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Poxvirus Infections in North American Pinnipeds

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Poxvirus Infections in North American Pinnipeds
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NOLLENS, HENDRIK HANS ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Animals ( jstor )
Antibodies ( jstor )
Infections ( jstor )
Lesions ( jstor )
Lions ( jstor )
Parapoxvirus ( jstor )
Poxviridae ( jstor )
Poxviridae infections ( jstor )
Seals ( jstor )
Seas ( jstor )

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University of Florida
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University of Florida
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Copyright Hendrik Hans Nollens. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2007

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POXVIRUS INFECTIONS IN NORTH AMERICAN PINNIPEDS By HENDRIK HANS NOLLENS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Hendrik Hans Nollens

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ACKNOWLEDGMENTS Funding for this work was provided in part by the UF College of Veterinary Medicine Batchelor Foundation, by a grant from the Florida Fish and Wildlife Conservation Commission, and by John H. Prescott Marine Mammal Rescue Assistance grant number NA03NMF4390410 to E.J. All samples were collected under NMFS Scientific Research Permit nos. 932-1489-00 and 1054-1731-00. Sample collection protocols were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC C403). I would like to thank the staff at The Marine Mammal Center (CA), the Marine Mammal Stranding Center (NJ), Animal Health Center (British Columbia), Mystic Aquarium (CT), Sea World of Orlando (FL), SeaWorld of San Diego (CA) and the Virginia Marine Science Museum (VA) for their participation in this study. I thank Dr. John Wise and Dr. Carie Goertz at the Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, for supplying the seal and sea lion cell cultures. Dr. Andy Mercer of the University of Otago, New Zealand, was very generous with his advice on virus isolation and interpretation of electron microscopy images. Dr. Robert DeLong of the National Marine Mammal Lab, Seattle WA, kindly provided me with sera from free-ranging sea lions. The cidofovir was provided by Chimerix, La Jolla CA. Special thanks go to Dr. Susan D’Costa, Dr. Cindy Prins, Dr. Sayuri Kato, Dr. Steve Cresawn, Dr. Nissin Moussatche, Brad Dilling, Travis Bainbridge, Dr. Marty iii

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Haulena, Denise Greig, Dr. Brian Stacy, Dr. Charles Courtney, Dr. Deke Beusse, Dr. Greg Bossart, Linda Green, Diane Duke, Sally O’Connell and Courtney Watkins for their assistance, in various ways, with this project. I am greatly indebted to the members of my supervisory committee: Dr. Elliott Jacobson, Dr. Richard Condit, Dr. Frances Gulland, Dr. Jorge Hernandez, Dr. Paul Klein and Dr. Mike Walsh. I thank them for their invaluable advice, ideas, support and above all their confidence in my ability all the way through this project. It truly has been a pleasure working with them. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES ............................................................................................................vii LIST OF FIGURES ...........................................................................................................ix ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION..........................................................................................................1 2 PARAPOXVIRUSES OF SEALS AND SEA LIONS MAKE UP A DISTINCT SUBCLADE WITHIN THE GENUS PARAPOXVIRUS...........................................6 Introduction...................................................................................................................6 Materials and Methods.................................................................................................8 Animals and Samples............................................................................................8 Polymerase Chain Reactions (PCR)......................................................................9 Viral Sequence Analyses.....................................................................................10 Results.........................................................................................................................13 Animals and Samples..........................................................................................13 Viral Sequence Analyses.....................................................................................13 Discussion...................................................................................................................15 3 PATHOLOGY AND PRELIMINARY CHARACTERIZATION OF A PARAPOXVIRUS ISOLATED FROM A CALIFORNIA SEA LION.....................27 Introduction.................................................................................................................27 Materials and Methods...............................................................................................28 Case Histories......................................................................................................28 Pathologic Studies...............................................................................................29 Cell Cultures........................................................................................................30 Virus Isolation.....................................................................................................30 Virus Morphology...............................................................................................31 Molecular Characterization of the Virus.............................................................32 Results.........................................................................................................................33 Pathologic Studies...............................................................................................33 Virus Isolation.....................................................................................................34 Virus Morphology...............................................................................................34 Molecular Characterization of the Virus.............................................................35 Discussion...................................................................................................................36 v

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4 SEROEPIDEMIOLOGY OF POXVIRUS INFECTIONS IN CAPTIVE AND FREE-RANGING CALIFORNIA SEA LIONS........................................................46 Introduction.................................................................................................................46 Methods......................................................................................................................48 Positive and Negative Reference Serum Samples...............................................48 ELISA Procedure.................................................................................................49 Diagnostic Performance (Sensitivity and Specificity) of ELISA........................53 Seroepidemiology of Parapoxvirus Infections....................................................53 Results.........................................................................................................................56 Diagnostic Performance (Sensitivity and Specificity) of ELISA........................56 Seroepidemiology of Parapoxvirus Infections....................................................56 Discussion...................................................................................................................58 5 RISK FACTORS ASSOCIATED WITH DEVELOPMENT OF POXVIRUS LESIONS IN HOSPITALIZED CALIFORNIA SEA LIONS...................................68 Introduction.................................................................................................................68 Materials and Methods...............................................................................................70 Results.........................................................................................................................73 Discussion...................................................................................................................74 6 THERAPEUTIC POTENTIAL OF CIDOFOVIR FOR THE TREATMENT OF PARAPOXVIRUS INFECTIONS OF PINNIPEDS..................................................84 Introduction.................................................................................................................84 Materials and Methods...............................................................................................87 Cell Cultures........................................................................................................87 Virus....................................................................................................................87 Compounds and Concentrations..........................................................................89 CPE Reduction Assays........................................................................................89 Virus Yield Assay................................................................................................90 Cytotoxicity Assays.............................................................................................91 Results.........................................................................................................................92 CPE Reduction Assays........................................................................................92 Virus Yield Assay................................................................................................93 Cytotoxicity Assays.............................................................................................93 Discussion...................................................................................................................94 7 CONCLUSIONS.........................................................................................................102 LIST OF REFERENCES.................................................................................................106 BIOGRAPHICAL SKETCH...........................................................................................113 vi

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LIST OF TABLES Table page 2-1. Host species, tentative designations and GenBank accession numbers for genomic sequences of pinniped poxvirus strains analyzed in this study.................19 2-2. Number of animals of each host species from which samples were received (N), number of animals in which a poxvirus was detected (Pos) and poxvirus strain detected using the Ortho, EACP, NACP and Para primer pairs..............................20 2-3. Genetic diversity of parapoxviruses of North American pinnipeds determined using average P distances calculated with MEGA (version 3.1).............................21 4-1. Sample size (N), number of animals with anti-parapoxviral antibodies (pos), antibody prevalence and 95% confidence interval (95% CI) for each age/gender category of California sea lions................................................................................65 4-2. Sample size (N), number of animals with anti-parapoxviral antibodies (pos), antibody prevalence (%) and 95% confidence interval (95% CI) for each sampling location/gender category of California sea lions......................................66 4-3. Sample size, number of animals with anti-parapoxviral antibodies (pos), and antibody prevalence (%) per month of sampling and age category of California sea lions....................................................................................................................67 5-1. Results of the univariable analysis of risk factors prior to admission associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility................................................................................................79 5-2. Results of the univariable analysis of risk factors at admission associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility.......................................................................................................................80 5-3. Results of the univariable analysis of risk factors during hospitalization associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility................................................................................................81 5-4. Results of the multivariable analysis of risk factors associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility.................83 vii

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6-1. Number of infected (CPE) and control (cytotoxicity) CZC-K and CZC-S wells displaying apparent CPE in the presence of the antiviral compounds at concentrations indicated...........................................................................................98 viii

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LIST OF FIGURES Figure page 2-1. Multiple alignment of the partial genomic sequences encoding the putative virion EEV protein (p42K) of the pinniped parapoxvirus strains detected in this study....22 2-1. Continued...................................................................................................................23 2-2. Phylogram depicting the relationship of the pinniped poxviruses to the published members of the genus Parapoxvirus........................................................................24 2-3. Phylogram depicting the relationship between the pinniped poxviruses of the Atlantic and the Pacific Ocean.................................................................................25 2-4. Phylogram depicting the relationship between the poxviruses of Pacific phocids and otariids...............................................................................................................26 3-1. Photomicrograph of a pox lesion (H & E stain, 4X).................................................41 3-2. Transmission electron micrographs of infected cell sea lion kidney cell cultures....42 3-3. Negative contrast electron micrograph......................................................................43 3-4. Multiple sequence alignment of the deduced amino acid sequence of part of the putative virion envelope antigen (p42K)..................................................................44 3-5. Phylogram depicting the relationship of SLPV-1 to the members of the genus Parapoxvirus............................................................................................................45 6-1. Sea lion parapoxvirus (SLPV-1) yield of infected CZC-S cells and incubated in the presence of 0.2, 2, 5 or 20 g/ml of cidofovir for five days...............................99 6-2. Effect of isatin-beta-thiosemicarbazone (A), rifampicin (B), acyclovir (C), cidofovir (D) and phosphonoacetic acid (E) on growth of early passage California sea lion kidney cells..............................................................................100 6-3. Effect of cidofovir (A) and phosphonoacetic acid (B) on growth of early passage California sea lion skin cells..................................................................................101 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy POXVIRUS INFECTIONS IN NORTH AMERICAN PINNIPEDS By Hendrik Hans Nollens December 2005 Chair: Elliott Jacobson Major Department: Veterinary Medicine Poxvirus infections are a common complication in the rehabilitation of stranded, diseased pinnipeds. Infections appear to be hospital-acquired and are especially common among juvenile pinnipeds. The objective of this study was to gain the information necessary for developing effective prevention and management strategies for poxvirus epizootics in marine mammal rehabilitation centers. During a molecular survey of pinniped poxviruses, six genetically distinct poxvirus strains were detected. Of these, one strain was isolated on California sea lion kidney cells inoculated with a skin nodule from a California sea lion (Zalophus californianus). The morphology of the poxvirions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative envelope phospholipase confirmed the assignment of all strains to the genus Parapoxvirus. One strain was detected in two host species, suggesting that pinniped parapoxviruses can be transmitted between species. To understand the epidemiology of this disease, an Enzyme-Linked Immunosorbent Assay was developed for determining exposure of California sea lions to parapoxviruses. A x

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seroepidemiological study in a rehabilitation center showed that previous exposure to a parapoxvirus does not confer protection against reinfection, and that the infections are sometimes hospital acquired. Analysis of 761 sera from free-ranging California sea lions indicated that 91% of sea lions are exposed to parapoxviruses at least once over the course of their lifetime, suggesting parapoxviruses are endemic in the California population. Therefore, the presence of pox lesions in fully rehabilitated California sea lions should not preclude their return to their wild population. A study aimed at identifying factors that predispose hospitalized California sea lions to acquiring poxvirus infections showed that sea lions that had previously been hospitalized were 43 times more likely to develop poxvirus lesions. Sea lions with elevated band neutrophil counts (> 0.69 x 10 3 bands/L) upon admission were 20 times less likely to develop poxvirus lesions than sea lions with counts within reference limits. Finally, the antiviral compound cidofovir was shown to have good in vitro antiviral activity pinniped parapoxviruses. Consequently, cidofovir has good therapeutic potential for the treatment of pinniped parapoxvirus infections in humans and animals. xi

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CHAPTER 1 INTRODUCTION The poxviruses (family Poxviridae) represent a unique family of large, double-stranded DNA viruses that have an entirely cytoplasmic life cycle. The family of poxviruses includes several viruses of medical and veterinary importance. In humans, the most notorious member, variola virus, causes smallpox and several domestic animal species are susceptible to infections with cowpox virus, sheeppox virus, and the virus that causes lumpy skin disease (Moss 1996). Poxviruses affecting wildlife include crocodilepoxvirus, fowlpoxvirus, buffalopoxvirus, squirrelpoxvirus, monkeypoxvirus, dolphinpoxvirus and the notorious rabbit and hare myxomavirus (Moss 1996). The poxvirus family is subdivided into the entomopoxvirus and chordopoxvirus subfamilies, which respectively infect insect and vertebrate hosts (Moss 1996). The chordopoxvirus subfamily is further divided into eight genera (Avipoxvirus, Capripoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Suipoxvirus, Yatapoxvirus and Parapoxvirus). The recognized members of the genus Parapoxvirus currently consist of orf virus, bovine papular stomatitis virus, pseudocowpoxvirus and parapoxvirus of red deer of New Zealand. The natural hosts of these viruses are small and large ruminants, but orf virus, bovine papular stomatitis virus and pseudocowpoxvirus also infect humans (Mercer et al. 1997). Tentative members of the parapoxvirus group include camel contagious ecthyma virus, chamois contagious ecthyma virus and a sealpox virus detected in harbor seals from the German North Sea (Moss 1996). 1

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2 The susceptibility of the members of the Pinnipedia, the order of mammals consisting of all seals (family Phocidae), sea lions (family Otariidae) and walruses (family Obodenidae), to poxviruses was first suspected in 1969 when a 1-year old California sea lion (Zalophus californianus) presented with pox-like lesions (Wilson et al. 1969). The presence of poxviruses has since been confirmed in harbor seals (Phoca vitulina) (Wilson et al. 1972), northern fur seals (Callorhinus ursinus) (Hadlow et al. 1980), grey seals (Halichoerus grypus) (Hicks and Worthy 1987), northern elephant seals (Mirounga angustirostris) (Hastings et al. 1989) and South American sea lions (Otaria flavescens) (Wilson and Poglayen-Neuwall 1971). The seal and sea lion parapoxviruses are considered zoonotic agents. Several animal handlers have acquired proliferative “sealpox” lesions on hands and fingers from contact with captive, lesion-bearing seals (Hicks and Worthy 1987, Clark et al. 2005). Affected pinnipeds develop raised nodules, reaching up to 3 cm in diameter, primarily on the head and neck, that may ulcerate and suppurate. In severe cases, lesions often become confluent and spread to the thorax, abdomen and flippers. The body of affected animals can be covered by up to several hundred nodules (Wilson and Poglayen-Neuwall 1971). The nodules are formed by epithelial cells containing large, eosinophilic, intracytoplasmic inclusions consisting of pox virions (Hadlow et al. 1980). Pox lesions are sometimes observed in free-ranging seals and sea lions, but they are especially common as a complication in the treatment of debilitated, stranded pinnipeds in specialized marine mammal rehabilitation centers. The poxvirus-related mortality in pinnipeds in marine mammal rehabilitation centers is typically low, except in cases where nodules develop on the eyes, nostrils or the oral cavity. However, morbidity can be high,

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3 especially in young animals. While mortality is typically low, poxvirus infections are likely to negatively affect the physiology, prognosis and length of hospitalization of animals in rehabilitation centers, thus adding to the cost of rehabilitating these animals. The pinniped poxviruses have been tentatively identified as belonging to both the genus Orthopoxvirus (Osterhaus et al. 1990) and Parapoxvirus (Simpson et al. 1994, Osterhaus et al. 1994, Nettleton et al. 1995). However, it has been suggested that these surface characteristics are preparation artifacts induced by osmotic stress imposed during negative staining and that in reality poxvirions have a smooth surface (Dubochet et al., 1994). While other evidence has since been presented suggesting that the appearance of the surface tubules is not artifactual (Wilton et al., 1995; Malkin et al., 2003; Heuser, 2005), sequencing of viral genes is the current method of choice for identifying poxviruses. Molecular investigations of the virus are limited to one account of a poxvirus outbreak in European harbor seals, in which the virus was classified as a new member of the parapoxviruses (Becher et al. 2002). In addition, only very limited information exists about the predisposing factors, the effects, effective therapeutics and the prevalence of poxvirus infections in free-ranging pinnipeds, as well as the epidemiology and prevention of poxvirus infections in pinnipeds in rehabilitation centers. Consequently, members of our team were contacted by the veterinary staff at marine mammal rehabilitation centers in California and several northeastern states to consider developing a collaborative research project to better understand annually recurring poxvirus epizootics in pinnipeds at their facilities and in the wild. Each year, up to 10% of several hundred harbor seals, elephant seals and Californian sea lions that are admitted to The Marine Mammal Center in California

