1tailed 420-bp PCR product, lane 2tailed 443-bp PCR product, lane 3untailed 420-bp PCR product, lane 4untailed 443-bp PCR product.

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65 P M V : U G G U C U G U U U C G A C C G A U C C C C A U C U U A U U G U C U A U U A C U A U U U A A U A G U C U G A D M V : U G G U C U G U U U C G A C C G A U C C C C A U C U U A U U G U C U A U U A C U A U U U A A U A G U A U G A R P V : U G G U C U G U U U C G A C C C A U U C C U A G C A A G A U A G U U A C U A A C A C U A A A U C G U G U G A P P R : U G G U U U G U U U C A A C C C A U U C C U A U C U A G A A U A U U A U U G A U A C C U G A C C G U U U G A M e V : U G G U U U G U U U C A A C C C A U U C C U A U C A A G U U A G U U A C U A G U A G A A G A U C A C G U G A C D V : U G G U C U G U U U C A A C C G A U U C C U A U C A A U U U A A U A A C U U A U A A A A U A A U U U U U G A P M V : A U C C U A A U U A C U A G G A U A G U U A A C C G U G U U C U C A A C C U A U U U C C G A G U G U U A G U D M V : A U C C U A A U U A C U A G G A U A G U U A A C C G U G U C C U A A A C C U A U U U C C A A G U G U C A G U R P V : A U C C U A A G U U C U A G G A U A G C U G A C C U C G U C C G A A U C C C G U G U C C A A G A A A U U U U P P R : A U C C U C A U U U C U A G G A U G A C A G C C C C C C U C C U C C U C C U C G U U C U A G A A A C U G A U M e V : A U C C U A A G U U C U A G G A U A A U A G U C C C U G U U C U C G U C C U A A U C C C U A U A G G C U C U C D V : A U C C C A G U U A C U A G G A U G G A A U U U C U U G U U C C G A U C C C A A G U C U G G A U G G U U A U 6 1218 24 30 36 42 48 54 60 6672 78 84 90 96 102 108 P M V : U G G U C U G U U U C G A C C G A U C C C C A U C U U A U U G U C U A U U A C U A U U U A A U A G U C U G A D M V : U G G U C U G U U U C G A C C G A U C C C C A U C U U A U U G U C U A U U A C U A U U U A A U A G U A U G A R P V : U G G U C U G U U U C G A C C C A U U C C U A G C A A G A U A G U U A C U A A C A C U A A A U C G U G U G A P P R : U G G U U U G U U U C A A C C C A U U C C U A U C U A G A A U A U U A U U G A U A C A U U C C U A G C A A G A U A G U U A C U A A C A C U A A A U C G U G U G A P P R : U G G U U U G U U U C A A C C C A U U C C U A U C U A G A A U A U U A U U G A U A C C U G A C C G U U U G A M e V : U G G U U U G U U U C A A C C C A U U C C U A U C A A G U U A G U U A C U A G U A G A A G A U C A C G U G A C D V : U G G U C U G U U U C A A C C G A U U C C U A U C A A U U U A A U A A C U U A U A A A A U A A U U U U U G A P M V : A U C C U A A U U A C U A G G A U A G U U A A C C G U G U U C U C A A C C U A U U U C C G A G U G U U A G U D M V : A U C C U A A U U A C U A G G A U A G U U A A C C G U G U C C U A A A C C U A U U U C C A A G U G U C A G U R P V : A U C C U A A G U U C U A G G A U A G C U G A C C U C G U C C G A A U C C C G U G U C C A A G A A A U U U U P P R : A U C C U C A U U U C U A G G A U G A C A G C C C C C C U C C U C C U C C U C G U U C U A G A A A C U G A U M e V : A U C C U A A G U U C U A G G A U A A U A G U C C C U G U U C U C G U C C U A A U C C C U A U A G G C U C U C D V : A U C C C A G U U A C U A G G A U G G A A U U U C U U G U U C C G A U C C C A A G U C U G G A U G G U U A U U C U C D V : A U C C C A G U U A C U A G G A U G G A A U U U C U U G U U C C G A U C C C A A G U C U G G A U G G U U A U 6 1218 24 30 36 42 48 54 60 6672 78 84 90 96 102 108 Figure 2-3. Nucleotide sequence comparison of the genomic promoter region of morbilliviruses. The GenBank accession numbers of the sequences used were: NC_005283-Dolphin morbillivirus (DMV ), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MeV), NC_006383-Peste-des-petits ruminant virus (PPR), and NC_006296-Rinderpest virus (RPV). P M V : A C C A G A C A A A G C U G G G U A U A G G U A C U U A A A U C A G A U U U U G U U U U U C U U A A U A A U D M V : A C C A G A C A A A G C U G G G U A U A G G U A C U U A A A U C G A A U C U U G U U U U U C U U A A U A A U R P V : A C C A G A C A A A G C U G G G G A U A G A A A C U U C A C A U C U U U G A A G U U U U C U U U A G U A U A P P R : A C C A G A C A A A G C U G G G A A U A G A U A C U U A U C C A A U A U G U A G U U U U C U U U A A U C U U M e V : A C C A G A C A A A G C U G G G A A U A G A A A C U U C G U A U U U U C A A A G U U U U C U U U A A U A U A C D V : A C C A G A C A A A G C U G G G U A U G A U A A C U U A U U A A U A A C C G U U G U U U U U U U U C G U A U P M V : U A C A A U C U G A U A C C A U C C U G G U A G G A C U A G G A G U A G G G A U C C G U U C U A U U A A U C D M V : U A U A A U C U G A U A U C A C U C U G A C A A G G A C A G G A G C A G G G G U C C A C U U C A U U A G U U R P V : U U A U U U C U A C A G U U U U A U U G A C C A G A U C A G G A U C A G G A A G C A G A C A G U U G C A G A P P R : U U G G A U C A U C A G U G G G G U C U U A U G G C G G A G G A A A A G G G G G C A G A U G U A U A G C U A M e V : U U G C A A A U A A U G C C U A A C C A C C U A G G G C A G G A U U A G G G U U C C G G A G U U C A A C C A C D V : A A C U A A G U U C A A U A G C A A U G A A U G G A A G G G G G C U A G G A G C C A G A C U A A C C U G U C 6 1218 24 30 36 42 48 54 60 6672 78 84 90 96 102 108 P M V : A C C A G A C A A A G C U G G G U A U A G G U A C U U A A A U C A G A U U U U G U U U U U C U U A A U A A U D M V : A C C A G A C A A A G C U G G G U A U A G G U A C U U A A A U C G A A U C U U G U U U U U C U U A A U A A U R P V : A C C A G A C A A A G C U G G G G A U A G A A A C U U C A C A U C U U U G A A G U U U U C U U U A G U A U A P P R : A C C A G A C A A A G C U G G G A A U A G A U A C U U A U C C A A U A U G U A G U U U U C U U U A A U C U U M e V : A C C A G A C A A A G C U G G G A A U A G A A A C U U C G U A U U U U C A A A G U U U U C U U U A A U A U A C D V : A C C A G A C A A A G C U G G G U A U G A U A A C U U A U U A A U A A C C G U U G U U U U U U U U C G U A U P M V : U A C A A U C U G A U A C C A U C C U G G U A G G A C U A G G A G U A G G G A U C C G U U C U A U U A A U C D M V : U A U A A U C U G A U A U C A C U C U G A C A A G G A C A G G A G C A G G G G U C C A C U U C A U U A G U U R P V : U U A U U U C U A C A G U U U U A U U G A C C A G A U C A G G A U C A G G A A G C A G A C A G U U G C A G A P P R : U U G G A U C A U C A G U G G G G U C U U A U G G C G G A G G A A A A G G G G G C A G A U G U A U A G C U A M e V : U U G C A A A U A A U G C C U A A C C A C C U A G G G C A G G A U U A G G G U U C C G G A G U U C A A C C A C D V : A A C U A A G U U C A A U A G C A A U G A A U G G A A G G G G G C U A G G A G C C A G A C U A A C C U G U C 6 1218 24 30 36 42 48 54 60 6672 78 84 90 96 102 108 Figure 2-4. Nucleotide sequence comparison of the antigenomic promoter region of morbilliviruses. The GenBank accession numbers of the sequences used were: NC_005283-Dolphin morbillivirus (DMV ), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MeV), NC_006383-Peste-des-petits ruminant virus (PPR), and NC_006296-Rinderpest virus (RPV). Amplification of the complete PMV N gene The sequence for the entire N gene of PMV was obtained from two overlapping PCR products (Figure 2-5) and deposited in the GenBank database under accession number AY949833. The results i ndicated that the N gene of PMV was 1572 nucleotides

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66 in length and translated into a protein of 524 amino acid residues. The N gene of PMV was identical in length to that of DMV, CDV and PDV, and slightly shorter than that of MV, PPRV and RPV. Sequence homologies (Tables 2-1, 2-2 and 2-3) and phylogenetic analyses (Figures 2-6 and 2-7) demonstrated that PMV was most closely related to DMV, with 93.7 and 95.8% amino acid identity and similarity, respectively. From the N gene nucleotide comparison of the three marine mammal morbilliviruses, it was observed that the PMV N gene had a nucleotide identity of 88.6% to that of DMV, but only 66.5% to the PDV homologue (Figure 2-8). The N ge ne of PMV clearly contained the four previously described regions common to al l morbillivirus N proteins (Figure 2-9). Figure 2-5. Gel electrophoresis of the overlapping PCR produc ts used to obtain the full N gene sequence of PMV. Primers 8A a nd N2 generated a product of ~1050-bp (lane 1), while primers P2 and N1 gene rated a product of ~1650-bp (lane 2).

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67 Table 2-1. Percent nucleotide identities of morbillivirus N genes. GenBank accession numbers are: NC_005283-Dolphin morb illivirus (DMV), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Pestedes-petits ruminant virus (PPRV) NC_006296-Rinderpest virus (RPV) and X75717-Phocine distemper virus (PDV). RPV MV PPRV CDV PDV DMV PMV RPV 100.0 MV 69.5 100.0 PPRV 67.0 66.3100.0 CDV 63.7 64.964.4100.0 PDV 63.4 63.264.677.5100.0 DMV 66.5 67.367.666.965.5100.0 PMV 66.3 68.767.867.766.588.6 100.0 Table 2-2. Percent amino acid identities of morbillivirus N genes. GenBank accession numbers are: NC_005283-Dolphin morb illivirus (DMV), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Pestedes-petits ruminant virus (PPRV) NC_006296-Rinderpest virus (RPV) and X75717-Phocine distemper virus (PDV). RPV MV PPRV CDV PDV DMV PMV RPV 100.0 MV 75.1 100.0 PPRV 74.4 73.6100.0 CDV 69.3 72.370.1100.0 PDV 68.3 68.568.865.3100.0 DMV 73.0 75.475.473.370.2100.0 PMV 73.4 76.676.873.070.393.7 100.0 Table 2-3. Percent amino acid similarities of morbillivirus N genes. GenBank accession numbers are: NC_005283-Dolphin morb illivirus (DMV), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Pestedes-petits ruminant virus (PPRV) NC_006296-Rinderpest virus (RPV) and X75717-Phocine distemper virus (PDV). RPV MV PPRV CDV PDV DMV PMV RPV 100.0 MV 83.1 100.0 PPRV 79.9 79.5100.0 CDV 75.6 79.777.2100.0 PDV 75.0 76.076.389.1100.0 DMV 80.1 80.882.278.777.3100.0 PMV 80.1 81.082.378.677.595.8 100.0

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68 A B Figure 2-6. Neighbor-Joining phyl ogenetic trees of the nuc leotide sequences of the complete N gene of morbilliviruses. Th e tree was generated by Clustal X slow and accurate function using Gonnet 250 resi due weight table, gap penalty of 15 and gap length penalty of 6.66. GenBank accession numbers are: NC_005283-Dolphin morbillivirus (DMV ), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-des-petits ruminant virus (PPRV), NC_006296-R inderpest virus (RPV) and X75717Phocine distemper virus (PDV). A) Radial tree where the numbers represent the percent confidence of 1000 bootstrap replications B) Phylogram with 0.1 divergence scale. 0.1 Diver g ence DMV PMV PPRV RPV MV CDV PDV PPRV RPV MV 99.2 96.8 CDV PDV 100.0 PMV DMV 100.0

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69 A B Figure 2-7. Neighbor-Joining phylog enetic trees of the deduced amino acid sequences from the complete N gene of morbilliviruses. The tree was generated by Clustal X slow and accurate function using Gonnet 250 residue weight table, gap penalty of 10 and gap length pe nalty of 0.10. GenBank accession numbers are: NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-des-petits ruminant virus (PPRV), NC_006296-R inderpest virus (RPV) and X75717Phocine distemper virus (PDV). A) Radi al tree where numbers represent the percent confidence of 1000 bootstrap re plications B) Phylogram with 0.1 divergence scale. 0.1 Divergence PMV DMV PPRV MV RPV CDV PPRV MV RPV 63.9 56.3 CDV PDV 100.0 DMV PMV 100.0 PDV

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70 1 50 100 PMV .......... .......... ......g... .......... .t........ ......ta.. .......... .......... .......... ........c. DMV .......... .......... ...a..tc.. .......... .c........ ......cc.. .......... .......... c......... .......... PDV .....c.gt. ....caaa.. c...agc... .....a.a.. ca.gg..aca .c.c..at.g gc.t....a. .t..c..g.. .......... ..a..a.... Consensus ATGGCGACAC TTCTTCGGAG TCTGGC-TTG TTCAAGAGGA A-AAAGATAG AACACC--TA ATTGCAGGTT CAGGAGGAGC AATAAGAGGG ATTAAGCATG 101 150 200 PMV .a........ a......... .......... .a...act.. .......... ........t. .......... .......... .......... ....c..g.. DMV ....cg.... .........c .......... .g.....c.. ...g...... .......... .gt....... .......... .......... .......a.. PDV .......... .ttga.c... .....c..ga gc.....a.. .......... c......... ....t..c.. .a.g.t...a ......g.ag .t.....t.. Consensus TTATTATAGT CCCAGTACCT GGTGATTCAT C-ATTGT-AC TAGATCAAGA TTATTGGACA GACTAGTAAG ACTTGCTGGT GACCCTTATA TAAGTGG-CC 201 250 300 PMV .......... ..t....... .......... g......... .....g.... .......... g........g .......... .......... .......... DMV t......... ..c....... .......a.. a.....g... .......... .......... .......... ........c. .......t.. .......... PDV ....t....c ..g..tc... ....c..... c......... ..a.....c. .tg.a..gt. ...t...a.. ....t..... .t..t...a. ...t..a.ag Consensus CAAGCTGACA GG-GTCATGA TCAGTATCCT -TCATTATTT GTTGAATCAC CCAGTCAACT AATACAGCGA ATCACTGATG ACCCAGACGT CAGCATCAGA 301 350 400 PMV .......... ..g....... ...a.....a .......... .......... .........g ..c....... .......... .......... ..t....... DMV ........a. .......... ...g.....c .......... .......... .........c ..t....... .......... .......... .......... PDV .....g.... ....a.cc.. .att..t..t acc.gc..t. .t..a..... .........t ..g.gct... .t.c...a.. t.....g.t. ...ggg.c.a Consensus TTAGTTGAGG TAATCCAAAG TGA-AAGTCCTATCAGGGC TCACTTTTGC ATCCAGAGGGC-AATATGG AGGATGAGGC GGATGACTAT TTCTCTATTC 401 450 500 PMV ......ca.. .......... .......... ..a....... .......... .......... .......... ...g...... .......... .......c.. DMV ......g... .......... .......... ..c.t..... .......... .......... .a........ ...a...... ..a....... .......... PDV tg.ac.a..g ttc.aa...t cat.atca.. tggg...... a..a..t... ..t...a.c. ....c..... ga.t...g.. ...c.g.... .t..ct..c. Consensus AAGCAG-GGA GGAAGGGGAC ACCAGAGGAA CC-ACTGGTT TGAGAACAAA GAGATAGTTG ACATTGAGGT TCA-GACCCA GAGGAATTCA ACATACTATT 501 550 600 PMV .......... ..c....... .......t.. .......... .......... .......... .......... ..t....... .......... .........c DMV .........t ..t....... .t......c. .t........ .......... .......... .......... ..c....c.. .......... ...a...... PDV a..g..a... ..g..t.... .......... g..t...... ..t..a.... .......c.. ...g.....t ..a..a.... .aa.a..... ......c... Consensus GGCATCTATC CT-GCACAAA TCTGGATCTT ACTAGCCAAG GCAGTCACTG CTCCAGATAC TGCAGCTGAC TC-GAGATGA GGCGGTGGAT TAAGTATACT 601 650 700 PMV .......... .......... .......... ..c....... .......a.. ...c...... .......... .......... .........a .......... DMV .......... ....a..g.. .......... ..t.....g. .......g.. ...t...... .......... .......c.. .......... .......... PDV .....ga.a. .....a.... a..a...a.a a.ga.t...a t....c.t.. .ata..t..g ..ca.a.... .t......t. g..t...... c....t.... Consensus CAGCAACGCC GTGTGGTAGG TGAGTTTCGG CT-GACAAAG GATGGTT-GA TGC-GTGAGA AATCGGATTG CGGAGGATCT ATCGTTGAGG AGATTCATGG Figure 2-8. Nucleotide comparison and consensus sequence for the full N gene of three morbilliviruses than infect marine mamma ls. GenBank accession numbers are: NC_005283-Dolphin morbilliv irus (DMV), X75717-Phocine distemper virus (PDV).

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71 71 701 750 800 PMV .a........ .......... .......... ....a..... .......... ..t....... .......... .......... .......... .......... DMV .......... .......... ..g..a.... ....g..... .......... .....g.... .......c.. .......... .....c.... ....t..... PDV .......... a......... ......t.t. .g..c..t.. ...a..a... .......... .c..t..... .....a.... ..a.....a. .t.....g.. Consensus TGGCATTAAT TTTAGATATC AAAAGGACAC CAGG-AACAA ACCCAGGATT GCCGAAATGA TATGCGATAT AGACACCTAT ATCGTAGAGG CAGGCCTTGC 801 850 900 PMV .......... .....c..t. .g.....a.. ...g...... ........g. .......... .........a .......... .c..g..c.. ...ct.g... DMV .......... .......... .......g.. .......... ........c. .......... .........g .......... .t..ag.... ...t..a... PDV a.....t... t......... ....t..c.. ......t... ........ac .t..tc.a.. ...g.....t ..a..g..g. .a..c..... a..a..t... Consensus TAGCTTCATC CTAACTATCA AATTCGG-AT CGAAACAATG TACCCGGC-T TAGGGTTGCA TGAATTCTCGGTGAATTAA C-AC-ATTGA GTC-CT-ATG 901 950 1000 PMV .......... .......... .......... .......... ....c..c.. ......t... .......... .......... .........a .....g.... DMV ..c.....c. .......... .......... .......... .a........ .......... .......... .......... .......... c..t.a.... PDV gt.t.a.... .......... t..a..g... .....t.... .......... g......g.. .....t..a. .......... c..t..t... ..g..t.... Consensus AATCTCTATC AACAGATGGG CGAGACTGCA CCGTACATGG TGATTCTTGA AAACTCAATT CAGAACAAGT TCAGTGCAGG TTCATACCCG TTACT-TGGA 1001 1050 1100 PMV .......... .......... .......... ..g.c..... .......... .......... .......... .......... ........gt .a........ DMV .......... .......... .....a.... .......... .......... .......... .......... c......... .......... .g........ PDV .t.....t.. ......c..g ..c....... .g.....c.. ...a..gt.a ..c..c.... .a........ ......a..t ..c...c... .t........ Consensus GCTATGCAAT GGGAGTTGGA GTTGAGCTTG AAAATTCAAT GGGTGGACTT AATTTTGGTC GTTCTTACTT TGACCCTGCA TATTTCAGAC T-GGTCAAGA 1101 1150 1200 PMV .......... .......... .......... .......... ..t....... .......... ...t.....t .......... .t........ .......... DMV .......... .......... .c........ ......g... ..c......c .......... ...c.....c .......... .c........ .......... PDV a......... .....t..t. ....a..... ...ta..t.t ..g..c.... .t..c..... caaa.....g ..gc....a. .g..a..a.. a.tat.aaga Consensus GATGGTCAGG AGATCAGCAG GTAAGGTGAG CTCATCACTA GC-GCAGAAT TAGGGATCAC AGC-GAGGAGCCAAACTTG T-TCCGAGAT TGCTGCGCAG 1201 1250 1300 PMV .......... .......... .......... .....g.g.. .......... .......... .......... ....a..gt. t...g....g .....t.... DMV .....t.... .......... ...a...... .....c.... .......... ...g...... .......... ....c..t.. c......... .......... PDV a.a.c...a. .t...a.a.c ........ct .....t.... ..tc...... ca.......a ..ct....a. ..aat..a.. acc..ac.a. a.act.cct. Consensus GCTAACGACG ACAGAGCTAA TAGGGCAATA GGTCC-AAAC AAAACCAGAT ATCATTTCTT CATCCTGACA GAGG-GA-GC -AGTACTCCA GGGAACATCC 1301 1350 1400 PMV ....t..... t..a..c..t .....t.... ..a.....g. .......... ..a...c... .......... .......... c..a...... ....g..... DMV t.c....... c........c .....c.... .......aa. .......... ..t...t.c. ....a...g. ....c..... .....c.... ....c..... PDV .c.t.a.c.t ga..tc...a ttccaaggag .t...a..t. ttccaatc.g .tcattg..g atcg.ct..c tgg.....ct ..t...g.tc .atca...g. Consensus CTAGCGCAAA -GAGGGTGAGGGTC-ACCC G-GATGAG-A GAGGGGGGAA CA-TGC-ATA CCAAGAGGTA CAAGTATAGA TCAGATATCA ACGA-TCTCA Figure 2-8. Continued.

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72 72 1401 1450 1500 PMV .......... c.c...t... ....gg..a. .t........ ...t..t... ...g...c.. ....g..... c........g ....g..... .......... DMV .c........ ..t....... .....a..g. .c......a. ...c...... a......t.. .......... t........a .......... .......... PDV a.gggacg.g ..ac.c..ga tcac.ca.t. gac....g.a a.gg....at .ac.a.gat. ..c.....at g..c.gtc.c .....a.... ..cagc.ca. Consensus GTAAAGACAC TC-AGACATT GATGA-CA-T C-GACAACGC TGA-GACCCA GTTAGTA-CC AAAAATCAGC -GAGGCATTGCCAAGATGA GAGCCATGGC 1501 1550 1573 PMV .......... .........a ..t....... cac....... .......... .c........ .......... ... DMV .......t.g .......... ..c...g... t.t...t... ..c....... ....t..... t.....t... .g. PDV ....a.t... a.tc.gtc.. ata.aa...g g.aggt.t.c .c..c.c... ......gg.. ...g...a.. ... Consensus CAAGCTACTA GAAAACCAAG GC-CGCATGA -G-CACCGCG CATGTTTATA ATGACAAAGA CCTACTCGGC TAA Figure 2-8. Continued. 1 50 100 PDV ..s.....s. ..ktreq... .......... .......l.. .......... .......m.. ..e....... .......... .......... .i.......k CDV ..s.....t. ...tr.q... .......... .......l.. .......... .......... ..k.n..... .......... .......... .i.......k MV .....r.... .........i t......... .......... .....t.... .......... n......... .a..g..... .......... .......... RPV ..s....... ...a...... .a........ .......... .....t.... ......km.. .......... .a........ .......... .........k PMV .....r.... ......rt.i ia........ .......... .....t.... ........a. ..y....... ..m....... ....s..... .......... DMV .....r.... ......rt.. ia........ .......... .......... ........a. ..y....... ..m....... ....s..... .......... PPRV .......... .......a.t .......... ..n....... .......... ........a. ....n.s... ..m..m.... .......... .......... Consensus MATLLKSLAL FKRNKDKPPL ASGSGGAIRG IKHVIIVPIP GDSSIVTRSR LLDRLVRLVG DPDISGPKLT GVLISILSLF VESPGQLIQR ITDDPDVSIR ------Variable region-----101 150 200 PDV .....p.in. tc........ .sl.a...e. .gtm.eg.k. hn.m..l... d......n.a .q........ .......... .......... ..m....... CDV .....p.in. ac........ .sl.s...e. .k.v.eg.ka qg.l..l... d......dna .q........ .......... .......... ..m....... MV .l.....dq. .......... tn.e....q. ..hd..s.s. qsrf...... ..s....... .g..mi.gt. .......... .......... ..l....... RPV .......d.t .......... ts......r. .tyee.nd.e e..sy....r d.q....... .g..mi..t. .......... .......... ..l....... PMV .......e.. l......... .n.e....d. ...qaaee.. t.g.n..... .......... .......... .......... .......... ..m....... DMV .......e.. l......... .n.e....d. ...qagee.. t.g.h..... ...e...... .......... .......... .......... ..t....... PPRV .......tr. .......... .dl.n...m. ..teg.s..g kkrin....r .......... ....m..... .......... .......... ..l....... Consensus LVEVVQS-KS QSGLTFASRG A-MDDEAD-Y FSI-DP-SGD –RQTGWFENK EIVDIEVQDP EEFNILLASI LAQIWILLAK AVTAPDTAAD SE-RRWIKYT Figure 2-9. Amino acid comparison and consensus sequence of th e full nucleocapsid gene of mo rbilliviruses. GenBank accession numbers of the N gene sequences used are: NC_005283-Dolphi n morbillivirus (DMV), AF3054 19-Canine distemper virus (CDV), AB012948-Measles virus (MV), NC _006383-Peste-des-petits ruminant virus (PPRV), NC_006296-Rinderpest virus (RPV) and X75717-Phocine distemper virus (PDV).

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73 73 201 250 300 PDV .......... mn.i...i.. .......... .......... ..s....... ........n. .......... .......... .......... .......... CDV .......... mn.i...i.. .......... .......... ..s....... ........n. .......... .......... .......... .......... MV .......... .erk...v.. .......... .......... .......... .......... .......... .......... .........a ...s.l.... RPV .......... .......t.. .......... .......... .......... .......... .......... .......... .........a ...s...... PMV .......... .......a.. .......... .......... .......... .......... .......... .......... .......... .......... DMV .......... .......a.. .......... .......... .......... .......... .......... .......... .......... .......... PPRV .......... .......a.. .......... ....s..... .......... ........n. .......... .......... .........a ...s...... Consensus QQRRVVGEFR LDKGWLD-VR NRIAEDLSLR RFMVALILDI KRTPGNKPRI AEMICDIDTY IVEAGLASFI LTIKFGIETM YPALGLHEFS GELTTIESLM 301 350 400 PDV v......... .......... .......... .......... .......... .......... .......... ........tf ...f...k.. .q.....vsr CDV m......... .......... .......... .......... .......... .......... .......... ........a. .......k.. .q......sk MV .......... .......... .......... .......... .......... .......... .......... ........t. .s.......d .r......mh RPV ........l. .......... .......a.. .......... .......... .......... .......... ........n. .s.....e.. .r......ay PMV .......... .......... .......... .......... ....d..... .......... .......... ........s. .........d ........a. DMV .......... .......... .......... .......... .......... .......... .......... ........s. .........d ........a. PPRV .....l..v. .......... .......a.. .......... .g........ .......... .......... ........vi .......... ........s. Consensus NLYQQMGETA PYMVILENSI QNKFSAGSYP LLWSYAMGVG VELENSMGGL NFGRSYFDPA YFRLGQEMVR RSAGKVSS-L AAELGITAEE AKLVSEIA-Q -------------------------------------------------Hypervariable C-termi nus---------------------------------------------401 450 500 PDV ..e...t..t ....s..t.. .s.rn.ap.q rlpp....tm ks.fq..dky snqli.d.ls .y.s.vq..e wd.srqitql tq.gdh...n d.q.mdg..k CDV ..e...i..t ....s..t.. .s.rs.va.q q.p.....nk rs.nq..dky pihfs.e.lp .y.p.vn..e w..sry.tqi iqdd.n...d dr..m..i.k MV ..e..is..v ..r....... ..hg.qs..e l..lggkedr rvkqsr..ar esyretgss. asderaahlp t..tpl...t a..s.q..qd srr..d..l. RPV .s...n..ts .......... ..rt.q.sea qhsaskkdea rapqvkk.tr tssks.khke ..dkepv..s am.tli...t tl.adt..le sk......l. PMV an...a...i ..r.n..... ...p.r..s. a.....n.ps an.gd.st.m .rg.nnai.. ..si..i.ts ..kdtp...g q.dnad..vg t......... DMV an...a...i ....n..... ...p.r.da. t.....n.lr an.gd.st.m krg.niat.k ..si..t.tt ..kdtl...e q.dntd..is i........k PPRV ag.e..a.gt ..r....... q.ktg...s. a.a.reg.ka aipngse..d .kqtrsg... .e.ps.l... .l.impedev sr.s.qn.re a.r.....f. Consensus TTDDRTNRAGPKQAQISFL HHE-DEGENS -PGT---I---E--GGERR--G-D-RPR GTT-DQ-SSLSE---DID-SE-G-DP--QKSAEALAR ----------------------501 529 PDV ..qlt...nq sdtngev.pa h..r...s* CDV ..mlt.m.sq p.tse.n.p. ....e..n* MV lqt..g...e ..sdt..pr. ...s...d* RPV lq...g..gd stl.n.slra .......n* PMV ......l..n .sshdt.ah. ........* DMV ......l..n ..prdv.ah. ........* PPRV lq.......d .ee.e.n.q. ........* Consensus MRAMAKILEQG-G-DTS-V YNDKDLLGFigure 2-9. Continued.

