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Evaluating the Clinical Significance of Alphaherpesviruses in Bottlenose Dolphins (Tursiops Truncatus)

Permanent Link: http://ufdc.ufl.edu/UFE0025101/00001

Material Information

Title: Evaluating the Clinical Significance of Alphaherpesviruses in Bottlenose Dolphins (Tursiops Truncatus)
Physical Description: 1 online resource (75 p.)
Language: english
Creator: Daniel, Heather
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: alphaherpesvirinae, bottlenose, delphinid, dolphin, herpesvirus, pcr, quantitative, tursiops
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: EVALUATING THE CLINICAL SIGNIFICANCE OF ALPHAHERPESVIRUSES IN BOTTLENOSE DOLPHINS TURSIOPS TRUNCATUS During the routine, conventional PCR-based, viral surveillance of a managed collection of bottlenose dolphins, two distinct alphaherpesviruses, tentatively named Delphinid herpesvirus 2 (DeHV-2) and Delphinid herpesvirus 8 (DeHV-8), were detected in the buffy coat of one animal. The 22-year-old, female bottlenose dolphin had a history of elevated transaminases and dehydrogenases, which were normalized following phlebotomy treatment, and a persistent lymphocytosis. Alphaherpesviruses are generally considered to persist in the sensory nerve ganglion or in circulating lymphocytes, thus changes in viral load in circulating blood cells was presumed to be indicative of infection with active virus replication. The presence of the virus was thus evaluated with a quantitative assay so the active virus replication event could be identified by the higher level of virus particles. Novel, specific TaqMan quantitative PCR assays were developed to measure the viral load of the alphaherpesviruses in the case dolphin. The complete blood counts (CBC) and serum chemistry parameters for the herpesvirus positive samples in the case dolphin were compared to those of the negative samples, in an attempt to correlate viral presence with changes in clinical blood parameters. These assays were also used in a cross-sectional survey to establish a baseline prevalence of the two alphaherpesviruses in a population of clinically healthy bottlenose dolphins. DeHV-2 has a higher presence in the population (3 animals) than DeHV-8 (which was only detectable in the case animal). The simple presence of DeHV-2 could not be associated with any specific blood parameter changes in the case animal. One positive case animal sample (Tt0719), however, had remarkable trends in four of the blood parameters analyzed: decreases in total white blood cell counts and serum chloride as well as increases in erythrocyte sedimentation rate and hematocrit. Similar trends were not identified surrounding any of the other four DeHV-2 qPCR positive samples. There were an insufficient number of DeHV-8 positives for comparison of blood parameters associated with positive and negative samples.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heather Daniel.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Nollens, Hendrik Hans.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025101:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025101/00001

Material Information

Title: Evaluating the Clinical Significance of Alphaherpesviruses in Bottlenose Dolphins (Tursiops Truncatus)
Physical Description: 1 online resource (75 p.)
Language: english
Creator: Daniel, Heather
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: alphaherpesvirinae, bottlenose, delphinid, dolphin, herpesvirus, pcr, quantitative, tursiops
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: EVALUATING THE CLINICAL SIGNIFICANCE OF ALPHAHERPESVIRUSES IN BOTTLENOSE DOLPHINS TURSIOPS TRUNCATUS During the routine, conventional PCR-based, viral surveillance of a managed collection of bottlenose dolphins, two distinct alphaherpesviruses, tentatively named Delphinid herpesvirus 2 (DeHV-2) and Delphinid herpesvirus 8 (DeHV-8), were detected in the buffy coat of one animal. The 22-year-old, female bottlenose dolphin had a history of elevated transaminases and dehydrogenases, which were normalized following phlebotomy treatment, and a persistent lymphocytosis. Alphaherpesviruses are generally considered to persist in the sensory nerve ganglion or in circulating lymphocytes, thus changes in viral load in circulating blood cells was presumed to be indicative of infection with active virus replication. The presence of the virus was thus evaluated with a quantitative assay so the active virus replication event could be identified by the higher level of virus particles. Novel, specific TaqMan quantitative PCR assays were developed to measure the viral load of the alphaherpesviruses in the case dolphin. The complete blood counts (CBC) and serum chemistry parameters for the herpesvirus positive samples in the case dolphin were compared to those of the negative samples, in an attempt to correlate viral presence with changes in clinical blood parameters. These assays were also used in a cross-sectional survey to establish a baseline prevalence of the two alphaherpesviruses in a population of clinically healthy bottlenose dolphins. DeHV-2 has a higher presence in the population (3 animals) than DeHV-8 (which was only detectable in the case animal). The simple presence of DeHV-2 could not be associated with any specific blood parameter changes in the case animal. One positive case animal sample (Tt0719), however, had remarkable trends in four of the blood parameters analyzed: decreases in total white blood cell counts and serum chloride as well as increases in erythrocyte sedimentation rate and hematocrit. Similar trends were not identified surrounding any of the other four DeHV-2 qPCR positive samples. There were an insufficient number of DeHV-8 positives for comparison of blood parameters associated with positive and negative samples.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heather Daniel.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Nollens, Hendrik Hans.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025101:00001


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EVAL UATING THE CLINICAL SIGNIFICA NCE OF ALPHAHERPESVIRUSES IN BOTTLENOSE DOLPHINS ( TURSIOPS TRUNCATUS ) By HEATHER TIFFANY DANIEL 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 2009 1

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2009 Heather Tiffany Daniel 2

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ACKNOWL EDGMENTS I would like to thank my advisor, Dr. He ndrik Nollens, for his guidance and help throughout this project and before its inception. Additionally, I am appreciative of my other committee members, Dr. Maureen Long and Dr. David Bloom who were supportive, enthusiastic, and very helpful. I would also like to thank my lab, Linda Archer, Dr. Jim Wellehan, Dr. Rebecca Rivera, and Dr Brian Stacy, for their continual help, expertise and moral support. I am indebted to Linda for all her help with this project and Jim was more helpful than Google and PubMed combinedthank you, thank you, thank you. In The Virologists Dilemma (1957), Dr. R obert J. Huebner discussed the conditions necessary for establishing a virus as cause of a specific human disease. While he mentions many important factors it is his ninth topic Financial support: A consideration so absolutely necessary that it deserves to be called a postulate that l eads me to this: I am very, very grateful for the significant funding, samples, and hard work from the US Navy Marine Mammal Program towards this project. I would specifically li ke to thank Dr. Stephanie Venn-Watson for the countless hours spen t analyzing and reanalyzing my data. I would also like to thank Dr. Tracey Goldstein for the Phocid HV-2 positive control, Dr. Graldine Lacave for the harbor seal sample, Dr. Judy St. Leger and Sarah LaMere for the orca sample, and Dr. John Sykes for the sea lion sample. Additionally, I would like to thank April Childress for processing the sea lion sample. I am very appreciative of the support from the Aquatic Animal Health program. I was very lucky to have the opportunity to work for the program and pursue a Master of Science. Thank you Dr. Ruth Francis-Floyd and Dr. Charlie Courtn ey for making that possible and thank you Dr. 3

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Iske Larkin for actually m aking the workload po ssible while keeping my sanity. I have made many friends in this program and I look forward to working with you in the future. Not only am I grateful for my newfound frie nds but also for those back home and my family. Without your constant harassment regard ing that pesky graduation date, I might have tried to get another season of student football tickets out of this degree. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 CHAP TER 1 INTRODUCTION................................................................................................................. .11 Herpesvirales ..........................................................................................................................11 Herpesvirus Latency ........................................................................................................12 The Subfamilies of Herpesvir idae ..........................................................................................12 Alphaherpesvirinae ..........................................................................................................12 Betaherpesvirinae ............................................................................................................13 Gammaherpesvirinae ......................................................................................................13 Herpesviruses of Humans .......................................................................................................13 Cetacean Herpesviruses ..........................................................................................................16 Criteria for Disease Causation ................................................................................................20 Correlating Clinical Signs with Bottlenose Dolphin Herpesviruses .......................................22 2 QUALITATIVE AND QUANTITATIVE PCR ASSAYS FOR THE DET ECTION OF HERPESVIRUSES.................................................................................................................2 5 Introduction .............................................................................................................................25 Materials and Methods ...........................................................................................................25 Sample Collection ...........................................................................................................25 Consensus PCR ...............................................................................................................26 Bayesian and Maximum Like lihood Phylogenetic Analysis ...........................................27 Quantitative PCR .............................................................................................................28 Results .....................................................................................................................................29 Consensus PCR ...............................................................................................................29 Bayesian and Maximum Like lihood Phylogenetic Analysis ...........................................30 Quantitative PCR .............................................................................................................31 Discussion ...............................................................................................................................32 3 USING QUANTITATIVE PCR TO I NVESTIGATE CORRELATIONS BETWEEN TW O BOTTLENOSE DOLPHIN HERPESVI RUSES AND HEALTH PARAMETERS...41 Introduction .............................................................................................................................41 Materials and Methods ...........................................................................................................42 Sample Collection ...........................................................................................................42 5

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Serum Chemistry and CBC .............................................................................................43 Quantitative PCR .............................................................................................................43 Specific PCR ...................................................................................................................44 Statistical Analysis ..........................................................................................................44 Results .....................................................................................................................................45 Quantitative PCR .............................................................................................................45 Specific PCR ...................................................................................................................45 Statistical analysis ...........................................................................................................45 Discussion ...............................................................................................................................46 4 DISCUSSION................................................................................................................... ......60 APPENDIX A TROUBLESHOOTING.........................................................................................................63 Inconsistent Amplifi cation of Standards ................................................................................63 Problem Identified ...........................................................................................................63 Troubleshooting Approach ..............................................................................................63 Materials and Methods ....................................................................................................63 Results .............................................................................................................................64 Discussion ........................................................................................................................64 Restriction Enzymes ...............................................................................................................65 Problem Identified ...........................................................................................................65 Troubleshooting Approach ..............................................................................................65 Materials and Methods ....................................................................................................65 Results .............................................................................................................................66 Discussion ........................................................................................................................67 SOURCES AND MANUFACTURERS ........................................................................................70 REFERENCES ..............................................................................................................................71 BIOGRAPHICAL SKETCH .........................................................................................................75 6

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LIST OF TABLES Table page 2-1 Proposed names, abbreviations, and sour ces for marine m ammal Dpol herpesviral sequences used in this study ..............................................................................................33 2-2 Sampling data and consensus herpesvirus PCR results for case study samples ................34 2-3 MUSCLE alignment of 19 alphaherp esvirus Dpol am ino acid sequences. .......................35 2-4 MUSCLE alignment of 20 gammaherpesvirus Dpol amino acid sequences. ....................36 3-1 Spectrophotometric analysis of DeHV-2 qPCR c ase study samples .................................49 3-2 Spectrophotometric analysis of DeHV-8 qPCR c ase study samples. ................................49 3-3 Spectrophotometric analysis of the cross-sectional survey samples. .................................50 3-4 DeHV-2 qPCR case study data ..........................................................................................52 3-6 DeHV-8 qPCR case study data ..........................................................................................55 3-7 DeHV-8 qPCR cross-sectional survey data .......................................................................56 A-1 Spectrophotometric analysis ..............................................................................................68 7

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LIST OF FI GURES Figure page 2-1 Bayesian phylogenetic tree of th e 19 predicted partial 58 am ino acid alphaherpesviral DNA-dependent DNA polymerase sequences based on MUSCLE alignment.. ..........................................................................................................................37 2-2 Bayesian phylogenetic tree of th e 20 predicted partial 55 am ino acid gammaherpesviral DNA-depe ndent DNA polymerase seque nces based on MUSCLE alignment. ...........................................................................................................................38 2-3 DeHV-2 10-fold serial dilutions in triplica te. ....................................................................39 2-4 DeHV-2 standard curve in triplicate. .................................................................................39 2-5 DeHV-8 10-fold serial dilutions in duplicate. ....................................................................40 2-6 DeHV-8 standard curve in duplicate. .................................................................................40 3-1 WBC counts surrounding sample Tt0719 collection date. ................................................58 3-2 ESR values surrounding the time of s ample Tt0719 collection date. ................................58 3-3 Hematocrit values surroundi ng sample Tt0719 collection date .........................................59 3-4 Sodium chloride values surr ounding sam ple Tt0719 collection date. ...............................59 A-1 DeHV-2 qPCR amplification comparison of cDNA and plasm id serial dilutions. ...........68 A-2 Gel electrophoresis illustrating the pl asm id after restriction enzyme digestion. ...............69 A-3 DeHV-2 qPCR amplification comparison of cDNA, plas mid, and RE serial dilutions ....69 8

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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 EVALUATING THE CLINICAL SIGNIFICA NCE OF ALPHAHERPESVIRUSES IN BOTTLENOSE DOLPHINS ( TURSIOPS TRUNCATUS ) By Heather Tiffany Daniel August 2009 Chair: Hendrik Nollens Major: Veterinary Medical Sciences During the routine, conventional PCR-based, viral surveillance of a managed collection of bottlenose dolphins, two distinct alphaherpesviruses, tentatively named Delphinid herpesvirus 2 (DeHV-2) and Delphinid herpesvirus 8 (DeHV-8), were detected in the buffy coat of one animal. The 22-year-old, female bottlenose dolphin had a history of elevated transaminases and dehydrogenases, which were normalized followi ng phlebotomy treatment, and a persistent lymphocytosis. Alphaherpesviruses are generally considered to persist in the sensory nerve ganglion or in circulating lymphocytes, thus chan ges in viral load in circulating blood cells was presumed to be indicative of in fection with active vi rus replication. The presence of the virus was thus evaluated with a quantitative assay so the active virus replic ation event could be identified by the higher level of virus particles. Novel, specific TaqMan quantitative PCR assa ys were developed to measure the viral load of the alphaherpesviruses in the case dolphin. The complete blood counts (CBC) and serum chemistry parameters for the herpesvirus positive samples in th e case dolphin were compared to those of the negative samples, in an attempt to correlate viral presence with changes in clinical blood parameters. These assays were also used in a cross-sectional survey to establish a baseline prevalence of the two alphaherpesviruses in a population of clinically healthy bottlenose 9

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10 dolphins. DeHV-2 has a higher presence in the popul ation (3 animals) than DeHV-8 (which was only detectable in the case animal ). The simple presence of DeHV2 could not be associated with any specific blood parameter changes in the case animal. One positive case animal sample (Tt0719), however, had remarkable trends in four of the blood parameters analyzed: decreases in total white blood cell counts and seru m chloride as well as increases in erythrocyte sedimentation rate and hematocrit. Similar trends were not identified surroundi ng any of the other four DeHV-2 qPCR positive samples. There were an insufficient number of DeHV-8 positives for comparison of blood parameters associated w ith positive and negative samples.