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4 (TMMC) develop pox lesions at the center (Hastings et al. 1989). Between 1 January and 31 December 2002, 275 stranded California sea lions were admitted for rehabilitation to TMMC. Eighteen of 275 (6.5%) sea lions developed poxvirus lesions. The prevalence of subclinical cases was unknown. The presence of complicating pox-like lesions is also commonly observed in rehabilitation centers in New Jersey, Maine and Connecticut as well (B. Schoelkopf, C. Goertz, J.L. Dunn, personal comm.). This prompted a study into the different aspects of poxvirus infections in pinnipeds, funded by the NOAA/NMFS John H. Prescott Marine Mammal Rescue Assistance Grant Program, with the following objectives: 1. To develop and validate a molecular diagnostic test (PCR) that will allow us to determine infection with poxvirus(es). 2. To identify the poxvirus(es) that infects pinnipeds from both the east and west coast of the United States, and to clarify their phylogenetic classification. 3. To isolate and characterize the pinniped poxvirus(es). 4. To develop and validate serological diagnostic test (indirect ELISA) that will allow us to determine either exposure to poxvirus(es). 5. To understand the factors involved in transmission of, and resistance to, poxvirus infections. 6. To estimate the prevalence and other epidemiologic features of poxvirus(es) in wild pinniped populations. 7. To identify the risk factors that predispose animals to contracting poxvirus infections during hospitalization. 8. To evaluate the in vitro therapeutic efficacy of different antiviral compounds against the poxviruses of pinnipeds. The results of this research will not only significantly increase the existing body of knowledge on poxvirus infections in pinnipeds, but will also allow us to develop recommendations to help prevent and manage poxvirus outbreaks in marine mammal

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5 rehabilitation centers and, if applicable, prevent introduction of poxviruses into wild pinniped populations. The eight objectives proposed above have been combined into a total of five chapters. Each chapter is written in the format of a stand-alone manuscript for publication. At the time of submission of this dissertation, each manuscript had been submitted for publication in peer-reviewed journals.

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CHAPTER 2 PARAPOXVIRUSES OF SEALS AND SEA LIONS MAKE UP A DISTINCT SUBCLADE WITHIN THE GENUS PARAPOXVIRUS Introduction The poxviruses (family Poxviridae) represent a unique family of large, double-stranded DNA viruses that have an entirely cytoplasmic life cycle. The family of poxviruses includes some major pathogens of humans, domestic animals and wildlife (Moss 1996). The poxvirus family is subdivided into the entomopoxvirus and chordopoxvirus (ChPV) subfamilies, which respectively infect insect and vertebrate hosts (Moss 1996). The ChPVs are further divided into eight genera (Avipoxvirus, Capripoxvirus, Leporipoxvirus, Molluscipoxvirus, Orthopoxvirus, Suipoxvirus, Yatapoxvirus and Parapoxvirus). The recognized members of the genus Parapoxvirus currently consist of Orf virus (OrfV), Bovine papular stomatitis virus (BPSV), Pseudocowpoxvirus (PCPV), Parapoxvirus of red deer in New Zealand (PVNZ) and Squirrel parapoxvirus (SPPV) (Fauquet and Mayo, 2005). The natural hosts of these viruses are wild and domestic ruminants, but OrfV, BPSV and PCPV are known to infect man as well (Mercer et al. 1997). Tentative members of the parapoxvirus group include Auzduk disease virus, Camel contagious ecthyma virus, Chamois contagious ecthyma virus, Sealpox virus and a parapoxvirus of California sea lions (Sea Lion Poxvirus-1) (Nollens et al. In press). The seal and sea lion parapoxviruses are considered zoonotic agents as animal handlers have 6

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7 acquired proliferative “sealpox” lesions on their hands after contact with captive, lesion-bearing seals (Hicks and Worthy 1987, Clark et al. 2005). In members of the Pinnipedia, the order of mammals consisting of all seals (family Phocidae), sea lions (family Otariidae) and walruses (family Obodenidae), poxvirus infections manifest clinically as proliferative lesions, 2-3 cm in diameter, on the skin or the mucosal surface of the mouth and nasal passages. Pox lesions are sometimes observed in free-ranging seals and sea lions, but they are especially common as a complication in the treatment of debilitated, stranded pinnipeds in specialized marine mammal rehabilitation centers. The pinniped poxviruses have been tentatively identified as belonging to both the genus Orthopoxvirus (Osterhaus et al. 1990) and Parapoxvirus (Simpson et al. 1994, Osterhaus et al. 1994, Nettleton et al. 1995), based on morphologic characteristics on positive and negative staining electron microscopy. Molecular investigations of pinniped poxviruses are limited to two accounts, in which two poxvirus strains from a European harbor seal (Phoca vitulina vitulina) (Becher et al. 2002, Nollens et al. In press) and a California sea lion (Zalophus californianus) (Nollens et al. In press) were tentatively classified as new members of the genus Parapoxvirus. Features that distinguish parapoxviruses (PPVs) from other poxvirus genera are the ovoid virion shape, the crisscross pattern on the particle surface, and the relatively high G-C content of the genome (Moss 1996, Delhon et al. 2004). The PPVs and orthopoxviruses (OPVs) have a common evolutionary ancestor and they consequently share features such as size, genome organization and general virion structure (Fleming et al. 1993, Mercer et al. 1995, Moss 1996, Delhon et al. 2004). However, only some of the orthopoxvirus and parapoxvirus surface antigens cross-react (Moss 1996), and the

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8 differing nucleic acid content of the genomes distinguishes them genetically (Fleming et al. 1993). The phylogenetic classification of the pinniped poxviruses is therefore important when developing molecular and serologic diagnostic assays. Additionally, it has been suggested that some parapoxviruses have the capability of causing persistent, recrudescing infections (Iketani et al. 2002). The phylogenetic classification of poxviruses of pinnipeds may therefore also be of importance for future epidemiological and ecological studies. Extensive molecular phylogenetic studies to resolve identity, taxonomy, evolutionary relationships and host range have not been performed on poxviruses of seals and sea lions. Here we report on a molecular survey of pinniped poxviruses. The tools for this survey consisted of panparapox and panorthopox PCR protocols that were designed to amplify highly conserved poxvirus genomic regions encoding either the p37K envelope phospolipase of orthopoxviruses or its equivalent in parapoxviruses or the hemagglutinin protein of orthopoxviruses. The aim of this study was to further clarify the phylogenetic identity of the pinniped poxviruses and at evaluating the role host range and geographic isolation may have on the evolutionary divergence of these viruses. Materials and Methods Animals and Samples A total of 110 clinical samples from 53 California sea lions (Zalophus californianus), 10 harbor seals (Phoca vitulina richardii; n =5; P.v.concolor, n = 5) and one grey seal (Halichoerus grypus) showing clinical signs of cutaneous poxvirus infections, were collected between 15 August 2002 and 14 June 2005 for PCR analysis (Table 2-1, Table 2-2). The samples consisted of 35 biopsy or necropsy samples, five paraffinized or formalinized tissues and 70 pox lesion swabs. All received samples were

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9 collected from stranded seals and sea lions that were admitted to specialized marine mammal rehabilitation centers [The California Marine Mammal Center (CA), Marine Mammal Stranding Center (NJ), Animal Health Center (British Columbia), Mystic Aquarium (CT), Sea World of Orlando (FL), SeaWorld of San Diego (CA) or the Virginia Marine Science Museum (VA)]. DNA was extracted from all pox lesion scabs and swabs, and from a 25 mg fragment of all tissue samples using a DNeasy Tissue Kit (Qiagen, Valencia, CA), following the manufacturer’s guidelines. Polymerase Chain Reactions (PCR) PCR amplifications were performed on each sample and using each of the following four primer sets: Para1/Para2, Ortho1/Ortho2, EACP1/EACP2 and NACP1/NACP2. A universal panparapox primer pair (Para1/Para2, GeneBank accession number U06671: sense 5’-3’: GTCGTCCACGATGAGCAGCT and antisense 5’-3’: TACGTGGGAAGCGCCTCGCT) were used to amplify part of the genomic region (B2L gene) encoding the putative virion envelope antigen (p42K) of parapoxviruses (Sullivan et al. 1994,Inoshima et al. 2000). The orthopoxvirus-specific primer pair Ortho1/Ortho2 (5’-CTGGTAGAAACACTACCAGAAAATATGGA-3’ and 5’-TCTTAATATGATACGCAGTGCTAACTGG-3’) was designed to amplify a 546 bp product of the genomic region (F13L) encoding the EEV phospholipase (p37K) of orthopoxviruses, the orthopoxvirus orthologue of the p42K of parapoxviruses. The primer sets EACP1/EACP2 and NACP1/NACP2 are panorthopox primers, designed to amplify the genomic region encoding the hemagglutinin protein (HA) of Old World and New World orthopoxviruses respectively (Ropp et al. 1995). An aliquot of 15 l of the extracted DNA of each sample was used in 100 l amplification reactions containing 10X PCR buffer, 1 mM MgCl2, 200 M of each dNTP, 1 M of each primer and two units of

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10 Taq polymerase (Roche Diagnostics Corporation, Indianapolis, IN). For each reaction, three wells containing DNA from wild type Orf virus (OrfV), vaccinia virus (Vac-WR) and no DNA were included as positive and negative controls. All four PCR protocols were optimized for yield and specificity using either VacV-WR or OrfV DNA as substrate. Final amplification conditions were as follows: incubation at 94C for 2.5 min, and 30 cycles of denaturation at 94C for 30 sec, 1 min annealing (57C for Para1/Para2, 54C for Ortho1/Ortho2, 53C for EACP1/EACP2 and 56C for NACP1/NACP2), and extension at 72C for 40 sec, followed by incubation at 72C for 10 min. All amplicons of controls and unknown samples were purified using a High Pure PCR Product Purification Kit (Roche Diagnostics Corporation, Indianapolis, IN) following the manufacturers instructions, and submitted to the Sequencing Core of the UF-ICBR for nucleotide sequencing. Each nucleotide position was sequenced at least three times in each direction and all primer sequences were excluded from the subsequent analyses. Vector NTI software (Informax, Frederick, MD) was used to analyze and construct contiguous nucleotide sequences. Viral Sequence Analyses A multiple sequence alignment of the generated nucleotide sequences was generated using ClustalW software. The aligned sequences were subsequently edited and analyzed using the MEGA software package version 3.1 (Kumar et al. 2004) (Figure 2-1). The derived amino acid sequences of each of the generated sequences were obtained using Vector NTI. Pairwise nucleotide and amino acid comparisons between all generated viral sequences and all available poxvirus sequences from seals and sea lions were conducted using MEGA 3.1 (Table 2-3). Available pinniped poxvirus sequences included Sea Lion Poxvirus-1 (DQ163058, SLPV-1), two Steller sea lion parapoxvirus

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11 sequences (AY952940, E.jubatus-V841; AY952946, E.jubatus-V1346), spotted seal (DQ073805.1, P.largha) and two harbor seal parapoxvirus sequences (AF414182.1, P.vitulina-NSea; AY952937.1 P.vitulina-V465) (Table 2-1). The nucleotide sequences of two poxviruses of a European grey seal (Halichoerus grypus) and a Weddell seal (Leptonychotes weddellii) (respective GenBank accession numbers AJ622901.1 and AJ622900.2 contain stop codons although the panparapox primers Para1/Para2 target the open reading frame of the orf virus B2L gene equivalent. As a result, these two sequences were excluded from this analysis. For each nucleotide and amino acid pair, the P distance, defined as the proportion of sites at which the two sequences to be compared differ, was calculated (Table 2-3). Additionally, the P distances within and between each of the following groups was calculated using Mega3.1: Atlantic poxviruses, Pacific poxviruses, Pacific phocid poxviruses and Pacific otariid poxviruses. No otariid species inhabit the Atlantic ocean. The P distance within a group was defined as the arithmetic average of all computed pairwise comparisons within that group. The P distance between groups was defined as arithmetic average of all computed inter-group pairwise comparisons (Kumar et al. 2004). To clarify the genetic relationships between pinniped poxviruses, published PPVs and other members of the ChPVs, phylogenetic trees were constructed from alignments of the translated envelope protein sequences (Figure 2-2). Sequences included in the analysis were Orf virus strains NZ2, SA00, IA82 and Iran (Orf-NZ2, UO6671.1; Orf-SA00, AY386264.1; Orf-IA82, AY386263; Orf-Iran, AY958203.1), bovine papular stomatitis virus strain BV-AR02 (BPSV, AY386265.1), pseudocowpox virus (PCPV, AY424972.1), red deer parapoxvirus (PVNZ, AB044794.1) and reindeer poxvirus

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12 (RndPV, AY453652). Previous pinniped parapoxvirus isolates included the North Sea harbor seal parapoxvirus (P.vit-NSea), the spotted seal parapoxvirus (P.largha) and a parapoxvirus detected in Steller sea lions (E.jubV1346). Also included were the protypal viruses of all other Chordopox genera: genus Orthopox: vaccinia virus strain WR, (VacV-WR, AY243312.1), squirrel poxvirus (SqPV, AY340985.1); genus Suipox: Swinepox virus isolate 17077-99 (SwPV, AF410153.1); genus Capripox: Sheeppox virus strain A (ShPV, AY077833.1); genus Molluscipoxvirus: Molluscum contagiosum virus subtype 1 (MCV, U60315.1); genus Leporipoxvirus: Myxoma virus strain Lausanne (MyxV, AF170726.2); genus Yatapoxvirus: Yaba monkey tumor virus (Yaba, AY386371.1) and genus Avipoxvirus: Fowlpox virus (FwlPV, AF198100.1). The amino acid sequences of the ChPVs were edited to correspond to the amino acid sequences of the PPVs. All phylogenetic analyses were performed with the PHYLIP package version 3.65 (Felsenstein 2005). Phylogenetic trees were inferred using the maximum-likelihood method. The chosen model of amino acid substitution was the Jones–Taylor–Thornton model with assumed constant rate of change. The maximum-likelihood analyses (with randomized input order and global rearrangements) were performed using the program PROML. The robustness of the phylogenetic analysis and significance of the branch order were determined by 100 data-set bootstrap resampling with the programs SEQBOOT and CONSENSE. Additionally, to evaluate the potential role of host range and geographic isolation on the evolutionary divergence of the detected pinniped poxviruses, phylogenetic trees were constructed using only the sequences derived from poxviruses of pinnipeds using the exact methodology outlined above (Figure 2-3 and Figure 2-4).

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13 Nucleotide sequence accession numbers The nucleotide sequences of SLPV-2, SLPV-3, GSPV-1, HSPV-1 and HSPV-2 generated in this study were deposited in the GenBank data library under the respective Accession Nos. DQ273137, DQ273138, DQ273134, DQ273135 and DQ273136. Results Animals and Samples In total, poxviral DNA was detected in 55 (50%) samples from 39 animals (Table 2-2). Twenty-two (63%) biopsy or necropsy samples, three (60%) paraffinized or formalinized tissues and 30 (43%) pox lesion swabs derived from 30 California sea lions, one grey seal, five West Atlantic harbor seals and three Pacific harbor seals were found to contain virus (Table 2-2). All 55 viral-derived amplicons were obtained using the Para1/Para2 panparapox primers. No poxviral DNA was detected using any of the three primersets specific for OPV (Ortho1/Ortho2, EACP1/EACP2 and NACP1/NACP2) (Table 2-2). Viral Sequence Analyses The multiple nucleotide sequence alignment showed that six distinct virus strains were detected in the 55 positive samples (Figure 2-1). One virus strain was only found in grey seals (Grey Seal Poxvirus-1; GSPV-1) (Table 2-1, Table 2-2, Figure 2-1). Similarly, two virus strains were only found in harbor seals of respectively the Western Atlantic (Harbor Seal Poxvirus-1; HSPV-1) and the Pacific (Harbor Seal Poxvirus-2; HSPV-2). Three virus strains were detected in California sea lions (Sea Lion Poxvirus-1,2,3; SLPV-1,2,3). All generated sequences were homogenous in length (554 bp). A total of 119 (22%) nucleotide positions were variable sites, defined as containing at least two types of nucleotides, but no hypervariable hot spot regions could be identified (Figure 2-1).

