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Detection and Molecular Characterization of Cetacean and Pinniped Poxviruses Associated with Cutaneous Lesions


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DETECTION AND MOLECULAR CHARAC TERIZATION OF CETACEAN AND PINNIPED POXVIRUSES ASSOCIATED WITH CUTANEOUS LESIONS By ALEXA JUSTINE BRACHT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Alexa Justine Bracht

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This document is dedicated to my husband, Ethan Sherman, for his support and patience through these long journeys, and to my loving mother, Camille, who holds my hand along the way.

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iv ACKNOWLEDGMENTS My greatest thanks are extended to my ment or, Dr. Carlos Romero, not only for his guidance throughout my project, but for gi ving me this great opportunity, when few others would have. His support and patience through some very trying times were truly appreciated. I would also like to thank my committee members, Dr. Ayalew Mergia, Dr. James Maruniak and Dr. Charles Manire, fo r their time, assistan ce and suggestions. I would like to express my gratitude to Dr. Ellis Greiner, for being my first liaison to the University of Florida, and for his strong support of my application to the graduate program. This project was supported by a grant from Harbor Branch Oceanographic Institution and Florida Fish and Wildlif e Commission through the Marine Mammal Animal Health Program of th e College of Veterinary Medi cine at the University of Florida, and would not have been possible without the cont ributions of collaborators affiliated with numerous zoological parks, a quariums, stranding networks, and the Alaska Department of Fish and Game. In particul ar, great thanks are extended to Dr. Kathy Burek, Dr. Cheryl Rosa, Dr. Ruth Ewing, Dr Forrest Townsend, Dr. Charles Manire, Dr. Gay Sheffield, Dr. Jeremiah Saliki, Dr. Ki mberlee Beckman and Mr. Bob Schoelkopf for collecting and contributing the we alth of samples that make this work possible. I would also like to acknowledge my fellow la b-mates, Kara Smolarek-Benson, Rebecca Woodruff, Rebecca Grant, and Shasta McClen ahan for their friendship, assistance and open ears throughout our time toge ther. Last but not least, I would like to thank my family for all of their support, particularly my mother Camille, who left me all of her

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v love, strength and persistence. I am exceptio nally grateful to my husband, Ethan, for his unending support and patience durin g these long two years, for coming along with me in the pursuit of my dreams, for putting up with al l of the time we have had to spend apart and for making the times we spend together wonderful.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES.............................................................................................................x ABSTRACT......................................................................................................................x ii CHAPTER 1 INTRODUCTION........................................................................................................1 Introduction to Poxviruses............................................................................................1 Poxvirus Infections of Cetaceans..................................................................................7 Poxvirus Infections of Pinnipeds................................................................................11 2 MATERIALS AND METHODS...............................................................................14 Sample Acquisition.....................................................................................................14 Histopathology and Electron Microscopy..................................................................15 Extraction of Total DNA............................................................................................15 General Conditions for PCR.......................................................................................16 Poxvirus PCR Targeting the DNA Polymerase Gene.........................................16 Poxvirus PCR Targeting the DNA Topoisomerase I Gene.................................17 Poxvirus PCR Targeting the Major Envelope Gene............................................17 Poxvirus PCR Targeting the Hemagg lutinin Gene of Orthopoxviuses...............17 Parapoxvirus PCR Targeting the DNA Polymerase Gene..................................18 Parapoxvirus PCR Targeting th e DNA Topoisomerase I Gene..........................18 Parapoxvirus PCR Targeting the Ma jor Envelope Protein Gene........................19 Gel Electrophoresis.....................................................................................................19 Cloning of amplified DNA fragments........................................................................19 DNA Sequencing and Sequence Analysis..................................................................20 Primer Specificity and Sensitivity Assays..................................................................22 Virus Isolation............................................................................................................23 3 RESULTS...................................................................................................................24 Summary of Positive Samples....................................................................................24 Histopathology and Electron Microscopy..................................................................24

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vii Detection of Poxviruses Targeting the DNA Polymerase Gene.................................25 Detection of Poxviruses Targeti ng the DNA Topoisomerase I Gene.........................25 PCR Targeting the Major Envelope Protein Gene of Orthopoxviruses.....................26 PCR Targeting the Orthopoxvirus Hemagglutinin Gene............................................26 Detection of Parapoxviruses Targeting the DNA Polymerase Gene..........................26 Detection of Parapoxviruses Targeti ng the DNA Topoisomerase I Gene..................27 Detection of Parapoxviruses Targeting the Major Envelope Protein Gene................28 Sequencing and Genetic Analysis..............................................................................28 DNA Polymerase.................................................................................................28 DNA Topoisomerase I.........................................................................................30 Major Envelope Protein Gene.............................................................................32 Phylogenetic Analysis................................................................................................32 Virus Isolation............................................................................................................33 4 DISCUSSION.............................................................................................................66 LIST OF REFERENCES............................................................................................82 BIOGRAPHICAL SKETCH......................................................................................88

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viii LIST OF TABLES Table page 3-1 Pair-wise comparisons of the nucle otide sequences of the DNA polymerase gene fragments of the cetacean poxvir us 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples......................................................................................................53 3-2 Pair-wise comparisons of the ami no acid sequences of the DNA polymerase gene fragments of the cetacean poxvir us 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples......................................................................................................54 3-3 Pair-wise comparisons of the ami no acid sequences of the DNA polymerase gene fragments of the cetacean poxvir us 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples......................................................................................................55 3-4 Pair-wise comparisons of the nucleot ide sequences of the DNA topoisomerase gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV2) samples.................................................................................................................56 3-5 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV2) samples.................................................................................................................57 3-6 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV2) samples. ...............................................................................................................58 3-7 Pair-wise comparisons of the nucle otide sequences of the DNA polymerase gene fragments of poxviruses of vari ous genera within Chordopoxvirinae.............59 3-8 Pair-wise comparisons of the ami no acid sequences of the DNA polymerase gene fragments of poxviruses of vari ous genera within the Chordopoxvirinae subfamily of viruses.................................................................................................60 3-9 Pair-wise comparisons of th e amino acid sequences of the DNA polymerase gene fragments of poxviruses of vari ous genera within the Chordopoxvirinae subfamily of viruses. ...............................................................................................61 3-10 Pair-wise comparisons of the nucleot ide sequences of the DNA topoisomerase gene fragments of poxviruses of vari ous genera within Chordopoxvirinae.............62

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ix 3-11 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase gene fragments of poxviruses of vari ous genera witin Chordopoxvirinae...............63 3-12 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase gene fragments of poxviruses of vari ous genera within Chordopoxvirinae.............64 3-13 Pair-wise comparisons of the nucleot ide sequences obtained from the major envelope protein gene fragments of marine parapoxviruses within Chordopoxvirinae.....................................................................................................65 3-14 Pair-wise comparisons of the amino acid sequences from the major envelope protein gene fragments of marine parapoxviruses within Chordopoxvirinae..........65 3-15 Pair-wise comparisons of the amino acid sequences of the major envelope protein of marine parapoxviruses.............................................................................65

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x LIST OF FIGURES Figure page 3-1 Typical tattoo lesions of cetaceans......................................................................33 3-2 Gross appearance of pox lesions associ ated with a poxvirus in a Steller sea lion (Eumetopias jubatus ). .............................................................................................34 3-3 Cutaneous pox lesions in a spotted seal (Phoca largha ) associated with spotted seal parapoxvirus......................................................................................................34 3-4 Histopathologic appearance of cutaneous lesions associated wtih Steller sea lion poxvirus....................................................................................................................35 3-5 Negatively stained poxvirus particle from cutaneous lesion of SSL observed by electron microscopy.................................................................................................35 3-6 Agarose gel electrophoresis of P CR amplified 543-546-bp fragments of the DNA polymerase gene of cetacean and Steller sea lion poxviruses. ......................36 3-7 Agarose gel electrophoresis of P CR amplified 344-bp fragments of the DNA topoisomerase gene of cetacean and Steller sea lion poxviruses.............................36 3-8 Agarose gel electrophoreses of PCR am plified fragments of the HA gene of orthopoxviruses........................................................................................................37 3-9 Agarose gel electrophoresis demonstr ating the PCR amplification of 536-bp parapox DNA polymerase gene fragments fr om lesions of different pinniped species. ....................................................................................................................37 3-10 Agarose gel electrophoresis demons trating the PCR sensitivity assay for primers CR540 and CR541......................................................................................38 3-11 Agarose gel electrophoresis demonstr ating the PCR amplification of 350-bp parapox DNA topoisomerase I gene fragment s from lesions of Steller sea lions (Eumetopias jubatus ) and harbor seals (Phoca vitulina )..........................................38 3-12 Agarose gel electrophoresis demons trating the PCR sensitivity assay for primers CR550 and CR551......................................................................................39

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xi 3-13 Agarose gel electrophoresis demons trating the PCR sensitivity assay for primers CR557 and CR558 .....................................................................................39 3-14 Agarose gel electrophoresis demonstr ating the PCR amplification of 252-bp parapox virus DNA topoisomerase I gene fragment from lesions of spotted seals (Phoca largha )..................................................................................................40 3-15 Agarose gel electrophoresis demons trating the PCR sensitivity assay for primers CR570 and CR571......................................................................................40 3-16 Agarose gel electrophoresis demonstr ating the PCR amplification of 594-bp parapox major envelope protein gene fr agments from lesions of different pinniped species.......................................................................................................41 3-17 Agarose gel electrophoresis demons trating the PCR sensitivity assay for primers CR339 and CR340......................................................................................41 3-18 Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the DNA polymerase gene fragments of poxviru ses identified in cutaneous lesions of cetaceans.................................................................................42 3-19. Multiple alignment of the amino aci d sequences deduced from the nucleotide sequences of the DNA polymerase gene fragment of poxviruses identified in cutaneous lesions of pinnipeds.................................................................................43 3-20 Multiple alignment of the amino acid sequences deduced from the nucleotide. Sequences of the DNA topoisomerase gene fragments of poxviruses identified in cutaneous lesions of cetaceans.............................................................................44 3-21 Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the DNA topoisomerase gene fragment of poxviruses and parapoxviruses identifie d in cutaneous lesions of pinnipeds. .................................45 3-22 Multiple alignment of the partial amino acid sequences predicted from the major envelope protein gene fragme nt of parapoxviruses identified in cutaneous lesions of pinnipeds.................................................................................46 3-23 Neighbor-Joining phylogenetic tree of th e deduced amino acid sequences of the DNA polymerase gene fragments from members of the Chordopoxvirinae subfamily of poxviruses...........................................................................................47 3-24 Neighbor-Joining phylogenetic tree of th e deduced amino acid sequences of the DNA topoisomerase gene fragments fr om members of the Chordopoxvirinae subfamily of poxviruses...........................................................................................49 3-25 Neighbor-Joining phylogenetic tree of th e deduced amino acid sequences of the Major envelope protein gene fragment s from members of the Chordopoxvirinae subfamily of poxviruses...........................................................................................51

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xii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science DETECTION AND MOLECULAR CHARAC TERIZATION OF CETACEAN AND PINNIPED POXVIRUSES ASSOCIATED WITH CUTANEOUS LESIONS By Alexa Justine Bracht August 2005 Chair: Carlos H. Romero Major Department: Veterinary Medicine Poxviruses are widespread and successful pathogens, known to infect a variety of vertebrates including, reptiles, birds, and over 30 species of mammals and several species of insects. Terrestrial p oxviruses encompass a variety of well known etiologic agents, that are currently classified into eight genera, within the Chordopoxvirinae subfamily. While significant advances have been made in understanding the genomic sequences of terrestrial poxviruses, little is known about marine poxviruses. DNA extracted from skin lesions of cetaceans in oceanaria and rehabi litation facilities as well as free-ranging cetaceans and pinnipeds was assayed by polymer ase chain reaction (PCR). Primers were designed to target gene fragments of three genes: DNA polym erase (DNApol), DNA topoisomerase (DNAtopo) and the major en velope protein (MEP) of poxviruses and parapoxviruses based on numerous DNA sequences available in the National Center for Biotechnology Information (NCBI) database. Targeting of the poxvirus DNApol gene yielded 543-bp fragments when swinepox ( SPV) and mule deer poxvirus (MDPV) DNA

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xiii were used as templates. Targeting of cetacean poxvirus (CPV) DNA yielded 546-bp amplicons that upon sequencing revealed th e existence of two distinct poxvirus sequences that were shown to be approxi mately 84% and 89% identical in their nucleotides and amino acid sequences, respec tively. These findings provide the first evidence of activity of at leas t two poxviruses in cetaceans, that we provisionally refer to as cetacean poxvirus -1 and 2 (CPV-1 and CPV2). Amplification of Steller sea lion (Eumetopias jubatus ) poxvirus (SSLPV) DNA yielded a 543-bp DNApol gene fragment with nucleotide identity ranges of 76-77% and amino acid identity ranges of 74 -78% when compared to homologous CPV-1 and CP V-2 fragments. Analyses of CPV-1 DNApol fragments showed closest amino acid identity to members of the orthopox genus (~81%), while CPV-2 had identities of ~ 83%. DNApol fragments amplified from parapox viruses from Steller s ea lions, spotted seals (Phoca largha ) and a harbor seal (Phoca vitulina ) were 536-bp in length and had closes t amino acid identities to members of the parapox genus (87-89%). PCR with poxvirus consensus primers that targeted the DNAtopo gene generated 344-bp amplicons using CPV-1, CPV-2, SSLPV, SPV and MDPV DNA as templates. Parapox DNAtopo fragments were amplified from the same set of pinnipeds and were 347, 350 and 252-bp in length. Consensus primers that target the MEP gene of parapoxvi ruses amplified 594-bp fragme nts from pinniped parapoxviruses, as well as from pseudocowpox viru s DNA. The describe d molecular assays based on PCR and direct sequencing of amplic ons have allowed us to identify several novel poxviruses and investigate the evolutiona ry relatedness of these viruses when compared to other well known terrestrial poxviruses of vertebrate s in the subfamily Chordopoxvirinae.

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1 CHAPTER 1 INTRODUCTION Introduction to Poxviruses The family Poxviridae contains the larges t known viruses of terr estrial and marine mammals that possess non-infectious, doubl e stranded DNA genomes that range in size from 130-380 kbp and replicate almost exclusiv ely in the cell cytoplasm (Moss, 2001). The basic poxvirus virion incorporates about 100 polypeptides and carries most of the compounds necessary for replication in the host cell. The non-infectious DNA genome (nucleosome) is arranged inside a core memb rane that is surrounded by lateral bodies and another outer membrane. Poxviruses encase th is outer membrane in yet another lipid bilayer, called the envelope, which func tions in host cell attachment (Buller and Palumbo, 1991; Moss, 2001). The Poxviri dae is divided into two subfamilies: Entomopoxvirinae, that comprises insect poxviruses, and Chordopoxvirinae, that includes all poxviruses of vertebrate s (Moss, 2001). Subfamily Chordopoxvirinae includes eight genera: orthopoxvirus, parapoxvirus, capri poxvirus, suipoxvirus, leporipoxvirus, yatapoxvirus, avipoxvirus and molluscipoxvirus. Poxviruses are highly adapted viruses infecting a large number of hosts, including insects, reptiles, birds, and over 30 mammalian species (Buller a nd Palumbo, 1991; Moss, 2001; Upton et al., 2003). Some members of the Orthopox genus, such as vari ola, the causative agent of smallpox virus, are endowed with high virule nce and were a scourge to mankind for at least two millennia until its eradication in 1977 (Moss, 2001). Highly invasive and virulent poxviruses of livestock of the Capripoxvirus genus, such as sheep pox, goat pox and

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2 lumpy skin disease viruses, and of the Orthopox genus, such as camel poxvirus, are currently restricted to some parts of the s ub-Saharan African contin ent, the Middle East and the Indian subcontinen t (Ireland and Binepal, 1998; Moss, 2001). Poxviruses replicate in the skin and mucosa producing lo calized or generalized lesions of variable gravity and duration (Bulle r and Palumbo, 1991). Localized lesions are seen in Molluscum contagiosum, pseudocowpox, and or f virus infections, whereas disseminated lesions are seen in ectromelia, cowpox, monkeypox, and the well known variola virus. (Moss, 2001; Upton et al., 2003). Some pa rapoxviruses and myxoma virus have the ability to display both disease patterns depending on the animal host (Buller and Palumbo, 1991). This study, fueled by the generation of gene tic data, would not have been possible without the fundamental technique, now implem ented in almost every molecular genetics laboratory, the polymerase chai n reaction, or PCR. Conceived by Kary Mullis in 1983 (Mullis et al., 1986), the idea revolutionized the fields of biotechnology and molecular biology, paving the way for development of ne w assays to diagnose medical disorders and a wide range of diseases (Schluger and Rom, 1995). PCR uses simple principles of DNA replication combined with the unique properties of a DNA polymerase from thermophilic bacteria to mimic the DNA re plication process in-vitro, exponentially amplifying copies of the targeted DNA (Bej et al., 1991). While the uses of PCR span the range of medical diagnoses, the relevanc e of this technique to virology is its facilitation of cloning and seque ncing of viral genes for the purposes of viral comparison and classification (Bej et al .,, 1991; Ropp et al., 1995; Elnifro et al., 2000). Prior to PCR technology, viruses were isolated in cell culture before any further analyses could occur

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3 (Levine, 2001). Identification and differentia tion of viruses before the implementation of PCR relied on less specific serologic a ssays such as, virus neutralization, hemagglutination and immuno fluorescence assa ys, later complemented by restriction endonuclease profiles of viral DNA resolved in agarose or polyacrylamide gels (Fleming et al., 1993; Robinson and Mercer, 1995; Ropp et al., 1995; Mangana-Vougiouka et al., 1999; Moss, 2001). These techniques require large amounts of viral DNA typically harvested from virus infected cultures. This approach hindered the study of viruses that do not readily grow in culture, such as papill oma viruses, or for that matter, poxviruses of marine mammals. The advent of PCR allowe d for the direct amplification of viral DNA and rapid genome sequencing (Moss, 2001). With the sudden accumulation of genome sequences from numerous viruse s representative of most families, detailed examination of genetic relationships escalated couple d with a new understanding of viral taxonomy. For example, upon genetic comparison, it was found that two genetic mutations were responsible for the antigenic difference be tween feline panleukopenia virus and canine parvovirus (Levine, 2001). Si nce the late 1980s, PCR protoc ols have been developed to detect a wide variety of human and animal viruses representing several viral families and genera including: hepatitis, papilloma, influenza, rhabdo-, re tro-, herpes-, calici-, adenoand pox-viruses, among numerous others (D e Rossi et al., 1988; Puchhammer-Stockl et al., 1990; Vandenvelde et al., 1990; Sacramento et al., 1991; De Leon et al., 1992; Hondo and Ito, 1992; Morishita et al., 1992; Vesy et al., 1993; Ropp et al., 1995; Heredia et al., 1996; Vantarakis and Papapetropoulou, 1999; Inoshima et al., 2000). Considering poxviruses in particular, PCR and genome sequencing has meant the evolution from sometimes vague histopathologic and electr on microscopic (EM) diagnoses to much

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4 more definitive genetic assays for poxvirus infection (Fleming et al., 1993; Ropp et al., 1995; Mangana-Vougiouka et al., 1999; Dama so et al., 2000; Gubser and Smith, 2002; Howsamani et al., 2004) Most poxviruses shar e a common ovoid or brick-like shape and measure 200 400 nm in length with tubules arranged in an irregular pattern on the envelope surface ( Buller and Palumbo, 1991; Mo ss, 2001). The exception to this is the genus parapox, which has a notable criss-cr oss tubule pattern on the envelope surface (Moss, 2001). Because of this common poxvi rus morphology, it is difficult to discern between genera of poxviruses when usi ng techniques like histopathology and EM. However, PCR and sequencing methods reveal not only the genus, but in most cases, species of the virus being ex amined (Ropp et al., 1995, Damaso et al., 2000; Becher et al., 2002; Howsamani et al., 2004). Beginning in 1990 with the sequence for vaccinia virusCopenhagen strain (Goebel et al., 1990), complete poxvirus genomes sequences have been generated using techniques de rived from PCR, rest riction endonuclease digestion, and basic DNA cloning and sequenci ng. The generation of complete genome sequences in pox virology reached a climax in the years 2000-2002 with the release of 14 complete genome sequences representi ng six genera within the sub-family Chordopoxvirinae and one in sub-family Entomopoxvirinae (Gubs er et al., 2004). Included in these were complete genome sequences of variola, vaccinia, monkeypox, camelpox, fowlpox, lumpy skin disease viru s, goatpox, sheeppox, swinepox, and Yabalike disease virus, as well as, Amsacta moorei entomopoxvirus (Goebel et al., 1990; Shchelkunov et al., 1995; Antoine et al., 1998; Shchelkunov et al., 2000; Tulman et al., 2001; Afonso et al., 2002; Gubser and Sm ith, 2002; Gubser et al., 2004). These sequencing advances have allowed for a be tter ability to define and understand the

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5 evolutionary relationships between the differe nt poxvirus genera. Comparing genes that have been identified as highly conserved can aid in new virus characterization and comparison. Attempts at obtaining a more global understanding of poxvirus genes and gene families have been made using th e Poxvirus Bioinformatic Resouce (PBR; www.poxvirus.org ) and have identified 49 conserved gene families in 21 complete poxvirus genomes (Upton et al., 2003). Previo us studies have elected phylogenetic analysis to be the best tool availabl e for characterizing poxv iruses known to date (Afonso et al., 2002; Becher et al., 2002, Gubser et al., 2004). Thes e studies present an easily comprehendible picture of the ancestr al lines of viruses, including possible progenitors for different lineages. The in crease in numbers of available sequences permitted attempts to create a more global understanding of poxvirus genetic relationships, mainly through the use of evolu tionary analysis. As different software programs emerged and improved over time, nume rous phylogenetic analyses have been conducted, initially on single genes or gene fr agments (Zanotto et a., 1996; Afonso et al., 2002; McGeoch et al., 2000; Gubser and Smit h, 2002; Hosamani et al., 2004; Tryland et al., 2005). Phylogenetic trees c onstructed using different i ndividual proteins can yield varying topologies, depending on the stringency of cons ervation of the DNA sequence for the specific gene (Gubser et al., 2004) More recently, phylogenetic studies using large fragments of the centra l region of the genome or the complete genome sequences have been published, revealing a more complete and accurate picture of poxvirus phylogeny (Fleming et al., 1993; Upton et al., 20 03; Delhon et al., 2004 ; Gubser et al., 2004,). The results of these studies served to verify what had been previously accepted in taxonomic classification of poxviruses by desi gnating the viruses in to groups that

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6 corresponded with the accepted poxvirus genera. Gubser et al. (2004) used the sequences of 26 poxviruses representing all poxvirus ge nera except parapoxviru s, in a phylogenetic study. They found that the general orga nization of the Chordopoxvirinae genome was conserved, specifically in regard to the cen tral region genes enc oding proteins for RNA and DNA replication, virion assembly and stru ctural proteins. This concurs with a previous study by Upton et al. (2003), wher e authors performed large scale genetic analysis on 21 complete poxvirus genomes. These authors used the Virus Genome Database to identify genes that are most highly conserved among the family Poxviridae, and determined that those genes were invol ved in DNA replication and transcription. Gubser et al. (2004) used these results to construct a tree inco rporating 17 of the 49 proteins that are conserved in all poxviruse s, and found that the vi ruses included in the subfamily Chordopoxvirinae could be divided into four main gr oups: the Molluscipox genus, the Avipox genus, the Orthopox ge nus, and a group containing the Yatapox, Capripox, Suipox and Leporipox genera. The tr ee suggests that the latter group includes viruses evolved more recently, and thus are gene tically more related to each other than to the first three groups. It should be noted that the parapoxvirus genus was excluded from this study, and it is speculated that it might clad into its own group, if similar analyses were repeated. Characterization of nove l poxviruses such as muledeer poxvirus, spectacled caiman poxvirus, Embu virus and Ca ntangalo virus that have not yet been assigned to a genus, may be aided by these ne w techniques. Equally mysterious are the poxviruses that have been known to infect va rious species of cetacea and pinnipedia. Though often and easily recognized by clinical means, these viruses have not been antigenically or molecularly characterized.

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7 Poxvirus Infections of Cetaceans Poxviruses have been known ( for over 30 ye ars), to affect various cetacean and pinniped species (Wilson et al., 1969; Wilson a nd Poglayen-Neuwall, 1971; Geraci et al., 1979; Baker, J.R., 1992a,b; Baker and Mart in, 1992; Van Bressem et al., 1993). Numerous studies have reporte d skin lesions asso ciated with poxvirus infections in cetaceans and parapoxvirus infections in pinnipe ds (Geraci et al., 1979; Osterhaus et al., 1990; Baker and Martin, 1992; Van Bressem et al., 1993; Nettleton et al., 1995). In cetaceans, poxvirus lesions are described as areas of hyperpig mentation of the skin with pinhole marks, termed tattoo or ring lesi ons (Geraci et al., 1979; Van Bressem et al., 1993). These lesions were reported to persist for months to years, and typically regress without treatment (Geraci et al., 1979, Sm ith, 1983). The appearance of poxviruses lesions in cetaceans seems different from that of poxvirus infections described in terrestrial vertebrates (Gerac i et al., 1979). Lesions associ ated with cetacean poxviruses remain relatively flat, and in some advanced st ages, form slightly da rk depressions in the center of the lesions (Geraci et al., 1979). Conversely, lesions associated with terrestrial poxviruses may form raised nodules in the sk in, and often advance to erupted pustules (Robinson and Mercer, 1995; Damaso et al., 2000; Moss, 2001; De lhon et al., 2004). Geraci etal. (1979), provided an explanation for the differenc e in clinical appearance and progression of lesions which involves the unique metabolic and m itotic rate of the epidermal cells of the cetacean integument. Further studies on the progression of the disease associated with cetacean pox virus infection have focused finding histopathological changes with presence of intracytoplasmic inclusions and on morphological characterization of the cet acean poxvirus using electron microscopy (Smith, 1983; Van Bressem, 1993). While the prevalence and conditions that participate

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8 or facilitate the occurrence of poxvirus inf ection in cetaceans have not been studied, Van Bressem et al., (1993), reported 8.1% and 30% prevalence of tattoo lesions in 74 dusky dolphins (Lagenorhynchus obscurus ) and 10 Burmeisters porpoises (Phocoena spinipinnis ), respectively, that were examined as fishing by-catch in 1990. The true prevalence of poxvirus infection in wild cetacean populations is unknown; however, Geraci et al., (1979) suggeste d an association of the occu rrence and severity of tattoo lesions, with animals under considerable envi ronmental stress or those exhibiting poor general health. The authors ci ted specific cases involving cap tive dolphins afflicted with lesions that improved under less stressful envi ronmental conditions (Geraci et al., 1979). In contrast to the abundance of sequence data for terrestria l poxviruses, even though the occurrence of poxviruses in marine mammals has been well documented for at least three decades (Wilson et al., 1969; Wils on and Poglayen-Neuwall, 1971; Geraci et al., 1979; Baker, J.R., 1992a,b; Baker and Martin, 1992; Van Bressem et al., 1993), almost no molecular data are available for marine poxviruses. Previous reports have described the pathogenicity and the gross and microscopic lesions after the infection of cetaceans and pinnipeds with marine poxviruse s. Most of these infections were diagnosed by demonstrating characteristic poxvirus particles by el ectron microscopy or the presence of acidophilic intr acytoplasmic inclusion bodies in sections of lesions by light microscopy (Flom and Houk, 1979; Geraci et al., 1979; Smith, 1983; Baker, 1992a; Van Bressem et al., 1993). The first publis hed description of poxvirus infection in captive and free-ranging cetaceans discussed ring and tattoo type lesions observed on seven bottlenose (Tursiops truncatus ) and one Atlantic white-sided dolphin (Lagenorynchus acutus ) (Geraci et al., 1979). Lesions we re noted to occur most often on

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9 the dorsal body, dorsal fin and flukes and pectoral flippers (Figure 3-1). Samples of the lesions were examined by light microsc opy and electron microscopy, which revealed eosinophilic intracytoplasmic inclusions contai ning virus particles w ith typical poxvirus morphology. The condition, termed dolphin po x, was found to vary in time course, severity and clinical appe arance and recurrence. Alt hough the author identifies a poxvirus as the causative agent for the obser ved lesions, associat ions between the environmental conditions, general animal health and stress level were also considered as possible reasons for the variations in dis ease progression. Concurrent studies with similar findings were reported in three mo re Atlantic bottlenose dolphins (Flom and Houk, 1979). While the pox lesions in both stud ies are reported to exist without causing any serious harm or consequence to the an imals health, one exceptional case was cited describing a dolphin that died after developing generalized lesions (Flom and Houk, 1979). Smith et al. (1983) reported an observa tion of two distinct regression patterns of the typical dolphin poxvirus tattoo lesions. The first regression patte rn consisted of the lesions becoming raised and edematous that, over time, became depressed and disappeared. The second regression pattern occurred following le sion biopsies where the lesions disappeared in zones surrounding the incision. Samples were taken from both raised and typical flat tattoo lesions, r eacted with dolphin sera and evaluated by immunoelectron microscopy. Positive reactivity occurred between the sera and the raised endematous lesions, but not w ith the flat tattoo lesion. Th e significance of this study is two fold: it is the first docu mentation of antibody response to a poxvirus recovered from dolphin lesions and secondly, it su ggests the possibility of two antigenically different poxviruses that cause lesions with dissimilar clinical appearance. Over the next ten

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10 years, numerous reports of poxvirus infecti ons in various cetacean species surfaced (Baker, 1992a,b; Baker and Martin, 1992; Van Br essem et al., 1993). Cetacean species in which infections with poxviruses have been previously reported include: Atlantic bottlenose dolphin (Geraci et al., 1979; Flom and Houk, 1979), Atlantic white-sided dolphin (Geraci et al., 1 979), common dolphin (Delphinus delphis ) (Britt and Howard, 1983), dusky dolphin (Lagenorynchus obscurus ) (Van Bressem et al., 1993), striped dolphin (Stenella coeruleoalba ) (Baker, 1992a), white beaked dolphin (Lagenorhynchus albirostris ) (Baker, 1992a,b) and Hectors dolph in (Cephalorhynchus hectori ) (Geraci et al., 1979; Baker, 1992a, b; Van Bressem et al., 1993, 1999). Similarly, poxvirus infections have previously been describe d in long finned pilot whales (Globocephala melaena ) (Baker, 1992a), killer whales (Orcina orca ), Burmeisters porpoise (Van Bressem et al., 1993), and harbor porpoises (Phocoena phocoena ) (Baker, 1992a,b; Baker and Martin, 1992; Van Bressem et al., 1993, 1999). Routine histological methods continued to provide the best descriptions of microscopi c changes in the lesions. Examinations of poxvirus lesions of a Burmeisters porpoise (Phocoena spinipinnis ) utilized transmission electron microscopy to hi ghlight an irregular a rrangement of tubules on the viral membrane, reminiscent of those s een in orthopoxviruses (Van Bressem et al., 1993). This was the first attempt to characteri ze the virus in order to assign it to one of the known genera. Little new information ha s been accumulated in respect to cetacean poxviruses since the early 1990s. The gr owing availability of DNA sequencing technologies has created opportunities to examine the genome of cetacean poxviruses, and how they situate within the subfamily Chordopoxvirinae.

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11 Poxvirus Infections of Pinnipeds Poxvirus infections have also been well documented in several pinniped species since 1969. These pinniped poxvirus lesions have a very different appearance from those seen in cetaceans and are typically raised nodul es in the skin (Figures 3-2 and 3-3).The first report of seal pox described pox lesions occurring in California sea lions (Zalophus californianus ) (Wilson et al., 1969). Closely follow ing that report was one describing an epizootic of a proliferative skin disease among captive California sea lions (Wilson et al., 1972). Histopathology and electron microscopy determined the causative agent to be a poxvirus, and a survey was initiated and sent out to 120 addresses in an attempt to understand more about the scope of this new virus (Wilson et al., 1972). Over the years these methods continued to be employed in identifying sealpox in fections of various species including; harbor seals (Phoca vitulina ) (Becher et al., 2002, Mller et al., 2003), grey seals (Halichoerus grypus ) (Hicks and Worthy, 1987; Osterhaus et al., 1990; Simpson et al., 1994; Nettleton et al., 1995), California sea lions (Wilson et al., 1969), South American sea lions (Otaria byronia ) (Wilson and Poglayen-Neuwall, 1971), Weddell seals (Leptonychotes weddellii ) (Tryland et al.2005) and northern fur seals (Callorhinus ursinus) (Hadlow et al., 1980). Because of the similarities to orf and bovine papular stomatitis (BPSV) virion morphology and lesion pathology, the seal poxv iruses were designated as probable members of the parapox sub-group (Esposito, 1991). While orf and BPSV were both known to be transmissible to humans (Bowman et al., 1981; Meechan and Macleod, 1992; Delhon et al., 2004), the zoonotic po tential of any marine mammal poxvirus was unknown. In 1987, a case report described two s eal handlers that developed lesions on their hands similar to milkers nodules that occurred while working with grey seals with

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12 typical seal pox lesions (Hicks and Worthy, 1987). Healing tim es varied and one handler experienced relapses over the ne xt several months. Negative staining of the virions from both the seals and the handlers suggest that the handlers nodular le sions were caused by the seal pox virus. The lesions described in South American sea lions were distinct from those previously described in California sea lio ns and harbor seals (Wilson and PoglayenNeuwall, 1971). The described lesions in Calif ornia sea lions and har bor seals proliferate outward, but the lesions of the South Ameri can sea lions proliferate downward into the dermal layer. The intracytoplasmic inclus ion bodies differed in morphology being large and oval shaped versus small and irregular. In addition, the virion of the South American sea lion pox virus appeared rectangular or brick shaped, versus the elongated or cylindrical shape normally associated with prev ious reports of seal pox virus shape. The results of this report suggested the existence of two poxviruses with the ability to infect pinnipeds. Similar observations were made in the reexamination of old formalinized samples from a stranded northern fur seal pup (Hadlow et al., 1980). Tissues preserved from an animal that was necropsied in 1951 were examined for poxvirus and found to resemble those reported in South American sea lions more than in California sea lions or Harbor seals. The suggestion of the existe nce of two pinniped poxviruses resurfaced in a report that outlined the isolation of both pa rapox and orthopox-like viral particles from lesions of a grey seal (Oster haus et al., 1990). The in vitr o culture of the orthopox-like virus was apparently, only possibl e in primary grey seal skin cells. However. no reports on the characterization of this poxvirus has appeared since. A parapoxvirus was later

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13 isolated from grey seal pox lesions using primary grey a nd harbor seal kidney cells (Osterhaus et al., 1994, Nettleton et al.,1995). The first mention of using the polymerase chain reaction (PCR) to test for pinniped poxvirus infection surfaced in 2002 (Becher et al., 2002). The PCR primers used were known to direct the amplificat ion of a segment of the major envelope protein gene and had been reported as a diagnostic tool for pa rapox infections of cattle, sheep and Japanese serows (Inoshima et al., 2000). Skin lesions from harbor seals were analyzed for parapoxvirus infection. Nucl eotide and amino acid sequences obtained from the DNA sequence of the amplified PCR fragments were compared against those of BPSV, pseudocowpox virus (PCPV), parapoxvirus of red deer in New Zealand (PVNZ) and orf virus (OV) and found to be significantly differe nt in both cases: <79% nucleotide identity and <77% amino acid identity. The author s suggested that the seal parapoxviruses constituted a separate species within th e genus Parapoxvirus (Bech er et al., 2002). Presently, sealpox is classifi ed as a tentative member of the parapox genus. It is evident that, while many advances have b een made since the early days of poxvirus detection in marine mammals, much is st ill unknown about the genomic organization and evolutionary relationships of these viruses.

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14 CHAPTER 2 MATERIALS AND METHODS Sample Acquisition Fresh and frozen skin lesions from 109 st randed, free-ranging and captive marine mammals were harvested and shipped to our laboratories between January, 2001 and March, 2005 for analyses of poxvirus infecti on. Lesion scrapings and biopsies from captive marine animals were provided by several amusement parks and aquariums from Florida, Texas, Portugal, and Hong Kong. Tissues from stranded and free-ranging animals were obtained from numerous partic ipants of the Southeast, Northeast and Alaska Stranding Networks, as well as the Al aska Department of Fish and Game. All samples collected from stranded marine mammals were obtained by licensed personnel from the Networks. These lesions were obtained from 92 cetaceans and 17 pinnipeds. Donor species were: Forty-two Atlantic bottl enose dolphin (Tursiops truncatus), twentytwo bowhead whales (Balaena mysticetus ), seven Indopacific bottlenose dolphin (Tursiops aduncus), four r ough-toothed dolphin (Steno bredan ensis), four pygmy sperm whales (Kogia breviceps), two killer whales (Orcina orca) (Dover, 1992), two shortfinned pilot whales (Globice phala macrorhynchus), three Rissos dolphin (Grampus griseus), one striped dolphin (Stenella coer uleoalba), one Pacific white-sided dolphin (Lagenorhynchus obliquidens ) one dwarf sperm whale (Kogia sima), one spinner dolphin (Stenella longirostris), one Pantropical spotted dolphi n (Stenella attenuata), one Harbor porpoise (Phocoena phocoena), fourteen Steller sea lions (Eumetopias jubatus), two spotted seals (Phoca largha), and one harbor seal (Phoca vitulina).