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CHAP TER 1 INTRODUCTION Herpesvirales Most vertebrate species investigated have yielded at least one, and usually several, herpesviruses.37 More than 200 herpesvirus species have been identified to date.37 When investigating the lineage of herpesviruses, many major subdivisions mirror the phylogenetic branching order of their hosts.37 Thus, herpesviruses have co-evolved with their hosts and tend to be host specific.37 Since there are almost 5,500 di fferent species of mammals43 alone, it can be expected that the number of herpesviruses that exist in nature well exceeds the 200 species discovered thus far. All herpesviruses fall w ithin the newly establ ished taxonomic order Herpesvirales.9 Herpesvirales consists of three families: Herpesviridae (which includes the herpesviruses of mammals, reptiles, and birds), Alloherpesviridae (which includes the herpesviruses of fish and amphibians), and Malacoherpesviridae (bivalve herpesviruses).9,37 Additionally, the family of Herpesviridae contains three subfamilies: Alphaherpesvirinae Betaherpesvirinae, and Gammaherpesvirinae .9,37 Herpesviruses were traditionally assigned a phylogenetic classification based on their morphology. Current criteria also include DNA sequence similarity, genome arrangement, and relatedness of viral proteins.37 Herpesvirus characteristics such as their envelope and icosahedral capsid are clearly visible by transmission electron microscopy.37 Herpesviruses are among the larger viruses with the diameter of mature virions ranging from 120 to 260 nm.37 Other physical properties include having a single, linear double-stranded DNA genome (ranging from 124 to 290 kb in length), an envelope with viral glycoprotein spikes, and the tegument, a proteinaceous matrix which surrounds the icosahedral capsid and interfaces with the lipid envelope.9,37 Four biological characteristics are shared by members of Herpesviridae. The first characteristic is self11

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generation of a large array of enzy mes involvi ng nucleic acid metabolism, DNA synthesis, and processing of proteins.37 Secondly, the synthesis of viral DNA and the capsid assembly occurs in the nucleus but the final processing of the virion occurs in the cytoplasm.37 Thirdly, production of the infectious progeny virus is associat ed with destruction of the infected cell.37 Lastly, and most notably, all of the herpesviruse s examined to date are able to en ter into a latent state in their natural host.37 Herpesvirus Latency The ability to remain latent in their natural host is a unique and funda mental characteristic of herpesviruses and has been greatly studied. Wh ile the exact mechanism for reactivation is still not fully understood37, much is known about the latent a nd active virus stat es. Latent virus genomes transform to closed, circular molecule s where only a few of th eir genes are expressed.37 While in latency, they retain their ability to replicate and they can cause disease upon reactivation.37 Infected cells can all be in a latent stat e however herpesvirus can also be latent in some cells and simultaneously active in others.37 When herpesvirus is ly tically active in only a subset of infected cells, there are usually no symptoms of the infection in the host.37 Yet, active proliferation causing disease in th e host can still occur while some infected cells remain latent.37 The ability of herpesviruses to en ter latent states and then reactivate allows for chronic infection. Chronic herpesvirus infections occur when infe ctious progeny are pres ent within the animal system37 thus the virus is actively proliferating in some infected cells. The Subfamilies of Herpesviridae Alphaherpesvirinae There are four genera within Alphaherpesvirinae Viruses within the genera Simplexvirus and Varicellovirus infect mammals, whereas those of the genera Mardivirus and Iltovirus infect avian hosts .9,37 While reptile herpesviruses are also present in the subfamily Alphaherpesvirinae, 12

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they have not been assigned a genus.9,37 Alphaherpesviruses ( HVs) share several biological characteristics including a relatively s hort reproduction cy cle (about 18-20 hrs41 but can be shorter), a variable host range, rapi d spread in cell culture, efficien t destruction of infected cells, and the capacity to establish latency in the sens ory ganglion of the cranial and spinal nerves.37 Betaherpesvirinae There are also four genera in Betaherpesvirinae: Cytomegalovirus, Muromegalovirus, Roseolovirus, and the newly recognized genus Proboscivirus.9 Additional betaherpesviruses ( HVs) exist that are unassigned to a genus within this subfam ily, but they are all mammalian herpesviruses.9,37 HVs have not been identified for re ptiles or birds to date. Biological characteristics shared by HVs include a long reproducti ve cycle (about 48-72 hrs)35, often a restricted host range, slow growth in culture, infected cells fre quently become enlarged, and the virus can establish latency in secretory glands, lymphoreticular cells, kidneys, and other tissues.37 Gammaherpesvirinae Two new genera have been added to Gammaherpesvirinae to make four genera in this subfamily as well: Lymphocryptovirus, Rhadinovirus, Macavirus and Percavirus .9 None of the reptilian or avian gammaherpesviruses ( HVs) have been assigned to a genus.9,37 Furthermore, about 20 unassigned mammalian HVs have been recognized.9,37 Biological characteristics for HV species are loosely described as having a limited host range within the order of their primary host and latency is fre quently seen in lymphoid tissue.37 Herpesviruses of Humans Humans are an excellent model of herpesvi rus infection because human herpesviruses belong to a variety of genera and are associated with a multit ude of clinical presentations. Herpesviruses from each subfamily within Herpesviridae are known to infect humans.9,37 The severity of symptoms from infection can range from subclinical infection to death.26,47,58 There 13

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are eight known herpesviruse s iden tified with humans as their primary hosts, Human herpesvirus 1 8 (HHV-1 8).9,37 The first three are HVs: herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and Varicell a-zoster virus (VZV). The fourth is a HV, EpsteinBarr virus (EBV). Th e next three are HVs: cytomegalovirus (CMV), HHV-6, and HHV-7. Lastly, the eighth is a HV, Kaposis sarcoma-associated herpesvirus (KSHV).9,37 Herpes simplex virus type 1 and 2 are common infections worldwide.58 A large-scale serosurvey of adults residing in the United States, indicated a seroprevalence of 57.7% and 17% for HSV-1 and HSV-2, respectively.58 HSV-1 is usually transmitte d via direct contact during childhood.58 In contrast, HSV-2 is almost always sexua lly transmitted and is the typical etiologic agent for genital herpes.58 Most HSV-1 and 2 infections ar e subclinical but they can cause ulcerative orogenital le sions as well as encephalitis and death.58 The severity of clinical symptoms following primary infection is directly indicative of the frequency and severity of reactivation symptoms.41 The seroprevalence for VZV infection of adults living in the United States is extremely high at 99.6%.26 Varicella primary infection typically results in the highly contagious childhood disease chickenpox.8 The classic symptom is developing a rash with fluid-filled vesicles surrounded by irregular erythema ma rgins on the head or trunk be fore spreading to cover the entire body.8 The virus establishes latency in the dor sal root ganglion of the spinal nerves.8 Reactivation of VZV causes a disease syndrome called shingles, or herpes zoster, most commonly affecting the elderly.8 With reactivation, the vesicular cutaneous lesions are typically well-delineated and follow one or more sensory nerve ganglia,8 instead of the full body rash seen in chickenpox. Varicella zoster vi rus is known to cause fatalities, and thus a vaccine was created which has been used routinely during childhood immuniza tions as of 1996.8,26 14

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Epstein-Barr virus, of the genus Lymphocryptovirus9,37, is a very successful virus infecting over 90% of humans worldwide.7 Both Burkitts lymphoma and infectious mononucleosis are associated with EBV.7,23 The virus maintains persistent infection in the B cells of the oropharynx.7 Epstein-Barr virus is most pathogenic in immunocompromised individuals and is typically not treated when diagnosed in those with uncomplicated disease.7 Cytomegalovirus seroprevalence for adults in the United States is 58.9% and it is transmitted by close contact.47 CMV is unique among herpesviruse s in that it can also be vertically transmitted.35 It is the leading cause of congenital illness, affecting 0.2-2.2% of all newborns.47 Symptoms from CMV infection in newbor ns include, vision and/or hearing loss, mental retardation, neurologic abnormalities, and death.47,48 Cytomegalovirus is usually a lifelong latent infection for adults and is only pathogenic in immunocompromised patients.48 However, CMV can also cause heterophile-neg ative mononucleosis-like infection in adults (attributes to 20 50% of the cases).23,49 Human herpesvirus 6 and 7 are both ubiquitous viruses.29,59 The seroprevalence of HHV6 ranges between 80% and 100% in adults under 40 years old.30 Furthermore, seroconversion typically occurs by age 2.29,59 Type 7 seroprevalence is estimated to be higher than 85% in the adult population with most seroc onverting as young children as well.29,59 Both HHV-6 and 7 are in the same genus, Roseolovirus and the pathogenesis of rose olovirus infections is not well characterized.59 Human herpesvirus 6 is known to be associated with exanthem subitum, a disease causing fever and rash in infants.59 Additionally, HHV-6 has been implicated as the cause of multiple sclerosis (MS),59 and it has been associated w ith some cases of heterophilenegative mononucleosis-like illness.23,49 15

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Kaposis sarcom a-associated herpesvirus, within the genus Rhadinovirus,9,37 is the rarest of all known human herpesviruses with an adul t seroprevalence in th e United States of 2.57.1%.14 As the name suggests, KSHV is the etiologic agent of Kaposis sarcom a, a histologically complex tumor containing multiple cell types within each lesion.20 Although rare, with the 1980s pandemic of human immunodeficiency vi rus (HIV), KSHV became the most common neoplasm complicating acquired i mmunodeficiency syndrome (AIDS).20 Seroprevalence data indisputably shows the unive rsal presence of herpesviruses. Most are common and widespread with infections causing minimal, usually mild disease like chickenpox. However, they are often also associated with mo re severe pathologies su ch as herpes simplex virus induced encephalitis. The seroprevalence of multiple herpesviruses in humans is not unusual. They are evolutionarily old viruses that have co-evolved with their host.37 Thus, it is not uncommon to find multiple species within alpha, beta-, and gammaherpesvirinae present in a host. For example, five herpesviruses (four HVs and 1 HV) have been identified in horses (and four more for the family Equidae ).9,37 Additionally, four herpesviruses (three HVs and one HV) have been identified in cows (and six more for the family Bovidae).9,37 This prevalence is comparable to the eight human herpesvi ruses and four additional viruses (one HV and three HVs) within the family Hominidae .37 Cetacean Herpesviruses The presence of herpesviruses in cetacean s (the group of animals consisting of all porpoises, dolphins, and whales) has been rec ognized since the late 1980s. In 1988, herpesviruslike viral particles were identif ied by electron microscopy (EM) in skin biopsies collected during the necropsy of a wild beluga whale ( Delphinapterus leucas ).32 The juvenile, female beluga whale stranded in the St. Lawrence Estuary, Canada.32 Biopsies were collected because the beluga had generalized dermatitis that consisted of circular, pale lesions.32 Pathologic findings 16

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also in cluded several abnormalities that could not be associated with herpesvirus infection including a perforated gastri c ulcer with acute exudative pe ritonitis, verminous bronchopnemonia, and chronic hepatitis.32 Herpesvirus-like viral pa rticles were identified by transmission electron microscopy (TEM) within skin biopsies of a second beluga whale with focal dermatitis in 1989.1 In this instance, the dermatitis developed in captivity a few months after the young adult, female beluga was colle cted in the Churchill River, Canada and transported to an oceanarium.1 In 1992, a herpesvirus was associated with an encephalitis case in a harbor porpoise ( Phocoena phocoena).25 A juvenile, female harbor porpoise was found stranded on the coast of Sweden in 1988.25 Necropsy findings describe white, flat and slightly raised foci on the skin but herpesvirus could not be identified by TEM or immunoperoxidase staining.25 However, a large amount of herpesvirus-like viral particles we re identified, by TEM, in the cerebral cortex tissues.25 Immunoperoxidase staining of these tissues showed cross-reactivity with HSV-1 and BHV-1 ( Bovine herpesvirus 1 an alphaherpesvirus) suggesting the herpesviral antigen was an alphaherpesvirus.25 In 1994, herpesvirus-like viral particles were again identified by TEM in the skin biopsies of two Peruvian dusky dolphins ( Lagenorhynchus obscures ) with raised black foci on their rostrum.50 These lesions were seen on two more animal s at the same time but viral particles were not detectable by TEM.50 In 1999, the first serologic evidence of exposure of cetaceans to herpesviruses was generated. Between 1995 and 1997, beluga whales in the St. Lawrence Estuary were tested by serum neutralization (n=13) and enzyme-label ed immunosorbent assay (ELISA; n=12) using BHV-1 antigen.34 The researchers concluded that an alpha herpesvirus was likely endemic to this 17