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14 The pairwise nucleotide and amino acid sequence comparisons between the all generated viral sequences and sequences from available poxvirus sequences from seals and sea lions showed that sequence HSPV-1 amplified from West Atlantic harbor seals was identical to sequence P.vitulina-V465 previously amplified from West Atlantic harbor seals (Table 2-1, Table 2-3). Additionally, SLPV-3, here amplified from California sea lions, was identical to sequence E.jubatus-V841 that was previously amplified from Steller sea lions (Table 2-1, Table 2-3). P.vitulina-V465 and E.jubatus-V841 are therefore represented by respectively HSPV-1 and SLPV-3 in all subsequent phylogenetic analyses. No virus strains were detected in multiple host species of the phocid family. The mean nucleotide and amino acid P distances between all pinniped poxviruses was 0.079 and 0.049 respectively. Of all pinniped poxviruses, P.vitulina-NSea and P.vitulina-V465 were most closely related to each other (nucleotide P = 0.002) and SLPV-2 was most divergent from all other pinniped poxviruses (Table 2-3). Pinniped poxviruses from the Atlantic ocean appeared least genetically diverse (nucleotide P within Atlantic = 0.018 0.004). Pinniped poxviruses from the Pacific ocean were even more divergent from each other (nucleotide P within Pacific = 0.096 0.009) than from those from the Atlantic (nucleotide P between Atlantic Pacific = 0.084 0.008). Phocid poxviruses of the Pacific were more closely related to each other (nucleotide P within Pacific phocid = 0.051 0.008) than to otariid poxviruses of the Pacific (nucleotide P between Pacific phocid – Pacific otariid = 0.093 0.009). The topology of a phylogenetic tree consisting of homologous sequences derived from all pinniped poxviruses in reference to all PPVs and the protypal members of all

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15 other ChPV genera showed that all six newly detected strains were most closely related to the known PPVs (Figure 2-2). This confirms their classification as members of the genus Parapoxvirus. The tree topology further showed that pinniped PPVs form a distinct, monophyletic clade within the genus Parapoxvirus with a node certainty of 96%. Of the known pinniped PPVs, SLPV-2 is most closely related to the root or ancestral pinniped PPV. The phylogenetic analysis of the nine pinniped PPVs alone demonstrated that PPVs from Atlantic pinnipeds (P.vitulina-NSea, HSPV-1, GSPV-1) were phylogenetically distant from most Pacific pinniped PPVs (P.largha, HSPV-2, E.jubV1346, SLPV-1, SLPV-2, SLPV-3) (Table 2-1, Figure 2-3). Similarly, PPVs from sympatric Pacific phocid (P.largha, HSPV-2) and otariid (E.jubV1346, SLPV-1, SLPV-2, SLPV-3) host species were also phylogenetically distant from each other (Figure 2-4). Discussion All pinniped poxviruses detected in this study were most closely related to the known PPVs. This confirms the earlier tentative classification of the poxviruses of seals and sea lions as members of the genus Parapoxvirus. Earlier phylogenetic classifications of the poxviruses of pinnipeds were usually solely based on the characteristics of surface tubules of purified virions on negative staining EM. However, it has been suggested that these surface characteristics are preparation artifacts induced by osmotic stress imposed during negative staining and that in reality poxvirions have a smooth surface (Dubochet et al. 1994). While other evidence has since been presented suggesting that the appearance of the surface tubules is not artifactual (Wilton et al. 1995, Malkin et al. 2003, Heuser 2005), sequencing of viral genes is the current method of choice for identifying poxviruses. In only one report were data on pinniped pox virion surface

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16 morphology supplemented with viral genomic sequence to unequivocally assign a poxvirus of pinnipeds to the genus Parapoxvirus (Nollens et al. In press). No orthopoxviruses were detected in any of the samples. It is possible that the genomic sequences of orthopoxviruses would be too divergent to be amplified by the degenerate pan-orthopox primer pairs used in this study, but because of the absence of appropriate controls, this can not be verified. It appears that the natural hosts of PPVs consist solely of ruminants and pinnipeds. Interestingly, ruminants are the closest extant terrestrial relatives of the cetacean group of marine mammals consisting of all whales, dolphins and porpoises. However, the poxviruses associated with whales and dolphins species are (tentatively) classified as orthopoxviruses (Van Bressem et al. 1993). In contrast, the closest terrestrial relatives of pinnipeds are the members of the suborder Ursidae and the other members of the Order Carnivora (Van Bressem et al. 1993), but the only reported cases of poxvirus infections in carnivores are accidental infections with orthopoxviruses, especially cowpox virus (Smith et al. 1999, Meyer et al. 1999, Pelkonen et al. 2003). The pinniped PPVs form a distinct, monophyletic clade within the genus Parapoxvirus (node certainty of 96%), thus setting the PPV of pinnipeds genetically apart from all other PPV. The International Committee on the Taxonomy of Viruses defines a virus species is a polythetic class of viruses that constitute a replicating lineage and occupy a particular ecological niche (Pringle 1991). Because of their genetic relatedness, their exclusive occurrence in the marine environment and their distinctive natural host range, the polythetic class of pinniped parapoxviruses could be recognized as a separate species within the genus Parapoxvirus.

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17 Phylogenetic analyses based on single genes may give rise to inaccurate tree topologies. In order to gather further support for the branching order observed in this study, we compared the results of the topology of phylogenetic tree generated in this study to a phylogenetic analysis of all ChPV, except the PPV, based on the sequence of 17 conserved poxvirus proteins (Gubser et al. 2004). Both phylogenetic analyses outline the same main groupings of ChPVs in the same branching order. Similarly, the topology of the phylogenetic tree generated here was nearly identical to that of a phylogenetic tree of PPVs and selected ChPVs that was generated using concatenated gene sequences encoding core and envelope proteins (Tikkanen et al. 2004). Parapoxviruses from Atlantic pinnipeds were phylogenetically distant from those of Pacific pinnipeds (Figure 2-3). The Atlantic and Pacific ocean basins are separated by the North and South American continent, but indirect contact between Atlantic and Pacific pinnipeds and occasional spill-over of their pathogens could occur via poxvirus infections of the Arctic ice seals (ringed seal (Phoca hispida), bearded seals (Erignathus barbatus), ribbon seals (Phoca fasciata), hooded seal (Cystophora cristata), harp seal (Phoca groenlandica)). Unfortunately, we were unable to obtain pox lesion samples from the Arctic ice seal species during this study and consequently have insufficient data on poxviruses of ice seals to verify this hypothesis. Parapoxviruses from sympatric phocids and otariids were also phylogenetically distant, suggesting that parapoxviruses are not commonly transmitted between phocids and otariids (Figure 2-4). Here, the effect of biological isolation (host range) and geographic isolation could be confounded, since no otariid species inhabit the North Atlantic ocean. To correct for this potential confounding effect, only the genetic relatedness of Pacific phocid and Pacific otariid PPV

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18 strains was evaluated (Figure 2-4). The calculated genetic distances (P distances) confirmed that the pinniped PPVs from the Pacific basin were genetically divergent from those from the Atlantic. Additionally, phocid PPVs of the Pacific were more closely related to each other than to the Pacific otariid PPVs. The genetic distances between these various groups is in agreement with the topology of the phylogenetic trees (Figure 2-3, Figure 2-4) and the hypothesis that both geographic isolation and host range are driving factors for the evolutionary divergence of the pinniped PPVs. Here we also present the first evidence that PPVs of pinnipeds are capable of infecting multiple pinniped host species. We detected one pinniped PPV strain (SLPV-3) in pox lesions obtained from two California sea lions that had previously been detected in pox lesions from a Steller sea lion (E.jubatus-V841; Table 2-1, Table 2-3). California sea lions and Steller sea lions are distinct species, but both are members of the family Otariidae. Steller sea lions inhabit the North Pacific coasts but occur as far south as the California coast. The geographic range of California sea lions is centered around the California coast but extends northward into Canada. Where the habitat of these two otariid species overlaps, direct and indirect inter-specific contact is possible. Regardless of the mode of transmission, this finding is in agreement with the abovementioned hypothesis that pinniped PPVs are capable of infecting multiple pinniped host species within one phylogenetic family.

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19 Table 2-1. Host species, tentative de signations and GenBa nk accession numbers for genomic sequences of pinniped poxvi rus strains analyzed in this study Pinniped host Poxvirus Family (Sub)species Common name Designation Accession Number Otariidae Eumatopias jubatus Steller sea lion E.jubatusV1346 AY952946.1 E.jubatus-V841 AY952940.1 Zalophus californianus californianus California sea lion SLPV-1 n/a SLPV-2 n/a SLPV-3 n/a Phocidae Phoca largha Spotted seal P.largha DQ073805.1 Phoca vitulina vitulina Harbor seal (East Atlantic) P.vitulina-NSea AF414182.1 Phoca vitulina concolor Harbor seal (West Atlantic) P.vitulina-V465 AY952937.1 Phoca vitulina concolor Harbor seal (West Atlantic) HSPV-1 n/a Phoca vitulina richardii Harbor seal (Pacific) HSPV-2 n/a Halichoerus grypus Grey seal GSPV-1 n/a n/a: Not available. These genomic sequences were generated as part of this study.

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20 Table 2-2. Number of animals of each host species from which samples were received (N), number of animals in which a poxvirus was detected (Pos) and poxvirus strain detected using the Ortho, EACP, NACP and Para primer pairs Para Host species N Pos Ortho EACP NACP SLPV-1 SLPV-2 SLPV-3 GSPV-1 HSPV-1 HSPV-2 California sea lion 53 30 16 12 2 Grey seal 1 1 1 Harbor seal (West Atlantic) 5 5 5 Harbor seal (Pacific) 5 3 3

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21 Table 2-3. Genetic diversity of parapoxviruses of North American pinnipeds determined using average P distances calculated with MEGA (version 3.1). The P distance calculated from the nucleotide sequence data is shown below the diagonal and the value calculated from predicted amino-acid sequences (AA) is shown above the diagonal. Identical parapoxviruses (P = .000) are indicated using bold font. Otariidae Phocidae E.jubatus-V1346 E.jubatus-V841 SLPV-1 SLPV-2 SLPV-3 P.largha P.vitulinaN Sea P.vitulina-V465 HSPV-1 HSPV-2 GSPV-1 E.jubatus-V1346 .038 .038 .082 .038 .038 .043 .038 .038 .038 .033 E.jubatus-V841 .056 .043 .082 .000 .065 .038 .033 .033 .065 .038 SLPV-1 .061 .063 .076 .043 .043 .027 .022 .022 .043 .005 SLPV-2 .153 .148 .161 .082 .076 .092 .087 .087 .076 .071 Otariidae SLPV-3 .056 .000 .063 .148 .065 .038 .033 .033 .065 .038 P.largha .045 .090 .067 .159 .090 .049 .043 .043 .011 .038 P.vitulina-NSea .074 .072 .034 .166 .072 .076 .005 .005 .049 .016 P.vitulina-V465 .072 .070 .032 .164 .070 .074 .002 .000 .043 .016 HSPV-1 .072 .070 .032 .164 .070 .074 .002 .000 .043 .016 HSPV-2 .045 .090 .063 .161 .090 .014 .069 .067 .067 .038 Phocidae GSPV-1 .065 .067 .011 .159 .067 .067 .034 .032 .032 .063

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22 GSPV-1 TAC CGG CGG CTC GCT AGC CAC TAT TAA AAA CCT GGG CGT GTA CTC CAC 48 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 48 HSPV-1 ... ... ... ... ... ... ... C.. ... ... .T. ... ... ... ... ... 48 HSPV-2 ... ... ... ... ... ... T.. C.. ... ... .T. ... ... ... ... ... 48 SLPV-3 ... T.. ... ... ... G.. ... C.. ... ... ... ... ... ... ... ... 48 SLPV-2 C.. ... ... ... ... G.. T.. C.. C.. G.. ... ... A.. ... ... G.. 48 GSPV-1 CAA CAA GCA CTT GGC TGT CGA CCT CAT GAA CAG GTA CAA CAC CTT TAG 96 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 96 HSPV-1 ... ... ... ... ... C.. T.. ... ... ... ... ... ... ... ... ... 96 HSPV-2 ... ... ... ... ... ... T.. ... T.. ... .C. ... ... ... T.. ... 96 SLPV-3 ... ... ... ... ... C.. ... ... ... ... .C. ... ... ... ... ... 96 SLPV-2 ... T.. ..G .C. ... CT. ... ... ... ... ... ... ... ... ... CG. 96 GSPV-1 CTC CAT GGT CGT GGA CCC CAA GCA GCC GTT TAC GCG CTT CTG CTG CGC 144 SLPV-1 ... ... ... T.. ... ... ... ... ... ... ... ... ... ... ... ... 144 HSPV-1 ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... 144 HSPV-2 ... ... ... T.. A.. ... ... ... ... ... ... ... A.. ... ... ... 144 SLPV-3 ... ... ... T.. ... ... ... ... ... ... C.. ... .A. ... ... ... 144 SLPV-2 G.. A.. ... ... A.. ... ... ... ... ... CG. A.. AC. ... T.. ... 144 GSPV-1 CAT GAT AAC GCC CAC AGC CAC GGA CTT CCA CAT GAA CCA CTC TGG CGG 192 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 192 HSPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 192 HSPV-2 ... ... C.. A.. ... G.. ... ... ... ... ... ... ... ... ... ... 192 SLPV-3 ... ... T.. ... ... G.. ... ... ... T.. ... .C. ... ... ... G.. 192 SLPV-2 ... C.. C.. T.. G.. G.. ... ... ... ... T.. ... T.. ... A.. ... 192 GSPV-1 CGG CGT TTT TTT CTC AGA CTC ACC AGA GCG CTT CTT GGG CTT CTA CCG 240 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 240 HSPV-1 ... ... ... ... ... G.. ... ... ... ... ... ... ... ... ... ... 240 HSPV-2 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 240 SLPV-3 ... ... ... C.. T.. ... ... ... ... ... ... ... ... ... ... ... 240 SLPV-2 T.. ... ... ... ... G.. ... C.. ... ... ... .C. ... A.. ... ... 240 GSPV-1 CAC GCT CGA CGA AGA CCT GGT GCT GCA CCG CAT CGA CGC TGC AGA AAA 288 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 288 HSPV-1 ... ... ... ... ... ... ... ... ... ... ... T.. ... ... GA. ... 288 HSPV-2 ... ... ... ... ... .T. ... C.. ... ... ... ... T.. ... G.. ... 288 SLPV-3 ... ... ... ... ... ... ... ... ... ... ... ... ... ... GA. ... 288 SLPV-2 ... ... ... ... ... ... ... .T. ... ... ... ... TT. C.. G.. ... 288 GSPV-1 CAG CAT TGA CCT CTC TCT GCT GTC TAT GGT GCC AGT GGT GCG CTC TGG 336 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 336 HSPV-1 ... ... ... ... ... G.. A.. ... ... ... ... ... ... ... ... ... 336 HSPV-2 ... ... ... ... ... G.. ... ... ... .C. ... ... ... ... ... ... 336 SLPV-3 ... ... C.. ... ... G.. ... ... C.. ... ... G.. ... ... ... C.. 336 SLPV-2 ... ... C.. T.. A.. A.. ... ... C.. ... ... G.. ... ... ... C.. 336 GSPV-1 CAG CGA GGT GTA CTA CTG GCC GCT GAT CAT GGA CGC GTT GCT GCG CGC 384 SLPV-1 ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... 384 HSPV-1 .G. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 384 HSPV-2 ... ... A.. .C. ... ... ... ... ... ... ... ... ... ... ... ... 384 SLPV-3 .G. ... ... ... ... ... ... ... ... ... ... ... ... ... ... A.. 384 SLPV-2 ... ..C C.. TC. ... ... ... A.. ... A.. ... ... .C. ... ... ... 384 Figure 2-1. Multiple alignment of the partial genomic sequences encoding the putative virion EEV protein (p42K) of the pinniped parapoxvirus strains detected in this study. This sequence alignment was generated using ClustalW and edited using MEGA3.1.