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15 Histopathology and Electron Microscopy A 6-mm punch biopsy was taken of two Ste ller sea lion skin lesi ons. One half of the biopsy was placed in 10% neutral buffered formalin and the other half frozen in dry ice and stored at C for DNA extraction a nd PCR analysis. Formalin fixed samples were embedded in paraffin, sectioned at 5 m, and stained with hematolyxlin and eosinophilic for evaluation by light microsc opy. Negative staining electron microscopy was also performed on formalin-fixed specimens. The samples were homogenized in distilled water in a Ten-Broeck grinder, clarified by centrifugati on at 4,000xg for 5 min, the supernatant removed to a clean tube a nd centrifuged at 12,000xg for 1 hr. The pellet was resuspended in 2% phosphotungstic acid solution at pH 6.8 c ontaining 0.01% bovine serum albumin and a drop of this suspension was applied to a carbon coated formvar film on a 400 mesh copper grid and the excess wicked away. The grid was examined with a Zeiss EM 109 microscope (Carl Zeis s, Inc., Thornwood, New York, USA). Extraction of Total DNA All samples were processed to obtain total DNA using the DNeasy kit (Qiagen, Valencia, California, USA) according to th e protocol indicated by the manufacturer. Briefly, 25 mg of tissue was cut into small pieces and combined with 180 l of lysis buffer ATL and 20 l of proteinase K. The tissues were incubated at 55 C until lysis was complete. DNA was precipitated by the addition of 200 l absolute ethanol and spun through the DNeasy Spin Column. After tw o washes with buffers AW1 and AW2, the DNA was eluted in 200 l buffer AE. The quality and content was evaluated by spectrophotometry using the Ultrospec 3000 (A mersham Biosciences Corp., Piscataway, New Jersey, USA). Each group of tissue samples was extracted along with a known negative sample to be used as a negative control for analysis.

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16 General Conditions for PCR Reaction tubes for PCR contained 200 nM of each primer, 100 M of each deoxynucleoside triphosphate (dNTP), 10 mM KCl, 10 mM (NH4)2 SO4, 20 mM TrisHCl, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8, 1 unit of Taq DNA polymerase (New England BioLabs, Beverly, Massachusetts, US A), and 500 ng of DNA template, in a final volume of 50 l. All PCR cyclings were perfor med in a PTC-100 thermal cycler (MJ Research, Inc., Waltham, Massac husetts, USA). Cycling conditions for the amplification of the DNA polymerase and DNA topoisomerase gene fragments of poxviruses were: Initial denaturation at 94 C for 1 min, followed by 39 cycles, each comprising of a denaturation step at 94 C for 30 sec, an annealing step at 45 C for 30 sec, and an elongation step at 72 C for 30 sec. The last cycle incl uded an extended elongation step at 72 C for 10 min. Cycling conditions for the am plification of the DNA polymerase gene of parapoxviruses were similar, except that the annealing temperature for the parapoxviruses was 61oC. The cycling conditions for the amplification of the DNA topoisomerase gene fragments of parapoxviruse s from Steller sea lions, harbor seals and spotted seals were also similar; howev er, the annealing temperatures were 53oC, 51oC and 58oC, respectively. Poxvirus PCR Targeting the DNA Polymerase Gene Oligonucleotide primers that target sequences within the DNA polymerase gene were designed based on sequences of lum py skin disease virus (LSDV) and swinepox virus (SPV) deposited in the GenBank databa se and from mule deer poxvirus (MDPV) sequenced in our laboratories (Accession number AY841895). These sequences were: Forward primer CR 422: 5 ATA CAG AGC TAG TAC ITT AAT AAA AG 3and

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17 reverse primer CR 421: 5CTA TTT TTA AAT CCC ATT AAA CC 3. MDPV or SPV DNA was used as a positive control, yielding DNA fragments of 543 base pairs (543-bp) in size. Negative tissues were used as negative controls. Poxvirus PCR Targeting the DNA Topoisomerase I Gene Oligonucleotide primers were designe d based on the sequences of homologous genes of LSDV, SPV and MDPV (AY841896). The primer sequences were: CR 432: 5 TAA TGG AAA CAA GTT TTT TTA T 3 and CR 433: 5 CCA AAA ATT ATA TAA AAA CG 3. These primers dire cted the amplifi cation of a 344-bp DNA fragment when SPV and MDPV genomic DNA wa s used as a positive control. Negative tissues were used as negative controls. Poxvirus PCR Targeting the Major Envelope Gene Oligonucleotide primers were designed based on the sequences of vaccinia, camelpox, monkeypox, variola, ectromelia and cowpox. Two forward primers were designed, the first one included the gene start codon, and the second was 42-bp internal to the start codon. The two forward primer sequences were: CR 597: 5 ATG TGG CCA TTT RYA TCR GY -3 and CR 598: 5 CTG GTA GAA ACA CTA CCA GAA AAT 3. The reverse primer sequence includ the stop codon and was designed as follows: CR596: 5TTA AAT TTT YAA CGA TTT ACT GT G GC -3. The expected sizes of the fragments generated by these prim ers were 1118-bp and 1076-bp respectively Vaccinia virus DNA was used as a positive c ontrol. Negative tissu es were used as negative controls.. Poxvirus PCR Targeting the Hemaggl utinin Gene of Orthopoxviuses PCR primers were designed to target the Hemagglutinin (HA) gene of orthopoxviruses based on the sequences of camelpox, vaccinia, monkeypox, cowpox,

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18 variola, and ectromelia. The primers ta rget the full HA gene and predict the amplification of a 1138-bp fragment from or thopox viruses. The primers were forward primer CR 619: 5GAT TTT CTA AAG TRY TTG GAR AGT TTT AT3 and reverse primer CR620: 5-GCT GTC TTT CCT IAA CCA GAT G -3. Vaccinia virus DNA was used as a positive control. DNA extracted from a negative tissues and a negative tube containing no DNA were used as negative controls. A previously described set of primers was also used to amplify the HA gene sequence of orthopoxviruses (Damaso et al., 2000). Parapoxvirus PCR Targeting the DNA Polymerase Gene Oligonucleotide primers that target genom ic sequences within the DNA polymerase gene of parapoxviruses were designed base d on genomic sequences of orf (NC_005336) and bovine papular stomatitis (NC_005337) viruse s that exist in the GenBank database. These primer sequences were: CR 54l: 5GCG AGC ACC TGC ATC AAG 3; CR 540: 5CTG TTI CGG AAG CCC ATG AG 3. Pseudocowpox virus DNA was used as a positive control. Negative tissues were used as negative controls.. Parapoxvirus PCR Targeting th e DNA Topoisomerase I Gene Oligonucleotide primers were first designe d based on the nucleotide sequences of the orf (NC_005336) and bovine papular stomatitis (NC_005337) virus DNA topoisomerase gene sequences from the Ge nBank database. These primers were: CR 550: 5 TCA TGG AGA CS A GCT TCT TCA TC 3(forward); CR 551: 5CCA GAA GTT GTA CAR RAA SGT GTA G 3(reverse). This primer set, however, did not amplify parapoxvirus sequences from DNA ex tracted from lesions of all species of marine mammals tested. Thus, a second prim er set was designed based on sequences obtained from the Steller sea lion parapoxviru s DNA topoisomerase gene fragment. The

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19 primer sequences were: CR 557: 5 TCA TGG AGA CGA GCT TCT TCA TC 3(forward); CR 558: 5 CCA GAA GTT GTA CAA GAA GGT GTA G 3(reverse). As these two sets of primers still did not amplify parapoxvirus DNA from spotted seals, a third set of primers had to be designed afte r performing a line up between the Steller sea lion and harbor seal parapoxvi ruses DNA topoisomerase gene fragments. These primer sequences were: CR 570: 5 GTC YTT AA C GCG AAT RCC AAA GC 3(forward); CR 571: 5AGC GGM ACW GTK GGY TTG CTC AC 3 (reverse). Pseudocowpox virus DNA was used as a positive control. Negative tissues were used as negative controls.. Parapoxvirus PCR Targeting the Major Envelope Protein Gene PCR was performed using previously publis hed consensus primers known to target the major envelope protein gene of para poxviruses (Inoshima et al., 2000). These primers were: FP-PPP-4: 5TAC GTG GG A AGC GCC TCG CT-3(forward); RP-PPP1: 5-GTC GTC CAC GAT GAG CAG CT-3(reve rse). This primer set directs the amplification of a 594-bp DNA fragment. Pseudocowpox virus DNA was used as a positive control. Negative tissues were used as negative controls.. Gel Electrophoresis Amplified DNA fragments were resolved by horizontal electrophoresis of 20-30 l of the PCR product in 1.0% agaros e containing ethidium bromide (0.5 g/ml), visualized under ultraviolet light and photographed using a gel docum entation system (Bio-Rad Laboratories, Inc., Hercules California, USA). Cloning of amplified DNA fragments To obtain the complete nucleotide sequen ce of all amplified DNA fragments, these were cloned into the bacterial plasmid vect or pCR 2.1 TOPO TA (Invitrogen, Carlsbad,

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20 California, USA) following the manuf acturers protocol. Competent E. coli DH5 alpha cells were transformed with vector-insert re actions and streaked on 2XYT agar medium containing ampicillin (100 g/ml). Colonies were grown ov ernight as minicultures, in 3 ml of 2XYT medium containing ampicillin (100 g/ml), while shaken at 270 rpm at 37 C. Plasmid DNA was extracted from 1.5 ml of the minicultures using a phenol-free method (Zhou et al., 1990). To screen fo r recombinant plasmids, plasmid DNAs were digested with restriction enzymes HindIII, EcoRI, ApaI, BamHI, and a combination of enzymes ApaI and BamHI. Recombinant plas mids containing the correct insert were purified for sequencing using the Aurum Pl asmid Mini Kit or the Plasmid Midi-Prep Kit (Bio-Rad Laboratories Inc., Hercul es, California, USA) according to the manufacturers protocol. In brief, this involved first pelle tting bacteria from 1.5 ml of bacterial culture by centrif ugation at 13,000 rpm for 30 seconds and then resuspending and lysing the pellet in the supplied buffer. A neutralization buffer was then added, and the cell debris was pelleted via centrifugation at 13,000 rpm for 10 minutes. The cleared supernatant was harvested and spun through th e supplied column and washed with the wash solution provided. Purified plasmid DNA was eluted in 50 l of elution solution, also provided in the kit. DNA Sequencing and Sequence Analysis Amplified DNA fragments that were st rong and uncontaminated with other fragments as observed after gel electrophores is were purified using the Wizard SV Gel and PCR Clean-up System (Promega Corpor ation, Madison, Wisconsin, USA). This protocol involved adding an equal volume of membrane binding solution to the PCR product and purifying the DNA by centrifuga tion through the supplied column. The

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21 column was washed with wash soluti on twice, and the DNA was eluted in 50 l of nuclease free water, quantified by spectropho tometry, and sequenced directly. Between 50 fmol of purified PCR products were sequ enced in duplicate in both directions using specific forward and reverse primers and the proprietary chemistry for the CEQ 2000XL sequencing instrument (Beckman-Coulte r Inc., Fullerton, California, USA). Chromatograms were manually reviewed fo r potential misreadings using the Chromas 2.3 software (Technelysium Pty Ltd., Tewantin, Queensland, Australia) and exported into the Seqed function of the University of Wisconsin Package Version 10.2 (Genetics Computer Group GCG, University of Wisconsin, Madison, Wisconsin, USA). Sequences were analyzed using the Gap, Translate and Lineup functions of this software and assembled using SeqMan, SeqEd and MegAlign (DNAStar, Lasergene software, Madison, Wisconsin, USA). The BL AST function of the National Center for Biotechnology Information (NCBI) was used to identify poxvirus sequences most closely related to those of marine mammal poxviruse s. Neighbor-joining phyl ogenetic trees were generated by PAUP 4.0 (Sinauer Associates Sunderland Massachusette, USA) software, using ClustalW slow and accurate function using Gonnet residue weight table, gap penalty of 11 and gap extension penalty of 0.2. The trees were based on the amino acid sequences deduced from the homologous DNA fragments of the DNA polymerase and DNA topoisomerase genes from members of the Chordopoxvirinae subfamily of poxviruses obtained from the GenBank re pository through the NCBI website. The GenBank accession numbers (in parentheses) for the viral sequences used in the genetic analysis were: Lumpy skin disease (AF409137), sheeppox (NC_004002), goatpox (AY077835), swinepox (NC_003389), canar ypox (AY318871), cetacean poxvirus-1

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22 (AY463004-AY463007), cetacean poxvirus-2 (AY846759, AY846760), fowlpox (NC_002188), Steller sea lion pox (AY424954, AY424955), ha rbor seal parapox (AY952937-AY952939, AF414182), spotted seal parapox (AY780676, AY780677, AY780678), Steller sea lion parapox ( AY952940-AY952984), Weddel sealpox (AJ622900), camelpox (AF438165), variola (NC_001611), rabbitpox (AY484669), monkeypox (NC_003310), mule deer pox (AY841895, AY841896), vaccinia (AY243312), ectromelia (NC_004105), pi geonpox (M88588), red deer parapox (AB044794), cowpox (AF482758), Yaba m onkey tumor (AY386371), rabbit myxoma (NC_001132, AAF14910), rabbit fibroma (N C_001266), orf (NC_005336), bovine papular stomatitis (NC_005337) and molluscum contagiosum (NC_001731). Primer Specificity and Sensitivity Assays The poxvirus DNA polymerase and DNA topoiso merase PCR assays were applied to swinepox, pseudocowpox, muledeerpox, CP V-1, CPV-2, SSLPV, HSPPV, SSPPV and SSLPPV DNA to determine primer specificity. Ten-fold serial dilutions ranging from 100 ng to 0.001 fg of pCRII-Topo 2.1 plasmid that contained the amplified 546-bp CPV1 DNA polymerase fragment or 344-bp DNA topoisomerase fragment were PCR amplified using primer set CR421/CR 422 and primer set CR432/CR433, respectively, to define the general sensitivity of these assays. The parapoxvirus DNA polymerase, DNA t opoisomerase, and major envelope protein gene PCR assays were applie d to pseudocowpox, CPV-1, CPV-2, SSLPPV, HSPPV, and SSPPV DNA to determine the primer specificity. Ten-fold serial dilutions ranging from 100 ng to 0.001fg of pCRII-T opo 2.1 plasmid containing the parapox DNA polymerase, DNA topoisomerase, or major enve lope protein gene fr agments, were PCR

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23 amplified using the respective primer set to determine the general sensitivity of the assays. Virus Isolation Numerous attempts to isolate pox and pa rapox viruses from marine mammal skin lesions were made. Fresh or frozen tissue sample were homogenized in a 2 ml glass Tenbroeck tissue grinder. One, five a nd ten percent dilutions were made using Dulbeccos modified medium (DMEM) cont aining antibiotic/antimycotic drugs. The dilutions were clarified vi a centrifugation at high speed (13,000 rpm for 1 minute) to reduce bacterial contamination. Tissue culture lines that were utilized in virus isolation attempts included: African green monkey ki dney (Vero), MadinDarby canine kidney (MDCK), Tursiops trucatus lung (TurtruLu), Tursips trucatus kidney (TurtruK), Phoca vitulina ovary (PhovituOv), and Phoca vitulina lung (PhoVitLu). D ilutions made from PCR positive cetacean and SSL pox skin lesions were innoculated onto Vero, MDCK, TurtruLu and TurtruK cell cu ltures. Dilutions made fr om pinniped pox and parapox lesions were innoculated on Vero, MDCK, PhovitO ad PhovitLu cell cultures. The inoculum was allowed to adsorb onto the ce ll monolayers for 2-3 hours, after which the monolayers were carefully rinsed with DM EM, fed with DMEM supplemented with 1.0 5.0% fetal bovine serum and then incubated 37 C in an atmosphere of 5% CO2. Innoculated and non-innoculate d cultures were checked daily for cytopathic effects (CPE), the maintenance medium was changed as needed, and discarded after 14 days, or two passages if no CPE was observed.

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24 CHAPTER 3 RESULTS Summary of Positive Samples Out of 109 fresh and frozen skin lesion samples tested, poxvirus positive results were determined for 10 cetacean lesions incl uding; four Indo-Pacifi c bottlenose dolphins, two rough-toothed dolphins, one striped dolph in, two Atlantic bottlenose dolphin and one bowhead whale (Figure 3-1). Three Steller sea lion skin samples also tested positive for poxvirus (Figure 3-2). Assays for parapoxvi rus yielded six positive results including lesions from three Steller sea lions, two spotted seals, and one harbor seal (Figure 3-3). Histopathology and Electron Microscopy Skin lesions of two Steller sea lion p ups were analyzed using histopathology and electron microscopy. Histology revealed a ma ss lesion within the dermis composed of large, polygonal epithelial cells. The mass wa s composed of broad cords of polygonal to round epithelial cells with sh arply delineated cytoplasmic borders. The nuclei were consistent in size, round to oval with 12 prominent nucle oli / nucleus, fine granular chromatin and 0-4 mitotic figures/high power field. Some nuclei contained 1-2 clear vacuoles. Many of these epit helial cells contained a sing le large brightly eosinophilic inclusion body (Figure 3-4). Scattered lym phocytes, plasma cells and neutrophils were present in the dermis surrounding the mass. On electron microscopy, virus particle s were smooth, rounded rectangles approximately 350 x 270 nm consistent w ith published reports of orthopox viruses (Moss, 2001) (Figure 3-5).

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25 Detection of Poxviruses Targeting the DNA Polymerase Gene Total DNA extracted from 10 cutaneous le sions from cetaceans and two lesions from Steller sea lions contained pox virus genomic DNA as evidenced by the amplification of DNA polymerase gene fragments of the expected size. Positive donor cetacean species were: Four Indo-Pacifi c bottlenose dolphins, two rough-toothed dolphins, one striped dolphin, two Atlantic bottlenose dolphin and one bowhead whale. Similarly, lesions harvested from three Stel ler sea lion pups also contained amplifiable poxvirus DNA polymerase gene sequences (Figur e 3-6). This PCR assay detected the DNA polymerase gene fragments of muled eer poxvirus, swine pox virus, cetacean poxvirus-1 (CPV-1), cetacean poxvirus-2 (C PV-2) and Steller sea lion poxvirus (SSLPV), but did not amplify the DNA polym erase gene fragments of pseudocowpox, Steller sea lion parapoxvirus (SSLPPV), harbor seal parapoxvirus (H SPPV), or spotted seal parapoxvirus ( SSPPV). Serial ten-fold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the CPV-1 DNA polymerase gene fragment were PCR amplified with primers CR 421 and CR422. The minimal amou nt of CPV-1 DNA detected was 1.0 fg. Detection of Poxviruses Targetin g the DNA Topoisomerase I Gene A total of seven lesions from cetaceans yielded positive PCR results when the poxvirus DNA topoisomerase gene was target ed. Positive cetacean species were: Two rough-toothed dolphins, two st riped dolphins, one Indo-Pa cific bottlenose dolphin, one Atlantic bottlenose dolphin and one bowh ead whale. DNA fragments corresponding in size to the DNA topoisomerase gene fragme nts were also amplified from total DNA extracted from lesions of three Steller s ea lion pups (Figure 3-7). This PCR assay detected the DNA topoisomerase gene fragme nts of muledeer poxvirus, Swinepox virus, CPV-1, CPV-2 and SSLPV, but did not amplif y the DNA topoisomerase gene fragments

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26 of pseudocowpox, SSLPPV, HSPPV, or SSPPV. Serial ten-fo ld dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the CPV-1 DNA toposiomerase fragment were PCR amplified with primers CR 421 and CR 422. The minimal amount of CPV-1 DNA detected was 1.0 fg. PCR Targeting the Major Envelope Protein Gene of Orthopoxviruses PCR was used to target the major envelope protein gene of ce tacean and pinniped poxviruses. While the primers amplified bands of the expected size, of approximately 1118-bp using cetacean poxvirus DNA, sequenc ing of the DNA fragments yielded nonpoxvirus DNA sequence. When vaccinia viru s DNA was used as template, the same primers drove the amplification of a fragme nt of the expected size (Figure 3-8). PCR Targeting the Orthopoxvirus Hemagglutinin Gene Cetacean and Steller sea lion poxvirus DNA te mplates were tested using primers designed to amplify the HA gene of orthopox viruses. Although the primers did not detect the presence of the HA gene in e ither cetacean, or Stel ler sea lion DNAs, the vaccinia virus DNA positive control validated th e PCR protocol amplifying a band at the expected size of 1138-bp (Figure 3-8). Detection of Parapoxviruses Targeting the DNA Polymerase Gene Parapoxvirus DNA polymerase gene fragments of the approximate expected size were amplified from total DNA extracted fr om biopsied or scraped skin lesions of pinnipeds. Donor species that yielded positive results were : Three Steller sea lions, two spotted seals and one harbor seal (Figur e 3-9). This PCR assay detected the DNA polymerase gene fragments of pseudocow pox, SSLPPV, HSPPV, or SSPPV, but did not amplify the DNA polymerase gene fragments of muledeer poxvirus, swinepox virus, CPV-1, CPV-2 and SSLPV. Serial ten-fold dilutions from 100ng to .001fg of Topo 2.1

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27 plasmid containing the SSPPV DNA polymerase gene fragme nt were PCR amplified with primers CR 540 and CR541. The minimal amount of target DNA detected was 0.1 fg (Figure 3-10). Detection of Parapoxviruses Targeting the DNA Topoisomerase I Gene PCR targeting the DNA topoisomerase gene of parapoxviruses usi ng the first set of primers (CR550 and CR551) amplified DNA fr agments approximately 350-bp in length when total DNA extracted from lesions of Ste ller sea lions was used as template (Figure 3-11). However, these primers did not amp lify DNA topoisomerase gene fragments from lesions of harbor or spotted seals. Seri al ten-fold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the SSLPPV DNA topoisomerase gene fragment were PCR amplified with primers CR 550 and CR551. The minimal amount of target DNA detected was 1.0 fg (Figure 3-12). A s econd set of primers (CR557 and CR558) was designed based on the Steller sea lion pa rapoxvirus DNA topoisomerase I sequence that successfully directed the amplification of a frag ment of the expected size from the harbor seal parapoxvirus lesion, but not from the spo tted seal lesion (Figur e 3-11). Serial tenfold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid contai ning the HSPPV DNA topoisomerase fragment were PCR amplified with primers CR557 and CR558. The minimal amount of target DNA detected was 0.1 fg (Figure 3-13). A third set of internal consensus primers (CR570 and CR571) based on the Steller sea li on and harbor seal parapoxvirus DNA topoisomerase I fragment seque nces directed the amplification of a fragment of approximately 250bp from the spotted s eal lesions (Figure 3-14). Serial tenfold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the SSPPV DNA topoisomerase fragment were PCR amplified with primers CR 570 and CR571. The minimal amount of target DNA detected was 1.0 fg (Figure 3-15).

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28 Detection of Parapoxviruses Targetin g the Major Envelope Protein Gene Oligonucleotide primers PPP-1 and PPP-4 (Inoshima et al.2000) known to amplify a 594-bp fragment within the major envelope gene of parapoxviruse s of herbivores and harbor seals, directed the amplification of DNA fragments of similar size using total DNA extracted from skin lesions harvested from three Steller sea li ons, two spotted seals and one harbor seal (Figure 3-16). Serial ten-fold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the HSPPV D NA topoisomerase fragment were PCR amplified with primers CR339 and CR340. The minimal amount of target DNA detected was 0.1 fg (Figure 3-17). Sequencing and Genetic Analysis DNA Polymerase Sequencing of amplified poxvirus DNA polymer ase gene fragments from lesions of 12 marine mammals revealed th at the fragments were 546-bp in length from 10 cetacean samples representing five species, while those amplified from two Steller sea lion lesions were 543-bp. Sequencing of DNA fragment s corresponding to the DNA topoisomerase I gene of poxviruses contained in lesions of cet aceans and Steller sea lions were 344-bp in length. Primers CR541 and CR540 directed the amplification of DNA fragments 536-bp in length, from the DNA polymerase gene of parapoxviruses contained in lesions of three Steller sea lions, two spotte d seals and one harbor seal Targeting of the DNA topoisomerase I gene of parapoxviruses w ith primers, CR550 and CR551, amplified DNA fragments of 347 or 350-bp when total DNA from lesions from three Steller sea lions was used as template. The second set of primers, CR557 and CR558, also directed the amplification of 350-bp DNA fragments when total DNA from the lesions of a third Steller sea lion and a harbor seal was used as a template. The third set of primers, CR570

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29 and CR571, directed the amplif ication of DNA topoisomerase I gene fragments 252-bp in length from parapoxvirus lesion s from two spotted seals. Targeting of the major envelope protein gene of parapoxviruses with primers validated with ruminant parapoxviruses (Inoshima et al., 2000) and ha rbor seal parapoxviruses (Becher et al., 2002; Mller et al., 2003), confirmed the universality of these primers for the amplification of parapoxvirus DNA contained in skin lesions of pinnipeds; in this case, Steller sea lions, spotted and harbor seals. Genetic analysis of the nucleotide sequences obtained from the DNA polymerase gene fragments of poxviruses of cetaceans ( 546-bp) demonstrated that nine of the 10 nucleotide sequences derived from cetacean pox viruses, shared identities greater than 93.0 and 97.2% at the nucleotide and amino acid level, respecti vely. We have tentatively grouped these nine poxviruses within a single group that we, herein refer toas cetacean poxvirus-1. The remaining cetacean poxvirus sample derived from a bowhead whale lesion was shown to be at least 84 and 89% identical at the nucleotide and amino acid level, respectively, when compared to ho mologous sequences from the other nine cetacean poxvirus-1 sequences (Tables 3-1 33). This virus was being provisionally named as cetacean poxvirus-2. The DNA polymer ase gene fragments (543-bp) amplified from cutaneous lesions of two Steller sea lion pups were 100 % identical to each other, and at least 76 and 81% identi cal at the nucleotide and amino acid level, respectively, when compared to homologous sequences of cetacean poxvirus-1. Similar comparisons to the homologous fragments from the bowhead whale (cetacean poxvirus-2) showed identities of 77 and 83% (Table 3-1 3-3). Genetic analysis of DNA polymerase gene fragments amplified from skin lesions of pinnipeds associated with parapoxviruses

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30 showed that the viruses contained in thes e lesions were members of the parapoxvirus genus and showed nucleotide and amino acid id entities greater than 98% when compared among themselves. Nucleotide and amino acid sequence comparisons of the DNA polymerase gene fragments of the Stelle r sea lion poxvirus and the Steller sea lion parapoxviruses showed, respectiv ely, identities of 55 and 61 %. The DNA polymerase fragments obtained from the cetacean and pinniped poxvirus DNA templates were compared with homologous fragments from ot her terrestrial poxviruses. These numerous pairwise comparisons were made to represen t the nucleotide identity, amino acid identity and amino acid similarity (Tables 3-1 3-3) of all these viruses. Multiple alignments were performed using the DNA polymerase fragments of CPV-1, CPV-2, SSLPV, SSLPPV, SSPPV, HSPPV (Figures 3-18 and 3-19). The multiple alignment comparing the CPV-1 and CPV-2 DNA polymerase fragme nts showed a clear difference between the CPV-1 and CPV-2 amino acid sequences (Figure 3-18). The multiple alignment comparing the SSLPV, SSLPPV, HSPPV, and SSPPV DNA fragments showed a clear difference between the pox and parapoxviru s amino acid sequences (Figure 3-19). DNA Topoisomerase I Genetic analysis of the DNA topoisomerase gene fragments (344-bp) of cetacean poxvirus demonstrated that six of the seve n positive samples had nucleotide and amino acid identities of at least 92 and 95%, re spectively (Tables 3-4, 3-5). These six poxviruses had all been included in the ceta cean poxvirus-1 type based on sequences of the DNA polymerase gene fragment. The seventh poxvirus corresponding to the bowhead whale poxvirus sample, had identiti es of 84 and 85% at the nucleotide and amino acid levels when compared to homologous sequences of cetacean poxvirus-1 (Tables 3-4 3-6). Based on sequences of the DNA polymerase gene fragment, the

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31 bowhead whale virus has been provisiona lly named cetacean poxvi rus-2. Poxvirus DNA topoisomerase fragments amplified from lesions of the three Steller sea lion pups were identical to each other with about 71 and 75% identity at the nucleotide and amino acid level, respectively, to homologous sequen ces of cetacean poxviruses-1. Similar comparison to homologous sequences of cet acean poxvirus-2 revealed identities of 72 and 77%. Genetic analysis of the DNA topoiso merase fragments of the Steller sea lion, spotted seal and harbor seal parapoxviruses demonstrated that they belong to the parapoxvirus genus. Pair-wise comparisons between the DNA t opoisomerase gene fragment sequences of the poxvirus from the two Steller sea li on pups and the homologous fragments from the Steller sea lion parapoxviruse s showed identities of 52-54 and 57% at the nucleotide and amino acid levels, respectively, clearly demo nstrating that these viruses are distinct members of separate genera within the Chordopoxvirinae subf amily (Tables 3-4 3-6). The genetic diversity of parapoxvi ruses of pinnipeds is reflected in the findings that the DNA polymerase gene fragment sequence the Steller sea lion and its homologue in the harbor seal parapoxviruses share 80-98 and 87-98% identity at the nucleotide and amino acid levels, respectively. This identity show s a similar pattern of about 80-99 and 8899% in the case of the spotted seal parapoxvirus. The DNA topoisomerase gene fragment of the harbor seal parapoxviruse s share 96 and 95% identities when compared to the homologous sequence from the s potted seal parapoxvirus. The DNA topoisomerase fragment sequences obtain ed from the cetacean and pinniped poxvirus DNA templates were compared with homol ogous fragments from other terrestrial poxviruses deposited in the GenBank database (Tables 3-43-6). These numerous

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32 pairwise comparisons established the nucleo tide identity, amino acid identity and amino acid similarity among the most relevant poxviruses. Multiple alignments were generated using the DNA topoisomerase gene fragme nt sequences of CPV-1, CPV-2, SSLPV, SSLPPV, SSPPV, HSPPV (Figures 3-15 and 3-18). The multiple aligment of the CPV-1 and CPV-2 DNA topoisomerase fragments dem onstrates a clear difference between the respective CPV-1 and CPV-2 amino acid sequences (Figure 3-20). The multiple alignment of the SSLPV, SSLPPV, HSPPV and SSPPV DNA topoisomerase gene fragments demonstrates a clear difference between the poxand parapoxvirus amino acid sequences (Figure 3-21). Major Envelope Protein Gene Sequence comparisons were performed with the nucleotide and deduced amino acid sequences of the major envelope gene fragme nts of the various pinni peds parapoxviruses. Nucleotide and amino acid sequences from the Steller sea lion major envelope fragment were, respectively, 93 and 98% identical to the homologous sequences from the harbor seal parapoxvirus and 93 and 96% identical to the homologous sequences of the spotted seal parapoxviruses (Tables 3-6 3-9). Seque nces of the major envelope protein gene fragments obtained from the pinniped para poxviruses were entered into a multiple alignment for simplified comparison (Figure 3-22). Phylogenetic Analysis Phylogenetic trees created using the am ino acid sequences of various species of marine mammal pox and parapox virus sequenc es plus numerous homologous fragments from DNA sequences of terrestrial poxviru ses demonstrate the ge netic relatedness of these virus fragments. The DNA polymerase and DNA topoisomerase phylograms indicate that the CPV-1 and CPV-2 viruses group together and form a unique branch,

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33 separate from the known poxvirus genera (F igures 3-23 and 3-24). The SSLPV also forms its own branch in both the DNA polym erase and DNA topoisomerase phylogragms (Figures 3-23 and 3-24). Th e phylogenetic tree constructed using the major envelope protein gene fragments amplified fo rm pinniped parapoxviruses and numerous homologous fragments from DNA sequences of terrestrial poxviruses demonstrates the placement of the HSPPV, SSPPV and SSPPV gene fragments into the branch including other terrestrial parapox viruses (Figure 3-25). Virus Isolation All attempts to isolate poxviruses from pi nniped and cetacean fresh and frozen skin lesions were unsuccessful. A B C Figure 3-1.Typical tattoo lesions of cetac eans. A and B) Skin lesions of a roughtoothed dolphin (Steno bredanenesis ). Photos taken by Dr. Charles Manire. C) Skin lesions of a bottlenose dolphin (Tursiops aduncus ) from a Hong Kong aquarium.

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34 Figure 3-2 Gross appearance of pox lesions associated with a poxvi rus in a Steller sea lion (Eumetopias jubatus ). Approximately 1 cm diameter raised smooth, hairless, often umbilicated, nodules were scattered across th e body. Photo supplied by Dr. Kathy Burek. Figure 3-3. Cutaneous pox lesi ons in a spotted seal (Pho ca largha) associated with spotted seal parapoxvirus. P hoto supplied by Dr. Kathy Burek.

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35 Figure 3-4 Histopathologic appearance of cuta neous lesions associated wtih Steller sea lion poxvirus, showing epithelia l cells containing acidophilic intracytoplasmic inclusion bodies (arrow). Slide supplied by Dr. Kathy Burek. Figure 3-5. Negatively stained poxvirus particle from cuta neous lesion of SSL observed by electron microscopy. The 'skew' pattern of orthopoxviruses is evident as opposed to the 'criss-cross' pattern of parapoxviruses. Photo supplied by Mr. Woody Fraser.