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population since 46% of the anim als were positiv e with the serum neutra lization test and 58% demonstrated reactivity by ELISA.34 The first cetacean herpesviral genomic sequences, derived from bottlenose dolphins ( Tursiops truncatus ), were not published until 2001.2 Two distinct alphaherpesviruses were reported in two separate, singl e bottlenose dolphin strandi ngs along the Atlantic coast.2 The herpesviruses were associated with acute necr otizing lesions on multiple organs by histology, electron microscopy (EM), and pol ymerase chain reaction (PCR).2 The first stranding occurred in 1995 where a juvenile, female bottlenose dolphin was found dead on Hilton Head Island, South Carolina.2 Herpesvirus particles were identified in the thymus by EM and unfixed lung tissue was used in two consensus PCRs to obtain partial sequence of the DNA-dependent DNA polymerase (Dpol) and terminase genes (GenBank accession numbers AF196646 and AF196647, respectively).2 In 1999, a second juvenile, female bottlenose dolphin was found alive, stranded on a beach in Dela ware, but died s hortly thereafter.2 Herpesvirus particles were identified by EM in tissues fr om the heart, snout, and tongue.2 No other tissues were examined. Efforts were also made to obtain Dpol and te rminase sequence from the heart tissue, following the same protocols as the first case, but only sequ encing of the partial Dpol gene was successful (GenBank accession number AF245443).2 For the purposes of clarity, these viruses are tentatively named Delphinid herpesvirus 1 and 2 (DeHV-1 and DeHV-2), respectively. Although not yet recognized by the Inte rnational Committee on Taxonomy of Viruses (ICTV), they are considered separate species because DeHV-1 (A F196646) has only a 61% amino acid identity to DeHV-2 (AF245443) in the highly conserved Dpol region. The proposed names are derived from the host family and order of virus discovery, in accordance with current ICTV naming conventions for herpesviruses.9,10 18

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In 2006, a third novel alphaherpesvirus was reported in a stranded bottlenose dolphin.31 This juvenile, male dolphin was found alive in 2004 along the west coas t of Florida and was transported to a rehabilitation facility.31 The dolphin had multiple attack wounds and hundreds of 1-3 mm raised, black papules distributed primarily over the dorsal and la teral portions of its body.31 These papules slowly progressed to superficia l, gray erosions and decreased over time.31 Meanwhile, herpesvirus-like pa rticles were identified using TEM on lesion biopsies.31 Molecular characterization of the viral part icles yielded a partial Dpol he rpesviral sequence via consensus PCR (GenBank accession number AF757301).31 This latest alphaherpes virus, tentatively named DeHV-3 in this study, has an amino acid identi ty of 70% to DeHV-1 (AF196646) and 60% to DeHV-2 (AF245443). The first cetacean gammaherpesviruses were also reported in 2006. Gammaherpesviruses were identified with consensus Dpol primers in mucosal lesion s of four separate cetacean species: a Rissos dolphin ( Grampus griseus ), a dwarf sperm whale ( Kogia sima ), a Blainvilles beaked whale (Mesoplodon densirostris ), and five Atlantic bottlenose dolphins.45,46 The majority of samples were from penile and vagi nal lesions. However, an identical HV Dpol sequence was identified from both an oral lesion and a penile lesion of one bottlenose dolphin.45,46 This dolphin was an adult male that stranded off Islamorada Key, Florida and later died of sepsis.45 Two more bottlenose dolphins also had the identical gammaherpesvirus Dpol sequence identified. This first bottlenose dolphin gammaherpesvirus will be referred to as DeHV-4 (GenBank accession number AY952777, additional identical sequence accession numbers AY952778, AY952779, AY949831). The other two bottlenose dolphins had a distinct gammaherpesviral sequence, 7-8 amino acids different from DeHV-4 (85-87% amino acid identity). This second gammaherpesvirus will be referred to as DeHV-5 (GenBank accession number AY952776, 19

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additional sequence, with 1 amino acid diffe rence, accession number DQ288667). Since Rissos dolphins are of the family Delphinidae, the gammaherpesviral sequence generated from that dolphin is tentatively named Delphinid herpesvirus 6 (DeHV-6; GenBank accession number DQ288666). Likewise, the gammaherpesvirus from the dwarf sperm whale is tentatively named Kogiid herpesvirus 1 (KoHV-1; AY949830) and the gammaher pesvirus from the Blainvilles beaked whale is tentatively named Ziphid herpesvirus 1 (ZiHV-1; AY949828). Moreover, in 2006, DeHV-1, 2, and 3 Dpol se quences were also obtained from two captive bottlenose dolphins with skin lesions. The viral genomic se quence amplified from a skin lesion of the first dolphin (AY949831) was only 2 amino acids different from DeHV-1. A blood sample from the second dolphin generated herp esviral sequence (DQ295064) identical to DeHV2, whereas a spleen sample generated sequen ce (DQ295063) only one ami no acid different from DeHV-3. Additionally, a third herpesvirus seque nce (AY608707) was amplified from a skin lesion of the dolphin which was 4 amino acids (9 3% amino acid identity) different from DeHV1. HSV-1 and 2 only have 5 amino acid differences in the homologous Dpol region so it is likely that sequence AY608707 represents a separate species. Thus, this alphaherpesvirus, AY608707, is tentatively named DeHV-7. Criteria for Disease Causation While the presence of herpesvirus in abnormal tissues is an essential argument for association it is not sufficient fo r establishing said herpesvirus as the cause of a specific lesion or disease. Surpassing association a nd fulfilling the requirements to es tablish causation is the spirit of the three Henle-Koch postulates.40 The first postulate states that the parasite causing the disease in question should occur in every case where the disease is present under circumstances which can account for the pathological and clinical course of the disease.40 Second, the agent cannot occur in any other disease as a fortuitous or non-pathogenic parasite.40 Third, after being 20

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isolated and grown in culture, experim ental exposure can induce identical disease in a normal host.40 While the scientific community embraced the postulates they were often found to be too stringent, especially since many pa thogens were difficult to culture.15,40 With the discovery of viruses, it became even more difficult to fulfill the Henle-Koch postulates when establishing etiol ogic viral agents for diseases.15,40 Not only are some viruses difficult to culture, but some viruses that co uld be cultured were found in both healthy and diseased individuals.15 Alfred Evans and others highlighted the limitations of the Henle-Koch criteria for viral agents.15 First, the same pathologic or c linical states can be produced by different etiological agents. Second, the causativ e agents may vary by geographic areas, age groups, or different patterns of host susceptibility Third, some diseases require the presence or action of two or more agents. Fourth, a si ngle agent may produce different clinical or pathological responses in differe nt settings. Fifth, any cause or set of causes usually produces a biological gradient of response fr om no detectable symptoms to m ild clinical signs to classic, recognized disease. Finally, the severity of host response following exposure will vary with host characteristics such as genetics, age, immunologi c status, socio-economic status, and exposure to other cofactors (infection, drugs, etc).15 Thus, it is necessary to consider multiple elements when proposing the etiology of a disease. Evans also stressed that even though it may not be possible to fulfill the Henle-Koch postulates in establis hing causation, there should be a justification which makes biological sense, using sound reasoning, for the proposed cause.15 These sentiments were echoed by David Fredri cks and David Relman in their assertion that Kochs postulates inspire scientific rigor to the process of establishing causality, but they are too stringent for todays times of sequence-base d identification of viral and microbial agents.19 Fredricks and Relman elaborated on the limitations of Kochs postulates for disease causation in 21

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light of modern molecular techniques. For instance, highly sensitive PCR assays, theoretically capable of detecting as few as one target molecule detect the presence of clinically irrelevant quantities of microbes. In rec ognition of the vastly growing da tabase of micr obial pathogens from sequence-based investigation, seven guide lines for establishing causation were proposed. First, nucleic acid sequence for the suspected caus e should be found in most cases of the disease and preferentially in tissues with evidenced pathology. Second, fewer, or no, copy numbers of pathogen-associated nucleic acid sequences should occur in host or tissues without disease. Third, with treatment, the copy number should decrease or beco me undetectable and as such increase during relapse. Fourt h, causation is compelling when se quence detection predates the disease or copy number correlates with the severity of the dis ease. Fifth, the nature of the microorganism should be consistent with known ch aracteristics or the ch aracteristics of those phylogentically-related. Sixt h, sequence from tissues should be i nvestigated at the cellular level, including efforts to demonstrate specific in situ hybridizatio n of the sequence to the tissue pathology. Seventh, sequence-based forms of evidence for causation should be reproducible. These guidelines were put forth to establish a compelling argument regarding disease etiology but are not intended for strict adherence.19 Correlating Clinical Signs with Bottlenose Dolphin Herpesviruses During the routine, conventional PCR-based, viral surveillance of a managed collection of bottlenose dolphins, two alphaherpesviruses were detected in the buffy coat of one animal. The 22-year-old, female bottlenose dolphin ha d a history of elevated transaminases and dehydrogenases, which were normalized followi ng phlebotomy treatment, and a persistent lymphocytosis. Furthermore, the dolphin did not ha ve any skin lesions, unlike the majority of the previous reports of herpesvirus in cetaceans. Thus, the clinical significance of this finding was unclear. Alphaherpesviruses are generally c onsidered to persist in the sensory nerve 22

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ganglion3,11,21,41 thus the presence in ci rculating blood cells was presumed to be indicative of infection with active virus replic ation. However, it has also been suggested that in some hosts, such as the horse, alphaherpesviruses can persist in the white cell fraction.6,44,55 Regardless of the site of persistence and/or repl ication, changes in the virus load in the circulating white blood cells, as measured using a quantitative PCR a ssay, reflect the level of replication of the herpesvirus. This virus load, or virus c opy number per sample, cannot be assessed using conventional PCR. Real-time quantitative PCR (qPCR) assays have been applied in diagnosing or monitoring herpesviral infections including the human herpesviruses22,27,36,48,54 and Equid herpesvirus 1 and 412 (EHV-1 and 4). Quantitative PCR has a faster turn-around time and a higher throughput than traditional PCR.27,48 The TaqMan qPCR method me asures the quantity of PCR products with a specific fluorogenic probe, specific primers, and real-time laser scanning in a 96-well plate.27,36 When these quantities are compared to known standards, the viral load in each sample can be determined. Therefore, TaqM an qPCR assays were developed to measure the viral load of the alphaherpesviruses in the case dolphin. The complete blood counts (CBC) and serum chemistry parameters for the herpesviru s positive samples in the case dolphin were compared to those of the negative samples, in an attempt to correlate viral presence with changes in clinical blood parameters. These assays were also used in a cross-sectio nal survey to establish a baseline prevalence of the two alphaherpes viruses in a population of clinically healthy bottlenose dolphins. The Fredricks-Relman criteria for establishing causality of disease require the association of viral load with disease.19 During the presence of clinical signs, a greater vi ral load, from lytically active herpesvirus, in the bloodstream is expected. For example, in the case study, time 23

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24 points with high viral loads could correlate with inflammation. Time points with low or undetectable viral loads would r eciprocally correlate with th e absence of inflammation and normal blood parameters. Following the causality of disease criteria, the case for DeHV-2 as the cause of inflammation would be strengthened if other dolphins in the population also had inflammation concurrent with increased DeHV-2 viral load. Thus, a qPCR assay was developed as a tool for investigating possible alphaherpes viral diseases because it can quantify the viral load in an animal.

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CHAP TER 2 QUALITATIVE AND QUANTITATIVE PCR A SSAYS FOR THE DETECTION OF HERPESVIRUSES Introduction Real-time quantitative PCR (qPCR) assays have been applied in diagnosing and monitoring herpesviral infections including the human herpesviruses22,27,36,48,54 and Equid herpesvirus 1 and 412 (EHV-1 and 4). Quantitative PCR has a faster turn-around time and a higher throughput than traditional PCR.27,48 The TaqMan qPCR method me asures the quantity of PCR products with a specific fluorogenic probe, specific primers, and real-time laser scanning in a 96-well plate.27,36 When these quantities are compared to known standards, the viral load in each sample can be determined. Alternative re al-time methods also exist that do not use a specific probe. Real-time detection during amp lification can be accomplished with SYBR Green, a fluorescent dye that will bind to any double-stranded DNA.36 However, there is increased nonspecific binding due to the broad nature of the dye.36 Two specific TaqMan qPCR assays were developed for the detection of Delphinid herpesvirus 2 and 8 (DeHV-2 and -8). Both of these assays target the DNA-dependent DNA polymerase (Dpol) region of the virus. Materials and Methods Sample Collection The case dolphin was part of a managed coll ection and was housed in a coastal, open ocean water enclosure. Sixteen serial blood samples were collected between July 12, 2004 and December 17, 2007 from the caudal peduncle vein of the case dolphin using a 20 or 21 gauge 1.5 inch Vacutainer needlea or from a fluke vein using a 21 ga uge 1 inch butterfly needle. An EDTA (K3) Vacutainer tube containing whole blood was us ed for collection of total white blood cell fraction (buffy coat). The blood tube was centrifuged at 3,000 rpm at 21C for 10 25

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m in. Approximately 100 l containing the buffy coat fraction, visible at th e interface of the red and white blood cell fraction, was co llected via gentle aspiration. An additional three samples were collected including a mucosal swab from a harbor seal ( Phoca vitulina ), blowhole exudate from an orca ( Orcinus orca ), and an eye swab from a California sea lion ( Zalophus californianus), and a consensus PCR for herpesvirus was performed. Consensus PCR DNA was extracted from the buffy coats via a commercial kit according to manufacturers instructions (DNeasy Blood and Tissue Kitb). PCR was performed on all sixteen case animal samples with nested consensus primers for the DNA-dependent-DNA polymerase (Dpol) of herpesviruses.51 Phocid herpesvirus 2 isolate, kindly provided by Dr. Tracey Goldstein, was used as a positive control in the consensus PCR and was also sequenced. Reactions were amplified in a Px2 thermal cyclerc using Platinum Taq DNA Polymerased in 30L reactions following manufacturers instructions. Amplification conditions were as follows: initial denaturation at 94C for 5 min; 45 cycles of amplification with each cycle consisting of denaturation at 94C for 30 s, annealing at 46C for 1 min, and elongation at 72C for 1 min; a final elongation step was performed at 72C fo r 7 min followed by a 4C hold. Secondary PCR products were resolved on a 1.5% agarose gel made up in 1X TBE and containing 1g of ethidium bromide per mL. Bands of expected si ze (170-315bp) were excised and purified using QIAquick Gel Extraction Kit.b Direct sequencing was performe d with the Big-Dye Terminase Kite using the second round primers, TGV and IYG.51 This was followed by adding 2L of 2.2% SDS to each forward and reverse 20 L sequencing reaction. Reactions were returned to the thermal cycler and heated to 98C for 5 min, 25 C for 10 min, and held at 4C. Unincorporated dye terminators were removed with the DyeEx 2.0 Spin Kitb and dried via vacuum centrifugef at 26