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23 GSPV-1 TGC TAT CAA CCG CAG CGT GCG CGT GCG CAT CAT CGT CAG CCA GTG GCG 432 SLPV-1 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 432 HSPV-1 ... ... ... ... ... ... ... ... ... ... T.. ... ... ... ... ... 432 HSPV-2 C.. ... T.. ... ... ... ... ... ... ... ... .A. ... ... ... ... 432 SLPV-3 C.. .G. .G. ... ... ... ... T.. ... ... ... .A. ... ... ... ... 432 SLPV-2 C.. G.. AG. ... ... ... A.. ... ... T.. ... .A. ... ... ... ... 432 GSPV-1 TAA CGC GGA TCC ACT GTC CGT GGC TGC AGT TCG TGC GCT GGA CAA CTT 480 SLPV-1 ... ... ... ... ... ... T.. ... ... ... ... ... ... ... ... ... 480 HSPV-1 ... ... ... C.. ... ... T.. ... ... ... ... ... ... ... ... ... 480 HSPV-2 ... ... ... ... G.. ... T.. A.. ... ... ... ... ... ... ... ... 480 SLPV-3 ... ... ... ... G.. ... ... ... ... ... ... C.. ... ... ... ... 480 SLPV-2 C.. ... ... ... G.. ... ... ... A.. G.. G.. C.. A.. ... TG. T.. 480 GSPV-1 TGG AGT GGG GCA TAT TGA CAT TAC TGC GCG CTG GTT CGC AAT TCC AGG 528 SLPV-1 ... ... ... ... ... C.. ... C.. ... ... ... ... ... ... ... ... 528 HSPV-1 ... ... ... ... .G. C.. ... C.. ... ... ... ... ... ... ... ... 528 HSPV-2 ... G.. ... ... CG. ... .G. ... ... ... ... ... ... .G. G.. ... 528 SLPV-3 ... ... ... ... ... C.. ... C.. C.. ... ... ... ... G.. A.. ... 528 SLPV-2 C.. ... ... ... C.. C.. ... C.. A.. ... ... ... T.. GG. A.. G.. 528 GSPV-1 CCG CGA CGA CGC ATC CAA CAA CAC TA 554 SLPV-1 ... ... ... ... G.. ... ... ... .. 554 HSPV-1 ... ... ... ... ... ... ... ... .. 554 HSPV-2 ... ... ... ... GG. ... ... ... .. 554 SLPV-3 ... ... ... ... G.. ... ... ... .. 554 SLPV-2 A.. T.. ... ... C.. T.. ... ... C. 554 Figure 2-1. Continued.

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24 Figure 2-2. Phylogram depicting the relationship of the pinniped poxviruses to the published members of the genus Parapox [Orf virus strains NZ2, SA00, IA82 and Iran (Orf-NZ2, UO6671.1; Orf-SA00, AY386264.1; Orf-IA82, AY386263; Orf-Iran, AY958203.1), bovine papular stomatitis virus strain BV-AR02 (BPSV, AY386265.1), pseudocowpox virus (PCPV, AY424972.1), red deer parapoxvirus (PVNZ, AB044794.1) and Reindeer poxvirus (RndPV, AY453652)]; previous pinniped parapoxvirus isolates [North Sea harbor seal parapoxvirus (P.vit-NSea), the spotted seal parapoxvirus (P.largha) and a Steller sea lion parapoxvirus (E.jubV1346)] and the protypal members of all other Chordopox genera [vaccinia virus strain WR, (VacV-WR, AY243312.1), squirrel poxvirus (SqPV, AY340985.1), Swinepox virus isolate 17077-99 (SwPV, AF410153.1), Sheeppox virus strain A (ShPV, AY077833.1), Molluscum contagiosum virus subtype 1 (MCV, U60315.1), Myxoma virus strain Lausanne (MyxV, AF170726.2), Yaba monkey tumor virus (Yaba, AY386371.1) and Fowlpox virus (FwlPV, AF198100.1)]. This phylogenetic tree is based on the 184 translated amino acids of the putative sea lion p42K fragment and homologous regions from the other poxviruses, and was generated according to the maximum likelihood method using the PHYLIP 3.65. Branch lengths are proportional to genetic distances. Numbers indicate the robustness of each node, defined as the percentage of 100 bootstrap replicates that support each interior branch.

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25 Figure 2-3. Phylogram depicting the relationship between the pinniped poxviruses of the Atlantic (P.vit-NSea, HSPV-1 and GSPV-1) and the Pacific Ocean (SLPV-1, SLPV-2, SLPV-3, HSPV-2, E.jubV1346, P.largha). This phylogenetic tree is based on the 184 translated amino acids of the putative sea lion p42K fragment and homologous regions from the other poxviruses, and was generated according to the maximum likelihood method using the PHYLIP 3.65. Branch lengths are proportional to genetic distances. Numbers indicate the robustness of each node, defined as the percentage of 100 bootstrap replicates that support each interior branch.

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26 Figure 2-4. Phylogram depicting the relationship between the poxviruses of Pacific phocids (HSPV-2, P.largha) and otariids (SLPV-1, SLPV-2, SLPV-3, E.jubV1346). This phylogenetic tree is based on the 184 translated amino acids of the putative sea lion p42K fragment and homologous regions from the other poxviruses, and was generated according to the maximum likelihood method using the PHYLIP 3.65. Branch lengths are proportional to genetic distances. Numbers indicate the robustness of each node, defined as the percentage of 100 bootstrap replicates that support each interior branch.

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CHAPTER 3 PATHOLOGY AND PRELIMINARY CHARACTERIZATION OF A PARAPOXVIRUS ISOLATED FROM A CALIFORNIA SEA LION Introduction The viral family Poxviridae consists of large, enveloped DNA viruses that are of veterinary and medical importance. The most pathogenic member, variola virus, causes smallpox in humans (Moss 1996). Members affecting wildlife include crocodilepox virus, fowlpox virus, buffalopox virus, squirrelpox virus, monkeypox virus, dolphinpox virus and the poxvirus that causes myxomatosis in rabbits and hares (Moss 1996). Parapoxviruses cause contagious pustular dermatitis or orf in sheep and goats, papular stomatitis and pseudocowpox in cattle and skin lesions in other animals including red deer, squirrels, reindeer, musk ox and camels (Reid 1998). Poxvirus infections have also been reported in aquatic mammals, including pinnipeds. The susceptibility of pinnipeds to pox infections was first suspected in 1969 when a one-year-old California sea lion (Zalophus californianus) was presented with pox-like lesions (Wilson et al. 1969). The presence of poxvirus has since been confirmed in cutaneous and mucosal nodular lesions from harbor seals (Phoca vitulina) (Wilson et al. 1972), northern fur seals (Callorhinus ursinus) (Hadlow et al. 1980), grey seals (Halichoerus grypus) (Hicks and Worthy 1987), northern elephant seals (Mirounga angustirostris) (Hastings et al. 1989) and South American sea lions (Otaria flavescens) (Wilson and Poglayen-Neuwall 1971). Based on electron microscopy of pox nodules in European and North American pinnipeds, several studies have suggested the involvement of both a parapoxvirus 27

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28 (Simpson et al. 1994, Osterhaus et al. 1994, Nettleton et al. 1995) and an orthopoxvirus (Osterhaus et al. 1990). Molecular investigations of the virus are limited to one account of a poxvirus outbreak in European harbor seals, in which the virus was classified as a new member of the parapoxviruses (Becher et al. 2002). Parapoxviruses and orthopoxviruses have a common evolutionary ancestor and they consequently share features such as size, genome organization and general virion structure (Fleming et al. 1993, Moss 1996). However, only some of the surface antigens cross-react (Moss 1996), and the differing nucleic acid content of the genomes differentiates them genetically (Fleming et al. 1993). The phylogenetic classification of the poxvirus is therefore also of importance when developing molecular and serologic diagnostic assays. It has been suggested that some parapoxviruses have the capability of causing persistent, recrudescing infections (Iketani et al. 2002). The phylogenetic classification of poxviruses of pinnipeds may therefore be of importance for future epidemiological and ecological studies as well. Molecular phylogenetic studies to resolve identity, taxonomy and host range have not been performed on poxviruses of seals and sea lions in the United States or elsewhere outside Europe. Here we describe the isolation and the electron microscopic appearance of a poxvirus (SLPV-1) of a California sea lion. Nucleic acid sequence of the gene encoding the p42K envelope protein of SLPV-1 is presented, which supports the classification of the sea lion poxvirus as a member of the genus Parapoxvirus. Materials and Methods Case Histories Three California sea lions (CSL No. 1, CSL No. 2, CSL No. 3) were recovered from the central California coastline between July 2002 and May 2003 and were

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29 transported to The Marine Mammal Center (TMMC) in Sausalito, California (37’N, 122’W), for rehabilitation. All animals were grouphoused in pens that were constructed of wire cyclone fencing bolted to an epoxy-coated concrete slab. Each pen contained a rough-textured fiberglass pool. The sea lions entered the pools by climbing a sand-textured fiberglass ramp. All pools were emptied daily and all surfaces cleaned with a dilute bleach solution and rinsed off with fresh water. Sea lion CSL No. 1, a yearling female, was admitted on 6 July 2002 with an abscess in the left inguinal area. On 14 August, two raised, firm, cutaneous nodules, 1.0 – 2.0 cm in diameter, were observed on the nares and upper lip of the animal, and a Dacron swab and a biopsy of the lesion were collected. The abscess was successfully treated and the animal was released on 24 August 2002. Sea lion CSL No. 2, a subadult female, was admitted on 8 July 2002 with evidence of sharkbite wounds to the head. Several raised, firm, ulcerated cutaneous nodules were observed on the right palpebra and skin overlying the cranium on 15 August 2002. A swab was collected from two of the lesions. The head wounds and cutaneous nodules gradually resolved and the sea lion was released on 29 Sept 2002. On 23 May 2003, a female California sea lion pup, CSL No. 3, was recovered for rehabilitation. Three raised, firm, nodular skin lesions, 2.0 – 3.0 cm in diameter, were noted on the head and neck during the physical examination upon admission. Four days later, the animal died and a necropsy was performed. Pathologic Studies The biopsy specimen from CSL No. 1 and all three pox-like lesions of CSL No. 3 that were collected on necropsy were bisected. Half of each nodule was processed for virologic studies (see below) and the remaining portion was fixed in neutral buffered 10% neutral buffered formalin for light microscopy. Samples from all major organ

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30 systems of CSL No. 3 were similarly fixed in 10% neutral buffered formalin for light microscopy. Subsequently, tissues were embedded in paraffin, sectioned at 6 m, stained with hematoxylin and eosin (HE), and examined with a light microscope. Cell Cultures Frozen early passage California sea lion skin cell cultures and California sea lion kidney cell cultures were obtained from the Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, and were grown to confluency in our laboratory. Additionally, modified African green monkey kidney cell cultures (BSC40) were obtained from the Condit Laboratory, University of Florida. The culture media consisted of 85% (volume:volume) DMEM-F12 (Cellgro, Herndon, VA), 15 % (volume:volume) cosmic fetal calf serum (Hyclone, Logan, UT), 2 mM L-glutamine (Cellgro, Herndon, VA), 0.1 mM Na-pyruvate (Cellgro, Herndon, VA), 50 IU penicillin/ml and 50 g streptomycin (Cellgro, Herndon, VA). All cultures were kept at 37.0C, 7.5% CO 2 and approximately 90% relative humidity. Virus Isolation All three swabs (CSL No. 1 and CSL No. 2) and the portion of each of the four nodules (CSL No. 1 and CSL No. 3) for virologic studies were kept frozen at C before overnight shipment to the University of Florida. For virus isolation, the three swabs were placed in 500 l of phosphate-buffered saline containing 500 IU/ml penicillin and 500 g/ml streptomycin (Cellgro, Herndon, VA). The swabs were pulse-vortexed for 30 sec, after which the swab was removed and the swab extract was filtered using a 0.450 m syringe filter (Fisher Scientific, Pittsburgh, PA). Approximately 75 mg of a tissue sample of both CSL No. 1 and CSL No. 3 was homogenized using a Dounce homogenizer in 500 l of phosphate-buffered saline containing 500 IU/ml penicillin and

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31 500 g/ml streptomycin (Cellgro, Herndon, VA). The homogenate was centrifuged at 2000 rpm for 20 min in a standard benchtop centrifuge, and the supernatant was filtered using a 0.450 m syringe filter (Fisher Scientific, Pittsburgh, PA). Next, 50 l aliquots of each of the filtrates were diluted to a total volume of 500 l in phosphate-buffered saline (PBS) for inoculation of the cell cultures. The three swab filtrates and the biopsy homogenate (CSL No. 1) were each used to inoculate two BSC40 cultures and four early passage California sea lion skin cell cultures. Two BSC40 cultures, four early passage California sea lion skin cell cultures, and four early passage California sea lion kidney cell cultures were inoculated with the pox lesion homogenate of CSL No. 3. After incubation for three hours at 37C, the cultures were washed and the tissue homogenate replaced by culture medium. All cultures were checked daily for cytopathic changes. For passaging, the cells were harvested using a scraper, and the cell suspension was centrifuged at 1,800 g for five min. Approximately 90% of the supernatant was removed after which the cells were resuspended in the remaining supernatant. One quarter of the cell concentrate (containing approximately 25% of the cell pellet and 2.5% of the supernatant) was freeze-thawed two times at -70C, sonicated for 2 min (Sonics VibraCell, Danbury, CT) and was used to inoculate the next passage. All cultures were blind passaged at least three times. Each second passage was tested for the presence of poxviral DNA using the PCR protocol described below. Virus Morphology After four passages, two cell cultures displaying a cytopathic effect were processed for transmission electron microscopy six days post inoculation. The monolayers were washed twice using PBS at room temperature and then fixed using a solution of 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, while the cells were adhered to the