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74 Detection of protein exp ression by Western blot Western blot analysis successfully de tected the expression of the N gene hypervariable C-terminal region of DMV, which corresponded to a 9240 kDa protein (Figure 2-10). No positive transformants were obtained for PMV. Figure 2-10. Expression of the N gene hypervar iable C-terminus of DMV in M15 cells as detected by Western blot. Lane 1DMV clone 1, Lane 2DMV clone 2. Analysis of Stranded Cetacean Tissues for Morbillivirus All 57 tissues tested from four cetacean species from recent mass stranding events in the southeast U.S. were negative for morbillivirus by PCR. Total RNAs extracted from the same tissues tested for the -actin housekeeping gene were all positive. Discussion Determining whether PMV and DMV should be considered two strains of CMV or individual virus species is no easy task, as much of the research has produced conflicting data. If the paramyxovirus nomenclature rules were applied to morbill iviruses, all viruses in that genus would belong to the same species based on high levels of serological crossreactivity, despite the distinct host range of each virus (Rima et al., 1995). All previous studies on cetace an morbilliviruses have c oncluded that they did not arise from any known morbillivirus, but repr esent an independent morbillivirus lineage (Barrett et al., 1993; Bolt et al., 1994). P hylogenetic analyses based on various gene sequences place DMV and PMV in a clad of th eir own, closer to MV and the ruminant

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75 morbilliviruses than to CDV and PDV (Ba rrett et al., 1993; Blixe nkrone-Mller et al., 1994; Bolt et al., 1994). Consequently, an epidemiological link between cetacean and pinniped morbillivirus outbreaks seems unlik ely. In support of these data, current evolutionary evidence suggests that cetacean s arose from land-based ruminants (Martin, 1990), whereas seals evolved from a terrestrial, otter-like ancestor (King, 1983). It also appears that morbilliviruses likely evolved with their host species, and the broadening host range may be due to migration of cetacean s in addition to spontaneous mutations of the viruses (Blixenkrone-Mller et al., 1994). Our results indicated that the N ge ne of PMV and DMV were 88.6 and 93.7% identical at the nucleotide and amino acid leve ls, respectively. Baron and Barrett (1995) reported the N gene homologies of four strains of RPV, and found amino acid identities to RPV-R of 99.2% for RPV-K, 93.1% for RPV-Kw, and 90.7% for RPV-L. The low homology of RPV-L was probably reflective of the fact that RPV-L is a lapinized strain, and is of geographically and temporally dist ant origin. It is know n, however, that the N proteins of morbilliviruses are much more c onserved over the first 400 amino acids. As a result, when only the first 400 amino acids were compared, the homologies between the four strains of RPV increased to approximately 99%. Similarly, the homology of PMV and DMV increased to 97.5% when only the first 400 amino acids of the N gene were compared. This region of the N protein is t hought to contain all th e necessary structural information for self-assembly into nucleocap sids, hence, the high sequence conservation, while the C-terminal tail is suggested to be i nvolved in replication. It is interesting to note that the hypervariable C-terminal homologi es also vary depending on the virus (Bolt et al., 1994). In this region, PMV and DM V differ by 21 amino acid residues, while

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76 CDV and PDV differ by 48. A maximum of 15 amino acid residue differences have been reported between the most divergent MV strain s. The residue homology of the cetacean morbilliviruses falls between that of two dist inct species and two very divergent strains, thus making conclusions about the relatedness of PMV and DMV very difficult. Previous research has indicated that the 3’ and 5’ terminal extragenic domains of different paramyxoviruses have only limited homology, whereas the ends (especially the first 18 bases) of individual viruses are ve ry similar (Baron and Barrett, 1995). These regions presumably contain th e promoter/landing site for th e viral polymerase, thus, high conservation is needed for transcription. Th e leader sequence of two strains of Sendai virus is relatively conserved, and a mutati on of only two nucleotid es at positions 20 and 24 results in a marked decrease of virus grow th and pathogenicity in mice (Fujii et al., 2002). Nucleotide sequence homologies for the genome and antigenome promoters of PMV and DMV were 95.3 and 80.6%, respectivel y. Homologies increased to 100% for both the leader and trailer when only the first 18 bases were analyzed. Our results thus indicate, that PMV a nd DMV are clearly related phylogenetically, and we suggest they should be regarded as two strains of CMV, based on high sequence homologies of the full N gene and the terminal extragenic regions. Phylogenetic analyses based on the full N gene of all morbilliviruses, including PMV, indicated the cetacean viruses form a distinct lineage, shown on the phylograms (Figures 2-6B and 2-7B) by considerable evolutionary divergence. As in previous studies, our results showed that CMV was closer in relation to MV and the rumi nant viruses than to the carnivore viruses. Based on multiple sequence homologies of the full N protein (Tables 2-1, 2-2, and 2-3), PMV was most closely related to DMV, followed by MV and PPRV, and was least

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77 related to PDV. In accordance with previous st udies, we concluded that there is indeed a substantial difference between morbilliviruses th at infect cetaceans and those that infect pinnipeds. For that reason, a biological association be tween cetacean and pinniped morbillivirus epizootics is highly improbable. Western blot analysis detected the expression of the N gene hypervariable C-te rminus of DMV. Positive transformants containing the homologous pr otein of PMV were not obtained, thus no genotypic comparisons could be made between these two strains of CMV. Other criteria, such as cell tropism, host range, pathogenesis and geographic localization, must also be considered when classifying viruses, as phylogenetic analysis alone seems insufficient (van de Bildt et al., 2004). With regard to these criteria, DMV and PMV are able to infect cells of the same species, with few exceptions, and cause similar lesions. On the other hand, DMV appears to have a broa der host range, and infection is more geographically widespread. More recently, a morbillivirus (PWMV) wa s identified in a stranded long-finned pilot whale ( Globicephala melas ) from New Jersey, and it wa s suggested that the virus might represent a third member of the CMV group (Taubenberger et al., 2000). Sequence fragments of the P and N gene obtained by RT-PCR showed a close, but distinct relationship to PMV and DMV. However, the fragments of the P and N genes that were analyzed were very small, 378and 230-bp respectively, thus the conclusions may be biased. Upon further sequence analys is of PWMV, we found that the nucleotide and amino acid identities to PMV and DMV were 85.7 and 98.7%, and 87.8 and 97.4%, respectively. It should also be noted th at the 76 amino acid residues of the N gene sequence of PWMV correspond to the highly co nserved third region of the N protein, and

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78 for that reason, the high degree of seque nce homology with PMV and DMV may be misleading. Much more sequence analysis is needed before any convincing conclusions can be made about the phylogeny of PWMV. Prior to the discovery of PWMV, Osterhau s et al. (1997) isolated a morbillivirus (MSMV) from tissues of Mediterranean monk seals ( Monachus monachus ) with respiratory distress in West Africa. Seru m antibodies to CDV were detected by an ELISA; however, based on polyclonal and m onoclonal antibody char acterization, MSMV was different from PMV and DMV. Usi ng a semi-nested PCR with group-specific primers for the genus Morbillivirus, 121-bp of the N gene were amplified, and a close relationship to DMV was found. Because th e sequence data remain unpublished, it is unknown what region of the N gene was amp lified. The use of Morbillivirus groupspecific primers suggests that, as with the PWMV, the highly conserved middle region of the N gene was the target of the PCR; ther efore, conclusions based on sequence analysis may be skewed. Also, the reliability of phylogenetic analys is based on only 121 nucleotides is questionable. A morbillivirus was also isolated from a Mediterranean monk seal that stranded in Greece (van de Bildt et al., 1999). Based on N and P gene sequence analyses and immunofluorescence patter ns, this virus was most closely related to PMV. These cases, along with the seropr evalence of morbillivirus antibodies found in polar bears, walruses and Florida manatees, strongly suggests that some interspecies transmission of morbilliviruses does occur in the wild. It has been reported that in the past few decades there has been a worldwide increase in diseases of marine organism s (Harvell et al., 1999). New diseases are appearing, not because new microorganism s are being introduced, but most likely

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79 because previous disease agents are changi ng. Many factors are hypothesized to play a role in emerging and resurging diseases, including host shifts, exposure of nave populations via translocation of pathogenic organisms, climate variability and human activity. All four factors may be of significant importance in regard to marine mammal morbillivirus outbreaks. Evidence of CDV host shifts, caused by pathogen spillover from domestic animals, has been proposed between sled dogs and Crabeater seals in Antarctica (Bengtson et al., 1991), as well as domestic dogs and Baikal seals in Siberia (Osterhaus et al., 1989). Host shifts may also be res ponsible for the repo rted prevalence of morbillivirus antibodies in polar bears and Florida manatees; howev er, both the infection and clinical disease in these two species have yet to be confirmed. Serological studies have shown that morbilliviruses are ubi quitous among pilot whal e populations, without much apparent prevalence of clinical disease or mortality. Pilot wh ales, as long distance vectors between America and Europe, are know n to mix with at least 13 other odontocete species, thus, providing numer ous opportunities for the intro duction of morbilliviruses into nave populations (Duignan et al., 1995a). Disease outbreaks occur when environm ental conditions either increase the prevalence or virulence of existing disease agen ts or facilitate a ne w disease (Harvell et al., 1999). Climate variability and human activ ity have been known to influence previous epidemics, for example, by enhancing the gl obal transport of marine species including pathogens or altering marine mammal be havior. One of the most prominent environmental factors is El Nio. This gl obal phenomenon causes shif ts in surface water temperature, which in turn a lters the water and atmospheric currents, and eventually food availability. It was reported that bottlenos e dolphins in southern California shifted home

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80 range over 500 km north in sear ch of food during an El Nio event (Reynolds III et al., 2000), and the 1982-1983 El Nio event caused wi despread mortality of pinnipeds in South America due to starvation (Trillmich et al ., 1991). Animals that travel outside their normal habitat have the potential to encount er new pathogens, while starvation may lead to immunosuppression and subsequent viral in fection. El Nio, as well as the opposing phenomenon La Nia, may have more direct influences on disease epidemiology through other large-scale oceanographic factors and pathogen transport. Very strong El Nio events were reported in 1982-1983, 1996-1997 and 2003-present, while weak events were seen in 1987-1988 and 2002-20 03. Severe morbillivirus ep izootics, such as those seen in 1987-1988 and 2002-2003, seem to be a ssociated with weak El Nio events, or those blocked by strong La Nia episodes. Prolonged El Nio events have been shown to increase the prevalence of other diseases of marine organisms, including coral bleaching and Dermo, a protozoan parasite disease of Eastern oysters ( Crassostrea virginica ) (Harvell et al., 1999). The a pparent relationship between these diseases and El Nio suggests that epidemics may be predicted fr om climate models based on the assumption that climate-mediated, physiological stress es may compromise host resistance and increase frequency of opportunist ic diseases. It would be in teresting to investigate the potential correlation between morb illivirus epizootics and such climatological episodes. Human activity has facilitated disease outbreaks in multiple ways, including the introduction of countless pollutants a nd habitat degradation. Environmental contaminants (particularly organochlorines and certain heavy metals) are known to bioaccumulate in top predators, such as many marine mammals (Geraci et al., 1999). Organochlorines have been shown to have immunosuppressive effects in laboratory

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81 animals (O’Hara and O’Shea, 2001); however; evidence for causal relationships between high contaminant levels and disease suscepti bility in the wild is inconclusive. In response to PDV, no difference was detected in harbor seals fed fish supplemented with polychlorinated biphenyls (PCBs) and those fed uncontaminated fish (Harder et al., 1992). Conversely, more recent studies showed that seals fed fish from polluted waters developed impaired immune responses, while t hose fed clean fish did not (Geraci et al., 1999). Similar contradictory results were al so obtained from research on cetaceans. Correlations were detected be tween organochlorine concentr ations and reduced immune responses of bottlenose dolphins in Florida (O’Hara and O’Shea, 2001). On the contrary, studies of harbor porpoises in Great Britain that died from infectious disease in comparison with those that died from trau ma revealed no significant differences in concentrations of organochlorines in the blubber. Pertaining to the 1987-1988 morbillivirus outbreak of bottlenose dolphins, so me samples infected with the virus also showed extremely high organochlorine concen trations, while othe rs were very low (Geraci et al., 1999). Cause and effect relationships between ma ss mortalities and a ny single factor are difficult to demonstrate; hence, it is proba ble that multiple factors play a role in morbillivirus epizootics of marine mammals. For example, brevetoxin was suspected to be a contributing cause in the DMV/PMV epiz ootic in Atlantic bot tlenose dolphins in 1987 and 1988 (Geraci et al., 1999). Sublethal exposure to th e toxin may have left the dolphins emaciated, exhausted, thermally st ressed and vulnerable to morbillivirus infections. Similarly, Mediterranean stri ped dolphin carcasses infected with DMV between 1990 and 1992 also contained high leve ls of organochlorines (Geraci et al.,

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82 1999). Finally, morbillivirus was detected in a number of Cape fur seals ( Arctocephalus pusillus pusillus ) that died in 1994, however the cause of death was presumed to be chronic starvation associated with the ongoing El Nio event (Geraci et al., 1999). The fundamentals of understa nding infectious diseases are the identification, isolation and characterization of the etiological agent. This information enables the development of specific diagnostic methods fo r epidemiological surveys and studies of host resistance. Much of this work has been done on marine mammal morbilliviruses, thus, the next logical step is treatment and pr evention. At this time, there is no effective treatment for morbillivirus infections; how ever, successful vaccines for RPV, PPRV, CDV and MV are widely employed to control mo rbillivirus agents in terrestrial domestic animals, including humans. Research has been done with vaccines against CDV in two species of pinnipeds, specifically grey and harbor seals. Attenuated CDV vaccines have been reported to produce antibodies in both species (Visser et al., 1989; Hughes et al., 1992); however, this method requires three separate vaccinations, which is time consuming and costly. A CDV vaccine using a recombinant vaccinia virus was given orally to harbor seals with unsuccessful resu lts (Van Bressem et al ., 1991). At present, no vaccination trials have been conducted on cetaceans. Although none of the tissues from cetaceans from recent mass stranding events in the southeastern U.S. tested positive for mo rbilliviruses in this study, these viruses should remain of high concern as both their geographi cal distribution and hos t range continue to expand. The mass mortality brought on by morb illivirus epizootics is damaging to all marine mammal populations, but can be especi ally devastating to endangered species. All knowledge gained about marine mammal morb illiviruses, including that of this study,

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83 aids in the understand ing of these devastating infections Appreciating the molecular and phylogenetic characteristics of these viruses br ings us one step closer to unearthing a successful preventative or remedial strategy.

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APPENDIX A MARINE MAMMAL SAMPLES

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85Table A-1. List of all lesion a nd tissue samples tested for herpesvirus genomes using the nested PCR approach. Strandedsing le animal stranding, UMEanimal was part of unusual mortality event, captureanimal was part of capture and release program, NAdata not available. Number Species Animal ID Location Date Sample Result v183 Globicephala macrorhynchus Captive NA NA skin lesion neg v210A Tursiops truncatus Captive NA NA oral lesion neg v223 Orcinus orca Captive NA NA skin lesion neg v224 Tursiops truncatus Captive NA NA oral lesion neg v225B Tursiops truncatus Captive NA NA skin lesion neg v226A, K231 Tursiops truncatus Captive NA NA skin lesion POS v228A Tursiops truncatus Captive NA NA pox-like lesion neg v240 Tursiops truncatus Captive NA NA oral lesion neg v262 Tursiops truncatus FB188 capture FL 11/2002 skin lesion neg v263 Tursiops truncatus CMA-02-21 stranded FL 10/2002 open sorerostrum neg v264 Tursiops truncatus CMA-02-21 stranded FL 10/2002 skin lesion neg v265 Tursiops truncatus CMA-02-21 stranded FL 10/2002 cerebellum neg v267 Tursiops truncatus CMA-02-21 stranded FL 10/2002 cerebrum neg v268 Tursiops truncatus CMA-02-21 stranded FL 10/2002 lung LN neg v269 Tursiops truncatus CMA-02-21 stranded FL 10/2002 brainstem neg v273 Tursiops truncatus CMA-02-21 stranded FL 10/2002 prescap LN neg v293 Kogia sima MML-0233 stranded FL 9/2002 mesenteric LN neg v298, K265 Kogia sima MML-0233 stranded FL 9/2002 genital lesion POS v300 Kogia sima MML-0233 stranded FL 9/2002 pulm LN neg v301B Kogia sima MML-0233 stranded FL 9/2002 prescap LN neg v386 Tursiops truncatus Captive NA NA oral lesion neg

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86Table A-1. Continued. Number Species Animal ID Location Date Sample Result v387 Tursiops truncatus Captive NA NA tongue lesion neg v388 Tursiops truncatus Captive NA NA tongue lesion neg v391 Tursiops truncatus MML-0229 stranded FL 8/2002 skinshark bite neg v447 Tursiops truncatus MML-0309 stranded FL 2/2003 skin lesion neg v470 Tursiops truncatus MML-0305 stranded FL 2/2003 skin lesion neg v557 Globicephala macrorhynchus FKMMRT 0305 NA NA pox-like lesion neg v558 Globicephala macrorhynchus FKMMRT 0305 NA NA pox-like lesion neg v585 Chelonia mydas Pappy ST0306 stranded FL 3/2003 fibropapilloma POS v587 Tursiops truncatus Captive NA NA oral lesion neg v614 Grampus griseus MARS 0303 NA NA skin lesion neg v626 Tursiops truncatus Captive NA NA oral lesion neg v634, K264 Tursiops truncatus Jacksonville stranded FL 9/2003 penile lesion POS v653 Tursiops truncatus MML-0313 stranded FL 4/2003 skin lesion neg v654 Lepiochelys kempii ST0109 stranded FL 1/2001 fibropapilloma POS v657 Lepiochelys kempii ST0109 stranded FL 1/2001 fibropapilloma POS v600 Lepiochelys kempii ST0109 stranded FL 1/2001 fibropapilloma POS v663 Phocoena phocoena 03-04Pp stranded NC 3/2003 skin lesion neg v682A Tursiops truncatus Captive NA NA skin lesion neg v683B Tursiops truncatus Captive NA NA skin lesion neg v702 Steno bredanensis MML-0237 stranded FL 12/2002 skin lesion neg v703 Steno bredanensis MML-0237 stranded FL 12/2002 skin lesion neg v711 Phocoena phocoena 03-04Pp stranded NC 3/2003 skin lesion neg v713 Chelonia mydas Simba stranded FL 11/2002 fibropapilloma POS v714 Chelonia mydas Coquina stranded FL NA fibropapilloma POS v715 Tursiops truncatus Captive NA NA oral lesion neg v716 Tursiops truncatus Captive NA NA oral lesion neg v722 Tursiops truncatus Captive NA NA penile lesion neg v724 Balaena mysticetus 99B4 capture AK 1999 skin lesion neg v725 Balaena mysticetus 97B7-1 capture AK 1997 skin lesion neg v726 Balaena mysticetus 97B7-2 capture AK 1997 skin lesion neg v727 Balaena mysticetus 99B12 capture AK 1999 skin lesion neg

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87Table A-1. Continued. Number Species Animal ID Location Date Sample Result v728 Balaena mysticetus 99KK2 capture AK 1999 skin lesion neg v729 Balaena mysticetus 98KK3 capture AK 1998 skin lesion neg v730 Balaena mysticetus 98KK3-2 capture AK 1998 skin lesion neg v731 Balaena mysticetus 98KK1 capture AK 1998 skin lesion neg v732 Balaena mysticetus 99B24 capture AK 1999 skin lesion neg v733 Kogia breviceps MARS 9905 NA NA mammary slit neg v734 Orcinus orca Captive NA NA skin lesion neg v735 Orcinus orca Captive NA NA skin lesion neg v736 Tursiops truncatus MML-9628 stranded FL 9/1996 skin lesion neg v737 Tursiops truncatus MML-9628 stranded FL 9/1996 skin lesion neg v738 Orcinus orca Captive NA NA skin papilloma neg v739 Orcinus orca Captive NA NA skin papilloma neg v740 Orcinus orca Captive NA NA skin papilloma neg v741 Orcinus orca Captive NA NA skin papilloma neg v742 Orcinus orca Captive NA NA skin papilloma neg v743 Orcinus orca Captive NA NA skin papilloma neg v744 Orcinus orca Captive NA NA skin papilloma neg v745 Orcinus orca Captive NA NA skin papilloma neg v746 Orcinus orca Captive NA NA skin papilloma neg v747 Orcinus orca Captive NA NA skin papilloma neg v748 Orcinus orca Captive NA NA skin papilloma neg v752 Caretta caretta Floater stranded FL NA tongue lesion neg v963 Balaena mysticetus 90B2 capture AK 1998 penile lesion neg v964 Balaena mysticetus 90B10 capture AK 1998 skin lesion neg v965 Balaena mysticetus 02-291-1 capture AK 2002 oral lesion neg v966 Balaena mysticetus 02-291-2 capture AK 2002 skin lesion neg v967 Balaena mysticetus 99B18 capture AK 1999 skin lesion neg v968 Balaena mysticetus 02-292-1 capture AK 2002 skin lesion neg v969 Balaena mysticetus 02-292-2 capture AK 2002 skin lesion neg v970 Balaena mysticetus 97-149-1 capture AK 1997 skin lesion neg v971 Balaena mysticetus 98KK3 capture AK 1998 skin lesion neg

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88Table A-1. Continued. Number Species Animal ID Location Date Sample Result v972 Balaena mysticetus 79B2 capture AK 1997 skin lesion neg v973 Balaena mysticetus 02KK3 capture AK 2002 skin lesion neg v974 Balaena mysticetus 99-035 capture AK 1999 lip lesion neg v975 Balaena mysticetus 99-102 capture AK 1999 skin lesion neg v976 Balaena mysticetus 99-044-A capture AK 1999 skin lesion neg v977 Balaena mysticetus 99-044-B capture AK 1999 skin lesion neg v978 Balaena mysticetus 99-108 capture AK 1999 oral lesion neg v979 Balaena mysticetus 00-143-1 capture AK 2000 skin lesion neg v980 Balaena mysticetus 00-143-2 capture AK 2000 skin lesion neg v981 Balaena mysticetus 00-146 capture AK 2000 skin lesion neg v1019 Tursiops truncatus Captive NA NA oral lesion neg v1025 Stenella attenuata FLGM011304-02 stranded FL 1/2004 skin lesion neg v1026 Tursiops truncatus FLPB102303-03 stranded FL 10/2003skin lesion neg v1027 Tursiops truncatus Captive NA NA oral lesion neg v1039 Kogia sima HUBBS 0336 NA NA skin lesion neg v1040 Tursiops truncatus HUBBS 0408 NA NA skin lesion neg v1164 Tursiops truncatus HUBBS 0435 UME FL 3/2004 spinal cord neg v1165 Tursiops truncatus HUBBS 0436 UME FL 3/2004 brain neg v1176 Tursiops truncatus HUBBS 0438 UME FL 3/2004 brain neg v1192 Tursiops truncatus HUBBS 0436 UME FL 3/2004 pox-like lesion neg v1193 Tursiops truncatus HUBBS 0439 UME FL 3/2004 pox-like lesion neg v1194 Tursiops truncatus HUBBS 0439 UME FL 3/2004 brain neg v1225 Tursiops truncatus SJP0426 UME FL 3/2004 brain neg v1229 Tursiops truncatus SJP0319-22 UME FL 3/2004 brain neg v1233 Tursiops truncatus SJP20 UME FL 3/2004 brain neg v1234 Tursiops truncatus SJP319-21 UME FL 3/2004 brain neg v1235 Tursiops truncatus MIA0411 UME FL 3/2004 brain neg v1270A Tursiops truncatus G040322-2tt UME FL 3/2004 skin lesion neg v1272 Chelonia mydas Pappy ST0306 stranded FL 3/2003 fibropapilloma POS v1273 Lepiochelys kempii ST0109 stranded FL 1/2001 fibropapilloma POS v1274 Lepiochelys kempii ST0109 stranded FL 1/2001 fibropapilloma POS

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89Table A-1. Continued. Number Species Animal ID Location Date Sample Result v1275 Chelonia mydas Coquina stranded FL NA fibropapilloma POS v1276 Chelonia mydas Coquina stranded FL NA fibropapilloma POS v1277 Chelonia mydas Coquina stranded FL NA fibropapilloma POS v1278 Chelonia mydas Simba stranded FL 11/2002fibropapilloma POS v1279 Chelonia mydas Simba stranded FL 11/2002fibropapilloma POS v1280 Chelonia mydas Simba stranded FL 11/2002fibropapilloma POS v1283 Tursiops truncatus SJB0323-27 UME FL 3/2004 brain neg v1288 Tursiops truncatus SJP0325-32 UME FL 3/2004 brain neg v1301 Tursiops truncatus SJP03-26-35 UME FL 3/2004 brain neg v1310 Tursiops truncatus SJP0325-32 UME FL 3/2004 skin lesion neg v1317 Tursiops truncatus SJP0325-32 UME FL 3/2004 skin lesion neg v1318 Tursiops truncatus SJP0325-32 UME FL 3/2004 skin lesion neg v1322 Tursiops truncatus SJP0325-32 UME FL 3/2004 skin lesion neg v1344, K156 Tursiops truncatus Captive NA NA penile lesion POS v1345A Tursiops truncatus MML-0407 stranded FL 5/2004 skin lesion neg v1346A, K167 Tursiops truncatus MML-0407 stranded FL 5/2004 rostral lesion POS v1347 Tursiops truncatus MML-0407 stranded FL 5/2004 skin lesion POS v1348 Tursiops truncatus MML-0407 stranded FL 5/2004 skin lesion POS v1349 Tursiops truncatus MML-0407 stranded FL 5/2004 skin lesion neg v1350 Tursiops truncatus MML-0407 stranded FL 5/2004 skin lesion POS v1353, K263 Tursiops truncatus Captive NA NA genital lesion POS v1422, K285 Mesoplodon densirostris OK 04050729 stranded NC 1/2004 penile lesion POS v1423 Caretta caretta Floater stranded FL NA oral lesion neg v1425 Grampus griseus GU0403 stranded NC 7/2004 skin lesion neg v1434 Tursiops truncatus OK 04070556 NA NA oral lesion neg v1435 Tursiops truncatus OK 04070556 NA NA oral lesion neg v1436 Tursiops truncatus OK 04070556 NA NA oral lesion neg v1445 Steno bredanensis MML-0414D UME FL 8/2004 kidney neg v1446 Steno bredanensis MML-0414D UME FL 8/2004 lung neg v1449 Steno bredanensis MML-0414D UME FL 8/2004 LN neg v1450 Steno bredanensis MML-0414D UME FL 8/2004 heart neg

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90Table A-1. Continued. Number Species Animal ID Location Date Sample Result v1451 Steno bredanensis MML-0414D UME FL 8/2004 brain neg v1454 Steno bredanensis MML-0414E UME FL 8/2004 brain neg v1455 Steno bredanensis MML-0414E UME FL 8/2004 kidney neg v1457 Steno bredanensis MML-0414E UME FL 8/2004 LN neg v1459 Steno bredanensis MML-0414E UME FL 8/2004 lung neg v1461 Steno bredanensis MML-0414E UME FL 8/2004 heart neg v1462 Kogia breviceps GA2004030 stranded GA 9/2004 lung neg v1463 Kogia breviceps GA2004030 stranded GA 9/2004 liver neg v1464 Kogia breviceps GA2004030 stranded GA 9/2004 spleen neg v1465 Kogia breviceps GA2004030 standed GA 9/2004 brain stem neg v1467 Kogia breviceps SC0437 UME SC 8/2004 lung neg v1487 Kogia breviceps SC0434 UME SC 8/2004 lung neg v1492 Tursiops truncatus SC0432 UME SC 8/2004 brain neg v1496 Kogia breviceps SC0433 UME SC 8/2004 lung neg v1499 Tursiops truncatus SC0431 capture SC 8/2004 lung neg v1500 Tursiops truncatus SC0431 capture SC 8/2004 cerebrum neg v1502 Tursiops truncatus SC0431 capture SC 8/2004 thymus neg v1503 Tursiops truncatus SC0441 UME SC 9/2004 lung neg v1504 Tursiops truncatus SC0441 UME SC 9/2004 kidney neg v1506 Tursiops truncatus SC0441 UME SC 9/2004 liver neg v1507 Tursiops truncatus Captive NA NA gingival mass neg v1508 Tursiops truncatus Captive NA NA placental lesions neg v1514 Tursiops truncatus KMS367 UME NC 9/2004 cerebrum neg v1519 Tursiops truncatus KMS367 UME NC 9/2004 lung neg v1521 Tursiops truncatus KMS367 UME NC 9/2004 cerebellum neg v1523 Grampus griseus GML028 NA NA cerebellum neg v1524 Grampus griseus GML028 NA NA cerebrum neg v1528 Grampus griseus GML028 NA NA penile wart neg v1529 Grampus griseus GML028 NA NA pox-like neg v1531 Grampus griseus GML028 NA NA penile nodule neg v1533 Grampus griseus KMS364 UME NC 9/2004 lung neg

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91Table A-1. Continued. Number Species Animal ID Location Date Sample Result v1546 Stenella coeruleoalba OK 04091932 Belgium NA pox-like lesion neg v1550 Kogia breviceps MARS0403 NA NA lung neg v1561 Kogia breviceps MARS0402 NA NA lung neg v1569 Tursiops truncatus MIA0414 NA NA skin lesion neg v1570 Tursiops truncatus MIA0414 NA NA skin lesion neg v1571 Tursiops truncatus MIA0414 NA NA skin lesion neg v1573 Tursiops truncatus MIA0414 NA NA lung neg v1611 Tursiops truncatus MARS0404 NA NA skin lesion neg v1612 Tursiops truncatus MARS0404 NA NA skin lesion neg v1614 Tursiops truncatus MARS0404 NA NA skin lesion neg v1616 Grampus griseus MMC02-2004 NA NA lung neg v1635 Kogia breviceps FKMMRT0403 NA NA lung neg v1644 Steno bredanensis GW04008A NA NA spleen neg v1645 Steno bredanensis GW04008A NA NA cerebellum neg v1646 Steno bredanensis GW04008A NA NA lung neg v1647 Steno bredanensis GW04008A NA NA medulla/cerebrumneg v1654 Steno bredanensis GW04009A NA NA spleen neg v1658 Steno bredanensis GW04009A NA NA lung neg v1661 Steno bredanensis GW04009A NA NA cerebellum neg v1662 Steno bredanensis GW04009A NA NA cerebrum neg v1693 Tursiops truncatus GA2004035 stranded GA 11/2004spleen neg v1694 Tursiops truncatus GA2004035 stranded GA 11/2004right lung neg v1695 Tursiops truncatus GA2004035 stranded GA 11/2004left lung neg v1696 Tursiops truncatus GA2004035 stranded GA 11/2004spleen neg v1697 Tursiops truncatus GA2004035 stranded GA 11/2004kidney neg v1698 Tursiops truncatus GA2004035 stranded GA 11/2004cerebrum neg v1699 Tursiops truncatus GA2004035 stranded GA 11/2004cerebellum neg v1700 Tursiops truncatus GA2004035 stranded GA 11/2004liver neg

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92Table A-1. Continued. Number Species Animal ID Location Date Sample Result v1775 Eumetopias jubatus V02-078 capture AK 3/2003 vesicular lesion Neg R218 Tursiops truncatus DP0101 stranded FL 6/2001 genital lesion neg R219 Tursiops truncatus DP0101 stranded FL 6/2001 lung neg R220 Tursiops truncatus DP0101 stranded FL 6/2001 tongue lesion neg

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APPENDIX B CETACEAN TISSUE SAMPLES