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36 M.M. 1 2 3 4 5 6 7 8 9 10 11 12 13 -14 15 16 M.M. Figure 3-6. Agarose gel electrophoresis of PCR amplified 543-546-bp fragments of the DNA polymerase gene of cetacean and Steller sea lion poxviruses using primers CR 421 and CR 422. M.M.: 1KB Plus Molecular Ladde r; Lane 1: Rough-t oothed dolphin (V365); Lane 2: Rough-toothed dolphin (GW010006D); Lane 3: Bottlenose dolphin (R127); Lane 4: Bottlenose dolphin (V466); Lane 5: Bottl enose dolphin (V550); Lane 6: Bottlenose dolphin (V551); Lane 7: Bottlenose dolphi n (MML0203); Lane 8: Bottlenose dolphin (OK04091932); Lane 9: Bottlenose dolphin (CMA0108); Lane 10: Bowhead whale (98KK3); Lane 11: Stelle r sea lion (SSL2001-279); Lane 12: Steller sea lion (SSL2000105); Lane 13: Steller sea lion (SSL2005-546); Lane 14: Positive control, MDPV; Lane 15: Negative tube, no DNA; Lane 16: Negative tissue (V1044) M.M. 1 2 3 4 5 6 7 8 9 10 11 12 13 M.M. Figure 3-7. Agarose gel electrophoresis of PCR amplified 344-bp fragments of the DNA topoisomerase gene of cetacean and Stelle r sea lion poxviruses using primers CR 432 and CR 433. M.M.: 1KB Plus Molecular Ladder; Lane 1: Rough-toothed dolphin (V365); Lane 2: Rough-toothed dolphin (GW010006D); Lane 3: Bottlenose dolphin (R127); Lane 4: Bottlenose dolphin (MML0203); Lane 5: Bottlenose dolphin (OK04091932); Lane 6: Bottlenose dolphin (CMA0108); Lane 7: Bowhead whale (98KK3); Lane 8: Steller sea lion (SSL2001-279); Lane 9: Steller sea lion (SSL2000-105); Lane 10: Steller sea lion (SSL2005-546); Lane 11: Negative water, no DNA; Lane 12: Negative tissue (V1044); Lane 13: Positive control, MDPV DNA

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37 Figure 3-8. Agarose gel electr ophoreses of PCR amplified fragments of the HA gene of orthopoxviruses using primers CR 619 and CR6 20 targeting a 1183-bp. M.M.: 1 KB Plus Molecular Ladder; Lane 1: Bottlenose dol phin CPV-1 (V1546); Lane 2: Bowhead whale CPV-2 (V730); Lane 3: St eller sea lion poxvirus (R227); Lane 4: Negative tissue (V1044); Lane 5: Negative water, no DNA; Lane 6: Vaccinia virus DNA Figure 3-9. Agarose gel electrophoresis demons trating the PCR amplification of 536-bp parapox DNA polymerase gene fragments from lesions of different pinniped species. M.M.: 1 KB Plus molecular ladder; Lane 1: Steller sea lion (SSL2003-450); Lane 2: Steller sea lion (SSL2003-451); Lane 3: Ste ller sea lion (SSL2004-495) ; Lane 4: Harbor seal (MMSC 03021); Lane 5: Spotted seal (D IO-136-03); Lane 6: S potted seal (DIO-11903); Lane 7: positive control, pseudocowpox virus DNA; Lane 8: negative control, no DNA template. M.M12345---61.0-kb 1.65-kb 650-bp 500-bp

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38 Figure 3-10. Agarose gel electrophoresis de monstrating the PCR sensitivity assay for primers CR540 and CR541 targeting the parapox DNA polymerase gene. pCR-II topo 2.1 plasmid vector containing the 536-bp fragme nt amplified from th e Spotted seal (DIO136-03) parapoxvirus DNA in 10-fold serial dilu tions. M.M: 1 Kb plus ladder. Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1.0 pg; Lane 7: 100 fg, Lane 8: 10 fg; Lane 9: 1.0 fg ; Lane 10: 0.1 fg; Lane 11: 0.01 fg; Lane 12: 0.001; Lane 13: negative control, no DNA. Figure 3-11. Agarose gel electrophoresis dem onstrating the PCR amp lification of 350-bp parapox DNA topoisomerase I gene fragment s from lesions of Steller sea lions (Eumetopias jubatus ) and harbor seals (Phoca vitulina ). M.M.: 1KB Plus molecular ladder; Lane 1: Steller sea lion (SSL2003450); Lane 2: Steller sea lion (SSL2003-451); Lane 3: Steller sea lion ( SSL2004-495); Lane 4: Harbor seal (MMSC 03021); Lane 5: positive control, pseudocowpox virus DNA; Lane 6: negative control, no DNA template. 650-bp 500-bp M.M 1 2 3 4 5 6 7 8 9 10 11 12 400-bp 300-bp

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39 Figure 3-12. Agarose gel electrophoresis de monstrating the PCR sensitivity assay for primers CR550 and CR551 targeting the St eller sea lion (SSL 2003-451) parapoxvirus DNA topoisomerase gene. pCR-II topo 2.1 plasmid vector containing the 350-bp fragment in 10-fold serial dilu tions. M.M: 1 Kb plus ladder. Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1.0 pg; Lane 7: 100 fg, Lane 8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg ; Lane 11: 0.01 fg; Lane 12: 0.001; Lane 13: negative control, no DNA. Figure 3-13. Agarose gel electrophoresis de monstrating the PCR sensitivity assay for primers CR557 and CR558 targeting the harb or seal DNA topoisomerase gene. pCR-II topo 2.1 plasmid vector containing th e 350-bp parapoxvirus DNA topoisomerase fragment in 10-fold serial d ilutions. M.M: 1 Kb plus ladd er. Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1.0 pg; Lane 7: 100 fg, Lane 8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg ; Lane 11: 0.01 fg; Lane 12: 0.001; Lane 13: negative control, no DNA. 400-bp 300-bp M.M 1 2 3 4 5 6 7 8 9 10 11 12 M.M 1 2 3 4 5 6 7 8 9 10 11 12 500-bp 650-bp

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40 Figure 3-14. Agarose gel electrophoresis dem onstrating the PCR amp lification of 252-bp parapox virus DNA topoisomerase gene fragment from lesions of spotted seals (Phoca largha ). M.M.: 100-bp molecular ladder; Lane 1: Spotted seal (DIO-136-03); Lane 2: Spotted seal (DIO-119-03); La ne 3: negative control, no DNA template; Lane 4: positive control, Steller sea lion (SSL2003-451). Figure 3-15. Agarose gel electrophoresis de monstrating the PCR sensitivity assay for primers CR570 and CR571 targeting the para poxvirus DNA topoisomerase gene. pCR-II topo 2.1 plasmid vector containing the 252-bp fr agment amplified from the Spotted seal (DIO-136-03) parapoxvirus DNA in 10-fold seri al dilutions. M.M: 1 Kb plus ladder. Lane 1: 10 ng; Lane 2: 1.0 ng; Lane 3: 100pg; Lane 4: 10 pg; Lane 5: 1.0 pg; Lane 6: 100 fg; Lane 7: 10 fg, Lane 8: 1.0 fg; Lane 9: 0.1 fg; Lane 10: 0.01 fg; Lane 11: 0.001 fg; Lane 12: negative control; Lane 13: M.M 600-bp 300-bp 200-bp 300-bp 200-bp M.M 1 2 3 4 5 6 7 8 9 10 11 12

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41 Figure 3-16. Agarose gel electrophoresis dem onstrating the PCR amp lification of 594-bp parapox major envelope protein ge ne fragment from lesions of different pinniped species. M.M.: 1 KB Plus molecular ladder; Lane 1: Steller sea lion (SSL2003-450); Lane 2: Steller sea lion (SSL2003-451); Lane 3: Ste ller sea lion (SSL2004-495) ; Lane 4: Harbor seal (MMSC 03021); Lane 5: Spotted seal (D IO-136-03); Lane 6: S potted seal (DIO-11903); Lane 7: positive control, pseudocowpox virus DNA; Lane 8: negative control, no DNA template. Figure 3-17. Agarose gel electrophoresis de monstrating the PCR sensitivity assay for primers CR339 and CR340 targeting the parapox major envelope protein gene. pCR-II topo 2.1 plasmid vector containing the 596-bp fr agment amplified from the Harbor seal (MMSC03021) parapoxvirus DNA in 10-fold seri al dilutions. M.M: 1 Kb plus ladder. Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1.0 ng; Lane 4: 100 pg ; Lane 5: 10 pg; Lane 6: 1.0 pg; Lane 7: 100 fg, Lane 8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg; Lane 11: 0.01 fg; Lane 12: 0.001; Lane 13: negative control, no DNA. M.M 1 2 3 4 5 6 7 8 9 10 11 12 1.0-Kb 500-bp 650-bp 650-bp 500-bp

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42 1 50 10 0 B. mysticetus -AK .......... .......... .........k l......... .......... .......... .......... .......... ..k..f.... .......... B. mysticetus -AK .......... .......... .........k l......... .......... .......... .......... .......... ..k..f.... .......... T. aduncus -HK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... T. aduncus -HK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... S. bredanensis -FL .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... T. truncatus -FL .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... S. bredanensis -FL .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... T. aduncus -HK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... S. coeruleoalba -FL .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... T. aduncus -HK .......... .......... .......... .......... ....r..... .......... .......... .......... s......... .......... S. coeruleoalba -PO .......... .......... ......y... .......... .......... .......... .......... .......... .......... .......... CONSENSUS YRASTLIKGP LLKLLLETKI ILYRSEHKQQ KLPYEGGKVF MPKQKMFSNN VLIFDYNSLY PNVCLFGNLS PETLVGVVVS NNVLELEINI QEIKKKFPS P 101 150 181 B.mysticetus -AK .......... .q......a. .......... .l..q..s.. ..c.....as kt........ .......... .......... B. mysticetus -AK .......... .q......a. .......... .l..q..s.. ..c.....as kt........ .......... .......... T. aduncus -HK .......... .......... .......... .......... .......... ...m...... .......... .......... T. aduncusHK .......... .......... .......... .......... .......... ...m...... .......... .......... S. bredanensis -FL .......... .......... .......... .......... .......... ...m...... .......... .......... T. truncatus -FL .......... .......... .......... .......... v......... .......... .......... .......... S. bredanensis -FL .......... .......... .......... .......... v......... .......... .......... .......... T. aduncus -HK .......... .......... .......... .......... v......... .......... .......... .......... S. coeruleoalba -FL .......... .......... .......... .......... v......... .......... .......... .......... T. aduncus -HK .......... .......... .......... .......... v......... .......... .......... .......... S. coeruleoalba -PO .......... .......... .......... .......... ....r..... .......... .i........ .......... Consensus RYIVVHCEPR FKNLISEISI FDREVEGTIP RILRRFLTER AKYKKLLKST NDCTEKAIYD SMQYTYKIVA NSVYGLMGFK N Figure 3-18. Multiple alignment of the am ino acid sequences deduced from the nucleo tide sequences of the DNA polymerase gene fragments of poxviruses identified in cutane ous lesions of cetaceans. AK = Alaska; FL = Florida; HK = Hong Kong; PO= Portugal.

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43 1 50 100 E. jubatus -AK .....l.... .l....etk. .ls..ek.qr .p....k.f. ......vnn. .......... ...l.g.... ........s. .k.es..nnq .llik..p.q E. jubatus -AK .....l.... .l....etk. .ls..ek.qr .p....k.f. ......vnn. .......... ...l.g.... ........s. .k.es..nnq .llik..p.q E. jubatus -AK ~~........ .......hk. ....a.t.s. ........l. .......... .......... .......... ........sr .......... .......... P. largha -AK ~~........ .......... .......... .......... .......... .......... .......... .......... .......... .......... P. largha -AK ~~........ .......... .......... .......... .......... .......... .......... .......... .......... .......... P. vitulina -NJ ~~........ .......... .....n.... .......... .......... .......... .......... .......... .......... .......... E. jubatus -AK ~~........ .......... .......... .......... .....h.... .......... .......... .......... .......... .......... E. jubatus -AK ~~........ .......... ........s. .......... .r..i..... .......... .......... .........d .......... ...r...... CONSENSUS YRASTCIKGP LMKLLLANRT VMVRSDVKTK YFFEGGRVMA PKQKMYDKHV LIFDYNSLYP NVCIYANLSP ETLVGVVVAN NRLDAEIAAV EIRQRFPAPR 101 150 180 E. jubatus -AK ..l.y..... tqf....... ..rte....l ..kk..ne.s y...ml.nsk .qkeks..d. .......i.. t.......k. E. jubatus -AK ..l.y..... tqf....... ..rte....l ..kk..ne.s y...ml.nsk .qkeks..d. .......i.. t.......k. E. jubatus -AK ..s.l..... ..f....... ....d..... .......... .......g.k .....n..d. .......... .......... P. largha -AK .......... .......... .......... .......... .........e .......... .......... .......... P. largha -AK .......... .......... .......... .......... .........e .......... .......... .......... P. vitulina -NJ .......... .......... .......... .......... .........e .......... .......... .......... E. jubatus -AK .......... s......... .......... .......... .........e .......... .......... .......... E. jubatus -AK ..t....... .d........ ....d..... .........t .........d s....d.... .......... .......... CONSENSUS FIAVPCEPRS PELVSEVAIF DREANGIIPM LLRSFLDARA KYKKLMKTATAVDREIFNS MQYTYKITAN SVYGLMGFRN Figure 3-19. Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the DNA polymerase gene fragment of poxviruses identified in cutaneous lesions of pinni peds. AK = Alaska; NJ = New Jersey.

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44 1 60 B. mysticetus -AK .......... .......... .......... .dik....i. ..i....... .....yn.r. B. mysticetus -AK .......... .......... .......... .dik....i. ..i....... .....yn.r. T. truncatusFL .......... .......... .......... .......... .......... .......... S. bredanensis -FL .......... .......... .......... .......... .......... .......... T. aduncus -HK .......... .......... .......... .......... .......... .......... S. coeruleoalba -FL .......... .......... .......... .......... .......... .......... S. bredanensis -FL .......... .......... .......n.. ..f....... ........a. .......... S. coeruleoalba -PO .......... .......... .......... ..i....... ..m....... .......... CONSENSUS METSFFIRTG KLRYLKENNT VGLLTLKSKH LTLTKDKLTI SFTGKDKVSH EFVIRRYDKL 61 114 B. mysticetus -AK .....k.a.. .d........ ...r...... nq...h.... .......... .... B. mysticetus -AK .....k.a.. .d........ ...r...... nq...h.... .......... .... T. truncatus -FL .......... .......... .......... .......... .......... .... S. bredanensis -FL .......... .......... .......... .......... .......... .... T. aduncus -HK .......... .......... .......... .......... .......... .... S. coeruleoalba -FL .......... .......... .......... .......... .......... .... S. bredanensis -FL .......... .......... .......... .q........ .......... .... S. coeruleoalba -PO .....k.... .d.......r .......... k......... .......... .... CONSENSUS YKPLIRLSKN KESECFLFDK LNENIIYKLI RPFGIRIKDL RTYGVNYTFL YNFW Figure 3-20. Multiple alignment of the amino acid sequences deduced from the nucleotide. Sequences of the DNA topoisomerase gene fragments of poxviruses identified in cutaneous lesions of cetaceans. AK = Alaska; FL = Florida; HK = Hong Kong; PO = Portugal.

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45 1 50 100 E. jubatus -AK .......... ..k.f..nn. ......q..n ihiek...k. f.t....... n.q.i..knn ...kp.lkii .nsk..sfi. .k.n..k..n l.km.y.h.. E. jubatus -AK .......... ..k.f..nn. ......q..n ihiek...k. f.t....... n.q.i..knn ...kp.lkii .nsk..sfi. .k.n..k..n l.km.y.h.. E. jubatus -AK .......... ..k.f..nn. ......q..n ihiek...k. f.t....... n.q.i..knn ...kp.lkii .nsk..sfi. .k.n..k..n l.km.y.h.. P. larghaAK ~~~~~~~~~~ ~~~~~~~... .......... .......d.. .......... .......... .......... ......q... .......... .......... P. largha -AK ~~~~~~~~~~ ~~~~~~~... .......... .......d.. .......... .......... .......... ......q... .......... .......... P. vitulina -NJ .......... .s........ .......... .......e.. ..k....... .......... .......... .......... .......... .......... E. jubatus -AK .......... .s...r.... .......... .......e.. ..k....... .......... .......... .......... t......... .......... E. jubatus -AK .......... .t........ .......... ...s...e.. ........r. ....a...g. ...s...... .......... d........m .......... E. jubatus -AK .......... .a...r.... ........r. ..rgd..da. l.......r. t...a..dg. ....v.e... ..ad...... h..g.k.... a......... Consensus METSFFIRIG KMRYEKESGT VGLLTLRNKH LSEAEGG-EI RVRFVGKDKV AHEFTVRNSQ RLFAALRRLW DPGAPERLLF NRLSERRVYA FMRRFGIR VK 101 116 E. jubatus -AK .......... ...... E. jubatus -AK .......... ...... E. jubatus -AK .......... ...... P. largha -AK .~~~~~~~~~ ~~~~~~ P. largha -AK .~~~~~~~~~ ~~~~~~ P. vitulinaNJ .......... ...... E. jubatus -AK .......... ....~~ E. jubatus -AK .......... .....~ E. jubatus -AK .......... .....~ Consensus DLRTYGVNYT FLYNFW Figure 3-21. Multiple alignment of the amino aci d sequences deduced from the nucleotide sequences of the DNA topoisomerase gene fragment of poxvi ruses and parapoxviruses identif ied in cutaneous lesions of pinnipeds. AK = Alaska; NJ = New Jersey.

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46 1 50 100 HSPPV-NJ .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... SSPPV-AK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... SSPPV2-AK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... SSLPPV1-AK .......... .......... .......... .......... ..a....... .i........ ......h... .......... .......... .......... SSLPPV2-AK .......... .......... .......... .......... .......... .i........ ......h... .......... .......... .......... SSLPPV3-AK .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... Consensus YVGSASLTGG SLATIKNLGV YSTNKHLAVD LMNRYNTFSS MVVDPKQPFT RFCCAMITPT ATDFHMNHSG GGVFFSDSPE RFLGFYRTLD EDLVLHRIDA 101 150 198 HSPPV-NJ .k........ ..v......g ..y....i.. .......... .......... .......... .......... ...i...... i......... ........ SSPPV-AK .e........ ..l......s ..h....v.. .......... .......... .......... .......... ...v...... v......... ........ SSPPV2-AK .e........ ..l......s ..h....v.. .......... .......... .......... .......... ...v...... v......... ........ SSLPPV1-AK .k........ ..v......g ..y....i.. .......d.. .......... .......... .......... ...i...... i......... ........ SSLPPV2-AK .k........ ..v......g ..y....i.. .......d.. .......... .......... .......... ...i...... i......... ........ SSLPPV3-AK .k........ ..y......s ..y....a.. .......... .......... .......... .......... ...v...... i......... ........ Consensus AKNSIDLSLL SMYPVVRSGEVYYWPLIMD ALLRAAINRS VRVRIIISQW RNADPLSVAA VRALDNFGVG HVD-TARWFA IPGRDDASNN TKLLIVDD Figure 3-22. Multiple alignment of the partial amino acid sequences predicted from the major envelope protein gene fragment o f parapoxviruses identif ied in cutaneous lesions of pinnipe ds. AK = Alaska; NJ = New Jersey.

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47 A. Figure 3-23. Neighbor-Joining phylogenetic tr ee of the deduced amino acid sequences of the DNA polymerase gene fragments from members of the Chordopoxvirinae subfamily of poxviruses. The tree generate d by Clustal X slow and accurate function using Gonnet 250 residue weight table, gap pe nalty of 11 and gap length penalty of 0.2. A) Format is a rectangular cladogram where the numbers represent the percent confidence of 1000 bootstrap repl ications. B) Radial format showing a .1 divergence scale representing 0.1 substitutions per site.

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48 B. Figure 3-23. Continued. 0. 1 SSLP V Variol a Camelpox Monkeypox Cowpox Vaccini a Ectromelia CPV1 CPV2 Y aba monkey tumor virus Goatpo x LSD V Sheeppox Muledeerpox Swinepox Rabbit Fibroma Rabbit myxoma Molluscum contagiousum Canarypox Fowlpo x SSLPPVv841 Or BPS V SSLPPVv1386 SSLPPVv842 SSP V HSPP V

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49 Figure 3-24. Neighbor-Joining phylogenetic tr ee of the deduced amino acid sequences of the DNA topoisomerase gene fragment s from members of the Chordopoxvirinae subfamily of poxviruses. The tree was ge nerated by Clustal X slow and accurate function using Gonnet 250 residue weight ta ble, gap penalty of 11 and gap length penalty of 0.2. A) Format is a rectangula r cladogram where the numbers represent the percent confidence of 1000 bootstrap repli cations. B) Radial format showing a .1 divergence scale representing 0.1 substitutions per site.

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50 Figure 3-24. Continued. 0.1 SSLPV CPV1 CPV2 Fowlpox Canarypox Molluscum contagiosum SSLPPVv841 SSLPPV v1386 BPSV Or f HSPPV SSPPV SSLPPV v842 Y aba monkey Tumor virus Rabbit fibroma Rabbit myxoma Muledee r Swine p ox Goatpox LSDV Sheeppox Variola Cowpox Monkeypox Ectromelia Vaccinia Camelpox

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51 Figure 3-25 Neighbor-Joining phylogenetic tr ee of the deduced amino acid sequences of the Major envelope protein gene frag ments from members of the Chordopoxvirinae subfamily of poxviruses. The tree was ge nerated by Clustal X slow and accurate function using Gonnet 250 residue weight ta ble, gap penalty of 11 and gap length penalty of 0.2. A) Format is a rectangula r cladogram where the numbers represent the percent confidence of 1000 bootstrap repli cations. B) Radial format showing a .1 divergence scale representing 0.1 substitutions per site

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52 Figure 3-25. Continued. 0.1 Molluscumcontagiosum Canarypox Fowlpox Pigeonpox Camelpox Variola Monkeypox Cowpox Ectromelia Vaccinia Yabamonkey tumorvirus S win epo x LSDV Sheeppox Rabbit Fibroma Rabbit Myxoma Weddell seal parapox SSLPPVv841 SSLPPVv842 HSPPV SSPPV SSLPPVv1386 Or f Pseudocowpox RedDeerParapox BPSV

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53Table 3-1. Pair-wise comparisons of the nuc leotide sequences obtained from the DNA pol ymerase gene fragments of the cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) sample s. Values correspond to percent identity between two nucleotide sequences. CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2 AJ28 Wiki R174 R164 V365 V1546 V466 V550 V551 V729 CPV-1 AJ28 100.0 CPV-1 Wiki 100.0 100.0 CPV-1 R174 100.0 100.0 100.0 CPV-1 R164 100.0 100.0 100.0 100.0 CPV-1 V365 96.3 96.3 96.3 96.3 100.0 CPV-1 V1546 93.0 93.0 93.0 93.0 91.9 100.0 CPV-1 V466 99.1 99.1 99.1 99.1 95.8 92.5 100.0 CPV-1 V550 96.2 96.2 96.2 96.2 99.5 92.1 95.6 100.0 CPV-1 V551 96.3 96.3 96.3 96.3 99.6 92.3 95.8 99.8 100.0 CPV-2 V729 84.4 84.41 84.4 84.4 84.4 84.1 83.5 84.6 84.4 100.0

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54Table 3-2. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences of DNA polymerase gene fragments of the cetacean poxvirus 1 (CPV-1) and cetacean p oxvirus 2 (CPV-2) samples. Values correspond to percent identity between two amino acid sequences. CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2 AJ28 Wiki R174 R164 V365 V1546 V466 V550 V551 V729 CPV-1 AJ28 100.0 CPV-1 Wiki 100.0 100.0 CPV-1 R174 100.0 100.0 100.0 CPV-1 R164 100.0 100.0 100.0 100.0 CPV-1 V365 98.9 98.9 98.9 98.9 100.0 CPV-1 V1546 97.2 97.2 97.2 97.2 97.2 100.0 CPV-1 V466 98.9 98.9 98.9 98.9 97.8 96.1 100.0 CPV-1 V550 98.9 98.9 98.9 98.9 100.0 97.2 97.8 100.0 CPV-1 V551 98.9 98.9 98.9 98.9 100.0 97.2 97.8 100.0 100.0 CPV-2 V729 89.0 89.0 89.0 89.0 89.0 87.9 87.8 89.0 89.0 100.0

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55Table 3-3. Pair-wise comparisons of the amino acid sequences deduced from the nucle otide sequences of the DNA polymerase gene fragments of the cetacean poxvirus 1 (CPV-1) and cetacean p oxvirus 2 (CPV-2) samples. Values correspond to percent similarity between two amino acid sequences. CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2 AJ28 Wiki R174 R164 V365 V1546 V466 V550 V551 V729 CPV-1 AJ28 100.0 CPV-1 Wiki 100.0 100.0 CPV-1 R174 100.0 100.0 100.0 CPV-1 R164 100.0 100.0 100.0 100.0 CPV-1 V365 98.9 98.9 98.9 98.9 100.0 CPV-1 V1546 98.9 98.9 98.9 98.9 98.9 100.0 CPV-1 V466 99.4 99.4 99.4 99.4 98.3 98.3 100.0 CPV-1 V550 98.9 98.9 98.9 98.9 100.0 98.9 98.3 100.0 CVP-1 V551 98.9 98.9 98.9 98.9 100.0 98.9 98.3 100.0 100.0 CPV-2 V729 92.3 92.3 92.3 92.3 92.3 92.3 91.7 92.3 92.3 100.0

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56Table 3-4. Pair-wise comparisons of the nuc leotide sequences obtained from the DNA t opoisomerase gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent identity between two nucleotide sequences. CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2 AJ28 Wiki R174 R164 V365 V1546 V729 CPV-1 AJ28 100.0 CPV-1 Wiki 100.0 100.0 CPV-1 R174 100.0 100.0 100.0 CPV-1 R164 100.0 100.0 100.0 100.0 CPV-1 V365 93.6 93.6 93.6 93.6 100.0 CPV-1 V1546 92.4 92.4 92.4 92.4 89.8 100.0 CPV-2 V729 84.3 84.3 84.3 84.3 84.9 86.0 100.0

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57 Table 3-5. Pair-wise comparisons of the amino acid sequences deduced from the nucle otide sequences of the DNA topoisomerase gene fragments of cetacean poxvirus 1 (C PV-1) and cetacean poxvirus 2 (CPV-2) samp les. Values correspond to percent identity between two amino acid sequences. CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2 AJ28 Wiki R174 R164 V365 V1546 V729 CPV-1 AJ28 100.0 CPV-1 Wiki 100.0 100.0 CPV-1 R174 100.0 100.0 100.0 CPV-1 R164 100.0 100.0 100.0 100.0 CPV-1 V365 96.5 96.5 96.5 96.5 100.0 CPV-1 V1546 94.7 94.7 94.7 94.7 92.1 100.0 CPV-2 V729 85.1 85.1 85.1 85.1 84.2 86.8 100.0

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58 Table 3-6. Pair-wise comparisons of the amino acid sequences deduced from the nucle otide sequences of the DNA topoisomerase gene fragments of cetacean poxvirus 1 (C PV-1) and cetacean poxvirus 2 (CPV-2) samp les. Values correspond to percent similarity between two amino acid sequences. CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2 AJ28 Wiki R174 R164 V365 V1546 V729 CPV-1 AJ28 100.0 CPV-1 Wiki 100.0 100.0 CPV-1 R174 100.0 100.0 100.0 CPV-1 R164 100.0 100.0 100.0 100.0 CPV-1 V365 96.5 96.5 96.5 96.5 100.0 CPV-1 V1546 99.1 99.1 99.1 99.1 95.6 100.0 CPV-2 V729 90.4 90.4 90.4 90.4 88.6 90.4 100.0

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59Table 3-7. Pair-wise comparisons of the nucleotide sequences of the DNA polymerase ge ne fragments of poxviruses of various genera within the Chordopoxvirinae subfamily of viruses. Values correspond to percent identity between two nucleotide sequences. CPV1 CPV-2 SSLPV SSLPPV SSL PPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Cetaceanpox-1 100.0 84.4 75.7 59.1 57.6 56.9 58.0 57.8 Cetaceanpox-2 84.4 100.0 77.0 59.0 55.2 54.3 56.7 56.5 Steller sealionpox 75.7 77.0 100.0 58.8 55.0 53.2 55.2 55.0 Camelpox 71.6 75.5 72.0 59.7 57.5 56.5 57.6 57.3 Cowpox 71.8 75.9 72.0 60.3 57.5 57.8 58.2 58.0 Monkeypox 72.6 76.1 72.4 60.1 57.5 57.1 57.6 57.3 Vaccinia 72.7 76.2 72.6 60.1 57.8 57.3 58.0 57.6 Ectromelia 72.0 76.1 71.6 60.1 58.8 58.0 59.0 58.6 Variola 71.6 74.8 71.8 60.8 58.4 57.5 58.6 58.2 Lumpy skin disease 74.0 74.4 74.0 61.2 56.7 56.9 56.7 56.3 Sheeppox 73.3 74.8 73.3 61.2 57.1 56.7 57.1 56.7 Goatpox 74.0 75.5 75.0 60.3 56.2 56.2 56.2 55.8 Muledeerpox 73.6 75.2 76.6 59.9 55.7 55.3 55.9 55.5 Swinepox 75.0 76.8 75.5 60.6 55.6 56.3 55.4 55.4 Rabbit fibroma 67.8 67.4 68.9 60.1 59.1 61.9 59.3 59.7 Rabbit myxoma 69.2 68.3 69.6 62.1 61.4 64.7 61.9 61.9 Yaba monkeypox 68.5 70.4 70.4 60.1 59.9 59.5 60.3 60.1 Orf 53.4 53.2 49.8 77.2 82.5 83.0 83.0 82.8 Bovine pap stom 53.4 51.6 50.6 77.3 81.3 83.8 81.9 81.5 Canarypox 40.4 41.5 40.6 36.1 36.9 36.8 32.2 32.3 Fowlpox 40.1 40.6 66.3 36.6 36.0 36.0 35.9 36.4 Molluscum conagiosum 50.5 51.6 48.3 57.3 60.4 62.7 60.4 60.4 Harbor sealparapox 57.8 56.5 55.0 79.7 98.3 84.0 99.1 100.0 Spotted sealparapox 58.0 56.7 55.2 79.5 98.7 84.3 100.0 99.1 Steller sealionparapox V841 59.1 59.0 58.8 100.0 78.9 77.1 79.5 79.7 Steller sealionparapox V842 57.6 55.2 55.0 78.9 100.0 84.3 98.7 98.3 Steller sealionparapox V1386 56.9 54.3 53.2 77.1 84.3 100.0 84.3 84.0

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60Table3-8. Pair-wise comparisons of the amino acid sequences deduced from the nucle otide sequence of the DNA polymerase gene fragments of poxviruses of various genera within the Chordopoxvirin ae subfamily of viruses. Values correspond to percent identity between two amino acid sequences. CPV1 CPV-2 SSLPV SSLPPV SSL PPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Cetaceanpox-1 100.0 89.0 74.4 60.7 62.4 60.7 62.4 62.4 Cetaceanpox-2 89.0 100.0 77.8 62.9 62.4 60.1 62.4 62.4 Steller sealionpox 74.4 77.8 100.0 62.4 61.2 59.6 61.2 61.2 Camelpox 80.0 82.2 76.1 65.2 62.9 62.9 63.5 63.5 Cowpox 80.6 82.8 77.2 66.3 64.1 64.0 64.6 64.6 Monkeypox 80.6 82.8 77.2 66.3 64.1 64.0 64.6 64.6 Vaccinia 80.6 82.8 77.2 66.3 64.1 64.0 64.6 64.6 Ectromelia 80.6 82.8 77.2 66.3 64.1 64.0 64.6 64.6 Variola 79.4 81.7 77.2 66.3 64.1 64.0 64.6 64.6 Lumpy skin disease 75.0 74.4 73.9 60.7 62.9 61.8 62.9 62.9 Sheeppox 73.9 73.3 72.8 60.1 62.4 61.2 62.4 62.4 Goatpox 74.4 75.6 74.4 60.1 62.4 61.2 62.4 62.4 Muledeerpox 75.0 75.0 73.2 60.8 62.1 59.6 61.5 61.5 Swinepox 72.2 73.9 71.7 62.9 62.4 61.8 62.4 62.4 Rabbit fibroma 74.3 72.1 72.1 60.5 60.5 59.9 60.5 60.5 Rabbit myxoma 78.3 76.1 76.7 62.9 64.1 63.5 64.1 64.1 Yaba monkeypox 75.0 75.6 73.9 62.9 59.6 59.0 60.1 60.1 Orf 62.2 63.9 60.6 86.5 88.8 86.5 88.8 88.2 Bovine pap stom 65.6 65.0 64.4 87.1 88.2 87.6 89.3 88.8 Canarypox 56.7 59.4 59.4 50.0 49.4 50.0 49.4 49.4 Fowlpox 58.3 60.6 61.1 50.6 51.7 51.7 51.1 51.1 Molluscum contagiosum 56.1 57.2 56.1 56.7 56.7 59.0 56.7 56.7 Harbor sealparapox 62.4 62.4 61.2 87.6 98.3 91.2 99.4 100.0 Spotted sealparapox 62.4 62.4 61.2 88.2 98.9 92.1 100.0 99.4 Steller sealionparapox V841 60.7 62.9 62.4 100.0 87.1 85.4 88.2 87.6 Steller sealionparapox V842 62.4 62.4 61.2 87.1 100.0 91.0 98.9 98.3 Steller sealionparapox V1386 60.7 60.1 59.6 85.4 91.0 100.0 92.1 91.6

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61Table 3-9. Pair-wise comparisons of the am ino acid sequences deduced from the nucleotid e sequences of the DNA polymerase gene fragments of poxviruses of various genera within the Chor dopoxvirinae subfamily of viruses. Values correspond to percent similarity between two amino acid sequences. CP V-1 CPV-2 SSLPV SSLPPV SSLPPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Cetaceanpox-1 100.0 92.3 81.1 74.7 74.2 74.2 74.7 74.2 Cetaceanpox-2 92.3 100.0 82.8 76.4 75.3 74.8 75.8 75.3 Steller sealionpox 81.1 82.8 100.0 78.1 75.8 74.7 76.4 75.8 Camelpox 84.4 85.0 82.2 77.5 75.3 76.4 76.4 75.8 Cowpox 85.0 85.6 83.3 78.7 76.4 77.5 77.5 77.0 Monkeypox 85.0 85.6 83.3 78.6 76.4 77.5 77.5 77.0 Vaccinia 85.0 85.6 83.3 78.6 76.4 77.5 77.5 77.0 Ectromelia 85.0 85.6 83.3 78.7 76.4 77.5 77.5 77.0 Variola 85.0 85.6 83.3 78.7 76.4 77.5 77.5 77.0 Lumpy skin disease 80.6 81.1 80.6 74.7 75.8 75.8 76.4 75.8 Sheeppox 79.4 80.0 79.4 73.6 74.7 74.7 75.3 74.7 Goatpox 80.0 80.6 80.0 74.8 75.8 75.8 76.4 75.8 Muledeerpox 80.4 80.4 81.0 75.3 76.5 75.3 76.5 75.9 Swinepox 81.1 82.2 80.6 75.3 74.7 75.3 75.3 75.3 Rabbit fibroma 79.9 78.8 78.2 72.3 72.9 72.9 73.4 72.9 Rabbit myxoma 83.9 82.8 81.1 75.8 75.8 76.4 76.4 75.8 Yaba monkeypox 81.7 81.7 81.1 74.2 71.9 73.6 73.0 72.5 Orf 73.9 75.6 73.9 91.6 92.7 92.7 93.3 92.7 Bovine pap stom 77.8 78.3 78.9 93.3 92.1 92.1 92.7 92.1 Canarypox 69.4 70.6 68.9 68.0 65.7 66.3 66.3 66.3 Fowlpox 68.3 69.4 70.6 67.4 66.3 65.7 66.3 66.3 Molluscum contagiosum 75.0 74.4 72.2 71.9 70.2 70.3 70.8 70.8 Harbor sealparapox 74.2 75.3 75.8 91.6 98.9 94.9 99.4 100.0 Spotted sealparapox 74.7 75.8 76.4 92.1 99.4 95.5 100.0 99.4 Steller sealionparapox V841 74.7 76.4 78.1 100.0 91.6 91.0 92.1 91.6 Steller sealionparapox V842 74.2 75.3 75.8 91.6 100.0 94.9 99.4 98.9 Steller sealionparapox V1386 74.2 74.8 74.7 91.0 94.9 100.0 95.5 94.9

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62Table 3-10. Pair-wise comparisons of the nucleotide sequences of the DNA topoisomerase gene fragments of poxviruses of variou s genera within the Chordopoxvirinae subfamily of viruses. Values correspond to percent identity between two nucleotide sequences. CPV1 CPV-2 SSLPV SSLPPV SSL PPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Cetaceanpox-1 100.0 84.3 70.9 51.7 53.5 53.2 47.6 53.8 Cetaceanpox-2 84.3 100.0 72.1 59.0 52.3 54.3 56.7 56.5 Steller sealionpox 70.9 72.1 100.0 51.7 54.4 52.6 48.0 52.6 Camelpox 64.5 68.0 70.1 55.2 55.5 54.9 52.0 55.5 Cowpox 63.4 67.2 70.6 55.2 54.9 55.5 51.2 54.9 Monkeypox 64.2 68.0 71.2 55.2 54.9 54.4 51.2 54.9 Vaccinia 64.5 68.0 70.1 55.2 55.5 54.9 52.0 55.5 Ectromelia 64.5 68.0 70.9 55.2 55.5 54.9 52.0 55.5 Variola 64.8 68.3 71.2 54.9 55.2 54.7 51.6 55.2 Lumpy skin disease 69.5 68.3 72.1 52.0 53.2 51.5 45.5 51.7 Sheeppox 68.6 68.6 70.9 52.0 53.5 52.3 45.9 52.0 Goatpox 69.2 68.0 71.5 52.3 53.2 51.5 45.5 51.7 Muledeerpox 66.9 68.0 72.7 53.8 55.5 55.2 50.8 54.7 Swinepox 73.0 72.4 73.0 55.5 55.5 53.8 51.6 55.2 Rabbit fibroma 64.2 63.1 65.4 55.5 54.9 57.3 50.0 54.9 Rabbit myxoma 63.6 65.1 65.7 57.3 57.8 60.2 51.6 57.6 Yaba monkeypox 69.6 68.0 69.4 54.6 53.8 51.5 47.6 53.5 Orf 50.0 50.0 47.1 72.6 82.3 82.3 81.0 83.7 Bovine pap stom 51.2 50.6 48.5 72.3 81.4 81.4 80.2 83.4 Canarypox 62.8 64.0 68.0 52.3 52.9 54.9 50.0 52.3 Fowlpox 61.0 61.3 68.0 53.8 54.4 52.0 50.0 53.8 Molluscum contagiosum 50.3 50.3 49.1 61.6 68.0 65.4 65.4 68.3 Harbor sealparapox 53.8 56.5 52.6 75.5 96.0 85.0 95.6 100.0 Spotted sealparapox 47.6 56.7 48.0 71.9 93.7 84.3 100.0 95.6 Steller sealionparapox V841 51.7 59.0 51.7 100.0 74.4 70.6 71.9 75.5 Steller sealionparapox V842 53.5 52.3 54.4 74.4 100.0 82.3 93.7 96.0 Steller sealionparapox V1386 53.2 54.3 52.6 70.6 82.3 100.0 84.3 85.0