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32 culture dish. Fixed cells were scraped, washed in 0.1 M cacodylate buffer, and then postfixed for 60 min at 4C in a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer and submitted to the University of Florida ICBR Electron Microscopy Core laboratory for transmission electron microscopy. Intracellular mature virions (IMV) were purified from lysates of infected cells by high-speed centrifugation through a 40% sucrose cushion followed by banding of the IMV on a 25-50% Na-diatrizoate density gradient as previously described (Robinson et al.. 1982). The virus was resuspended in 10 mM Tris, pH 8.0. An aliquot of the purified virus was fixed in 2% formaldehyde solution and submitted to the Electron Microscopy Core of the University of Florida Interdisciplinary Center for Biotechnology Research (UF-ICBR) for negative staining electron microscopy. Molecular Characterization of the Virus DNA was extracted from all three pox lesion swabs (CSL No. 1, CSL No. 2), from a 25 mg fragment of all four tissue samples (CSL No. 1, CSL No. 3) and from each second passage of the virus cultures, using a DNeasy Tissue Kit (Qiagen, Valencia, CA), following the manufacturer’s guidelines. A universal parapox primer pair (Para1/Para2, GenBank accession number U06671: sense 5’-3’: GTCGTCCACGATGAGCAGCT and antisense 5’-3’: TACGTGGGAAGCGCCTCGCT) were used to amplify part of the genomic region encoding the putative virion envelope antigen (p42K) of parapoxviruses (Sullivan et al., 1994; Inoshima et al., 2000). An aliquot of 15 l of the extracted DNA was used in 100 l amplification reactions containing 10X PCR buffer, 1 mM MgCl 2 , 200 M of each dNTP, 1 M of each primer and two units of Taq polymerase (Roche Diagnostics Corporation, Indianapolis, IN). DNA from wild type orf virus was included reaction as positive control for the PCR reaction. Two wells containing respectively no

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33 DNA and vaccinia DNA (Vac-WR) served as negative controls Amplification conditions were: incubation at 94C for 2.5 min, and 30 cycles of denaturation at 94C for 30 sec, annealing at 57C, and extension at 72C for 40 sec, followed by incubation at 72C for 10 min. All amplicons (controls and unknown samples) were purified using a High Pure PCR Product Purification Kit (Roche Diagnostics Corporation, Indianapolis, IN) following the manufacturers instructions, and submitted to the Sequencing Core of the UF-ICBR for nucleotide sequencing. Each nucleotide position was sequenced at least three times in each direction. Vector NTI software (Informax, Frederick, MD) was used to perform the analysis of the sequences. The ClustalX software was used to generate the multiple sequence alignment of the deduced amino acid sequence and to construct a phylogenetic tree according to the maximum likelihood method. The robustness of the phylogenetic analysis and significance of the branch order were determined by bootstrap analysis carried out on 100 replicates using the PHYLIP software. Results Pathologic Studies At necropsy, sea lion CSL No. 3 was 81 cm in length and weighed 12.5 kg. The blubber thickness measured at the level of the sternum was 13 mm. Three, raised, cutaneous nodules, measuring between 2.0 – 3.0 cm in diameter, were noted on the forehead and left abdomen of the sea lion. On section the nodules were firm, congested, focally ulcerated and the epidermis appeared thickened. Internally, severe atrophy of the renal and cardiac fat depots and a diffuse, moderate hemorrhagic gastritis was observed. No evidence of clinically significant viral, bacterial or parasitic infections was found. Based on the animal’s low bodyweight and the fat atrophy, death was attributed to malnutrition.

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34 On histological section diffuse necrosis of the outer layers of the epidermis was observed throughout the skin nodule (Figure 3-1). There was acanthosis and ballooning degeneration of epidermal cells within the stratum spinosum. With hematoxylin and eosin staining, many of ballooned cells contained large, coalescing eosinophilic intracytoplasmic inclusions. In one area there was ulceration of the epidermis with an accumulation of overlying necrotic cellular debris containing large numbers of bacterial colonies. In the dermis there were focal areas of necrosis. The dermis contained diffuse infiltrates of mixed inflammatory cells including small round cells and neutrophils that subtended the epidermal basement membrane and in some areas, infiltrated epidermal follicular and rete pegs. Virus Isolation All attempts to isolate the virus onto BSC40 African green monkey kidney cell cultures or in the early passage California sea lion skin cell cultures were unsuccessful. Cytopathic changes suggestive of a poxvirus infection developed within 24 hours of inoculation on all four early passage California sea lion kidney cell cultures. The cytopathic changes consisted of generalized, diffuse rounding of the cells. No cell lysis or plaque formation was observed. Lysates of the infected cell cultures were passaged seven times and portions of each passage were stored at -80C. Virus Morphology Positive staining transmission electron microscopy revealed the presence of poxvirus particles in the sea lion kidney cell cultures (Figure 3-2). Mature virions, immature virus particles, crescents and viroplasm were observed. Mature virions were brick-shaped, although somewhat elongated. The virions had a length and width of approximately 300 nm and 170 nm respectively. The structure of the mature virions

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35 consisted of an electron-dense core surrounded by the viral envelope. The cores of mature virions were ovoid to lanceolate and only occasionally dumbbell shaped. On the negative stained preparation (Figure 3-3) the virus particles appeared oval or cylindrical with convex ends. The negative staining also revealed a regularly arranged surface tubule pattern. No smooth capsular type virions were observed. Some smaller, rounded virions that did not show the typical parapox surface morphology were also present. Molecular Characterization of the Virus TBLASTX results of the 594 bp nucleotide sequence showed the highest score with the harbor seal parapoxvirus from Northern Europe (SPV, AF414182.1). At the nucleotide level, the sea lion parapoxvirus sequence was 96.6% and 81% identical to the corresponding sequence of SPV and to Orf virus OV-SA200 (ORFV-SA200, AY386264.1) respectively. The nucleotide sequence derived from the sea lion poxvirus corresponds to nucleotide positions 11606-11022 of the Orf virus genome (strain OV-SA200). A multiple sequence alignment of the deduced amino acid sequence of the sea lion parapoxvirus with the known sequences of other parapoxviruses is presented in Figure 3-4. At the amino acid level the sea lion poxvirus sequence was 97.3% and 76.1% identical to the harbor seal poxvirus from Northern Europe and to Orf virus OV-SA200 respectively. Further, the sea lion poxvirus amino acid sequence was 77% identical to the corresponding amino acid sequences of bovine papular stomatitis virus strain BV-AR02 (BPSV, AY386265.1) and pseudocowpox virus (PCPV, AY424972.1), and 76% identical to the amino acid sequences of the red deer parapoxvirus (PVNZ, AB044794.1) and the Orf virus strains IA82 (ORFV-I82, AY386263.1) and NZ2 (ORFV-NZ2, OVU06671).

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36 The evolutionary relationship of the sea lion poxvirus with the other parapoxviruses and with the prototypal member of the other genera of chordopoxviruses is depicted using a phylogenetic tree (Figure 3-5). The phylogenetic analysis of the 184 translated amino acids of the putative major envelope antigen of parapoxviruses shows that the sea lion poxvirus is most closely related to the harbor seal poxvirus from Europe. While the poxvirus of the California sea lions and the European sealpox virus are most closely related to the members of the genus Parapoxvirus, they do make up a distinct phylogenetic subgroup within the parapoxviruses (Figure 3-5). Discussion In this study we report nodular dermal changes in California sea lions at a rehabilitation facility. Electron microscopic examination of a poxvirus isolated from such a skin nodule showed particles with morphologic characteristics of parapoxviruses. A partial sequence of the gene encoding the viral major envelope protein confirmed the assignment to the subgroup of parapoxviruses and showed that the sea lion parapoxvirus was most closely related to a parapoxvirus of harbor seals of the North Sea. Although a poxvirus infection in a pinniped was first described in 1969 (Wilson et al. 1969) and several cases have been reported since, reports on the exact nature of the etiologic agents have been conflicting. In most instances the poxvirus was classified as a parapoxvirus (Wilson and Sweeney 1970, Wilson et al. 1972, Hicks and Worthy 1987, Simpson et al. 1994, Osterhaus et al. 1994, Nettleton et al. 1995, Muller et al. 2003). In one case, a mixed infection of a parapoxvirus and an orthopoxvirus was reported (Osterhaus et al. 1990). The classification of the poxviruses in all these cases was based solely on the characteristics of surface tubules of purified virions on negative staining EM. However, it has been suggested that these surface characteristics are preparation

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37 artifacts induced by osmotic stress imposed during negative staining and that in reality poxvirions have a smooth surface (Dubochet et al., 1994). While other evidence has since been presented suggesting that the appearance of the surface tubules is not artifactual (Wilton et al., 1995; Malkin et al., 2003; Heuser, 2005), sequencing of viral genes is the current method of choice for identifying poxviruses. Only in one poxvirus outbreak in harbor seals from the North Sea was the poxvirus classified as a parapoxvirus based on sequence data that were obtained directly from the pox lesion (Becher et al. 2002). The difficulty in isolating marine mammal poxviruses has been one of the greatest impediments for the characterization of these viruses. These viruses have only been isolated on early passage kidney cell cultures derived from pinnipeds (Osterhaus et al. 1990, Osterhaus et al. 1994, Nettleton et al. 1995), whereas attempts to isolate poxviruses from pinnipeds using primary rabbit kidney cells, canine kidney cells, and African green monkey kidney cells (Vero) have failed (Osterhaus et al. 1990). Similarly, our attempts to isolate the poxvirus from California sea lions onto modified African green monkey kidney cells (BSC40) failed, but virus isolation was successfully carried out using early passage California sea lion kidney cells. Our study is the first providing DNA sequences for a pinniped poxvirus that has been cultured. The sequence analysis data was complemented by positive staining and negative staining electron microscopic findings. On the negative stained preparation the regularly arranged surface tubule structure, a hallmark feature of parapox virions (Moss 1996), was visible. The phylogenetic analysis of the amino acid sequence of the putative major envelope antigen p42K confirmed that the SLPV-1 is most closely related to the

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38 parapoxviruses. All observed virions were of the structured mulberry type (Wilson and Sweeney 1970). Some shorter, more rounded virions were also observed in the negative stain preparation, in addition to those with the more typical parapox morphology. These atypical viral particles were observed in other studies (Simpson et al. 1994) and are considered either immature or disrupted virions. Based on part of the open reading frame of the p42K surface protein, SLPV-1 is most closely related to the European harbor seal poxvirus. However, they are only 97.3% identical at the amino acid level and thus are distinct. The phylogenetic analysis of the amino acid sequence of SLPV-1 indicated that the European harbor seal poxvirus and SLPV-1 should be classified within the genus Parapoxvirus. The degree of identity between the amino acid chain translated from the target gene between two Orf virus strains and between SLPV-1 and the harbor seal poxvirus from Northern Europe was 97% in both cases. In order to determine whether SLPV-1 is a novel parapoxvirus species, a more extensive study of the SLPV-1 genome sequence was initiated and is ongoing. In the absence of the complete viral genome sequence, host range is one of the main criteria for provisionally defining virus species within the genus Parapoxvirus (Pringle 1991). The natural host range of this poxvirus of California sea lions is presently unknown. Guadalupe fur seals (Arctocephalus townsendi), northern fur seals (Callorhinus ursinus), northern or Steller sea lions (Eumetopias jubatus), Pacific harbor seals (P. vitulina richardsii) and northern elephant seals (Mirounga angustirostris) frequent the California coast as well and poxvirus infections have been reported in several of these species (Hadlow et al. 1980, Hastings et al. 1989). To date we have been unable to obtain pox lesion samples from these sympatric pinniped species for molecular

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39 studies. Pacific harbor seals, northern elephant seals and California sea lions are often housed in rehabilitation centers at the same time. A previous report of a poxvirus outbreak in this facility did suggest transmission of the same poxvirus from harbor seals to California sea lions and northern elephant seals (Hastings et al. 1989). The transmission of the poxvirus between species during that outbreak was however unconfirmed, as electron microscopy images were not obtained from the poxvirus(es) of neither the affected California sea lions nor the northern elephant seals, and no viral sequence was generated to confirm that the same strain of poxvirus was affecting all three species. The high degree of amino acid sequence conservation between the European harbor seal poxvirus and the novel California sea lion poxvirus was noteworthy. Not only are the two poxvirus strains apparently geographically isolated, but Atlantic harbor seals (P. vitulina vitulina) and California sea lions (Z. californianus) respectively belong to the families Phocidae and Otariidae, and they are therefore taxonomically very distinct hosts. If the parapoxviruses of pinnipeds are able to infect multiple host species, viruses circulating in the Atlantic basin may be able to enter the Pacific (and vice versa) via infection of the Arctic ice seals (harp seal, hooded seal). As a result, the sea lion and harbor seal parapoxvirus strains may have diverged much more recently which would account for the high degree of genetic identity between the two viruses. Differences were seen in the nature and distribution of the lesions caused by the poxviruses in Atlantic harbor seals and California sea lions, whereas the histological appearance was very similar. SLPV-1 of California sea lions was associated with nodular skin lesions of head and neck that were occasionally focally ulcerated. The most obvious

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40 pathology observed during the poxvirus outbreak in harbor seals were ulcerating to verrucose oral lesions and ulcerated cutaneous lesions of flippers, chest, neck and perineum (Muller et al. 2003). This divergent clinical presentation may be due to different virus-host interactions or due to the use of different husbandry procedures. Muller et al. (2003) suggested that the parapoxvirus may have been transmitted between the harbor seals via contaminated feeding tubes leading to oral and mucosal lesions, whereas the source of infection for the California sea lions was unknown.

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41 Figure 3-1. Photomicrograph of a pox lesion (H & E stain, 4X). Diffuse necrosis was observed in the outer layers of the epidermis. The dermis contains focal areas of necrosis and diffuse infiltrates of mixed inflammatory cells. Inset (H & E stain, 40X): ballooning degeneration of epidermal cells within the stratum spinosum. Many ballooned cells contained large, coalescing eosinophilic intracytoplasmic inclusions (arrowheads).

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42 Figure 3-2. Transmission electron micrographs of infected cell sea lion kidney cell cultures. Mature virions (M), immature virus particles (I), crescents (C) and viroplasm (VP) of the California sea lion poxvirus can be observed. The cores of mature virions were ovoid to lanceolate and only occasionally dumbbell shaped.

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43 Figure 3-3. Negative contrast electron micrograph. The virus particles appeared oval or cylindrical with convex ends. The negative staining also revealed the regularly arranged surface tubule spiral; consistent with parapoxvirus morphology (mulberry type).

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44 SLPV-1 TGGSLATIKN LGVYSTNKHL AVDLMNRYNT FSSMVVDPKQ PFTRFCCAMI 50 SPV .......... .......... .......... .......S.. .......... 50 Orf-NZ82 ....VS.... ..L....... .W........ .Y..I.E..V ....L...IV 50 BPSV ....ISN... .......R.. .T........ .Y..I.E..V ..S.L..... 50 Orf-IA82 ....VS.... ..L....... .W........ .Y..I.E..V ....L...VV 50 Orf-SA00 ....VS.... ..L....... .W........ .Y..I.E..V ....L...VV 50 PCPV ....VS.... ..L....... .A........ .Y..I.E..V ....L...IV 50 PVNZ ....IQ.... ..L.....R. .A........ .F..I.E..I ....M..S.. 50 SLPV-1 TPTATDFHMN HSGGGVFFSD SPERFLGFYR TLDEDLVLHR IDAAENSIDL 100 SPV .......... .......... .......... .......... ....K..... 100 Orf-NZ82 .....N..LD .......... .......... .......... .EN.K..... 100 BPSV ........LD .A........ A..K...... .......... ..S.K..... 100 Orf-IA82 .....N..LD .......... .......... .......... .EN.K..... 100 Orf-SA00 .....N..L. .......... .......... .......... .EN.K..... 100 PCPV ........L. .......... .......... .......... .EN.N..... 100 PVNZ ........L. .A...I.... A..K...... .......... .NS.V..... 100 SLPV-1 SLLSMVPVVR SGSEVYYWPL ITDALLRAAI NRSVRVRIIV SQWRNADPLS 150 SPV .......... ..G....... .M........ .......... .......... 150 Orf-NZ82 ........IK HA.A.E...Q .I........ ..G....V.I TE.K...... 150 BPSV ....L...I. HADR.E...R .M........ D......V.. TE.K...... 150 Orf-IA82 ........IK HA.A.E...R .I........ D.G....V.I TE.K...... 150 Orf-SA00 ........IK HAGA.E...R .I........ ..G....V.I TE.K...... 150 PCPV ........IK HA.A.E...Q .I........ ..G......T TE.K...... 150 PVNZ ....I...I. HA.SME...A .M.......V E.G....V.. TE.K...... 150 SLPV-1 VAAVRALDNF GVGHIDITAR WFAIPGRDDA SNNT 184 SPV .......... ....V..... .......... .... 184 Orf-NZ82 .S.A.S..D. ...SV.MSV. K.VV...... A... 184 BPSV .S.A.T.ND. ...S...ST. L.S....... A... 184 Orf-IA82 .S.A.S..D. ...SV.MSV. K.VV...... A... 184 Orf-SA00 .S.A.S..D. ...SV.MSV. K.VV...... A... 184 PCPV LS.A.S.ND. ...SV.MSV. K.V....... A... 184 PVNZ ...A.T.ND. ...S...S.. L.S....... A... 184 Figure 3-4. Multiple sequence alignment of the deduced amino acid sequence of part of the putative virion envelope antigen (p42K). The sequence from the sea lion parapoxvirus (SLPV-1) is compared to the corresponding sequence of published parapoxviruses. These sequences are: Orf virus strains SA200 (ORFV-SA00), IA82 (ORFV-I82), NZ2 (ORFV-NZ2), bovine papular stomatitis virus strain BV-AR02 (BPSV), pseudocowpox virus (PCPV), red deer parapoxvirus (PVNZ) and the European harbor seal poxvirus (SPV). This sequence alignment was generated using the ClustalX software.