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94 Table B-1. List of all tissue samples test ed for morbillivirus nucleic acids using PCR targeting the phos phoprotein (P) gene. Strandedsingle animal stranding, UMEanimal was pa rt of unusual mortality event, capturean imal was part of capture and release program, NAdata not available. Number Species Animal ID Location Date Sample Result v1444 Steno bredanensis MML-0414D UME FL 8/2004 Spleen neg v1446 Steno bredanensis MML-0414D UME FL 8/2004 Lung neg v1449 Steno bredanensis MML-0414D UME FL 8/2004 LN neg v1451 Steno bredanensis MML-0414D UME FL 8/2004 Brain neg v1452 Steno bredanensis MML-0414E UME FL 8/2004 Spleen neg v1454 Steno bredanensis MML-0414E UME FL 8/2004 Brain neg v1457 Steno bredanensis MML-0414E UME FL 8/2004 LN neg v1459 Steno bredanensis MML-0414E UME FL 8/2004 Lung neg v1462 Kogia breviceps GA2004030 Stranded GA 9/2004 Lung neg v1463 Kogia breviceps GA2004030 Stranded GA 9/2004 Liver neg v1464 Kogia breviceps GA2004030 Stranded GA 9/2004 Spleen neg v1465 Kogia breviceps GA2004030 Stranded GA 9/2004 brain stem neg v1467 Kogia breviceps SC0437 UME SC 8/2004 Lung neg v1473 Kogia breviceps 2004089SC UME SC 8/2004 Lung neg v1477 Kogia breviceps SC0439 UME SC 9/2004 Lung neg v1482 Kogia breviceps 2004088SC UME SC 8/2004 Lung neg v1487 Kogia breviceps SC0434 UME SC 8/2004 lung neg v1489 Kogia breviceps SC0432 UME SC 8/2004 lung neg v1492 Kogia breviceps SC0432 UME SC 8/2004 brain neg v1496 Kogia breviceps SC0433 UME SC 8/2004 lung neg v1499 Tursiops truncatus SC0431 capture SC 8/2004 lung neg

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95Table B-1. Continued. Number Species Animal ID Location Date Sample Result v1500 Tursiops truncatus SC0431 capture SC 8/2004 cerebrum neg v1503 Tursiops truncatus SC0441 UME SC 9/2004 lung neg v1505 Tursiops truncatus SC0441 UME SC 9/2004 spleen neg v1509 Tursiops truncatus KMS365 UME NC 9/2004 liver neg v1510 Tursiops truncatus KMS365 UME NC 9/2004 pancreas neg v1511 Tursiops truncatus KMS365 UME NC 9/2004 thyroid neg v1512 Tursiops truncatus KMS365 UME NC 9/2004 LN neg v1513 Tursiops truncatus KMS365 UME NC 9/2004 pre-scap LN neg v1514 Tursiops truncatus KMS367 UME NC 9/2004 cerebrum neg v1515 Tursiops truncatus KMS367 UME NC 9/2004 lung LN neg v1516 Tursiops truncatus KMS367 UME NC 9/2004 spleen neg v1519 Tursiops truncatus KMS367 UME NC 9/2004 lung neg v1521 Tursiops truncatus KMS367 UME NC 9/2004 cerebellum neg v1522 Grampus griseus GML028 NA NA lung neg v1523 Grampus griseus GML028 NA NA cerebellum neg v1524 Grampus griseus GML028 NA NA cerebrum neg v1527 Grampus griseus GML028 NA NA spleen neg v1537 Grampus griseus KMS364 UME NC 9/2004 spleen neg v1550 Kogia breviceps MARS0403 NA NA lung neg v1561 Kogia breviceps MARS0402 NA NA lung neg v1573 Tursiops truncatus MIA0414 NA NA lung neg v1610 Tursiops truncatus MARS0404 NA NA lung neg v1616 Grampus griseus MMC02-2004 NA NA lung neg v1635 Kogia breviceps FKMMRT0403 NA NA lung neg v1644 Steno bredanensis GW04008A NA NA spleen neg v1645 Steno bredanensis GW04008A NA NA cerebellum neg v1646 Steno bredanensis GW04008A NA NA lung neg v1647 Steno bredanensis GW04008A NA NA medulla/cerebrumneg v1654 Steno bredanensis GW04009A NA NA spleen neg v1658 Steno bredanensis GW04009A NA NA lung neg v1661 Steno bredanensis GW04009A NA NA cerebellum neg

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96Table B-1. Continued. Number Species Animal ID Location Date Sample Result v1662 Steno bredanensis GW04009A NA NA cerebrum neg v1694 Tursiops truncatus GA2004035 stranded GA 11/2004 right lung neg v1695 Tursiops truncatus GA2004035 stranded GA 11/2004 left lung neg v1698 Tursiops truncatus GA2004035 stranded GA 11/2004 cerebrum neg v1699 Tursiops truncatus GA2004035 stranded GA 11/2004 cerebellum neg

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97 LIST OF REFERENCES Baker, J.R. 1992. Skin disease in wild cet aceans from British waters. Aquat. Mam. 18.1:27-32. Banyard, A.C. 2003. The roles of the leader a nd trailer regions of rinderpest virus in determining pathogenicity: PhD thesis, University of Reading. Pirbright Institute for Animal Health, UK. Baron, M.D., and T. Barrett. 1995. Sequencing and analysis of the nucleocapsid (N) and polymerase (L) genes and the terminal extrag enic domains of the vaccine strain of rinderpest virus. J. Gen. Virol. 76:593-602. Barr, B., J.L. Dunn, M.D. Daniel, and A. Ba nford. 1989. Herpes-like viral dermatitis in a beluga whale ( Delphinapterus leucas ). J. Wildl. Dis. 25:608-611. Barrett, T., J. Crowther, A.D.M.E. Osterhau s, S.M. Subbarao, J. Groen, I. Haas, I.V. Mamaev, A.M. Titenko, I.K.G. Visser, and C.J. Bostock. 1992. Molecular and serological studies on the recent seal virus epizootics in Europe and Siberia. Sci. Total Environ. 115:117-132. Barrett, T., I.K.G. Visser, L. Mamaev, L. Goatley, M.-F. van Bressem, and A.D.M.E. Osterhaus. 1993. Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus. Virology 193:1010-1012. Bengtson, J.L., P. Boveng, U. Franzn, P. Have, M.P. Heide-Jrgensen, and T.J. Hrknen. 1991. Antibodies to canine distempe r virus in Antarctic seals. Mar. Mam. Sci. 7:85-87. Blanchard, T.W., N.T. Santia go, T.P. Lipscomb, R.L. Garber, W.E. McFee, and S. Knowles. 2001. Two novel alphaherpesviruses associated with fatal disseminated infections in Atlantic bottlenose dolphins. J. Wildl. Dis. 37:297-305. Blixenkrone-Mller, M., G. Bolt, E. Gottschalck, and M. Kenter. 1994. Comparative analysis of the gene encoding the nucl eocapsid protein of dolphin morbillivirus reveals its distant evolutionary relati onship to measles virus and ruminant morbilliviruses. J. Gen. Virol. 75: 2829-2834. Bolt, G., M. Blixenkrone-Mller, E. Gottsch alck, R.G.A. Wishaupt M.J. Welsh, J.A.P. Earle, and B.K. Rima. 1994. Nucleotide and deduced amino acid sequences of the matrix (M) and fusion (F) protein genes of cetacean morbilliviruses isolated from a porpoise and a dolphin. Virus Res. 34:291-304.

PAGE 110

98 Buchanan, J.S., and R.H. Richards. 1982. Herpes-type virus diseases of marine organisms. Proc. R. Soc. Edin. 81B:151-168. Carter, S.D., D.E. Hughes, V.J. Taylor, and S.C. Bell. 1992. Immune responses in common and grey seals during the seal ep izootic. Sci. Total. Environ. 115:83-91. Curry, S.S., D.R. Brown, J.M. Gaskin, E.R. Jacobson, L.M. Ehrhart, S. Blahak, L.H. Herbst, and P.A. Klein. 2000. Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater. J. Wildl. Dis. 36:792-797. Davison, A.J. 1992. Channel catfish virus: A ne w type of herpesvirus. Virology 186:9-14. Davison, A.J. 1998. The genome of salmonid herpesvirus 1. J. Virol. 72:1974-1982. Diallo, A., T. Barrett, M. Barbron, G. Me yer, and P.C. Lefvre. 1994. Cloning of the nucleocapsid protein gene of peste-des-pe tits-ruminants virus: relation to other morbilliviruses. J. Gen. Virol. 75:233-237. Domingo, M., J. Visa, M. Pumarola, A.J. Marc o, I. Ferrer, R. Rabanal, and S. Kennedy. 1992. Pathological and immunocytoc hemical studies of morbillivirus infection in striped dolphins ( Stenella coeruleoalba ). Vet. Pathol. 29: 1-10. Duignan, P.J. 1999. Morbillivirus infections of marine mammals. In : Fowler, M.E., and R.E. Miller (Eds.) Zoo and Wild Animal Medicine: Current Therapy 4 W.B. Saunders, Philadelphia. Pp. 497-501. Duignan, P.J., C. House, J.R. Geraci, G. Ba rly, H.G. Copland, M.T. Walsh, G.D. Bossart, C. Cray, S. Sadove, D.J. St. Aubin, and M. Moore. 1995a. Morbillivirus infection in two species of pilot whales ( Globicephala sp.) from the western Atlantic. Mar. Mam. Sci. 11:150-162. Duignan, P.J., C. House, M.T. Walsh, T. Campbell, G.D. Bossart, N. Duffy, P.J. Fernandes, B.K. Rima, S. Wright, and J.R. Geraci. 1995b. Morbillivirus infection in manatees. Mar. Mam. Sci. 11:441-451. Duignan, P.J., J.T. Saliki, D.J. St. Aubin, J. A. House, and J.R. Geraci. 1994. Neutralizing antibodies to phocine distemper in Atlantic walruses ( Odobenus rosmarus rosmarus ) from Arctic Canada. J. Wildl. Dis. 30:90-94. Ehlers, B., K. Borchers, C. Grund, K. Frlich, H. Ludwig, and H.-J. Buhk. 1999. Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers. Virus Genes 18:211-220. Fijan, N.N., T.L. Wellborn, Jr., and J.P. Naft el. 1970. An acute viral disease of channel catfish. U.S. Dept. of Interior Bur. S port Fish and Wildlife. Tech. Paper 43:11.

PAGE 111

99 Follmann, E.H., G.W. Garner, J.F. Evermann, and A.J. McKiernan. 1996. Serological evidence of morbillivirus in fection in polar bears ( Ursus maritimus ) from Alaska and Russia. Vet. Rec. 138:615-618. Frey, H.R., B. Liess, L. Haas, H. Lehman, and H.J. Marschall. 1989. Herpesvirus in harbor seals ( Phoca vitulina ): isolation, partia l characterization a nd distribution. J. Vet. Med. B 36:699-708. Fujii, Y., T. Sakaguchi, K. Kiyotani, C. Hu ang, N. Fukuhara, Y. Egi, and T. Yoshida. 2002. Involvement of the leader sequence in sendai virus path ogenesis revealed by recovery of a pathogenic field isolat e from cDNA. J. Virol. 76:8540-8547. Geraci, J.R., J. Harwood, and V.J. Lounsbur y. 1999. Marine mammal die-offs: causes, investigations, and issues. In : Twiss Jr., J.R., and R.R. Reeves (Eds.). Conservation and Management of Marine Mammals Smithsonian Institute Press. Pp. 367-395. Goldstein, T., F.M.D. Gulland, B.M. Aldridge, J.T. Harvey, T. Rowles, D.M. Lambourn, S.J. Jeffries, L. Measures, P.K. Yochem, B. S. Stewart, R.J. Small, D.P. King, J.L. Scott, and J.A.K. Mazet. 2003a. Antibodies to phocine herpesvirus-1 are common in North American harbor seals ( Phoca vitulina ). J. Wildl. Dis. 39:487-494. Goldstein, T., F.M.D. Gulland, R.C. Braun, G. A. Antonelis, L.M. Dalton, T. Rowles, J.A.K. Mazet, B.M. Aldridge, and J.L. Sc ott. 2003b. Molecular indentification of a novel gammaherpesvirus in Hawaiian monk seals ( Monachus schauinslandii ). Proceedings of the International Associ ation for Aquatic Animal Medicine. Pp. 130-132. Goldstein, T., J.A.K. Mazet, F.M.D. Gulland, T. Rowles, J.T. Harvey, S.G. Allen, D.P. King, B.M. Aldridge, and J.L. Scott. 2004. The transmission of phocine herpesvirus-1 in rehabilitating and free-ranging Pacifi c harbor seals ( Phoca vitulina ) in California. Vet. Microbiol. 103:131-141. Greenblatt, R.J., S.L. Quackenbush, R.N. Ca sey, J. Rovnak, G.H. Balazs, T.M. Work, J.W. Casey, and C.A. Su tton. 2005. Genomic variation of the fibropapillomaassociated marine turtle herpesvirus acr oss seven geographic areas and three host species. J. Virol. 79:1125-1132. Greenblatt, R.J., T.M. Work, G.H. Balzs, C.A. Sutton, R.N. Casey, and J.W. Casey. 2004. The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesviru s found latently infecting skin tumors on Hawaiian green turtles ( Chelonia mydas ). Virology 321:101-110 Greenwood, A.G., R.J. Harrison, and H.W. Whitting. 1974. Functional and pathological aspects of the skin of marine mammals. In : Harrison, R.J. (Ed.) Functional Anatomy of Marine Mammals Academic Press, London. Pp. 73-111.

PAGE 112

100 Griffin, D.E. 2001. Measles virus. In : Knipe, D.M. and P.M. Howley (eds.). Fields Virology, vol. 1 Lippincott, Williams and Wilkins publishers, Philadelphia, Pennsylvania. Pp. 1401-1441. Grillo, V., E.C.M. Parsons, and J.H. Shrimp ton. 2001. A review of sewage pollution and cetaceans: a Scottish perspective. Proceedings from the 53rd Meeting of the International Whaling Commission. Harder, T.C., M. Harder, H. Vos, K. Kul onen, S. Kennedy-Stoskopf, B. Liess, M.J.G. Appel, and A.D.M.E. Osterhaus. 1996. Ch aracterization of p hocid herpesvirus-1 and -2 as putative alphaand gammah erpesviruses of North American and European pinnipeds. J. Gen. Virol. 77:27-35. Harder, T.C., T. Williams, W. Leibold, and B. Liess. 1992. Investigations on course and outcome of phocine distemper viru s infection in harbour seals ( Phoca vitulina ) exposed to polychlorinated biphenyls. J. Vet. Med. 39:19-31. Harding, K.C., T. Hrknen, and H. Caswe ll. 2002. The 2002 European seal plague: epidemiology and population conseq uences. Ecol. Letters 5:727-732. Harvell, C.D., K. Kim, J.M. Burkholder, R.R. Colwell, P.R. Epstein, D.J. Grimes, E.E. Hofmann, E.K. Lipp, A.D.M.E. Osterhaus, R.M. Overstreet, J. W. Porter, G.W. Smith, and G.R. Vasta. 1999. Emerging marine diseasesclimate links and anthropogenic factors. Science 285:1505-1510. Harzen, S. and B.J. Brunnick. 1997. Skin disorders in bottlenose dolphins ( Tursiops truncatus ) resident in the Sado estuary, Portugal. Aquat. Mam. 23:59-68. Hedrick, R.P., O. Gilad, S. Yun, J.V. Spange nberg, G.D. Marty, R.W. Nordhausen, M.J. Kebus, H. Bercovier, and A. Elder. 2000. A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp. J. Aquat. Anim. Health 12:44-57. Herbst, L., A. Ene, M. Su, R. Desalle, and J. Lenz. 2004. Tumor outbreaks in marine turtles are not due to recent herpes virus mutations. Curr. Biol. 14:R697-R699. Herbst, L.H., E.R. Jacobson, R. Moretti, T. Brown, J.P. Sandberg, and P.A. Klein. 1995. Experimental transmission of green turtle fibropapillomatosis using cell-free tumor extracts. Dis. Aquat. Org. 22:1-12. Herbst, L.H., and P.A. Klein. 1995. Green tu rtle fibropapillomatosis: challenges to assessing the role of environmental cof actors. Environ. Health Perspect. 103:Suppl 4. Hughes, D.E., S.D. Carter, I. Robinson, D.D. Clarke, and C.J. Clarke. 1992. Anti-canine distemper virus antibodies in common a nd grey seals. Vet. Rec. 130:449-450.

PAGE 113

101 Jacobson, E.R., J.M. Gaskin, M. Roelke, E.C. Greiner, and J. A llen. 1986. Conjunctivitis, tracheitis, and pneumonia associated with he rpesvirus infection in green sea turtles. J.A.V.M.A. 189:1020-1023. Jacobson, E.R., J.L. Mansell, J.P. Sundberg, L. Hajjar, M.E. Reichmann, L.M. Ehrhart, M. Walsh, and F. Murru. 1989. Cutaneous fibropapillomas of green turtles ( Chelonia mydas ). J. Comp. Pathol. 101:39-52. Jensen, T., M. van de Bildt, H.H. Dietz, T. H. Andersen, A.S. Hammer, T. Kuiken, and A. Osterhaus. 2002. Another phocine distem per outbreak in Europe. Science 297:209. Kennedy, S. 1998. Morbillivirus infections in aquatic mammals. J. Comp. Pathol. 119:201-225. Kennedy, S., T. Kuiken, P.D. Jepson, R. Deaville M. Forsyth, T. Barrett, M.W.G. van de Bildt, A.D.M.E. Osterhaus, T. Eybat ov, C. Duck, A. Kydrymanov, I. Mitrofanov, and S. Wilson. 2000. Mass die-off of Caspian seals caused by canine distemper virus. Emerg. Infect. Dis. 6:637-639. Kennedy, S., I.J. Lindstedt, M.M. McAliske y, S.A. McConnell, and S.J. McCullough. 1992. Herpesviral encephalitis in a harbor porpoise ( Phocoena phocoena ). J. Zoo. Wildl. Med. 23:374-379. Kennedy, S., J.A. Smyth, P.F. Cush, S.J. McCullough, G.M. Allan, and S. McQuaid. 1988. Viral distemper now found in porpoises. Nature 336:21. Kennedy-Stoskopf, S. 2001. Viral diseases. In : Dierauf, L.A., and F.M.D. Gulland (Eds.). CRC Handbook of Marine Mammal Medicine CRC Press, Boca Raton, Florida. Pp. 285-307. Kennedy-Stoskopf, S., M.K. Stoskopf, M. A. Eckhaus, and J.D. Strandberg. 1986. Isolation of a retrovirus a nd a herpesvirus from a captiv e California sea lion. J. Wildl. Dis. 22:156-164. Kimura, T., M. Yoshimizu, M. Tanaka, and H. Sannohe. 1981. Studies on a new virus (OMV) Oncorhynchus masou I. Characteristics and pa thogenicity. Fish Pathol. 15:143-147. King, D.P., M.C. Hure, T. Goldstein, B.M. Aldridge, F.M.D. Gulland, J.T. Saliki, E.L. Buckles, L.J. Lowenstine, and J.L. Stott. 2002. Otarine herpesvirus-1: a novel gammaherpesvirus associated with urogeni tal carcinoma in California sea lions ( Zalophus californianus ). Vet. Microbiol. 86:131-137. King, D.P., R. Parselles, F.M.D. Gulland, J.-M Lapointe, L.J. Lowenstine, D.A. Ferrick, and J.L. Stott. 1998. Antigenic and nucleo tide characterization of a herpesvirus isolated from Pacific harbor seals ( Phoca virulina richardsii ). Arch. Virol. 143:2021-2027.

PAGE 114

102 King, J.E. 1983. Seals of the World. Cornell University Press, New York. Lackovich, J.K., D.R. Brown, B.L. Homer, R.L. Garber, D.R. Mader, R.H. Moretti, A.D. Patterson, L.H. Herbst, J. Oros, E.R. Jacobson, S.S. Curry, and P.A. Klein. 1999. Association of herpesvirus with fibr opapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida. Dis. Aquat. Org. 37:8997. Landsberg, J.H., G.H. Balazs, K.A. Steidi nger, D.G. Baden, T.M. Work, and D.J. Russell. 1999. The potential role of natura l tumor promoters in marine turtle fibropapillomatosis. J. Aquat. Anim. Health 11:199-210. Lebich, M., T.C. Harder, H.-R. Frey, I.K.G. Visser, A.D.M.E. Osterhaus, and B. Liess. 1994. Comparative immunological characteriza tion of type-specific and conserved B-cell epitopes of pinniped, felid and canid herpesviruses. Arch. Virol. 136:335347. Lipscomb, T.P., P.L. Habecker, D.M. Damback, and R. Schoelkopf. 1996a. Genital herpes infection in a male harbor por poise. Proceedings of the International Association for Aquatic An imal Medicine. P. 17. Lipscomb, T.P., S. Kennedy, D. Moffett, A. Krafft, B.A. Klaunberg, J.H. Lichy, G.T. Regan, G.A.J. Worthy, and J.K. Taubenberg er. 1996b. Morbilliviral epizootic in Atlantic bottlenose dolphins ( Tursiops truncatus ) from the Gulf of Mexico. J. Vet. Diag. Invest. 8:283-290. Lipscomb, T.P., F.Y. Schulman, D. Moffett, and S. Kennedy. 1994. Morbilliviral disease in Atlantic bottle-nosed dolphins ( Tursiops truncatus ) from the 1987-1988 epizootic. J. Wildl. Dis. 30:567-571. Lipscomb, T.P., D.P. Scott, R.L. Garber, A.E. Krafft, M.M. Tsai, J.H. Lichy, J.K. Taubenberger, F.Y. Schulman, and F. M.D. Gulland. 2000. Common metastatic carcinoma of California sea lions ( Zalophus californianus ): evidence of genital origin and association with novel gamma herpesvirus. Vet. Pathol. 37:609-617. Lu, Y., Y. Wang, G. Yu, A.A. Aguirre, G.H. Balazs, V.R. Nerurkar, and R. Yanagihara. 2000. Detection of herpesviral sequences in tissues of green turtles with fibropapilloma by polymerase chain r eaction. Arch. Virol. 145:1885-1893. Lu, Y. and Q. Yu. 2000. Detection of green turtle herpesviral se quence in saddleback wrasse Thalassoma duperrey : A possible mode of transmission of green turtle fibropapilloma. J. Aquat. Anim. Health 12:58-63. Mahy, B., T. Barrett, S. Evans, E.C. Ande rson, and C.J. Bostock. 1988. Characterization of seal morbillivirus. Nature. 336:115. Martin, A.R. 1990. The Encyclopedia of Whales and Dolphins Salamander Books Ltd., London.

PAGE 115

103 Martineau, D., A. Lagac, P. Bland, R. Higgins, D. Armstrong, and L.R. Shugart. 1988. Pathology of stranded beluga whales ( Delphinapterus leucas ) from the St. Lawrence Estuary, Qubec, Cana da. J. Comp. Path. 98:287-311. McGeoch, D.J., S. Cook, A. Dolan, F.E. Jamieson, and E.A.R. Telford. 1995. Molecular phylogeny and evolutionary timescale for th e family of mammalian herpesviruses. J. Mol. Biol. 247:443-458. Mikaelian, I., M.-P. Tremblay, C. Montpetit, S.V. Tessaro, H.J. Cho, C. House, L. Measures, and D. Martineau. 1999. Seroprevalen ce of selected viral infections in a population of beluga whales ( Delphinapterus leucas ) in Canada. Vet. Rec. 144:5051. Murphy, F.A., E.P.J. Gibbs, M.C. Horzinec, and M.J. Studdert. 1999. Chapter 18: Herpesviridae, In : Veterinary Virology, 3rd ed ., Academic Press, New York. Pp. 301-326. O’Hara, T.M., and T.J. O’Shea. 2001. Toxicology. In : Dierauf, L.A., and F.M.D. Gulland (Eds.). CRC Handbook of Marine Mammal Medicine CRC Press, Boca Raton, Florida. Pp. 471-520. Osterhaus, A.D.M.E., J. Groen, H. Niesters, M. van de Bildt, B. Martina, L. Vedder, J. Vos, H. van Egmond, B.A. Sidi, and M. E.O. Barham. 1997. Morbillivirus in monk seal mass mortality. Nature 388:838-839. Osterhaus, A.D.M.E., J. Groen, F.G.C.M. UytdeHaag, I.K.G. Visser, M.W.G. van de Bildt, A. Bergman, and B. Klingeborn. 1989. Distemper virus in Baikal seals. Nature 388:209-210. Osterhaus, A.D.M.E., H. Yang, H.E.M. Spijk ers, J. Groen, J.S. Teppema, and G. van Steenis. 1985. The isolation and partial ch aracterization of a highly pathogenic herpesvirus from the harbor seal ( Phoca vitulina ). Arch. Virol. 86:239-251. Quackenbush, S.L., R.N. Casey, R.J. Murcek, T.A. Paul, T.M. Work, C.J. Limpus, A. Chaves, L. duToit, J.V. Perez, A.A. Aguirre, T.R. Spraker, J.A. Horrocks, L.A. Vermeer, G.H. Balazs, and J.W. Casey. 2001. Quantitative analysis of herpesvirus sequences from normal tissue and fibropapillo mas of marine turtles with real-time PCR. Virology 287:105-111. Quackenbush, S.L., T.M. Work, G.H. Balazs, R.N. Casey, J. Rovnak, A. Chaves, L. duToit, J.D. Baines, C.R. Parrish, P.R. Bowser, and J.W. Casey. 1998. Three closely related herpesviruses are associat ed with fibropapillomatosis in marine turtles. Virology 246:392-399. Rebell, H., A. Rywlin, and H. Haines. 1975. Herp esvirus-type agent associated with skin lesions of green sea turtles in aqua culture. Am. J. Vet. Res. 36:1221-1224.

PAGE 116

104 Reynolds III, J.E., R.S. Wells, and S.D. Elde. 2000. The Bottlenose Dolphin: biology and conservation University Press of Florida, Gainesville, Florida. P. 123. Rima, B.K., R.G.A. Wishaupt, M.J. Welsh, and J.A.P. Earle. 1995. The evolution of morbilliviruses: a comparison of nucleocap sid gene sequences including a porpoise morbillivirus. Vet. Microbiol. 44:127-134. Roizmann, B., M.B. Kovler, R.C. Desrosiers B. Fleckenstein, C. Lopez, A.C. Minson, and M.J. Studdert. 1992. The family Herpesviridae : an update. Arch. Virol. 123:426-449. Roizmann, B., and P.E. Pellet. 2001. The fa mily herpesviridae: A brief introduction. In : Knipe, D.M. and P.M. Howley (Eds.). Fields Virology, vol. 2 Lippincott, Williams and Wilkins publishers, Philadel phia, Pennsylvania. Pp. 2381-2397. Taubenberger, J.K., M.M. Tsai, T.J. Atkin, T.G. Fanning, A.E. Krafft, R.B. Moeller, S.E. Kodsi, M.G. Mense, and T.P. Lipscom b. 2000. Molecular genetic evidence of a novel morbillivirus in a long-finned pilot whale ( Globicephala melas ). Emerg. Infect. Dis. 6:42-45. Trillmich, F., K.A. Ono, D.P. Costa, R.L. De Long, S.D. Feldkamp, J.M. Francis, R.L. Gentry, C.B. Heath, B.J. LeBoeuf, P. Ma jluf, and A.E. York. 1991. The effects of El Nio on pinniped populations in the eastern pacific. In : Trillman, F. and K.A. Ono (Eds.). Pinnipeds and El Nio: respons es to environmental stress SpringerVerlag, Berlin, Germany. Pp. 247-270. Van Bressem, M.F., J. De Meurichy, G. Chappuis, D. Spehner, M.P. Kieny, and P.P. Pastoret. 1991. Attemp to vaccinate orally harbour seals against phocid distemper. Vet. Rec. 129:362. Van Bressem, M.-F., K. Van Waerebeek, A. Garcia-Godos, D. Dekegel, and P.P. Pastoret. 1994. Herpes-like virus in dusky dolphins, Lagenorhynchus obscurus from coastal Peru. Mar. Mam. Sci. 10:354-359. Van Bressem, M.-F., K. Van Waerebeek, and J.A. Raga. 1999. A review of virus infections of cetaceans and the potential impact of morbilliviruses, poxviruses and papillomaviruses on host population dyna mics. Dis. Aquat. Org. 38:53-65. Van de Bildt, M.W.G., T. Kuiken, a nd A.D.M.E. Osterhaus. 2004. Cetacean morbilliviruses are phyloge netically divergent. Arc h. Virol. Published online November 18, 2004. Van de Bildt, M.W.G., E.J. Vedder, B.E.E. Martina, B.A. Sidi, A.B. Jiddou, M.E.O. Barham, E. Androukaki, A. Komnenou, H.G.M. Niesters, and A.D.M.E. Osterhaus. 1999. Morbilliviruses in Mediterranean monk seals. Vet. Microbiol. 69:19-21.

PAGE 117

105 VanDevanter, D.R., P. Warrener, L. Bennett, E.R. Schultz, S. Coulter, R.L. Garber, and T.M. Rose. 1996. Detection and analysis of diverse herpesviral species by consensus primer PCR. J. Clin. Microbiol. 34:1666-1671. Visser, I.K.G., M.-F. Van Bressem, R.L. de Swart, M.W.G. van de Bildt, H.W. Vos, R.W.J. van der Heijden, J.T. Saliki, C. r vell, P. Kitching, T. Kuiken, T. Barrett, and A.D.M.E. Osterhaus. 1993. Characteriza tion of morbilliviruses isolated from dolphins and porpoises in Eur ope. J. Gen. Virol. 74:631-641. Visser, I.K.G., M.W.G. van de Bildt, H.N. Brugge, P.J.H. Reijnders, E.J. Vedder, J. Kuiper, P. de Vries, J. Groen, H.C. Walvoort, F.G.C.M. UytdeHaag, and A.D.M.E. Osterhaus. 1989. Vaccination of harbour seals ( Phoca vitulina ) against phocid distemper with two different inactivated canine distemper virus (CDV) vaccines. Vaccine 7:521-526. Wilson, B., H. Arnold, G. Bearzi, C.M. Fortuna R. Gaspar, S. Ingram, C. Liret, S. Pribani A.J. Read, V. Ridoux, K. Schneider K.W. Urian, R.S. Wells, C. Wood, P.M. Thompson, and P.S. Hammond. 1999. Epidermal diseases in bottlenose dolphins: impacts of natural and anthr opogenic factors. Proc. R. Soc. Lond. 226:1077-1083. Wolf, K., and R.W. Darlingt on. 1971. Channel catfish virus: A new herpesvirus of ictalurid fish. J. Virol. 8:525-533. Wolf, K., R.W. Darlington, W. G. Taylor, M.C. Quimby, and T. Nagabayashi. 1978. Herpesvirus salmonis : characterization of a new pa thogen of rainbow trout. J. Virol. 27:659-666. Yu, Q., N. Hu, Y. Lu, V.R. Nerurkar, a nd R. Yanagihara. 2001. Rapid acquisition of entire DNA polymerase gene of a nove l herpesvirus from green turtle fibropapilloma by a genomic walking t echnique. J. Virol. Meth. 91:183-195. Yu, Q., Y. Lu, V.R. Nerurkar, and R. Yana gihara. 2000. Amplificati on and analysis of DNA flanking known sequences of a novel he rpesvirus from green turtles with fibropapilloma. Arch. Virol. 145:2669-2676.