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63Table 3-11. Pair-wise comparisons of the amino acid sequences deduced from the nucle otide sequences of the DNA topoisomerase gene fragments of poxviruses of various genera within the Chordopoxvirinae subfamily of viruses. Values correspond to percent identity between two amino acid sequences. CPV1 CPV-2 SSLPV SSLPPV SSL PPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Cetaceanpox-1 100.0 85.1 60.5 56.1 57.0 57.9 46.4 57.9 Cetaceanpox-2 85.1 100.0 67.5 55.3 56.1 57.0 45.1 57.0 Steller sealionpox 60.5 82.8 100.0 57.0 57.0 57.0 46.3 58.9 Camelpox 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0 Cowpox 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0 Monkeypox 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0 Vaccinia 64.6 66.8 69.0 57.5 62.8 61.9 57.3 63.7 Ectromelia 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0 Variola 65.8 67.5 70.2 57.9 62.3 61.4 56.1 63.2 Lumpy skin disease 63.2 64.9 69.3 55.3 59.6 58.8 48.8 59.6 Sheeppox 63.2 64.9 69.3 55.3 59.6 58.8 48.8 59.6 Goatpox 63.2 65.8 69.3 55.3 59.7 58.8 48.8 59.6 Muledeerpox 62.3 64.0 69.3 57.0 61.4 60.5 52.4 62.3 Swinepox 64.9 65.8 67.5 58.8 59.6 59.6 52.4 60.5 Rabbit fibroma 62.3 62.3 64.9 58.8 57.8 57.0 48.8 58.8 Rabbit myxoma 64.0 62.3 64.9 58.7 58.8 57.0 48.8 58.8 Yaba monkeypox 63.2 60.5 62.3 57.9 59.6 57.9 50.0 60.5 Orf 55.3 54.4 53.5 79.1 89.7 91.4 85.7 87.9 Bovine pap stom 58.7 56.1 55.3 80.9 89.7 92.2 87.0 90.5 Canarypox 57.1 57.9 64.0 54.5 56.1 55.3 50.0 56.1 Fowlpox 55.3 51.8 61.4 52.6 56.1 55.3 46.3 56.1 Molluscum contagiosum 56.1 55.3 55.3 61.4 62.3 62.3 58.5 61.4 Harbor sealparapox 57.9 57.0 58.9 79.1 97.4 91.4 95.2 100.0 Spotted sealparapox 46.4 45.1 46.3 75.9 95.2 89.3 100.0 95.2 Steller sealionparapox V841 56.1 55.3 57.0 100.0 80.9 81.6 75.9 79.1 Steller sealionparapox V842 57.0 56.1 57.0 80.9 100.0 91.4 95.2 97.4 Steller sealionparapox V1386 57.9 57.0 57.0 81.6 91.4 100.0 89.3 91.4

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64Table 3-12. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences from the DNA topoisomerase gene fragments of poxviruse s of various genera within the Chordopoxvi rinae subfamily of viruses. Values correspond to percent similarity be tween two amino acid sequences. CP V-1 CPV-2 SSLPV SSLPPV SSL PPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Cetaceanpox-1 100.0 90.4 75.4 67.5 66.7 67.5 57.3 65.8 Cetaceanpox-2 90.4 100.0 77.2 68.4 66.7 67.5 57.3 65.8 Steller sealionpox 75.4 82.8 100.0 65.8 70.2 70.2 62.2 71.1 Camelpox 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6 Cowpox 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6 Monkeypox 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6 Vaccinia 75.2 77.0 76.1 70.8 73.5 73.5 70.7 74.3 Ectromelia 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6 Variola 76.3 78.1 77.2 71.1 72.8 72.8 69.5 73.7 Lumpy skin 73.7 72.8 75.4 70.2 71.9 71.9 65.9 71.9 Sheeppox 73.7 72.8 75.4 70.2 71.9 71.9 65.9 71.9 Goatpox 73.7 72.8 75.4 71.1 71.9 71.9 48.8 71.9 Muledeerpox 72.8 72.8 75.4 68.4 70.2 69.3 64.6 71.1 Swinepox 75.4 74.5 79.8 73.7 73.7 72.8 68.3 74.6 Rabbit fibroma 71.9 74.6 72.8 71.1 68.4 66.7 59.8 68.4 Rabbit myxoma 71.9 74.6 72.8 71.1 68.4 66.7 59.8 68.4 Yaba monkeypox 70.0 70.2 73.7 69.3 70.2 69.3 63.4 71.1 Orf 67.5 67.5 70.2 86.1 93.1 94.8 90.5 93.1 Bovine pap stom 69.3 68.4 70.2 87.0 92.2 94.0 91.7 92.2 Canarypox 70.2 68.4 74.6 70.2 71.9 71.9 65.9 71.9 Fowlpox 69.3 65.8 73.7 68.4 70.2 70.2 63.4 70.2 Molluscum 68.4 67.5 70.2 69.3 71.9 71.9 69.5 71.1 Harbor sealparapox 65.8 75.3 75.8 86.1 99.1 94.0 98.8 100.0 Spotted sealparapox 57.3 75.8 76.4 80.7 97.6 91.7 100.0 98.8 Steller sealionparapox V841 67.5 76.4 78.1 100.0 86.1 86.0 80.7 86.1 Steller sealionparapox V842 66.7 75.3 75.8 86.1 100.0 94.0 97.6 99.1 Steller sealionparapox V1386 67.5 74.8 74.7 86.0 94.0 100.0 91.7 94.0

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65 Table 3-13. Pair-wise comparisons of th e nucleotide sequences obtained from the major envelope protein gene fragments of marine parapoxviruses within the Chordopoxvirinae subfamily of viruse s.Values correspond to percent identity between two nucleotide sequences. SSLPPV SSLPPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Harbor sealparapox 93.4 93.3 93.3 93.1 100.0 Spotted sealparapox 91.6 91.4 95.8 100.0 93.1 Steller sealionparapox V841 100.0 99.8 94.8 91.6 93.4 Steller sealionparapox V842 99.8 100.0 94.9 91.4 93.3 Steller sealionparapox V1386 91.6 91.4 100.0 95.8 93.3 Table 3-14. Pair-wise comparisons of th e amino acid sequences deduced from the nucleotide sequences from the major e nvelope protein gene fragments of marine parapoxviruses within the Chor dopoxvirinae subfamily of viruses. Values correspond to percent identity between two amino acid sequences. SSLPPV SSLPPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Harbor sealparapox 97.0 96.5 96.0 96.0 100.0 Spotted sealparapox 93.9 93.4 100.0 100.0 96.0 Steller sealionparapox V841 100.0 99.5 93.9 93.9 97.0 Steller sealionparapox V842 99.5 100.0 93.4 96.5 96.5 Steller sealionparapox V1386 93.9 93.4 100.0 96.0 96.0 Table 3-15. Pair-wise comparisons of th e amino acid sequences deduced from the nucleotide sequences of the major e nvelope protein gene fragments of marine parapoxviruses within th e Chordopoxvirinae subfamily of viruses.Values correspon d to percent similarity between two amino acid sequences. SSLPPV SSLPPV SSLPPV SSPPV HSPPV V841 V842 V1386 V688 V465 Harbor sealparapox 98.5 98.0 98.5 98.5 100.0 Spotted sealparapox 97.0 96.5 100.0 100.0 98.5 Steller sealionparapox V841 100.0 99.5 97.0 97.0 98.5 Steller sealionparapox V842 99.5 100.0 96.5 98.0 98.0 Steller sealionparapox V1386 97.0 96.5 100.0 98.5 98.5

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66 CHAPTER 4 DISCUSSION All of the cetacean poxvirus skin lesions examined in the present study conformed with the typical tattoo lesion appearance. Some lesions were over 2.0 cm in diameter, while some samples of lesions consisted of 8 mm diameter skin biopsies. We cannot make any conclusions regarding the specific stage of infectio n represented in each lesion, other than to observe that so me lesions showed more definite hyperpigmentation of the skin, or more clearly defined edges su rrounding the lesion. We did not find any association between cetacean lesion app earance and positive pox PCR results, or poxvirus DNA sequences obtained. Pinniped pa rapoxviruses are associated with skin lesions that resemble those reported for other terrestrial parapoxviruse s such as those that are seen in orf, pseudocowpox, and bovine papul ar stomatitis, both histologically and in patterns of disease progression (Wilson et al., 1972; Hadlow et al., 1980; Hicks and Worthy, 1987). The prevalence of parapoxvi rus infection in pinnipeds remains unreported; however, skin lesi ons associated with these infections are frequently encountered in both stranded pinnipeds brought into rehabilitation centers and in captive pinnipeds (Wilson et al., 1969; Wilson et al., 1972; Hadlow et al., 1980; Osterhaus et al., 1990; Simpson et al., 1994; Muller et al., 2003 ). Hicks and Worthy, (1987), reported that five of 11 recently weaned grey seal (Halochoerus grypus ) pups collected for a nutritional study developed parapox lesions after 1 4 weeks in captivity. The appearance of these lesions in animals that appeared otherwise h ealthy at the time of co llection, suggests that

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67 while the pups may be exposed to the para poxvirus in the wild population, the viral infection may be exacerbated under stressful conditions brought on in captivity. Pinniped skin lesions examined in this study were co llected during Steller se a lion capture-release studies or by members of the Alaska Depa rtment of Fish and Game and the Marine Mammal Stranding Center in New Jersey. A gross distinction betw een the appearance of lesions associated with Stel ler sea lion poxvirus and lesions associated with pinniped parapoxvirus, could not be made. While histopathology and electron micr oscopy are useful in confirming the presence of typical microscopi c poxvirus lesions and in the vi sualization of viral particles in cetacean lesions (Flom and Houk, 1979; Ge raci et al., 1979; Smith, 1983; Baker, 1992a,b; Van Bressem et al., 1993), they offer little information about the type of poxvirus involved. The primary objective of this study was to develop a diagnostic strategy based on extraction of total DNA from lesions, PCR assay using the extracted DNA as template, and sequencing of the amplifie d fragments, to detect and characterize poxviruses in cutaneous lesions of cetaceans a nd pinnipeds. The first step in creating a PCR protocol was to design oligonucleotide pr imers that would anneal to targeted genes in the template viral DNA present in cuta neous lesions. Problems encountered in designing primers to target ce tacean and pinniped poxvirus gene s, stemmed from the lack of any available genetic data pertaining to marine mammal poxviruses. Despite the absence of sound antigenic and molecular da ta, most previous work using electron microscopy on cetacean poxviruses has repeatedly implicated them as members of the orthopoxvirus genus. In addition, one study re ported a mixed parapox and orthopox virus infection in a grey seal, based on the same techniques (Osterhaus et al., 1990). In the

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68 present study, histopathologic examination of the two Steller sea lion skin lesions revealed a similar appearance to lesions of the northern fur seal and South American sea lions, characterized by dermal nodules of hyperp lastic epithelial cell s versus the raised plaque-like lesions described in harbor a nd grey seal lesions (Wilson and PoglayenNeuwall, 1971; Wilson et al., 1972; Hicks and Worthy, 1987; Osterhaus et al., 1990, 1994). Electron microscopy performed on the tw o Steller sea lion skin lesions revealed the presence of poxvirus virions with mor phologic characteristics consistent with published reports of orthopox viruses (Moss, 2001). However, sequencing of amplified fragments showed that most lik ely, these viruses are species specific poxviruses of Steller sea lions and not orthopoxviruses. Hi stopathologic and electron microscopic examination of the 10 positive cetacean skin lesions was not performed due to poor sample quality and in general, improper sample preservation. Because of their high level of conserva tion within the Chordopoxvirinae, the DNA polymerase and DNA topoisomerase I gene s were targeted for the design of oligonucleotide primers for PCR. Specificall y, nucleotide sequences within regions of the open reading frame of these genes that we re highly conserved within members of the Orthopox, Suipox, and Capripox gene ra were targeted with consensus primers to drive the amplification of approximately 543-bp in the case of the DNA polymerase gene and 344-bp for the DNA topoisomerase I gene. Sequences of the Orthopox, Suipox, and Capripox genera were obtained from the Ge nBank database and through the website of the National Center for Biotechnology Info rmation (NCBI). The amount of sequence generated by fragments of the above sizes is usually sufficient to characterize viruses molecularly and assign them to proper virus ge nera and species, when derived from genes

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69 that exhibit high levels of conservation (Ropp et al., 1995; Zanotto et al., 1996; Becher et al., 2002; McGeoch et al., 2000). Most pinniped poxviruses have long been considered probable members of the Parapox genus. Inoshima et al., (2000), vali dated consensus primers that target a 596-bp gene fragment of the major envelope protei n of parapoxviruses in ungulates. Using these primers in a PCR protocol, initially with suboptimal annealing temperatures, we were able to identify parapoxvirus positive samples from pinniped skin lesions and confirm the usefulness of the primers. A nucleotid e alignment of orf and BPS viruses DNA sequences available in the NCBI was used to design PCR primers targeting a 536-bp fragment of the DNA polymerase gene and a 350-bp fragment of the DNA topoisomerase of parapoxviruses. These prim ers effectively amplified the respective genes of pinniped parapox viruses, confirming the diagnoses made using the major envelope protein gene primers. Our results expand the molecular di agnosis tools as applicable to parapoxvirus, and make possible a wider genetic analysis comprising two more genes. PCR protocols were developed using these primers at subopt imal annealing temperatures in order to maximize the chances of amplifying the cetacean and SSL poxvirus and pinniped poxvirus genes. Once each primer set was test ed for reactivity using positive cetacean and/or pinniped poxvirus DNA, each protoc ol was optimized to produce a single amplicon, usually by raising the annealing temp erature of PCR until the desired reactivity was obtained. Positive samples were identified by the presence of a single amplicon of the expected size. DNA sequence was obtained by two methods; Firstly, cleaning of the PCR product followed by direct sequencing, an d/or secondly, sequencing of the cloned

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70 PCR product in the bacterial plasmid vector, PCR-Topo2.1. The first method was used when the amplified fragments were unique and allowed for the rapid diagnosis of poxvirus infection, and for verifying DNA se quences obtained from cloned products, when disparities between two or more clon ed sequences were found. All samples that yielded positive results were later cloned, to obtain full sequences and to preserve valuable DNA products, as the amount of tota l DNA obtained from lesions was usually small and rapidly exhausted after multiple uses. Ten cetacean skin lesions were found to contain amplifiable poxvirus DNA using the PCR protocols and DNA sequencing strate gies described above The identified positive samples represented two different groups of cetacean poxviruses, provisionally referred to as CPV-1 and CPV-2. Viruse s in the CPV-1 corresponded to the poxvirus DNA polymerase and DNA topoisomerase sequen ces obtained from four species of dolphins while the CPV-2 virus corre sponded to the DNA polymerase and DNA topoisomerase sequences of the bowhead whale (Balaena mysticetus ) poxvirus. The same PCR protocols also amplified poxvirus DNA from two Steller sea lion skin lesions indicating the existence of a unique and most likely, specie s specific, Steller sea lion poxvirus (SSLPV). The three PCR assays for pinniped parapoxvirus allowed the identification of six positiv e skin lesion samples harvested from one harbor seal (HSPPV), two spotted seals (SSLPPV) and th ree Steller sea lions (SSLPPV). Although none of the Steller sea lions examined in th is study showed evidence of a dual infection of both pox and parapoxviruses, we speculate that a dual infection could occur. Mammalian species that have been document ed to be afflicted with multiple poxvirus species, belonging to different genera, in clude cattle,sheep an d camels (Robinson and

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71 Mercer, 1995; Inoshima et al., 2000; Mo ss, 2001). In cattle, infections with pseudocowpox virus, a member of the Para pox genus, and cowpox virus, a member of the Orthopox genus, have been observed (Pic kup et al., 1982; Buller and Palumbo, 1991). In sheep, orf virus, of the Parapox genus and sheeppox, of the Capripox genus, have been observed (Inoshima et al., 2000; Hosa mani et al., 2004). In camels, camelpox, a notable member of the Orthopox genus, has been observed, as well as camel parapox virus (Robinson and Mercer, 1995; Gubser and Smith, 2002). Nucleotide sequences and their deduced amino acid sequences obtained from all poxvirus positive samples were entered into the GenBank database and compared using pairwise and multiple alignment functions fr om the GCG Wisconsin Package. Pairwise comparisons were made between sequences obtai ned from each targeted gene of each of the cetacean and pinniped pox and parapox vi ruses, to sequences available in the GenBank and available in the NCBI database representing several terrestrial poxviruses within the Chordopoxvirinae. Considering first the DNA polymerase co mparisons, the cetacean poxviruses share the highest homology among themselves, with a nucleotide identity of 84.4% (Table 3-7) and an amino acid identity of 89.0% (Table 38). The nucleotide id entities described in Table 3-1 indicate that both CPV-1 and CPV2 share the second clos est identities to the SSL poxvirus, with identities of 75.7 and 77.0%. Following the SSL pox virus, CPV-1 and CPV-2 are most closely related to me mbers of the Orthopox genus, with nucleotide identities ranging from 71.6 to 76.2% (Table 37). These viruses may have evolved from a common ancestor as species sp ecific marine poxviruses, prior to the evolution of some of the terrestrial orthopoxviruses such as cam elpox and some strains of the variola virus

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72 (Afonso et al., 2002, Gubser and Smith, 2002). The cetacean poxviruses and SSL poxvirus shared the least homology with me mbers of the Avipox and Parapox genera, with nucleotide identities below 53.2% (Table 3-7). These findings are supported by a previous phylogenetic study demonstrating th e distant relationship of the orthopoxviruses to the avipoxviruses (Gubser et al., 2004). The pinniped pa rapox viruses shared highest nucleotide identities among themselves (Table 3-7). Notable are the nucleotide identities of 98.3 and 98.7% of one Steller sea lion, (V 842), when compared to the harbor and spotted seal sequences (Table 3-7). The significance and inte rpretation of the identities are difficult to ascertain, as Steller sea lions and spotted s eals inhabit northwest Pacific waters, while the harbor seal originated from northeast Atlantic waters. SSL V842 shared only 78.9 and 84.3% nucleotide identity to the other two SSL sequen ces. The nucleotide and amino acid identit ies between the harbor and spotted seals are above 98% (Table 3-7). These results suggest th at the SSPPV, HSPPV and SSLPPV may have originated from a common ancestor and, diverged as they evolve d with their host species. The pinniped parapoxviruses shar e the least homology to avip ox viruses, as would be expected based on previous phylogenetic anal ysis of Chordopoxvirinae (Gubser et al., 2004). The amino acid identities represented in Table 3-8 show slightly different homologies. This is due to the nature of flex ibility or degeneracy in the protein or amino acid code. In the nucleotide comparisons each discrepancy between two nucleotide sequences is reported as a difference, whereas in translation to a protein sequence, a nucleotide substitution may be silent, causing no amino acid change, and thus no difference between the two sequences. Results from Table 3-8 indicate that the cetacean poxviruses are most homologous to each other, with the next clos est homology being to

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73 the orthopox viruses, followed by the SSL poxvi rus. The Avipox and Parapox genera are consistently, the least homol ogous to the cetacean poxviruses. Protein identities of the pinniped parapoxviruses are high est to orf and BPSV, with the exception of the harbor and spotted seal poxviruses that share 99.4% identity to each other (Table 3-8). Variations in the 3 Steller sea lion DNA polym erase sequences are apparent in the protein identities with ranges from 85.4 to 91.0% (Tab le 3-8), suggesting th e existence of more than one strain or type of Steller se a lion parapoxvirus. Comparisons among DNA polymerase protein similarities are reported in Table 3-9. Protein similarity comparisons offer a means to weigh the significance of observed amino acid differences. For example, the substitution of a basic amino acid for an acidic amino acid may cause a more significant functional change than a ba sic to basic amino acid substitution. The relevance of viewing the protein similarities of the gene sequences reported in Table 3-9 is simply to ascertain the significance of the amino acid differences indicated by the protein identities in Table 3-8 (Needlema n and Wunsch, 1970). The homology patterns observed by looking at protein similarities ag ree with those reported for the protein identities, and warrant no further discussion. Considering next, the DNA topoisomerase gene comparisons, the overall nucleotide identities are lower than those observed in the DNA polymerase comparisons, indicating a lesser degree of conservati on in the DNA topoisomerase gene when compared to the DNA polymerase gene, with in the Chordopoxvirinae (T able 3-10). The CPV-1 and CPV-2 fragments share 84.3% nucleo tide identity with each other, followed by identities to swinepox virus of 72.4 to 73.0% (Table 3-10). The next closest homology is to SSL poxvirus followed by member s of the Capripox genus (Table 3-10).

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74 The difference in the pattern of homol ogy between the DNA topoisomerase and DNA polymerase genes demonstrates the variability in gene evolution. Vi ral genes evolve at varying rates, depending on need to adapt to new host or environmental stresses (Upton et al., 2003; Gubser et al., 2004;). The pi nniped parapoxviruses are consistently closest in homology to orf BPSV and to each ot her (Table 3-10). Steller sea lion, V842, demonstrated a higher homology to the harbor and spotted seal sequences, than to the other (V841 and V1386) SSL sequences (Table 3-10), as seen in the DNA polymerase gene comparisons. The variance of the DNA topoisomerase amino acid identities from the DNA polymerase amino acid identities mimi cs these differences in the nucleotide identity tables. Pairwise comparisons of th e amino acid identities i ndicate homologies of CPV-1 and CPV-2 to the Orthopox genus rangi ng from 64.6 to 67.5% (Table 3-11). CPV-1 shows only 60.5% amino acid identity to SSL poxvirus, while CPV-2 shows an amino acid identity to SSL poxvirus of 82.8% (T able 3-11). These different identities represent the differences in the evolutionary rates be tween the DNA polymerase and DNA topoisomerase genes examined in this st udy. However, these results indirectly confirm the differences between the cetacean po xviruses and indicate that CPV-2 is more closely related to the SSL poxvirus. The major envelope protein gene (MEP) pairwise comparisons were made using exclusively the pinniped parapox gene sequences generated in this study. Attempts to amplify the MEP gene of cetacean and SSL poxviruses were unsuccessful, limiting the scope of the comparisons. The problems enc ountered in amplifying the MEP gene from the cetacean and SSL poxviruse s stem from the degree of variation found between these novel poxviruses and other terrestrial poxviruses. The MEP gene of poxviruses is more

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75 variable than the DNA polymerase and DNA topoi somerase genes (Upton et al., 2003). Primers designed based on the available MEP DNA sequences of other terrestrial poxviruses, most likely did not amplify th e cetacean or SSL poxvirus MEP due to the greater degree of variation within the gene The MEP gene comparisons demonstrate nucleotide and amino acid id entities ranging from 91.4 to 99.8% (Table 3-13), and 93.4 to 99.5%, respectively (Table 3-14). The variance observe d in the DNA polymerase and topoisomerase gene sequence comparisons were absent in the MEP comparisons and the homologies in the latter we re more uniform. The MEP gene of poxviruses is typically more variable than those invol ved in DNA replication, as it is involved in host specificity, viral adhesion to the host cell, and possibly ev asion of host immunity (Smith et al., 2002). Partial nucleotide and deduced amino acid sequences have been used to make a distinction between different species of parapoxvirus, such as orf BPSV and pseudocowpox (Inoshima et al., 2000). Our resu lts showed less variation in the pinniped parapoxvirus MEP gene fragments than the variation reported be tween homologous MEP gene fragments of orf, BPSV, and pseudocow pox (Inoshima et al., 2000 ; Becher et al., 2002; Delhon et al., 2004). These results may be due to the specific region of the gene amplified by the MEP PCR primers. Certai n areas of the MEP gene are likely more conserved in DNA sequence, such as t hose encoding the hypdrophobic regions of the protein, found within the envelope lipid bilayer (Silverman, 2005). In addition, the poxviruses of marine mammals may not have su ccumbed to the same selective pressures encountered over hundreds of years by the te rrestrial poxviruses, such as vaccination, husbandry and environmental conditions that s timulate genetic evolution and mutation in the viral genome. The high degree of conser vation observed in the pinniped parapoxvirus

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76 MEP sequences can be understood after consid ering the nature of these consensus PCR primers, designed to amplify the MEP gene fragment of all parapoxviruses. Overall, it can be inferred, based on pair wise comparisons, th at CPV-1, CPV-2 and SSLPV are most closely related to th e orthopoxviruses, and that the pinniped parapoxviruses are most closely related to the known terrestrial parapoxviruses of ruminants. Phylogenetic trees were construc ted using the deduced amino acid sequences, to further determine the genetic relatedne ss of the marine mammal poxviruses to known virus members of the Chordopoxvirinae. The phylogenetic studies described in Upton et al. (2003), and G ubser et al. (2004), provided new insight into novel methods of an alysis for uncharacterized poxviruses, such as those described in this thesis. In the present study, phylogenetic analysis was performed based on partial proteins of the DNA polymerase, DNA topoisomerase and major envelope protein genes of several members of the Chordopoxvirinae, including CPV-1, CPV-2, SSLPV, SSLPPV SSPPV, and HSPPV (Fi gures 3-23A&B, 3-24A&B and 3-25A&B). The bootstrapped cladogram and the radi al divergence tree representing the DNA polymerase protein sequences indicate that th e cetacean poxviruses form a distinct genus within the Chordopoxvirinae, separate fr om the Orthopox genus and from SSLPV, indicating a species specific poxvi rus.. The SSLPV falls into a clad by itself, outside of the Orthopox lineage group. The pinniped para poxviruses group, as expected, within the Parapox genus (Figure 3-23A). These results were reiterated in the divergence tree, revealing the ancestry of the DNA poly merase gene fragments within the Chordopoxvirinae. This tree cl early showed genetic divergence from the ancestor

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77 branch, of SSLPV first, followed by th e cetacean poxviruses, and finally, the differentiation of the orthopoxviruses (Figure 2-23B). Among the parapoxviruses, the SSLPPV sequences show three different poin ts of divergence. SSLPPV(V841) diverged first, followed by orf and BPSV. SSLPPV(V1386), SSLPPV(V842), SSPPV, and HSPPV are branched together; however, SSLPPVv1386 diverges from the branch by itself. These results strongly suggest the ex istence of three differe nt SSL parapoxviruses, supporting conclusions drawn from the pairwise comparison tables. These are the first sequences of SSL parapoxviruses ever obt ained for the DNA polymerase gene. Phylogenetic trees constructed based on pa rtial proteins of the DNA topoisomerase gene indicate that CPV-1 and CPV-2 form a group separate from any other, as does SSLPV strongly suggesting that the viruses c ould be assigned to new genera within the Chordopoxvirinae subfamily of viruses (Fig ure 3-24A). All SSLPPVs, HSPPV, and SSPPV clad inside the parapox group. The radial divergence tr ee representing the DNA topoisomerase protein fragments differ s from the DNA polymerase divergence tree (Figure 3-24B). The topoisomerase diverg ence tree shows the orthopox viruses as having a separate lineage from CPV-1, CPV2 and SSL PV, rather than the three groups diverging from a single branch. SSLPV is depicted closer to the Orthopox group, where as the DNA polymerase tree depicted the cetacean poxviruses closer to the Orthopox group. These results are further exem plification that the DNA topoisomerase I gene may have evolved at a different ra te and direction than th e DNA polymerase gene. The final phylogenetic analyses perfor med were based on the partial protein sequences of the major envelope protein (M EP) (Figures 3-25A and 3-25B). Poxvirus MEP sequences were not obtained, and ar e, consequently, absent in the MEP

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78 phylogenetic trees. Examination of the ra dial divergence tree indicates a closer relationship between the SSLPPV MEP fr agments than those seen for the DNA polymerase and DNA topoisomerase genes (Fig ure 3-25B). These results agree with results obtained from the pairwise comparison tables. The radial tr ee (3-25B) also shows clear divergence of the pi nniped parapox group, including a recently published sequence for the Weddel seal (Tryland et al., 2005), and from the other parapox species, namely orf, BPS, pseudocowpox, and parapox of red deer viruses. Clear definitions for nucleotide and/or am ino acid identity requirements necessary for the assignment of novel poxviruses to an appropriate genus are currently lacking. In the case of the orthopoxviruses, specifically variola, vacc inia and cowpox, the nucleotide identities are >90% (Goebel et al., 1990; G ubser et al., 2004). The newly identified cetacean poxviruses, CPV-1 and CPV-2, shar e only 84% nucleotide identity in the targeted regions of the DNA polymerase and DNA topoisomerase genes, and their nucleotide or amino acid identities with a ny members of the known poxvirus genera are even lower. Phylogenetic and evolutiona ry analysis of the DNA polymerase and DNA topoisomerase gene fragments show that although the cetacean poxviruses and the members of the orthopoxvirus genus originate from a comm on node, there is a clear divergence of the cetacean poxviruses into a unique branch. It is clear from both the bootstrapped and divergence phylograms, that th ere is a greater degree of divergence between the members of the Capripox ge nus, namely goatpox, sheeppox and lumpy skin disease viruses, than is observed between th e two cetacean poxviruses. We infer that the cetacean poxviruses, as evidenced by thei r genetic isolation from all of the known

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79 poxvirus genera, as well as the formation of a unique branch in th e phylogenetic trees, should constitute a new genus within the Chordopoxvirinae subfam ily of viruses. The nucleotide and/or amino acid identity requirements for the classification of poxviruses as strains, species and genera vary depending on several factors. These factors include the gene from which the DNA sequence data was derived, the poxvirus genus under consideration and the length of the available DNA sequence. Becher et al. (2002), suggested the inclusion of sealpox viru s as a new species of parapoxvirus based on nucleotide identities that ranged from 75 -79% when a 594-bp fragment of the major envelope protein was compared with homologous fragments from other ungulate parapoxviruses. Damaso et al. (2000), conc luded that an emerge nt poxvirus, Cantagalo virus, constituted a strain of vaccinia virus and not a se parate species of orthopoxvirus based on 98% nucleotide identity of a 950-bp fragment of the hemagglutinin gene when compared to other vaccinia viruses. DNA sequences derived from highly conserved genes or regions within a gene may have different requiremen ts for the classification of strain, species and genus than genes or gene regions possessi ng lesser degrees of conservation. For the purposes of this study, we consider DNA polymerase and DNA topoisomerase gene sequences that possess a nucleotide and amino acid difference >10% when compared to homologous sequences of te rrestrial poxviruses, as indication of a new genus within Chordopoxvirinae. Pairwise comparisons showing 90-100% nucleotide and amino acid identity between poxviruses within a genus are considered separate strains. Our pairwise comparisons suggest the existenc e of more than one strain of parapoxvirus occurring in Steller sea lions.

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80 Whenever possible, skin lesions that yiel ded a positive PCR result were inoculated onto cell culture to attempt vi rus isolation. Numerous attempts have been made in our laboratories and by others (VanBressem et al., 1999), however, to date, there are no reports of a cetacean or Steller sea lion poxvi rus being successfully isolated in cell culture. While parapoxviruses have been isol ated from pinnipeds in primary cell culture of pinniped tissues (Osterhaus et al., 1990; Osterhaus et al., 1994; Nettlleton et al., 1995), attempts to isolate the virus from the positive samples diagnosed in our laboratories were unsuccessful. We attribute these difficulties to an apparent specificity of the cetacean poxviruses to grow only in cetacean skin cells, and suspect that the cell lines that were available in this study, did not adequately support viral growth. Another possible cause for the difficulty in growing these cetacean poxviruses include the low amounts of viable poxvirus that may have been contained in th e few available skin lesion samples. The parapoxvirus isolated by Nettleton et al. (1995) was grown in primary grey seal kidney cells, and was passed weekly over 25 days. Os terhaus et al. (1990) reported the use of primary harbor seal kidney cells to isolat e an orthopox and a parapoxvirus from grey seal skin lesions. However, the orthopoxvirus was lost after several passages in culture. While poxviruses have been thoroughly reporte d in cetaceans and pinnipeds and repeated attempts to isolate those viruses have been made, relatively few successes, if any, are reported. Possible explanations for the difficul ties in isolating this virus are poor sample quality, use of improper cell lines, and l ack of ideal media and tissue culturing conditions, in general. Only in recent years, has there been an advancement in the un derstanding of the genetic characteristics and e volutionary relationships of poxviruses, enabled by the

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81 sequencing of complete poxvirus genomes a nd improvements in phylogenetic analysis. The genetic properties and phylogenetic relati onships of poxviruses th at affect marine mammals are still relativel y unknown, as these viruses are difficult to isolate and typically, are found in samples th at are not readily accessible. Further efforts to isolate poxviruses from cetacean and pinniped skin lesi ons are necessary for the advancement in the characterization of these viruses. The isolation of marine ma mmal poxviruses would permit the complete sequencing of the viral ge nome, development of new assays such as ELISA, and the simple detection of antibody respons es in infected animals. It would also be possible to target full genes and develop a more detailed understa nding of the structure and function of the proteins they encode. Th e PCR assays developed as part of this study will help to rapidly identify cetacean and pinniped pox and parapox viruses that afflict cetaceans and pinnipeds. The DNA sequences generated from poxvirus and parapoxvirus after the various PCR assays reported here, constitute a significant advancement in the molecular genetics of marine poxviruses and represent the first known report of comprehensive sets of nucleotide and amino acid sequences of novel poxviruses of cetacean and pinniped poxand parapoxviruses.

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85 MOSS, B. 2001. Poxviruses. In Fields Virology, 4th ed., Vol.2, D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman and S. E. Straus (eds.) Lippincott Williams & Wilkins pub lishers, Philaadelphia, Pennsylvania, pp. 2849-2884. MLLER, G., GRTERS, S., SIEBERT, U., ROSENBERGER, T., DRIVER, J., KNIG, M., BECHER, P., HETZEL, U ., AND BAUMGRTNER, W. 2003. Parapox infection in harbor seals (Phoca vitulina) from the German North Sea. Veterinary Pathology 40: 445-454. MULLIS, K., FALOONA, F., SCHARF, S., SAIKI, R., HORN, G., AND ERLICH, H. 1986. Specific enzymatic amplification of DNA in vitro : The polymerase chain reaction. Cold Spring Harbor Sympos ium on Quantitative Biology LI: 263-273 NEEDLEMAN, S. B., AND WUNSCH, C. D. 1970. A general method applicable to the search for similarities in the amino aci d sequence of two proteins. Journal of Molecular Biology 48(3): 443-453 NETTLETON, P.F., MUNRO, R., POW, I ., GILRAY, J., GRAY, E.W., AND REID, H.W. 1995. Isolation of a para poxvirus from a grey seal (Halichoerus grypus). The Veterinary Record 137:562-564 OSTERHAUS, A.D.M., BROEDERS, H.W.J., VISSER, I.K.G., TEPPEMA, J.S., AND VEDDER, E.J. 1990. Isolation of an orthopoxvi rus from pox-like lesions of a grey seal (Halichoerus grypus). Veterinary Record 127: 91-92 OSTERHAUS, A. D. M. E., BROEDERS, H. W. J., VISSER, I. K. G., TEPPEMA, J. S.,AND KUIKEN, T. 1994. Isolation of a parapoxvirus from pox-like lesions in grey seals. Veterinary Record 135: 601-602 PICKUP, D. J., BASTIA, D., STONE, H. O ., AND JOKLIK, W. K. 1982. Sequence of terminal regions of cowpox virus DNA: arrangement of repeated and unique sequence elements. Proceedings of the Nati onal Academy of Sciences of the United Stated of America. (79)23: 7112-7116 PUCHHAMMER-STOCKL, E., POPOW-KRAUPP, T., HEINZ, F. X., MANDL, C. W. AND KUNZ, C. 1990. Establishment of PCR for the early diagnosis of herpes simplex encephalitis. Journal of Medical Virology 32(2): 77-82 ROBINSON, A. J., AND MERCER, A., A. 1995. Parapox of Red Deer: Evidence for its inclusion as a new member in the ge nus parapoxvirus. Virology 208: 812-815 ROPP, S. L., JIN, Q., KNIGHT J. C., MASSUNG, R. F., AND ESPOSITO, J. J. 1995. PCR Strategy for identification and differenctiation of small pox and other orthopoxviruses. Journal of Clinic al Microbiology 33(8): 2069-2076

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88 BIOGRAPHICAL SKETCH Alexa Justine Bracht, the only child of Camille and Susan Bracht, was born in Boston, MA, 1977. Her family returned to New York in 1978, where Alexa spent the next 17 years of her youth. She began her college career at the University of MassachusettsAmherst, where she majored in animal science. Exposed to many new animal husbandry experiences at UMASS, including vaccinating a herd of cattle at Greensborough county jail, delivering twin lamb s (Lucy and Ethel), and castrating quite a few squealing piglets, Alexa decided her futu re would hold a life dedicated to animal service. Upon graduation with a Bachelor of Science, she re located to Brigantine, NJ, for a job as a marine mammal stranding technician at the Marine Mamm al Stranding Center. After countless seal rehabilitations and many cetacean strandings, she and her significant other Ethan, decided it was time to move on to bigger and sunnier places, and found them selves in Palm Coast, FL. After a year of wo rking at a fine veterinary hospital, Alexa embarked on the pursuit of highe r education as a graduate student at the University of Florida. Thrilled to find a professor involved in Florid as marine mammal stranding network, she joined the virology lab of Dr. Carlos Romero and began a study of poxvirus infection in cetaceans and pinnipe ds. Over two and a half year s, she gained a wealth of knowledge in virology, learned valuable mo lecular techniques, and made fabulous lifelong friends. After graduati on, she plans to take a year off from her weekly, and sometimes daily, commute from Gainesville to Palm Coast, and re turn home to her husband, where they will await the ar rival of their new baby together.