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45 Figure 3-5. Phylogram depicting the relationship of SLPV-1 to the members of the genus Parapox: Orf virus NZ2 (ORFV-NZ2), bovine papular stomatitis virus BV-AR02 (BPSV), pseudocowpox virus (PCPV), red deer parapoxvirus (PVNZ) and the European harbor seal poxvirus (SPV). Also included are the protypal viruses of all other Chordopox genera: genus Orthopox: vaccinia virus strain WR, (VACV-WR, AY243312.1), squirrel poxvirus (SQPV, AY340985.1); genus Suipox: Swinepox virus isolate 17077-99 (SWPV, AF410153.1); genus Capripox: Sheeppox virus strain A (SHPV, AY077833.1); genus Molluscipoxvirus: Molluscum contagiosum virus subtype 1 (MCV, U60315.1); genus Leporipoxvirus: Myxoma virus strain Lausanne (MYXV, AF170726.2); genus Yatapoxvirus: Yaba monkey tumor virus (YABA, AY386371.1) and genus Avipoxvirus: Fowlpox virus (FWLPV, AF198100.1). This phylogenetic tree was generated according to the maximum likelihood method using ClustalX software. Branch lengths are proportional to genetic distances. Numbers indicate the percentage of 100 bootstrap replicates that support each interior branch.

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CHAPTER 4 SEROEPIDEMIOLOGY OF POXVIRUS INFECTIONS IN CAPTIVE AND FREE-RANGING CALIFORNIA SEA LIONS Introduction The viral family Poxviridae includes several viruses of veterinary and medical importance. Poxviruses affect domestic and wild mammals, reptiles and birds. In humans, the most notorious member, variola virus, causes smallpox (Moss 1996, Murphy et al. 1999). Aquatic mammals also are susceptible to poxvirus infections. The susceptibility of pinnipeds to pox infections was first suspected in 1969 when a 1-yr-old sea lion was presented with pox-like lesions (Wilson et al. 1969). The presence of a poxvirus has since been confirmed in harbor seals (Phoca vitulina) (Wilson et al. 1972), northern fur seals (Callorhinus ursinus) (Hadlow et al. 1980), grey seals (Halichoerus grypus) (Hicks and Worthy 1987), northern elephant seals (Mirounga angustirostris) (Hastings et al. 1989), South American sea lions (Otaria flavescens) (Wilson and Poglayen-Neuwall 1971) and, more recently, in California sea lions (Zalophus californianus) (Nollens et al. In press). Pox lesions in pinnipeds are seen as raised cutaneous nodules, reaching up to 3 cm in diameter (Hastings et al. 1989), which may ulcerate and suppurate. Pox lesions develop primarily on the head and neck, but the thorax, abdomen and flippers can be affected. In severe cases, the body of affected animals can be covered by up to several hundred nodules (Wilson and Poglayen-Neuwall 1971). Histologically, the pox nodules are formed by epithelial hyperplasia and hypertrophy, with epithelial cells often 46

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47 containing large, eosinophilic, intracytoplasmic inclusions consisting of pox virions (Hadlow et al. 1980). Mortality in captive pinnipeds is typically low, except in cases where nodules develop on the eyes or in the oral cavity or nostrils, where the infection impairs feeding or respiration. However, the morbidity of poxvirus infections is typically high, especially in young animals. Captive pinnipeds with pox lesions are unsuitable for display, and secondary parapoxvirus infections are likely to negatively affect the physiology, prognosis and length of hospitalization of animals in rehabilitation centers. The seal poxvirus is also a zoonosis, and is therefore of concern to animal care staff (Hicks and Worthy 1987). Several early reports are available on the light microscopic and electron microscopic appearance of the pox lesions in different species of pinnipeds (reviewed in Muller et al. 2003). More recent studies have confirmed parapoxviruses as the etiologic agent of this disease (Nollens et al. In press, Osterhaus et al. 1990, Osterhaus et al. 1994). Previous knowledge on the epidemiology of poxvirus infections in sea lions is limited to one study conducted in hospitalized sea lions in rehabilitation center in California (Nollens et al. In press). In that study, readmission to a rehabilitation center, long duration of hospitalization and high hospital caseload were identified as risk factors that may predispose California sea lions to acquiring poxvirus infections during hospitalization. While this epidemiologic information is relevant for formulation, implementation, and evaluation of infection control measures to reduce risk of nosocomial infections in hospitalized sea lions, the prevalence of subclinical poxvirus in free-ranging California sea lions is not known. Furthermore, no serological test for detection of anti-parapoxviral antibodies has been developed. Therefore, the objectives of

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48 this study were to (1) develop and validate an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against poxvirus in California sea lions, (2) describe the antibody response of affected and unaffected sea lions to parapoxviruses during rehabilitation, and (3) estimate the seroprevalence of parapoxvirus infections in wild California sea lions populations. Methods Positive and Negative Reference Serum Samples A panel of 24 positive and 26 negative reference samples was selected from the California sea lion serumbank at The Marine Mammal Center (TMMC) (37’N, 122’W) to optimize and evaluate the diagnostic performance of the ELISA. The 24 positive reference sera were collected from 24 CSL that had been found stranded on the central California coast between 6 July 2002 and 19 Sept 2004 and were hospitalized in TMMC for rehabilitation. Poxvirus infections in California sea lions typically manifest as prominent dermal nodules, 1-3 cm in diameter, that occur on the head, neck or flippers of the sea lions. The presence of these dermal pox lesions is pathognomonic for the presence of a poxvirus (Hastings et al., 1989). Sea lions with dermal pox lesions were classified as “affected”. The 24 CSL developed poxvirus lesions between 1 and 87 days (Median = 20, Min = 1, Max = 87) after hospitalization in TMMC. One serum sample was collected from each of the 24 sea lions on average 12 days after first clinical signs (FCS) (Median = 3, Min = 1, Max = 65). The negative reference sera were collected from 26 CSL fetuses that were aborted between 5 July 1998 and 3 July 2003 by adult female CSL affected by domoic acid toxicosis. This group of 26 CSL was classified as “unaffected”. All sera were kept frozen at -80C until analysis.

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49 ELISA Procedure Antigen production. A parapoxvirus of California sea lions (Sea lion poxvirus 1; SLPV-1) was isolated in our laboratory in early passage California sea lion kidney cells (Nollens et al. In press). Purified SLPV-1 was used as antigen in the whole virus-based indirect ELISA system. For purification, the virus was propagated in early passage California sea lions kidney cells. When more than 90% of the monolayer showed cytopathic effect, the cells and supernatants were harvested and freeze-thawed twice and the virus was purified as previously described (Nollens et al. In press). Briefly, intracellular mature virions (IMV) were purified from the infected cell lysates by high-speed centrifugation through a 40% sucrose cushion followed by banding of the IMV on a 25-50% Na-diatrizoate density gradient. The virus was resuspended in 10 mM Tris (pH 8.2) and stored at -80C until analysis. For use in the ELISA, the virus was thawed, sonicated for 3 min and inactivated in 2% paraformaldehyde for 30 min at room temperature. The protein concentration of the inactivated virus concentrate was determined using a modified Bradford assay for ELISA, after which the inactivated virus was diluted in phosphate-buffered saline (PBS) to the desired coating concentration. Selection of secondary detection reagent. The ability of a panel of alkaline phosphatase labeled secondary reagents [n = 19; protein G (Zymed, San Francisco, CA); protein A, goat anti-chicken whole IgG pAb, goat anti-mouse IgG Fab pAb, goat anti-mouse whole IgG pAb, goat anti-rat whole IgG pAb, goat anti-guinea pig Ig pAb, goat anti-rabbit whole IgG pAb, goat anti-hamster IgG (heavy and light chain) pAb, rabbit anti-horse whole Ig pAb, rabbit anti-cow whole Ig pAb, rabbit anti-goat whole Ig pAb, donkey anti-sheep Ig pAb (Sigma, St. Louis, MO); goat anti-rabbit IgG Fab pAb, goat anti-rabbit IgG Fc pAb, donkey anti-rabbit IgG (heavy and light chain) pAb (Pierce,

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50 Rockford, IL); rabbit anti-dolphin whole IgG (Bethyl laboratories, Montgomery, TX); rabbit anti-dolphin whole IgG, rabbit anti-bat whole IgG (University of Florida, Gainesville, FL)] to react with California sea lions immunoglobulins was evaluated using ELISA. Wells of a high protein binding microplate (Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) were coated with 50 l of whole California sea lions serum diluted 1:1,000 in 1x phosphate buffered saline (PBS), and were left to adsorb overnight at 4C. After this and each subsequent step, all wells were washed three times with PBS/Az and 0.05% PBS-T using an automated microplate washer (Biotek Instruments, Winooski, VT). After washing, the wells were blocked with 1% bovine serum albumin (BSA; Roche Diagnostics, Indianapolis, IN), after which each of the 22 secondary reagents [diluted 1:2000 in 1% BSA in PBS with 0.02% azide (PBS/Az)] was applied to each of two wells. Each step of the ELISA was left to incubate with gentle agitation (Nutator; Adams, Fisher Scientific, Pittsburgh, PA) for 1 hr at room temperature. Finally, 1.0 mg/ml -1 P-Nitrophenyl Phosphate (PNPP; Sigma, St. Louis, MO) substrate was added and the absorbance at 405 nM (OD 405 ) was recorded after 30 min using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA). Of the 22 reagents tested, protein G (Zymed, San Francisco, CA), protein A, goat anti-chicken Ig pAb, goat anti-rabbit Ig pAb, rabbit anti-horse Ig pAb, goat anti-guinea pig Ig pAb, rabbit anti-cow Ig pAb and donkey anti-sheep Ig pAb (Sigma, St. Louis, MO) appeared to crossreact with California sea lion serum in ELISA format. The highest reactivity was observed using protein A (OD 405 = 4.173), rabbit anti-cow Ig pAb (OD 405 = 4.100), donkey anti-sheep Ig pAb (OD 405 = 4.037) and protein G (OD 405 = 2.653).

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51 Next, the specificity of protein A, rabbit anti-cow Ig pAb, donkey anti-sheep Ig pAb and protein G for immunoglobulins of California sea lions was evaluated via by Na+ dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). California sea lion serum, diluted 1:10 in PBS, was separated (45 min at 200 V) by SDS-PAGE under denaturing conditions, with a precast 10% (wt:vol) polyacrylamide bis-tris Nupage gel and morpholinepropanesulfonic acid (MOPS) running buffer (Invitrogen, Carlsbad, CA). Purified mouse and bovine IgG, diluted 1:10 in PBS, were included as reference sera. The protein fractions from all lanes were electrophoretically transferred (60 min at 30 V) to a nitrocellulose sheet (Invitrogen, Carlsbad, CA) using a Novex Western transfer apparatus. Tris-glycine buffer (Invitrogen, Carlsbad, CA) in 20% methanol was used as the transfer buffer. After transfer the nitrocellulose was blocked overnight using 5% nonfat dry milk in PBS/Az at room temperature. The nitrocellulose blot was washed with PBS-T and placed into a Fast Blot-Developer (Pierce, Rockford, Ill). A total of 900 l of each of the four secondary reagents was loaded into a channel each and was left to incubate for 60 min at room temperature with gentle agitation (Nutator; Adams, Fisher Scientific, Pittsburgh, PA). Rabbit anti-mouse IgG polyclonal antibody (pAb) and rabbit anti-bovine IgG pAb were loaded into the mouse and bovine IgG reference lanes. After washing three times with PBS-Az, the blot was developed with NBT-BCIP (nitroblue tetrazolium 5-bromo-4-chloro-3 inolyphosphate p-toluidine) substrate following the manufacturer’s instructions (Sigma, St. Louis, MO). All four reagents reacted with a band comparable in size with the heavy chain of mouse and bovine IgG (55kDa). Overall, AP labeled protein A appeared to have the highest affinity for immunoglobulins of California sea lions on ELISA and reacted strongly with a band of approximately

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52 55kDa on the SDS-PAGE blot. Consequently Protein A was selected as the secondary detection reagent for the ELISA. Optimization of ELISA parameters. Three randomly selected positive and three randomly selected negative reference serum samples were used to optimize the ELISA conditions. All assay parameters were varied (working volume: 50 – 100 l; coating concentration: 1 – 15 g/ml; California sea lion serum dilution: 1:20-1:2,500; developing time: 15 60 min), and the assay conditions with the highest ratio of the mean OD 405 of positive reference serum samples (n = 3) to the mean OD 405 of negative reference serum samples (n = 3) were selected. The conditions of the optimized ELISA protocol were as follows. Wells of high protein binding microplate (Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) were coated with 50 l of inactivated whole virus (SLPV-1) at 2 g/ml in PBS, and were left to adsorb overnight at 4C. After this and each subsequent step, all wells were washed three times with PBS/Az and 0.05% PBS-T using an automated microplate washer (Biotek Instruments, Winooski, VT). After washing, all wells were blocked with 1% bovine serum albumin (BSA) (Roche Diagnostics, Indianapolis, IN) in PBS/Az, after which the California sea lion sera were applied (1:500 in 1% BSA in PBS/Az) to each of two wells. AP-labeled protein A (1:2000 in 1% BSA in PBS/Az) was applied as the secondary reagent for the detection of bound sea lion antibodies. Each step of the ELISA was left to incubate with gentle agitation (Nutator; Adams, Fisher Scientific, Pittsburgh, PA) for 1 hr at room temperature. Finally, 1.0 mg/ml P-Nitrophenyl Phosphate (PNPP; Sigma, St. Louis, MO) substrate was added. The OD 405 was recorded 30 min after addition of the substrate using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA). To correct for variation between ELISA