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1,400 rpm for 25-30 min. Sequencing reactions were submitted to the University of Florida Interdisciplinary Center for Biotechnology Research and analyzed on ABI 3130 DNA sequencers.g Primer sequences were edited out prio r to further analysis. Sequences were confirmed via translated nucleot ide-translated nucleotide and protein BLAST search in the National Center for Biotechnology Information (NCBI) database ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Bayesian and Maximum Likelihood Phylogenetic Analysis A m ultiple sequence alignment computer program (MUSCLE13) was used to align a 5864 amino acid predicted homologous region of 19 alphaherpesvirus DNA-dependent DNA polymerase sequences. Similarly, MUSCLE was us ed to align a 55-60 amino acid predicted homologous region of 20 gammaherpesvirus DN A-dependent DNA polymerase sequences. Two separate analyses were run because the predic ted amino acid region was too small for the two groups to be analyzed together. References and GenBank accession numbers for the Delphinid herpesviruses are shown in Table 2-1. The MU SCLE alignments were analyzed with MrBayes 3.142 with gamma-distributed rate variation plus a proportion of invariant sites, and mixed amino acid substitution models. Four chains were run and statistical convergence was assessed by looking at the standard deviation of split frequenc ies as well as potential scale reduction factors of parameters. The first 10% of 1,000,000 ite rations were discarded as a burn-in. Iguanid herpesvirus 2 (GenBank accession number AY236869) was designated as the outgroup for the alphaherpesvirus alignment due to its early divergence from other herpesviruses.33,56 Likewise, Elephantid herpesvirus 1 (GenBank accession number AF322977) was designated as the outgroup for the gammaherpesvirus alignment. Maximum likelihood (ML) analys es of each amino acid alignment was performed using computer based phylogenic modeling software, PHYLIP (Phylogeny Inference Package, Version 27

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3.66),17 and by running each alignment in ProML (p rotein maximum likelihood) with amino acid substitution models Jones-Taylor-Thornton24 and Probability Matrix from Blocks (PMB).52 Both models were set for global rearrangements, five replications of rando m input order, less rough, gamma plus invariant rate dist ributions, and unrooted. The value for the alpha of the gamma distribution was taken from the Ba yesian analysis, and the pro portion of invariant sites was directly taken from the data set. The alignments were also us ed to create data subsets for bootstrap analysis to test the strength of the tree topology (100 re-samplings),16 which were analyzed using the amino acid substituti on model producing the most likely tree. Quantitative PCR Prior to qPCR analysis, sample concentration and purity (260/280 absorbance ratio) was tested using 1.5L of DNA extract by spectrophotometry (NanoDrop 8000c). Specific primers and probes (forward primer, DeHV-2 MGB 50F [5 -GGG AAA TCT GAA GTT AAA CGC TCT A-3 ]; reverse primer, DeHV-2 MGB 113R [5 -TGA GAA CTC GCC AGT ATT TGC A3 ]; probe, DeHV-2 76T [6 FA M-CGT TGG CCA TCG GT-MGBNF Q]; forward primer, DeHV8 MGB 69F [5-TTA GCA CGC GCG ATT ACC T-3]; revers e primer, DeHV-8 MGB 143R [5-CGC ACA CGT TCG CAA AGT-3]; probe, DeHV-8 104T [6 FAM-ACC CGT GAA CAG CTG-MGBNFQ]) targeting the D pol gene were designed for Delphinid herpesvirus 2 and 8 via commercial software (Primer Express software v2.0) and synthesized.g A 0.9M working dilution was used for the primers and 0.25M for the probe in each 20L reaction using a commercial universal qPCR mix (TaqMan Fast Universal PCR Master Mix 2X).g The maximum amount of DNA template equaled 7uL or 100ng per well. A 7500 Fast Real-Time PCR Systemg was used to amplify the reactions with cycling conditions as follows: initial denaturation at 95C for 20 s; 50 cycles of 95C for 3 s followed by 60C for 30 s. Data 28

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collection w as at the annealing/extension pha se where the fluorescence crosses the cycle threshold line (Ct), set at 0.3. Analytical sensitivity of the qPCR assay was tested using a 10-fold serial dilution of cDNA, nested PCR product from the case dolp hin. For DeHV-2, the 10-fold serial dilution started at 2 million copies (1:105 dilutions) and continue d to 2 copies (1:1011). For DeHV-8, the 10-fold serial dilution started at 2 million copies (1:105 dilutions) and continued to 20 copies (1:1010) followed by two 2-fold dilutions to 5 copies The virions per L ra tio (viral load) was calculated by multiplying the sample concentra tion by Avogadros number and dividing this total by the total of the amplic on length (224bp and 172bp for DeHV2 and DeHV-8, respectively) multiplied by the average base pair weight (656.6x109). The resulting mean Ct values for the serial dilutions were used to generate the standard curve. Results Consensus PCR Herpesviral DNA was detected in two samples, labeled Tt0625 and Tt0724, using the consensus PCR assay (Table 2-1). Protein BLAST (BLASTp) analysis of resulting sequence from Tt0625 showed a 100% amino acid identity to DeHV-2 (GenBank accession number AF245443). Sample Tt0724 contained a herpesviral sequence most closely related to DeHV-3 (AY757301) with an 87% amino acid identity. Since the Dpol region is highly conserved, this sequence likely represents a new bottlenose dolphin herpesvirus and is tentatively named DeHV8. The partial Dpol sequence of DeHV-8 was submitted to GenBank (accession number bankit1249098). The remaining 14 case study sample s were negative on consensus PCR (Table 2-2). Genetically distinct herpesvi ral sequences were detected in the harbor seal, orca, and California sea lion samples. The translated amino acid sequence from the harbor seal sample was 29

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61% iden tical to a ly mphocryptovirus of a golden-handed tamarin ( Saguinas midas ; GenBank accession number AY166692). The orca sample had an amino acid identity of 87% to DeHV-1 (AF196646). BLASTp analysis of the California sea lion sequence showed a 70% identity to Mustelid herpesvirus 1 (MusHV-1; AF275657). The partial D pol sequence of PhHV-2 was also most similar to MusHV-1 with a 72% amino acid identity. These novel DPol herpesviral sequences have been submitted to GenBank under the following accession numbers: Delphinid HV 8 (DeHV-8, from Tursiops truncatus ; bankit1249098), DeHV-9 (from Orcinus orca ; bankit1249103), Otariid HV 2 (OtHV-2, from Zalophus californianus bankit1248734), Phocid HV 2 (PhHV-2, bankit1249104), and PhHV-5 (from Phoca vitulina ; bankit1249107). Bayesian and Maximum Likelihood Phylogenetic Analysis The MUSCLE alignments of the 19 alphahe rpesviral and 20 gammaherpesviral Dpol sequences are shown in Table 2-3 and 2-4, resp ectively. Bayesian analysis concluded the Wag model of amino acid substitution was most probable with a posterior probability of 0.930 for the alphaherpesvirus alignment. For the gammaherpesvirus alignment, the Jones model was most probable with a posterior probability of 0.768 followed by the Wag model with a posterior probability of 0.232. ML analysis showed the most likely tree, for both alignments, was from the JTT model of amino acid substitution and these pa rameters were used in the bootstrap analysis. The Bayesian tree of the alphaherpesvirus MUSCLE alignment is shown in Figure 2-1 along with the bootstrap values from ML anal ysis. The delphinid alphaherpesviruses were genetically diverse and formed three dist inct phylogenetic clades. DeHV-1 and DeHV-7 clustered as a monophyletic group with DeHV-9, the orca herpesvirus. Additionally, DeHV-3 and DeHV-8 clustered together. DeHV-2 formed a monotypic clade and did not cluster with any of the other alphaherpesvirus us ed in the analysis. None of the delphinid alphaherpesviruses clearly associated with an esta blished alphaherp esviral genus. 30

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The Bayesian tree of the gamm aherpesvir us MUSCLE alignment is shown in Figure 2-2 along with the bootstrap values from ML anal ysis. All of the cetac ean gammaherpesviruses formed a monophyletic group. Within the cetac ean gammaherpesvirus clade, ML bootstrap values were weak thus the br anching order could not be relia bly determined. None of the delphinid gammaherpesviruses clustered with a species in an established gammaherpesvirus genus. The novel California sea lion herpesvirus (OtHV2) and three of the phocid herpesviruses (PhHV-2, PhHV-3, and PhHV-4) formed two distin ct phylogenetic clades (Figure 2-2). OtHV-1 and PhHV-5 each formed a distinct monotypic clad e. None of the pinniped gammaherpesviruses clustered with an established gammaherpesviral genus. The analysis al so shows that EHV-7 clusters with the other Equi d herpesviruses in the genus Percavirus. The ML bootstrap values, however, were weak for this cluste r so the confidence interval for branch order within this cluster is low. Quantitative PCR The DeHV-2 qPCR assay accurately detected 2 to 2 million virus copies (see Figure 2-3). To reduce false positives, the last serial dilution, 2 copies, was discarded and the detection limit was conservatively set at 10 copies (Ct = 35.90). The standard curve (see Figure 2-4) for the DeHV-2 qPCR assay had a slope of -3.30 and a high correlation coefficient (R2) of 0.994. The DeHV-8 qPCR assay accurately detected 5 to 2 million virus copies (see Figure 2-5). Again, the last serial dilution, 5 copies, was disc arded and the detection li mit was conservatively set at 10 copies (Ct = 36.67). The standard curve (see Fi gure 2-6) for the DeHV-8 qPCR assay had a slope of -3.1 and a R2 of 0.990. 31

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Discussion Two bottlen ose dolphin herpesviruses, one of which is a novel alphaherpesviruses, were identified by consensus and specific PCR in the case dolphin. Add itionally, three novel herpesviruses (DeHV-8, DeHV-9, OtHV-2) were identified and one novel Dpol herpesviral sequence was generated from the PhHV-2 cont rol isolate by consensus PCR. Current ICTV (International Committee on Taxonom y of Viruses) naming conventions for herpesviruses use host family and order of virus discovery.9,10 Each of these novel herpesviruses ( Delphinid herpesvirus 8 Delphinid herpesvirus 9 Otariid herpesvirus 2 and Phocid herpesvirus 5 ) was tentatively named in accordance with these guid elines. Bayesian and ML phylogenic analysis supports the classification of the isolates as novel alphaand gammaherpesviruses. It is unclear, based on the limited Dpol sequences, whether the delphinid and pinniped herpesviruses belong to established genera or represent novel genera. Mo re viral genomic sequence would be needed to further clarify their phylogenetic placement. The DeHV-2 and DeHV-8 qPCR assays should prove useful as primary screening tools for these alphaherpesviruses in blood samples from bo ttlenose dolphins. The qPCR assays are faster and have a higher throughput than traditional PCR. The DeHV-2 qPCR assay has a greater amplification intensity and a better slope comp ared to the DeHV-8 qPCR assay. Nevertheless, both qPCR assays are very sensitive with a lower detection limit of 10 virus copies. 32

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Table 2-1. P roposed names, abbreviations, and sources for marine mammal Dpol herpesviral sequences used in this study Name Abbreviation Species Reference GenBank accession number Delphinid HV 1 DeHV1 Tursiops truncatus Blanchard et al (2001) AF196646 Delphinid HV 2 DeHV2 Tursiops truncatus Blanchard et al (2001) AF245443 Delphinid HV 3 DeHV3 Tursiops truncatus Manire et al (2006) AY757301 Delphinid HV 4 DeHV4 Tursiops truncatus Smolarek Benson et al (2006) AY952777 Delphinid HV 5 DeHV5 Tursiops truncatus Smolarek Benson et al (2006) AY952776 Delphinid HV 6 DeHV6 Grampus griseus Smolarek Benson et al (2006) DQ288666 Delphinid HV 7 DeHV7 Tursiops truncatus Smolarek Benson et al (2006) AY608707 Delphinid HV 8 DeHV8 Tursiops truncatus This study bankit12490 98 Delphinid HV 9 DeHV9 Orcinus orca This study bankit12491 03 Kogiid HV 1 KoHV-1 Kogia sima Smolarek Benson et al (2006) AY949830 Otariid HV 1 OtHV-1 Zalophus californianus King et al (2002) AF236050 Otariid HV 2 OtHV-2 Zalophus californianus This study bankit12487 34 Phocid HV 1 PhHV-1 Phoca vitulina King et al (1998) U92269 Phocid HV 2 PhHV-2 Phoca vitulina This study bankit12491 04 Phocid HV 3 PhHV-3 Monachus schauinslandi Goldstein, Gulland, et al (2006) DQ093191 Phocid HV 4 PhHV-4 Mirounga angustirostris Goldstein, Lowenstine, et al (2006) DQ183057 Phocid HV 5 PhHV-5 Phoca vitulina This study bankit12491 07 Ziphid HV 1 ZiHV-1 Mesoplodon densirostris Smolarek Benson et al (2006) AY949828 ______________________________________________________________________________ 33