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Title: Detection and Molecular Characterization of Cetacean and Pinniped Poxviruses Associated with Cutaneous Lesions
Physical Description: Mixed Material
Copyright Date: 2008

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DETECTION AND MOLECULAR CHARACTERIZATION OF CETACEAN AND
PINNIPED POXVIRUSES ASSOCIATED WITH CUTANEOUS LESIONS













By

ALEXA JUSTINE BRACHT


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

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Alexa Justine Bracht


































This document is dedicated to my husband, Ethan Sherman, for his support and patience
through these long journeys, and to my loving mother, Camille, who holds my hand
along the way.















ACKNOWLEDGMENTS

My greatest thanks are extended to my mentor, Dr. Carlos Romero, not only for his

guidance throughout my project, but for giving me this great opportunity, when few

others would have. His support and patience through some very trying times were truly

appreciated. I would also like to thank my committee members, Dr. Ayalew Mergia, Dr.

James Maruniak and Dr. Charles Manire, for their time, assistance and suggestions. I

would like to express my gratitude to Dr. Ellis Greiner, for being my first liaison to the

University of Florida, and for his strong support of my application to the graduate

program. This project was supported by a grant from Harbor Branch Oceanographic

Institution and Florida Fish and Wildlife Commission through the Marine Mammal

Animal Health Program of the College of Veterinary Medicine at the University of

Florida, and would not have been possible without the contributions of collaborators

affiliated with numerous zoological parks, aquariums, stranding networks, and the Alaska

Department of Fish and Game. In particular, great thanks are extended to Dr. Kathy

Burek, Dr. Cheryl Rosa, Dr. Ruth Ewing, Dr. Forrest Townsend, Dr. Charles Manire, Dr.

Gay Sheffield, Dr. Jeremiah Saliki, Dr. Kimberlee Beckman and Mr. Bob Schoelkopf for

collecting and contributing the wealth of samples that make this work possible. I would

also like to acknowledge my fellow lab-mates, Kara Smolarek-Benson, Rebecca

Woodruff, Rebecca Grant, and Shasta McClenahan for their friendship, assistance and

open ears throughout our time together. Last but not least, I would like to thank my

family for all of their support, particularly my mother Camille, who left me all of her









love, strength and persistence. I am exceptionally grateful to my husband, Ethan, for his

unending support and patience during these long two years, for coming along with me in

the pursuit of my dreams, for putting up with all of the time we have had to spend apart

and for making the times we spend together wonderful.















TABLE OF CONTENTS

page

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

LIST O F TA B LE S ...................... ............ ............. ............. viii

LIST OF FIGURES ................................... ...... ... ................. .x

ABSTRACT ........ .............. ............. .. ...... .......... .......... xii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Introduction to Poxviruses ................................................... ................... ......
Poxvirus Infections of C etaceans........................................................................... 7
Poxvirus Infections of Pinnipeds ...................................................... ..... .......... 11

2 M ATERIALS AND M ETHOD S ........................................ ......................... 14

Sam ple A acquisition ............................................. ...................... 14
Histopathology and Electron M icroscopy ...................................... ............... 15
Extraction of Total DNA .............. ............................... ...............15
G general Conditions for PCR ...................... .. ............................ .... ............... 16
Poxvirus PCR Targeting the DNA Polymerase Gene.................................. 16
Poxvirus PCR Targeting the DNA Topoisomerase I Gene..............................17
Poxvirus PCR Targeting the Major Envelope Gene................. ..................17
Poxvirus PCR Targeting the Hemagglutinin Gene of Orthopoxviuses ..............17
Parapoxvirus PCR Targeting the DNA Polymerase Gene .............................18
Parapoxvirus PCR Targeting the DNA Topoisomerase I Gene ..........................18
Parapoxvirus PCR Targeting the Major Envelope Protein Gene........................ 19
G el E lectrophoresis............... .......................................... ........ ... 19
Cloning of am plified DN A fragm ents .................................. ............ .................. 19
DN A Sequencing and Sequence A nalysis....................................... .....................20
Prim er Specificity and Sensitivity A ssays....................................... ............... 22
V irus Isolation .........................................................................23

3 R E S U L T S .......................................................................... 2 4

Sum m ary of Positive Sam ples ........................................................ ............. 24
Histopathology and Electron M icroscopy ...................................... ............... 24









Detection of Poxvimses Targeting the DNA Polymerase Gene..............................25
Detection of Poxviruses Targeting the DNA Topoisomerase I Gene.........................25
PCR Targeting the Maj or Envelope Protein Gene of Orthopoxviruses ...................26
PCR Targeting the Orthopoxvirus Hemagglutinin Gene................. ... ........... 26
Detection of Parapoxviruses Targeting the DNA Polymerase Gene.......................26
Detection of Parapoxviruses Targeting the DNA Topoisomerase I Gene..................27
Detection of Parapoxviruses Targeting the Major Envelope Protein Gene ...............28
Sequencing and G enetic A analysis ........................................ ......................... 28
DNA Polymerase .......... .... ........................ .. ...... 28
D N A Topoisom erase I................................................ ............................. 30
M ajor Envelope Protein G ene ..................................... ........................ .......... 32
Phylogenetic A analysis ...................... ................ ................. .... ....... 32
V irus Isolation .........................................................................33

4 D ISCU SSION ............................................................... ... .... ......... 66

L IST O F R E F E R E N C E S ......................................... .............................................82

BIOGRAPH ICAL SKETCH .......................................................... ............... 88















LIST OF TABLES

Table p

3-1 Pair-wise comparisons of the nucleotide sequences of the DNA polymerase
gene fragments of the cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2
(C P V -2) sam ples.. ......................................................................53

3-2 Pair-wise comparisons of the amino acid sequences of the DNA polymerase
gene fragments of the cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2
(C PV -2) sam ples.. ..................... .. ........................ .. .... ..... .. ........... 54

3-3 Pair-wise comparisons of the amino acid sequences of the DNA polymerase
gene fragments of the cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2
(C P V -2) sam ples.. ......................................................................55

3-4 Pair-wise comparisons of the nucleotide sequences of the DNA topoisomerase
gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-
2 ) sa m p le s ..................................................................... 5 6

3-5 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase
gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-
2 ) sa m p le s ..................................................................... 5 7

3-6 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase
gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-
2) sam ples. ........................................................ ................. 58

3-7 Pair-wise comparisons of the nucleotide sequences of the DNA polymerase
gene fragments of poxviruses of various genera within Chordopoxvirinae............59

3-8 Pair-wise comparisons of the amino acid sequences of the DNA polymerase
gene fragments of poxviruses of various genera within the Chordopoxvirinae
subfam ily of viruses. ...... ................... ........... ..................... .................. 60

3-9 Pair-wise comparisons of the amino acid sequences of the DNA polymerase
gene fragments of poxviruses of various genera within the Chordopoxvirinae
subfamily of viruses. ........................... ......... ................ .. ............... 61

3-10 Pair-wise comparisons of the nucleotide sequences of the DNA topoisomerase
gene fragments of poxviruses of various genera within Chordopoxvirinae............62









3-11 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase
gene fragments of poxviruses of various genera within Chordopoxvirinae ..............63

3-12 Pair-wise comparisons of the amino acid sequences of the DNA topoisomerase
gene fragments of poxviruses of various genera within Chordopoxvirinae............64

3-13 Pair-wise comparisons of the nucleotide sequences obtained from the major
envelope protein gene fragments of marine parapoxviruses within
Chordopoxvirinae ........... ...... .......... ....... .. ...... .... ..... ..............65

3-14 Pair-wise comparisons of the amino acid sequences from the major envelope
protein gene fragments of marine parapoxviruses within Chordopoxvirinae ..........65

3-15 Pair-wise comparisons of the amino acid sequences of the major envelope
protein of marine parapoxviruses................... ..... ... .. ................ ..65
















LIST OF FIGURES


Figure pge

3-1 Typical "tattoo" lesions of cetaceans ........................................... ............... 33

3-2 Gross appearance of pox lesions associated with a poxvirus in a Steller sea lion
(Eum etopias jubatus). ............................................ ...... ...... ......34

3-3 Cutaneous pox lesions in a spotted seal (Phoca largha) associated with spotted
seal parapoxvirus..........................................................................................34

3-4 Histopathologic appearance of cutaneous lesions associated wtih Steller sea lion
poxvirus..................................... .................. .............. ........... 35

3-5 Negatively stained poxvirus particle from cutaneous lesion of SSL observed by
electron m icroscopy.. ....................................... ........ ...............35

3-6 Agarose gel electrophoresis of PCR amplified 543-546-bp fragments of the
DNA polymerase gene of cetacean and Steller sea lion poxviruses. ....................36

3-7 Agarose gel electrophoresis of PCR amplified 344-bp fragments of the DNA
topoisomerase gene of cetacean and Steller sea lion poxviruses ...........................36

3-8 Agarose gel electrophoreses of PCR amplified fragments of the HA gene of
orthopoxviruses ................................. ............... .. ............37

3-9 Agarose gel electrophoresis demonstrating the PCR amplification of 536-bp
parapox DNA polymerase gene fragments from lesions of different pinniped
sp ecies. .............................................................................37

3-10 Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
prim ers CR540 and CR541 .............................................................................. 38

3-11 Agarose gel electrophoresis demonstrating the PCR amplification of 350-bp
parapox DNA topoisomerase I gene fragments from lesions of Steller sea lions
(Eumetopias jubatus) and harbor seals (Phoca vitulina) .......................................38

3-12 Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR550 and CR551 ........................................... .................... 39









3-13 Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
prim ers CR557 and CR558 ............................................................................. 39

3-14 Agarose gel electrophoresis demonstrating the PCR amplification of 252-bp
parapox virus DNA topoisomerase I gene fragment from lesions of spotted
seals (Phoca largha).................. ...................................... .. ............ 40

3-15 Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR570 and CR571. ........................................... .................... 40

3-16 Agarose gel electrophoresis demonstrating the PCR amplification of 594-bp
parapox major envelope protein gene fragments from lesions of different
pinniped species.. ....................................................................... 4 1

3-17 Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
prim ers CR 339 and CR 340. ............................................ ............................. 41

3-18 Multiple alignment of the amino acid sequences deduced from the nucleotide
sequences of the DNA polymerase gene fragments of poxviruses identified in
cutaneous lesions of cetaceans.. ...............................................................................42

3-19. Multiple alignment of the amino acid sequences deduced from the nucleotide
sequences of the DNA polymerase gene fragment of poxviruses identified in
cutaneous lesions of pinnipeds. ....... ......................................... .....................43

3-20 Multiple alignment of the amino acid sequences deduced from the nucleotide.
Sequences of the DNA topoisomerase gene fragments of poxviruses identified
in cutaneous lesions of cetaceans........................................ ....... .. ......... ...... 44

3-21 Multiple alignment of the amino acid sequences deduced from the nucleotide
sequences of the DNA topoisomerase gene fragment of poxviruses and
parapoxviruses identified in cutaneous lesions of pinnipeds ...............................45

3-22 Multiple alignment of the partial amino acid sequences predicted from the
major envelope protein gene fragment of parapoxviruses identified in
cutaneous lesions of pinnipeds. ....... ......................................... .....................46

3-23 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the
DNA polymerase gene fragments from members of the Chordopoxvirinae
subfam ily of poxviruses.. ................... ...... .............................................. ...... ...47

3-24 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the
DNA topoisomerase gene fragments from members of the Chordopoxvirinae
subfam ily of poxviruses.. ................. ... ........................................................... 49

3-25 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the
Major envelope protein gene fragments from members of the Chordopoxvirinae
subfam ily of poxviruses.. ................................ ................................. ....................... 51














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

DETECTION AND MOLECULAR CHARACTERIZATION OF CETACEAN AND
PINNIPED POXVIRUSES ASSOCIATED WITH CUTANEOUS LESIONS

By

Alexa Justine Bracht

August 2005

Chair: Carlos H. Romero
Major Department: Veterinary Medicine

Poxviruses are widespread and successful pathogens, known to infect a variety of

vertebrates including, reptiles, birds, and over 30 species of mammals and several species

of insects. Terrestrial poxviruses encompass a variety of well known etiologic agents,

that are currently classified into eight genera, within the Chordopoxvirinae subfamily.

While significant advances have been made in understanding the genomic sequences of

terrestrial poxviruses, little is known about marine poxviruses. DNA extracted from skin

lesions of cetaceans in oceanaria and rehabilitation facilities as well as free-ranging

cetaceans and pinnipeds was assayed by polymerase chain reaction (PCR). Primers were

designed to target gene fragments of three genes: DNA polymerase (DNApol), DNA

topoisomerase (DNAtopo) and the major envelope protein (MEP) of poxviruses and

parapoxviruses based on numerous DNA sequences available in the National Center for

Biotechnology Information (NCBI) database. Targeting of the poxvirus DNApol gene

yielded 543-bp fragments when swinepox (SPV) and mule deer poxvirus (MDPV) DNA









were used as templates. Targeting of cetacean poxvirus (CPV) DNA yielded 546-bp

amplicons that upon sequencing revealed the existence of two distinct poxvirus

sequences that were shown to be approximately 84% and 89% identical in their

nucleotides and amino acid sequences, respectively. These findings provide the first

evidence of activity of at least two poxviruses in cetaceans, that we provisionally refer to

as cetacean poxvirus -1 and 2 (CPV-1 and CPV-2). Amplification of Steller sea lion

(Eumetopias jubatus) poxvirus (SSLPV) DNA yielded a 543-bp DNApol gene fragment

with nucleotide identity ranges of 76-77% and amino acid identity ranges of 74 -78%

when compared to homologous CPV-1 and CPV-2 fragments. Analyses of CPV-1

DNApol fragments showed closest amino acid identity to members of the orthopox genus

(-81%), while CPV-2 had identities of -83%. DNApol fragments amplified from

parapox viruses from Steller sea lions, spotted seals (Phoca largha) and a harbor seal

(Phoca vitulina) were 536-bp in length and had closest amino acid identities to members

of the parapox genus (87-89%). PCR with poxvirus consensus primers that targeted the

DNAtopo gene generated 344-bp amplicons using CPV-1, CPV-2, SSLPV, SPV and

MDPV DNA as templates. Parapox DNAtopo fragments were amplified from the same

set of pinnipeds and were 347, 350 and 252-bp in length. Consensus primers that target

the MEP gene of parapoxviruses amplified 594-bp fragments from pinniped para-

poxviruses, as well as from pseudocowpox virus DNA. The described molecular assays

based on PCR and direct sequencing of amplicons have allowed us to identify several

novel poxviruses and investigate the evolutionary relatedness of these viruses when

compared to other well known terrestrial poxviruses of vertebrates in the subfamily

Chordopoxvirinae.














CHAPTER 1
INTRODUCTION

Introduction to Poxviruses

The family Poxviridae contains the largest known viruses of terrestrial and marine

mammals that possess non-infectious, double stranded DNA genomes that range in size

from 130-380 kbp and replicate almost exclusively in the cell cytoplasm (Moss, 2001).

The basic poxvirus virion incorporates about 100 polypeptides and carries most of the

compounds necessary for replication in the host cell. The non-infectious DNA genome

(nucleosome) is arranged inside a core membrane that is surrounded by lateral bodies and

another outer membrane. Poxviruses encase this outer membrane in yet another lipid

bilayer, called the envelope, which functions in host cell attachment (Buller and

Palumbo, 1991; Moss, 2001). The Poxviridae is divided into two subfamilies:

Entomopoxvirinae, that comprises insect poxviruses, and Chordopoxvirinae, that includes

all poxviruses of vertebrates (Moss, 2001). Subfamily Chordopoxvirinae includes eight

genera: orthopoxvirus, parapoxvirus, capripoxvirus, suipoxvirus, leporipoxvirus,

yatapoxvirus, avipoxvirus and molluscipoxvirus. Poxviruses are highly adapted viruses

infecting a large number of hosts, including insects, reptiles, birds, and over 30

mammalian species (Buller and Palumbo, 1991; Moss, 2001; Upton et al., 2003). Some

members of the Orthopox genus, such as variola, the causative agent of smallpox virus,

are endowed with high virulence and were a scourge to mankind for at least two

millennia until its eradication in 1977 (Moss, 2001). Highly invasive and virulent

poxviruses of livestock of the Capripoxvirus genus, such as sheep pox, goat pox and









lumpy skin disease viruses, and of the Orthopox genus, such as camel poxvirus, are

currently restricted to some parts of the sub-Saharan African continent, the Middle East

and the Indian subcontinent (Ireland and Binepal, 1998; Moss, 2001). Poxviruses

replicate in the skin and mucosa producing localized or generalized lesions of variable

gravity and duration (Buller and Palumbo, 1991). Localized lesions are seen in

Molluscum contagiosum, pseudocowpox, and orf virus infections, whereas disseminated

lesions are seen in ectromelia, cowpox, monkeypox, and the well known variola virus.

(Moss, 2001; Upton et al., 2003). Some parapoxviruses and myxoma virus have the

ability to display both disease patterns depending on the animal host (Buller and

Palumbo, 1991).

This study, fueled by the generation of genetic data, would not have been possible

without the fundamental technique, now implemented in almost every molecular genetics

laboratory, the polymerase chain reaction, or PCR. Conceived by Kary Mullis in 1983

(Mullis et al., 1986), the idea revolutionized the fields of biotechnology and molecular

biology, paving the way for development of new assays to diagnose medical disorders

and a wide range of diseases (Schluger and Rom, 1995). PCR uses simple principles of

DNA replication combined with the unique properties of a DNA polymerase from

thermophilic bacteria to mimic the DNA replication process in-vitro, exponentially

amplifying copies of the targeted DNA (Bej et al., 1991). While the uses of PCR span

the range of medical diagnoses, the relevance of this technique to virology is its

facilitation of cloning and sequencing of viral genes for the purposes of viral comparison

and classification (Bej et al.,, 1991; Ropp et al., 1995; Elnifro et al., 2000). Prior to PCR

technology, viruses were isolated in cell culture before any further analyses could occur









(Levine, 2001). Identification and differentiation of viruses before the implementation of

PCR relied on less specific serologic assays such as, virus neutralization,

hemagglutination and immune fluorescence assays, later complemented by restriction

endonuclease profiles of viral DNA resolved in agarose or polyacrylamide gels (Fleming

et al., 1993; Robinson and Mercer, 1995; Ropp et al., 1995; Mangana-Vougiouka et al.,

1999; Moss, 2001). These techniques require large amounts of viral DNA typically

harvested from virus infected cultures. This approach hindered the study of viruses that

do not readily grow in culture, such as papilloma viruses, or for that matter, poxviruses of

marine mammals. The advent of PCR allowed for the direct amplification of viral DNA

and rapid genome sequencing (Moss, 2001). With the sudden accumulation of genome

sequences from numerous viruses representative of most families, detailed examination

of genetic relationships escalated coupled with a new understanding of viral taxonomy.

For example, upon genetic comparison, it was found that two genetic mutations were

responsible for the antigenic difference between feline panleukopenia virus and canine

parvovirus (Levine, 2001). Since the late 1980's, PCR protocols have been developed to

detect a wide variety of human and animal viruses representing several viral families and

genera including: hepatitis, papilloma, influenza, rhabdo-, retro-, herpes-, calici-, adeno-

and pox-viruses, among numerous others (De Rossi et al., 1988; Puchhammer-Stockl et

al., 1990; Vandenvelde et al., 1990; Sacramento et al., 1991; De Leon et al., 1992; Hondo

and Ito, 1992; Morishita et al., 1992; Vesy et al., 1993; Ropp et al., 1995; Heredia et al.,

1996; Vantarakis and Papapetropoulou, 1999; Inoshima et al., 2000). Considering

poxviruses in particular, PCR and genome sequencing has meant the evolution from

sometimes vague histopathologic and electron microscopic (EM) diagnoses to much









more definitive genetic assays for poxvirus infection (Fleming et al., 1993; Ropp et al.,

1995; Mangana-Vougiouka et al., 1999; Damaso et al., 2000; Gubser and Smith, 2002;

Howsamani et al., 2004) Most poxviruses share a common ovoid or brick-like shape and

measure 200 400 nm in length with tubules arranged in an irregular pattern on the

envelope surface (Buller and Palumbo, 1991; Moss, 2001). The exception to this is the

genus parapox, which has a notable criss-cross tubule pattern on the envelope surface

(Moss, 2001). Because of this common poxvirus morphology, it is difficult to discern

between genera of poxviruses when using techniques like histopathology and EM.

However, PCR and sequencing methods reveal not only the genus, but in most cases,

species of the virus being examined (Ropp et al., 1995, Damaso et al., 2000; Becher et

al., 2002; Howsamani et al., 2004). Beginning in 1990 with the sequence for vaccinia

virus- Copenhagen strain (Goebel et al., 1990), complete poxvirus genomes sequences

have been generated using techniques derived from PCR, restriction endonuclease

digestion, and basic DNA cloning and sequencing. The generation of complete genome

sequences in pox virology reached a climax in the years 2000-2002 with the release of 14

complete genome sequences representing six genera within the sub-family

Chordopoxvirinae and one in sub-family Entomopoxvirinae (Gubser et al., 2004).

Included in these were complete genome sequences of variola, vaccinia, monkeypox,

camelpox, fowlpox, lumpy skin disease virus, goatpox, sheeppox, swinepox, and Yaba-

like disease virus, as well as, Amsacta moorei entomopoxvirus (Goebel et al., 1990;

Shchelkunov et al., 1995; Antoine et al., 1998; Shchelkunov et al., 2000; Tulman et al.,

2001; Afonso et al., 2002; Gubser and Smith, 2002; Gubser et al., 2004). These

sequencing advances have allowed for a better ability to define and understand the









evolutionary relationships between the different poxvirus genera. Comparing genes that

have been identified as highly conserved can aid in new virus characterization and

comparison. Attempts at obtaining a more global understanding of poxvirus genes and

gene families have been made using the Poxvirus Bioinformatic Resouce (PBR;

www.poxvirus.org) and have identified 49 conserved gene families in 21 complete

poxvirus genomes (Upton et al., 2003). Previous studies have elected phylogenetic

analysis to be the best tool available for characterizing poxviruses known to date

(Afonso et al., 2002; Becher et al., 2002, Gubser et al., 2004). These studies present an

easily comprehendible picture of the ancestral lines of viruses, including possible

progenitors for different lineages. The increase in numbers of available sequences

permitted attempts to create a more global understanding of poxvirus genetic

relationships, mainly through the use of evolutionary analysis. As different software

programs emerged and improved over time, numerous phylogenetic analyses have been

conducted, initially on single genes or gene fragments (Zanotto et a., 1996; Afonso et al.,

2002; McGeoch et al., 2000; Gubser and Smith, 2002; Hosamani et al., 2004; Tryland et

al., 2005). Phylogenetic trees constructed using different individual proteins can yield

varying topologies, depending on the stringency of conservation of the DNA sequence

for the specific gene (Gubser et al., 2004). More recently, phylogenetic studies using

large fragments of the central region of the genome or the complete genome sequences

have been published, revealing a more complete and accurate picture of poxvirus

phylogeny (Fleming et al., 1993; Upton et al., 2003; Delhon et al., 2004; Gubser et al.,

2004,). The results of these studies served to verify what had been previously accepted in

taxonomic classification of poxviruses by designating the viruses into groups that









corresponded with the accepted poxvirus genera. Gubser et al. (2004) used the sequences

of 26 poxviruses representing all poxvirus genera except parapoxvirus, in a phylogenetic

study. They found that the general organization of the Chordopoxvirinae genome was

conserved, specifically in regard to the central region genes encoding proteins for RNA

and DNA replication, virion assembly and structural proteins. This concurs with a

previous study by Upton et al. (2003), where authors performed large scale genetic

analysis on 21 complete poxvirus genomes. These authors used the Virus Genome

Database to identify genes that are most highly conserved among the family Poxviridae,

and determined that those genes were involved in DNA replication and transcription.

Gubser et al. (2004) used these results to construct a tree incorporating 17 of the 49

proteins that are conserved in all poxviruses, and found that the viruses included in the

subfamily Chordopoxvirinae could be divided into four main groups: the Molluscipox

genus, the Avipox genus, the Orthopox genus, and a group containing the Yatapox,

Capripox, Suipox and Leporipox genera. The tree suggests that the latter group includes

viruses evolved more recently, and thus are genetically more related to each other than to

the first three groups. It should be noted that the parapoxvirus genus was excluded from

this study, and it is speculated that it might clad into its own group, if similar analyses

were repeated. Characterization of novel poxviruses such as muledeer poxvirus,

spectacled caiman poxvirus, Embu virus and Cantangalo virus that have not yet been

assigned to a genus, may be aided by these new techniques. Equally mysterious are the

poxviruses that have been known to infect various species of cetacea and pinnipedia.

Though often and easily recognized by clinical means, these viruses have not been

antigenically or molecularly characterized.









Poxvirus Infections of Cetaceans

Poxviruses have been known ( for over 30 years), to affect various cetacean and

pinniped species (Wilson et al., 1969; Wilson and Poglayen-Neuwall, 1971; Geraci et al.,

1979; Baker, J.R., 1992a,b; Baker and Martin, 1992; Van Bressem et al., 1993).

Numerous studies have reported skin lesions associated with poxvirus infections in

cetaceans and parapoxvirus infections in pinnipeds (Geraci et al., 1979; Osterhaus et al.,

1990; Baker and Martin, 1992; Van Bressem et al., 1993; Nettleton et al., 1995). In

cetaceans, poxvirus lesions are described as areas of hyperpigmentation of the skin with

pinhole marks, termed "tattoo" or "ring" lesions (Geraci et al., 1979; Van Bressem et al.,

1993). These lesions were reported to persist for months to years, and typically regress

without treatment (Geraci et al., 1979, Smith, 1983). The appearance of poxviruses

lesions in cetaceans seems different from that of poxvirus infections described in

terrestrial vertebrates (Geraci et al., 1979). Lesions associated with cetacean poxviruses

remain relatively flat, and in some advanced stages, form slightly dark depressions in the

center of the lesions (Geraci et al., 1979). Conversely, lesions associated with terrestrial

poxviruses may form raised nodules in the skin, and often advance to erupted pustules

(Robinson and Mercer, 1995; Damaso et al., 2000; Moss, 2001; Delhon et al., 2004).

Geraci etal. (1979), provided an explanation for the difference in clinical appearance and

progression of lesions which involves the unique metabolic and mitotic rate of the

epidermal cells of the cetacean integument. Further studies on the progression of the

disease associated with cetacean poxvirus infection have focused finding

histopathological changes with presence of intracytoplasmic inclusions and on

morphological characterization of the cetacean poxvirus using electron microscopy

(Smith, 1983; Van Bressem, 1993). While the prevalence and conditions that participate









or facilitate the occurrence of poxvirus infection in cetaceans have not been studied, Van

Bressem et al., (1993), reported 8.1% and 30% prevalence of tattoo lesions in 74 dusky

dolphins (Lagenorhynchus obscurus) and 10 Burmeister's porpoises (Phocoena

spinipinnis), respectively, that were examined as fishing by-catch in 1990. The true

prevalence of poxvirus infection in wild cetacean populations is unknown; however,

Geraci et al., (1979) suggested an association of the occurrence and severity of tattoo

lesions, with animals under considerable environmental stress or those exhibiting poor

general health. The authors cited specific cases involving captive dolphins afflicted with

lesions that improved under less stressful environmental conditions (Geraci et al., 1979).

In contrast to the abundance of sequence data for terrestrial poxviruses, even

though the occurrence of poxviruses in marine mammals has been well documented for at

least three decades (Wilson et al., 1969; Wilson and Poglayen-Neuwall, 1971; Geraci et

al., 1979; Baker, J.R., 1992a,b; Baker and Martin, 1992; Van Bressem et al., 1993),

almost no molecular data are available for marine poxviruses. Previous reports have

described the pathogenicity and the gross and microscopic lesions after the infection of

cetaceans and pinnipeds with marine poxviruses. Most of these infections were

diagnosed by demonstrating characteristic poxvirus particles by electron microscopy or

the presence of acidophilic intracytoplasmic inclusion bodies in sections of lesions by

light microscopy (Flom and Houk, 1979; Geraci et al., 1979; Smith, 1983; Baker, 1992a;

Van Bressem et al., 1993). The first published description of poxvirus infection in

captive and free-ranging cetaceans discussed ring and tattoo type lesions observed on

seven bottlenose (Tursiops truncatus) and one Atlantic white-sided dolphin

(Lagenorynchus acutus) (Geraci et al., 1979). Lesions were noted to occur most often on









the dorsal body, dorsal fin and flukes and pectoral flippers (Figure 3-1). Samples of the

lesions were examined by light microscopy and electron microscopy, which revealed

eosinophilic intracytoplasmic inclusions containing virus particles with typical poxvirus

morphology. The condition, termed "dolphin pox", was found to vary in time course,

severity and clinical appearance and recurrence. Although the author identifies a

poxvirus as the causative agent for the observed lesions, associations between the

environmental conditions, general animal health and stress level were also considered as

possible reasons for the variations in disease progression. Concurrent studies with

similar findings were reported in three more Atlantic bottlenose dolphins (Flom and

Houk, 1979). While the pox lesions in both studies are reported to exist without causing

any serious harm or consequence to the animals' health, one exceptional case was cited

describing a dolphin that died after developing generalized lesions (Flom and Houk,

1979). Smith et al. (1983) reported an observation of two distinct regression patterns of

the typical dolphin poxvirus tattoo lesions. The first regression pattern consisted of the

lesions becoming raised and edematous that, over time, became depressed and

disappeared. The second regression pattern occurred following lesion biopsies where the

lesions disappeared in zones surrounding the incision. Samples were taken from both

raised and typical flat tattoo lesions, reacted with dolphin sera and evaluated by

immunoelectron microscopy. Positive reactivity occurred between the sera and the raised

endematous lesions, but not with the flat tattoo lesion. The significance of this study is

two fold: it is the first documentation of antibody response to a poxvirus recovered from

dolphin lesions and secondly, it suggests the possibility of two antigenically different

poxviruses that cause lesions with dissimilar clinical appearance. Over the next ten









years, numerous reports of poxvirus infections in various cetacean species surfaced

(Baker, 1992a,b; Baker and Martin, 1992; Van Bressem et al., 1993). Cetacean species in

which infections with poxviruses have been previously reported include: Atlantic

bottlenose dolphin (Geraci et al., 1979; Flom and Houk, 1979), Atlantic white-sided

dolphin (Geraci et al., 1979), common dolphin (Delphinus delphis) (Britt and Howard,

1983), dusky dolphin (Lagenorynchus obscurus) (Van Bressem et al., 1993), striped

dolphin (Stenella coeruleoalba) (Baker, 1992a), white beaked dolphin (Lagenorhynchus

albirostris) (Baker, 1992a,b) and Hectors dolphin (Cephalorhynchus hectori) (Geraci et

al., 1979; Baker, 1992a, b; Van Bressem et al., 1993, 1999). Similarly, poxvirus

infections have previously been described in long finned pilot whales (Globocephala

melaena) (Baker, 1992a), killer whales (Orcina orca), Burmeister's porpoise (Van

Bressem et al., 1993), and harbor porpoises (Phocoena phocoena) (Baker, 1992a,b; Baker

and Martin, 1992; Van Bressem et al., 1993, 1999). Routine histological methods

continued to provide the best descriptions of microscopic changes in the lesions.

Examinations of poxvirus lesions of a Burmeister's porpoise (Phocoena spinipinnis)

utilized transmission electron microscopy to highlight an irregular arrangement of tubules

on the viral membrane, reminiscent of those seen in orthopoxviruses (Van Bressem et al.,

1993). This was the first attempt to characterize the virus in order to assign it to one of

the known genera. Little new information has been accumulated in respect to cetacean

poxviruses since the early 1990's. The growing availability of DNA sequencing

technologies has created opportunities to examine the genome of cetacean poxviruses,

and how they situate within the subfamily Chordopoxvirinae.









Poxvirus Infections of Pinnipeds

Poxvirus infections have also been well documented in several pinniped species

since 1969. These pinniped poxvirus lesions have a very different appearance from those

seen in cetaceans and are typically raised nodules in the skin (Figures 3-2 and 3-3).The

first report of seal pox described pox lesions occurring in California sea lions (Zalophus

californianus) (Wilson et al., 1969). Closely following that report was one describing an

epizootic of a proliferative skin disease among captive California sea lions (Wilson et al.,

1972). Histopathology and electron microscopy determined the causative agent to be a

poxvirus, and a survey was initiated and sent out to 120 addresses in an attempt to

understand more about the scope of this new virus (Wilson et al., 1972). Over the years

these methods continued to be employed in identifying sealpox infections of various

species including; harbor seals (Phoca vitulina) (Becher et al., 2002, Miller et al., 2003),

grey seals (Halichoerus grypus) (Hicks and Worthy, 1987; Osterhaus et al., 1990;

Simpson et al., 1994; Nettleton et al., 1995), California sea lions (Wilson et al., 1969),

South American sea lions (Otaria byronia) (Wilson and Poglayen-Neuwall, 1971),

Weddell seals (Leptonychotes weddellii) (Tryland et al.2005) and northern fur seals

(Callorhinus ursinus) (Hadlow et al., 1980).

Because of the similarities to orf and bovine popular stomatitis (BPSV) virion

morphology and lesion pathology, the seal poxviruses were designated as probable

members of the parapox sub-group (Esposito, 1991). While orf and BPSV were both

known to be transmissible to humans (Bowman et al., 1981; Meechan and Macleod,

1992; Delhon et al., 2004), the zoonotic potential of any marine mammal poxvirus was

unknown. In 1987, a case report described two seal handlers that developed lesions on

their hands similar to milker's nodules that occurred while working with grey seals with









typical seal pox lesions (Hicks and Worthy, 1987). Healing times varied and one handler

experienced relapses over the next several months. Negative staining of the virions from

both the seals and the handlers suggest that the handlers' nodular lesions were caused by

the seal pox virus.

The lesions described in South American sea lions were distinct from those

previously described in California sea lions and harbor seals (Wilson and Poglayen-

Neuwall, 1971). The described lesions in California sea lions and harbor seals proliferate

outward, but the lesions of the South American sea lions proliferate downward into the

dermal layer. The intracytoplasmic inclusion bodies differed in morphology being large

and oval shaped versus small and irregular. In addition, the virion of the South American

sea lion pox virus appeared rectangular or brick shaped, versus the elongated or

cylindrical shape normally associated with previous reports of seal pox virus shape. The

results of this report suggested the existence of two poxviruses with the ability to infect

pinnipeds. Similar observations were made in the reexamination of old formalinized

samples from a stranded northern fur seal pup (Hadlow et al., 1980). Tissues preserved

from an animal that was necropsied in 1951 were examined for poxvirus and found to

resemble those reported in South American sea lions more than in California sea lions or

Harbor seals. The suggestion of the existence of two pinniped poxviruses resurfaced in a

report that outlined the isolation of both parapox and orthopox-like viral particles from

lesions of a grey seal (Osterhaus et al., 1990). The in vitro culture of the orthopox-like

virus was apparently, only possible in primary grey seal skin cells. However. no reports

on the characterization of this poxvirus has appeared since. A parapoxvirus was later









isolated from grey seal pox lesions using primary grey and harbor seal kidney cells

(Osterhaus et al., 1994, Nettleton et al.,1995).