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106 BIOGRAPHICAL SKETCH Kara Ann Smolarek Benson was born on April 3, 1980, in Buffalo, New York, as the first of two daughters to Dave and Judy Sm olarek. After high school Kara retired as a snow bunny and relocated to Melbourne, Florida, in search of sun and surf. There she attended the Florida Instit ute of Technology. She gradua ted with honors in 2002 with a Bachelor of Science degree in marine biol ogy and an awesome tan. She then pursued higher education as a Gator at the Univers ity of Florida, where she became a dolphin herpes aficionado. Her graduate studies t ook her to England for two months where she researched porpoise morbillivirus and learned the excitement of Eur opean football. Upon graduation from UF she will be moving to Oa hu, Hawaii, to be with her husband, Brock, and pursue a career in marine mammal health and conservation.


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MOLECULAR IDENTIFICATION AND GENETIC CHARACTERIZATION OF
CETACEAN HERPESVIRUSES AND PORPOISE MORBILLIVIRUS

















By

KARA ANN SMOLAREK BENSON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Kara Ann Smolarek Benson

































I dedicate this to my best friend and husband, Brock, who has always believed in me.















ACKNOWLEDGMENTS

First and foremost I thank my mentor, Dr. Carlos Romero, who once told me that

love is fleeting but herpes is forever. He welcomed me into his lab with very little

experience and I have learned so much from him over the past few years. Without his

excellent guidance, this project would not have been possible.

I thank my parents, Dave and Judy Smolarek, for their continual love and support.

They taught me the importance of hard work and a great education, and always believed

that I would be successful in life.

I would like to thank Dr. Tom Barrett for the wonderful opportunity to study

porpoise morbillivirus in his laboratory at the Institute for Animal Health in England, and

Dr. Romero for making the trip possible. I especially thank Dr. Ashley Banyard for

helping me accomplish all the objectives of the project, and all the wonderful people at

the IAH for making a Yankee feel right at home in the UK.

I thank Alexa Bracht and Rebecca Woodruff who have been with me in Dr.

Romero's lab since the beginning. Their continuous friendship and encouragement have

kept me sane even in the most hectic of times. I thank the newest members of the lab,

Shasta McClennahan and Rebecca Grant, for their input and support over the past few

months. I also thank Linda Thomas and Mia Shandell for all their help.

I would like to thank my committee members, Dr. David Bloom, Dr. Donald

Forrester and Dr. Charles Manire, for their guidance and expertise.









This project would not have been possible without the assistance of the many

people and organizations that sent samples, especially Dr. Ruth Ewing, Dr. Charles

Manire, Dr. Jeremiah Saliki and Dr. Forrest Townsend. Big thanks go to all of them!

They keep this laboratory going.

I would like to thank the computer guys at CSM for solving all my computer

mishaps, and although they could not retrieve any of my lost thesis information when my

computer crashed, I know they gave it their all.

Last but not least, I thank my sister Ali, my Floridian significant other Maura, and

my husband Brock for always believing in me, even when I did not believe in myself.

Finally, this work was supported by a grant from Harbor Branch Oceanographic

Institution and Florida Fish and Wildlife Commission through the Marine Animal Health

Program of the College of Veterinary Medicine at the University of Florida.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................................... ........ .. .............. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABSTRACT ........ .............. ............. ...... ...................... xi

CHAPTER

1 CETACEAN HERPESVIRU SES ........................................ .......................... 1

In tro d u ctio n .................................................................................................... ..... .
M materials an d M eth o d s ........................................................................ ................. 12
Sample Acquisition and DNA Extraction ................................. ............... 12
Nested PCR Targeting the DNA Polymerase Gene ........................................13
Cloning, Sequencing and Sequence Analysis ............................................. 15
PCR Targeting the DNA Terminase Gene ............. ...............................16
Results ............. ............................................. ............ 17
Nested PCR Targeting the DNA Polymerase Gene ........................................17
PCR Targeting the DNA Terminase Gene ................... ......................... 38
D discussion ............... ..... .. .......... ......... .. ............ ............ 40

2 PORPOISE MORBILLIVIRUS ................................. ........................... 50

In tro d u ctio n ...................................... ................................................ 5 0
M materials and M methods ....................................................................... ..................56
A analysis of PM V ..................................................... ... .. ............ 56
Reverse transcription................ ....... ........ ............... 56
RACE and amplification of the terminal extragenic domains ...................57
Amplification of the complete PMV N gene ..............................................58
Amplification of the C-terminus of the PMV N gene..............................60
Detection of N protein expression by Western blot..............................60
Analysis of Stranded Cetacean Tissues for Morbillivirus................................61
Sample acquisition, RNA extraction and reverse transcription ...................61
PCR targeting the P gene ........................................ ......... ............... 61
PCR targeting the -actin gene.................................... ...... ............... 62
R e su lts............................................................................................................. 6 3









Analysis of PM V .................... ........... ...... ............ ......63
RACE and amplification of the terminal extragenic domains .................63
Amplification of the complete PMV N gene ................... ...... ............65
Detection of protein expression by W western blot................................ ...74
Analysis of Stranded Cetacean Tissues for Morbillivirus...............................74
D discussion ..................................... .................. ............... ........... 74

APPENDIX

A MARINE MAMMAL SAMPLES................................. ...................84

B CETACEAN TISSUE SAM PLES ........................................ ........................ 93

LIST OF REFEREN CES ............................................................................. 97

BIOGRAPHICAL SKETCH ............................................................. ............... 106
















LIST OF TABLES


Table p

1-1. Percent nucleotide identities of DNA polymerase gene fragments among
members of the family Herpesviridae. ......................................... ...............31

1-2. Percent amino acid identities of DNA polymerase gene fragments among
members of the family Herpesviridae. ......................................... ...............32

1-3. Percent amino acid similarities of DNA polymerase gene fragments among
members of the family Herpesviridae. ......................................... ...............33

2-1. Percent nucleotide identities of morbillivirus N genes. ................. .................67

2-2. Percent amino acid identities of morbillivirus N genes. .........................................67

2-3. Percent amino acid similarities of morbillivirus N genes. ............. ... ................67

A-1. List of all samples tested for herpesvirus using the nested PCR approach .............85

B-1. List of all samples tested for morbillivirus nucleic acid using PCR targeting the
phosphoprotein gene. ....................................... .......................... 94
















LIST OF FIGURES


Figure pge

1-1. Schematic of the location of the primers used in the nested PCR targeting the
herpesvirus DNA polym erase gene ....................................................................... 14

1-2. Gel electrophoresis of herpesvirus nested PCR products.................. ............. 17

1-3. Wart-like lesion on the genital slit of a female dwarf sperm whale.........................18

1-4. Genital lesions of two captive Atlantic bottlenose dolphins.............. .....................19

1-5. Blainville's beaked w hale penile lesion. ........................................ .....................20

1-6. Atlantic bottlenose dolphin dermatitis lesions. .............................. ......... ...... .21

1-7. Epithelial cells from a skin lesion of a bottlenose dolphin. ....................................22

1-8. Atlantic bottlenose dolphin with lesions on the tongue and the penis....................23

1-9. Gel electrophoresis of large DNA polymerase fragments from cetacean
gam m aherpesviruses ...................... ................ ................. .... ....... 25

1-10. Multiple alignments of the nucleotide sequences of the DNA polymerase gene
fragments of cetacean herpesviruses.. ........................................... ............... 27

1-11. Multiple alignments of the deduced amino acid sequences of the DNA
polymerase gene fragments ofcetacean herpesviruses .........................................30

1-12. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the
herpesvirus DNA polym erase gene ....................................................................... 34

1-13. Neighbor-Joining phylogenetic trees of the nucleotide sequences of the DNA
polymerase gene of cetacean herpesviruses. ................................. .................36

1-14. Neighbor-Joining phylogenetic trees of the deduced amino acid sequences of the
DNA polymerase gene of cetacean herpesviruses. .............................................37

1-15. Comparison of the nucleotide sequences of the DNA terminase gene fragment
of cetacean alphaherpesviruses. ........................................ ......................... 39









1-16. Comparison of the deduced amino acid sequences of the DNA terminase gene
fragm ent of cetacean alphaherpesviruses ........................................ ....................39

2-1. Schematic of the primers used to sequence the full nucleocapsid gene of
P M V ............................................................................... 59

2-2. Gel electrophoresis of tailed and untailed PCR products.................. ...............64

2-3. Nucleotide sequence comparison of the genomic promoter region of
morbilliviruses............................................. ......... 65

2-4. Nucleotide sequence comparison of the antigenomic promoter region of
morbilliviruses............................................. ......... 65

2-5. Gel electrophoresis of the overlapping PCR products used to obtain the full N
gene sequence of PM V ............................................................................. ....... 66

2-6. Neighbor-Joining phylogenetic trees of the nucleotide sequences of the complete
N gene of m orbilliviruses ............................ ......... ..................... ...............68

2-7. Neighbor-Joining phylogenetic trees of the deduced amino acid sequences from
the complete N gene of morbilliviruses. ............................................................. 69

2-8. Nucleotide comparison and consensus sequence for the full N gene of three
morbilliviruses than infect marine mammals. ................... .............. .......... 70

2-9. Amino acid comparison and consensus sequence of the full nucleocapsid gene
of m orbilliviruses. .......................................................................................... .......72

2-10. Expression of the N gene hypervariable C-terminus of DMV as detected by
W western blot. .........................................................................74















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MOLECULAR IDENTIFICATION AND GENETIC CHARACTERIZATION OF
CETACEAN HERPESVIRUSES AND PORPOISE MORBILLIVIRUS


By

Kara Ann Smolarek Benson

August 2005

Chair: Carlos H. Romero
Major Department: Veterinary Medicine

Extracted DNA from tissues and lesions of captive and stranded cetaceans was

analyzed for the presence of herpesvirus genomes by nested and direct polymerase chain

reactions (PCR) using consensus primers. The targeted sequence corresponded to a

region of the DNA polymerase gene containing multiple conserved amino acid motifs.

Herpesvirus genomic DNA fragments were amplified by nested PCR, from nine lesions

out of 118 lesion samples from eight animals, while none of the stranded cetacean tissue

samples were positive for herpesvirus using the same technique. Sequence analysis of the

small DNA fragments indicated that alpha or gamma herpesviruses were present in the

positive cetacean lesions. Alphaherpesvirus DNA was detected in skin lesions of two

Atlantic bottlenose dolphins (Tursiops truncatus), while gammaherpesvirus DNA was

amplified from genital lesions from three Atlantic bottlenose dolphins, one dwarf sperm

whale (Kogia sima) and one Blainville's beaked whale (Mesoplodon densirostris), as

well as one oral lesion from an Atlantic bottlenose dolphin. Longer amplicons were









obtained using direct PCR from the six genital gammaherpesviruses, but could not be

amplified from lesions containing alphaherpesviruses. Upon sequencing and phylogenetic

analysis of cetacean herpesviruses DNA polymerase gene fragments, they were shown to

be most similar to Varicelloviruses and Rhadinoviruses, respectively. Furthermore, these

analyses showed that the cetacean herpesviruses were genetically distinct from known

herpesviruses of fish, marine turtles and pinnipeds. These findings strongly indicate that

these herpesviruses are cetacean specific and most likely coevolved with their hosts.

The terminal extragenic domains and the complete nucleocapsid (N) gene of

porpoise morbillivirus (PMV) were sequenced to better understand the phylogenetic

relationship between PMV and dolphin morbillivirus (DMV). Furthermore, tissue

samples from four species of cetaceans that were involved in recent mass stranding

events in the southeast U.S. were tested by PCR for the presence of morbillivirus nucleic

acid using consensus primers that target the phosphoprotein (P) gene. High sequence

homologies of the terminal extragenic domains and the complete N gene of PMV and

DMV were suggestive of a common cetacean morbillivirus, with two strains: PMV and

DMV. Phylogenetic analyses confirmed that PMV and DMV were closely related and

that the cetacean morbilliviruses represent an independent morbillivirus lineage, which

likely evolved with their hosts. In agreement with previous studies, our results have

indicated that the cetacean morbilliviruses are more closely related to rinderpest virus,

peste-des-petits ruminant virus and measles virus than they are to canine distemper virus

and phocine distemper virus of carnivores. None of the tissue samples from recently

stranded cetaceans in waters of the US Southeast coasts were positive for morbillivirus

by PCR.














CHAPTER 1
CETACEAN HERPESVIRUSES

Introduction

Herpesvimses are enveloped viruses that vary in size from 120 to 200 nm in

diameter. They have a unique tegument which surrounds an icosahedral nucleocapsid

that is approximately 100 nm in diameter. The genome is composed of a single, linear,

double stranded DNA molecule ranging in size from 125 to 235 Kbp (Kilo base pairs).

They encode between 70 (the smallest genome) and 200 (the largest genome) genes

(Roizmann and Pellet, 2001). Herpesviruses have two categories of genes: Those needed

for viral replication (immediate early and early genes) and structural proteins (late genes).

Some also produce a heterologous set of optional genes. Replication of new viral DNA

and virus assembly occur in the nucleus. Herpesviruses obtain their envelope by budding

through the inner layer of the nuclear envelope of the host cell and the integrity of the

envelope appears to be essential for infectivity (Roizmann and Pellet, 2001).

Landmarks of herpesvirus infection include latency and reactivation. All known

herpesviruses undergo persistent infection with periodic or continuous shedding of

infectious virus. Viral reactivation is often caused by stress, intercurrent disease, trauma,

hormonal irregularities, immunosuppression or waning immunity (Murphy et al., 1999).

Eosinophilic intranuclear inclusion bodies are characteristic of herpesvirus infection and

are the result of late degenerative changes, as well as condensation and margination of

chromatin. Some herpesvirus infections also produce syncytia resulting from the fusion

of infected cells with neighboring infected or uninfected cells. In general, herpesviruses









have a narrow host range and transmission requires close contact, usually between

mucosal surfaces (Murphy et al., 1999).

Herpesviruses are divided into three main subfamilies, Alpha-, Beta- and

Gammaherpesvirinae, which were originally based on biological characteristics, but more

recently, with gene content and sequence similarity (Roizmann and Pellet, 2001). Most

alphaherpesviruses grow rapidly, lyse infected cells and establish latent infections in

sensory ganglia of the central nervous system. Many produce localized infections of the

skin and mucosas of the respiratory or genital tract, while general infection is

characterized by necrosis of organs and tissues. The latent genome of alphaherpesviruses

is essentially silent except for the production of a latency-associated transcript (LAT).

The subfamily Betaherpesvirinae comprises the cytomegaloviruses, which may remain

latent in secretary glands, lymphoreticular tissues and kidneys. Betaherpesvirus

replication is slow and cell lysis does not occur until several days after infection.

Gammaherpesviruses are lymphotropic and become latent in lymphocytes. They often

have a narrow host range and some can be linked to oncogenic transformation of

lymphocytes. These viruses typically cause chronic infection lasting several months

before clinical recovery. Gammaherpesviruses replicate in lymphoblastoid cells, with

different viruses being specific for either B or T lymphocytes. These viruses can also

enter a lytic stage in which they can cause cell death without the production of virions.

Herpesviruses are evolutionarily old and have been found in almost every species

of bird and mammal that has been investigated, as well as reptiles, amphibians and

mollusks (Murphy et al., 1999). Molecular phylogenetic studies suggest that there are at

least three distinct lineages of herpesviruses (mammalian/avian, fish/amphibian and









invertebrate) that arose approximately 180 to 220 million years ago (McGeoch et al.,

1995), and because each virus is unique, they are believed to have coevolved with their

host species (Murphy et al., 1999). Herpesviruses infect a variety of aquatic vertebrates

including teleost (bony) fish, chelonians (turtles and tortoises), pinnipeds (seals and sea

lions) and cetaceans (dolphins, porpoises and whales) (Roizmann et al., 1992; Kennedy-

Stoskopf, 2001).

The most studied fish herpesviruses include salmonid herpesvirus-1 (SalHV-1),

salmonid herpesvirus-2 (SalHV-2), channel catfish virus (CCV) and koi herpesvirus

(KHV). Wolf et al. (1978) isolated SalHV-1 from rainbow trout (Oncorhynchua mykiss),

and the virus was shown to induce syncytia and intranuclear inclusions when inoculated

into other salmonid fish cell lines. Structurally, the genome of SalHV-1 is characteristic

of the Varicellovirus genus of the subfamily Alphaherpesvirinae. However, no genetic

relationship was found between SalHV-1 and mammalian herpesviruses leading to the

conclusion that this genome structure evolved independently in both the mammalian and

fish herpesvirus lineages (Davison, 1998). SalHV-2 was first isolated from landlocked

masu salmon (Oncorhynchus masou) in Japan (Kimura et al., 1981). SalHV-2 is most

closely related to SalHV-1, but is serologically distinct and has a wider host range. CCV

was isolated during epizootics of an acute hemorrhagic disease of high mortality in young

channel catfish (Ictaluruspunctatus) (Fijan et al., 1970) and was classified as a

herpesvirus based upon the presence of intranuclear inclusions and syncytia as well as its

virus particle morphology (Wolf and Darlington, 1971). Initially classified as an

alphaherpesvirus based on virion morphology, complete genome analysis found no

relationship with mammalian herpesviruses; thus, CCV was placed in a new subfamily,









Ictalurivirus (Davison, 1992). A herpesvirus was also isolated from adult koi (Cyprinus

carpio), a strain of common carp that suffered mass mortality during outbreaks in the

mid-Atlantic region of the U.S. and in Israel (Hedrick et al., 2000). KHV was

distinguished from other known herpesviruses of carp, namely carp pox (Cyprinid

herpesvirus), by indirect fluorescent antibody tests.

Herpesviruses infect many species of chelonians, including marine turtles, of which

all seven species are either endangered or threatened. Herpesviruses have been

associated with two diseases of mariculture-reared green turtles (Chelonia mydas) in the

Cayman Islands. Lung-eye-trachea disease (LETD) associated herpesvirus was isolated

from juveniles with conjunctivitis, tracheitis and severe pneumonia (Jacobson et al.,

1986). Gray-patch disease (GPD) was also concluded to be of herpesvirus etiology based

on virus particle morphology and the presence of intranuclear inclusion bodies (Rebell et

al., 1975). Fibropapillomatosis (FP) is a neoplastic disease that causes external

fibropapillomas of the skin, eyes, oral cavity and carapace, as well as internal fibromas of

the visceral organs. Although the disease itself is usually not fatal, the large tumors can

interfere with locomotion, vision, swallowing, and breathing, and can also affect organ

function. FP was first described over 60 years ago and has been rapidly increasing in

prevalence worldwide. Until recently, the etiology of FP was unknown, but multiple

studies have suggested that a virus is involved in the pathogenesis of this disease.

Papillomavirus was first suspected, but Southern blot hybridization of green turtle

fibropapillomas failed to detect any papillomavirus DNA (Jacobson et al., 1989).

Multiple studies have now suggested that a herpesvirus (FPHV) is the etiologic agent that

causes FP; however, definitive evidence of the involvement of papillomavirus, or any









other agent, is not yet available. Polymerase chain reaction (PCR) has demonstrated

herpesvirus genomic DNA in fibropapillomas of four species of marine turtles including

greens, loggerheads (Caretta caretta), olive ridleys (Lepidochelys olivacea) and Kemp's

ridleys (Lepidochelys kempii) (Quackenbush et al., 1998; Lackovich et al., 1999; Lu et

al., 2000; Yu et al., 2000; Quackenbush et al., 2001; Yu et al., 2001; Herbst et al., 2004).

In addition to the external tumors, various visceral organs, such as kidney, heart, lung and

brain, have also resulted in herpesvirus DNA amplification. An extensive study was

performed to investigate the genomic variation of FPHV across seven geographic areas

and three host species (Greenblatt et al., 2005). All variants tested showed greater than

96% nucleotide sequence conservation. Sequence variations correlated with geographic

location but not with species. Phylogenetic analysis of a 23 Kbp fragment suggested that

FPHV was an alphaherpesvirus, but not a member of any of the currently established

genera. Natural transmission is still largely unknown; however Herbst et al. (1995)

demonstrated that FP could be experimentally transmitted to young captive-reared green

turtles using cell-free fibropapilloma extracts prepared from free-ranging turtles with

spontaneous disease. Fibropapillomas developed in all recipients and were histologically

indistinguishable from tumors of free-ranging turtles. To date, no FPHV has been

isolated from any species of marine turtle.

In pinnipeds, three herpesviruses have been characterized to date, including phocid

herpesvirus-1 (PhHV-1), phocid herpesvirus-2 (PhHV-2) and otarine herpesvirus-1

(OtHV-1). PhHV-1 was first seen in young harbor seals (Phoca vitulina) in captivity in

The Netherlands and was associated with acute pneumonia, focal hepatitis, general

depression and high mortality (Osterhaus et al., 1985). Upon isolation, electron









microscopy (EM) revealed herpesvirus-like particles and intranuclear inclusions. The

virus was also shown to be antigenically related to canine herpesvirus (CHV) and feline

herpesvirus (FHV), and was eventually classified as a member of the Alphaherpesvirinae.

A similar epizootic was seen in harbor seals being rehabilitated in California (King et al.,

1998). Phylogenetic analysis of sequenced DNA fragments from the Pacific isolate

showed greater than 95% identity in translated amino acids after comparisons with the

Atlantic-European isolate (Harder et al., 1996). PhHV-1 has also been associated with

mass mortality of free-ranging harbor seals in northwestern Europe in 1988 (Frey et al.,

1989). Goldstein et al. (2004) provided evidence that PhHV-1 can be transmitted

between harbor seals through direct contact with oro-nasal secretions containing shedding

virus. PhHV-2 has been isolated from lung tissue of a juvenile captive California sea lion

(Zalophus californianus) that was simultaneously infected with a retrovirus (Kennedy-

Stoskopf et al., 1986) and from leukocytes of a harbor seal from the German Wadden Sea

(Lebich et al., 1994). At present, PhHV-2 has not been associated with clinical disease.

Harder et al. (1996) performed antigenic and genetic analyses to further characterize

PhHV-1 and -2. Herpesviruses were isolated from four harbor seals from Long Island as

well as one harbor seal and one grey seal (Halichoerus grypus) from European waters.

PCR was performed with partially degenerate primers based on conserved regions of the

glycoprotein B and D genes of CHV, FHV and equine herpesvirus-1. Fragments

amplified from PhHV-1 showed a high degree of homology with CHV and other

members of the genus Varicellovirus. No DNA amplification occurred with PhHV-2.

However, based on sequence analysis of two EcoRI fragments, PhHV-2 was classified as

a gammaherpesvirus, most closely related to equine herpesvirus-2. Serological studies









have shown that herpesvirus infections occur in at least 11 species of pinnipeds (for

review, see Kennedy-Stoskopf, 2001) and antibodies have been reported in as many as

99% of subadult and adult harbor seals in North American populations (Goldstein et al.,

2003a).

OtHV-1 has been associated with urogenital carcinoma of California sea lions that

stranded on the central California coast. Metastatic carcinoma was found in sublumbar

lymph nodes of ten sea lions, and upon histological examination, intraepithelial neoplasia

was found in the genital tracts of all ten animals (Lipscomb et al., 2000). Eosinophilic

intranuclear inclusions were seen in one animal, herpesvirus-like particles were seen in

two others, and another was positive for Epstein-Barr virus (HHV-4) by

immunohistochemical staining. Analysis of herpesvirus DNA sequences of the

polymerase and terminase genes obtained by PCR allowed for the classification of this

virus as a gammaherpesvirus in the genus Rhadinovirus. Samples were also tested for

papillomavirus by Southern blot and PCR; all samples were negative by both methods.

King et al. (2002) designed an OtHV-1 specific PCR and demonstrated that the virus was

present not only in tumors, but in brain and muscle tissue as well. Because viral DNA

was evident in all tumors examined, it was hypothesized that OtHV-1 may be a factor in

oncogenesis. Phylogenetic analysis of large fragments of the polymerase and terminase

genes showed a clear grouping with other gammaherpesviruses; however OtHV-1 was

distinct from PHV-2.

Herpesvirus infections have been suspected to occur in three families of cetaceans,

namely, Phocoenidae (porpoises), Monodontidae (belugas and narwhals) and Delphinidae

(dolphins, killer whales, pilot whales and relatives) (Van Bressem et al., 1999) for at least









three decades. Although there are only a handful of documented cases, past reports have

indicated that these viruses are important pathogens that may be associated with localized

infections of the skin and mucosas as well as systemic infections.

Barr et al. (1989) described a focal necrotizing dermatitis in a young adult female

beluga whale (Delphinapterus leucas) from the Churchill River, Manitoba, Canada. Pale,

depressed lesions with irregular borders on the dorsal ridge and lateral side of the whale

were first seen approximately 3.5 months after capture. Histologically, many

eosinophilic intranuclear inclusion bodies were seen in epithelial cells and herpesvirus-

like particles were demonstrated by transmission electron microscopy (TEM). The

lesions were transient in nature, lasting approximately eight months, and appeared to be

confined to the skin as no other health problems were noticed in the animal. It was

believed that the virus was either latent at capture or that it was transmitted from other

beluga whales following capture. Once in captivity, the beluga whale also had contact

with Atlantic bottlenose dolphins (Tursiops truncatus), California sea lions and harbor

seals, all of which are susceptible to herpesvirus infections, although infections across

species are rare events. A similar, yet more severe, viral dermatitis was reported in a

free-ranging juvenile female beluga whale that stranded in the St. Lawrence Estuary,

Quebec, Canada (Martineau et al., 1988). Lesions were found covering the body, were

most apparent on the head and were described as pale, circular, depressed areas outlined

by a narrow dark rim. Epithelial cells contained eosinophilic intranuclear inclusion

bodies shown by EM and viral particles similar in morphology to those of herpesviruses

were also seen. A second young female beluga from the same area was also reported as

having widespread dermatitis, which was grossly and microscopically similar to the first.









However, no viral particles were observed in the second animal suggesting that lesions of

the first animal may have been at an earlier stage of infection. Despite extensive

dermatitis, it was determined that herpesvirus infection was not the cause of death of

either whale. Beluga whales in the St. Lawrence estuary tested for cetacean herpesvirus

seroprevalence using bovine herpesvirus-1 (BHV-1) as the indicator in a serum

neutralization test and a blocking enzyme-linked immunosorbent assay (ELISA) were

found to be 46 and 58 % antibody positive, respectively (Mikaelian et al., 1999). The

presence of antibodies against BHV-1 was interpreted as previous infection of beluga

whales with a closely related cetacean alphaherpesvirus.

Skin lesions associated with herpesvirus-like particles have been also reported in

free-ranging sexually immature dusky dolphins (Lagenorhynchus obscurus) from

Peruvian coastal waters (Van Bressem et al., 1994). Skin lesions consisting of a few

black points on the rostrum were noticed in three dusky dolphins, while a fourth had

lesions dispersed all over its body. The epithelial cells of two dusky dolphins were

shown by TEM to contain virus particles with morphological features similar to those of

herpesviruses. No virus particles were detected in the other two dolphins, as the lesions

may have been in a convalescent stage. Although the clinical signs resembled those of

alphaherpesvirus infection, antigenic or molecular characterization was not performed.

The virus strain involved in these cases showed a skin tropism and seemed to be only

mildly pathogenic, as no other evidence of disease was observed. It should be noted that

the dusky dolphin is a highly sociable species and can congregate in supergroups of 700

to 800 individuals, which may facilitate easy direct transmission of this virus.









Although the presence of herpesvirus or herpesvirus-like particles was not

confirmed, skin lesions in a killer whale (Orcinus orca), a striped dolphin (Stenella

coeruleoalba) and three harbor porpoises (Phocoenaphocoena) were also reported to be

the result of herpesvirus infection. Greenwood et al. (1974) reported a killer whale that

developed a widespread rash of small vesicles, which was described as closely

resembling a varicella infection. Varicella zoster, an alphaherpesvirus commonly known

as chickenpox, is caused by human herpesvirus-3. Baker (1992) surveyed skin lesions in

wild cetaceans from British waters and reported possible herpesvirus infections in one

striped dolphin and three harbor porpoises. The diagnosis of the four cases of

herpesvirus infection was based on the similarity of gross pathology to the herpesvirus

infections seen in beluga whales (Martineau et al., 1988; Barr et al., 1989). Histological

examination of lesion samples revealed the presence of large eosinophilic intranuclear

inclusions in epithelial cells. A herpesvirus has been described also in association with

genital lesions on the penile mucosa of an adult harbor porpoise found on the New Jersey

coast (Lipscomb et al., 1996a). Histologically, epithelial cells contained many

eosinophilic intranuclear inclusion bodies and immunohistochemical stainings for herpes

simplex-1 and -2 were positive.

More serious disease in the form of herpesvirus encephalitis was diagnosed in a

juvenile female harbor porpoise that stranded off the coast of Sweden (Kennedy et al.,

1992). Skin lesions, which were morphologically similar to those of beluga whales (Barr

et al., 1989; Martineau et al., 1988) were found on the head, thorax and abdomen of the

animal, however, there was no histological or immunohistochemical evidence to support

a herpesvirus etiology. Several tissues were examined for histopathologic lesions but









acidophilic intranuclear inclusions were seen only in the cerebral cortex. Herpesvirus-

like particles were observed by EM in affected neurons and herpesviral antigen was

detected by immunoperoxidase staining of the cerebral cortex. Immunologic cross-

reactivity between the presumed porpoise herpesvirus and antisera to pseudorabies virus,

human herpesvirus-1 and bovine herpesvirus-1 indicated that the porpoise virus was most

likely an alphaherpesvirus. The presence of encephalitis in this porpoise indicates that

fatal herpesviral disease in free-ranging cetaceans is possible.