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Table 2-2. Sampling data and consensus herpesvirus PCR results for case study samples Sample ID Collection Date Consensus PCR _____ ______________________________________________________ Tt0625 11/8/2006 Positive Tt0631 12/13/2006 Negative Tt0634 12/20/2006 Negative Tt0701 11/22/2006 Negative Tt0718 10/20/2005 Negative Tt0719 8/17/2006 Negative Tt0720 10/25/2006 Negative Tt0721 1/10/2007 Negative Tt0724 2/7/2007 Positive Tt0730 2/21/2007 Negative Tt0748 2/28/2007 Negative Tt0777 6/26/2007 Negative Tt0804 12/17/2007 Negative Tt0901 7/12/2004 Negative Tt0902 8/2/2004 Negative Tt0903 9/22/2006 Negative _____________________________________________________________________________________________________________________ 34

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Table 2-3. MUSCLE al ignment of 19 alphahe rpesvirus Dpol amino acid sequences.* IgHV2 AHGYLPCLSIAASITSIGRTMLLKTRDFIHTSWATRENLCSSVSTLPLETVGPD-----YSMKV GaHV1 MHGMLPCLEVASTVTAIGRDMLLRTKAHIEKEWRSGNQFAEKFLPGSERIQLNQ-----YSVRV PsHV1 MNGMLPCLEVAATVTAIGRDMLLKTKQYIEENWREYSNIRERFFPAMAHEGVPQ-----YSVAV GaHV2 SNGLLPCIDVAATVTTIGRNMLLTVRDYIHKQWGTRDALLREFPNLSNFMRPED-----YSVSV MeHV1 ANGMLPCIDVAASVTTIGRNMLLTVRDYIHDQWGDKSSIMCKFPELENFMQNKE-----YSVDV HHV3 AQGFLPCLYVAATVTTIGRQMLLSTRDYIHNNWAAFERFITAFPDIESSVLSQK----AYEVKV DeHV2 AHGLLPCLPVAATVTTIGRDMLLRTRQYLHDRWPTVERLTSDFPEVVSMFIPNT----EYSIRV PhHV1 SNGLLPCLHIAATVTTIGRSMLLATQSYVESRWATRELLEKDFPGSSSIAIPKK----SYSVNI BHV2 QRGLLPCLPVAATVTTIGRDMLLATRDYVHSRWVSFDGLVMDFPEAAAIRGEGE-----YSMRI HHV1 QHGLLPCLHVAATVTTIGREMLLATREYVHARWAAFEQLLADFPEAADMRAPGP-----YSMRI HHV2 QHGLLPCLHVAATVTTIGRDMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGP-----YSMRI DeHV9 AQGLLPCLHIAATVTTIGRDMLLQTRDYLHTHWATAERLVEDFDGAAAALLTAP-SAPPYSIHV DeHV1 AHGLLPCLQIAATVTTIGRDMLLRTRDYLHAHWATAERLVADFDGAAAALLASP-PAPPYSIHV DeHV7 AQGLLPCLQIAATVTTIGRDMLLQTRDYLHARWATAERLVADFDGAAAALLSSP-PAPPYSIHV DeHV3 AQGLLPCLPIAATVTTIGRDMLLSTRDYLHSRWATREQLAADFGDAYASPAPISPSASPYSIRV DeHV8 AQGLLPCLPIAATVTTIGRDMLLSTRDYLHSRWATREQLVADFANVCASPAPGPPSASLYSIRV EHV3 -NGLLPCLRIAATVTTIGRDMLLGTRDYVHARWATRELLEANFPEARAHRADGP-----YSVRV EHV1 ANGLLPCLRIAATVTTIGRDMLLKTRDYVHSRWATRELLEDNFPGAIGFRNHKP-----YSVRV EHV9 ANGLLPCLRIAATVTTIGRDMLLKTRDYVHSRWATRELLEDNFPGATAFRNHKP-----YSVRV _____________________________________________________________________________________________________________________ Sequences retrieved from GenBank include: Bovine HV 2 (BHV-2; AF181249), Delphinid HV 1 (DeHV-1; AF196646), DeHV-2 (AF245443) DeHV-3 (AY757301), DeHV-7 (AY608707), DeHV-8 (bankit1249098), DeHV-9 ( Orcinus orca ; bankit1249103), Equid HV 1 (EHV-1; NC_001491), EHV-3 (AF514779), EHV-9 (NC_011644), Gallid HV 1 (GaHV-1; AF168792), GaHV-2 (NC_002229), Human HV 1 (HHV-1; X14112), HHV-2 (Z86099), HHV-3 (AB059831), Iguanid HV 2 (IgHV-2; AY236869), Meleagrid HV 2 (MeHV-1; NC_002641), Phocid HV 1 (PhHV-1; U92269), and Psittacid HV 1 (PsHV-1; NC_005264). IgHV-2 is shown first as the data outgroup. 35

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Table 2-4. MUSCLE al ignment of 20 gammahe rpesvirus Dpol amino acid sequences.* ElHV1 SKGMFPCLAIAESVTAQGRQLLAVTKQYICDRFNDWTFLTQIA-PE--LVDCPVDSNRFKIDV PhHV3 SGGMFPCVKIAETVTLQGRTMLDRSKHFI-EQLSAETLEAVTG-KT--I-QRENDAS-FKV-PhHV4 SGGMFPCVKIAETVTLQGRTMLDRSKHFI-EQLSAETLEAATG-KT--I-QREDDAC-FK--OvHV2 ASGMLPCLMIAETVTLQGRTMLEKTKQFV-ENLDVQSLQQICPTQTLKIHAQHPTPR-FTV-HHV4 ANGLFPCLSIAETVTLQGRTMLERAKAFV-EALSPANLQALAP-SPDAWAPLNPEGQ-LRV-PhHV5 SSGLLPCIKIAETITLQGRTMLEKSKLFI-ENLSFMEVQNLVP-SY--E-LQTGNGN-FRI-OtHV1 ATGIMPCLKIAETVTLQGRTMLEQSRQFI-EAIGVDDVSTLMK-QR--V-VAAPTAR-LHV-TrHV1 SSGLLPCIKIAETVTFQGRAMLEKSKRFV-EAITPERLRELVP-EP--F-SHEPGAR-FQ--KoHV1 ASGLLPCLKIAETVTSQGRCMLERSKKFI-EAINHSKLEELIG-RA--VPDTCENAD-FRV-ZiHV1 ASGLMPCLKIAETVTLQGRCMLERSKKFI-EAIDYRRLEELIG-QT--VTDADENSE-FKV-DeHV5 ASGLLPCLKIAETVTLQGRRMLERSKKFI-EAIDRRKLEELVG-HV--VAGADGDAE-FKV-DeHV6 ASGLMPCLKIAETVTLQGRCMLERSKMFI-EAINHRRLEELIG-HA--VADADRDAE-FKV-DeHV4 ASGLLPCLKIAETVTLQGRRMLERSKRFI-EAINHRRLEELIG-HA--VAGADGNAE-FRV-BHV4 ASGILPCIPIAETVTLQGRTMLEKSKAFV-EMITPERLSDIVS-YP--V-PCDPDAS-FRV-HHV8 ASGILPCLNIAETVTLQGRKMLERSQAFV-EAISPERLAGLLR-RP--I-DVSPDAR-FKV-EHV2 ASGILPCLKIAETVTFQGRRMLENSKRYI-EGVTPEGLADILG-RR--V-ECAPDAS-FKV-EHV5 ASGILPCLKIAETVTFQGRRMLERSKRYI-EAVTPEGLAAILQ-RP--VAACDPEAS-FKV-EHV7 ASGILPCLKIAETVTFQGRRMLERSKRYI-EAVTPEGLAAILH-RP--V-ACAPDAS-FKV-PhHV2 ASGILPCLKIAETVTFEGRRMLDRSKKFI-EDISPVDLERLLC-RP--I-TCAPDAN-FRV-OtHV2 SSGILPCLKIAETVTFEGRRMLERSKKFI-EDISPLDLERLLS-RP--V-VCSEDAN-FRV _____________________________________________________________________________________________________________________ Sequences retrieved from GenBank include: Bovine HV 4 (BHV-4; AF318573), Delphinid HV 4 (DeHV-4; AY952777), DeHV-5 (AY952776), DeHV-6 ( Grampus griseus ; DQ288666), Elephantid HV 1 (ElHV-1; AF322977), Equid HV 2 (EHV-2; U20824), EHV-5 (AF141886), EHV-7 (EU165547), Human HV 4 (HHV-4; DQ279927), HHV-8 (U93872), Kogiid HV 1 (KoHV-1; AY949830), Otariid HV 1 (OtHV-1; AF236050), OtHV-2 (bankit1248734), Ovine HV 2 (OvHV-2; DQ198083), Phocid HV 2 (PhHV-2; bankit1249104), PhHV-3 (DQ093191), PhHV-4 (DQ183057); PhHV-5 (bankit1249107), Trichechid HV 1 (TrHV-1; DQ238847), and Ziphid HV 1 (ZiHV-1; AY949828). ElHV-1 (GenBank accession number AF322977) is shown first as the data outgroup. 36

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Figure 2-1. Bayesian phylogenetic tree of the 19 predicted partial 58 amino acid alphaherpesviral DNA-dependent DNA polymerase sequences based on MUSCLE alignment Bayesian posterior probab ilities of branchings as percentages are in bold and ML bootstrap values for branching, ba sed on 100 re-sam plings, are given below them. Iguanid HV 2 (GenBank accession number AY236869) was used as the outgroup. Herpesvirus genera are delineated brackets. Areas of multifurcation are marked by arcs. Bottlenose dolphin herpesviru ses used in quantitative PCR assays are bolded. Sequences retrieved from GenB ank include: Bovine HV 2 (BHV-2; AF181249), Delphinid HV 1 (DeHV-1; AF196646), DeHV-2 (AF245443), DeHV-3 (AY757301), DeHV-7 (AY608707), DeHV-8 (bankit1249098), DeHV-9 (Orcinus orca; bankit1249103), Equid HV 1 (EHV-1; NC_001491), EHV-3 (AF514779), EHV-9 (NC_011644), Gallid HV 1 (G aHV-1; AF168792), GaHV-2 (NC_002229), Human HV 1 (HHV-1; X14112), HHV2 (Z86099), HHV-3 (AB059831), Iguanid HV 2 (IgHV-2; AY236869), Meleagrid HV 2 (MeHV-1; NC_002641), Phocid HV 1 (PhHV-1; U92269), and Psittacid HV 1 (PsHV-1; NC_005264). 37

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Figure 2-2. Bayesian phylogenetic tree of the 20 predicted partial 55 amino acid gammaherpesviral DNA-depe ndent DNA polymerase seque nces based on MUSCLE alignment. Bayesian posterio r probabilities of branchings as percentages are in bold and ML bootstrap values for branching, ba sed on 100 re-samplings, are given below them. Elephantid HV 1 (GenBank accession number AF322977) was used as the outgroup. Herpesvirus genera are delineated brackets. Areas of multifurcation are marked by arcs. Sequences retrieved from GenBank include: Bovine HV 4 (BHV-4; AF318573), Delphinid HV 4 (DeHV-4; AY952777), DeHV-5 (AY952776), DeHV-6 (Grampus griseus; DQ288666), Elephantid HV 1 (ElHV-1; AF322977), Equid HV 2 (EHV-2; U20824), EHV-5 (AF141886), EHV-7 (EU165547), Human HV 4 (HHV-4; DQ279927), HHV-8 (U93872), Kogiid HV 1 (KoHV-1; AY949830), Otariid HV 1 (OtHV-1; AF236050), OtHV-2 (bankit 1248734), Ovine HV 2 (OvHV-2; DQ198083), Phocid HV 2 (PhHV-2; bankit 1249104), PhHV-3 (DQ093191), PhHV-4 (DQ183057); PhHV-5 (bankit1249107), Tric hechid HV 1 (TrHV-1; DQ238847), and Ziphid HV 1 (ZiHV-1; AY949828). 38

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RN Cycle Figure 2-3. DeHV-2 10-fold serial dilutions in triplica te. Cycle threshold line set at 0.3. Ct Value log(Virus C opy Number) Figure 2-4. DeHV-2 standard curve in trip licate. Cycle thresh old line set at 0.3. 39

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RN Cycle Figure 2-5. DeHV-8 10-fold serial dilutions in duplicate. Cycle threshold line set at 0.3. Ct Value log(Virus C opy Number) Figure 2-6. DeHV-8 standard curve in dupl icate. Cycle threshold line set at 0.3. 40

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CHAP TER 3 USING QUANTITATIVE PCR TO INVEST IGATE CORRELATIONS BETWEEN TWO BOTTLENOSE DOLPHIN HERPESVIRU SES AND HEALTH PARAMETERS Introduction Herpesvirus infections are being reported with increased frequency in cetaceans. Currently, four alphaherpesviruses and two gammah erpesvirus have been reported in bottlenose dolphins. Alphaherpesvirus particles have been identified by electron microscopy (EM) and polymerase chain reaction (PCR) in skin lesions of bottlenose dolphins.2,46 An alphaherpesvirus was also identified, by EM and immunoperoxidase st aining, in cerebral cortex tissues of a harbor porpoise with encephalitis.25 Herpesviruses must be lytically active to cause disease.37 While herpesviruses can be actively proliferating in some cells and latent in others, clinical signs ar e usually only apparent when the virus is lytically active in the majority of cells.37 The amount of viral DNA circulating in the white blood cells correlates with the level of virus replication at the site of infection. Thus, an alphaherpesvirus is more activ e and clinical signs can be exp ected when a greater number of viral particles are in the bloodstream. During the routine, conventional PCR-based, viral surveillance of a managed collection of bottlenose dolphins, two alphaherpesviruses (t entatively named DeHV-2 and DeHV-8) were detected in the buffy coat of one animal. The 22-year-old, female bottlenose dolphin had a history of elevated transaminases and dehydr ogenases, which were normalized following phlebotomy treatment, and a persistent lymphoc ytosis. Furthermore, the dolphin did not have any skin lesions, unlike the majority of the previously reported cases of herpesvirus infections in cetaceans. Thus, the clinical significance of th is finding was unclear. Al phaherpesviruses are generally considered to persis t in the sensory nerve ganglion3,11,21,41 thus the presence in circulating blood cells was presumed to be indicative of infection with ac tive virus replication. 41