The first mention of using the polymerase chain reaction (PCR) to test for pinniped

poxvirus infection surfaced in 2002 (Becher et al., 2002). The PCR primers used were

known to direct the amplification of a segment of the major envelope protein gene and

had been reported as a diagnostic tool for parapox infections of cattle, sheep and Japanese

serows (Inoshima et al., 2000). Skin lesions from harbor seals were analyzed for

parapoxvirus infection. Nucleotide and amino acid sequences obtained from the DNA

sequence of the amplified PCR fragments were compared against those of BPSV,

pseudocowpox virus (PCPV), parapoxvirus of red deer in New Zealand (PVNZ) and orf

virus (OV) and found to be significantly different in both cases: <79% nucleotide identity

and <77% amino acid identity. The authors suggested that the seal parapoxviruses

constituted a separate species within the genus Parapoxvirus (Becher et al., 2002).

Presently, "sealpox" is classified as a tentative member of the parapox genus. It is

evident that, while many advances have been made since the early days of poxvirus

detection in marine mammals, much is still unknown about the genomic organization and

evolutionary relationships of these viruses.














CHAPTER 2
MATERIALS AND METHODS

Sample Acquisition

Fresh and frozen skin lesions from 109 stranded, free-ranging and captive marine

mammals were harvested and shipped to our laboratories between January, 2001 and

March, 2005 for analyses of poxvirus infection. Lesion scrapings and biopsies from

captive marine animals were provided by several amusement parks and aquariums from

Florida, Texas, Portugal, and Hong Kong. Tissues from stranded and free-ranging

animals were obtained from numerous participants of the Southeast, Northeast and

Alaska Stranding Networks, as well as the Alaska Department of Fish and Game. All

samples collected from stranded marine mammals were obtained by licensed personnel

from the Networks. These lesions were obtained from 92 cetaceans and 17 pinnipeds.

Donor species were: Forty-two Atlantic bottlenose dolphin (Tursiops truncatus), twenty-

two bowhead whales (Balaena mysticetus), seven Indopacific bottlenose dolphin

(Tursiops aduncus), four rough-toothed dolphin (Steno bredanensis), four pygmy sperm

whales (Kogia breviceps), two killer whales (Orcina orca) (Dover, 1992), two short-

finned pilot whales (Globicephala macrorhynchus), three Risso's dolphin (Grampus

griseus), one striped dolphin (Stenella coeruleoalba), one Pacific white-sided dolphin

(Lagenorhynchus obliquidens), one dwarf sperm whale (Kogia sima), one spinner

dolphin (Stenella longirostris), one Pantropical spotted dolphin (Stenella attenuata), one

Harbor porpoise (Phocoena phocoena), fourteen Steller sea lions (Eumetopias jubatus),

two spotted seals (Phoca largha), and one harbor seal (Phoca vitulina).









Histopathology and Electron Microscopy

A 6-mm punch biopsy was taken of two Steller sea lion skin lesions. One half of

the biopsy was placed in 10% neutral buffered formalin and the other half frozen in dry

ice and stored at -700 C for DNA extraction and PCR analysis. Formalin fixed samples

were embedded in paraffin, sectioned at 5 rm, and stained with hematolyxlin and

eosinophilic for evaluation by light microscopy. Negative staining electron microscopy

was also performed on formalin-fixed specimens. The samples were homogenized in

distilled water in a Ten-Broeck grinder, clarified by centrifugation at 4,000xg for 5 min,

the supernatant removed to a clean tube and centrifuged at 12,000xg for 1 hr. The pellet

was resuspended in 2% phosphotungstic acid solution at pH 6.8 containing 0.01% bovine

serum albumin and a drop of this suspension was applied to a carbon coated formvar film

on a 400 mesh copper grid and the excess wicked away. The grid was examined with a

Zeiss EM 109 microscope (Carl Zeiss, Inc., Thornwood, New York, USA).

Extraction of Total DNA

All samples were processed to obtain total DNA using the DNeasy kit (Qiagen,

Valencia, California, USA) according to the protocol indicated by the manufacturer.

Briefly, 25 mg of tissue was cut into small pieces and combined with 180 ul of lysis

buffer ATL and 20 ld of proteinase K. The tissues were incubated at 550C until lysis was

complete. DNA was precipitated by the addition of 200 [l absolute ethanol and spun

through the DNeasy Spin Column. After two washes with buffers AW1 and AW2, the

DNA was eluted in 200 [l buffer AE. The quality and content was evaluated by

spectrophotometry using the Ultrospec 3000 (Amersham Biosciences Corp., Piscataway,

New Jersey, USA). Each group of tissue samples was extracted along with a known

negative sample to be used as a negative control for analysis.









General Conditions for PCR

Reaction tubes for PCR contained 200 nM of each primer, 100 gM of each

deoxynucleoside triphosphate (dNTP), 10 mM KC1, 10 mM (NH4)2 SO4, 20 mM Tris-

HC1, 2 mM MgSO4, 0.1% Triton X-100 at pH 8.8, 1 unit of Taq DNA polymerase (New

England BioLabs, Beverly, Massachusetts, USA), and 500 ng of DNA template, in a final

volume of 50 pl. All PCR cycling were performed in a PTC-100 thermal cycler (MJ

Research, Inc., Waltham, Massachusetts, USA). Cycling conditions for the amplification

of the DNA polymerase and DNA topoisomerase gene fragments of poxviruses were:

Initial denaturation at 940C for 1 min, followed by 39 cycles, each comprising of a

denaturation step at 940C for 30 sec, an annealing step at 450C for 30 sec, and an

elongation step at 720C for 30 sec. The last cycle included an extended elongation step at

720C for 10 min. Cycling conditions for the amplification of the DNA polymerase gene

of parapoxviruses were similar, except that the annealing temperature for the

parapoxviruses was 61C. The cycling conditions for the amplification of the DNA

topoisomerase gene fragments of parapoxviruses from Steller sea lions, harbor seals and

spotted seals were also similar; however, the annealing temperatures were 53C, 5 1C

and 58C, respectively.

Poxvirus PCR Targeting the DNA Polymerase Gene

Oligonucleotide primers that target sequences within the DNA polymerase gene

were designed based on sequences of lumpy skin disease virus (LSDV) and swinepox

virus (SPV) deposited in the GenBank database and from mule deer poxvirus (MDPV)

sequenced in our laboratories (Accession number AY841895). These sequences were:

Forward primer CR 422: 5'- ATA CAG AGC TAG TAC ITT AAT AAA AG 3'and









reverse primer CR 421: 5'- CTA TTT TTA AAT CCC ATT AAA CC 3'. MDPV or

SPV DNA was used as a positive control, yielding DNA fragments of 543 base pairs

(543-bp) in size. Negative tissues were used as negative controls.

Poxvirus PCR Targeting the DNA Topoisomerase I Gene

Oligonucleotide primers were designed based on the sequences of homologous

genes ofLSDV, SPV and MDPV (AY841896). The primer sequences were: CR 432: 5'-

TAA TGG AAA CAA GTT TTT TTA T 3' and CR 433: 5'- CCA AAA ATT ATA

TAA AAA CG 3'. These primers directed the amplification of a 344-bp DNA

fragment when SPV and MDPV genomic DNA was used as a positive control. Negative

tissues were used as negative controls.

Poxvirus PCR Targeting the Major Envelope Gene

Oligonucleotide primers were designed based on the sequences of vaccinia,

camelpox, monkeypox, variola, ectromelia and cowpox. Two forward primers were

designed, the first one included the gene start codon, and the second was 42-bp internal to

the start codon. The two forward primer sequences were: CR 597: 5' ATG TGG CCA

TTT RYA TCR GY -3' and CR 598: 5' CTG GTA GAA ACA CTA CCA GAA AAT -

3'. The reverse primer sequence include the stop codon and was designed as follows:

CR596: 5'- TTA AAT TTT YAA CGA TTT ACT GTG GC -3'. The expected sizes of

the fragments generated by these primers were 1118-bp and 1076-bp respectively

Vaccinia virus DNA was used as a positive control. Negative tissues were used as

negative controls..

Poxvirus PCR Targeting the Hemagglutinin Gene of Orthopoxviuses

PCR primers were designed to target the Hemagglutinin (HA) gene of

orthopoxviruses based on the sequences of camelpox, vaccinia, monkeypox, cowpox,









variola, and ectromelia. The primers target the full HA gene and predict the

amplification of a 1138-bp fragment from orthopox viruses. The primers were forward

primer CR 619: 5'- GAT TTT CTA AAG TRY TTG GAR AGT TTT AT- 3' and reverse

primer CR620: 5'-GCT GTC TTT CCT IAA CCA GAT G -3'. Vaccinia virus DNA was

used as a positive control. DNA extracted from a negative tissues and a negative tube

containing no DNA were used as negative controls.

A previously described set of primers was also used to amplify the HA gene

sequence of orthopoxviruses (Damaso et al., 2000).

Parapoxvirus PCR Targeting the DNA Polymerase Gene

Oligonucleotide primers that target genomic sequences within the DNA polymerase

gene of parapoxviruses were designed based on genomic sequences of orf (NC_005336)

and bovine popular stomatitis (NC_005337) viruses that exist in the GenBank database.

These primer sequences were: CR 541: 5'- GCG AGC ACC TGC ATC AAG 3'; CR

540: 5'- CTG TTI CGG AAG CCC ATG AG 3'. Pseudocowpox virus DNA was used

as a positive control. Negative tissues were used as negative controls..

Parapoxvirus PCR Targeting the DNA Topoisomerase I Gene

Oligonucleotide primers were first designed based on the nucleotide sequences of

the orf (NC_005336) and bovine popular stomatitis (NC_005337) virus DNA

topoisomerase gene sequences from the GenBank database. These primers were: CR

550: 5' TCA TGG AGA CSA GCT TCT TCA TC 3'(forward); CR 551: 5'- CCA

GAA GTT GTA CAR RAA SGT GTA G 3'(reverse). This primer set, however, did

not amplify parapoxvirus sequences from DNA extracted from lesions of all species of

marine mammals tested. Thus, a second primer set was designed based on sequences

obtained from the Steller sea lion parapoxvirus DNA topoisomerase gene fragment. The









primer sequences were: CR 557: 5' TCA TGG AGA CGA GCT TCT TCA TC -

3'(forward); CR 558: 5' CCA GAA GTT GTA CAA GAA GGT GTA G 3'(reverse).

As these two sets of primers still did not amplify parapoxvirus DNA from spotted seals, a

third set of primers had to be designed after performing a line up between the Steller sea

lion and harbor seal parapoxviruses DNA topoisomerase gene fragments. These primer

sequences were: CR 570: 5' GTC YTT AAC GCG AAT RCC AAA GC 3'(forward);

CR 571: 5'- AGC GGM ACW GTK GGY TTG CTC AC 3'(reverse). Pseudocowpox

virus DNA was used as a positive control. Negative tissues were used as negative

controls..

Parapoxvirus PCR Targeting the Major Envelope Protein Gene

PCR was performed using previously published consensus primers known to target

the major envelope protein gene of parapoxviruses (Inoshima et al., 2000). These

primers were: FP-PPP-4: 5'- TAC GTG GGA AGC GCC TCG CT-3'(forward); RP-PPP-

1: 5'-GTC GTC CAC GAT GAG CAG CT-3'(reverse). This primer set directs the

amplification of a 594-bp DNA fragment. Pseudocowpox virus DNA was used as a

positive control. Negative tissues were used as negative controls..

Gel Electrophoresis

Amplified DNA fragments were resolved by horizontal electrophoresis of 20-30 tl

of the PCR product in 1.0% agarose containing ethidium bromide (0.5 [tg/ml), visualized

under ultraviolet light and photographed using a gel documentation system (Bio-Rad

Laboratories, Inc., Hercules, California, USA).

Cloning of amplified DNA fragments

To obtain the complete nucleotide sequence of all amplified DNA fragments, these

were cloned into the bacterial plasmid vector pCR 2.1 TOPO TA (Invitrogen, Carlsbad,









California, USA) following the manufacturer's protocol. Competent E. coli DH5 alpha

cells were transformed with vector-insert reactions and streaked on 2XYT agar medium

containing ampicillin (100 [tg/ml). Colonies were grown overnight as minicultures, in 3

ml of 2XYT medium containing ampicillin (100 [tg/ml), while shaken at 270 rpm at

37C. Plasmid DNA was extracted from 1.5 ml of the minicultures using a phenol-free

method (Zhou et al., 1990). To screen for recombinant plasmids, plasmid DNAs were

digested with restriction enzymes HindIII, EcoRI, Apal, BamHI, and a combination of

enzymes Apal and BamHI. Recombinant plasmids containing the correct insert were

purified for sequencing using the AurumTM Plasmid Mini Kit or the Plasmid Midi-Prep

Kit (Bio-Rad Laboratories Inc., Hercules, California, USA) according to the

manufacturer's protocol. In brief, this involved first pelletting bacteria from 1.5 ml of

bacterial culture by centrifugation at 13,000 rpm for 30 seconds and then resuspending

and lysing the pellet in the supplied buffer. A neutralization buffer was then added, and

the cell debris was pelleted via centrifugation at 13,000 rpm for 10 minutes. The cleared

supernatant was harvested and spun through the supplied column and washed with the

wash solution provided. Purified plasmid DNA was eluted in 50 tl of elution solution,

also provided in the kit.

DNA Sequencing and Sequence Analysis

Amplified DNA fragments that were strong and uncontaminated with other

fragments as observed after gel electrophoresis were purified using the Wizard SV Gel

and PCR Clean-up System (Promega Corporation, Madison, Wisconsin, USA). This

protocol involved adding an equal volume of membrane binding solution to the PCR

product and purifying the DNA by centrifugation through the supplied column. The









column was washed with wash solution twice, and the DNA was eluted in 50 ul of

nuclease free water, quantified by spectrophotometry, and sequenced directly. Between

50-100 fmol of purified PCR products were sequenced in duplicate in both directions

using specific forward and reverse primers and the proprietary chemistry for the CEQ

2000XL sequencing instrument (Beckman-Coulter Inc., Fullerton, California, USA).

Chromatograms were manually reviewed for potential misreadings using the Chromas

2.3 software (Technelysium Pty Ltd., Tewantin, Queensland, Australia) and exported into

the Seqed function of the University of Wisconsin Package Version 10.2 (Genetics

Computer Group GCG, University of Wisconsin, Madison, Wisconsin, USA).

Sequences were analyzed using the Gap, Translate and Lineup functions of this

software and assembled using SeqMan, SeqEd and MegAlign (DNAStar, Lasergene

software, Madison, Wisconsin, USA). The BLAST function of the National Center for

Biotechnology Information (NCBI) was used to identify poxvirus sequences most closely

related to those of marine mammal poxviruses. Neighbor-joining phylogenetic trees were

generated by PAUP 4.0 (Sinauer Associates, Sunderland Massachusette, USA) software,

using ClustalW slow and accurate function using Gonnet residue weight table, gap

penalty of 11 and gap extension penalty of 0.2. The trees were based on the amino acid

sequences deduced from the homologous DNA fragments of the DNA polymerase and

DNA topoisomerase genes from members of the Chordopoxvirinae subfamily of

poxviruses obtained from the GenBank repository through the NCBI website. The

GenBank accession numbers (in parentheses) for the viral sequences used in the genetic

analysis were: Lumpy skin disease (AF409137), sheeppox (NC_004002), goatpox

(AY077835), swinepox (NC_003389), canarypox (AY318871), cetacean poxvirus-1









(AY463004-AY463007), cetacean poxvirus-2 (AY846759, AY846760), fowlpox

(NC_002188), Steller sea lion pox (AY424954, AY424955), harbor seal parapox

(AY952937-AY952939, AF414182), spotted seal parapox (AY780676, AY780677,

AY780678), Steller sea lion parapox (AY952940-AY952984), Weddel sealpox

(AJ622900), camelpox (AF438165), variola (NC_001611), rabbitpox (AY484669),

monkeypox (NC_003310), mule deer pox (AY841895, AY841896), vaccinia

(AY243312), ectromelia (NC_004105), pigeonpox (M88588), red deer parapox

(AB044794), cowpox (AF482758), Yaba monkey tumor (AY386371), rabbit myxoma

(NC_001132, AAF14910), rabbit fibroma (NC_001266), orf(NC_005336), bovine

popular stomatitis (NC_005337) and molluscum contagiosum (NC_001731).

Primer Specificity and Sensitivity Assays

The poxvirus DNA polymerase and DNA topoisomerase PCR assays were applied

to swinepox, pseudocowpox, muledeerpox, CPV-1, CPV-2, SSLPV, HSPPV, SSPPV and

SSLPPV DNA to determine primer specificity. Ten-fold serial dilutions ranging from

100 ng to 0.001 fg of pCRII-Topo 2.1 plasmid that contained the amplified 546-bp CPV-

1 DNA polymerase fragment or 344-bp DNA topoisomerase fragment were PCR

amplified using primer set CR421/CR 422 and primer set CR432/CR433, respectively, to

define the general sensitivity of these assays.

The parapoxvirus DNA polymerase, DNA topoisomerase, and major envelope

protein gene PCR assays were applied to pseudocowpox, CPV-1, CPV-2, SSLPPV,

HSPPV, and SSPPV DNA to determine the primer specificity. Ten-fold serial dilutions

ranging from 100 ng to 0.001fg of pCRII-Topo 2.1 plasmid containing the parapox DNA

polymerase, DNA topoisomerase, or major envelope protein gene fragments, were PCR









amplified using the respective primer set to determine the general sensitivity of the

assays.

Virus Isolation

Numerous attempts to isolate pox and parapox viruses from marine mammal skin

lesions were made. Fresh or frozen tissue sample were homogenized in a 2 ml glass

Tenbroeck tissue grinder. One, five and ten percent dilutions were made using

Dulbecco's modified medium (DMEM) containing antibiotic/antimycotic drugs. The

dilutions were clarified via centrifugation at high speed (13,000 rpm for 1 minute) to

reduce bacterial contamination. Tissue culture lines that were utilized in virus isolation

attempts included: African green monkey kidney (Vero), Madin- Darby canine kidney

(MDCK), Tursiops trucatus lung (TurtruLu), Tursips trucatus kidney (TurtruK), Phoca

vitulina ovary (PhovituOv), and Phoca vitulina lung (PhoVitLu). Dilutions made from

PCR positive cetacean and SSL pox skin lesions were innoculated onto Vero, MDCK,

TurtruLu and TurtruK cell cultures. Dilutions made from pinniped pox and parapox

lesions were innoculated on Vero, MDCK, PhovitO ad PhovitLu cell cultures. The

inoculum was allowed to adsorb onto the cell monolayers for 2-3 hours, after which the

monolayers were carefully rinsed with DMEM, fed with DMEM supplemented with 1.0

5.0% fetal bovine serum and then incubated 370C in an atmosphere of 5% CO2.

Innoculated and non-innoculated cultures were checked daily for cytopathic effects

(CPE), the maintenance medium was changed as needed, and discarded after 14 days, or

two passages if no CPE was observed.














CHAPTER 3
RESULTS

Summary of Positive Samples

Out of 109 fresh and frozen skin lesion samples tested, poxvirus positive results

were determined for 10 cetacean lesions including; four Indo-Pacific bottlenose dolphins,

two rough-toothed dolphins, one striped dolphin, two Atlantic bottlenose dolphin and one

bowhead whale (Figure 3-1). Three Steller sea lion skin samples also tested positive for

poxvirus (Figure 3-2). Assays for parapoxvirus yielded six positive results including

lesions from three Steller sea lions, two spotted seals, and one harbor seal (Figure 3-3).

Histopathology and Electron Microscopy

Skin lesions of two Steller sea lion pups were analyzed using histopathology and

electron microscopy. Histology revealed a mass lesion within the dermis composed of

large, polygonal epithelial cells. The mass was composed of broad cords of polygonal to

round epithelial cells with sharply delineated cytoplasmic borders. The nuclei were

consistent in size, round to oval with 1- 2 prominent nucleoli / nucleus, fine granular

chromatin and 0-4 mitotic figures/high power field. Some nuclei contained 1-2 clear

vacuoles. Many of these epithelial cells contained a single large brightly eosinophilic

inclusion body (Figure 3-4). Scattered lymphocytes, plasma cells and neutrophils were

present in the dermis surrounding the mass.

On electron microscopy, virus particles were smooth, rounded rectangles

approximately 350 x 270 nm consistent with published reports of orthopox viruses

(Moss, 2001) (Figure 3-5).









Detection of Poxviruses Targeting the DNA Polymerase Gene

Total DNA extracted from 10 cutaneous lesions from cetaceans and two lesions

from Steller sea lions contained poxvirus genomic DNA as evidenced by the

amplification of DNA polymerase gene fragments of the expected size. Positive donor

cetacean species were: Four Indo-Pacific bottlenose dolphins, two rough-toothed

dolphins, one striped dolphin, two Atlantic bottlenose dolphin and one bowhead whale.

Similarly, lesions harvested from three Steller sea lion pups also contained amplifiable

poxvirus DNA polymerase gene sequences (Figure 3-6). This PCR assay detected the

DNA polymerase gene fragments of muledeer poxvirus, swinepox virus, cetacean

poxvirus-1 (CPV-1), cetacean poxvirus-2 (CPV-2) and Steller sea lion poxvirus

(SSLPV), but did not amplify the DNA polymerase gene fragments of pseudocowpox,

Steller sea lion parapoxvirus (SSLPPV), harbor seal parapoxvirus (HSPPV), or spotted

seal parapoxvirus ( SSPPV). Serial ten-fold dilutions from 100 ng to 0.001fg of Topo 2.1

plasmid containing the CPV-1 DNA polymerase gene fragment were PCR amplified with

primers CR 421 and CR422. The minimal amount of CPV-1 DNA detected was 1.0 fg.

Detection of Poxviruses Targeting the DNA Topoisomerase I Gene

A total of seven lesions from cetaceans yielded positive PCR results when the

poxvirus DNA topoisomerase gene was targeted. Positive cetacean species were: Two

rough-toothed dolphins, two striped dolphins, one Indo-Pacific bottlenose dolphin, one

Atlantic bottlenose dolphin and one bowhead whale. DNA fragments corresponding in

size to the DNA topoisomerase gene fragments were also amplified from total DNA

extracted from lesions of three Steller sea lion pups (Figure 3-7). This PCR assay

detected the DNA topoisomerase gene fragments of muledeer poxvirus, Swinepox virus,

CPV-1, CPV-2 and SSLPV, but did not amplify the DNA topoisomerase gene fragments









of pseudocowpox, SSLPPV, HSPPV, or SSPPV. Serial ten-fold dilutions from 100 ng

to 0.001fg of Topo 2.1 plasmid containing the CPV-1 DNA toposiomerase fragment were

PCR amplified with primers CR 421 and CR422. The minimal amount of CPV-1 DNA

detected was 1.0 fg.

PCR Targeting the Major Envelope Protein Gene of Orthopoxviruses

PCR was used to target the major envelope protein gene of cetacean and pinniped

poxviruses. While the primers amplified bands of the expected size, of approximately

1118-bp using cetacean poxvirus DNA, sequencing of the DNA fragments yielded non-

poxvirus DNA sequence. When vaccinia virus DNA was used as template, the same

primers drove the amplification of a fragment of the expected size (Figure 3-8).

PCR Targeting the Orthopoxvirus Hemagglutinin Gene

Cetacean and Steller sea lion poxvirus DNA templates were tested using primers

designed to amplify the HA gene of orthopox viruses. Although the primers did not

detect the presence of the HA gene in either cetacean, or Steller sea lion DNAs, the

vaccinia virus DNA positive control validated the PCR protocol amplifying a band at the

expected size of 1138-bp (Figure 3-8).

Detection of Parapoxviruses Targeting the DNA Polymerase Gene

Parapoxvirus DNA polymerase gene fragments of the approximate expected size

were amplified from total DNA extracted from biopsied or scraped skin lesions of

pinnipeds. Donor species that yielded positive results were: Three Steller sea lions, two

spotted seals and one harbor seal (Figure 3-9). This PCR assay detected the DNA

polymerase gene fragments of pseudocowpox, SSLPPV, HSPPV, or SSPPV, but did not

amplify the DNA polymerase gene fragments of muledeer poxvirus, swinepox virus,

CPV-1, CPV-2 and SSLPV. Serial ten-fold dilutions from 100ng to .001fg of Topo 2.1









plasmid containing the SSPPV DNA polymerase gene fragment were PCR amplified

with primers CR 540 and CR541. The minimal amount of target DNA detected was 0.1

fg (Figure 3-10).

Detection of Parapoxviruses Targeting the DNA Topoisomerase I Gene

PCR targeting the DNA topoisomerase gene of parapoxviruses using the first set of

primers (CR550 and CR551) amplified DNA fragments approximately 350-bp in length

when total DNA extracted from lesions of Steller sea lions was used as template (Figure

3-11). However, these primers did not amplify DNA topoisomerase gene fragments from

lesions of harbor or spotted seals. Serial ten-fold dilutions from 100 ng to 0.001fg of

Topo 2.1 plasmid containing the SSLPPV DNA topoisomerase gene fragment were PCR

amplified with primers CR 550 and CR551. The minimal amount of target DNA

detected was 1.0 fg (Figure 3-12). A second set of primers (CR557 and CR558) was

designed based on the Steller sea lion parapoxvirus DNA topoisomerase I sequence that

successfully directed the amplification of a fragment of the expected size from the harbor

seal parapoxvirus lesion, but not from the spotted seal lesion (Figure 3-11). Serial ten-

fold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the HSPPV DNA

topoisomerase fragment were PCR amplified with primers CR557 and CR558. The

minimal amount of target DNA detected was 0.1 fg (Figure 3-13). A third set of internal

consensus primers (CR570 and CR571) based on the Steller sea lion and harbor seal

parapoxvirus DNA topoisomerase I fragment sequences directed the amplification of a

fragment of approximately 250-bp from the spotted seal lesions (Figure 3-14). Serial ten-

fold dilutions from 100 ng to 0.001fg of Topo 2.1 plasmid containing the SSPPV DNA

topoisomerase fragment were PCR amplified with primers CR 570 and CR571. The

minimal amount of target DNA detected was 1.0 fg (Figure 3-15).









Detection of Parapoxviruses Targeting the Major Envelope Protein Gene

Oligonucleotide primers PPP-1 and PPP-4 (Inoshima et al.2000) known to amplify

a 594-bp fragment within the major envelope gene of parapoxviruses of herbivores and

harbor seals, directed the amplification of DNA fragments of similar size using total

DNA extracted from skin lesions harvested from three Steller sea lions, two spotted seals

and one harbor seal (Figure 3-16). Serial ten-fold dilutions from 100 ng to 0.001fg of

Topo 2.1 plasmid containing the HSPPV DNA topoisomerase fragment were PCR

amplified with primers CR339 and CR340. The minimal amount of target DNA detected

was 0.1 fg (Figure 3-17).

Sequencing and Genetic Analysis

DNA Polymerase

Sequencing of amplified poxvirus DNA polymerase gene fragments from lesions of

12 marine mammals revealed that the fragments were 546-bp in length from 10 cetacean

samples representing five species, while those amplified from two Steller sea lion lesions

were 543-bp. Sequencing of DNA fragments corresponding to the DNA topoisomerase I

gene of poxviruses contained in lesions of cetaceans and Steller sea lions were 344-bp in

length. Primers CR541 and CR540 directed the amplification of DNA fragments 536-bp

in length, from the DNA polymerase gene of parapoxviruses contained in lesions of three

Steller sea lions, two spotted seals and one harbor seal. Targeting of the DNA

topoisomerase I gene of parapoxviruses with primers, CR550 and CR551, amplified

DNA fragments of 347 or 350-bp when total DNA from lesions from three Steller sea

lions was used as template. The second set of primers, CR557 and CR558, also directed

the amplification of 350-bp DNA fragments when total DNA from the lesions of a third

Steller sea lion and a harbor seal was used as a template. The third set of primers, CR570









and CR571, directed the amplification of DNA topoisomerase I gene fragments 252-bp in

length from parapoxvirus lesions from two spotted seals. Targeting of the major

envelope protein gene of parapoxviruses with primers validated with ruminant

parapoxviruses (Inoshima et al., 2000) and harbor seal parapoxviruses (Becher et al.,

2002; Miller et al., 2003), confirmed the universality of these primers for the

amplification of parapoxvirus DNA contained in skin lesions of pinnipeds; in this case,

Steller sea lions, spotted and harbor seals.

Genetic analysis of the nucleotide sequences obtained from the DNA polymerase

gene fragments of poxviruses of cetaceans (546-bp) demonstrated that nine of the 10

nucleotide sequences derived from cetacean poxviruses, shared identities greater than

93.0 and 97.2% at the nucleotide and amino acid level, respectively. We have tentatively

grouped these nine poxviruses within a single group that we, herein refer toas cetacean

poxvirus-1. The remaining cetacean poxvirus sample derived from a bowhead whale

lesion was shown to be at least 84 and 89% identical at the nucleotide and amino acid

level, respectively, when compared to homologous sequences from the other nine

cetacean poxvirus-1 sequences (Tables 3-1 3-3). This virus was being provisionally

named as cetacean poxvirus-2. The DNA polymerase gene fragments (543-bp) amplified

from cutaneous lesions of two Steller sea lion pups were 100% identical to each other,

and at least 76 and 81% identical at the nucleotide and amino acid level, respectively,

when compared to homologous sequences of cetacean poxvirus-1. Similar comparisons

to the homologous fragments from the bowhead whale (cetacean poxvirus-2) showed

identities of 77 and 83% (Table 3-1 3-3). Genetic analysis of DNA polymerase gene

fragments amplified from skin lesions of pinnipeds associated with parapoxviruses









showed that the viruses contained in these lesions were members of the parapoxvirus

genus and showed nucleotide and amino acid identities greater than 98% when compared

among themselves. Nucleotide and amino acid sequence comparisons of the DNA

polymerase gene fragments of the Steller sea lion poxvirus and the Steller sea lion

parapoxviruses showed, respectively, identities of 55 and 61 %. The DNA polymerase

fragments obtained from the cetacean and pinniped poxvirus DNA templates were

compared with homologous fragments from other terrestrial poxviruses. These numerous

pairwise comparisons were made to represent the nucleotide identity, amino acid identity

and amino acid similarity (Tables 3-1 3-3) of all these viruses. Multiple alignments

were performed using the DNA polymerase fragments of CPV-1, CPV-2, SSLPV,

SSLPPV, SSPPV, HSPPV (Figures 3-18 and 3-19). The multiple alignment comparing

the CPV-1 and CPV-2 DNA polymerase fragments showed a clear difference between

the CPV-1 and CPV-2 amino acid sequences (Figure 3-18). The multiple alignment

comparing the SSLPV, SSLPPV, HSPPV, and SSPPV DNA fragments showed a clear

difference between the pox and parapoxvirus amino acid sequences (Figure 3-19).

DNA Topoisomerase I

Genetic analysis of the DNA topoisomerase gene fragments (344-bp) of cetacean

poxvirus demonstrated that six of the seven positive samples had nucleotide and amino

acid identities of at least 92 and 95%, respectively (Tables 3-4, 3-5). These six

poxviruses had all been included in the cetacean poxvirus-1 type based on sequences of

the DNA polymerase gene fragment. The seventh poxvirus corresponding to the

bowhead whale poxvirus sample, had identities of 84 and 85% at the nucleotide and

amino acid levels when compared to homologous sequences of cetacean poxvirus-1

(Tables 3-4 3-6). Based on sequences of the DNA polymerase gene fragment, the









bowhead whale virus has been provisionally named cetacean poxvirus-2. Poxvirus DNA

topoisomerase fragments amplified from lesions of the three Steller sea lion pups were

identical to each other with about 71 and 75% identity at the nucleotide and amino acid

level, respectively, to homologous sequences of cetacean poxviruses-1. Similar

comparison to homologous sequences of cetacean poxvirus-2 revealed identities of 72

and 77%. Genetic analysis of the DNA topoisomerase fragments of the Steller sea lion,

spotted seal and harbor seal parapoxviruses demonstrated that they belong to the

parapoxvirus genus.

Pair-wise comparisons between the DNA topoisomerase gene fragment sequences

of the poxvirus from the two Steller sea lion pups and the homologous fragments from

the Steller sea lion parapoxviruses showed identities of 52-54 and 57% at the nucleotide

and amino acid levels, respectively, clearly demonstrating that these viruses are distinct

members of separate genera within the Chordopoxvirinae subfamily (Tables 3-4 3-6).

The genetic diversity of parapoxviruses of pinnipeds is reflected in the findings that the

DNA polymerase gene fragment sequence the Steller sea lion and its homologue in the

harbor seal parapoxviruses share 80-98 and 87-98% identity at the nucleotide and amino

acid levels, respectively. This identity shows a similar pattern of about 80-99 and 88-

99% in the case of the spotted seal parapoxvirus. The DNA topoisomerase gene

fragment of the harbor seal parapoxviruses share 96 and 95% identities when compared

to the homologous sequence from the spotted seal parapoxvirus. The DNA

topoisomerase fragment sequences obtained from the cetacean and pinniped poxvirus

DNA templates were compared with homologous fragments from other terrestrial

poxviruses deposited in the GenBank database (Tables 3-4- 3-6). These numerous









pairwise comparisons established the nucleotide identity, amino acid identity and amino

acid similarity among the most relevant poxviruses. Multiple alignments were generated

using the DNA topoisomerase gene fragment sequences of CPV-1, CPV-2, SSLPV,

SSLPPV, SSPPV, HSPPV (Figures 3-15 and 3-18). The multiple aligment of the CPV-1

and CPV-2 DNA topoisomerase fragments demonstrates a clear difference between the

respective CPV-1 and CPV-2 amino acid sequences (Figure 3-20). The multiple

alignment of the SSLPV, SSLPPV, HSPPV and SSPPV DNA topoisomerase gene

fragments demonstrates a clear difference between the pox- and parapox- virus amino

acid sequences (Figure 3-21).

Major Envelope Protein Gene

Sequence comparisons were performed with the nucleotide and deduced amino acid

sequences of the major envelope gene fragments of the various pinnipeds parapoxviruses.

Nucleotide and amino acid sequences from the Steller sea lion major envelope fragment

were, respectively, 93 and 98% identical to the homologous sequences from the harbor

seal parapoxvirus and 93 and 96% identical to the homologous sequences of the spotted

seal parapoxviruses (Tables 3-6 3-9). Sequences of the major envelope protein gene

fragments obtained from the pinniped parapoxviruses were entered into a multiple

alignment for simplified comparison (Figure 3-22).

Phylogenetic Analysis

Phylogenetic trees created using the amino acid sequences of various species of

marine mammal pox and parapox virus sequences plus numerous homologous fragments

from DNA sequences of terrestrial poxviruses demonstrate the genetic relatedness of

these virus fragments. The DNA polymerase and DNA topoisomerase phylograms

indicate that the CPV-1 and CPV-2 viruses group together and form a unique branch,








separate from the known poxvirus genera (Figures 3-23 and 3-24). The SSLPV also

forms its own branch in both the DNA polymerase and DNA topoisomerase phylogragms

(Figures 3-23 and 3-24). The phylogenetic tree constructed using the major envelope

protein gene fragments amplified form pinniped parapoxviruses and numerous

homologous fragments from DNA sequences of terrestrial poxviruses demonstrates the

placement of the HSPPV, SSPPV and SSPPV gene fragments into the branch including

other terrestrial parapox viruses (Figure 3-25).

Virus Isolation

All attempts to isolate poxviruses from pinniped and cetacean fresh and frozen skin

lesions were unsuccessful.

4V.R 4 1 h F E


i 1 1 ..1 -1111






A










C

Figure 3-1.Typical "tattoo" lesions of cetaceans. A and B) Skin lesions of a rough-
toothed dolphin (Steno bredanenesis). Photos taken by Dr. Charles Manire. C) Skin
lesions of a bottlenose dolphin (Tursiops aduncus) from a Hong Kong aquarium.
































Figure 3-2 Gross appearance of pox lesions associated with a poxvirus in a Steller sea
lion (Eumetopias jubatus). Approximately 1 cm diameter raised smooth, hairless, often
umbilicated, nodules were scattered across the body. Photo supplied by Dr. Kathy Burek.


Figure 3-3. Cutaneous pox lesions in a spotted seal (Phoca largha) associated with
spotted seal parapoxvirus. Photo supplied by Dr. Kathy Burek.





























Figure 3-4 Histopathologic appearance of cutaneous lesions associated wtih Steller sea
lion poxvirus, showing epithelial cells containing acidophilic intracytoplasmic inclusion
bodies (arrow). Slide su lied b Dr. Kath Burek.


















Figure 3-5. Negatively stained poxvirus particle from cutaneous lesion of SSL observed
by electron microscopy. The 'skew' pattern of orthopoxviruses is evident as opposed to
the 'criss-cross' pattern of parapoxviruses. Photo supplied by Mr. Woody Fraser.


















M.M. 1 2 3 4 5 6 7 8 9 10 11 12 13 -- 14 15 16M.M.