More recently, two novel alphaherpesviruses have been described in association

with disseminated infection in Atlantic bottlenose dolphins that stranded along the East

coast of the U.S. (Blanchard et al., 2001). Two juvenile female bottlenose dolphins, one

found on Hilton Head Island, South Carolina (case 1), the other on Prime Hook Beach,

Delaware (case 2), presented with acute necrotizing lesions in multiple organ systems,

which was the leading cause of death in both cases. Examination of various tissues by

TEM revealed the presence of herpesvirus-like particles in the nucleus and enveloped

virions in the cytoplasm. PCR targeting the herpesvirus DNA polymerase and DNA

terminase genes confirmed the diagnosis of alphaherpesvirus infection. Case 1 yielded a

189-bp product from lung tissue while case 2 yielded a 180-bp product from heart tissue

of a highly conserved region of the DNA polymerase gene (GenBank accession numbers

AF196646 and AF245443, respectively). A partial terminase gene sequence of 375-bp

was also obtained from case 1 (GenBank accession number AF196647). Phylogenetic

analyses of these herpesvirus genome fragments revealed that they were most closely

related to each other and not very closely related to herpesviruses found in other marine

animals, namely, harbor seals, sea lions and green sea turtles. Because the dolphins









presented with symptoms characteristic of both herpesvirus and morbillivirus infection,

such as syncytial cells, intranuclear inclusions and lymphoid depletion, reverse

transcriptase PCR (RT-PCR) was also performed to detect possible morbillivirus;

however, results were negative. This was the first report of disseminated herpesvirus

infection in cetaceans as well as the first molecular evidence of the existence of

alphaherpesviruses in cetaceans.

The objective of this research was to develop a diagnostic assay for herpesviruses

infecting tissues and lesions of captive and free-ranging cetaceans. A previously

described PCR targeting highly conserved regions of the herpesvirus DNA polymerase

gene (VanDevanter et al., 1996) was selected as a starting point because it has been

shown to amplify herpesvirus DNA in other aquatic animals. Utilizing this same

technique, FPHV sequences were obtained from three species of marine turtles

(Quackenbush et al., 1998), and a novel gammaherpesvirus was identified in Hawaiian

monk seals (Monachus schauinslandi) (Goldstein et al., 2003b). The results of the assays

with cetacean samples were then used for the genetic identification and molecular

characterization of these novel cetacean herpesviruses.

Materials and Methods

Sample Acquisition and DNA Extraction

Lesion and tissue samples were obtained from stranded cetaceans from Florida,

Georgia, North and South Carolina, Texas and Alaska waters. Scrapings or biopsy

lesions were also collected from live cetaceans at wildlife parks and rehabilitation centers

at various locations throughout Florida and California. In total, 118 lesion samples

encompassing 12 cetacean species were analyzed for herpesvirus genomes; 87 from skin

lesions and 31 from mucosal lesions. Seventy-two tissue samples, mostly from lung and









brain, were also tested from five cetacean species. In addition to cetacean samples,

lesions from three species of marine turtles and one pinniped were also tested. A

comprehensive listing of all samples tested is presented in Appendix A.

Total DNA was extracted from all samples using the DNeasy Tissue Kit (Qiagen

Inc., Valencia, California, USA) according to the manufacturer's protocol. Briefly, the

sample was crushed in a microfuge tube with a sterile pestle and allowed to lyse

overnight in lysis buffer at 550C. DNA was precipitated by the addition of absolute

ethanol and applied to a spin column, washed and eluted in 200 il of elution buffer. The

total DNA content and quality of the eluted DNA were determined using the Ultrospec

3000 spectrophotometer (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA).

Nested PCR Targeting the DNA Polymerase Gene

Herpesvirus DNA was amplified using published degenerate primers designed to

target a region of the DNA polymerase gene of herpesviruses that corresponds to highly

conserved amino acid motifs (VanDevanter et al., 1996; Ehlers et al., 1999). These

primers are known to direct the amplification of DNA polymerase gene fragments 215 to

235-bp in length for most herpesviruses and 315-bp for cytomegaloviruses. A nested

PCR assay was performed with two forward and one reverse primers in the first reaction,

and one forward and one reverse primer in the second reaction. Primer sequences for the

first reaction were: DFA-5'- GAY TTY GCI AGY YTI TAY CC -3' (forward), ILK-5'-

TCC TGG ACA AGC AGC ARI YSG CIM TIA A -3' (forward), KG1-5'- GTC TTG

CTC ACC AGI TCI ACI CCY TT -3' (reverse). Primers for the second reaction were:

TGV-5'- TGT AAC TCG GTG TAY GGI TTY ACI GGI GT -3'(forward), IYG-5'- CAC

AGA GTC CGT RTC ICC RTA IAT -3' (reverse). (See Figure 1-1.) Total DNA

extracted from cell monolayers of Madin Darby canine kidney (MDCK) cultures infected









with canine herpesvirus (CHV) and from cell monolayers of tortoise heart (TH) cultures

infected with the tortoise herpesvirus-1 (THV-1) were used as positive templates for

PCR. In the first PCR, approximately 500 ng of sample DNA was used as template. The

first PCR mixture contained 400 nM of each primer, 100 pM of each dNTP, 10 mM KC1,

10 mM (NH4)2 SO4, 20 mM Tris-HC1, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1

unit of Taq DNA polymerase (New England BioLabs Inc., Beverly, Massachusetts,

USA). All PCRs were run in a PTC-100 Programmable Thermal Cycler (MJ Research,

Inc., Waltham, Massachusetts, USA). Cycling conditions for the first and second PCR

were: Initial incubation at 940C for 2 min followed by 55 cycles at 940C for 20 sec, 46C

for 30 sec, and 720C for 30 sec. A final extension step at 720C for 10 min finished the

cycling. In the second PCR, mixtures were identical to those in the first PCR but

contained 400 nM of each of the second reaction primers and 2 pl of the first PCR

product as DNA template. Approximately 20 [tl from the second PCR were resolved by

horizontal gel electrophoresis in 1.0% agarose containing ethidium bromide (0.5 ug/ml)

and the DNA fragments were visualized by UV light transillumination and photographed

using a gel documentation system. Amplified DNA fragments of the predicted size were

purified using the MinElute PCR Purification Kit (Qiagen Inc., Valencia, California,

USA).

IMorA MotiB MOpifC
3P --)kl 4-
OFA4 K TV K31i

Figure 1-1. Schematic of the location of the primers used in the nested PCR targeting the
herpesvirus DNA polymerase gene (VanDevanter et al., 1996).

In order to obtain additional nucleotide sequence, samples that were positive for

herpesvirus DNA by the nested PCR were used subsequently for the amplification of a









larger fragment of the DNA polymerase gene, approximately 700-bp in length, using

primers DFA and KG1. The PCR mixture contained approximately 500 ng of sample

DNA, 400 nM of each primer, 100 [M of each dNTP, 2 mM MgC12, IX Expand High

Fidelity buffer and 3.3 units of Expand High Fidelity enzyme mix (Roche Applied

Science, Indianapolis, Indiana, USA). Cycling conditions were: Initial denaturing at

940C for 2 min followed by 40 cycles at 940C for 30 sec, variable annealing temperature

for 30 sec and extension at 720C for 2 min. A final extension step at 720C for 10 min

finished the cycling. The annealing temperature varied between 420 and 470C depending

on the species of DNA template being assayed. If the amplification of the 700-bp

fragment was unsuccessful, a third PCR was attempted with primers DFA and IYG to

amplify approximately 500-bp of herpesvirus DNA. The Expand High Fidelity PCR

system (Roche Applied Science, Indianapolis, Indiana, USA) was used in the PCR

mixture and all the quantities were the same as above. Cycling conditions were: Initial

denaturing at 940C for 1 min followed by 40 cycles at 940C for 30 sec, variable annealing

temperature for 30 sec, extension at 720C for 1 min and a final extension step at 720C for

10 min. As above, the annealing temperature varied between 420 and 47C depending on

the species of DNA template being assayed. Amplified DNA fragments of the predicted

size from both subsequent PCRs were purified using the MinElute PCR Purification Kit

(Qiagen Inc., Valencia, California, USA) or excised after gel electrophoresis in 1.2%

low-melting-point agarose and purified using the MinElute Gel Extraction Kit (Qiagen

Inc., Valencia, California, USA).

Cloning, Sequencing and Sequence Analysis

Purified DNA fragments were cloned into the plasmid vector pCR2.1-TOPO T/A

(Invitrogen, Carlsbad, California, USA). Two clones for each sample were selected for









plasmid purification and sequenced in duplicate from both ends using the CEQ 2000 XL

(Beckman Coulter Inc., Fullerton, California, USA) sequencing instrument, following the

manufacturer's protocol. Briefly, the 20 [tl sequencing reaction contained approximately

100 fmol of total DNA, 2 [tl of either the forward or reverse M13 primer, 4 [tl DTCS

Quick Start Master Mix, 1 [tl sequencing buffer and ultra pure water. Cycling conditions

consisted of 50 cycles at 960C for 20 sec, 500C for 20 sec and 600C for 4 min following

an initial denaturation step at 960C for 2 min. Exported chromatograms were manually

reviewed using the Chromas 2.3 software (Technelysium Pty Ltd., Tewantin,

Queensland, Australia). Genetic analyses were performed using the functions Seqed,

Gap, Translate, Lineup, Pileup and Pretty of the University of Wisconsin Package

Version 10.2, Genetics Computer Group (GCG), Madison, Wisconsin, USA. Multiple

sequence alignments and phylogenetic analyses were performed using PAUP* 4.0

(Sinauer Associates, Sunderland, Massachusetts, USA). The BLAST function of the

National Center for Biotechnology Information (NCBI) website

(http://www.ncbi.nlm.nih.gov/) was used to identify herpesvirus sequences more closely

related to those of the cetacean herpesviruses.

PCR Targeting the DNA Terminase Gene

Samples positive for herpesvirus polymerase gene DNA by the nested PCR were

tested subsequently for cetacean herpesvirus terminase DNA. Primers were designed

from the terminase sequence (GenBank accession number AF 196647) of an Atlantic

bottlenose dolphin herpesvirus (Blanchard et al., 2001); 5'- ACC AAC ACC GGC AAA

GCT A -3' (forward), 5'- CAC GTA CAC GAA CAG TTC C -3' (reverse). The PCR

mixture contained approximately 500 ng of sample DNA, 400 atM of each primer, 100









mM of each dNTP, 10 mM KC1, 10 mM (NH4)2 S04, 20 mM Tris-HC1, 2 mM MgSO4,

0.1% Triton X-100 at pH 8.8 and 1 unit of Taq DNA polymerase (New England BioLabs

Inc., Beverly, Massachusetts, USA). Cycling conditions were: Initial incubation at 940C

for 2 min followed by 40 cycles at 940C for 30 sec, 500C for 30 sec, and 720C for 30 sec.

DNA fragments of the expected size were purified, cloned, sequenced and analyzed as

previously described.

Results

Nested PCR Targeting the DNA Polymerase Gene

The degenerate primer nested PCR approach, as previously described for the

detection of herpesvirus genomic sequences of terrestrial mammals, was also successful

in the amplification of DNA polymerase gene fragments from total DNA from cutaneous

and mucosal lesions of cetaceans. Fragments of the herpesvirus DNA polymerase gene

were amplified from nine lesion samples from three species of cetaceans, as well as 15

fibropapilloma samples from two species of turtles (Figure 1-2). Positive cetacean

samples are described in detail below.









Figure 1-2. Gel electrophoresis of herpesvirus nested PCR products; lane 1- K311
bottlenose dolphin penile lesion, lane 2- K263 bottlenose dolphin vaginal
lesion, lane 3- K310 bottlenose dolphin penile lesion, lane 4- K308 bottlenose
dolphin tongue lesion, lane 5- K264 bottlenose dolphin penile lesion, lane 6-
K265 dwarf sperm whale vaginal lesion, lane 7- K285 Blainville's beaked
whale penile lesion, lane 8- K167 bottlenose dolphin skin lesion, lane 9- K231
bottlenose dolphin skin lesion, lane 10- green turtle fibropapilloma, lane 11-
Kemp's ridley turtle fibropapilloma, lane 12- negative control, lane 13-
positive control, CHV.









An adult female dwarf sperm whale (Kogia sima) that had repeatedly stranded in

Manatee County, Florida was euthanized in a deteriorating condition. Sample K265 was

taken from a wart-like lesion located on the genital slit and tested positive for herpesvirus

DNA. Several lymph nodes from this animal were also tested, but were all negative by

the nested PCR approach.




















Figure 1-3. Wart-like lesion (arrow) on the genital slit of a female dwarf sperm whale
(K265). Photo provided by Dr. Nelio B. Barros.

Two Atlantic bottlenose dolphins from a Florida wildlife park presented with

genital lesions. Sample K311 was a scraping of a plaque on the penile mucosa of an

adult male (Figure 1-4A) and K263 was a scraping of vaginal sores of an adult female

(Figure 1-4B and C). Evidence of sexual activity between these two animals was

apparent as they have produced offspring.



















A C

















B

Figure 1-4. Genital lesions of two captive Atlantic bottlenose dolphins. A) Penile lesions
(K311) B) vaginal sores C) vaginal sores 10X.

An adult male Blainville's beaked whale (Mesoplodon densirostris) was found

stranded on Kure Beach, North Carolina in January, 2004 with a papilloma-like penile

lesion (K285) (Figure 1-5A). Penile epithelial cells contained 4 to 6 I intranuclear

inclusions shown by hematoxylin and eosin (H&E) staining (Figure 1-5B).

























A W '



















B

Figure 1-5. Blainville's beaked whale (K285) A) Penile lesion B) H&E stain showing
multiple cells that contain intranuclear inclusions (arrows). Bar = 20 im.
Photos provided by Dr. Jeremiah T. Saliki.

A stranded juvenile male Atlantic bottlenose dolphin was admitted to the Dolphin

and Whale Hospital, Mote Marine Laboratory and Aquarium, Sarasota, Florida with mild

dermatitis on the rostrum, head, dorsal fins, flanks, peduncle and flukes (Figure 1-6).

Lesions were described as hundreds of 1 to 3 mm, spherical, raised, black papules and









biopsies were taken 1 to 2 months post-admission (K167). Lesions regressed slowly and

were almost completely resolved by 3 months post-admission. Histologically, epithelial

cells contained intranuclear inclusions (Figure 1-7A) and herpesvirus-like virions were

shown by TEM (Figure 1-7B).

















A

















B

Figure 1-6. Atlantic bottlenose dolphin dermatitis lesions (K167) on the A) rostrum and
B) skin. Photos provided by Dr. Charles A. Manire.
























A rmi ui ",. ... :-;-"w.," ,.- ....ii


























Figure 1-7. Epithelial cells from a skin lesion of a bottlenose dolphin (K167). A)
Intranuclear inclusion bodies (arrows) seen with H&E staining. B)
Transmission electron micrograph illustrating non-enveloped nucleocapsids
(arrowheads) in the nucleus and an enveloped virion (arrow) in the cytoplasm
adjacent to the nuclear membrane (nm). Uranyl acetate and lead citrate, bar
250 nm. Images provided by Dr. Michael Kinsel.









In June 2001, an adult male Atlantic bottlenose dolphin stranded offshore of

Islamorada Key, Florida. Reddened ulcerations were widely apparent on the tongue

(K308) (Figure 1-8A) and plaques were seen on the penile mucosa (K310) (Figure 1-8B).

Total DNA extracted from the penile and oral lesions were both positive for herpesvirus

by PCR. Amphophilic intranuclear inclusions and syncytia were observed in a cluster of

vessels in the lung; however DNA extracted from lung tissue yielded negative results by

nested PCR. This animal was severely debilitated and immunocompromised, and

ultimately died of sepsis.


















A FB B .

Figure 1-8. Atlantic bottlenose dolphin with lesions on A) the tongue and B) the penis.
Photos provided by Dr. Ruth Ewing.

Sample K231 was obtained from a juvenile male Atlantic bottlenose dolphin from a

wildlife park in Florida. The animal had multiple skin lesions on the right lateral side,

which were described as 1mm black pinpoints that could be palpated. Histological

changes were not suggestive of a herpesvirus infection.









Sample K264 was obtained during necropsy from a penile lesion of an Atlantic

bottlenose dolphin that stranded along the coast of Jacksonville, Florida.

Sequencing of the DNA fragments amplified from cetacean lesions after the nested

PCR approach demonstrated that they ranged in size from 222 to 244 nucleotides in

length, and translation of these nucleotide sequences, resulted in DNA polymerase

fragments that ranged in size from 73 to 80 amino acid residues. The subsequent PCR

with primers DFA and KG1 amplified 731-bp amplicons that translated into proteins

composed of 243 amino acid residues, from six animals (Figure 1-9). These primers

were used also to amplify a 737-bp, 245 amino acid residue fragment from CHV. This

sequence has been deposited in the GenBank database under the accession number

AY949827. Larger fragments from the remaining samples that were positive for the

small DNA polymerase fragment could not be obtained by any of the PCR approaches

attempted. All cetacean herpesvirus sequences have been deposited in the GenBank

database. Accession numbers for individual samples are: K285 Blainville's beaked whale

penile lesion- AY949828, K265 dwarf sperm whale vaginal slit lesion- AY949830, K311

bottlenose dolphin penile lesion- AY949831, K231 bottlenose dolphin skin lesion-

AY949832, K264 bottlenose dolphin penile lesion- AY952776, K263 bottlenose dolphin

vaginal lesion- AY952777, K310 bottlenose dolphin penile lesion- AY952778, K308

bottlenose dolphin tongue lesion- AY952779, K167 bottlenose dolphin skin lesion-

AY757301.

Multiple sequence alignments of the nucleotide and amino acid sequences derived

from eight positive cetacean samples indicated that viruses that could be placed in two

virus groups were present (Figures 1-10 and 1-11). The sequence from sample K308 was









excluded from the alignment as it was identical to the sequence of a separate lesion

(K310) from the same animal. Nucleotide and amino acid homologies with multiple

known herpesviruses indicated that cetacean herpesviruses fell within the Alpha- and

Gammaherpesvirinae subfamilies (Tables 1-1, 1-2 and 1-3). Although genetic variability

was observed within the alpha and gammaherpesvirus sequences, phylogenetic analyses

clearly indicated that both alphaherpesviruses and gammaherpesviruses infect cetaceans

(Figures 1-12, 1-13 and 1-14).










Figure 1-9. Gel electrophoresis of large DNA polymerase fragments from cetacean
gammaherpesviruses; lane 1- K263 bottlenose dolphin vaginal lesion, lane 2-
K311 bottlenose dolphin penile lesion, lane 3- K310 bottlenose dolphin penile
lesion, lane 4- K264 bottlenose dolphin penile lesion, lane 5- Blainville's
beaked whale penile lesion, lane 6- K265 dwarf sperm whale vaginal lesion,
lane 7- negative control, lane 8- positive control, CHV.

The nested PCR approach using degenerate consensus primers has demonstrated

that cutaneous lesions from cetaceans are associated with alphaherpesvirus infection

while genital lesions, and in one case, an oral lesion, are associated with

gammaherpesvirus infection. Alphaherpesvirus genomic DNA was amplified from two

cutaneous lesions from Atlantic bottlenose dolphins (K167 and K231). Bottlenose

dolphin K231 appeared to be infected with the same virus identified by Blanchard et al.

(2001) that caused a disseminated infection in an Atlantic bottlenose dolphin from South

Carolina in 1995 (GenBank accession number AF 196646). Nucleotide and amino acid

identities between the two dolphin alphaherpesviruses were 98.9 and 96.8%, respectively.









Tissue samples for bottlenose dolphin K231 could not be tested for disseminated

infection as the dolphin is still alive. Gammaherpesvirus genomic DNA was amplified

from penile lesion samples from three Atlantic bottlenose dolphins (K311, K310 and

K264) and one Blainville's beaked whale (K285), as well as two vaginal lesions from an

Atlantic bottlenose dolphin (K263) and a dwarf sperm whale (K265). In addition to the

penile lesion (K310), one bottlenose dolphin had ulcerations on the tongue (K308), and

upon comparison of the sequences from both lesions, it was determined that the same

virus was present in the oral and genital mucosas. Of the cetacean tissue samples from

stranded animals that were assayed none were positive for herpesvirus using the nested

PCR approach.

A pair of cetacean-specific primers was designed to amplify the small DNA

polymerase gene fragment of both alpha and gammaherpesviruses, while a second pair

was designed to amplify the large DNA polymerase gene fragment from the

gammaherpesviruses. Unfortunately, neither set of primers were consistently successful.

Sequences obtained from the turtle fibropapilloma samples were 237-bp in length

and translated into polypeptides of 79 amino acid residues. Upon comparison with FPHV

sequences from the NCBI website, all amino acid homologies were greater than 96%,

clearly indicating the presence of FPHV in our samples.

































































Figure 1-10.


Multiple alignments of the nucleotide sequences of the DNA polymerase gene fragments of cetacean herpesviruses.

BND- bottlenose dolphin, BBW- Blainville's beaked whale, DSW- dwarf sperm whale. K311- penile lesion, K263-

vaginal lesion, K310- penile lesion, K264- penile lesion, K285- penile lesion, K265- vaginal slit lesion, K167- skin

lesion, K231- skin lesion.


.t. . . .c . .. . . . . . . . . . . . . . . . . . . . .
t . . . . . . . . . . . . . . . . . . . . . . . .
. ^ .C\ . c"c1 . . . . . . . . C . . . . . . . . . . . . .


ct....... .......... ..g....... .c..... ..t .......... ....... ...
.......... .. t ....... .......... .......... .......... ... a ..... t ..... t ..t .
.......... ct ....... .......... .. g ....... c ..... t .. ......... t ...... c ...


......... tt ....... ... t .... g .
i......t.. t..... t..c .at...ac.c


. . . . . . . . . . . . . . . . . . . . . . . . . I I I I I I I


.... g ..... ... a ..... c ..... t .... t ..... t .. a ..... c ... ..a ..... t .g ..t .....
cc.aa ..... ... c..a ... .......... .g ..t ..g .. ... ..... t ..a..a .... t ........


t..t ...... ..... t .... a..c ...t .
t ..... c..a .g...at... t..g .....


. . . . . . . . . . . . . . . . . . . I .. . . . . .
. . . . . . . . . . . . . . . . . . . a .. . . . . .
. . . . . . . . . . . . . . . . . . . a .. . . . . .
. . . . . a . . . . . . . . c .g c .c . . g . . . . . .
... t ..... t ....... aa .... t ..... .... g .... t ..a ..... t .at .g ..... ... a ...... ..... g .... .... t ..t .. ...... c ...
... ...... ........ a .... t...t a ..t C -...t ..c.....t. .a..c ..... c..a ..... t ..... .... .......... ... a ......










































































.......... ........ g. .......... .......... .......... ..........

.......... ........ .......... .......... .. ...... ..........
. . . . . a . . . . . a . . . . .


BBWK285 ..t.a..... ....... a. ..... a ........ c. ......... a ........ t ..g ....... ..........

DSWK265 ..t.t...a. ........ t. a ..... t... .... a .... .... t..a.. .... cca... .. g ...... ....... a..

BNDK167 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

BNDK231 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus TTCACGGACC CCATAAAGCT GGAGGCGGAA AAGACTTTTA AGTGCTTGCT CCTCTTGACC AA-AAGAGAT ACATTGGGAT








Figure 1-10. Continued.


. . . . . . . . . . . . . . . ..I I I I I I I I I I I I


S.t .......

..... c. t .

. t .c ....


t ..... t..

.t..g..t..
..... a ....


a..t ......

t ........ a

. . . .


. .a ...... a

S.c..t. .g.

ca ...... a.


. . .t .


. . . .


c. a ......

a ..... t ...

.cc.. c..a


.....t ....

S.a..t..g.

.c...c..a.


. . .
.... t .....

a. at.....

.c..ca ....


..a. .g ....

...... c.c.

t.actc..ca


... .. ............. .......... .......... .......... ..........
. . g . . . . . . . . . . . . . . . . . . . . . . .
. .a . . . . . . . . . . . . . . . . . . . . . . . .


.t ..g .....
.acc c. .
.aaac. a


.......... .. g. .. t ...

...... a ... ..... t ..tt

.t.ac.ct.. tcatt aa.t


.g .....t ..
ca.a...t. .

a.ac. .aa. c


......... t

a .........

a.ctac.a c.


.a. .ga. t.

.... g ...c.
..a.ttta..


.a.a.a...

.ac...ata.

cacc..tacc


t..t.aa...

gtg. .a. ....

..t.ccccac


. .t t .

t..c.tc..aC


aa..caacat



cct. caact


. . . . L . . . . . .

. . . . t . . . . . .

. . . . t . . . . . .


.a..c .....

.ttc...a. .

.cac.atc. .
























BBWK285 ....... g.. ......... .......... .
DSWK265 ....... g .. .......... .......... .
BNDK167 .......... ...........
BNDK167 ---------- ---------- ---------- -
BNDK231- ---------- ---------- -
Consensus TAATGAA-GG CGTCGACCTG GTGAGCAAGA C



Figure 1-10. Continued.




















BN DK 2 63 .......... ......... ......... ......... ......... ......... ......... ......... .........
BN DK 31 0 e ......... ...... ... .......... .......... .......... .......... .......... .......... .......... ..........
BNDK264 .................... ..... ... ...... ..... .......... .........
BBWK285 .......... .......... ... ...l.. ..... .......... .. h ... .......... ........ .. ..........

DSWK265 .......... .......... .. he.i .... n .tpe..d .. .......... .......... r ....... ........g .......... ..........
BNDK167 ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
BNDK231 ------ ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
Consensus DFASLYPSII QAHNLCYSTL IPDGEMHRHP TLLKGDFETF HISSGPVHFV KKHVTYSLLS KLLATWLAKR KAIRRELSQC SDPQLKTILD KQQLAIKVTC

101 150 200
BNDK311 .......... .......... .......... ..... r.... .......... .......... .......... .......... .......... ..........
BN DK 2 63 .......... .......... .......... ..... r .... .......... .......... .......... .......... .......... ..........
BN DK 31 0 .......... .......... .......... ..... r .... .......... .......... .......... .......... .......... ..........
BNDK264 .......... .......... .......... .....k.... ...dr.k ... ....v..... .d........ .k.a ...... .......... .... k .....
BBWK285 .......... ...m...... ........ c. ..... k .... ...dy..... ... qt.td.. e.s ....... .k ...... ......... a...k.....
DSWK265 ...... .......... .... s... c. .....k.... ..... sk... ... r..pdtc e ........ d .......... .......... ... tn.....
BNDK167 .s........ q ...... p.. a...ti..d. .1strd.lhs r.atreq.aa df.d.y.spa pis.s.s..s i......... ---------- ----------
BNDK231 .s........ q..p...q.. a...ti..d. .l.trd.lh. h.atae..va df.dga.a.1 las.p.p..s ih........ ---------- ----------
Consensus NAVYGFTGVA SGLLPCLKIA ETVTLQGRRM LERSK-FIEA -WINHRRLEE LIGHAVAGAD GNAP-A-PYE FRVVYGDTDS LFIECRGYSL DSVSEFCDAL

201 250
BNDK311 ......... .......... .......... .......... .......... .
BN DK263 ......... .......... .......... .......... .......... .
BN DK310 .......... .......... .......... .......... .......... .
BNDK264 .a ..... a .......... .......... .......... .......... .
BBWK285 ..... s ...k .......... .......... .......... .......... .
DSWK265 ..... n...m e ......... ......p... ....... n .. .......... .
BNDK167 ---------- ---------- ---------- ---------- ---------- -
BNDK231 --------- ---------- ---------- ---------- ---------- -
Consensus ASVTSGTLFT DPIKLEAEKT FKCLLLLTKK RYIGILSTDK ILMKGVDLVS K


Figure 1-11. Multiple alignments of the deduced amino acid sequences of the DNA polymerase gene fragments ofcetacean

herpesviruses. BND- bottlenose dolphin, BBW- Blainville's beaked whale, DSW- dwarf sperm whale. K311- penile

lesion, K263- vaginal lesion, K310- penile lesion, K264- penile lesion, K285- penile lesion, K265- vaginal slit lesion,

K167- skin lesion, K231- skin lesion.