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53 plates, the mean OD 405 of each sample was divided by the OD 405 of positive reference serum California sea lion 5814b. Diagnostic Performance (Sensitivity and Specificity) of ELISA The sensitivity (Se, proportion of exposed California sea lions with positive test results), specificity (Sp, proportion of California sea lions that have not been exposed with negative test results), positive predictive values (the fraction of California sea lions that test positive that have truly been exposed to poxviruses) and negative predictive values (the fraction of California sea lions that test negative that have truly not been exposed to poxviruses) of the ELISA were calculated at different cut-off points using MedCalc 4.15c software (Frank Schoonjans). Selection of cut-off point to identify highest performance (combination of high Se and high Sp) was made by means of receiver-operator characteristic (ROC) curves based on the corrected OD405 of the 24 positive and 26 negative reference samples using MedCalc 4.15c software (Frank Schoonjans). Seroepidemiology of Parapoxvirus Infections To evaluate the antibody response of affected and unaffected California sea lions to parapoxviruses during rehabilitation, 56 serum samples were collected opportunistically from 26 affected yearling and subadult California sea lions at The Marine Mammal Center (TMMC), Sausalito, CA. The 56 serum samples consisted of 20 initial samples (collected from 16 sea lions prior to FCS), 18 acute samples (collected from 18 sea lions on the first day of clinical signs) and 18 convalescent samples (collected from 15 sea lions 1 day after FCS). On average 2.2 samples (Median = 2, Min = 2, Max = 4) were available for each affected sea lion. The 20 samples collected from all 16 affected sea lions prior to FCS were collected on average 22 days after admission (Median = 19, Min

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54 = 1, Max = 64) and 25 days prior to FCS (Median = 17, Min = 1, Max = 85). The 18 convalescent sera collected from all 15 affected sea lions were collected on average 22 days after FCS (Median = 14, Min = 2, Max = 65). A complete set of paired sera, consisting of at least one initial serum (prior to FCS) and at least one convalescent serum sample (1 day after FCS), was available for eight sea lions. The initial (N = 10) and convalescent (N =8) sera were collected from these eight sea lions on average 24 days (Median = 16, Min = 1, Max = 69) prior to FCS and 17 days (Median = 14, Min = 3, Max = 44) after FCS. Additionally, 164 serum samples were collected opportunistically from 74 unaffected yearling, subadult and adult California sea lions, starting with a sample obtained within six days of admission and ending with samples just prior to release. On average 2.2 samples (median = 2, min = 2, max = 5) were available for each unaffected sea lion. All 100 California sea lions included in this study had been found stranded on the central California coast between 27 Jan 2002 and 19 Sept 2004 and were hospitalized in TMMC for rehabilitation. The presence of anti-parapoxviral antibodies in all serum samples was determined using indirect ELISA described above. Reference sera CSL5814b and F5200 were included on each ELISA plate as positive and negative controls respectively. Each serum sample was analyzed in duplicate and the corrected OD405 was calculated for each sample as described above. The sea lion age classes were defined as follows: pup, <1 yr; yearling, 1 to <2 yrs; subadult male, 2 to 8 yrs; subadult female, 2 to 5 years; adult male, >8 yrs; and adult female >5 yrs. Free-ranging sea lions. A total of 551 serum samples from 67 weaned (6-12 mo-old) California sea lion pups, 130 yearlings (1-2 yr old), 188 juveniles/subadults and 166

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55 adult California sea lions were selected from the serum bank at TMMC (Table 1). Only serum samples that were collected within six days of admission to TMMC and from animals that had not previously been admitted to a rehabilitation center were included in the survey. Sea lions that had been in captivity less than 6 days were considered serologically representative of the free-ranging population. All 551 California sea lions had been found stranded on the central California coast between 3 Jan 2000 and 24 Aug 2004 and were hospitalized in TMMC for rehabilitation. In addition, a second group of serum samples from 150 pre-weanling pups (2-4 mo old) and 60 adult male California sea lions from ongoing population monitoring programs were selected and tested for poxvirus antibodies. The sera from the pre-weanling California sea lions were collected on San Miguel Island, CA (34’N, 120’W) during Sept 2001, 2002 and 2003 and during Oct 2000 and 2004. The sera from adult male sea lions were collected in Shilshole Bay, WA (4741’N, 122’W) during April and Oct 2001 and May 2003. Forty-two (47%) of the adult males were sampled just prior to the annual breeding season (April June). Seventy-seven (56%) of the adult female sea lions were sampled during their hospitalization at TMMC shortly after the breeding season (July Sept), whereas only 24 (18%) of the adult females was sampled just prior to the breeding season (April – June) (Table 1). Each serum sample was analyzed in duplicate and the corrected OD405 was calculated for each sample as described above. The prevalence of circulating antibodies amongst the age categories and sampling locations were compared via a 2 test using Statistix software (Analytical Software, Tallahassee, FL).

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56 Results Diagnostic Performance (Sensitivity and Specificity) of ELISA Based on the ROC analysis of the corrected OD405 of the positive and negative reference sera, a positive:negative cut-off value of 0.10 was selected. California sea lion serum samples for which the corrected OD405 was 0.10 were considered to have no antibodies against SLPV-1 (negative). Samples for which the corrected OD405 was >0.10 were considered to contain antibodies against SLPV-1 (positive or exposed), whereas samples for which the corrected OD405 was 0.10 were considered not to contain detectable antibodies against SLPV-1 (negative or not exposed). Using this cut-off point, the Se was 100% (95% CI = 86 to 100%) and the Sp was 100% (95% CI = 87 to 100%). The calculated area under the curve (AUC) was 1.0 (95% CI = 0.92 to 1.00), indicating that the test accurately identified all affected and unaffected California sea lions used in the evaluation. Based on the lower 95% confidence limits of the sensitivity and specificity and based on a 91% prevalence of parapoxvirus antibodies in California sea lions, the predictive values for a positive and a negative result were 98% and 39% respectively. Seroepidemiology of Parapoxvirus Infections Captive sea lions. A rise in circulating anti-parapoxviral antibodies was observed in all sea lions (n = 8) for which at least one initial serum (collected prior to FCS) and at least one convalescent serum sample (1 day after FCS) was available. The intensity and the time of onset of the humoral immune response varied, but a rise in circulating antibodies was detectable as early as two days before FCS in three sea lions. The rise in circulating antibodies was detectable in all convalescent sera collected > 1 day after FCS. On average and compared to the corresponding initial serum sample, a 1.8-fold increase

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57 in circulating antibodies was observed in sera collected from sea lions on the first day of clinical signs (N = 10 sea lions; = 1.8-fold increase, SD = 0.6, Min = 1.2, Max = 3.0). On average and compared to the corresponding initial serum sample, a 3.6-fold increase in the circulating antibody level was observed in all convalescent sera collected from sea lions > 1 day after FCS (n = 8 sea lions; = 3.6-fold increase, SD = 2.3, Min = 1.7, Max = 7.6). The corrected OD405 of all serum samples (n = 20) collected from affected California sea lions prior to developing pox lesions were greater than 0.1 and were considered positive. Compared to sera collected upon admission, the level of circulating antibodies in two affected California sea lions had not yet increased at 24 and 39 days after admission to the hospital. These two animals developed clinical pox disease after 70 and 58 days respectively after admission to the hospital. Subsequently, a 2-3 fold increase in the level of circulating antibodies was observed in sera collected on day 73 and 75 respectively of hospitalization. The corrected OD405 of 63 (85%) serum samples collected from the 74 unaffected California sea lions upon admission were greater than 0.1 and were considered positive. Of the 74 unaffected sea lions, five (7%) sea lions also had 3.6 fold increased levels of circulating antibodies (mean = 4.3-fold increase, SD = 0.7, Min = 3.6, Max = 5.0) prior to release than at admission. Of these five sea lions, three were maintained at the rehabilitation center for 31, 32 and 88 days after the rise in circulating antibodies was first detected and no pox lesions were observed. Free-ranging sea lions. A total of 761 California sea lions were included in the survey (Table 1). The sample consisted of 418 males (55%) and 342 (45%) females. Overall, the seroprevalence of anti-parapoxviral antibodies in the sampled and tested

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58 population of free-ranging California sea lions was 91% (95% CI = 89 to 93%). The prevalence of seropositive sea lions was not different between females (92%) and males (89%) (Table 1). The proportion of seropositive adult male sea lions (75%) was higher than the proportion of seropositive adult females (86%), whereas the proportion of seropositive female sea lions was lower than that of males in all other age categories. The prevalence of seropositive sea lions was lower in animals from Shilshole Bay, WA (71%), than that of any other location. In the univariable analysis, seroprevalence was significantly different within age groups, origin and month of sampling (p < 0.05). In the multivariable analysis, using logistic regression, the odds of exposure to parapoxviruses was higher in males, non-adult sea lions, sea lions from counties other than Shilhole Bay, and sea lions captured during October-December. Discussion Our study results indicate that the newly developed ELISA test is a useful tool for the serologic diagnosis of poxvirus infections in California sea lions. The area under the ROC curve was 1.0, indicating that the ELISA is highly accurate. The ROC curve is a plot of the Se against the Sp of a given test and, as a result, the area under the curve (AUC) is a measure of overall performance of a diagnostic test at multiple cut-off points (Greiner et al., 2003). The AUC values range between 0 and 1, with 1 representing a hypothetical perfect test (i.e. 100% sensitive, 100% specific). An AUC value of 0.5 indicates that the test is not informative, no better than a coin toss. When a ROC analysis was applied to the ELISA for the detection of anti-parapoxviral antibodies in sea lions, a cut-off point of 0.10 was associated with a test Se and Sp of 100%. The lower and upper limits of the 95% confidence intervals for sensitivity and specificity were xx to 100% and xx to 100%, respectively, indicating that the precision of the calculated point estimates

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59 for both parameters was low, because our sample size was limited to 26 affected CSL and 26 non-affected sea lions; therefore, our study results must be interpreted with caution. While the sensitivity and specificity are important parameters used to measure the accuracy of a diagnostic test, in the field, clinicians and wildlife managers are interested in knowing if a sea lion that tests positively to a serologic test is indeed infected, or if a negative test is indicative of absence of infection. These test characteristics are known as positive and negative predictive values, respectively, and they are influenced by disease prevalence. For example, based on an observed disease prevalence of 91% and a test sensitivity of 86% and a test specificity of 87%, the positive and negative predictive values are 99% and 39% respectively. If this ELISA test were used in a subpopulation of sea lions were the observed disease prevalence was 50%, the positive predictive value would decrease from 99% to 87%, and the negative predictive value would increase from 39% to 86% respectively (MedCalc 4.15c ). The onset and the intensity of the humoral response of California sea lions to parapoxviruses were variable, but a rise in the level of antibodies was detectable in all California sea lions with clinical signs. In some cases, anti-parapoxviral antibodies were detected even before clinical signs were first observed. These findings are very similar to the serologic observations of infections with orf virus (a.k.a. scabby mouth, contagious ecthyma; the protypal parapoxvirus) in sheep: the onset and intensity of the antibody response is variable, an antibody response is present in all cases and, above all, the appearance of antibodies coincides with the appearance of first clinical signs (Dales and Pogo, 1981; Haig et al., 1997). Based on the high similarity between the anti-parapoxviral antibody dynamics of sheep and sea lions, it is likely that the length of the

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60 incubation period (i.e. the period between infection and first clinical signs) is also similar. Based on experimental transmission studies, the incubation period of orf virus in sheep was determined to be approximately seven days (Dales and Pogo, 1981; Haig et al., 1997). Similar observations have been recorded in sheep, in which the timing and intensity of the antibody response to parapoxviruses are equally variable. This suggests that the incubation period of parapoxvirus infections in California sea lions is also approximately seven days. Previous exposure of California sea lions to parapoxviruses does not necessarily protect against reinfection. All serum samples that were collected prior to clinical disease were seropositive and indicated prior exposure to a parapoxvirus. Similarly, the acquired immunity of sheep to parapoxviruses is short-lived and reinfection can occur (Haig et al., 1997). The mechanism behind this weak acquired protection of sheep is poorly understood, but the characteristic localized nature of a parapoxvirus infection may be in part responsible for this. Parapoxviruses typically do not cause a secondary, generalized viremia. If infections are acute and restricted to epidermal cells, the immune response may provide less protection. Localized infections may also go undetected for longer by the immune system and pre-existing immunity may therefore have limited effect (Haig et al., 1997). Conversely, some studies have suggested that parapoxviruses do cause a secondary viremia, but this remains unconfirmed (Iketani et al., 2002). Regardless of the mechanism, the inability of the sea lions to be protected following exposure to parapoxviruses suggests that the usefulness of a vaccine may be as ambiguous as orf virus vaccines are in sheep. The efficacy of parapoxvirus vaccines for sheep is disputed as in some cases they fail to confer protection against a challenge infection, in some cases

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61 even against homologous virus (Buddle et al., 1984; Pye, 1990). Vaccine strain differences, adaptation of the vaccine virus to cell culture and the antigenic diversity wild orf virus strains have been suggested as a cause for these vaccination failures (Buddle et al., 1984; Pye, 1990). However, a potent anamnestic immune response of sheep to orf virus reinfection has been reported (Haig et al., 1997). This anamnestic response does leave lambs susceptible to a second infection, but the lesions are smaller and heal more rapidly (Nettleton et al., 1996). However, we do not know if the clinical signs of sea lions become less severe as the animals are repeatedly infected. Two affected and five unaffected sea lions were exposed to a parapoxvirus and developed anti-parapoxviral antibodies during their hospitalization. Likewise, the appearance of clinical signs after 60 or more days does indicate that the infection was acquired during hospitalization, as previous estimates of incubation period range between 2-5 weeks (Hastings et al., 1989). Additionally, no known poxvirus has the ability to establish a latent infection and reactivation of a previous infection is therefore not likely. It may therefore be possible to reduce the incidence of poxvirus infections in pinnipeds in rehabilitation centers using prophylactic measures. These measures should be aimed at minimizing the direct and indirect contact between animals, both during the incubation period and during clinical disease. Further, we observed a rise in circulating antibodies in five unaffected animals as well. Of these five sea lions, three were maintained at the rehabilitation center for over 1 mo after the rise in circulating antibodies was first detected and no pox lesions were observed. This indicates that subclinical parapoxvirus infections can occur in California sea lions, suggesting all contact between unaffected animals should be minimized as well. Furthermore, as parapoxviruses have the ability to

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62 persist in the environment for months to years (Haig et al., 1997) and are resistant to desiccation, thorough cleaning of the hospital pens is advised. Effective cleaning practices should consist of the thorough scrubbing of the pens to remove all organic material, followed by bleaching or washing with detergents with known virucidal activity. A total of 693 of 761 (91%) of the sampled and tested study population of California sea lions tested positive to poxvirus antibodies. This high seroprevalence is likely a reflection of the typical highly infectious nature of poxviruses and the high rate of contact amongst wild California sea lions (Odell, 1975). Overall, male sea lions were more likely to have been exposed to parapoxviruses than female sea lions. Adult male California sea lions occupy separate bachelor colonies near the northern end of the range of their species (e.g. Shilshole Bay, WA), but each spring all reproductively active animals congregate in the California Channel Islands (e.g. San Miguel Island, CA) for pupping and subsequent breeding (Odell DK, 1975). The exact reasons for the lower antibody prevalence in adult males are unclear. Male California sea lions return to their batchelor colonies shortly after breeding and have little contact with pup and yearling animals. If, in free-ranging animals, the virus circulates particularly among young sea lions, adult males would be less frequently exposed. Alternatively, the lower antibody prevalence in adult males could be accounted for by an inherent bias in the sample set used for this serosurvey. Most adult male sea lions (47%) were sampled just prior to the breeding season, whereas the majority of adult female sea lions (56%) was sampled shortly after the breeding season. If the virus circulates during the annual breeding congregation and if the antibodies are short-lived, antibody levels of adult male sea lions

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63 may have dropped below the detection limit by the time of sampling. Unfortunately, due to the low number of samples collected during some parts of the year, our sample set does not allow for further exploration of a seasonal effect on antibody prevalence. Ideally, a randomly selected sample set with stratification for age, gender, season and location should have been employed in this serosurvey. In the absence of such a stratified sample a convenience sample was used and inherent biases can therefore not be discounted. Also, because our sample set is not weighted to reflect the population structure of California sea lions, the overall seroprevalence estimate needs to be interpreted with caution. Although our data do indicate that the prevalence of anti-parapoxviral antibodies in free-ranging California sea lions is high, the exact seroprevalence estimate of 91% may not apply to the free-ranging California sea lion population. The high seroprevalence in nursing sea lion pups (98-100%) was remarkable. The detected antibodies may be maternal antibodies, or sea lions may be exposed to poxviruses within the first few weeks of delivery. It appears more likely that the detected antibodies are maternal antibodies, as poxvirus lesions have not been reported in free-ranging, nursing California sea lion pups. The decreasing antibody prevalence with increasing age of sea lions < 1 yr old supports this hypothesis. However, serial samples collected within the first several months after birth would be needed to evaluate whether these antibodies in dependent California sea lions pups are maternally derived. Finally, exposure to parapoxviruses appears to occur frequently in the wild. As a result, the release of captive sea lions infected with parapoxvirus into the wild should not increase the frequency of exposure of free-ranging sea lions to the virus. Therefore, the

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64 presence of pox lesions in fully rehabilitated California sea lions should not preclude their return to the wild.