Figure 3-6. Agarose gel electrophoresis of PCR amplified 543-546-bp fragments of the
DNA polymerase gene of cetacean and Steller sea lion poxviruses using primers CR 421
and CR 422. M.M.: 1KB Plus Molecular Ladder; Lane 1: Rough-toothed dolphin (V365);
Lane 2: Rough-toothed dolphin (GW010006D); Lane 3: Bottlenose dolphin (R127); Lane
4: Bottlenose dolphin (V466); Lane 5: Bottlenose dolphin (V550); Lane 6: Bottlenose
dolphin (V551); Lane 7: Bottlenose dolphin (MML0203); Lane 8: Bottlenose dolphin
(OK04091932); Lane 9: Bottlenose dolphin (CMA0108); Lane 10: Bowhead whale
(98KK3); Lane 11: Steller sea lion (SSL2001-279); Lane 12: Steller sea lion (SSL2000-
105); Lane 13: Steller sea lion (SSL2005-546); Lane 14: Positive control, MDPV; Lane
15: Ne ative tube, no DNA; Lane 16: Ne ative tissue (V1044)









M.M. 1 2 3 4 5 6 7 8 9 10 11 12 13 M.M.

Figure 3-7. Agarose gel electrophoresis of PCR amplified 344-bp fragments of the DNA
topoisomerase gene of cetacean and Steller sea lion poxviruses using primers CR 432 and
CR 433. M.M.: 1KB Plus Molecular Ladder; Lane 1: Rough-toothed dolphin (V365);
Lane 2: Rough-toothed dolphin (GW010006D); Lane 3: Bottlenose dolphin (R127); Lane
4: Bottlenose dolphin (MML0203); Lane 5: Bottlenose dolphin (OK04091932); Lane 6:
Bottlenose dolphin (CMA0108); Lane 7: Bowhead whale (98KK3); Lane 8: Steller sea
lion (SSL2001-279); Lane 9: Steller sea lion (SSL2000-105); Lane 10: Steller sea lion
(SSL2005-546); Lane 11: Negative water, no DNA; Lane 12: Negative tissue (V1044);
Lane 13: Positive control, MDPV DNA










1.65-kb-

1.0-kb--













Figure 3-8. Agarose gel electrophoreses of PCR amplified fragments of the HA gene of
orthopoxviruses using primers CR 619 and CR620 targeting a 1183-bp. M.M.: 1 KB Plus
Molecular Ladder; Lane 1: Bottlenose dolphin CPV-1 (V1546); Lane 2: Bowhead whale
CPV-2 (V730); Lane 3: Steller sea lion poxvirus (R227); Lane 4: Negative tissue
(V1044); Lane 5: Ne ative water, no DNA; Lane 6: Vaccinia virus DNA


650-bp--

500-bp--







Figure 3-9. Agarose gel electrophoresis demonstrating the PCR amplification of 536-bp
parapox DNA polymerase gene fragments from lesions of different pinniped species.
M.M.: 1 KB Plus molecular ladder; Lane 1: Steller sea lion (SSL2003-450); Lane 2:
Steller sea lion (SSL2003-451); Lane 3: Steller sea lion (SSL2004-495); Lane 4: Harbor
seal (MMSC 03021); Lane 5: Spotted seal (DIO-136-03); Lane 6: Spotted seal (DIO-119-
03); Lane 7: positive control, pseudocowpox virus DNA; Lane 8: negative control, no
DNA template.








650-bp---



500-bp--






Figure 3-10. Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR540 and CR541 targeting the parapox DNA polymerase gene. pCR-II topo 2.1
plasmid vector containing the 536-bp fragment amplified from the Spotted seal (DIO-
136-03) parapoxvirus DNA in 10-fold serial dilutions. M.M: 1 Kb plus ladder. Lane 1:
100 ng; Lane 2: 10 ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1.0 pg;
Lane 7: 100 fg, Lane 8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg; Lane 11: 0.01 fg; Lane 12:
0.001; Lane 13: ne ative control, no DNA.






400-bp--

300-bp--



Figure 3-11. Agarose gel electrophoresis demonstrating the PCR amplification of 350-bp
parapox DNA topoisomerase I gene fragments from lesions of Steller sea lions
(Eumetopias jubatus) and harbor seals (Phoca vitulina). M.M.: 1KB Plus molecular
ladder; Lane 1: Steller sea lion (SSL2003-450); Lane 2: Steller sea lion (SSL2003-451);
Lane 3: Steller sea lion (SSL2004-495); Lane 4: Harbor seal (MMSC 03021); Lane 5:
positive control, pseudocowpox virus DNA; Lane 6: negative control, no DNA template.















400-bp --- --, M -A --L ............
400-bp--






Figure 3-12. Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR550 and CR551 targeting the Steller sea lion (SSL2003-451) parapoxvirus
DNA topoisomerase gene. pCR-II topo 2.1 plasmid vector containing the 350-bp
fragment in 10-fold serial dilutions. M.M: 1 Kb plus ladder. Lane 1: 100 ng; Lane 2: 10
ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1.0 pg; Lane 7: 100 fg, Lane
8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg; Lane 11: 0.01 fg; Lane 12: 0.001; Lane 13:
negative control no DNA


650-bp-
500-bp-


.W9 S


Figure 3-13. Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR557 and CR558 targeting the harbor seal DNA topoisomerase gene. pCR-II
topo 2.1 plasmid vector containing the 350-bp parapoxvirus DNA topoisomerase
fragment in 10-fold serial dilutions. M.M: 1 Kb plus ladder. Lane 1: 100 ng; Lane 2: 10
ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1.0 pg; Lane 7: 100 fg, Lane
8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg; Lane 11: 0.01 fg; Lane 12: 0.001; Lane 13:
negative control, no DNA.














600-bp--


300-bp--

200-bp--



Figure 3-14. Agarose gel electrophoresis demonstrating the PCR amplification of252-bp
parapox virus DNA topoisomerase gene fragment from lesions of spotted seals (Phoca
largha). M.M.: 100-bp molecular ladder; Lane 1: Spotted seal (DIO-136-03); Lane 2:
Spotted seal (DIO-119-03); Lane 3: negative control, no DNA template; Lane 4: positive
control, Steller sea lion (SSL2003-451).







300-bp-
200-bp-



Figure 3-15. Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR570 and CR571 targeting the parapoxvirus DNA topoisomerase gene. pCR-II
topo 2.1 plasmid vector containing the 252-bp fragment amplified from the Spotted seal
(DIO-136-03) parapoxvirus DNA in 10-fold serial dilutions. M.M: 1 Kb plus ladder.
Lane 1: 10 ng; Lane 2: 1.0 ng; Lane 3: 100pg; Lane 4: 10 pg; Lane 5: 1.0 pg; Lane 6:
100 fg; Lane 7: 10 fg, Lane 8: 1.0 fg; Lane 9: 0.1 fg; Lane 10: 0.01 fg; Lane 11: 0.001 fg;
Lane 12: negative control; Lane 13: M.M.















650-bp---O
500-bp--

Figure 3-16. Agarose gel electrophoresis demonstrating the PCR amplification of 594-bp
parapox major envelope protein gene fragment from lesions of different pinniped species.
M.M.: 1 KB Plus molecular ladder; Lane 1: Steller sea lion (SSL2003-450); Lane 2:
Steller sea lion (SSL2003-451); Lane 3: Steller sea lion (SSL2004-495); Lane 4: Harbor
seal (MMSC 03021); Lane 5: Spotted seal (DIO-136-03); Lane 6: Spotted seal (DIO-119-
03); Lane 7: positive control, pseudocowpox virus DNA; Lane 8: negative control, no
DNA tem late.


1.0-Kb--

650-bp--
500-bp-




Figure 3-17. Agarose gel electrophoresis demonstrating the PCR sensitivity assay for
primers CR339 and CR340 targeting the parapox major envelope protein gene. pCR-II
topo 2.1 plasmid vector containing the 596-bp fragment amplified from the Harbor seal
(MMSC03021) parapoxvirus DNA in 10-fold serial dilutions. M.M: 1 Kb plus ladder.
Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1.0 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6:
1.0 pg; Lane 7: 100 fg, Lane 8: 10 fg; Lane 9: 1.0 fg; Lane 10: 0.1 fg; Lane 11: 0.01 fg;
Lane 12: 0.001; Lane 13: negative control, no DNA.
























B. mysticetus-AK
B. mysticetus-AK
T. aduncus-HK
T. aduncus-HK
S. bredanensis-FL
T. truncatus-FL
S. bredanensis-FL
T. aduncus-HK
S. coeruleoalba-FL
T. aduncus-HK
S. coeruleoalba-PO
CONSENSUS





B.mysticetus-AK
B. mysticetus-AK
T. aduncus-HK
T. aduncus-HK
S. bredanensis-FL
T. truncatus-FL
S. bredanensis-FL
T. aduncus-HK
S. coeruleoalba-FL
T. aduncus-HK
S. coeruleoalba-PO
Consensus


1 50 100
........ ........ ......... k 1 ......... .......... .... ....... .. ......... .. k ..f .... ..........
.......... .........k l......... .......... .......... .......... ........ .. k..f .... ..........


YRASTLIKGP LLKLLLETKI


.r... ....... ........r..... ..... .................... s............
. . . . . . r . . . . . . . . s . . . . .
. . y . . . . . . . . . . . . . . . . . . .
ILYRSEHKQQ KLPYEGGKVF MPKQKMFSNN VLIFDYNSLY PNVCLFGNLS PETLVGVVVS NNVLELEINI QEIKKKFPSP


101


150


.......... .q ...... a. .......... ..q ..s.. ..c.....as kt......... ......... ..........
.......... .q ...... a. .......... ..q ..s.. ..c.....as kt......... ......... ..........


....... .......... .... ..... ...m .. .. .... .........
......... ....... m. .. ... .... .......... .......... .
.... ... .......... .......... ... m...... .............. .......... .
....... .......... v ................... ...... .


.......... ....... .. .... .... v ......... ........... .........
.......... ........ .... .... v ......... ........... .........
........ ......... .... .... v ......... ........... .........
.......... .......... ..... .. ..... .... v .. ......... ........ . ..
....RR.. .....FI.. .....FP.. ..RF....... .. r L..... .........N. .DCD SA NK N.......
RYIVVHCEPR FKNLISEISI FDREVEGTIP RILRRFLTER AKYKKLLKST NDCTEKAIYD SMQYTYKIVA NSVYGLMGFK N


Figure 3-18. Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the DNA polymerase gene

fragments of poxviruses identified in cutaneous lesions of cetaceans. AK = Alaska; FL = Florida; HK = Hong Kong; PO= Portugal.


. .. . .. .
. .. . . .
.. . . . .
. .. . . .






















1 50 100
E. jubatus-AK .....1.... .1...etk. .Is..ek.qr .p.... k.f ......vnn. .......... .. l.g.... ........s. .k.es..nnq .llik..p.q
E. jubatus-AK .....1.... .1...etk. .Is..ek.qr .p.... k.f. ......vnn ......... ...l.g... ........ s. .k.es..nnq .11ik..p.q
E. jubatus-AK ~~........ .......hk. ....a.t.s. ........ .... ......... .. .........sr ......... ..........
P. largha-AK .... .......... ........ .
P. largha-AK .... .......... ........ .
P. vitulina-NJ ~~....... ..... ..... n .........
E. jubatus-AK ~~........ ........ ................... ..... h .... .......... .......... .......... ........ ..........
E. jubatus-AK ~~........ .... .... ......... .......... .r..i .... .......... .......... ......... d ........ ... .r......
CONSENSUS YRASTCIKGP LMKLLLANRT VMVRSDVKTK YFFEGGRVMA PKQKMYDKHV LIFDYNSLYP NVCIYANLSP ETLVGVVVAN NRLDAEIAAV EIRQRFPAPR


E. jubatus-AK
E. jubatus-AK
E. jubatus-AK
P. largha-AK
P. largha-AK
P. vitulina-NJ
E. jubatus-AK
E. jubatus-AK
CONSENSUS


101
S. .y .....
.1l .y.....
..s.1 .....


. .. .


tqf .......
tqf .......
..f .......
. . . .


150 180
..rte .... 1 ..kk..ne.s y...ml.nsk .qkeks..d. .......i.. t....... k.
..rte .... 1 ..kk..ne.s y...ml.nsk .qkeks..d. .......i.. t....... k.
....d ..... ....... ... .......g .k .....n ..d. .......... ..........
.......... ......... ......... .e ...... .. ..........
... .............. ............e ...... ..... .....


......... .......... ........... .. ........ e ...... ..........
.......... s......... ...... ....... ...... ... e ....... ..... .... .
..t ....... d ........ ....d ..... .........t .........d s .... d .... .......... .........
FIAVPCEPRS PELVSEVAIF DREANGIIPM LLRSFLDARA KYKKLMKTA- TAVDREIFNS MQYTYKITAN SVYGLMGFRN


Figure 3-19. Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the

DNA polymerase gene fragment of poxviruses identified in cutaneous lesions of pinnipeds. AK = Alaska; NJ = New Jersey.




























B. mysticetus-AK
B. mysticetus-AK
T. truncatus-FL
S. bredanensis-FL
T. aduncus-HK
S. coeruleoalba-FL
S. bredanensis-FL
S. coeruleoalba-PO
CONSENSUS


........ ..... .......... dik .... i ..i ....... .....yn .r .
........ ..... .......... dik .... i ..i ....... .....yn .r .





........... ... .......n.. ..f............ ........a...
.......... .......... .......... ..i ......... ..m.........
METSFFIRTG KLRYLKENNT VGLLTLKSKH LTLTKDKLT SFTGKDKVSH EFVIRRYDKL. a
. . . . . . . . i . . m . . . . .
METSFFIRTG KLRYLKENNT VGLLTLKSKH LTLTKDKLTI SFTGKDKVSH EFVIRRYDKL


61 114
B. mysticetus-AK .....k.a.. .d........ ...r...... nq....h.... .......... ....
B. mysticetus-AK .....k.a.. .d........ ...r...... nq....h.... .......... ....
T. truncatus-FL .......... ..... .......... .
S. bredanensis-FL .......... ..... .......... .
T. aduncus-HK .......... ..... .......... .
S. coeruleoalba-FL .......... ..... .......... .
S. bredanensis-FL .......... .......... ...... ..........
S. coeruleoalba-PO ..... k.... .d. .......r .......... k........ .......... ....
CONSENSUS YKPLIRLSKN KESECFLFDK LNENIIYKLI RPFGIRIKDL RTYGVNYTFL YNFW


Figure 3-20. Multiple alignment of the amino acid sequences deduced from the nucleotide. Sequences of the

DNA topoisomerase gene fragments of poxviruses identified in cutaneous lesions of cetaceans.

AK = Alaska; FL = Florida; HK = Hong Kong; PO = Portugal.




























E. jubatus -AK
E. jubatus -AK
E. jubatus -AK
P. largha-AK
P. largha-AK
P. vitulina-NJ
E. jubatus -AK
E. jubatus -AK
E. jubatus -AK
Consensus



E. jubatus -AK
E. jubatus -AK
E. jubatus -AK
P. largha-AK
P. largha-AK
P. vitulina-NJ
E. jubatus -AK
E. jubatus -AK
E. jubatus -AK
Consensus


1














METSFFIRIG


101














DLRTYGVNYT
. . . .
. . . .
. . . .
----------


.k.f. .nn.
.k.f. .nn.
.k.f. .nn.



.s ........
.s...r....
.t ........
.a...r....
KMRYEKESGT


...... q..n
.... q..n
.... q..n





.r..


. .... VGTRNKH
........ r .
VGLLTLRNKH


ihiek...k.
ihiek...k.
ihiek...k.
.......d ..
.......d ..
.......e ..
.......e ..
. .s...e..
.rgd..da.
LSEAEGG-EI


50
f.t....... n.q.i..knn
f.t....... n.q.i..knn
f.t....... n.q.i..knn




..k ....... ..........

..k ....... ....a.. .g.
........ r ..... a ... g .
1....... r. t...a..dg.
RVRFVGKDKV AHEFTVRNSQ


116














FLYNFW


Figure 3-21. Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the DNA

topoisomerase gene fragment of poxviruses and parapoxviruses identified in cutaneous lesions of pinnipeds.

Alaska; NJ = New Jersey.


...kp.kii
...kp.lkii
...kp.lkii
..........






... v.e....

RLFAALRRLW


.nsk..sfi.
.nsk..sfi.
.nsk..sfi.
.q....
.q...
......q ...

...... q....



..ad......
DPGAPERLLF


.k.n..k..n
.k.n..k..n
.k.n..k..n





t.........
t .........
d........ m
h..g.k ....
NRLSERRVYA


100
1.km.y.h..
1.km.y.h..
1.km.y.h..








a..........
FMRRFGIRVK























1 50 100

HSPPV-NJ
SSPPV -AK .......... ............... .......... .......... ....
SSPPV2-AK .......... ............... .......... .......... ....
SSLPPV1-AK .......... .......... .......... .......... .. a....... i ........ ......h..... ............................
SSLPPV2-AK .......... ......... ............... ... ............. .i ............. .......... .... ....... .........
SSLPPV3-AK
Consensus YVGSASLTGG SLATIKNLGV YSTNKHLAVD LMNRYNTFSS MVVDPKQPFT RFCCAMITPT ATDFHMNHSG GGVFFSDSPE RFLGFYRTLD EDLVLHRIDA


101 150 198

HSPPV-NJ .k....... .. g ... .. ...... .. .. ....... .......... ........... .... .. i ...... i......... ........
SSPPV-AK .e........ .. 1 ......s ..h....v.. ... ...... ........... ......... .......... ... ...... v ... ...... ........
SSPPV2-AK .e........ .. 1 ......s ..h....v.. ... ...... ........... ......... .......... ... ...... v ... ...... ........
SSLPPV1-AK .k....... .v. ..... g ..y... i.. .......d.. ........... ......... ....... ... i ..... i..... .. ..
SSLPPV2-AK .k....... .v. ..... g ..y... i.. .......d.. ........... ......... ....... ... i ..... i..... .. ..
SSLPPV3-AK .k........ ..y.. .s ..y...a ........... ........ .......... ............ .v...... i........... ........
Consensus AKNSIDLSLL SMYPVVRSG- EVYYWPLIMD ALLRAAINRS VRVRIIISQW RNADPLSVAA VRALDNFGVG HVD-TARWFA IPGRDDASNN TKLLIVDD


Figure 3-22. Multiple alignment of the partial amino acid sequences predicted from the major envelope protein gene fragment of

parapoxviruses identified in cutaneous lesions of pinnipeds. AK = Alaska; NJ = New Jersey.











HSPPV


SSSLPPW842
_SSLPPW13A Parapox
Orf








I c3hopo Vacxinia
Ya mcnrey YIataopox
EZ2





34 LSI Cap-tip x




Swinepo 1 ipox





Rabbit Fibr am -
CPW ^





C42 LSDV







Rabbitmyxima
MallusciL cornagiousmn
Canaryp A-ipox

Failpox

A.
Figure 3-23. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the DNA polymerase gene fragments from members of the Chordopoxvirinae
subfamily of poxviruses. The tree generated by Clustal X slow and accurate function
using Gonnet 250 residue weight table, gap penalty of 11 and gap length penalty of 0.2.
A) Format is a rectangular cladogram where the numbers represent the percent
confidence of 1000 bootstrap replications. B) Radial format showing a .1 divergence
scale representing 0.1 substitutions per site.













Canarypox
SFowlpo


SSLPPVv841 Or SSLPPVv1386

I BPS I SSP
USPP
I / BSLPPVv842


Swinepox.

Muledeerpox


V Goatpo
x


Yaba monkey
tumor virus


CPV-
2


Variol
a


Camelpox
Vaccini
Ccaepox
Monkeypox


CPV-
1


0.
1


Figure 3-23. Continued.


SSLP
V








49



Camelpox

Monkeypox
Vaccinia
Orthopox
Ectromelia

Cowpox

Variola

.~~~~~____~~~~~_____________________aba
monkey }Yatapox
Muledeer pox

Rabbit ibroma }
s- F.... Leponpox
Rabbit myxoma
Swinepox > Suipox
913
Goatpox
10.0 I- gpLSDV
SCapnpox
741
Sheeppox
SSLPV teller sea lion
E CPV-1I 1
0Co PV- Cetacean pox
CPV-2 J

64.9 Fowipox
so. Avip ox
Canarypox
Molluscum
contagiosum Moillusclp ox
',I' SSLPPV v841
BPSV
10.0 50. Orf

SSSLPPV 1386 Parapox

HSPPV
SSPPV
SSLPPV v842

Figure 3-24. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the DNA topoisomerase gene fragments from members of the Chordopoxvirinae
subfamily of poxviruses. The tree was generated by Clustal X slow and accurate
function using Gonnet 250 residue weight table, gap penalty of 11 and gap length
penalty of 0.2. A) Format is a rectangular cladogram where the numbers represent the
percent confidence of 1000 bootstrap replications. B) Radial format showing a .1
divergence scale representing 0.1 substitutions per site.











Goatpox
Sheeppox LSDV


Camelpox
Ectromelia


Yaba monkey
Tumor virus











SSLPPV
v842



Orf v1386
SSPPV BPSV


SSLPV


- CPV1

CPV2





Fowlpox


1 Molluscum contagiosum
SSLPPVv841


0.1


Figure 3-24. Continued.












BPSV
RedDeerParapox

Orf

I Pseudocowpox
Weddell seal
I nirannox


S100.0

|54.SI


1100.0


100.0


SSLPPVu841 > Parapox
93.7
SSSLPPVu842
HSPPVHJ

HSPPV
62.8
SSPPV
996.
SSLPPVu1386
Molluscun 1
contagiosman Molluscipox
6.7 Canarypox

Fowlpox Avopox
1000
PigeonpoxJ

Carnelpox

I Vlariola
Monkewpox
Orthopox
876 Cowpoan

,.5 Ectrao nelia

Vaccnia
Yaba monkey Yatapox
TiT or urus i
.inepox 3.- Suipox


as i ---I ? Capripox
!3 IL Sheeppo -

Rabbit Fibroilna
SRabbit Myximaf Leporipox


Figure 3-25 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the Major envelope protein gene fragments from members of the Chordopoxvirinae
subfamily of poxviruses. The tree was generated by Clustal X slow and accurate
function using Gonnet 250 residue weight table, gap penalty of 11 and gap length
penalty of 0.2. A) Format is a rectangular cladogram where the numbers represent the
percent confidence of 1000 bootstrap replications. B) Radial format showing a .1
divergence scale representing 0.1 substitutions per site


I












SSLPPVv1386
HSPPV
SSPPV SSLPPVv842 Orf
Weddell seal SSLPPVv841 Pseudocowpox
parapox '/ ,RedDeerParapox


Rabbit Fibroma


- Molluscumcontagiosum



Canarypox

Fowlpox
Pigeonpox


SwineDox '
Yabamonkey
tumorvirus
Monkeypox
Variola Camelpox
Ectromelia
0.1 Vaccinia
Cowpox


Figure 3-25. Continued.


Rabbit













Table 3-1. Pair-wise comparisons of the nucleotide sequences obtained from the DNA polymerase gene fragments of the cetacean
poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent identity between two
nucleotide sequences.

CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2
AJ28 Wiki R174 R164 V365 V1546 V466 V550 V551 V729

CPV-1 AJ28 100.0
CPV-1 Wiki 100.0 100.0
CPV-1 R174 100.0 100.0 100.0
CPV-1 R164 100.0 100.0 100.0 100.0
CPV-1 V365 96.3 96.3 96.3 96.3 100.0
CPV-1 V1546 93.0 93.0 93.0 93.0 91.9 100.0
CPV-1 V466 99.1 99.1 99.1 99.1 95.8 92.5 100.0
CPV-1 V550 96.2 96.2 96.2 96.2 99.5 92.1 95.6 100.0
CPV-1 V551 96.3 96.3 96.3 96.3 99.6 92.3 95.8 99.8 100.0
CPV-2 V729 84.4 84.41 84.4 84.4 84.4 84.1 83.5 84.6 84.4 100.0













Table 3-2. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences ofDNA polymerase gene
fragments of the cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent
identity between two amino acid sequences.

CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2
AJ28 Wiki R174 R164 V365 V1546 V466 V550 V551 V729

CPV-1 AJ28 100.0
CPV-1 Wiki 100.0 100.0
CPV-1 R174 100.0 100.0 100.0
CPV-1 R164 100.0 100.0 100.0 100.0
CPV-1 V365 98.9 98.9 98.9 98.9 100.0
CPV-1 V1546 97.2 97.2 97.2 97.2 97.2 100.0
CPV-1 V466 98.9 98.9 98.9 98.9 97.8 96.1 100.0
CPV-1 V550 98.9 98.9 98.9 98.9 100.0 97.2 97.8 100.0
CPV-1 V551 98.9 98.9 98.9 98.9 100.0 97.2 97.8 100.0 100.0
CPV-2 V729 89.0 89.0 89.0 89.0 89.0 87.9 87.8 89.0 89.0 100.0













Table 3-3. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences of the DNA polymerase gene
fragments of the cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent
similarity between two amino acid sequences.

CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2
AJ28 Wiki R174 R164 V365 V1546 V466 V550 V551 V729

CPV-1 AJ28 100.0
CPV-1 Wiki 100.0 100.0
CPV-1 R174 100.0 100.0 100.0
CPV-1 R164 100.0 100.0 100.0 100.0
CPV-1 V365 98.9 98.9 98.9 98.9 100.0
CPV-1 V1546 98.9 98.9 98.9 98.9 98.9 100.0
CPV-1 V466 99.4 99.4 99.4 99.4 98.3 98.3 100.0
CPV-1 V550 98.9 98.9 98.9 98.9 100.0 98.9 98.3 100.0
CVP-1 V551 98.9 98.9 98.9 98.9 100.0 98.9 98.3 100.0 100.0
CPV-2 V729 92.3 92.3 92.3 92.3 92.3 92.3 91.7 92.3 92.3 100.0













Table 3-4. Pair-wise comparisons of the nucleotide sequences obtained from the DNA topoisomerase gene fragments of cetacean
poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent identity between two
nucleotide sequences.

CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2
AJ28 Wiki R174 R164 V365 V1546 V729

CPV-1 AJ28 100.0
CPV-1 Wiki 100.0 100.0
CPV-1 R174 100.0 100.0 100.0
CPV-1 R164 100.0 100.0 100.0 100.0
CPV-1 V365 93.6 93.6 93.6 93.6 100.0
CPV-1 V1546 92.4 92.4 92.4 92.4 89.8 100.0
CPV-2 V729 84.3 84.3 84.3 84.3 84.9 86.0 100.0














Table 3-5. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences of the DNA topoisomerase
gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent
identity between two amino acid sequences.

CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2
AJ28 Wiki R174 R164 V365 V1546 V729

CPV-1 AJ28 100.0
CPV-1 Wiki 100.0 100.0
CPV-1 R174 100.0 100.0 100.0
CPV-1 R164 100.0 100.0 100.0 100.0
CPV-1 V365 96.5 96.5 96.5 96.5 100.0
CPV-1 V1546 94.7 94.7 94.7 94.7 92.1 100.0
CPV-2 V729 85.1 85.1 85.1 85.1 84.2 86.8 100.0














Table 3-6. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences of the DNA topoisomerase
gene fragments of cetacean poxvirus 1 (CPV-1) and cetacean poxvirus 2 (CPV-2) samples. Values correspond to percent
similarity between two amino acid sequences.

CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-1 CPV-2
AJ28 Wiki R174 R164 V365 V1546 V729

CPV-1 AJ28 100.0
CPV-1 Wiki 100.0 100.0
CPV-1 R174 100.0 100.0 100.0
CPV-1 R164 100.0 100.0 100.0 100.0
CPV-1 V365 96.5 96.5 96.5 96.5 100.0
CPV-1 V1546 99.1 99.1 99.1 99.1 95.6 100.0
CPV-2 V729 90.4 90.4 90.4 90.4 88.6 90.4 100.0

00













Table 3-7. Pair-wise comparisons of the nucleotide sequences of the DNA polymerase gene fragments of poxviruses of various
genera within the Chordopoxvirinae subfamily of viruses. Values correspond to percent identity between two nucleotide
sequences.


CPV-1 CPV-2 SSLPV SSLPPV
V841
Cetaceanpox-1 100.0 84.4 75.7 59.1
Cetaceanpox-2 84.4 100.0 77.0 59.0
Steller sealionpox 75.7 77.0 100.0 58.8
Camelpox 71.6 75.5 72.0 59.7
Cowpox 71.8 75.9 72.0 60.3
Monkeypox 72.6 76.1 72.4 60.1
Vaccinia 72.7 76.2 72.6 60.1
Ectromelia 72.0 76.1 71.6 60.1
Variola 71.6 74.8 71.8 60.8
Lumpy skin disease 74.0 74.4 74.0 61.2
Sheeppox 73.3 74.8 73.3 61.2
Goatpox 74.0 75.5 75.0 60.3
Muledeerpox 73.6 75.2 76.6 59.9
Swinepox 75.0 76.8 75.5 60.6
Rabbit fibroma 67.8 67.4 68.9 60.1
Rabbit myxoma 69.2 68.3 69.6 62.1
Yaba monkeypox 68.5 70.4 70.4 60.1
Orf 53.4 53.2 49.8 77.2
Bovine pap stom 53.4 51.6 50.6 77.3
Canarypox 40.4 41.5 40.6 36.1
Fowlpox 40.1 40.6 66.3 36.6
Molluscum conagiosum 50.5 51.6 48.3 57.3
Harbor sealparapox 57.8 56.5 55.0 79.7
Spotted sealparapox 58.0 56.7 55.2 79.5
Steller sealionparapox V841 59.1 59.0 58.8 100.0
Steller sealionparapox V842 57.6 55.2 55.0 78.9
Steller sealionparapox V1386 56.9 54.3 53.2 77.1


SSLPPV
V842
57.6
55.2
55.0
57.5
57.5
57.5
57.8
58.8
58.4
56.7
57.1
56.2
55.7
55.6
59.1
61.4
59.9
82.5
81.3
36.9
36.0
60.4
98.3
98.7
78.9
100.0
84.3


SSLPPV SSPPV HSPPV


V1386
56.9
54.3
53.2
56.5
57.8
57.1
57.3
58.0
57.5
56.9
56.7
56.2
55.3
56.3
61.9
64.7
59.5
83.0
83.8
36.8
36.0
62.7
84.0
84.3
77.1
84.3
100.0


V688
58.0
56.7
55.2
57.6
58.2
57.6
58.0
59.0
58.6
56.7
57.1
56.2
55.9
55.4
59.3
61.9
60.3
83.0
81.9
32.2
35.9
60.4
99.1
100.0
79.5
98.7
84.3


V465
57.8
56.5
55.0
57.3
58.0
57.3
57.6
58.6
58.2
56.3
56.7
55.8
55.5
55.4
59.7
61.9
60.1
82.8
81.5
32.3
36.4
60.4
100.0
99.1
79.7
98.3
84.0













Table3-8. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequence of the DNA polymerase gene
fragments of poxviruses of various genera within the Chordopoxvirinae subfamily of viruses. Values correspond to
percent identity between two amino acid sequences.


CPV-1 CPV-2 SSLPV SSLPPV
V841
Cetaceanpox-1 100.0 89.0 74.4 60.7
Cetaceanpox-2 89.0 100.0 77.8 62.9
Steller sealionpox 74.4 77.8 100.0 62.4
Camelpox 80.0 82.2 76.1 65.2
Cowpox 80.6 82.8 77.2 66.3
Monkeypox 80.6 82.8 77.2 66.3
Vaccinia 80.6 82.8 77.2 66.3
Ectromelia 80.6 82.8 77.2 66.3
Variola 79.4 81.7 77.2 66.3
Lumpy skin disease 75.0 74.4 73.9 60.7
Sheeppox 73.9 73.3 72.8 60.1
Goatpox 74.4 75.6 74.4 60.1
Muledeerpox 75.0 75.0 73.2 60.8
Swinepox 72.2 73.9 71.7 62.9
Rabbit fibroma 74.3 72.1 72.1 60.5
Rabbit myxoma 78.3 76.1 76.7 62.9
Yaba monkeypox 75.0 75.6 73.9 62.9
Orf 62.2 63.9 60.6 86.5
Bovine pap stom 65.6 65.0 64.4 87.1
Canarypox 56.7 59.4 59.4 50.0
Fowlpox 58.3 60.6 61.1 50.6
Molluscum contagiosum 56.1 57.2 56.1 56.7
Harbor sealparapox 62.4 62.4 61.2 87.6
Spotted sealparapox 62.4 62.4 61.2 88.2
Steller sealionparapox V841 60.7 62.9 62.4 100.0
Steller sealionparapox V842 62.4 62.4 61.2 87.1
Steller sealionparapox V1386 60.7 60.1 59.6 85.4


SSLPPV
V842
62.4
62.4
61.2
62.9
64.1
64.1
64.1
64.1
64.1
62.9
62.4
62.4
62.1
62.4
60.5
64.1
59.6
88.8
88.2
49.4
51.7
56.7
98.3
98.9
87.1
100.0
91.0


SSLPPV
V1386
60.7
60.1
59.6
62.9
64.0
64.0
64.0
64.0
64.0
61.8
61.2
61.2
59.6
61.8
59.9
63.5
59.0
86.5
87.6
50.0
51.7
59.0
91.2
92.1
85.4
91.0
100.0


SSPPV
V688
62.4
62.4
61.2
63.5
64.6
64.6
64.6
64.6
64.6
62.9
62.4
62.4
61.5
62.4
60.5
64.1
60.1
88.8
89.3
49.4
51.1
56.7
99.4
100.0
88.2
98.9
92.1


HSPPV
V465
62.4
62.4
61.2
63.5
64.6
64.6
64.6
64.6
64.6
62.9
62.4
62.4
61.5
62.4
60.5
64.1
60.1
88.2
88.8
49.4
51.1
56.7
100.0
99.4
87.6
98.3
91.6













Table 3-9. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences of the DNA polymerase


gene fragments of poxviruses of various genera within the
to percent similarity between two amino acid sequences.


CPV-1 CPV-2 SSLPV


Cetaceanpox-1
Cetaceanpox-2
Steller sealionpox
Camelpox
Cowpox
Monkeypox
Vaccinia
Ectromelia
Variola
Lumpy skin disease
Sheeppox
Goatpox
Muledeerpox


Swinepox
Rabbit fibroma
Rabbit myxoma
Yaba monkeypox
Orf
Bovine pap stom
Canarypox
Fowlpox
Molluscum contagiosum
Harbor sealparapox
Spotted sealparapox
Steller sealionparapox V841
Steller sealionparapox V842
Steller sealionparapox V1386


00.0
92.3
81.1
84.4
85.0
85.0
85.0
85.0
85.0
80.6
79.4
80.0
80.4
81.1
79.9
83.9
81.7
73.9
77.8
69.4
68.3
75.0
74.2
74.7
74.7
74.2
74.2


92.3
100.0
82.8
85.0
85.6
85.6
85.6
85.6
85.6
81.1
80.0
80.6
80.4
82.2
78.8
82.8
81.7
75.6
78.3
70.6
69.4
74.4
75.3
75.8
76.4
75.3
74.8


81.1
82.8
100.0
82.2
83.3
83.3
83.3
83.3
83.3
80.6
79.4
80.0
81.0
80.6
78.2
81.1
81.1
73.9
78.9
68.9
70.6
72.2
75.8
76.4
78.1
75.8
74.7


SSLPPV
V841
74.7
76.4
78.1
77.5
78.7
78.6
78.6
78.7
78.7
74.7
73.6
74.8
75.3
75.3
72.3
75.8
74.2
91.6
93.3
68.0
67.4
71.9
91.6
92.1
100.0
91.6
91.0


Chordopoxvirinae subfamily of viruses. Values correspond


SSLPPV
V842
74.2
75.3
75.8
75.3
76.4
76.4
76.4
76.4
76.4
75.8
74.7
75.8
76.5
74.7
72.9
75.8
71.9
92.7
92.1
65.7
66.3
70.2
98.9
99.4
91.6
100.0
94.9


SSLPPV SSPPV HSPPV


V1386
74.2
74.8
74.7
76.4
77.5
77.5
77.5
77.5
77.5
75.8
74.7
75.8
75.3
75.3
72.9
76.4
73.6
92.7
92.1
66.3
65.7
70.3
94.9
95.5
91.0
94.9
100.0


V688
74.7
75.8
76.4
76.4
77.5
77.5
77.5
77.5
77.5
76.4
75.3
76.4
76.5
75.3
73.4
76.4
73.0
93.3
92.7
66.3
66.3
70.8
99.4
100.0
92.1
99.4
95.5


V465
74.2
75.3
75.8
75.8
77.0
77.0
77.0
77.0
77.0
75.8
74.7
75.8
75.9
75.3
72.9
75.8
72.5
92.7
92.1
66.3
66.3
70.8
100.0
99.4
91.6
98.9
94.9


1













Table 3-10. Pair-wise comparisons of the nucleotide sequences of the DNA topoisomerase gene fragments of poxviruses of various
genera within the Chordopoxvirinae subfamily of viruses. Values correspond to percent identity between two nucleotide
sequences.