Table 1-1. Percent nucleotide identities of DNA polymerase gene fragments among members of the family Herpesviridae. GenBank
accession numbers for the virus sequences used are the same as in Figure 1-12.
BBW K285 BND K264 BND K310 BND K311 BND K263 DSW K265 BND K167 BND K231
Human herpesvirus-8 62.9 66.3 66.1 66.1 66.2 64.0 51.2 50.2
Human herpesvirus-4 58.6 66.2 63.5 64.2 63.7 59.4 56.6 57.1
CA sea lion gammherpesvirus 60.8 59.4 60.2 60.4 60.1 60.2 47.4 44.8
Bovine herpesvirus-4 65.1 63.4 63.2 63.6 63.4 63.0 45.1 41.1
Ovine herpesvirus-2 64.6 67.8 66.1 66.3 66.1 63.7 49.6 53.3
Porcine lymphotropic herpesvirus-1 61.4 57.5 59.4 59.2 59.2 60.1 49.0 45.9
Equine herpesvirus-2 65.1 72.9 71.0 71.6 71.2 64.4 53.4 50.6
Beaked whale K285 100.0 80.8 82.8 83.3 83.2 76.3 40.6 54.1
Bottlenose dolphin K264 80.8 100.0 91.5 91.8 92.2 77.4 54.9 56.4
Bottlenose dolphin K263 83.2 92.2 99.3 99.6 100.0 78.4 57.8 58.1
Bottlenose dolphin K311 83.3 91.8 99.0 100.0 99.6 78.5 57.8 58.1
Bottlenose dolphin K310 82.8 91.5 100.0 99.0 99.3 78.4 57.8 58.1
Dwarf sperm whale K265 76.3 77.4 78.4 78.5 78.4 100.0 48.4 49.0
Dolphin alphaherpesvirus SC95 42.3 52.9 49.2 49.2 49.2 46.0 73.0 98.9
Dolphin alphaherpesvirus DE99 41.0 38.5 38.9 38.9 38.9 36.1 55.6 55.6
Bottlenose dolphin K167 40.6 54.9 57.8 57.8 57.8 48.4 100.0 78.8
Bottlenose dolphin K231 54.1 56.4 58.1 58.1 58.1 49.0 78.8 100.0
Human herpesvirus-1 52.4 59.3 56.3 56.7 56.6 53.4 71.6 71.6
Human herpesvirus-2 50.6 57.0 55.9 56.3 56.1 51.9 72.5 72.1
Green turtle FP herpesvirus 52.6 56.1 54.5 54.8 55.2 52.8 62.9 59.1
Bovine herpesvirus-1 52.6 58.9 58.5 58.6 58.8 53.4 69.8 69.0
Suid herpesvirus-1 52.7 61.6 59.5 59.7 59.6 57.2 61.5 75.0
Marek's disease virus 50.2 49.2 50.4 50.6 50.6 52.3 56.8 56.3
Canine herpesvirus 54.5 47.6 50.1 50.3 50.5 54.6 53.9 50.9
Phocine herpesvirus-1 40.7 35.7 37.7 38.0 37.7 38.1 55.1 55.1
Feline herpesvirus-1 53.1 51.2 51.2 51.4 51.4 53.9 60.3 57.8
Equine herpesvirus-1 53.8 55.5 55.6 55.9 55.5 53.7 72.9 68.1
Human herpesvirus-5 50.7 53.0 52.6 53.0 52.8 51.3 58.6 47.5
Murid herpesvirus-4 51.0 56.3 55.2 55.1 55.5 51.5 57.6 58.8
Porcine cytomegalovirus 53.9 54.8 53.0 53.4 53.5 53.1 54.1 56.2
Elephant herpesvirus-1 55.1 53.2 53.5 54.0 54.0 52.7 47.5 52.4
Koi herpesvirus 42.2 44.7 42.4 42.4 42.2 39.9 ND ND
Channel catfish virus 37.2 41.4 39.2 37.7 39.0 39.6 42.4 41.9
Salmonid herpesvirus-1 34.9 36.6 39.5 39.3 39.3 38.2 ND ND














Table 1-2. Percent amino acid identities of DNA polymerase gene fragments among members of the family Herpesviridae. GenBank
accession numbers for the virus sequences used are the same as in Figure 1-12.
BBW K285 BND K264 BND K310 BND K311 BND K263 DSW K265 BND K167 BND K231
Human herpesvirus-8 70.7 70.7 69.8 70.7 70.7 70.2 53.5 58.0
Human herpesvirus-4 63.0 63.4 63.8 64.6 64.6 62.6 52.7 51.2
CA sea lion gammherpesvirus 64.4 63.6 62.0 63.2 63.2 62.0 42.9 46.0
Bovine herpesvirus-4 68.9 69.3 68.5 69.3 69.3 68.3 59.2 53.5
Ovine herpesvirus-2 68.1 66.8 66.8 67.6 67.6 67.6 45.3 46.7
Porcine lymphotropic herpesvirus-1 64.5 65.1 64.5 63.8 63.8 66.9 41.2 42.6
Equine herpesvirus-2 69.4 70.7 69.0 69.8 69.8 67.8 56.3 56.3
Beaked whale K285 100.0 90.9 91.4 92.2 92.2 86.4 45.1 47.9
Bottlenose dolphin K264 90.9 100.0 93.0 93.8 93.8 84.8 48.6 51.4
Bottlenose dolphin K263 92.2 93.8 99.2 100.0 100.0 87.7 50.0 52.8
Bottlenose dolphin K311 92.2 93.8 99.2 100.0 100.0 87.7 50.0 52.8
Bottlenose dolphin K310 91.4 93.0 100.0 99.2 99.2 86.8 50.0 52.8
Dwarf sperm whale K265 86.4 84.8 86.8 87.7 87.7 100.0 48.6 50.7
Dolphin alphaherpesvirus SC95 36.5 42.9 41.3 41.3 41.3 38.1 71.4 96.8
Dolphin alphaherpesvirus DE99 34.5 41.1 36.2 36.2 36.2 43.4 65.0 61.7
Bottlenose dolphin K167 45.1 48.6 50.0 50.0 50.0 48.6 100.0 77.2
Bottlenose dolphin K231 47.9 51.4 52.8 52.8 52.8 50.7 77.2 100.0
Human herpesvirus-1 50.4 51.9 51.3 51.7 51.7 50.6 72.0 68.0
Human herpesvirus-2 50.4 51.3 52.9 53.4 53.4 50.0 70.7 66.7
Green turtle FP herpesvirus 49.8 51.7 50.8 51.3 51.3 50.4 64.5 60.5
Bovine herpesvirus-1 50.4 50.6 53.8 54.2 54.2 52.5 68.4 67.1
Suid herpesvirus-1 50.4 51.7 51.7 52.1 52.1 52.1 73.6 69.4
Marek's disease virus 47.7 46.5 45.6 46.1 46.1 47.9 60.0 57.3
Canine herpesvirus 51.0 50.0 49.0 49.4 49.4 51.5 63.2 57.9
Phocine herpesvirus-1 48.8 49.7 48.5 49.1 49.1 49.7 65.3 61.3
Feline herpesvirus-1 51.7 50.8 50.8 51.3 51.3 52.1 68.4 64.5
Equine herpesvirus-1 51.0 52.3 50.6 51.0 51.0 50.8 76.0 70.7
Human herpesvirus-5 48.5 47.7 47.7 48.5 48.5 48.1 50.0 48.1
Murid herpesvirus-4 45.2 46.0 44.4 45.2 45.2 44.8 53.3 46.7
Porcine cytomegalovirus 46.7 46.9 45.2 46.1 46.1 46.9 51.4 45.9
Elephant herpesvirus-1 52.7 51.3 51.9 52.7 52.7 50.4 47.9 44.0
Koi herpesvirus 29.0 30.0 35.3 34.8 34.8 36.4 ND ND
Channel catfish virus 27.2 25.3 23.3 23.7 23.7 24.9 20.8 21.1
Salmonid herDesvirus-1 27.8 25.9 25.9 25.9 25.9 30.8 ND ND














Table 1-3. Percent amino acid similarities of DNA polymerase gene fragments among members of the family Herpesviridae. GenBank
accession numbers for the virus sequences used are the same as in Figure 1-12.
BBW K285 BND K264 BND K310 BND K311 BND K263 DSW K265 BND K167 BND K231
Human herpesvirus-8 78.9 77.7 78.9 78.9 78.9 79.3 59.2 60.9
Human herpesvirus-4 70.4 71.2 71.2 71.2 71.2 69.1 59.5 55.4
CA sea lion gammherpesvirus 70.6 70.4 69.9 69.9 69.9 69.3 50.8 54.0
Bovine herpesvirus-4 78.0 77.6 77.6 77.6 77.6 78.8 64.8 60.6
Ovine herpesvirus-2 75.5 73.4 74.3 74.3 74.3 74.7 50.7 57.3
Porcine lymphotropic herpesvirus-1 75.7 75.7 75.7 75.7 75.7 78.8 51.5 52.9
Equine herpesvirus-2 77.7 77.3 77.3 77.3 77.3 77.3 62.0 60.6
Beaked whale K285 100.0 93.4 94.2 94.2 94.2 90.1 57.7 56.3
Bottlenose dolphin K264 93.4 100.0 96.3 96.3 96.3 88.1 54.2 56.9
Bottlenose dolphin K263 94.2 96.3 100.0 100.0 100.0 90.5 55.6 59.7
Bottlenose dolphin K311 94.2 96.3 100.0 100.0 100.0 90.5 55.6 59.7
Bottlenose dolphin K310 94.2 96.3 100.0 100.0 100.0 90.5 55.6 59.7
Dwarf sperm whale K265 90.1 88.1 90.5 90.5 90.5 100.0 55.6 57.7
Dolphin alphaherpesvirus SC95 44.4 47.6 47.6 47.6 47.6 47.6 71.4 96.8
Dolphin alphaherpesvirus DE99 48.3 51.8 46.6 46.6 46.6 56.6 68.3 65.0
Bottlenose dolphin K167 57.7 54.2 55.6 55.6 55.6 55.6 100.0 77.2
Bottlenose dolphin K231 56.3 56.9 59.7 59.7 59.7 57.7 77.2 100.0
Human herpesvirus-1 61.3 60.7 60.4 60.8 60.8 60.7 78.7 74.7
Human herpesvirus-2 61.8 60.0 61.8 62.2 62.2 59.2 77.3 73.3
Green turtle FP herpesvirus 58.5 60.0 59.6 60.0 60.0 59.6 73.7 69.7
Bovine herpesvirus-1 60.0 58.9 61.3 61.2 61.2 60.0 73.7 72.4
Suid herpesvirus-1 61.0 61.0 61.0 61.4 61.4 61.9 77.8 73.6
Marek's disease virus 57.7 56.0 55.6 56.0 56.0 57.9 68.0 66.7
Canine herpesvirus 59.8 58.8 58.1 58.5 58.5 60.3 71.1 64.5
Phocine herpesvirus-1 56.5 58.1 56.9 56.9 56.9 58.8 68.0 65.3
Feline herpesvirus-1 60.0 57.9 57.5 57.9 57.9 58.8 72.4 68.4
Equine herpesvirus-1 61.1 60.3 59.8 60.2 60.2 60.4 77.3 74.7
Human herpesvirus-5 60.3 59.0 59.8 59.8 59.8 59.4 58.8 55.7
Murid herpesvirus-4 55.2 55.2 55.2 55.2 55.2 54.0 64.0 57.3
Porcine cytomegalovirus 56.7 56.0 55.6 55.6 55.6 56.4 59.5 54.1
Elephant herpesvirus-1 62.3 59.2 61.5 61.5 61.5 60.0 60.3 52.0
Koi herpesvirus 38.4 40.0 45.6 44.2 44.2 47.5 ND ND
Channel catfish virus 37.1 36.9 34.7 34.7 34.7 35.5 30.6 26.3
Salmonid herpesvirus-1 31.5 29.6 29.6 29.6 29.6 38.5 ND ND












Figure 1-12. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the herpesvirus DNA polymerase gene. The
tree was generated by Clustal X slow and accurate function using Gonnet 250 residue weight table, gap penalty of 11, gap
length penalty of 0.20, and 1000 bootstrap replications. The bar indicates a 0.1 divergence scale. The GenBank accession
numbers of the DNA polymerase gene fragments used for the construction of this tree are: AF196646-Dolphin
alphaherpesvirus SC95, AF245443-Dolphin alphaherpesvirus DE99, AF181249-Bovine herpesvirus-2, AB070848-Human
herpesvirus-1, AY038367-Human herpesvirus-2, AF299109-Olive ridley turtle FP herpesvirus, AY646894-Kemp's ridley
turtle FP herpesvirus, AF239684-Green turtle FP herpesvirus, AY646888-Loggerhead turtle FP herpesvirus, NC_001847-
Bovine herpesvirus-1, NC_006151-Suid herpesvirus-1, AF147806-Marek's disease virus, AY464052-Equine herpesvirus-
1, AF030027-Equine herpesvirus-4, NC_001348-Human herpesvirus-3, U92269-Phocid herpesvirus-1, AJ224971-Feline
herpesvirus-1, NC_006273-Human herpesvirus-5, AY186194-Rhesus cytomegalovirus, AF268040-Porcine
cytomegalovirus, AY529146-Murid herpesvirus-4, NC_000898-Human herpesvirus-6, AF005477-Human herpesvirus-8,
AF250886-Gorilla rhadinovirus-1, AF290600-Gorilla lymphocryptovirus-1, AJ507799-Human herpesvirus-4, AF159033-
Macaca mulatta gammavirus, AF029302-Rhesus monkey rhadinovirus, AY270026-Baboon gammaherpesvirus,
AF236050-CA sea lion gammaherpesvirus, NC_002665-Bovine herpesvirus-4, AF287948-Black rhinoceros herpesvirus,
AF083424-Ateline herpesvirus-3, NC_001350-Saimiriine herpesvirus-2, AF376034-Badger herpesvirus, NC_001650-
Equine herpesvirus-2, AF327831-Ovine herpesvirus-2, NC_002531-Alcelaphine herpesvirus-1, AF118399-Porcine
lymphotropic herpesvirus-1, AF 118401-Porcine lymphotropic herpesvirus-2, AF327830-Bovine lymphotropic herpesvirus,
AF322977-Elephant herpesvirus-1, AB047545-Tortoise herpesvirus, AY572853-Koi herpesvirus, NC_001493-Channel
catfish virus, AF023673-Salmonid herpesvirus-1, AY949827-Canine herpesvirus. BND- bottlenose dolphin, BBW-
Blainville's beaked whale, DWS- dwarf sperm whale, K311- penile lesion, K263- vaginal lesion, K310- penile lesion,
K264- penile lesion, K265- vaginal lesion, K285- penile lesion, K167- skin lesion, K231- skin lesion.
















Humanherpesvirus5
Rhesuscytomegaloviru s
Muridherpesvirus4
M u-id-rpe- Pa rcinecytomegalovirus
Humanherpesvirus6
Elephantherpesvirusl
CAsealio ngamma herpesvirus
HumanherpesvirusS
Gorillarhadin ovirusl
Baboongammaherpesvirus
SMacacamulattagammavirus
Rhesusmo n keyrh adin o virus
Badgerherpesvirus
Equineherpesvirus2
B ovineherpesvirus4
Blackrhino cerosherpesvirus
Atelineherpesvirus3
Saimiriineherpesvirus2
D SWK265
BBWK2B 5
BNDK264
BNDK310
BNDK2_63
BNDK311
Bavinelymphotropic
Ovineherpesvirus2
Alcelaphineherpesvirusl
Porcinelymphotropicl
Porcinelymphotropic2
Gorillalymphocryptovirusl
Humanherpesvirus4
To rtoiseherpesvirus
OliveridleyturtleFPherpesvirus
GreenturtleFPherpesvirus
Kempsridleytu rtleFPherpesvirus
Log gerheadtu rtleFPh erpesviru s
B 1 DolphinalphaherpesvirusDE.99
BNDK167
_I- DolphinalphaherpesvirusSC95
BNDK231
B ovineherpesvirusl
Suidherpesvirusl
Equineherpesvirusl
Equineherpesvirus4
Felineherpesvirusl
Phocineherpesvirus1l
Canineherpesvirus
B ovineh erpesvirus2
Humanherpesvirusl
Hu man herpesvirus2
M a reksd iseasevirus
Human herpesvirus3
Sa Imon id herpesvirusl
Koiherpesvirus
0.1 Channelcatfishvirus












BNDK310
BNDK311 BNDK263



100.0
BNDK264

96.0
BBWK285
82.0


100.0 9
DSWK265



BNDK167
BNDK231
A


BNDK263

BNDK310

BNDK311

BNDK264

BBWK285

DSWK265

BNDK231

B 0.1 Divergence BNDK167


Figure 1-13. Neighbor-Joining phylogenetic trees of the nucleotide sequences of the
DNA polymerase gene of cetacean herpesviruses. The tree was generated by
Clustal X slow and accurate function using Gonnet 250 residue weight table,
gap penalty of 15 and gap length penalty of 6.66. BND- bottlenose dolphin,
BBW- Blainville's beaked whale, DWS- dwarf sperm whale, K311- penile
lesion, K263- vaginal lesion, K310- penile lesion, K264- penile lesion, K265-
vaginal lesion, K285- penile lesion, K167- skin lesion, K231- skin lesion. A)
Radial tree where the numbers represent the percent confidence of 100
bootstrap replications B) Phylogram with 0.1 divergence scale.










BNDK311
BNDK263


BNDK310
90.0




100.0
99.0 BBNDK264

100.0 / BBWK285

BNDK167



A BNDK231 DSWK265





BNDK263

BNDK311

BNDK310

BNDK264

BBWK285

DSWK265

BNDK231

0.1 Divergence BNDK167
B

Figure 1-14. Neighbor-Joining phylogenetic trees of the deduced amino acid sequences of
the DNA polymerase gene of cetacean herpesviruses. The tree was generated
by Clustal X slow and accurate function using Gonnet 250 residue weight
table, gap penalty of 11 and gap length penalty of 0.20. BND- bottlenose
dolphin, BBW- Blainville's beaked whale, DWS- dwarf sperm whale, K311-
penile lesion, K263- vaginal lesion, K310- penile lesion, K264- penile lesion,
K265- vaginal lesion, K285- penile lesion, K167- skin lesion, K231- skin
lesion. A) Radial tree where the numbers represent the percent confidence of
100 bootstrap replications B) Phylogram with 0.1 divergence scale.









PCR Targeting the DNA Terminase Gene

Negative results were obtained for the PCR targeting the terminase gene for five of

the six samples tested, namely K167, K263, K265, K285 and K311. Only skin lesion

sample K231 yielded a positive result. Upon sequencing, it was demonstrated that the

sequence of K231 corresponded to an alphaherpesvirus that differed from the sequence

obtained from a bottlenose dolphin from South Carolina that had died with disseminated

herpesvirus infection (GenBank accession number AF196647) by two nucleotides at

positions 208 and 294 (Figure 1-15). The deduced amino acid sequences of both

terminase gene fragments were identical (Figure 1-16). The DNA terminase gene

fragment obtained from a skin lesion of bottlenose dolphin K231 was deposited in the

GenBank database under accession number AY949829.





































Figure 1-15. Comparison of the nucleotide sequences of the 375-bp partial DNA terminase gene fragment of an alphaherpesvirus
amplified from a skin lesion of an Atlantic bottlenose dolphins (K231)- skin lesion, AF196647- lung, disseminated
infection (Blanchard et al., 2001).



1 50 100

A F1 96647 .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........
Consensus TNTGKASTSF LFNLKYSSDD LLNVVTYICD EHMDRVRVHT NATACSCYVL NKPVFITMDA SMRNTAEMFL PNSFMQEIIG GGSADPAAGG DGPVFTKAAA

101 125
K231 .......... .......... .....
AF196647 .......... .......... .....
Consensus DQFLLYRPST TTRRGAMAEE LFVYV



Figure 1-16. Comparison of the deduced amino acid sequences of the partial DNA terminase gene fragment from two Atlantic
bottlenose dolphins. K231- skin lesion, AF196647- lung, disseminated infection (Blanchard et al., 2001).









Discussion

Applying PCR technology, we have been able to demonstrate the existence of

both alpha and gamma herpesviruses in several species of cetaceans in which they cause,

respectively, local or generalized cutaneous lesions, and localized genital and oral

lesions. It is not known at this point whether the herpesvirus DNA present in these

lesions corresponds to latent or to actively replicating virus, as in the few instances in

which virus isolation was attempted, the results were unsuccessful. When accessible,

central nervous and lymphoid tissues were also tested in the form of tissue homogenates

after a positive PCR result was obtained from a lesion; however, herpesvirus genomic

DNA was not detected in any tissue tested, most likely indicating virus absence.

Mesenteric, pulmonary and prescapular lymph node samples were tested from dwarf

sperm whale K265 in addition to the genital lesion. A localized gammaherpesvirus

infection was demonstrated in the lesion but not in the lymph nodes.

Gammaherpesviruses are known to become latent in lymph tissue, thus, this infection was

most likely active. Certain gammaherpesviruses, such as human herpesvirus-8 (HHV-8),

which causes Kaposi's sarcoma in AIDS patients, and OtHV-1, have the potential for

oncogenic transformation of host cells, but no evidence of this was seen in any of the

cetacean cases presented in this study.

All cetacean tissue samples tested by nested PCR were negative for herpesvirus

genomic DNA. Although no systemic infection was found during this study, Blanchard

et al. (2001) recently used PCR to demonstrate disseminated fatal herpesvirus infections

in two Atlantic bottlenose dolphins. These viruses were unknown previously, thus, it was

speculated that the natural host of such viruses could be a species outside the order

Cetacea, and the cases described could represent atypical infections in an aberrant host.









The herpesviruses found in the cetaceans of our study, as well as those found in the

bottlenose dolphins described by Blanchard et al. (2001), appeared to be distinct from

known herpesviruses of other marine animals, such as pinnipeds and marine turtles.

Sequence homologies and phylogenetic analyses demonstrated that the cetacean

herpesviruses identified during this study were more closely related to other cetacean

herpesviruses of the respective subfamily than to other known herpesviruses. Given the

lower sequence homologies of cetacean herpesviruses with other mammalian

herpesviruses, it seems unlikely that these viruses arose from a known virus that spread

into a new cetacean host. Our study supports the hypothesis that the herpesviruses

identified in various cetaceans are specific for the cetaceans examined and, most likely,

coevolved with their hosts.

The deduced amino acid sequences of the small DNA polymerase fragment of

alphaherpesviruses from skin lesions from two Atlantic bottlenose dolphins (K231 and

K167) shared 77.2% identity, while the two alphaherpesviruses identified by Blanchard

et al. (2001) shared 65.0%. These findings suggest that there may be several species of

cetacean alphaherpesvirus. As with other herpesviruses, such as those associated with FP

of marine turtles, the cetacean herpesviruses could vary based on species and/or

geographical location, but extensive epidemiological studies are needed to make any

definitive conclusions. Our findings, nevertheless, have shown that a stranded bottlenose

dolphin from South Carolina (GenBank accession number AF 196646) and a captive

bottlenose dolphin in Florida (K231) were infected with the same virus. As further

support, bottlenose dolphin K231 yielded the only positive PCR result for the terminase

gene, similar to the bottlenose dolphin from South Carolina. If the terminase gene of









cetacean alphaherpesvimses maintains as much conservancy/variation as the DNA

polymerase gene, it is not surprising that the primers used did not amplify sequences

from any other sample. From these findings it can be concluded that the same

alphaherpesvirus can cause localized skin lesions as well as deadly disseminated

infections.

The six cetacean gammaherpesviruses identified in this study showed variations

in deduced amino acid sequences of DNA polymerase gene fragments of 84.8 to 100%,

which is much less than the sequence variation observed for the cetacean

alphaherpesviruses (> 61.7%). The high degree of sequence homologies between species

indicates that cetacean gammaherpesviruses may not be species specific, but rather

family or order specific. For example, the four bottlenose dolphin sequences were more

similar to each other than to the two whale sequences. The amino acid identities of the

DNA polymerase gene fragment of the gammaherpesvirus from bottlenose dolphin K263

when compared with the homologous fragments from the other three bottlenose dolphins

ranged from 93.8 to 100%, while the identities to beaked whale K285 and dwarf sperm

whale K265 were 92.2 and 87.7%, respectively. This suggests that cetacean

gammaherpesvirus variants could be correlated with the species they infect. On the other

hand, bottlenose dolphins K263, K310 and K311 inhabited the same general area in the

Florida Keys, thus suggesting that geographic variants are also possible.

Recent guidelines for differentiating herpesvirus species include separation based

on serologic and restriction endonuclease cleavage methods, as well as the occupation of

different ecological niches (Roizmann et al., 1992). The cetacean herpesviruses

identified in this study appear to occupy similar niches throughout both alpha- and









gammaherpesvimses. This observation along with the relatively comparable sequence

homologies suggests that there are two subfamilies of cetacean herpesviruses; cetacean

alphaherpesviruses and gammaherpesvirus, both with several strains encompassing either

different viral strains or species.

Based on phylogenetic analysis and multiple sequence homologies, it was

determined that cetacean herpesviruses were most closely related to bovine herpesvirus-

1, suid herpesvirus-1 and equine herpesvirus-1 and -4 (genus Varicellovirus) in the case

of alphaherpesviruses, and to saimiriine herpesvirus-2, ateline herpesvirus-3 and bovine

herpesvirus-4 (genus Rhadinovirus) in the case of gammaherpesvimses. Similar analysis

also revealed that cetacean herpesviruses are least similar to the fish herpesviruses KHV,

SalHV-1 and CCV, with the majority of amino acid identities below 30%, thus

supporting the theory of the existence of three distinct herpesvirus lineages

(mammalian/avian, fish/amphibian and invertebrate).

The cetacean-specific primers that were designed based on the small DNA

polymerase gene fragment of both cetacean alphaherpesviruses and gammaherpesviruses

were, essentially unsuccessful in PCR amplification, most likely due to the high

variability among the target sequences. Furthermore, cetacean-specific primers designed

from the large DNA polymerase fragment sequences of the gammaherpesviruses did not

give consistent results, most likely due to the viral gene diversity among the species

tested. As expected, these primers did not drive the amplification of the long fragment of

the cetacean alphaherpesviruses. Cetacean-specific primers are essential not only to

increase the specificity and sensitivity of the assay, but also to reduce the risk of error.

The nested PCR, which has two rounds of reactions, is cumbersome and time consuming,









and can lead to a greater possibility for mistakes and cross-contamination. There is an

urgent need for the availability of more cetacean herpesvirus sequences to design better

cetacean-specific herpesvirus primers.

Herpesviruses are generally transmitted via direct contact of mucosal surfaces,

and the gregarious nature of many cetacean species would favor this type of transmission.

The cetacean alphaherpesviruses detected in skin lesions were probably transmitted

through skin abrasions, which are similar to alphaherpesvirus infections of terrestrial

mammals including humans, namely BHV-2, which causes mammillitis, and HHV-1,

which produces both facial and oral lesions. Two captive dolphins from the same park,

one male (K311) and one female (K263), were both diagnosed with genital infections

from identical gammaherpesviruses (100% amino acid identity). Sexual activity between

these two animals was apparent as they have produced offspring, which strongly

indicates that the cetacean gammaherpesviruses are sexually transmitted. This mode of

transmission is unique, as very few gammaherpesvirus infections are transmitted through

genital contact. One example is the Kaposi's sarcoma-associated herpesvirus (HHV-8) in

humans, and perhaps OtHV-1, given that it has a genital origin. In one case, identical

gammaherpesvirus sequences were obtained from both a genital (K310) and oral (K308)

lesion of a bottlenose dolphin. It is interesting that oral lesions were also present in this

animal, as no other gammaherpesviruses have been shown to cause lesions of the oral

mucosas.

Although the possible modes of transmission for herpesviruses among cetaceans

are still largely unknown, vertical transmission and the incorporation of unidentified

vectors cannot be ruled out. A recent study suggested that PhHV-1 may be vertically









transmitted, as adult female harbor seals were found to be shedding virus in vaginal

secretions and premature newborn pups showed evidence of early infection (Goldstein et

al., 2004). Tentative candidate vectors for the spread of FP among marine turtles have

been identified recently. Greenblatt et al. (2004) performed quantitative PCR on an

assortment of parasites of Hawaiian green turtles and found that marine leeches

(Ozobranchus spp.) carried fibropapilloma-associated herpesviral loads sufficient to

cause infection. There is evidence that leeches may serve as mechanical vectors for other

pathogenic agents, including hepatitis B virus in humans, several parasites of fish and a

blood protozoan of turtles. PCR has also been used to detect FPHV sequences in the

snout, gill and liver of a reef cleaner fish, the saddleback wrasse (Thalassoma duperrey)

(Lu and Yu, 2000). Toxic benthic dinoflagellates (Prorocentrum spp.) have been

suggested as yet another cofactor, since they have a worldwide distribution and are

epiphytic on seagrasses that are normally consumed by green turtles. These

dinoflagellates are known to produce a tumor promoter, okadaic acid (OA), which has

been detected also in tissues of Hawaiian green turtles, suggesting a potential role of OA

in the etiology ofFP (Landsberg et al., 1999).

Herpesviruses, by virtue of being enveloped viruses, are generally unstable

outside the host, however, research has shown that LETD-associated herpesvirus of

marine turtles can remain infectious as long as 120 hours in natural and artificial sea

water at 230 C (Curry et al., 2000). It has been suggested that both FPHV and GPD-

associated herpesvirus may also be able to survive for extended periods of time under

harsh environmental conditions. The possibility remains that all marine herpesviruses,

including those of cetaceans, may also be transmitted directly through sea water.









Although the prevalence of cetacean herpesvirus is not well documented, it is

possible that false negative diagnoses may have been made in the past, thus, overlooking

potential herpesvirus infections. The characteristic papillomatous lesions of marine

turtles with FP and the urogenital carcinoma of California sea lions were both originally

thought to be caused by papillomaviruses. However, negative results for papillomavirus

using Southern blot hybridization (Jacobson et al., 1989) and PCR (Lipscomb et al.,

2000) have detracted from these theories. Other likely misdiagnoses could include carp

pox, a herpesvirus that infects farmed carp, which is also associated with papillomatous

lesions (Buchanan and Richards, 1982) and cetacean genital lesions, which can be

papilloma- and wart-like in appearance. The mild dermatitis lesions of bottlenose

dolphin K167 was initially thought to resemble early poxvirus infection. Additional

herpesviruses, cetacean or otherwise, may remain undiscovered due to a misdiagnosis of

the etiological agent.

Treatment for herpesvirus infections generally includes antiviral drugs, commonly

acyclovir, ganciclovir and cidofovir, which block herpesvirus replication. Bottlenose

dolphin K311 was treated with acyclovir for penile lesions without any improvement.

Although acyclovir is often used to treat genital herpes in humans, the cause is an

alphaherpesvirus, whereas gammaherpesviruses seem to be the cause of genital lesions in

cetaceans. The difference in host species could also be a factor on how effective the

treatment will be. It is unknown how the treatment in this case was administered, and no

other accounts of treatment for herpesvirus infections in cetaceans have been reported.

Mammalian herpesvirus disease can often be linked with environmental stress;

therefore, the correlation between skin disorders and various natural and anthropogenic









factors is now being investigated for cetaceans. Environmental contaminants are

commonly believed to influence the development of disease in wild populations. Marine

mammals, as long-lived top level predators, can accumulate high concentrations of

chemicals in their tissues, which can then be passed to offspring through the milk. An

extensive study was performed by Wilson et al. (1999) in which bottlenose dolphin

populations were monitored in ten areas spanning the globe. Skin lesion prevalence and

severity was determined using photograph-identification of the dorsal fin and correlations

were examined between multiple factors. Epidermal lesions were found in all ten

populations in frequencies of 63 to 100%. High prevalence of epidermal disease was

found to be related to low water temperature and low salinity. It was suggested that low

salinity causes cellular damage to the epidermis by disrupting the electrolyte balance,

thus, weakening the ability to shield the animal from infectious agents. Likewise, low

water temperature may limit blood flow to the skin and impede pathways for immune

protection. Surprisingly, no significant correlations were found between prevalence or

severity of lesions and contaminants, namely organochlorine compounds and trace

metals; however, only four populations were included as the other six lacked baseline

toxicological data.

A similar study was performed in the Sado estuary, Portugal, where skin disorders

of bottlenose dolphins were monitored over two time periods. Harzen and Brunnick

(1997) concluded that 85% of the long term residents had skin disorders after the second

study period, and most of the disorders appeared continuous and nonfatal. Lesion

samples were not taken; therefore, the etiological agents involved could not be identified.

However, skin disorders that matched the description of pox and herpes virus infections









by Baker (1992) were noted. Periods of bloom were also reported, in which the skin

lesions were more distinct. These bloom periods could begin quickly and then enter into

remission, but the reasons behind this remained unclear. One important factor that needs

further research is that of habitat degradation. Areas where habitats are being destroyed,

such as the Sado estuary, often experience excessive nutrient enrichment or

euthrophication that can harm plant growth, which in turn may lead to the damage of the

entire food web. Contaminant-induced immune suppression can also occur. Anecdotal

reports suggested that FP of marine turtles is most prevalent in near-shore habitats, such

as estuaries and lagoons that have been negatively impacted by human occupation

(Herbst and Klein, 1995). Human activity has also directly added to the concentration of

pathogens in the oceans, primarily though sewage discharge and storm water run-off In

Scotland, an alarming number of pathogens were identified in domestic sewage that was

discharged into coastal waters. Specifically, viruses, including herpes, were found in

concentrations of 10,000 to 10,000,000 per liter of raw sewage (Grillo et al., 2001).