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However, it has also been suggested that in som e hosts, such as the horse, alphaherpesviruses can persist in the white cell fraction.6,44,55 Regardless of the site of pe rsistence and/or replication, changes in the virus load in the circulating white blood cells, as measured using a quantitative PCR assay, reflect the level of re plication of the herpesvirus. This virus load, or virus copy number per sample, cannot be asse ssed using conventional PCR. Real-time quantitative PCR (qPCR) assays have been applied in diagnosing or monitoring herpesviral infections including the human herpesviruses22,27,36,48,54 and Equid herpesvirus 1 and 412 (EHV-1 and 4). Quantitative PCR has a faster turn-around time and has a higher throughput than traditional PCR.27,48 The TaqMan qPCR method me asures the quantity of PCR products with a specific fluorogenic probe, specific primers, and real-time laser scanning in a 96-well plate.27,36 When these quantities are compared to known standards, the viral load in each sample can be determined. Therefore, TaqM an qPCR assays were developed to measure the DeHV-2 and DeHV-8 viral load in the case do lphin. The complete blood counts (CBC) and serum chemistry parameters for the herpesviru s positive samples in the case dolphin were compared to those of the negative samples, in an attempt to correlate viral presence with changes in clinical blood parameters. These assays were also used in a cross-sectio nal survey to establish a baseline prevalence of the two alphaherpes viruses in a population of clinically healthy bottlenose dolphins. Materials and Methods Sample Collection Sixteen serial blood samples were collect ed between July 12, 2004 and December 17, 2007 from the case dolphin, a 22-ye ar-old (at the start of the study) female bottlenose dolphin with persistent lymphocytosis. As part of a cross-sectional survey, an additional 55 blood samples, collected from 55 clinically healt hy bottlenose dolphins, were collected randomly 42

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between July 2nd and Septem ber 25, 2007. Daily food consumption and behavioral abnormalities were noted for each animal. Blood was collected into one Vacutainer serum separator tube (SST) and two Vacutainer EDTA (K3) tube for serum chemistries, complete blood counts (CBC) and buffy coat collection, respectively. Serum Chemistry and CBC The SST samples for serum chemistry analys is were centrifuged within two hours of collection. Centrifugation was performed at 3,000 rpm at 21C for 10 minutes. Fibrin clots were removed and serum was transferred to a 5 ml plastic submission tube. One EDTA Vacutainer tube containing whole blood was sh ipped on ice via courier to Ques t Diagnostic Laboratories in San Diego, California. Automated CBC analyses were conducted by the reference laboratory with the Coulter LH 1500 Series.h The Fisherbrand Dispette 2,c, correlating with the Westergren method, was used to determine sixt y minute erythrocyte sedimentation rate (ESR) from 1ml EDTA whole blood. Quantitative PCR For qPCR analysis, 16 buffy coat samples from the case dolphin and 55 samples from the clinically healthy animals were extracted, as de scribed in Chapter 2. All samples were run in triplicate and a mean Ct value was calculated. Eu karyotic 18S rRNA (20X)g was used as a positive control for each sample in a separate well. Each specific qPCR assay was performed as described in Chapter 2. Using a standard cu rve and a reference sample on each plate for comparison, a Ct value between 1 and 35.90 was considered positive for DeHV-2 and between 1 and 36.67 for DeHV-8. Alternatively, a Ct value greater than or equal to 35.90, and 36.67 for the DeHV-8 qPCR assay, as well as samples that did not yield a detectable level of fluorescence were considered negative. 43

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Specific PCR Specific DeHV-8 prim ers were designed (f orward primer, DeHV8-F2b [5TTG CTC CCG TGT CTG CCG AT-3]; re verse primer, DeHV8-R2b [5AGT AGA GAG ACG CGG AAG GAG G-3]) base d on the Dpol herpesviral sequence detected in the case dolphin via consensus PCR. A Px2 thermal cycler was used to amplify 50L reactions using Platinum Taq DNA Polymerase and following manufacturer s instructions. DeHV-8 specific PCR amplification conditions were as follows: initial denaturation at 94C for 5 min; 45 cycles of amplification with each cycle consisting of denatu ration at 94C for 30 s, annealing at 65C for 1 min, and elongation at 72C for 1 min; a final el ongation step was performed at 72C for 10 min followed by a 4C hold. The amplification process was repeated in a second 50L reaction PCR. The final PCR products were resolved on a 1.5% agarose gel made up in 1X TBE and containing 1g of ethidium bromide per mL. The DeHV-8 assay amplifies a 172bp band. Bands of expected size were excised and prepared for direct seque ncing as described in Chapter 2. The specific primers were used in the sequencing reactions. Statistical Analysis Sixteen parameters for the CBC, including ESR, and 25 parameters for the serum chemistry, were analyzed for each sample. Data were analyzed by use of computer software.i Analyses of variance (ANOVAs) were conducted to compare mean clinicopathologic values of DeHV-2 positive and DeHV-2 negative samples. DeHV-2 case animal positives were also compared to clinicpathologic values of the th ree most recent blood draws preceding and the three immediately following the DeHV-2 qPCR positive. Significance for all analysis was set at 0.05. 44

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Results Quantita tive PCR DNA concentration and purity data for all sample s is shown in Tables 3-1 3. Five of the 16 case animal samples (31.2%) were DeHV-2 qPCR positive (see Table 3-4). For the crosssectional survey, 2 out of 54 samples (3.7%) were DeHV-2 qPCR positive (see Table 3-5). One of the cross-sectional survey samples was excluded from the analysis because the eukaryotic 18S reaction control was negative. Only one case animal sample (6.3%) was positive using the DeHV-8 qPCR assay (see Table 3-6). Additionally, there were no DeHV-8 qPCR positive crosssectional survey samples (see Table 3-7). Specific PCR The amino acid sequence of sample Tt0721 was 100% identical to the DeHV-8 amino acid sequence (from sample Tt0724 using consensu s PCR primers) with the specific DeHV-8 primers. Statistical analysis The case animal, a female bottlenose dolphi n, had a mean age of 24.2 during the study (range 22.1 25.0). There were no significant di fferences when comparing mean CBC and serum chemistry values between DeHV-2 qPCR positive and DeHV-2 qPCR negative samples. For samples with detectable DeHV-2, the 41 CBC and serum chemistry parameters were plotted with the animals six most recent blood profile s spanning before and af ter the positive date. When assessing trend CBC and serum chemistry values on days surrounding one of the five positive samples (Tt0719), there was an associat ed decrease in overall WBC counts (see Figure 3-1) and an increase in erythr ocyte sedimentation rate (ESR; see Figure 3-2) and hematocrit (HCT; see Figure 3-3). Also noteworthy was a decr ease in serum chloride (see Figure 3-4). Similar trends were not identified surroundi ng any of the other four DeHV-2 qPCR positive 45

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sam ples. The cross-sectional survey sample s were composed of 23 females (42.6%) and 31 males. Animals ages ranged from 0.1 to 50.3 years old with a mean of 23.7 years old. There were no significant differences between ge nder or age of the DeHV-2 qPCR positive and negative cross-sectiona l survey dolphins (P = 0.1 and 0.4, respectively). Only two animals in the cross-sectional survey were DeHV-2 qPCR positive so statistical analysis was not performed. Discussion Unlike DeHV-8, DeHV-2 was detected in th e cross-sectional su rvey of the study population. Despite an identical calculated detection limit (10 virus copies), DeHV-2 was detected in two other dolphins whereas DeHV-8 was only detected in the case dolphin and then only in one sample. The DeHV-2 qPCR assay prove d to be more sensitive than traditional consensus PCR (see Chapter 2) with three additional DeHV-2 qPCR positive case animal samples. Additionally, low DeHV-8 viral load is suggested by comparison of the DeHV-8 qPCR assay to the traditional PCR assays. With consensus PCR only one sample, Tt0724, was positive however with qPCR only sample Tt0721 was positive and that positive was confirmed with specific PCR. This suggests that even in our case dolphin active DeHV-8 viral load was minimal and virus replication infrequent. Di ffering levels of prevalence for distinct alphaherpesviruses in a population is common in other species. Seropr evalence of the three human alphaherpesvirus, human simplex virus 1, 2 a nd Varicella-Zoster, is 57.7%58, 17%58, and 99.6%26, respectively, in adults in the United States. Thus, it is not une xpected to see varying prevalence between these two viruses. Prevalence of DeHV-2 and 8 will likely differ between dolphin populations as well. A serological survey in China of Bovine herpesvirus 1 (BHV-1) showed a nationwide seroprevalence of 35.8%, while the prevalence for individual provinces ra nged from 12.1% to 77.8%.60 This highlights the need for attaining baseline values for dis tinct populations. These 46

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baseline prevalences are needed for the clin ic al interpretations of the detection of an alphaherpesvirus. The simple presence of DeHV-2 could not be associated with any specific blood parameter changes in the case animal. One posi tive case animal sample (Tt0719), however, had noteworthy changes in four of the blood parameters analyzed (total WBC count, ESR, hematocrit, and serum chloride). However, these changes were only associ ated with one of the five DeHV-2 positive samples and they can therefore not be correlated with the increased circulating herpesviral load. Even if unrelated to DeHV-2 replication, the observed changes do suggest a clinical event because they were ofte n significant differences and had values outside the normal reference ranges for the population. The total WBC count for females between 10 and 30 years old in this population ranges from 5, 381 11,336 cells/L.53 While the case animal did not significantly exceed this range, her WBC count was about 4,000cells/L higher 24 days prior and 11 days after the positive sample date. This is clearly a significant decr ease in WBCs. The decrease in WBCs was coupled with a significant increase in ESR (normal range: 0-19mm/h)53 to 45mm/h. An increase of that proportion is highly suggestive of inflammation.28,39 An increase in the hematocrit (47%) was also noted in Sample Tt0719 which also exceeded the normal range of 38 46% for animals in this age group within the population.53 The HCT increase suggests the animal was less hydrated at the time of sample collection than normal.4 The fourth affected para meter was serum chloride. The normal range for the population is between 115 and 125mEq/L. Sample Tt0719 had a chloride level of only 113mEq/L and the animals chloride level further dropped to 111mEq/L 11 days later before slowly returning to normal. The hypochloremia could be an indicator of 47

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m etabolic alkalosis, as associated with il eus in cattle and horses, or an effect of vomiting/diarrhea.5 Even if a true clinical disease process was pr esent at the time of the sample collection, the association of clinical signs with active herpesviral proliferati on cannot be confirmed. It remains unclear whether the disease process demanded an immunologic response that created an opportunity for latent DeHV-2 to proliferate, whether DeHV-2 proliferation was responsible for the subsequent abnormalities in the homeostasi s of the body, or whether the two events are causally linked at all. With only one DeHV-8 positive sample, statistical associations between DeHV-8 viral load and clinical signs could not be completed. Despite the more frequent detection of DeHV-2 in the study population, clinical sy mptoms could not be associated with DeHV-2 presence. Our data demonstrate that herpesviruses are co mmon in bottlenose dolphins and one should be cautious in attributing clinical signs with he rpesvirus detection as they are often present asymptomatically. 48

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Table 3-1. Spectrophotometric analysis of DeHV-2 qPCR case study samples DNA purity expressed as the absorbance at 260 nm :280 nm. Sample ID Collection Date ng/l 260 280 Tt0625 11/8/2006 13.05 1.67 Tt0631 12/13/2006 32.02 1.83 Tt0634 12/20/2006 93.57 1.97 Tt0701 11/22/2006 22.35 1.84 Tt0718 10/20/2005 0.70 0.80 Tt0719 8/17/2006 8.60 1.70 Tt0720 10/25/2006 11.45 1.63 Tt0721 1/10/2007 6.94 1.64 Tt0724 2/7/2007 102.07 1.99 Tt0730 2/21/2007 11.53 1.89 Tt0748 2/28/2007 9.11 1.62 Tt0777 6/26/2007 187.66 2.06 Tt0804 12/17/2007 119.01 1.98 Tt0901 7/12/2004 12.22 1.47 Tt0902 8/2/2004 11.81 1.50 Tt0903 9/22/2006 7.36 1.55 _____________________________________________________________________________________________________________________ Table 3-2. Spectrophotometric analysis of DeHV-8 qPCR case study samples. DNA purity expressed as the absorbance at 260 nm :280 nm. Sample ID Collection Date ng/l 260 280 Tt0625 11/8/2006 20.08 1.73 Tt0631 12/13/2006 32.02 1.83 Tt0634 12/20/2006 93.57 1.97 Tt0701 11/22/2006 22.35 1.84 Tt0718 10/20/2005 0.70 0.80 Tt0719 8/17/2006 4.28 1.48 Tt0720 10/25/2006 11.45 1.63 Tt0721 1/10/2007 6.94 1.64 Tt0724 2/7/2007 260.54 1.99 Tt0730 2/21/2007 8.94 2.36 Tt0748 2/28/2007 9.11 1.62 Tt0777 6/26/2007 206.73 2.08 Tt0804 12/17/2007 119.01 1.98 Tt0901 7/12/2004 12.22 1.47 Tt0902 8/2/2004 11.81 1.50 Tt0903 9/22/2006 7.36 1.55 _____________________________________________________________________________ 49