CPV-1 CPV-2 SSLPV SSLPPV SSLPPV SSLPPV SSPPV HSPPV
V841 V842 V1386 V688 V465
Cetaceanpox-1 100.0 84.3 70.9 51.7 53.5 53.2 47.6 53.8
Cetaceanpox-2 84.3 100.0 72.1 59.0 52.3 54.3 56.7 56.5
Steller sealionpox 70.9 72.1 100.0 51.7 54.4 52.6 48.0 52.6
Camelpox 64.5 68.0 70.1 55.2 55.5 54.9 52.0 55.5
Cowpox 63.4 67.2 70.6 55.2 54.9 55.5 51.2 54.9
Monkeypox 64.2 68.0 71.2 55.2 54.9 54.4 51.2 54.9
Vaccinia 64.5 68.0 70.1 55.2 55.5 54.9 52.0 55.5
Ectromelia 64.5 68.0 70.9 55.2 55.5 54.9 52.0 55.5
Variola 64.8 68.3 71.2 54.9 55.2 54.7 51.6 55.2
Lumpy skin disease 69.5 68.3 72.1 52.0 53.2 51.5 45.5 51.7
Sheeppox 68.6 68.6 70.9 52.0 53.5 52.3 45.9 52.0
Goatpox 69.2 68.0 71.5 52.3 53.2 51.5 45.5 51.7
Muledeerpox 66.9 68.0 72.7 53.8 55.5 55.2 50.8 54.7
Swinepox 73.0 72.4 73.0 55.5 55.5 53.8 51.6 55.2
Rabbit fibroma 64.2 63.1 65.4 55.5 54.9 57.3 50.0 54.9
Rabbit myxoma 63.6 65.1 65.7 57.3 57.8 60.2 51.6 57.6
Yaba monkeypox 69.6 68.0 69.4 54.6 53.8 51.5 47.6 53.5
Orf 50.0 50.0 47.1 72.6 82.3 82.3 81.0 83.7
Bovine pap stom 51.2 50.6 48.5 72.3 81.4 81.4 80.2 83.4
Canarypox 62.8 64.0 68.0 52.3 52.9 54.9 50.0 52.3
Fowlpox 61.0 61.3 68.0 53.8 54.4 52.0 50.0 53.8
Molluscum contagiosum 50.3 50.3 49.1 61.6 68.0 65.4 65.4 68.3
Harbor sealparapox 53.8 56.5 52.6 75.5 96.0 85.0 95.6 100.0
Spotted sealparapox 47.6 56.7 48.0 71.9 93.7 84.3 100.0 95.6
Steller sealionparapox V841 51.7 59.0 51.7 100.0 74.4 70.6 71.9 75.5
Steller sealionparapox V842 53.5 52.3 54.4 74.4 100.0 82.3 93.7 96.0
Steller sealionparapox V1386 53.2 54.3 52.6 70.6 82.3 100.0 84.3 85.0













Table 3-11. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences of the DNA topoisomerase
gene fragments of poxviruses of various genera within the Chordopoxvirinae subfamily of viruses. Values correspond to
percent identity between two amino acid sequences.

CPV-1 CPV-2 SSLPV SSLPPV SSLPPV SSLPPV SSPPV HSPPV
V841 V842 V1386 V688 V465
Cetaceanpox-1 100.0 85.1 60.5 56.1 57.0 57.9 46.4 57.9
Cetaceanpox-2 85.1 100.0 67.5 55.3 56.1 57.0 45.1 57.0
Steller sealionpox 60.5 82.8 100.0 57.0 57.0 57.0 46.3 58.9
Camelpox 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0
Cowpox 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0
Monkeypox 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0
Vaccinia 64.6 66.8 69.0 57.5 62.8 61.9 57.3 63.7
Ectromelia 64.9 66.7 69.3 57.9 63.2 62.3 57.3 64.0
Variola 65.8 67.5 70.2 57.9 62.3 61.4 56.1 63.2
Lumpy skin disease 63.2 64.9 69.3 55.3 59.6 58.8 48.8 59.6
Sheeppox 63.2 64.9 69.3 55.3 59.6 58.8 48.8 59.6
Goatpox 63.2 65.8 69.3 55.3 59.7 58.8 48.8 59.6
Muledeerpox 62.3 64.0 69.3 57.0 61.4 60.5 52.4 62.3
Swinepox 64.9 65.8 67.5 58.8 59.6 59.6 52.4 60.5
Rabbit fibroma 62.3 62.3 64.9 58.8 57.8 57.0 48.8 58.8
Rabbit myxoma 64.0 62.3 64.9 58.7 58.8 57.0 48.8 58.8
Yaba monkeypox 63.2 60.5 62.3 57.9 59.6 57.9 50.0 60.5
Orf 55.3 54.4 53.5 79.1 89.7 91.4 85.7 87.9
Bovine pap stom 58.7 56.1 55.3 80.9 89.7 92.2 87.0 90.5
Canarypox 57.1 57.9 64.0 54.5 56.1 55.3 50.0 56.1
Fowlpox 55.3 51.8 61.4 52.6 56.1 55.3 46.3 56.1
Molluscum contagiosum 56.1 55.3 55.3 61.4 62.3 62.3 58.5 61.4
Harbor sealparapox 57.9 57.0 58.9 79.1 97.4 91.4 95.2 100.0
Spotted sealparapox 46.4 45.1 46.3 75.9 95.2 89.3 100.0 95.2
Steller sealionparapox V841 56.1 55.3 57.0 100.0 80.9 81.6 75.9 79.1
Steller sealionparapox V842 57.0 56.1 57.0 80.9 100.0 91.4 95.2 97.4
Steller sealionparapox V1386 57.9 57.0 57.0 81.6 91.4 100.0 89.3 91.4













Table 3-12. Pair-wise comparisons of the amino acid sequences deduced from the nucleotide sequences from the DNA
topoisomerase gene fragments of poxviruses of various genera within the Chordopoxvirinae subfamily of viruses. Values
correspond to percent similarity between two amino acid sequences.

CPV-1 CPV-2 SSLPV SSLPPV SSLPPV SSLPPV SSPPV HSPPV
V841 V842 V1386 V688 V465
Cetaceanpox-1 100.0 90.4 75.4 67.5 66.7 67.5 57.3 65.8
Cetaceanpox-2 90.4 100.0 77.2 68.4 66.7 67.5 57.3 65.8
Steller sealionpox 75.4 82.8 100.0 65.8 70.2 70.2 62.2 71.1
Camelpox 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6
Cowpox 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6
Monkeypox 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6
Vaccinia 75.2 77.0 76.1 70.8 73.5 73.5 70.7 74.3
Ectromelia 75.4 77.2 76.3 71.1 73.7 73.7 70.7 74.6
Variola 76.3 78.1 77.2 71.1 72.8 72.8 69.5 73.7
Lumpy skin 73.7 72.8 75.4 70.2 71.9 71.9 65.9 71.9
Sheeppox 73.7 72.8 75.4 70.2 71.9 71.9 65.9 71.9
Goatpox 73.7 72.8 75.4 71.1 71.9 71.9 48.8 71.9
Muledeerpox 72.8 72.8 75.4 68.4 70.2 69.3 64.6 71.1
Swinepox 75.4 74.5 79.8 73.7 73.7 72.8 68.3 74.6
Rabbit fibroma 71.9 74.6 72.8 71.1 68.4 66.7 59.8 68.4
Rabbit myxoma 71.9 74.6 72.8 71.1 68.4 66.7 59.8 68.4
Yaba monkeypox 70.0 70.2 73.7 69.3 70.2 69.3 63.4 71.1
Orf 67.5 67.5 70.2 86.1 93.1 94.8 90.5 93.1
Bovine pap stom 69.3 68.4 70.2 87.0 92.2 94.0 91.7 92.2
Canarypox 70.2 68.4 74.6 70.2 71.9 71.9 65.9 71.9
Fowlpox 69.3 65.8 73.7 68.4 70.2 70.2 63.4 70.2
Molluscum 68.4 67.5 70.2 69.3 71.9 71.9 69.5 71.1
Harbor sealparapox 65.8 75.3 75.8 86.1 99.1 94.0 98.8 100.0
Spotted sealparapox 57.3 75.8 76.4 80.7 97.6 91.7 100.0 98.8
Steller sealionparapox V841 67.5 76.4 78.1 100.0 86.1 86.0 80.7 86.1
Steller sealionparapox V842 66.7 75.3 75.8 86.1 100.0 94.0 97.6 99.1
Steller sealionparapox V1386 67.5 74.8 74.7 86.0 94.0 100.0 91.7 94.0










Table 3-13. Pair-wise comparisons of the nucleotide sequences obtained from the
major envelope protein gene fragments of marine parapoxviruses within the
Chordopoxvirinae subfamily of viruses.Values correspond to percent
identity between two nucleotide sequences.

SSLPPV SSLPPV SSLPPV SSPPV HSPPV
V841 V842 V1386 V688 V465

Harbor sealparapox 93.4 93.3 93.3 93.1 100.0
Spotted sealparapox 91.6 91.4 95.8 100.0 93.1
Steller sealionparapox V841 100.0 99.8 94.8 91.6 93.4
Steller sealionparapox V842 99.8 100.0 94.9 91.4 93.3
Steller sealionparapox V1386 91.6 91.4 100.0 95.8 93.3

Table 3-14. Pair-wise comparisons of the amino acid sequences deduced from the
nucleotide sequences from the major envelope protein gene fragments of
marine parapoxviruses within the Chordopoxvirinae subfamily of viruses.
Values correspond to percent identity between two amino acid sequences.


SSLPPV SSLPPV SSLPPV SSPPV HSPPV
V841 V842 V1386 V688 V465

Harbor sealparapox 97.0 96.5 96.0 96.0 100.0
Spotted sealparapox 93.9 93.4 100.0 100.0 96.0
Steller sealionparapox V841 100.0 99.5 93.9 93.9 97.0
Steller sealionparapox V842 99.5 100.0 93.4 96.5 96.5
Steller sealionparapox V1386 93.9 93.4 100.0 96.0 96.0

Table 3-15. Pair-wise comparisons of the amino acid sequences deduced from the
nucleotide sequences of the major envelope protein gene fragments of
marine parapoxviruses within the Chordopoxvirinae subfamily of
viruses.Values correspond to percent similarity between two amino acid
sequences.


SSLPPV SSLPPV SSLPPV SSPPV HSPPV
V841 V842 V1386 V688 V465

Harbor sealparapox 98.5 98.0 98.5 98.5 100.0
Spotted sealparapox 97.0 96.5 100.0 100.0 98.5
Steller sealionparapox V841 100.0 99.5 97.0 97.0 98.5
Steller sealionparapox V842 99.5 100.0 96.5 98.0 98.0
Steller sealionparapox V1386 97.0 96.5 100.0 98.5 98.5














CHAPTER 4
DISCUSSION


All of the cetacean poxvims skin lesions examined in the present study conformed

with the typical tattoo lesion appearance. Some lesions were over 2.0 cm in diameter,

while some samples of lesions consisted of 8 mm diameter skin biopsies. We cannot

make any conclusions regarding the specific stage of infection represented in each lesion,

other than to observe that some lesions showed more definite hyperpigmentation of the

skin, or more clearly defined edges surrounding the lesion. We did not find any

association between cetacean lesion appearance and positive pox PCR results, or

poxvirus DNA sequences obtained. Pinniped parapoxviruses are associated with skin

lesions that resemble those reported for other terrestrial parapoxviruses such as those that

are seen in orf, pseudocowpox, and bovine popular stomatitis, both histologically and in

patterns of disease progression (Wilson et al., 1972; Hadlow et al., 1980; Hicks and

Worthy, 1987). The prevalence of parapoxvirus infection in pinnipeds remains

unreported; however, skin lesions associated with these infections are frequently

encountered in both stranded pinnipeds brought into rehabilitation centers and in captive

pinnipeds (Wilson et al., 1969; Wilson et al., 1972; Hadlow et al., 1980; Osterhaus et al.,

1990; Simpson et al., 1994; Muller et al., 2003). Hicks and Worthy, (1987), reported that

five of 11 recently weaned grey seal (Halochoerus grypus) pups collected for a nutritional

study developed parapox lesions after 1 4 weeks in captivity. The appearance of these

lesions in animals that appeared otherwise healthy at the time of collection, suggests that









while the pups may be exposed to the parapoxvirus in the wild population, the viral

infection may be exacerbated under stressful conditions brought on in captivity. Pinniped

skin lesions examined in this study were collected during Steller sea lion capture-release

studies or by members of the Alaska Department of Fish and Game and the Marine

Mammal Stranding Center in New Jersey. A gross distinction between the appearance of

lesions associated with Steller sea lion poxvirus and lesions associated with pinniped

parapoxvirus, could not be made.

While histopathology and electron microscopy are useful in confirming the

presence of typical microscopic poxvirus lesions and in the visualization of viral particles

in cetacean lesions (Flom and Houk, 1979; Geraci et al., 1979; Smith, 1983; Baker,

1992a,b; Van Bressem et al., 1993), they offer little information about the type of

poxvirus involved. The primary objective of this study was to develop a diagnostic

strategy based on extraction of total DNA from lesions, PCR assay using the extracted

DNA as template, and sequencing of the amplified fragments, to detect and characterize

poxviruses in cutaneous lesions of cetaceans and pinnipeds. The first step in creating a

PCR protocol was to design oligonucleotide primers that would anneal to targeted genes

in the template viral DNA present in cutaneous lesions. Problems encountered in

designing primers to target cetacean and pinniped poxvirus genes, stemmed from the lack

of any available genetic data pertaining to marine mammal poxviruses. Despite the

absence of sound antigenic and molecular data, most previous work using electron

microscopy on cetacean poxviruses has repeatedly implicated them as members of the

orthopoxvirus genus. In addition, one study reported a mixed parapox and orthopox virus

infection in a grey seal, based on the same techniques (Osterhaus et al., 1990). In the









present study, histopathologic examination of the two Steller sea lion skin lesions

revealed a similar appearance to lesions of the northern fur seal and South American sea

lions, characterized by dermal nodules of hyperplastic epithelial cells versus the raised

plaque-like lesions described in harbor and grey seal lesions (Wilson and Poglayen-

Neuwall, 1971; Wilson et al., 1972; Hicks and Worthy, 1987; Osterhaus et al., 1990,

1994). Electron microscopy performed on the two Steller sea lion skin lesions revealed

the presence of poxvirus virions with morphologic characteristics consistent with

published reports of orthopox viruses (Moss, 2001). However, sequencing of amplified

fragments showed that most likely, these viruses are species specific poxviruses of Steller

sea lions and not orthopoxviruses. Histopathologic and electron microscopic

examination of the 10 positive cetacean skin lesions was not performed due to poor

sample quality and in general, improper sample preservation.

Because of their high level of conservation within the Chordopoxvirinae, the DNA

polymerase and DNA topoisomerase I genes were targeted for the design of

oligonucleotide primers for PCR. Specifically, nucleotide sequences within regions of

the open reading frame of these genes that were highly conserved within members of the

Orthopox, Suipox, and Capripox genera were targeted with consensus primers to drive

the amplification of approximately 543-bp in the case of the DNA polymerase gene and

344-bp for the DNA topoisomerase I gene. Sequences of the Orthopox, Suipox, and

Capripox genera were obtained from the GenBank database and through the website of

the National Center for Biotechnology Information (NCBI). The amount of sequence

generated by fragments of the above sizes is usually sufficient to characterize viruses

molecularly and assign them to proper virus genera and species, when derived from genes









that exhibit high levels of conservation (Ropp et al., 1995; Zanotto et al., 1996; Becher et

al., 2002; McGeoch et al., 2000).

Most pinniped poxviruses have long been considered probable members of the

Parapox genus. Inoshima et al., (2000), validated consensus primers that target a 596-bp

gene fragment of the major envelope protein of parapoxviruses in ungulates. Using these

primers in a PCR protocol, initially with suboptimal annealing temperatures, we were

able to identify parapoxvirus positive samples from pinniped skin lesions and confirm the

usefulness of the primers. A nucleotide alignment of orf and BPS viruses DNA

sequences available in the NCBI was used to design PCR primers targeting a 536-bp

fragment of the DNA polymerase gene and a 350-bp fragment of the DNA topoisomerase

of parapoxviruses. These primers effectively amplified the respective genes of pinniped

parapox viruses, confirming the diagnoses made using the major envelope protein gene

primers. Our results expand the molecular diagnosis tools as applicable to parapoxvirus,

and make possible a wider genetic analysis comprising two more genes. PCR protocols

were developed using these primers at suboptimal annealing temperatures in order to

maximize the chances of amplifying the cetacean and SSL poxvirus and pinniped

poxvirus genes. Once each primer set was tested for reactivity using positive cetacean

and/or pinniped poxvirus DNA, each protocol was optimized to produce a single

amplicon, usually by raising the annealing temperature of PCR until the desired reactivity

was obtained.

Positive samples were identified by the presence of a single amplicon of the

expected size. DNA sequence was obtained by two methods; Firstly, cleaning of the

PCR product followed by direct sequencing, and/or secondly, sequencing of the cloned









PCR product in the bacterial plasmid vector, PCR-Topo2.1. The first method was used

when the amplified fragments were unique and allowed for the rapid diagnosis of

poxvirus infection, and for verifying DNA sequences obtained from cloned products,

when disparities between two or more cloned sequences were found. All samples that

yielded positive results were later cloned, to obtain full sequences and to preserve

valuable DNA products, as the amount of total DNA obtained from lesions was usually

small and rapidly exhausted after multiple uses.

Ten cetacean skin lesions were found to contain amplifiable poxvirus DNA using

the PCR protocols and DNA sequencing strategies described above. The identified

positive samples represented two different groups of cetacean poxviruses, provisionally

referred to as CPV-1 and CPV-2. Viruses in the CPV-1 corresponded to the poxvirus

DNA polymerase and DNA topoisomerase sequences obtained from four species of

dolphins while the CPV-2 virus corresponded to the DNA polymerase and DNA

topoisomerase sequences of the bowhead whale (Balaena mysticetus) poxvirus. The

same PCR protocols also amplified poxvirus DNA from two Steller sea lion skin lesions

indicating the existence of a unique and most likely, species specific, Steller sea lion

poxvirus (SSLPV). The three PCR assays for pinniped parapoxvirus allowed the

identification of six positive skin lesion samples harvested from one harbor seal

(HSPPV), two spotted seals (SSLPPV) and three Steller sea lions (SSLPPV). Although

none of the Steller sea lions examined in this study showed evidence of a dual infection

of both pox and parapoxviruses, we speculate that a dual infection could occur.

Mammalian species that have been documented to be afflicted with multiple poxvirus

species, belonging to different genera, include cattle,sheep and camels (Robinson and









Mercer, 1995; Inoshima et al., 2000; Moss, 2001). In cattle, infections with

pseudocowpox virus, a member of the Parapox genus, and cowpox virus, a member of

the Orthopox genus, have been observed (Pickup et al., 1982; Buller and Palumbo, 1991).

In sheep, orf virus, of the Parapox genus, and sheeppox, of the Capripox genus, have

been observed (Inoshima et al., 2000; Hosamani et al., 2004). In camels, camelpox, a

notable member of the Orthopox genus, has been observed, as well as camel parapox

virus (Robinson and Mercer, 1995; Gubser and Smith, 2002).

Nucleotide sequences and their deduced amino acid sequences obtained from all

poxvirus positive samples were entered into the GenBank database and compared using

pairwise and multiple alignment functions from the GCG Wisconsin Package. Pairwise

comparisons were made between sequences obtained from each targeted gene of each of

the cetacean and pinniped pox and parapox viruses, to sequences available in the

GenBank and available in the NCBI database, representing several terrestrial poxviruses

within the Chordopoxvirinae.

Considering first the DNA polymerase comparisons, the cetacean poxviruses share

the highest homology among themselves, with a nucleotide identity of 84.4% (Table 3-7)

and an amino acid identity of 89.0% (Table 3-8). The nucleotide identities described in

Table 3-1 indicate that both CPV-1 and CPV-2 share the second closest identities to the

SSL poxvirus, with identities of 75.7 and 77.0%. Following the SSL pox virus, CPV-1

and CPV-2 are most closely related to members of the Orthopox genus, with nucleotide

identities ranging from 71.6 to 76.2% (Table 3-7). These viruses may have evolved from

a common ancestor as species specific marine poxviruses, prior to the evolution of some

of the terrestrial orthopoxviruses such as camelpox and some strains of the variola virus









(Afonso et al., 2002, Gubser and Smith, 2002). The cetacean poxviruses and SSL

poxvirus shared the least homology with members of the Avipox and Parapox genera,

with nucleotide identities below 53.2% (Table 3-7). These findings are supported by a

previous phylogenetic study demonstrating the distant relationship of the orthopoxviruses

to the avipoxviruses (Gubser et al., 2004). The pinniped parapox viruses shared highest

nucleotide identities among themselves (Table 3-7). Notable are the nucleotide identities

of 98.3 and 98.7% of one Steller sea lion, (V842), when compared to the harbor and

spotted seal sequences (Table 3-7). The significance and interpretation of the identities

are difficult to ascertain, as Steller sea lions and spotted seals inhabit northwest Pacific

waters, while the harbor seal originated from northeast Atlantic waters. SSL V842

shared only 78.9 and 84.3% nucleotide identity to the other two SSL sequen ces. The

nucleotide and amino acid identities between the harbor and spotted seals are above 98%

(Table 3-7). These results suggest that the SSPPV, HSPPV and SSLPPV may have

originated from a common ancestor and, diverged as they evolved with their host species.

The pinniped parapoxviruses share the least homology to avipox viruses, as would be

expected based on previous phylogenetic analysis of Chordopoxvirinae (Gubser et al.,

2004). The amino acid identities represented in Table 3-8 show slightly different

homologies. This is due to the nature of flexibility or degeneracy in the protein or amino

acid code. In the nucleotide comparisons, each discrepancy between two nucleotide

sequences is reported as a difference, whereas in translation to a protein sequence, a

nucleotide substitution may be silent, causing no amino acid change, and thus no

difference between the two sequences. Results from Table 3-8 indicate that the cetacean

poxviruses are most homologous to each other, with the next closest homology being to









the orthopox viruses, followed by the SSL poxvirus. The Avipox and Parapox genera are

consistently, the least homologous to the cetacean poxviruses. Protein identities of the

pinniped parapoxviruses are highest to orf and BPSV, with the exception of the harbor

and spotted seal poxviruses that share 99.4% identity to each other (Table 3-8).

Variations in the 3 Steller sea lion DNA polymerase sequences are apparent in the protein

identities with ranges from 85.4 to 91.0% (Table 3-8), suggesting the existence of more

than one strain or type of Steller sea lion parapoxvirus. Comparisons among DNA

polymerase protein similarities are reported in Table 3-9. Protein similarity comparisons

offer a means to weigh the significance of observed amino acid differences. For

example, the substitution of a basic amino acid for an acidic amino acid may cause a

more significant functional change than a basic to basic amino acid substitution. The

relevance of viewing the protein similarities of the gene sequences reported in Table 3-9

is simply to ascertain the significance of the amino acid differences indicated by the

protein identities in Table 3-8 (Needleman and Wunsch, 1970). The homology patterns

observed by looking at protein similarities agree with those reported for the protein

identities, and warrant no further discussion.

Considering next, the DNA topoisomerase gene comparisons, the overall

nucleotide identities are lower than those observed in the DNA polymerase comparisons,

indicating a lesser degree of conservation in the DNA topoisomerase gene when

compared to the DNA polymerase gene, within the Chordopoxvirinae (Table 3-10). The

CPV-1 and CPV-2 fragments share 84.3% nucleotide identity with each other, followed

by identities to swinepox virus of 72.4 to 73.0% (Table 3-10). The next closest

homology is to SSL poxvirus followed by members of the Capripox genus (Table 3-10).









The difference in the pattern of homology between the DNA topoisomerase and DNA

polymerase genes demonstrates the variability in gene evolution. Viral genes evolve at

varying rates, depending on need to adapt to new host or environmental stresses (Upton

et al., 2003; Gubser et al., 2004;). The pinniped parapoxviruses are consistently closest

in homology to orf, BPSV and to each other (Table 3-10). Steller sea lion, V842,

demonstrated a higher homology to the harbor and spotted seal sequences, than to the

other (V841 and V1386) SSL sequences (Table 3-10), as seen in the DNA polymerase

gene comparisons. The variance of the DNA topoisomerase amino acid identities from

the DNA polymerase amino acid identities mimics these differences in the nucleotide

identity tables. Pairwise comparisons of the amino acid identities indicate homologies of

CPV-1 and CPV-2 to the Orthopox genus ranging from 64.6 to 67.5% (Table 3-11).

CPV-1 shows only 60.5% amino acid identity to SSL poxvirus, while CPV-2 shows an

amino acid identity to SSL poxvirus of 82.8% (Table 3-11). These different identities

represent the differences in the evolutionary rates between the DNA polymerase and

DNA topoisomerase genes examined in this study. However, these results indirectly

confirm the differences between the cetacean poxviruses and indicate that CPV-2 is more

closely related to the SSL poxvirus.

The major envelope protein gene (MEP) pairwise comparisons were made using

exclusively the pinniped parapox gene sequences generated in this study. Attempts to

amplify the MEP gene of cetacean and SSL poxviruses were unsuccessful, limiting the

scope of the comparisons. The problems encountered in amplifying the MEP gene from

the cetacean and SSL poxviruses stem from the degree of variation found between these

novel poxviruses and other terrestrial poxviruses. The MEP gene of poxviruses is more









variable than the DNA polymerase and DNA topoisomerase genes (Upton et al., 2003).

Primers designed based on the available MEP DNA sequences of other terrestrial

poxviruses, most likely did not amplify the cetacean or SSL poxvirus MEP due to the

greater degree of variation within the gene. The MEP gene comparisons demonstrate

nucleotide and amino acid identities ranging from 91.4 to 99.8% (Table 3-13), and 93.4

to 99.5%, respectively (Table 3-14). The variance observed in the DNA polymerase and

topoisomerase gene sequence comparisons were absent in the MEP comparisons and the

homologies in the latter were more uniform. The MEP gene of poxviruses is typically

more variable than those involved in DNA replication, as it is involved in host specificity,

viral adhesion to the host cell, and possibly evasion of host immunity (Smith et al., 2002).

Partial nucleotide and deduced amino acid sequences have been used to make a

distinction between different species of parapoxvirus, such as orf, BPSV and

pseudocowpox (Inoshima et al., 2000). Our results showed less variation in the pinniped

parapoxvirus MEP gene fragments than the variation reported between homologous MEP

gene fragments of orf, BPSV, and pseudocowpox (Inoshima et al., 2000; Becher et al.,

2002; Delhon et al., 2004). These results may be due to the specific region of the gene

amplified by the MEP PCR primers. Certain areas of the MEP gene are likely more

conserved in DNA sequence, such as those encoding the hypdrophobic regions of the

protein, found within the envelope lipid bilayer (Silverman, 2005). In addition, the

poxviruses of marine mammals may not have succumbed to the same selective pressures

encountered over hundreds of years by the terrestrial poxviruses, such as vaccination,

husbandry and environmental conditions that stimulate genetic evolution and mutation in

the viral genome. The high degree of conservation observed in the pinniped parapoxvirus









MEP sequences can be understood after considering the nature of these consensus PCR

primers, designed to amplify the IMEP gene fragment of all parapoxviruses.

Overall, it can be inferred, based on pairwise comparisons, that CPV-1, CPV-2 and

SSLPV are most closely related to the orthopoxviruses, and that the pinniped

parapoxviruses are most closely related to the known terrestrial parapoxviruses of

ruminants. Phylogenetic trees were constructed using the deduced amino acid sequences,

to further determine the genetic relatedness of the marine mammal poxviruses to known

virus members of the Chordopoxvirinae.

The phylogenetic studies described in Upton et al. (2003), and Gubser et al. (2004),

provided new insight into novel methods of analysis for uncharacterized poxviruses, such

as those described in this thesis. In the present study, phylogenetic analysis was

performed based on partial proteins of the DNA polymerase, DNA topoisomerase and

major envelope protein genes of several members of the Chordopoxvirinae, including

CPV-1, CPV-2, SSLPV, SSLPPV, SSPPV, and HSPPV (Figures 3-23A&B, 3-24A&B

and 3-25A&B).

The bootstrapped cladogram and the radial divergence tree representing the DNA

polymerase protein sequences indicate that the cetacean poxviruses form a distinct genus

within the Chordopoxvirinae, separate from the Orthopox genus and from SSLPV,

indicating a species specific poxvirus.. The SSLPV falls into a clad by itself, outside of

the Orthopox lineage group. The pinniped parapoxviruses group, as expected, within the

Parapox genus (Figure 3-23A). These results were reiterated in the divergence tree,

revealing the ancestry of the DNA polymerase gene fragments within the

Chordopoxvirinae. This tree clearly showed genetic divergence from the ancestor









branch, of SSLPV first, followed by the cetacean poxviruses, and finally, the

differentiation of the orthopoxviruses (Figure 2-23B). Among the parapoxviruses, the

SSLPPV sequences show three different points of divergence. SSLPPV(V841) diverged

first, followed by orf and BPSV. SSLPPV(V1386), SSLPPV(V842), SSPPV, and

HSPPV are branched together; however, SSLPPVv1386 diverges from the branch by

itself. These results strongly suggest the existence of three different SSL parapoxviruses,

supporting conclusions drawn from the pairwise comparison tables. These are the first

sequences of SSL parapoxviruses ever obtained for the DNA polymerase gene.

Phylogenetic trees constructed based on partial proteins of the DNA topoisomerase

gene indicate that CPV-1 and CPV-2 form a group separate from any other, as does

SSLPV strongly suggesting that the viruses could be assigned to new genera within the

Chordopoxvirinae subfamily of viruses (Figure 3-24A). All SSLPPVs, HSPPV, and

SSPPV clad inside the parapox group. The radial divergence tree representing the DNA

topoisomerase protein fragments differs from the DNA polymerase divergence tree

(Figure 3-24B). The topoisomerase divergence tree shows the orthopox viruses as having

a separate lineage from CPV-1, CPV2 and SSLPV, rather than the three groups diverging

from a single branch. SSLPV is depicted closer to the Orthopox group, where as the

DNA polymerase tree depicted the cetacean poxviruses closer to the Orthopox group.

These results are further exemplification that the DNA topoisomerase I gene may have

evolved at a different rate and direction than the DNA polymerase gene.

The final phylogenetic analyses performed were based on the partial protein

sequences of the major envelope protein (MEP) (Figures 3-25A and 3-25B). Poxvirus

MEP sequences were not obtained, and are, consequently, absent in the MEP









phylogenetic trees. Examination of the radial divergence tree indicates a closer

relationship between the SSLPPV MEP fragments than those seen for the DNA

polymerase and DNA topoisomerase genes (Figure 3-25B). These results agree with

results obtained from the pairwise comparison tables. The radial tree (3-25B) also shows

clear divergence of the pinniped parapox group, including a recently published sequence

for the Weddel seal (Tryland et al., 2005), and from the other parapox species, namely

orf, BPS, pseudocowpox, and parapox of red deer viruses.

Clear definitions for nucleotide and/or amino acid identity requirements necessary

for the assignment of novel poxviruses to an appropriate genus are currently lacking. In

the case of the orthopoxviruses, specifically variola, vaccinia and cowpox, the nucleotide

identities are >90% (Goebel et al., 1990; Gubser et al., 2004). The newly identified

cetacean poxviruses, CPV-1 and CPV-2, share only 84% nucleotide identity in the

targeted regions of the DNA polymerase and DNA topoisomerase genes, and their

nucleotide or amino acid identities with any members of the known poxvirus genera are

even lower. Phylogenetic and evolutionary analysis of the DNA polymerase and DNA

topoisomerase gene fragments show that although the cetacean poxviruses and the

members of the orthopoxvirus genus originate from a common node, there is a clear

divergence of the cetacean poxviruses into a unique branch. It is clear from both the

bootstrapped and divergence phylograms, that there is a greater degree of divergence

between the members of the Capripox genus, namely goatpox, sheeppox and lumpy skin

disease viruses, than is observed between the two cetacean poxviruses. We infer that the

cetacean poxviruses, as evidenced by their genetic isolation from all of the known









poxvirus genera, as well as the formation of a unique branch in the phylogenetic trees,

should constitute a new genus within the Chordopoxvirinae subfamily of viruses.

The nucleotide and/or amino acid identity requirements for the classification of

poxviruses as strains, species and genera vary depending on several factors. These

factors include the gene from which the DNA sequence data was derived, the poxvirus

genus under consideration and the length of the available DNA sequence. Becher et al.

(2002), suggested the inclusion of sealpox virus as a new species of parapoxvirus based

on nucleotide identities that ranged from 75 -79% when a 594-bp fragment of the major

envelope protein was compared with homologous fragments from other ungulate

parapoxviruses. Damaso et al. (2000), concluded that an emergent poxvirus, Cantagalo

virus, constituted a strain of vaccinia virus and not a separate species of orthopoxvirus

based on 98% nucleotide identity of a 950-bp fragment of the hemagglutinin gene when

compared to other vaccinia viruses. DNA sequences derived from highly conserved

genes or regions within a gene, may have different requirements for the classification of

strain, species and genus than genes or gene regions possessing lesser degrees of

conservation. For the purposes of this study, we consider DNA polymerase and DNA

topoisomerase gene sequences that possess a nucleotide and amino acid difference >10%

when compared to homologous sequences of terrestrial poxviruses, as indication of a new

genus within Chordopoxvirinae. Pairwise comparisons showing 90-100% nucleotide and

amino acid identity between poxviruses within a genus are considered separate strains.

Our pairwise comparisons suggest the existence of more than one strain of parapoxvirus

occurring in Steller sea lions.









Whenever possible, skin lesions that yielded a positive PCR result were inoculated

onto cell culture to attempt virus isolation. Numerous attempts have been made in our

laboratories and by others (VanBressem et al., 1999), however, to date, there are no

reports of a cetacean or Steller sea lion poxvirus being successfully isolated in cell

culture. While parapoxviruses have been isolated from pinnipeds in primary cell culture

of pinniped tissues (Osterhaus et al., 1990; Osterhaus et al., 1994; Nettlleton et al., 1995),

attempts to isolate the virus from the positive samples diagnosed in our laboratories were

unsuccessful. We attribute these difficulties to an apparent specificity of the cetacean

poxviruses to grow only in cetacean skin cells, and suspect that the cell lines that were

available in this study, did not adequately support viral growth. Another possible cause

for the difficulty in growing these cetacean poxviruses include the low amounts of viable

poxvirus that may have been contained in the few available skin lesion samples. The

parapoxvirus isolated by Nettleton et al. (1995), was grown in primary grey seal kidney

cells, and was passed weekly over 25 days. Osterhaus et al. (1990) reported the use of

primary harbor seal kidney cells to isolate an orthopox and a parapoxvirus from grey seal

skin lesions. However, the orthopoxvirus "was lost" after several passages in culture.

While poxviruses have been thoroughly reported in cetaceans and pinnipeds and repeated

attempts to isolate those viruses have been made, relatively few successes, if any, are

reported. Possible explanations for the difficulties in isolating this virus are poor sample

quality, use of improper cell lines, and lack of ideal media and tissue culturing

conditions, in general.

Only in recent years, has there been an advancement in the understanding of the

genetic characteristics and evolutionary relationships of poxviruses, enabled by the









sequencing of complete poxvirus genomes and improvements in phylogenetic analysis.

The genetic properties and phylogenetic relationships of poxviruses that affect marine

mammals are still relatively unknown, as these viruses are difficult to isolate and

typically, are found in samples that are not readily accessible. Further efforts to isolate

poxviruses from cetacean and pinniped skin lesions are necessary for the advancement in

the characterization of these viruses. The isolation of marine mammal poxviruses would

permit the complete sequencing of the viral genome, development of new assays such as

ELISA, and the simple detection of antibody responses in infected animals. It would also

be possible to target full genes and develop a more detailed understanding of the structure

and function of the proteins they encode. The PCR assays developed as part of this study

will help to rapidly identify cetacean and pinniped pox and parapox viruses that afflict

cetaceans and pinnipeds. The DNA sequences generated from poxvirus and parapoxvirus

after the various PCR assays reported here, constitute a significant advancement in the

molecular genetics of marine poxviruses and represent the first known report of

comprehensive sets of nucleotide and amino acid sequences of novel poxviruses of

cetacean and pinniped pox- and parapoxviruses.















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