Natural environmental fluctuations may also play a role in herpesvirus

prevalence. Under artificial culture conditions, stress, crowding and in some cases,

increased temperature have been shown to enhance the risk of herpesvirus outbreaks

among marine organisms, namely teleost fish, turtles, oysters and blue crabs (Buchanan

and Richards, 1982). Similarly, thermal stress has been shown to exacerbate herpesvirus

infection in hatchling green turtles (Herbst and Klein, 1995). It has been suggested that

temperature variation causes a shift in balance between virus replication and the

efficiency of host defense mechanisms.






49


Much more research is needed on cetacean herpesvirus infections including; mode

of transmission, species susceptibility, host defense mechanisms and identification of

cofactors that aid in the spread of these viruses. This information along with the

phylogenetic comparisons made in the present study should lead to a better understanding

of the epidemiology of cetacean herpesviruses and may result in practical methods of

control and prevention in cetaceans in captivity, those undergoing rehabilitation and

possibly, in the not too distant future, even those of wild populations.














CHAPTER 2
PORPOISE MORBILLIVIRUS

Introduction

Morbilliviruses are members of the family Paramyxoviridae; a family whose

members have caused more morbidity and mortality than any other group of diseases in

history (Murphy et al., 1999). All paramyxoviruses have genomes consisting of a single

molecule of negative-sense, single-stranded RNA that is 15 to 16 Kbp (Kilo base pairs) in

length. The pleomorphic (spherical or filamentous) virions are enveloped, covered with

large peplomers and contain a "herringbone-shaped" helically symmetrical nucleocapsid.

Glycoprotein peplomers aid in cell attachment and pathogenesis of paramyxovirus

infection. The two glycoproteins of morbilliviruses are the fusion (F) and the

hemagglutinin (H) proteins, which, unlike that of other paramyxoviruses, lack

neuraminidase activity. Morbilliviruses encode only six genes, which are located in the

genome in the following order: 3' N-P-M-F-H-L 5' (Griffin, 2001). The phosphoprotein

(P) gene is spliced into three separate proteins, namely, P, V and C. Replication is

entirely cytoplasmic and newly synthesized negative-sense RNA associates with

nucleocapsid (N) protein and transcriptase to form nucleocapsids. As the virions mature,

viral glycoproteins are incorporated into the host cell plasma membrane allowing the

matrix (M) protein to associate with the host cell membrane. Mature virions are released

by budding and thus, obtain their envelope from the host cell plasma membrane.

Characteristics of morbillivirus infection include the production of acidophilic

intracytoplasmic and intranuclear inclusions as well as the formation of syncytia. In









general, morbillivirus infection presents itself mainly as a respiratory disease of

vertebrates, and most viruses have a relatively narrow host range (Murphy et al., 1999).

Aerosol transmission is probably the most important route of infection for

morbilliviruses, but indirect contact cannot be ruled out.

Up until 1988, only four morbilliviruses were known, all of which infected

terrestrial mammals, namely, human measles virus (MV), bovine rinderpest virus (RPV),

peste-des-petits ruminant virus of goats and sheep (PPRV) and canine distemper virus

(CDV). In the past 15 years, various epizootics in marine mammals have demonstrated

the existence of three new morbilliviruses that have become known as phocine distemper

virus (PDV), porpoise morbillivirus (PMV) and dolphin morbillivirus (DMV). (For

review see Kennedy, 1998 and Duignan, 1999.) Between 1987 and 1988, it was

estimated that more than half of the Atlantic bottlenose dolphin (Tursiops truncatus)

population along the Atlantic coast of the U.S. died (Lipscomb et al., 1994). Initially

thought to be caused by red tide toxins, retrospective studies showed that dolphins were

infected with both PMV and DMV. At approximately the same time, a CDV strain,

which is believed to have originated in terrestrial carnivores, killed thousands of Baikal

seals (Phoca siberica) in Siberia (Barrett et al., 1992). In the spring of 1988, over 18,000

harbor seals (Phoca vitulina) died in northwestern Europe (Mahy et al., 1988). Pup

abortion was widespread and adult seals presented with CDV-like lesions, however, upon

virus isolation it was determined that a new morbillivirus, PDV, was the cause of the

mass-mortality. Grey seals (Halichoerus grypus) were also infected, but with relatively

low mortality. Between 1990 and 1992, thousands of Mediterranean striped dolphins

(Stenella coeruleoalba) died from a DMV outbreak that began in Spain and spread









eastward to Greece and Turkey (Domingo et al., 1992). CDV was implicated as the

cause of thousands of Caspian seal (Phoca caspica) deaths in the summer of 2000 in

Kazakhstan, Azerbaijan and Turkmenistan (Kennedy et al., 2000). The origin of the

virus was unknown, but there were local anecdotal reports of contact between Caspian

seals and terrestrial carnivores. The last major documented epizootic occurred in

European harbor seals infected with PDV in 2002 (Harding et al., 2002). The death toll

was comparable to that of the PDV outbreak in 1988, and sequence analysis of P gene

fragments from seal tissues from 1988 and 2002 showed greater than 97% identity

(Jensen et al., 2002). Smaller scale die-offs from PMV have also been reported in harbor

porpoises (Phocoenaphocoena) in Ireland (Visser et al., 1993) and Atlantic bottlenose

dolphins in the Gulf of Mexico (Lipscomb et al., 1996b). Serological studies have shown

that numerous cetacean and pinniped species from every major ocean possess

morbillivirus-neutralizing antibodies. Antibodies against CDV were retrospectively

detected in Crabeater seals (Lobodon carcinophagus) from Antarctica, suggesting that a

morbillivirus epizootic may have occurred in 1955 (Bengtson et al., 1991). The list of

seropositive species also includes walruses (Odobenus rosmarus rosmarus) from

northwest Canada (Duignan et al., 1994), polar bears (Ursus maritimus) from Alaska and

Russia (Follmann et al., 1996), and Florida manatees (Trichechus manatus latirostris)

(Duignan et al., 1995b).

Clinical signs and lesions of infected marine mammals are similar to those of CDV

and are mainly seen in the lung, central nervous system and lymphoid tissues. Affected

tissues often present with acidophilic intranuclear and intracytoplasmic inclusion bodies,

and syncytia are commonly found in cetaceans, but not in pinnipeds. Signs commonly









include bronchointerstitial pneumonia, respiratory distress, encephalitis, neurological

disturbances, ulcerations of the skin and buccal mucosa, and abortion. Morbilliviruses

induce immunosuppression; therefore, infected animals are much more susceptible to

secondary infections by bacteria and opportunistic parasites. These same signs are seen

in pinnipeds as well as cetaceans, but to a lesser extent. The social behavior of many

marine mammals probably favors horizontal transmission. Anti-CDV maternally-derived

antibodies have been reported in immature grey seals (Carter et al., 1992). Possible

reservoirs for marine mammal morbilliviruses include harp seals (Phoca groenlandica)

and pilot whales (Globicephala species). Both species are gregarious, migratory and

numerous, which would facilitate lateral transmission of these viruses. For example, harp

seals from Greenland, with inapparent or subacute infections, were suspected of

introducing PDV to northwestern Europe in 1988. In the western Atlantic, it was

reported that 86% of stranded pilot whales had morbillivirus-neutralizing titers,

suggesting that this whale species is in continuous contact with the virus and that

immunity or natural resistance may have prevented a serious outbreak from occurring

(Duignan et al., 1995a). It is suspected that recent epizootics are the result of virus

transfer to immunologically-naive populations.

There is much debate over where to place these new marine mammal

morbilliviruses in the paramyxovirus phylogeny. Nucleotide sequence comparisons of

PDV and other morbilliviruses indicate that PDV is most closely related to CDV, but

should be regarded as a separate species. Cross reactivity studies of DMV and PMV with

monoclonal antibodies against known morbilliviruses revealed that the two cetacean

viruses are more closely related antigenically to RPV and PPRV of ruminants than to









distemper of carnivores (Visser et al., 1993). Researchers are still undecided as to

whether PMV and DMV are separate viral species, or simply different strains of a

common cetacean morbillivirus (CMV). Previous attempts to establish the relatedness of

DMV and PMV have concentrated on sequence homologies of various genes.

Polymerase chain reaction (PCR) utilizing universal morbillivirus P gene primers

generated fragments 429-bp in length (Barrett et al., 1993). The amount of nucleotide

variation between the P gene fragments of PMV and DMV was approximately 10%,

which is similar to that observed between geographically distinct isolates of RPV. In a

similar evaluation, the complete F proteins of PMV and DMV were compared and found

to be 94% identical (Bolt et al., 1994). The results of these two studies indicate a close

sequence relationship between DMV and PMV suggesting that they should be considered

two variants of CMV. Recently, van de Bildt et al. (2004) sequenced the hypervariable

carboxyterminal (C-terminal) end of the N gene, and the complete H protein of both

PMV and DMV. The divergence between the N gene C-terminal sequences of the two

cetacean morbilliviruses was 18.3%. This was greater than 12.4% divergence between

the two most distantly related strains of MV. The sequence divergence of the complete H

gene gave similar results. PMV and DMV showed 15.5% divergence, while the MV

strains only demonstrated 6.7%; thus, it was suggested that PMV and DMV be

considered two different species of morbillivirus. Based on results of Western blot

analysis of the N protein and other differences in biological properties, Visser et al.

(1993) concurred with the theory of two separate cetacean morbillivirus species.

Areas of interest of the morbillivirus genome with relevance to this study include

the N protein and the genome (leader) and antigenome (trailer) promoter regions. All









non-segmented, negative strand RNA viruses produce a nucleocapsid protein that serves

to encapsidate viral RNA, thus, protecting it from cellular proteases as well as being

involved in allowing the viral polymerase access to the RNA for transcription and

replication. Multiple alignments of the N genes ofMV, RPV, PPRV, CDV and PDV

defined four regions within the N gene with varying degrees of homology (Diallo, et al.,

1994). Region I (amino acid residues 1-222) was quite well conserved with identities

between 75 and 83%, region II (residues 123-144) had homologies below 40%, and

region III (residues 145 to 420) was the most conserved with 85 to 90% homology.

Region IV (residues 421 to the end) contained the hypervariable C-terminus with less

than 30% identity. The hypervariable region of MV, which is located outside of the

nucleocapsid structure, has been shown to contain linear epitopes recognized by

monoclonal antibodies, and has been used to separate MV into several different

genotypes (Griffin, 2001). The biological significance of this area is uncertain, but this

portion of the N gene may evolve rapidly, possibly indicating the lack of function or

structural constraint. The N gene product is the most abundant viral protein and one of

the most variable in all morbilliviruses. The variation in sequence is impressive when

compared to the high degree of conservation seen in other morbillivirus genes (van de

Bildt et al., 2004). The N gene sequence dissimilarity between the two most distantly

related MV strains is -7%, and increases to 12.4% when the comparison is limited to the

456-bp of the C-terminal end. The N gene was chosen in this study, as it had been

sequenced in full for all morbilliviruses, except PMV, and for the fact that the

hypervariable 5' end may possibly be used to distinguish infections by PMV and DMV.

Recent studies with morbilliviruses, as well as a number of other negative strand RNA









viruses, have highlighted the importance of short untranslated regions at either end of the

full length single stranded genome as playing a major role in virus pathogenesis. These

regions, termed the genome and antigenome promoters, generally take up less than 3% of

the total genome length, but as promoters and regulators of both virus transcription and

replication, they have been shown to play vital roles in viral pathogenesis, although these

events are not fully understood (Banyard et al., 2003; Fujii et al., 2002).

The objectives of the present study were to amplify and sequence the 3' and 5'

extragenic regions, as well as the complete nucleocapsid gene of PMV in order to better

understand its phylogenetic relationship to DMV and other morbilliviruses. The

hypervariable C-terminal end of the N gene of the two cetacean morbilliviruses was

expressed in order to aid in the distinction of PMV from DMV. Furthermore, cetacean

tissues from recent mass stranding events were tested by PCR for the presence of

morbillivirus nucleic acid in order to help determine the cause of the mass strandings, as

well as the possible presence of morbillivirus in the southeastern U.S throughout 2004.

Materials and Methods

Analysis of PMV

Reverse transcription

Porpoise morbillivirus (Belfast strain) RNA extracted from infected Vero cell

cultures was kindly donated by Dr. Jeremiah T. Saliki from the Oklahoma State Animal

Diagnostic Laboratory. The Belfast strain of PMV was isolated from a harbor porpoise

that stranded on the coast of Northern Ireland (Kennedy et al., 1988). Complementary

DNA (cDNA) was prepared by reverse transcription (RT) reactions using gene specific

primers based on the sequence of the closely related DMV (GenBank accession number

NC_005283). Briefly, 13.5 ptl of RNA and 2.7 [iM of primer were heated at 700C for 10









min and snapped-cooled on ice. The final reactions contained 50 mM Tris-HCl at pH

8.3, 75 mM KC1, 3 mM MgCl2, 10 mM DTT, 333 iM dNTP and 40 units RNase out.

Reactions were incubated at 250C for 10 min followed by 2 min at 420C. Three hundred

units of Superscript II (Invitrogen, Carlsbad, California, USA) were added, and the

reactions were incubated at 420C for 1 hr and inactivated at 700C for 15 min. Specific

primers 8A and P2 were used to obtain cDNA for PCR targeting the complete N gene.

Primers N2 and L were used to obtain cDNA to be used in the RACE technique for the

amplification of the genome and antigenome promoter regions, respectively. Primer

sequences were: 8A-5'- ACC AGA CAA AGC TGG CTA GGG GT -3' (forward), P2-

5'- GTC GGG TTG CAC CAC CTG TC -3' (reverse), N2-5'- CTG AAC TTG TTC TTC

TGG ATT GAG TTC T -3' (reverse), L-5'- TCG CGT CTG GAT CAG AGG -3'

(forward).

RACE and amplification of the terminal extragenic domains

The genome and antigenome promoter regions were obtained using the RACE

(Rapid Amplification of cDNA Ends) technique as described previously by Baron and

Barrett (1995). Ten microliters of cDNA were added to 11.25 [l ultra-pure water and

0.25 M NaOH, and incubated at 500C for 30 min. Acetic acid (0.3 M) was added and the

mixture was purified using the GFX PCR DNA and Gel Band Purification Kit

(Amersham Biosciences UK Limited, Little Chalfont, Buckinghamshire, UK). The

tailing reactions contained 10 [il cDNA, 25 mM Tris-HCl at pH 8.3, 37.5 mM KC1, 1.5

mM MgCl2, 5 mM DTT, 1 jg BSA, 0.1 mM dCTP, and 19.8 units TdT (Promega UK

Ltd., Southampton, UK). The mixture was heated to 370C for 5 min followed by 65C

for 10 min. The result of the RACE technique produced run-off transcripts that were

tailed with cytosine residues. The tailed cDNAs were subsequently used in a PCR









containing 5 il of cDNA, 50 mM KC1, 10 mM Tris-HCl at pH 9.0, 0.1% Triton X-100,

1.5 mM MgC12, 100 IM of each dNTP, 2.5 units Taq (Promega UK Ltd., Southampton,

UK), 20 pM DMV specific internal primer and 25 pM of poly-Gn primer (5'- GGG GGG

GGG GAC CA -3'). The DMV specific internal reverse primers used for leader and

trailer amplification were: 5'- TAC TTC AAC TAA TCT GAT GCT -3', 5'- TCG CGT

CTG GAT CAG AGG -3', respectively. Cycling conditions were: Initial incubation at

95C for 2 min followed by 34 cycles at 950C for 45 sec, 55C for 45 sec, and 720C for 1

min 30 sec. A final extension step at 720C for 5 min finished the cycling. The RACE

generated PCR products were purified using the GFX PCR DNA and Gel Band

Purification Kit (Amersham Biosciences UK Limited, Little Chalfont, Buckinghamshire,

UK) and sequenced in their entirety on both strands using the DMV specific primers

described above. DNA sequencing was carried out using the Beckman automated

CEQ8000 (Beckman Coulter Limited, Buckinghamshire, UK) sequencer as per the

manufacturer's instructions.

Amplification of the complete PMV N gene

Porpoise morbillivirus cDNA was used in two PCRs to obtain the full N gene.

Primers 8A and N2 directed the amplification of approximately 1050-bp, while primers

P2 and N1 directed the amplification of approximately 1650-bp with an overlap of

approximately 230-bp (Figure 2-1). Primer sequences were: 8A-5'- ACC AGA CAA

AGC TGG CTA GGG GT -3' (forward), N1- 5'- ATA AAC CAA GGA TCG CTG AAA

TGA T -3' (forward), N2-5'- CTG AAC TTG TTC TTC TGG ATT GAG TTC T -3'

(reverse), P2-5'- GTC GGG TTG CAC CAC CTG TC -3' (reverse). The PCR mixture

contained 2 [l of PMV cDNA, 400 nM of each primer, 100 iM of each dNTP, 2 mM

MgC12, IX Expand High Fidelity buffer and 3.3 units of Expand High Fidelity enzyme









mix (Roche Applied Science, Indianapolis, Indiana, USA). Cycling conditions were:

Initial incubation at 940C for 2 min followed by 40 cycles at 940C for 30 sec, 450C for 30

sec, and 720C for 3 min, followed by a final extension step at 720C for 10 min.

Approximately 20 [tl of the PCR products were resolved by horizontal gel electrophoresis

in 1.0% agarose containing ethidium bromide (0.5 ug/ml) and the DNA fragments were

visualized by UV light transillumination and photographed using a gel documentation

system.

8A N1 N2 P2




Leader N gene P gene


Figure 2-1. Schematic of the primers used to sequence the full nucleocapsid gene of
PMV. Note that this figure is not drawn to scale.

Amplified DNA fragments of the predicted size were purified using the MinElute

PCR Purification Kit (Qiagen Inc., Valencia, California, USA) and cloned into the

plasmid vector pCR2.1-TOPO T/A (Invitrogen, Carlsbad, California, USA). Two clones

for each sample were selected for plasmid purification and sequenced in duplicate from

both ends using the CEQ 2000 XL (Beckman Coulter Inc., Fullerton, California, USA)

sequencing instrument following the manufacturer's protocol as previously described in

Chapter 1. Exported chromatograms were manually reviewed using the Chromas 2.3

software (Technelysium Pty Ltd., Tewantin, Queensland, Australia). Genetic analyses

and multiple sequence alignments were performed using the programs Seqed, Gap,

Bestfit, Translate, Lineup, Pileup and Pretty of the University of Wisconsin Package

Version 10.2, Genetics Computer Group (GCG), Madison, Wisconsin, USA.









Phylogenetic analyses were performed using PAUP* 4.0 (Sinauer Associates,

Sunderland, Massachusetts, USA) and the National Center for Biotechnology

Information (NCBI) website was used to obtain sequences for all other morbilliviruses.

Amplification of the C-terminus of the PMV N gene

PCR was used to amplify the hypervariable C-terminus of the N gene of both PMV

and DMV. The PCR procedure using Taq polymerase (Promega UK Ltd., Southampton,

UK) was the same as above, and the primers used were: PMVDMVForward-3'- AAC

GAA TGG ATC CGG TCA AGA GAT GGT CAG GAG A -5', PMVReverse-3'- AAC

CGT AAG CTT ATT AGC CGA GTA GGT CTT TGT CGT TAT -5', DMVReverse-3'-

TTC GCC CAA GCT TAT CAG CCA AGT AGA TCT TTA TCA TTA T -5'. The PCR

products were approximately 480-bp, which corresponded to amino acid residues 400 to

529 in the hypervariable C-terminus of the N gene of both viruses. The GFX PCR DNA

and Gel Band Purification Kit (Amersham Biosciences UK Ltd., Little Chalfont,

Buckinghamshire, UK) was used to purify multiple PCR products from each virus before

digestion with BamHI and HindIII, and ligation into the bacterial expression vector pQE-

30 (Qiagen Ltd., Crawley, West Sussex, UK). The vector-insert construct was used to

transform competent M15 E. coli cells.

Detection of N protein expression by Western blot

Positive transformants, those that contained the 6xHis tagged morbillivirus protein,

were expressed in M15 cells. Protein expression was induced by IPTG and detected by

Western blot after Tris Tricine gel electrophoresis, and protein transfer onto a

nitrocellulose membrane. Primary and secondary antibodies used were HisTag

monoclonal antibodies (EMD Biosciences, Inc., Novagen Brand, Madison, Wisconsin,

USA) and peroxidase labeled anti-mouse immunoglobulin antibodies (Amersham









Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK). ECL Plus Western

Blotting Detection Reagent (Amersham Biosciences UK Ltd., Little Chalfont,

Buckinghamshire, UK) was then used to detect the truncated proteins corresponding to

the N-gene C-terminus.

Analysis of Stranded Cetacean Tissues for Morbillivirus

Sample acquisition, RNA extraction and reverse transcription

Tissue samples were obtained from cetaceans that mass stranded on the coasts of

Florida, Georgia, North and South Carolina, and Texas throughout 2004. In total, 57

tissue samples, mostly from spleen, brain and lung, from four cetacean species were

analyzed for morbillivirus genomes. A comprehensive listing of all samples tested is

presented in Appendix B.

Total cellular RNA was extracted from all samples using Trizol (Invitrogen,

Carlsbad, California, USA). Briefly, chloroform was added to crushed tissue. After

centrifugation, the top aqueous phase was collected and RNA was precipitated with

propanol, washed with 70% ethanol, and eluted in 100 itl of RNase-free water. The total

RNA content and quality of the eluted RNA was determined using the Ultrospec 3000

spectrophotometer (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). Total

RNA was used in RT reactions as previously described; however, 1.5 tg of random

hexamer primers were used as a substitute for DMV specific primers.

PCR targeting the P gene

PCR using universal morbillivirus primers designed to amplify a region of the P

gene was used to detect genomic morbillivirus nucleic acid in tissues of recently stranded

cetaceans. These primers are known to direct the amplification of a P gene fragment 429-

bp in length (Barrett et al., 1993). Primer sequences were: 5'- ATG TTT ATG ATC









ACA GCG GT -3' (forward), 5'- ATT GGG TTG CAC CAC TTG TC -3' (reverse).

These primers had been thoroughly tested in our laboratories and shown to be very

efficient at amplifying P gene fragments of DMV, PMV, MV, PDC and CDV

(unpublished results). Total cDNA extracted from monolayers of Vero cell cultures

infected with the Edmonton strain of MV was used as a positive control for PCR.

Five microliters of cDNA were used as amplification template, and the PCR

mixture contained 400 nM of each primer, 100 iM of each dNTP, 10 mM KC1, 10 mM

(NH4)2 S04, 20 mM Tris-HC1, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1 unit of

Taq DNA polymerase (New England BioLabs Inc., Beverly, Massachusetts, USA).

Cycling conditions were: Initial incubation at 940C for 30 sec followed by 40 cycles at

940C for 30 sec, 56C for 30 sec, and 720C for 1 min. A final extension step at 720C for 5

min finished the cycling. Approximately 20 [tl of the PCR products were resolved by

horizontal gel electrophoresis in 1.0% agarose containing ethidium bromide (0.5 ug/ml)

and the DNA fragments were visualized by UV light transillumination and photographed

using a gel documentation system.

PCR targeting the P-actin gene

To insure the integrity and amplification of the RNA, an internal control PCR was

implemented on cDNA produced with random primers. Primers targeting the

mammalian p-actin housekeeping gene were used to amplify a 275-bp fragment from

cetaceans. Primer sequences were: FP-5'- GAG AAG CTG TGC TAC GTC GC -3', RP-

5'- CCA GAC AGC ACT GTG TTG GC -3'. The PCR mixture contained 5 il cDNA,

400 nM of each primer, 100 |M of each dNTP, 10 mM KC1, 10 mM (NH4)2 SO4, 20 mM

Tris-HC1, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8 and 1 unit of Taq DNA









polymerase (New England BioLabs Inc., Beverly, Massachusetts, USA). Cycling

conditions were 40 cycles at 940C for 30 sec, 600C for 30 sec, and 720C for 30 sec.

Approximately 20 [tl of the PCR products were resolved by horizontal gel electrophoresis

in 1.0% agarose containing ethidium bromide (0.5 ug/ml) and the DNA fragments were

visualized by UV light transillumination and photographed using a gel documentation

system.

Results

Analysis of PMV

RACE and amplification of the terminal extragenic domains

The poly-Gn primer amplifies 10 G bases to the 3' end of the product, thus, if the

RACE technique was successful, the tailed PCR product will be 10 nucleotides longer

than the untailed PCR product (Figure 2-2). Upon sequencing the RACE generated PCR

products, it was determined that the genome promoter region was 107-bp long, directly

followed by the ATG start codon that begins the N gene open reading frame. Similarly,

the antigenome promoter region was 108-bp long and directly followed the L gene TAA

stop codon. The leader and trailer sequences of PMV have been deposited into the

GenBank database under accession numbers AY949833 and AY949834, respectively.

Nucleotide sequence comparisons between PMV and DMV showed 95.3% identity for

the genome promoter and 80.6% identity for the antigenome promoter. Comparisons of

all morbillivirus genome and antigenome promoter regions, with the exception of PDV,

are shown in Figures 2-3 and 2-4, respectively.









2 3


amw-


500-bp
A 400-bp


Figure 2-2. Gel electrophoresis of PCR products; lane 1- tailed 420-bp PCR product, lane
2- tailed 443-bp PCR product, lane 3- untailed 420-bp PCR product, lane 4-
untailed 443-bp PCR product.


i~La!











6 12 18 24 30 36 42 48 54
GI G M GM. CICM.UUAU. GUC U.AC U.UU AUA. G CM.
G. MGM C C UUAU .GUC U. ACMU. UUA UA. GA .
G. C .UU UG. AGA .AGU MAACC.UA AUC. G G .
A. CM .U U UAGAA.UAU U.GA C.CUG CC. GUM .
UAGU.AGU AG .AAG C.ACG .
G .U UU AUU. AAUAC UA A.AA UU .. UU .


72 78 84 90 96
,. GUUAA GUU.UCAA U.AUU
. GUUAA. C AAA UAU.
A.GCUGA UC AAU CGUG
G. ACAGC CCC CU UCG
.AUAGU CUMU.UCGU UAAUC.C
G.GAAUUU. IU .CGAU CAAGI. I


102 108
\GU.GUUAG.
\GU.GUCAG
\GA.AAUUU
GAA.ACUGA
\UA.GGCUC
GAU.GGUUA.


Figure 2-3. Nucleotide sequence comparison of the genomic promoter region of
morbilliviruses. The GenBank accession numbers of the sequences used were:
NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine distemper
virus (CDV), AB012948-Measles virus (MeV), NC_006383-Peste-des-petits
ruminant virus (PPR), and NC_006296-Rinderpest virus (RPV).


12 18 24
C.
GC UBBBJ Aj IUAB HUM
IC UGG. UA .
U.
GC UGGG AUA U


30 36 42 48
AA. UCAGAU UUUIl.l UC
AA.UCGAAU.CUUM.UCM.I
AC.AUCUUU. GA .CUM.C
JC.CAAUAU GUAM.CUM.
GU.AUUUUC. AACUM.
JU.AAUAAC CGUUG.UU U(


60
fCACU.
UAUU.
AUUU.
UGGAU.
UGC A.
AACU .


66 72 78 84 90 96
UA.CCAUCC.UGGUAG. ACU U, GAU C. i
UA.UCACUC.UGACAA. GAC. GC GGU C.A
AG. UUUUAU. UGACCA. AUC JC AAGIA.I
AG.UGGGGU.CUUAUG. CGG GGG I
UG. CCUAAC.CACCUA. GGC. UUT GUU C
AA.UAGCAA.UGAAUG. AAGG .JGCU .U AGC1A.H


102 108


Figure 2-4. Nucleotide sequence comparison of the antigenomic promoter region of
morbilliviruses. The GenBank accession numbers of the sequences used were:
NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine distemper
virus (CDV), AB012948-Measles virus (MeV), NC_006383-Peste-des-petits
ruminant virus (PPR), and NC_006296-Rinderpest virus (RPV).


Amplification of the complete PMV N gene


The sequence for the entire N gene of PMV was obtained from two overlapping


PCR products (Figure 2-5) and deposited in the GenBank database under accession


number AY949833. The results indicated that the N gene of PMV was 1572 nucleotides


60 66
PHI.


G U .









in length and translated into a protein of 524 amino acid residues. The N gene of PMV

was identical in length to that of DMV, CDV and PDV, and slightly shorter than that of

MV, PPRV and RPV. Sequence homologies (Tables 2-1, 2-2 and 2-3) and phylogenetic

analyses (Figures 2-6 and 2-7) demonstrated that PMV was most closely related to DMV,

with 93.7 and 95.8% amino acid identity and similarity, respectively. From the N gene

nucleotide comparison of the three marine mammal morbilliviruses, it was observed that

the PMV N gene had a nucleotide identity of 88.6% to that of DMV, but only 66.5% to

the PDV homologue (Figure 2-8). The N gene of PMV clearly contained the four

previously described regions common to all morbillivirus N proteins (Figure 2-9).












Figure 2-5. Gel electrophoresis of the overlapping PCR products used to obtain the full N
gene sequence of PMV. Primers 8A and N2 generated a product of -1050-bp
(lane 1), while primers P2 and N1 generated a product of-1650-bp (lane 2).