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Table 3-3. S pectrophotometric analysis of the cross-sectional survey samples. DNA purity expressed as the absorbance at 260 nm :280 nm. Sample ID Collection Date Media* ng/L 260 280 Tt0812 8/23/2007 EDTA 19.85 1.63 Tt0813 8/7/2007 EDTA 16.70 1.62 Tt0814 7/24/2007 EDTA 18.45 1.53 Tt0815 7/20/2007 EDTA 19.62 1.61 Tt0816 7/2/2007 EDTA 17.75 1.68 Tt0817 7/27/2007 EDTA 11.25 1.43 Tt0818 7/2/2007 EDTA 45.40 1.92 Tt0819 8/7/2007 EDTA 17.50 1.62 Tt0820 7/25/2007 EDTA 8.72 2.04 Tt0821 8/14/2007 EDTA 21.24 1.76 Tt0822 9/21/2007 EDTA 22.22 1.94 Tt0823 7/12/2007 EDTA 20.03 1.89 Tt0824 9/13/2007 EDTA 14.51 2.04 Tt0825 7/5/2007 EDTA 31.44 1.96 Tt0826 8/29/2007 EDTA 7.61 1.56 Tt0827 7/13/2007 EDTA 40.00 1.86 Tt0828 9/4/2007 EDTA 14.74 1.81 Tt0829 8/8/2007 EDTA 31.78 1.91 Tt0830 9/6/2007 EDTA 21.98 2.04 Tt0831 8/24/2007 EDTA 16.61 2.01 Tt0832 7/5/2007 EDTA 31.21 1.88 Tt0833 7/11/2007 EDTA 26.43 1.93 Tt0834 7/28/2007 EDTA 26.13 1.83 Tt0835 8/23/2007 EDTA 13.82 1.83 Tt0836 7/5/2007 EDTA 12.78 1.67 Tt0837 7/5/2007 EDTA 20.27 1.84 Tt0838 8/9/2007 EDTA 5.33 1.85 Tt0839 9/19/2007 EDTA 20.33 1.97 Tt0840 8/7/2007 EDTA 12.70 1.77 Tt0841 9/4/2007 EDTA 12.57 1.85 Tt0842 7/12/2007 EDTA 9.68 1.86 Tt0843 8/22/2007 EDTA 17.86 1.89 Tt0844 9/24/2007 EDTA 19.27 1.94 Tt0845 9/11/2007 EDTA 47.00 1.85 Tt0846 9/20/2007 EDTA 10.43 1.99 Tt0847 7/2/2007 EDTA 8.89 1.69 Tt0848 7/18/2007 EDTA 10.62 1.72 Tt0849 9/11/2007 EDTA 26.69 1.88 Tt0850 8/3/2007 EDTA 28.91 1.94 50

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Table 3-3. C ontinued Sample ID Collection Date Media* ng/L 260 280 Tt0851 8/30/2007 EDTA 29.44 1.85 Tt0852 7/2/2007 EDTA 13.78 1.98 Tt0853 9/13/2007 EDTA 6.40 1.74 Tt0854 7/5/2007 EDTA 25.46 1.79 Tt0855 7/16/2007 EDTA 25.03 2.04 Tt0856 8/10/2007 EDTA 13.20 1.97 Tt0857 8/21/2007 EDTA 40.88 1.91 Tt0858 9/25/2007 EDTA 20.53 1.86 Tt0859 9/12/2007 EDTA 11.89 2.01 Tt0860 7/31/2007 Na Hep 16.69 1.85 Tt0861 8/10/2007 EDTA 10.34 1.67 Tt0862 8/7/2007 EDTA 10.24 1.81 Tt0863 8/22/2007 EDTA 10.29 2.14 Tt0864 7/27/2007 EDTA 13.28 1.95 Tt0865 7/2/2007 EDTA 31.39 1.92 Tt0866 8/21/2007 EDTA 10.20 1.61 ______________________________________________________________________________ *Media refers to the anticoagulant us ed in the blood collection tubes. 51

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Table 3-4. D eHV-2 qPCR case study data Sample ID Collection Date ng DNA* Ct Value Viral Presence Tt0625 11/8/2006 91.35 34.9 + Tt0631 12/13/2006 100.90 36.8 ND Tt0634 12/20/2006 93.57 35.6 ND Tt0701 11/22/2006 100.60 35.0 ND Tt0718 10/20/2005 4.90 35.90 + Tt0719 8/17/2006 60.20 33.3 + Tt0720 10/25/2006 80.20 34.6 ND Tt0721 1/10/2007 48.58 36.4 ND Tt0724 2/7/2007 102.07 35.4 ND Tt0730 2/21/2007 80.71 32.0 + Tt0748 2/28/2007 63.77 36.5 ND Tt0777 6/26/2007 187.66 33.4 ND Tt0804 12/17/2007 119.01 33.4 ND Tt0901 7/12/2004 85.54 36.0 ND Tt0902 8/2/2004 82.67 32.4 + Tt0903 9/22/2006 51.52 35.8 ND __________________________________________________________________________________________________ *DNA equals the total DNA of the sample used in each well of the qPCR. Ct value for each of the three sample rep licates was averaged to calculate the Ct value. + indicates positive viral load (the Ct valu e for the sample was before the 35.90 detection limit); ND = The Ct value was higher than the 35.90 detection limit and thus not detectable. ( 52

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Table 3-5. D eHV-2 qPCR cross-sectional survey data Sample ID Collection Date ng DNA* Ct Value Viral Presence Tt0812 8/23/2007 99.25 35.5 ND Tt0813 8/7/2007 100.20 ND ND Tt0814 7/24/2007 101.48 38.4 ND Tt0815 7/20/2007 98.10 ND ND Tt0817 7/27/2007 78.75 ND ND Tt0818 7/2/2007 102.15 42.3 ND Tt0819 8/7/2007 96.25 44.6 ND Tt0820 7/25/2007 61.04 40.2 ND Tt0821 8/14/2007 100.89 39.0 ND Tt0822 9/21/2007 99.99 33.8 ND Tt0823 7/12/2007 100.15 35.8 ND Tt0824 9/13/2007 101.57 ND ND Tt0825 7/5/2007 94.32 33.4 ND Tt0826 8/29/2007 53.27 ND ND Tt0827 7/13/2007 100.00 32.4 ND Tt0828 9/4/2007 103.18 ND ND Tt0829 8/8/2007 103.29 33.2 ND Tt0830 9/6/2007 98.91 35.90 ND Tt0831 8/24/2007 99.66 37.0 ND Tt0832 7/5/2007 101.43 34.8 ND Tt0833 7/11/2007 99.11 33.0 ND Tt0834 7/28/2007 97.99 38.8 ND Tt0835 8/23/2007 96.74 ND ND Tt0836 7/5/2007 89.46 37.1 ND Tt0837 7/5/2007 101.35 35.6 ND Tt0838 8/9/2007 37.31 42.9 ND Tt0839 9/19/2007 101.65 37.4 ND Tt0840 8/7/2007 88.90 42.5 ND Tt0841 9/4/2007 87.99 38.8 ND Tt0842 7/12/2007 67.76 36.5 ND Tt0843 8/22/2007 98.23 39.6 ND Tt0844 9/24/2007 96.35 ND ND Tt0845 9/11/2007 94.00 32.4 ND Tt0846 9/20/2007 73.01 ND ND Tt0847 7/2/2007 62.23 ND ND Tt0848 7/18/2007 74.34 ND ND Tt0849 9/11/2007 100.09 39.6 ND Tt0850 8/3/2007 101.19 35.6 + Tt0851 8/30/2007 103.04 36.0 + Tt0852 7/2/2007 96.46 ND ND 53

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Table 3-5. C ontinued Sample ID Collection Date ng DNA* Ct Value Viral Presence Tt0853 9/13/2007 44.80 ND ND Tt0854 7/5/2007 101.84 40.4 ND Tt0855 7/16/2007 100.12 34.5 ND Tt0856 8/10/2007 92.40 ND ND Tt0857 8/21/2007 102.20 33.0 ND Tt0858 9/25/2007 102.65 40.2 ND Tt0859 9/12/2007 83.23 ND ND Tt0860 7/31/2007 100.14 ND ND Tt0861 8/10/2007 72.38 ND ND Tt0862 8/7/2007 71.68 43.0 ND Tt0863 8/22/2007 72.03 ND ND Tt0864 7/27/2007 92.96 36.4 ND Tt0865 7/2/2007 102.02 ND ND Tt0866 8/21/2007 71.40 ND ND _________________________________________________________________________________________ *ng DNA equals the total sample DNA used in each well of the qPCR. Ct for each of the three sample replicat es was averaged to calculate the Ct value. + indicates positive viral load (the Ct valu e for the sample was before the 35.90 detection limit); ND = Ct was not detected for any sample replicate or the Ct value was greater than the 35.90 (10 copies) detection limit. 54

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Table 3-6. DeHV-8 qPCR case study data Sample ID Collection Date ng DNA* Ct Value Viral Presence Tt0625 11/8/2006 104.00 ND ND Tt0631 12/13/2006 100.90 43.5 ND Tt0634 12/20/2006 93.57 ND ND Tt0701 11/22/2006 100.60 39.7 ND Tt0718 10/20/2005 4.90 38.6 ND Tt0719 8/17/2006 29.96 ND ND Tt0720 10/25/2006 80.20 ND ND Tt0721 1/10/2007 48.58 36.5 + Tt0724 2/7/2007 260.54 39.6 ND Tt0730 2/21/2007 62.58 ND ND Tt0748 2/28/2007 63.77 ND ND Tt0777 6/26/2007 206.73 ND ND Tt0804 12/17/2007 119.01 ND ND Tt0901 7/12/2004 85.54 ND ND Tt0902 8/2/2004 82.67 ND ND Tt0903 9/22/2006 51.52 ND ND _________________________________________________________ *ng DNA equals the total sample DNA used in each well of the qPCR. Ct for each of the three sample replicat es was averaged to calculate the Ct value. + indicates positive viral load (the Ct value for the sample was before the 36.67 detection limit) ND = Ct was not detected for any sample replicate or the Ct value was greater than the 36.67 (10 copies) detection limit. 55

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Table 3-7. D eHV-8 qPCR cross-sectional survey data Sample ID Collection Date ng DNA* Ct Value Viral Presence Tt0812 8/23/2007 99.25 ND ND Tt0813 8/7/2007 100.20 ND ND Tt0814 7/24/2007 101.48 ND ND Tt0815 7/20/2007 98.10 ND ND Tt0817 7/27/2007 78.75 43.6 ND Tt0818 7/2/2007 102.15 ND ND Tt0819 8/7/2007 96.25 ND ND Tt0820 7/25/2007 61.04 ND ND Tt0821 8/14/2007 100.89 ND ND Tt0822 9/21/2007 99.99 ND ND Tt0823 7/12/2007 100.15 ND ND Tt0824 9/13/2007 101.57 43.1 ND Tt0825 7/5/2007 94.32 ND ND Tt0826 8/29/2007 53.27 44.6 ND Tt0827 7/13/2007 100.00 ND ND Tt0828 9/4/2007 103.18 41.6 ND Tt0829 8/8/2007 103.29 39.3 ND Tt0830 9/6/2007 98.91 ND ND Tt0831 8/24/2007 99.66 39.7 ND Tt0832 7/5/2007 101.43 ND ND Tt0833 7/11/2007 99.11 38.4 ND Tt0834 7/28/2007 97.99 ND ND Tt0835 8/23/2007 96.74 48.7 ND Tt0836 7/5/2007 89.46 ND ND Tt0837 7/5/2007 101.35 ND ND Tt0838 8/9/2007 37.31 42.5 ND Tt0839 9/19/2007 101.65 39.4 ND Tt0840 8/7/2007 88.90 47.3 ND Tt0841 9/4/2007 87.99 40.3 ND Tt0842 7/12/2007 67.76 39.7 ND Tt0843 8/22/2007 98.23 ND ND Tt0844 9/24/2007 96.35 ND ND Tt0845 9/11/2007 94.00 ND ND Tt0846 9/20/2007 73.01 43.1 ND Tt0847 7/2/2007 62.23 38.6 ND Tt0848 7/18/2007 74.34 ND ND Tt0849 9/11/2007 100.09 ND ND Tt0850 8/3/2007 101.19 ND ND Tt0851 8/30/2007 103.04 ND ND Tt0852 7/2/2007 96.46 ND ND Tt0853 9/13/2007 44.80 40.4 ND 56

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Table 3-7. C ontinued Sample ID Collection Date ng DNA* Ct Value Viral Presence Tt0854 7/5/2007 101.84 ND ND Tt0855 7/16/2007 100.12 ND ND Tt0856 8/10/2007 92.40 ND ND Tt0857 8/21/2007 102.20 ND ND Tt0858 9/25/2007 102.65 ND ND Tt0859 9/12/2007 83.23 43.8 ND Tt0860 7/31/2007 100.14 ND ND Tt0861 8/10/2007 72.38 ND ND Tt0862 8/7/2007 71.68 38.7 ND Tt0863 8/22/2007 72.03 ND ND Tt0864 7/27/2007 92.96 ND ND Tt0865 7/2/2007 102.02 ND ND Tt0866 8/21/2007 71.40 ND ND _________________________________________________________________ *ng DNA equals the total sample DNA used in each well of the qPCR. Ct for each of the three sample replicat es was averaged to calculate the Ct value. + indicates positive viral load (the Ct value for the sample was before the 36.67 detection limit) ND = Ct was not detected for any sample replicate or the Ct value was greater than the 36.67 (10 copies) detection limit. 57

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Figure 3-1. WBC counts surrounding sample Tt0719 collection date. Figure 3-2. ESR values surrounding sam ple Tt 0719 collection date. Dashed line marks high normal. 58

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59 Figure 3-3. He matocrit values surrounding sample Tt0719 collection date. Dashed line marks high normal. Figure 3-4. Sodium chloride va lues surrounding sample Tt0719 colle ction date. Dashed-dot line marks low normal.

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CHAP TER 4 DISCUSSION Two bottlenose dolphin herpesviruses, one of which is a novel alphaherpesviruses, were identified by consensus PCR in the case an imal. Additionally, three novel marine mammal herpesviruses were identified (DeHV-8, De HV-9, OtHV-2) and one nove l Dpol herpesviral sequence from the PhHV-2 control isolate was generated by consensus PCR. Bayesian and ML phylogenic analysis supports the classification of the is olates as novel alphaand gammaherpesviruses. It is unclear, based on the limited Dpol sequences, whether the novel delphinid and pinniped herpesviruses belong to established genera or represent novel genera.. None of the delphinid herpesviru ses clearly associate with any es tablished genus either. Further studies assessing phylogenetic relationships amongst marine mammal herpesviruses should aim to attain more sequence data for phylogenetic analysis. More complete genome segments for comparison should yield greater phylogenetic resolution18 and will allow for the alphaand gammaherpesviruses to be included in the same Bayesian and ML analysis. The novel qPCR assays developed for DeHV-2 and DeHV-8 are faster and have a higher throughput than traditional PCR. Both qPCR assays are very sensitive and had a wide detectable range with a lower limit of 10 virus copies. The assays were successful in detecting lytically active alphaherpesviruses as shown by the detection of changes in vi ral load in the case animal at varying time points during the study. The simple presence of DeHV-2 could not be associated with any specific blood parameter changes in the case animal. One posi tive case animal sample (Tt0719), however, had remarkable trends in four of the blood parame ters analyzed: decreases in total WBC counts and serum chloride as well as increases in ESR and HCT. These tr ends are not well supported as relating to the increased herpesvi ral load since they were only present with one of the five 60

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positiv es. Yet, the trend changes do suggest a clin ical event because they were often significant differences and had values outside the normal reference ranges for the population. One way to demonstrate the probability of inflammation would be to run fibrinogen or acute-phase protein levels on sample Tt0719.28,39 It would also be helpful to have a more thorough analysis of animals eating habits to better grasp the theories of dehydrati on and metabolic alkalosis. The majority of water intake for bottlenose dolphins is the pre-formed and metabolic water in their food.57 If the animal was not eating normally or regur gitating food the clinical significance of the changes would be greatly increased. Even if a true clinical disease process was present it remains unclear whether the disease process demanded an immunologic response that created an opportunity for latent DeHV-2 to proliferate, whether DeHV-2 proliferation was responsible for the subsequent abnormalities in the homeostasi s of the body, or whether the two events are causally linked at all. While an attempt was made to compare the blood parameters associated with samples that did or did not contain detectable DeHV-2 levels, there were very few positives samples available. There were an insufficient number of positives for statistical analysis of DeHV-2 and 8 within the population and insufficient DeHV-8 positive dates in the case animal. Future work should focus on collecting more positive samp les with high and varying viral loads for comparisons. This would allow for a more robust an alysis of the positives and negatives as well as for quantitative correlations between virus lo ad and changes in blood parameters. If changes in blood parameters can be correlated with chan ges in viral load, one of the Fredricks-Relman criteria would be satisfied and a case for establishing causality of disease would be strengthened. The qPCR assays detected a higher baseline prevalence of DeHV-2 (3 animals) than DeHV-8 (only the case dolphin). Attaining baseline values for di stinct populations can assist 61

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with clinica l interpretations of virus presence as they are expect ed to vary between populations. Additionally, with five more bottlenose dolphin herpesviruses identified thus far, population prevalence rates for the other do lphin herpesviruses should also be investigated. Herpesviruses are common in bottlenose dolphins and one should be cautious in at tributing clinical signs with herpesvirus detection as they are often present asym ptomatically. Assessment of the clinical significance of herpesvirus presen ce should be completed before use of potentially harmful antivirals for treatment in unstudied species. 62

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APPENDIX A TROUBLE SHOOTING Inconsistent Amplification of Standards Problem Identified The Tursiops truncatus herpesvirus 2 (DeHV-2) complementary deoxyribonucleic acid (cDNA) serially diluted standards, made from the consensus Polymerase Chain Reaction (PCR) product, was amplifying inconsistently in th e quantitative PCR (qPCR) assay. While the amplification efficiency remained acceptable ( Rn > 3.5), threshold values shifted. For example, the cycle threshold (Ct)value of 2 million copies which equaled 18 on 1/11/08, was delayed to cycle 20.5 the next day. Troubleshooting Approach Degradation of the qPCR probe and/or th e template cDNA was the anticipated cause. Troubleshooting approaches included the following: Making smaller aliquots of the probe, to reduce the number of freeze/thaw cycles. Cloning of the DeHV-2 DNA-dependent DNA polymerase (Dpol) cDNA into a plasmid to increase stability and constructi ng standard curve serial dilutions using purified plasmid DNA rather than cDNA. For consistency between the DeHV-2 and DeHV-8 assays, we planned to construct a plasmid based standard curve serial dilution for DeHV-8 as well. Materials and Methods Purified cDNA, from secondary consensus PCR product, was cloned into the pDrive Cloning Vector using a PCR Cloningplus Kit.b Five DeHV-2 clones were selected for plasmid purification using the PureLink Quick Plasmid Miniprep Kit.d Twenty-four clones were selected for DeHV-8 plasmid purification. Insertion of the sequence was confirmed by PCR of 63

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the purified plasm ids and sequencing the resulting bands. M13 forward (-20) and M13 reverse primers were used with the Platinum Taq DNA Polymerasec in 10L reactions following manufacturers instru ctions with cycler conditions as follo ws: initial denaturation at 94C for 5 min; then 40 cycles of denaturation at 94C for 30 s, immediately followed by annealing at 46C for 45 s, and extension at 72C for 45 s; fina l extension was carried out at 72C for 10 min followed by a 4C hold. Sequencing of the purif ied plasmids (using the M13 primers) and creation of the serial dilutions followed the same protocol outlined in Chapter 2. The plasmid serial dilutions were then run in tandem w ith a freshly amplified cDNA serial dilution. Results Making smaller aliquots of the probe did ma ke the amplification more consistent. However, the variability in Ct value for each dilution was still not within acceptable limits. Thus, creating plasmids from the cDNA was pursued. The purified plasmids yielded a higher DNA c oncentration and higher purity ratios than the cDNA (see Table A-1). All five DeHV-2 clone s were confirmed via sequencing to contain the insert but none of the DeHV-8 clones containe d inserts. The DeHV-2 clone with the highest concentration of DNA (Tt0625-1pp shown in Table A-1) was then used to create the serial dilutions. Once used in the qPCR, the plasmid dilu tions, which were calcul ated to have similar virions/L as the cDNA dilutions, consistently generated Ct values about 2 cycles behind the cDNA counterpart (see Figure A-1). Discussion The difference in length between the DeHV2 and DeHV-8 Dpol segment is 43bp. Since both products are small, 224bp for DeHV-2 and 172bp for DeHV-8, this difference is significant and believed to be the reason fo r the lack of insertion into th e plasmid vector. While the cloning kit used did not specify a minimum PCR product size, other kits are known to have a minimum 64

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of 200bp. Addition ally, after qPCR analysis, sample Tt0724 had a viral copy number so low that it could not be detected. The low amount of templa te may have also contributed to the inability to insert the DeHV-8 sequence into the plasmid. Restriction Enzymes Problem Identified The Ct values of the plasmid standards lagged 2 cycles behind those of the cDNA dilutions, despite similar concentrations and less protein in the plasmid product, it was postulated that the secondary or tertiary structure of th e plasmid was inhibiting the binding of the primers and probe to the insert. The effects of secondary and tertiary structure ar e being recognized with increasing importance. In epigenetic studies of twins, DNA methylation has caused disease, such as Beckwith-Wiedemann syndrome, in one tw in while the identical twin is unaffected.38 Troubleshooting Approach A restriction enzyme was utilized to cut the plasmid in an attempt to linearize it. A linear plasmid should resolve hindrance issues from se condary and tertiary structures. The sequence was thus analyzed for a restriction enzy me and site that would only cut once. Materials and Methods Recovery of plasmids stored in glycerol. DeHV-2 plasmids were stored, individually, in 5 mLs of glycerol at -80C. To recover the plas mids, 0.5 mL of frozen stored plasmid was added to 5 mLs LB broth and placed in an incubated sh aker overnight. It took eight more nights before one of the five tubes turned cloudy and was spre ad onto an LB agar plate to grow up for two days in the incubator and then plaque purified (as described above). Sinc e the recovery of the plasmids was poor, white colonies were also picked for storage as well as purification. To store the plasmids, one white colony was put into 5 mLs LB broth and shaken in an incubator for three hours before adding 5 mLs of glycerol, dividing into 2 mL aliquots and storing at -80C. 65

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Restriction enz yme digestion. Purified plasmids underwent spectrophotometric analysis (as described in Chapter 2) to assess purity and concentration prior to use in the restriction enzyme assay. The first plasmid, Tt0625-2pp, used in the restriction enzyme digestion had a concentration of 2g (5 U BamH I used) while 4g was used for the second plasmid, Tt06253pp, (10 U BamH I used). BamH Id was chosen for the digestion because it only cuts once, just outside of the insert. Digestion was performed, according to manufacturers instructions, using the buffer supplied with the restriction enzy me in 60L reactions. A Px2 thermal cyclerb was used to incubate the reactions at 37C for 60 min then to heat th e reactions to 60C for 10 min to stop the digestion. The product was resolved on an agarose gel and gel extr acted for purification (as described in Chapter 2). Spectrophotmeteric an alysis was then performed prior to creating a standard curve for qPCR (as described in Chap ter 2). Lastly, the restriction enzyme serial dilutions (RE serial dilutions) we re run concurrently with the pl asmid and cDNA serial dilutions. Results The digested plasmids had much lower con centrations and higher protein presence than prior to digestion as well as compared to the cDNA (see Table A-1). Since the restriction enzyme digestion produced a cleaner band with only 2g of purified plasmid in the reaction (see Figure A-2), Tt0625-1re was used to creat e the dilutions. The results from all three serial dilutions, run simultaneously, are shown in Figure A-3. The pl asmid dilutions and the cDNA dilutions are almost identical to the previous run except for a misfire of the last cDNA standard. Thus, the plasmid standard at this dilution, 1:108, actually has a lower Ct value (more virions/L) than the 1:108 cDNA standard. At every other dilution, the plasmid rises about 2 cycles after its cDNA counterpart. The RE serial dilu tion amplification was poor even though it had similar virions/L at each dilution factor as the others. The first Ct value for the RE serial dilution is approximately 20 cycles behind the first Ct value of the two other standards. Furthermore, all the RE standards 66

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cam e up after the 1:108 dilution, the last of the four 10-fo ld dilutions shown, of the other two standards. Discussion The significant decrease in the recovery and purity of the DNA after the restriction enzyme digestion clearly inhibited th e qPCR reaction. A different purif ication method post-digestion, such as a column-based method that would bypass the agarose gel step, may preserve more of the yield and purity but it seems dubious to expect a significant change in recovery when almost 90% of the DNA was lost and 40% of the purity. Therefore, attempts to make plasmid based standards were abandoned. 67

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Table A-1. Spectrophotom etric analysis Sample ID Sample type ng/L 260/280 ____________________________________________________________ Tt0625-1c cDNA (PCR product) 26.0 1.83 Tt0625-2c cDNA (PCR product) 20.8 1.73 Tt0625-1pp purified plasmid 82.8 1.96 Tt0625-2pp purified plasmid 90.9 1.94 Tt0625-3pp purified plasmid 103.6 2.03 Tt0625-1 re BamH I digested plasmid 12.3 1.54 Tt0625-2re BamH I digested plasmid 7.5 1.28 ______________________________________________________________________________ Delta Rn C ycle Figure A-1. DeHV-2 qPCR amplification comparis on of cDNA and plasmid serial dilutions. Threshold line shown at 0.3. 68

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69 1,500 bp Lane 1 Lane 2 Lane 3 Lane 4 Figure A-2. Gel electrophoresis illustrating the plasm id after restriction enzyme digestion. Lane 1&2 = Tt0625-1re, Lane 3&4 = Tt06252re, Outside lanes = Benchtop 100bp DNA ladder. j Delta Rn Cycle Figure A-3. DeHV-2 qP CR amplification comparison of cDNA, plasmid, and RE serial dilutions (RE serial dilution reflects the purified plasm id digested by the restriction enzyme BamH I). Threshold line shown at 0.3.

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SOUR CES AND MANUFACTURERS a. Becton, Dickinson, and Co., Franklin Lakes, NJ. b. Qiagen Inc., Valencia, CA. c. Thermo Fisher Scientific, Inc., Waltham, MA. d. Invitrogen Corp., Carlsbad, CA. e. PerkinElmer Inc., Branchburg, NJ. f. Eppendorf AG, Hamburg, Germany. g. Applied Biosystems Inc., Foster City, CA. h. Beckman Coulter Inc., Fullerton, CA. i. PROC GLM Overview, SAS Online Doc, version 8, SAS Institute Inc., Cary, NC. j. Promega Corp., Madison, WI. 70

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75 BIOGRAPHICAL SKETCH Heather Daniel was born and raised in Tampa, FL. She was a girl scout for thirteen years and earned the gold award as a senior. She received the Hillsborough Community College Honors Institute Presidential scholarship and gradua ted with an Associate of Arts degree in 2003. Heather graduated from the University of Flor ida in 2007 with a dual Bachelor of Science, majoring in Wildlife, Ecology & Conservation as well as Animal Sciences. She graduated, for the second time from University of Florida, in 2009 with a Master of Science from the College of Veterinary Medicine. She currently works full-tim e at the college in Aquatic Animal Health.