Table 2-1. Percent nucleotide identities of morbillivirus N genes. GenBank accession
numbers are: NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine
distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-
des-petits ruminant virus (PPRV), NC_006296-Rinderpest virus (RPV) and
X75717-Phocine distemper virus (PDV).
RPV MV PPRV CDV PDV DMV PMV


RPV
MV
PPRV
CDV
PDV
DMV
PMV


100.0
69.5
67.0
63.7
63.4
66.5
66.3


100.0
66.3
64.9
63.2
67.3
68.7


100.0
64.4
64.6
67.6
67.8


100.0
77.5
66.9
67.7


100.0
65.5
66.5


100.0
88.6


100.0


Table 2-2. Percent amino acid identities of morbillivirus N genes. GenBank accession
numbers are: NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine
distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-
des-petits ruminant virus (PPRV), NC_006296-Rinderpest virus (RPV) and
X75717-Phocine distemper virus (PDV).
RPV MV PPRV CDV PDV DMV PMV


RPV
MV
PPRV
CDV
PDV
DMV
PMV


100.0
75.1
74.4
69.3
68.3
73.0
73.4


100.0
73.6
72.3
68.5
75.4
76.6


100.0
70.1
68.8
75.4
76.8


100.0
65.3
73.3
73.0


100.0
70.2
70.3


100.0
93.7


100.0


Table 2-3. Percent amino acid similarities of morbillivirus N genes. GenBank accession
numbers are: NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine
distemper virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-
des-petits ruminant virus (PPRV), NC_006296-Rinderpest virus (RPV) and
X75717-Phocine distemper virus (PDV).
RPV MV PPRV CDV PDV DMV PMV


RPV
MV
PPRV
CDV
PDV
DMV
PMV


100.0
83.1
79.9
75.6
75.0
80.1
80.1


100.0
79.5
79.7
76.0
80.8
81.0


100.0
77.2
76.3
82.2
82.3


100.0
89.1
78.7
78.6


100.0
77.3
77.5


100.0
95.8


100.0










PMV
DMV



PDV /100oo.o
100.0

96.8
CDV
PPRV
99.2



RPV
MV
A






DMV

PMV

PPRV

-RPV



CDV

0.1 Divergence PDV
B




Figure 2-6. Neighbor-Joining phylogenetic trees of the nucleotide sequences of the
complete N gene of morbilliviruses. The tree was generated by Clustal X slow
and accurate function using Gonnet 250 residue weight table, gap penalty of
15 and gap length penalty of 6.66. GenBank accession numbers are:
NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine distemper
virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-des-petits
ruminant virus (PPRV), NC_006296-Rinderpest virus (RPV) and X75717-
Phocine distemper virus (PDV). A) Radial tree where the numbers represent
the percent confidence of 1000 bootstrap replications B) Phylogram with 0.1
divergence scale.










DMV
PMV



PDV 100.0
100.0

56.3
CDV 5PPRV
63.9



MV
RPV
A





PMV

DMV

PPRV

MV

RPV

CDV

0.1 Divergence
B PDV




Figure 2-7. Neighbor-Joining phylogenetic trees of the deduced amino acid sequences
from the complete N gene of morbilliviruses. The tree was generated by
Clustal X slow and accurate function using Gonnet 250 residue weight table,
gap penalty of 10 and gap length penalty of 0.10. GenBank accession numbers
are: NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine distemper
virus (CDV), AB012948-Measles virus (MV), NC_006383-Peste-des-petits
ruminant virus (PPRV), NC_006296-Rinderpest virus (RPV) and X75717-
Phocine distemper virus (PDV). A) Radial tree where numbers represent the
percent confidence of 1000 bootstrap replications B) Phylogram with 0.1
divergence scale.





























































DMV ... ...... ... a..g.. .......... ..t ..... g .......g.. .. .......... ..........
PDV .....ga.a. ..... a .... a..a...a.a a.ga.t...a t .... c.t.. .ata..t..g ..ca.a .... .t ......t. g..t...... c .... ....
Consensus CAGCAACGCC GTGTGGTAGG TGAGTTTCGG CT-GACAAAG GATGGTT-GA TGC-GTGAGA AATCGGATTG CGGAGGATCT ATCGTTGAGG AGATTCATGG


Figure 2-8. Nucleotide comparison and consensus sequence for the full N gene of three morbilliviruses than infect marine mammals.

GenBank accession numbers are: NC_005283-Dolphin morbillivirus (DMV), X75717-Phocine distemper virus (PDV).


.......... .......... ...a..tc .. .......... .c ........ ......cc .. .......... .......... ......... ........ .
. ...... c t ..... .a., c...a. c ... .....a.a.. ca......aca .c.c..at.. c c.t....a. .t..c.. .. .......... ..a..a....















































.......... .......... ..... a .... .......... .......... .......... ......... c ......... .......... g ........
.t ..... t .. ...... c..g ..c ....... g..... c.. ... a..gt.a ..c..c .... .a........ ...... a..t ..c...c... t ........
GCTATGCAAT GGGAGTTGGA GTTGAGCTTG AAAATTCAAT GGGTGGACTT AATTTTGGTC GTTCTTACTT TGACCCTGCA TATTTCAGAC T-GGTCAAGA

c


Figure 2-8. Continued.


.......... ......... .. .. a. ... ...g ..... .......... .....g .... ....... .. .......... ..... ... .... .. ..
.......... a ......... ......t.t. ... .... ... .. ... .......... ..t ..... ..... .... .. a ..... a. ..... ..













































Figure 2-8. Continued.


------Variable region------


Figure 2-9. Amino acid comparison and consensus sequence of the full nucleocapsid gene of morbilliviruses. GenBank accession

numbers of the N gene sequences used are: NC_005283-Dolphin morbillivirus (DMV), AF305419-Canine distemper virus

(CDV), AB012948-Measles virus (MV), NC_006383-Peste-des-petits ruminant virus (PPRV), NC_006296-Rinderpest

virus (RPV) and X75717-Phocine distemper virus (PDV).


........ .. t ...... ..... a ..g .c ...... a ... ...... a ...... t .. ..........
a. CC ..ac.c.. a tca cc.ca.t. S (....-.a a. ( .... ..at .ac (..at. ..c ..... at


..... r ....
S......r ....
..S... .. .


S. .a ....
...... rt.i


t ......... ........ ..... t .


ia ........ .... .... ..... t .


. . km. .
........ a .


n . . .


. v .......


.a..g....
.a. ....
a ........
M .m .......


. . . .
. . . .
. . . .
. S . .


. . . .
......... k
. . . .


.1 ... dq. .......... tn.e .... q. ..hd..s.s.
....... d.t .......... ts ...... r. .tyee.nd. e
....... e.. 1 ......... .n.e .... d. ..qaaee..
....... e.. 1 ......... .n.e .... d. ..qagee..
... ... tr. .......... .dl.n...m. ..teg.s..g


qsrf ...... .. ..... .. g ..mi .gt .......... .......... .. .......
e ..sy .... r d .q ....... g ..mi ..t .......... .......... .. .......
t.g.n ..... .......... .......... ......... .......... ..m.....
t .g .h ..... ... e ...... .......... .......... .......... .. t .......
kkrin .... r .......... .... m ..... .......... .......... .. 1 .......





















































---------------------------------------------------- Hypervariable C-terminus------------------------------- ----
401 450 500
..e...t..t ....s..t.. .s.rn.ap.q rlpp .... tm ks.fq..dky snqli.d.ls .y.s.vq..e wd.srqitql tq.gdh...n d.q.mdg..k
..e...i..t ....s..t.. .s.rs.va.q q.p..... nk rs.nq..dky pihfs.e.lp .y.p.vn..e w..sry.tqi iqdd.n...d dr..m..i.k
..e..is..v ..r....... ..ha.as..e 1..laackedr rvkasr..ar esvretass. asderaahlD t..tol...t a..s.a..ad srr..d..l.


Figure 2-9. Continued.


.......... mn .i ... .. .......... .......... .. s ....... ........ n .......... .......... .......... ..........
.......... mn .i ...i .. .......... .......... .. s ....... ........ n .......... .......... .......... ..........
.......... erk ...v .. .......... .......... .......... .......... ........ .......... ......... a ...s .1 ....
.......... ....... t .. .......... .......... .......... .......... .......... .......... ......... a ...s ......
. . . . .. a . . . . . . . . . . . . . . . . . . . . .
. . . . .. a . . . . . . . . . . . . . . . . . . . . .
.......... ....... a .. .......... .... s .... .......... ........ n .......... .......... ......... a s ......
QQRRVVGEFR LDKGWLD-VR NRIAEDLSLR RFMVALILDI KRTPGNKPRI AEMICDIDTY IVEAGLASFI LTIKFGIETM YPALGLHEFS GELTTIESLM




301 350 400
v ......... .......... .......... .......... .......... .......... .......... ........ tf ...f...k.. .q ..... vsr
m......... .......... .......... .......... .......... ....... .. .......... ........ a ....... k.. .q...... sk
.......... .......... .......... .......... .......... .......... .......... ........ t .s ....... d .r ...... m h
........ 1 .......... ...... a .. .......... ........ .. .......... .......... ...... n .s ..... e.. .r......ay
.......... .......... .......... .......... .... d ... .......... .......... ........ s. ......... d ........ a .


.mlt.m. s
lqt. .g...
la ... a..a


p.tse.n.p.
..sdt. .pr.
stl.n.slra









Detection of protein expression by Western blot

Western blot analysis successfully detected the expression of the N gene

hypervariable C-terminal region of DMV, which corresponded to a 9240 kDa protein

(Figure 2-10). No positive transformants were obtained for PMV.











Figure 2-10. Expression of the N gene hypervariable C-terminus of DMV in M15 cells as
detected by Western blot. Lane 1- DMV clone 1, Lane 2- DMV clone 2.

Analysis of Stranded Cetacean Tissues for Morbillivirus

All 57 tissues tested from four cetacean species from recent mass stranding events

in the southeast U.S. were negative for morbillivirus by PCR. Total RNAs extracted

from the same tissues tested for the P-actin housekeeping gene were all positive.

Discussion

Determining whether PMV and DMV should be considered two strains of CMV or

individual virus species is no easy task, as much of the research has produced conflicting

data. If the paramyxovirus nomenclature rules were applied to morbilliviruses, all viruses

in that genus would belong to the same species based on high levels of serological cross-

reactivity, despite the distinct host range of each virus (Rima et al., 1995).

All previous studies on cetacean morbilliviruses have concluded that they did not

arise from any known morbillivirus, but represent an independent morbillivirus lineage

(Barrett et al., 1993; Bolt et al., 1994). Phylogenetic analyses based on various gene

sequences place DMV and PMV in a clad of their own, closer to MV and the ruminant









morbilliviruses than to CDV and PDV (Barrett et al., 1993; Blixenkrone-Moller et al.,

1994; Bolt et al., 1994). Consequently, an epidemiological link between cetacean and

pinniped morbillivirus outbreaks seems unlikely. In support of these data, current

evolutionary evidence suggests that cetaceans arose from land-based ruminants (Martin,

1990), whereas seals evolved from a terrestrial, otter-like ancestor (King, 1983). It also

appears that morbilliviruses likely evolved with their host species, and the broadening

host range may be due to migration of cetaceans in addition to spontaneous mutations of

the viruses (Blixenkrone-Moller et al., 1994).

Our results indicated that the N gene of PMV and DMV were 88.6 and 93.7%

identical at the nucleotide and amino acid levels, respectively. Baron and Barrett (1995)

reported the N gene homologies of four strains of RPV, and found amino acid identities

to RPV-R of 99.2% for RPV-K, 93.1% for RPV-Kw, and 90.7% for RPV-L. The low

homology of RPV-L was probably reflective of the fact that RPV-L is a lapinized strain,

and is of geographically and temporally distant origin. It is known, however, that the N

proteins of morbilliviruses are much more conserved over the first 400 amino acids. As a

result, when only the first 400 amino acids were compared, the homologies between the

four strains of RPV increased to approximately 99%. Similarly, the homology of PMV

and DMV increased to 97.5% when only the first 400 amino acids of the N gene were

compared. This region of the N protein is thought to contain all the necessary structural

information for self-assembly into nucleocapsids, hence, the high sequence conservation,

while the C-terminal tail is suggested to be involved in replication. It is interesting to

note that the hypervariable C-terminal homologies also vary depending on the virus (Bolt

et al., 1994). In this region, PMV and DMV differ by 21 amino acid residues, while









CDV and PDV differ by 48. A maximum of 15 amino acid residue differences have been

reported between the most divergent MV strains. The residue homology of the cetacean

morbilliviruses falls between that of two distinct species and two very divergent strains,

thus making conclusions about the relatedness of PMV and DMV very difficult.

Previous research has indicated that the 3' and 5' terminal extragenic domains of

different paramyxoviruses have only limited homology, whereas the ends (especially the

first 18 bases) of individual viruses are very similar (Baron and Barrett, 1995). These

regions presumably contain the promoter/landing site for the viral polymerase, thus, high

conservation is needed for transcription. The leader sequence of two strains of Sendai

virus is relatively conserved, and a mutation of only two nucleotides at positions 20 and

24 results in a marked decrease of virus growth and pathogenicity in mice (Fujii et al.,

2002). Nucleotide sequence homologies for the genome and antigenome promoters of

PMV and DMV were 95.3 and 80.6%, respectively. Homologies increased to 100% for

both the leader and trailer when only the first 18 bases were analyzed.

Our results thus indicate, that PMV and DMV are clearly related phylogenetically,

and we suggest they should be regarded as two strains of CMV, based on high sequence

homologies of the full N gene and the terminal extragenic regions. Phylogenetic analyses

based on the full N gene of all morbilliviruses, including PMV, indicated the cetacean

viruses form a distinct lineage, shown on the phylograms (Figures 2-6B and 2-7B) by

considerable evolutionary divergence. As in previous studies, our results showed that

CMV was closer in relation to MV and the ruminant viruses than to the carnivore viruses.

Based on multiple sequence homologies of the full N protein (Tables 2-1, 2-2, and 2-3),

PMV was most closely related to DMV, followed by MV and PPRV, and was least









related to PDV. In accordance with previous studies, we concluded that there is indeed a

substantial difference between morbilliviruses that infect cetaceans and those that infect

pinnipeds. For that reason, a biological association between cetacean and pinniped

morbillivirus epizootics is highly improbable. Western blot analysis detected the

expression of the N gene hypervariable C-terminus of DMV. Positive transformants

containing the homologous protein of PMV were not obtained, thus no genotypic

comparisons could be made between these two strains of CMV. Other criteria, such as

cell tropism, host range, pathogenesis and geographic localization, must also be

considered when classifying viruses, as phylogenetic analysis alone seems insufficient

(van de Bildt et al., 2004). With regard to these criteria, DMV and PMV are able to

infect cells of the same species, with few exceptions, and cause similar lesions. On the

other hand, DMV appears to have a broader host range, and infection is more

geographically widespread.

More recently, a morbillivirus (PWMV) was identified in a stranded long-finned

pilot whale (Globicephala melas) from New Jersey, and it was suggested that the virus

might represent a third member of the CMV group (Taubenberger et al., 2000).

Sequence fragments of the P and N gene obtained by RT-PCR showed a close, but

distinct relationship to PMV and DMV. However, the fragments of the P and N genes

that were analyzed were very small, 378- and 230-bp respectively, thus the conclusions

may be biased. Upon further sequence analysis of PWMV, we found that the nucleotide

and amino acid identities to PMV and DMV were 85.7 and 98.7%, and 87.8 and 97.4%,

respectively. It should also be noted that the 76 amino acid residues of the N gene

sequence of PWMV correspond to the highly conserved third region of the N protein, and









for that reason, the high degree of sequence homology with PMV and DMV may be

misleading. Much more sequence analysis is needed before any convincing conclusions

can be made about the phylogeny of PWMV.

Prior to the discovery of PWMV, Osterhaus et al. (1997) isolated a morbillivirus

(MSMV) from tissues of Mediterranean monk seals (Monachus monachus) with

respiratory distress in West Africa. Serum antibodies to CDV were detected by an

ELISA; however, based on polyclonal and monoclonal antibody characterization, MSMV

was different from PMV and DMV. Using a semi-nested PCR with group-specific

primers for the genus Morbillivirus, 121-bp of the N gene were amplified, and a close

relationship to DMV was found. Because the sequence data remain unpublished, it is

unknown what region of the N gene was amplified. The use of Morbillivirus group-

specific primers suggests that, as with the PWMV, the highly conserved middle region of

the N gene was the target of the PCR; therefore, conclusions based on sequence analysis

may be skewed. Also, the reliability of phylogenetic analysis based on only 121

nucleotides is questionable. A morbillivirus was also isolated from a Mediterranean

monk seal that stranded in Greece (van de Bildt et al., 1999). Based on N and P gene

sequence analyses and immunofluorescence patterns, this virus was most closely related

to PMV. These cases, along with the seroprevalence of morbillivirus antibodies found in

polar bears, walruses and Florida manatees, strongly suggests that some interspecies

transmission of morbilliviruses does occur in the wild.

It has been reported that in the past few decades there has been a worldwide

increase in diseases of marine organisms (Harvell et al., 1999). New diseases are

appearing, not because new microorganisms are being introduced, but most likely









because previous disease agents are changing. Many factors are hypothesized to play a

role in emerging and resurging diseases, including host shifts, exposure of naive

populations via translocation of pathogenic organisms, climate variability and human

activity. All four factors may be of significant importance in regard to marine mammal

morbillivirus outbreaks. Evidence of CDV host shifts, caused by pathogen spillover from

domestic animals, has been proposed between sled dogs and Crabeater seals in Antarctica

(Bengtson et al., 1991), as well as domestic dogs and Baikal seals in Siberia (Osterhaus et

al., 1989). Host shifts may also be responsible for the reported prevalence of

morbillivirus antibodies in polar bears and Florida manatees; however, both the infection

and clinical disease in these two species have yet to be confirmed. Serological studies

have shown that morbilliviruses are ubiquitous among pilot whale populations, without

much apparent prevalence of clinical disease or mortality. Pilot whales, as long distance

vectors between America and Europe, are known to mix with at least 13 other odontocete

species, thus, providing numerous opportunities for the introduction of morbilliviruses

into naive populations (Duignan et al., 1995a).

Disease outbreaks occur when environmental conditions either increase the

prevalence or virulence of existing disease agents or facilitate a new disease (Harvell et

al., 1999). Climate variability and human activity have been known to influence previous

epidemics, for example, by enhancing the global transport of marine species including

pathogens or altering marine mammal behavior. One of the most prominent

environmental factors is El Niho. This global phenomenon causes shifts in surface water

temperature, which in turn alters the water and atmospheric currents, and eventually food

availability. It was reported that bottlenose dolphins in southern California shifted home









range over 500 km north in search of food during an El Nifio event (Reynolds III et al.,

2000), and the 1982-1983 El Nifio event caused widespread mortality of pinnipeds in

South America due to starvation (Trillmich et al., 1991). Animals that travel outside their

normal habitat have the potential to encounter new pathogens, while starvation may lead

to immunosuppression and subsequent viral infection. El Niho, as well as the opposing

phenomenon La Nifia, may have more direct influences on disease epidemiology through

other large-scale oceanographic factors and pathogen transport. Very strong El Nifio

events were reported in 1982-1983, 1996-1997 and 2003-present, while weak events

were seen in 1987-1988 and 2002-2003. Severe morbillivirus epizootics, such as those

seen in 1987-1988 and 2002-2003, seem to be associated with weak El Nifio events, or

those blocked by strong La Nifia episodes. Prolonged El Nifio events have been shown to

increase the prevalence of other diseases of marine organisms, including coral bleaching

and Dermo, a protozoan parasite disease of Eastern oysters (Crassostrea virginica)

(Harvell et al., 1999). The apparent relationship between these diseases and El Nifio

suggests that epidemics may be predicted from climate models based on the assumption

that climate-mediated, physiological stresses may compromise host resistance and

increase frequency of opportunistic diseases. It would be interesting to investigate the

potential correlation between morbillivirus epizootics and such climatological episodes.

Human activity has facilitated disease outbreaks in multiple ways, including the

introduction of countless pollutants and habitat degradation. Environmental

contaminants (particularly organochlorines and certain heavy metals) are known to

bioaccumulate in top predators, such as many marine mammals (Geraci et al., 1999).

Organochlorines have been shown to have immunosuppressive effects in laboratory









animals (O'Hara and O'Shea, 2001); however; evidence for causal relationships between

high contaminant levels and disease susceptibility in the wild is inconclusive. In

response to PDV, no difference was detected in harbor seals fed fish supplemented with

polychlorinated biphenyls (PCBs) and those fed uncontaminated fish (Harder et al.,

1992). Conversely, more recent studies showed that seals fed fish from polluted waters

developed impaired immune responses, while those fed clean fish did not (Geraci et al.,

1999). Similar contradictory results were also obtained from research on cetaceans.

Correlations were detected between organochlorine concentrations and reduced immune

responses of bottlenose dolphins in Florida (O'Hara and O'Shea, 2001). On the contrary,

studies of harbor porpoises in Great Britain that died from infectious disease in

comparison with those that died from trauma revealed no significant differences in

concentrations of organochlorines in the blubber. Pertaining to the 1987-1988

morbillivirus outbreak of bottlenose dolphins, some samples infected with the virus also

showed extremely high organochlorine concentrations, while others were very low

(Geraci et al., 1999).

Cause and effect relationships between mass mortalities and any single factor are

difficult to demonstrate; hence, it is probable that multiple factors play a role in

morbillivirus epizootics of marine mammals. For example, brevetoxin was suspected to

be a contributing cause in the DMV/PMV epizootic in Atlantic bottlenose dolphins in

1987 and 1988 (Geraci et al., 1999). Sublethal exposure to the toxin may have left the

dolphins emaciated, exhausted, thermally stressed and vulnerable to morbillivirus

infections. Similarly, Mediterranean striped dolphin carcasses infected with DMV

between 1990 and 1992 also contained high levels of organochlorines (Geraci et al.,









1999). Finally, morbillivirus was detected in a number of Cape fur seals (Arctocephalus

pusilluspusillus) that died in 1994, however the cause of death was presumed to be

chronic starvation associated with the ongoing El Nifio event (Geraci et al., 1999).

The fundamentals of understanding infectious diseases are the identification,

isolation and characterization of the etiological agent. This information enables the

development of specific diagnostic methods for epidemiological surveys and studies of

host resistance. Much of this work has been done on marine mammal morbilliviruses,

thus, the next logical step is treatment and prevention. At this time, there is no effective

treatment for morbillivirus infections; however, successful vaccines for RPV, PPRV,

CDV and MV are widely employed to control morbillivirus agents in terrestrial domestic

animals, including humans. Research has been done with vaccines against CDV in two

species of pinnipeds, specifically grey and harbor seals. Attenuated CDV vaccines have

been reported to produce antibodies in both species (Visser et al., 1989; Hughes et al.,

1992); however, this method requires three separate vaccinations, which is time

consuming and costly. A CDV vaccine using a recombinant vaccinia virus was given

orally to harbor seals with unsuccessful results (Van Bressem et al., 1991). At present,

no vaccination trials have been conducted on cetaceans.

Although none of the tissues from cetaceans from recent mass stranding events in

the southeastern U.S. tested positive for morbilliviruses in this study, these viruses should

remain of high concern as both their geographical distribution and host range continue to

expand. The mass mortality brought on by morbillivirus epizootics is damaging to all

marine mammal populations, but can be especially devastating to endangered species.

All knowledge gained about marine mammal morbilliviruses, including that of this study,






83


aids in the understanding of these devastating infections. Appreciating the molecular and

phylogenetic characteristics of these viruses brings us one step closer to unearthing a

successful preventative or remedial strategy.















APPENDIX A
MARINE MAMMAL SAMPLES














Table A-i. List of all lesion and tissue samples tested for herpesvirus genomes using the nested PCR approach. Stranded- single
animal stranding, UME- animal was part of unusual mortality event, capture- animal was part of capture and release


p!
Number
v183
v210A
v223
v224
v225B
v226A, K231
v228A
v240
v262
v263
v264
v265
v267
v268
v269
v273
v293
v298, K265
v300
v301B
v386


program, NA- data not available.
Species
Globicephala macrorhynchus
Tursiops truncatus
Orcinus orca
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Kogia sima
Kogia sima
Kogia sima
Kogia sima
Tursiops truncatus


Animal ID
Captive
Captive
Captive
Captive
Captive
Captive
Captive
Captive
FB188
CMA-02-21
CMA-02-21
CMA-02-21
CMA-02-21
CMA-02-21
CMA-02-21
CMA-02-21
MML-0233
MML-0233
MML-0233
MML-0233
Captive


Location


NA
NA
NA
NA
NA
NA
NA
NA
capture FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
NA


Date


NA
NA
NA
NA
NA
NA
NA
NA
11/2002
10/2002
10/2002
10/2002
10/2002
10/2002
10/2002
10/2002
9/2002
9/2002
9/2002
9/2002
NA


Sample


skin lesion
oral lesion
skin lesion
oral lesion
skin lesion
skin lesion
pox-like lesion
oral lesion
skin lesion
open sore- rostrum
skin lesion
cerebellum
cerebrum
lung LN
brainstem
prescap LN
mesenteric LN
genital lesion
pulm LN
prescap LN
oral lesion


Result


neg
neg
neg
neg
neg
POS a
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
POS y
neg
neg
neg














Table A-1. Continued.


Number
v387
v388
v391
v447
v470
v557
v558
v585
v587
v614
v626
v634, K264
v653
v654
v657
v600
v663
v682A
v683B
v702
v703
v711
v713
v714
v715
v716
v722
v724
v725
v726
v727


Species
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Globicephala macrorhynchus
Globicephala macrorhynchus
Chelonia mydas
Tursiops truncatus
Grampus griseus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Lepiochelys kempii
Lepiochelys kempii
Lepiochelys kempii
Phocoena phocoena
Tursiops truncatus
Tursiops truncatus
Steno bredanensis
Steno bredanensis
Phocoena phocoena
Chelonia mydas
Chelonia mydas
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus


Animal ID
Captive
Captive
MML-0229
MML-0309
MML-0305
FKMMRT 0305
FKMMRT 0305
Pappy ST0306
Captive
MARS 0303
Captive
Jacksonville
MML-0313
ST0109
ST0109
ST0109
03-04Pp
Captive
Captive
MML-0237
MML-0237
03-04Pp
Simba
Coquina
Captive
Captive
Captive
99B4
97B7-1
97B7-2
99B12


Location
NA
NA
stranded FL
stranded FL
stranded FL
NA
NA
stranded FL
NA
NA
NA
stranded FL
stranded FL
stranded FL
stranded FL
stranded FL
stranded NC
NA
NA
stranded FL
stranded FL
stranded NC
stranded FL
stranded FL
NA
NA
NA
capture AK
capture AK
capture AK
capture AK


Date
NA
NA
8/2002
2/2003
2/2003
NA
NA
3/2003
NA
NA
NA
9/2003
4/2003
1/2001
1/2001
1/2001
3/2003
NA
NA
12/2002
12/2002
3/2003
11/2002
NA
NA
NA
NA
1999
1997
1997
1999


Sample
tongue lesion
tongue lesion
skin- shark bite
skin lesion
skin lesion
pox-like lesion
pox-like lesion
fibropapilloma
oral lesion
skin lesion
oral lesion
penile lesion
skin lesion
fibropapilloma
fibropapilloma
fibropapilloma
skin lesion
skin lesion
skin lesion
skin lesion
skin lesion
skin lesion
fibropapilloma
fibropapilloma
oral lesion
oral lesion
penile lesion
skin lesion
skin lesion
skin lesion
skin lesion


Result
neg
neg
neg
neg
neg
neg
neg
POS
neg
neg
neg
POS y
neg
POS
POS
POS
neg
neg
neg
neg
neg
neg
POS
POS
neg
neg
neg
neg
neg
neg
neg














Table A-1. Continued.
Number Species
v728 Balaena mysticetus
v729 Balaena mysticetus
v730 Balaena mysticetus
v731 Balaena mysticetus
v732 Balaena mysticetus
v733 Kogia breviceps
v734 Orcinus orca
v735 Orcinus orca
v736 Tursiops truncatus
v737 Tursiops truncatus
v738 Orcinus orca
v739 Orcinus orca
v740 Orcinus orca
v741 Orcinus orca
v742 Orcinus orca
v743 Orcinus orca
v744 Orcinus orca
v745 Orcinus orca
v746 Orcinus orca
v747 Orcinus orca
v748 Orcinus orca
v752 Caretta caretta
v963 Balaena mysticetus
v964 Balaena mysticetus
v965 Balaena mysticetus
v966 Balaena mysticetus
v967 Balaena mysticetus
v968 Balaena mysticetus
v969 Balaena mysticetus
v970 Balaena mysticetus
v971 Balaena mysticetus


Animal ID
99KK2
98KK3
98KK3-2
98KK1
99B24
MARS 9905
Captive
Captive
MML-9628
MML-9628
Captive
Captive
Captive
Captive
Captive
Captive
Captive
Captive
Captive
Captive
Captive
Floater
90B2
90B10
02-291-1
02-291-2
99B18
02-292-1
02-292-2
97-149-1
98KK3


Location
capture AK
capture AK
capture AK
capture AK
capture AK
NA
NA
NA
stranded FL
stranded FL
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
stranded FL
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK


Date
1999
1998
1998
1998
1999
NA
NA
NA


Sample
skin lesion
skin lesion
skin lesion
skin lesion
skin lesion
mammary slit
skin lesion
skin lesion


9/1996 skin lesion
9/1996 skin lesion


NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1998
1998
2002
2002
1999
2002
2002
1997
1998


skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
skin papilloma
tongue lesion
penile lesion
skin lesion
oral lesion
skin lesion
skin lesion
skin lesion
skin lesion
skin lesion
skin lesion


Result














Table A-1. Continued.


Number
v972
v973
v974
v975
v976
v977
v978
v979
v980
v981
v1019
v1025
v1026
v1027
v1039
v1040
v1164
v1165
v1176
v1192
v1193
v1194
v1225
v1229
v1233
v1234
v1235
v1270A
v1272
v1273
v1274


Species
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Balaena mysticetus
Tursiops truncatus
Stenella attenuata
Tursiops truncatus
Tursiops truncatus
Kogia sima
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Tursiops truncatus
Chelonia mydas
Lepiochelys kempii
Lepiochelys kempii


Animal ID
79B2
02KK3
99-035
99-102
99-044-A
99-044-B
99-108
00-143-1
00-143-2
00-146
Captive
FLGM011304-02
FLPB102303-03
Captive
HUBBS 0336
HUBBS 0408
HUBBS 0435
HUBBS 0436
HUBBS 0438
HUBBS 0436
HUBBS 0439
HUBBS 0439
SJP0426
SJP0319-22
SJP20
SJP319-21
MIA0411
G040322-2tt
Pappy ST0306
ST0109
ST0109


Location
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
capture AK
NA
stranded FL
stranded FL
NA
NA
NA
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
UME FL
stranded FL
stranded FL
stranded FL


Date
1997
2002
1999
1999
1999
1999
1999
2000
2000
2000
NA
1/2004
10/2003
NA
NA
NA
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2004
3/2003
1/2001
1/2001


Sample
skin lesion
skin lesion
lip lesion
skin lesion
skin lesion
skin lesion
oral lesion
skin lesion
skin lesion
skin lesion
oral lesion
skin lesion
skin lesion
oral lesion
skin lesion
skin lesion
spinal cord
brain
brain
pox-like lesion
pox-like lesion
brain
brain
brain
brain
brain
brain
skin lesion
fibropapilloma
fibropapilloma
fibropapilloma


Result
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
POS
POS
POS