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65 Table 4-1. Sample size (N), number of animals with anti-parapoxviral antibodies (pos), antibody prevalence and 95% confidence interval (95% CI) for each age/gender category of California sea lions Age Gender N Pos Prevalence (%) 95% CI p gender p age class Overall prevalence 761 693 91.1 89.1-93.1 Adult Female 137 118 86.1 80.3-91.9 ref Male 89 67 75.3 66.3-84.3 0.06 Total 226 185 81.9 76.9-86.9 ref Juvenile/ Female 33 29 87.9 76.8-99.0 ref Subadult Male 155 149 96.1 93.1-99.1 0.14 Total 188 178 94.7 91.5-97.9 0.01 Yearling Female 57 51 89.5 81.5-97.5 ref (1-2 yr) Male 73 70 95.9 91.4-100 0.28 Total 130 121 93.1 88.7-97.5 0.01 Weanling pup Female 32 29 90.6 80.5-100 ref (6-12 mo) Male 35 33 94.3 86.6-100 0.91 Total 67 62 92.5 86.2-98.8 0.06 Pre-weanling pup Female 83 80 96.4 92.4-100 ref (2-4 mo) Male 67 67 100.0 100-100 0.33 Total 150 147 98.0 95.5-100 0.01

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66 Table 4-2. Sample size (N), number of animals with anti-parapoxviral antibodies (pos), antibody prevalence (%) and 95% confidence interval (95% CI) for each sampling location/gender category of California sea lions Origin / sampling location Gender N Pos Prevalence (%) 95% CI p location Shilshole Bay, WA Female 0 Male 56 Total 56 40 71.4 59.6-83.2 ref Female 9 Mendocino, Sonoma, Marin County Male 41 Total 50 45 90.0 81.7-98.3 0.03 Female 22 Alameda, Contra Costa, San Francisco,San Mateo County Male 59 Total 81 76 93.8 88.5-99.1 0.01 Santa Cruz County Female 42 Male 55 Total 97 87 89.7 83.7-95.7 0.01 Monterey County Female 78 Male 106 Total 184 175 95.1 92.0-98.2 0.01 Female 108 San Luis Obispo, Santa Barbara, Los Angeles County Male 31 Total 139 121 87.1 81.5-92.7 0.02 San Miguel Island Female 83 Male 67 Total 150 147 98.0 95.8-100 0.01

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67 Table 4-3. Sample size, number of animals with anti-parapoxviral antibodies (pos), and antibody prevalence (%) per month of sampling and age category of California sea lions July Sept Oct Dec Jan March April June Age/ gender N Pos (%) N Pos (%) N Pos (%) N Pos (%) Adult male 12 9 (75) 30 25 (83) 5 4 (80) 42 29 (69) Adult female 77 67 (87) 30 29 (97) 6 3 (50) 24 19 (79) Adult 89 76 (85) 60 54 (90) 11 7 (64) 66 48 (73) Juvenile/ Subadult 92 85 (92) 61 61 (100) 8 8 (100) 27 24 (89) Yearling (1-2 yr) 54 47 (87) 16 16 (100) 5 4 (80) 55 54 (98) Weanling (6-12 mo) n.a. n.a. n.a. n.a. 2 1 (50) 65 61 (94) Preweanling (2-4 mo) 107 106 (99) 43 41 (60) n.a. n.a. n.a. n.a. Overall prevalence 342 314 (92) 180 172 (96) 26 20 (77) 213 187 (88) n.a. = Not available. No California sea lions belonging to that age category are available during those months.

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CHAPTER 5 RISK FACTORS ASSOCIATED WITH DEVELOPMENT OF POXVIRUS LESIONS IN HOSPITALIZED CALIFORNIA SEA LIONS Introduction The presence of poxviruses has been confirmed in free-ranging pinnipeds from the northern and southern Atlantic and Pacific oceans, including California sea lions (Zalophus californianus) (Wilson et al. 1969, Hastings et al. 1989), harbor seals (Phoca vitulina) (Wilson et al. 1972, Muller et al. 2003), northern fur seals (Callorhinus ursinus) (Hadlow et al. 1980), grey seals (Halichoerus grypus) (Hicks and Worthy 1987, Osterhaus et al. 1990, Simpson et al. 1994, Osterhaus et al. 1994, Nettleton et al. 1995), northern elephant seals (Mirounga angustirostris) (Hastings et al. 1989) and South American sea lions (Otaria flavescens) (Wilson and Poglayen-Neuwall 1971). Poxvirus-induced lesions are pathognomonic. Typically, the lesions are raised, firm cutaneous nodules ( 3 cm in diameter), which may ulcerate and suppurate. Poxvirus lesions develop primarily on the head and neck of affected pinnipeds, but lesions have also been reported (Muller et al. 2003) on the oral mucosa and in the nasal passages and perineal area. In severe cases, cutaneous lesions can become confluent and spread to the thoracic and abdominal regions as well as the flippers. The body of severely affected pinnipeds can be covered by as many as several hundred nodules (Wilson and Poglayen-Neuwall 1971). Unless the lesions develop on the eyes or in the nostrils or oral cavity, the poxvirus-related mortality rate among captive pinnipeds is usually low (Muller et al. 2003); however, the poxvirus-related morbidity rate can be high, especially among young 68

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69 animals (Hastings et al. 1989). The morbidity and mortality rates associated with poxvirus infections in wild pinnipeds are unknown. When diseased pinnipeds are found stranded on beaches, they are often transferred to specialized marine mammal clinics for rehabilitation with the intention of subsequent release. Poxvirus infections are a common complication in the rehabilitation of these stranded pinnipeds (Wilson et al. 1969, Hastings et al. 1989). Poxvirus infections are likely to negatively affect the overall health of the animals and duration of hospitalization, and consequently increase the cost of maintaining these animals in rehabilitation centers. Poxviruses from pinnipeds are also potential zoonotic agents (Hicks and Worthy 1987). Poxvirus infections are therefore also of concern to marine mammal rehabilitation workers. To our knowledge, very little information is available about the susceptibility of pinnipeds to this common hospital-acquired disease or the factors involved in the transmission and prevention of nosocomial infection; no studies have addressed the epidemiologic features of hospital-acquired poxvirus infections in pinniped rehabilitation centers. The purpose of the study of this report was to identify risk factors that may predispose captive California sea lions to development of cutaneous poxvirus nodules during hospitalization in a rehabilitation center. A better understanding of the predisposing factors for poxvirus infections would be useful in the development of strategies to prevent and manage poxvirus outbreaks in marine mammal rehabilitation centers, which would ultimately contribute to provision of better patient care for hospitalized seals and sea lions.

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70 Materials and Methods Hospital records of 275 stranded California sea lions admitted for rehabilitation to The Marine Mammal Center (TMMC) in Sausalito, California, between 1 January and 31 December, 2002 were considered for inclusion in the study. Sea lions (n = 65) that were hospitalized for < 24 hours were not included in the study, because the medical records of these animals were often incomplete. This investigation was designed as a retrospective case-control study. All California sea lions (n = 18) that developed at least one pathognomonic, cutaneous, raised pox nodule during hospitalization were classified as cases. A random sample of 72 California sea lions that did not develop poxvirus lesions during hospitalization was selected by use of a random number table; these sea lions were classified as controls (control-to-case ratio, 4:1). All data analyzed in the study were gathered from the TMMC medical records of the case and control sea lions. All potential risk factors were categorized as risk factors prior to admission, clinical findings at the time of admission, or clinical findings, procedures, and treatments during hospitalization. Risk factors prior to admission included month of admission, county of stranding (Sonoma, Marin, and Alameda; San Mateo; Santa Cruz; Monterey; San Luis Obispo and Santa Barbara), sex, age group (pup, yearling, subadult, or adult), and whether the sea lion had previously been admitted to TMMC (readmitted animal). Age classes were defined as follows: pup, < 1 year; yearling, 1 to < 2 years; subadult male, 2 to 8 years; subadult female, 2 to 5 years; adult male, > 8 years; and adult female, > 5 years. Clinical findings recorded at the time of admission included primary clinical finding or problem (trauma, fracture, or entanglement; domoic acid intoxication; infectious disease; malnutrition; neoplasia; or other problems); body condition (considered normal, mildly underweight, or moderately underweight or emaciated); hospital caseload (the total

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71 number of California sea lions hospitalized at TMMC at the time of admission to TMMC); sea surface temperature (data obtained from the National Oceanic and Atmospheric Administration satellites and information service, measured at coordinates 37N, 122.5W); total serum protein concentration; and counts of WBCs, neutrophils, band neutrophils, eosinophils, and lymphocytes. Clinical findings, procedures, and treatments during hospitalization included use of quarantine, duration of hospitalization (days), housing history (pen B, C, D, E, G, H, or I), signs of anorexia (defined as no spontaneous food consumption for a period > 24 hours), clinical procedures (feeding via orogastric tube, anesthesia, ultrasonography, radiography, and surgery), the use of anti-inflammatory (non-steroidal anti-inflammatory drugs and corticosteroids) and anti-convulsive (phenobarbital) treatments, final diagnosis (trauma, fracture, entanglement; domoic acid intoxication; infectious disease; malnutrition; neoplasia; other diagnosis) and discharge status (alive or died during treatment). The number of days after admission that poxvirus nodules were first noted was recorded. Only exposure data (prior to admission, at admission, and during hospitalization) prior to development of pox lesions were included in the analyses. A logistic regression analysis approach was used to model the probability of hospitalized sea lions developing pox lesions as a function of the risk factors evaluated in this study. First, crude odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for each potential risk factor. Continuous variables (caseload at admission, sea surface temperature, and duration of hospitalization) were categorized into two, three, or four groups on the basis of their frequency distributions. Clinicopathologic variables were categorized as low, within reference limits, or high, based on reference values for stranded weanling sea lions (Bossart et al. 2001). Adjacent

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72 categories of multinomial variables (month of admission, county of origin, primary clinical finding, body condition, and final diagnosis) were collapsed whenever it was biologically reasonable to do so and when those categories had similar stratum-specific odds for developing pox lesions. Initial screening of potential risk factors was performed by use of univariable logistic regression. A univariable level of significance of p 0.20 was required for a potential risk factor to be entered in a starting model. Variables that passed the initial univariable screening were grouped into three subsets for further analysis (risk factors prior to admission, risk factors at admission, and risk factors during hospitalization). A backward stepping approach was used to identify multivariable models for each of the three subsets (critical p value for retention 0.10). Variables retained in the multivariable models of the three subsets were then included in a single model (critical p value for retention 0.10). Variables that passed the multivariable screening were used to develop the final multivariable model. To identify the best fitting final multivariable model, a backward model selection procedure was used in a sequential fashion starting with a full model. A model with hierarchical structure was specified by adding terms for biologically plausible interactions between independent variables. The variables for age, month of admission, hospital caseload, and duration of hospitalization were included as required variables in the final model, because they can influence the probability of hospitalized sea lions developing poxvirus lesions. The goodness of fit of the multivariable model was explored by use of the Hosmer-Lemeshow goodness-of-fit 2 statistic. In the final model, adjusted OR and 95% CI were reported. In this study, we used the OR as an epidemiologic measure of association between a risk factor and development of pox lesions. Thus, if a factor was not associated with risk of developing

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73 pox lesions, the OR was 1. Risk factors had an OR > 1 and protective factors had an OR < 1. The greater the departure from 1 (either larger or smaller), the stronger the association was between the factor and the odds of developing pox lesions. The upper and lower limits of the 95% CI indicate that we can be 95% confident in the assertion that the true OR falls within this interval. Results Between 1 January and 31 December, 2002, 275 stranded California sea lions were admitted for rehabilitation to TMMC. The 275 stranded California sea lions included six pups, 106 yearlings, 88 subadults, and 75 adults. Of these, 15 (5%) California sea lions had previously been admitted. Eighteen of 275 (6.5%) sea lions developed poxvirus nodules. Of the 90 California sea lions included in this study, there were 44 (49%) females and 46 (51%) males; 70 (78%) were subadults or adults. No pups or adults were diagnosed with pox lesions; only yearlings and subadults were affected. The mean SE number of days between admission to the hospital and development of pox lesions was 25 18 days. The duration of hospitalization of cases (median, 47 days; range, 4 to 83 days) and control sea lions (median, 20 days; range, 1 to 174 days) differed significantly (p < 0.01). Three of the 18 cases and three of the 72 control sea lions had previously been admitted to TMMC (Table 5-1). These three readmitted cases developed pox lesions 23 and 42 days after their initial release from TMMC. Among the 18 cases, 14 were discharged alive; among the 72 control sea lions, 39 were discharged alive. This difference between the groups was not significant (p = 0.07; Table 5-3). Sixteen of the 35 variables examined in univariable analyses had p values 0.20 and were included in the multivariable analysis (Table 5-1, Table 5-2, Table 5-3). Age, month of admission, hospital caseload, and duration of hospitalization were forced into

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74 the final model because they can influence the probability of hospitalized sea lions developing poxvirus lesions. The two other variables that were retained in the final model were readmission status and number of band neutrophils (Table 5-4). Addition of 2-way interaction terms did not contribute to the final model for risk of developing pox lesions; therefore, these terms were removed from the model. The p value of the Hosmer-Lemeshow statistic was 0.77, which supported the overall goodness-of-fit of the model. In the multivariable analysis, California sea lions that had previously been admitted to TMMC were 43 times as likely to develop poxvirus lesions than sea lions admitted for the first time (OR = 43.4; 95% CI = 1.6 to 1,129.5; p = 0.02). California sea lions with high counts of band neutrophils (> 0.69 X 103 bands/L) at the time of the initial examination were 20 times less likely to develop pox lesions than sea lions with band neutrophil counts that were within reference limits (OR = 0.05; 95% CI = 0.0 to 0.6; p = 0.02). Discussion The objective of the study of this report was to identify risk factors that may predispose California sea lions to the development of poxvirus lesions during hospitalization in a rehabilitation center, thereby providing data that might be used to improve patient care for hospitalized seals and sea lions. The results of our study suggest that sea lions with a history of prior hospitalization were more likely to develop poxvirus lesions during hospitalization at the rehabilitation center. Sea lions with high counts of circulating banded neutrophils at the time of admission were less likely to develop poxvirus lesions. The variables of age, month of admission, hospital caseload, and duration of hospitalization were retained in the final logistic regression model because the morbidity rate associated with poxvirus infections is higher in younger than in older

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75 pinnipeds (Hastings et al. 1989), the summer months are associated with a high hospital caseload, and the duration of hospitalization can affect the probability of sea lions developing poxvirus lesions. With regard to California sea lions, the caseload at TMMC is typically highest between June and August of each year. During those months, harbor seals and northern elephant seals are often also hospitalized at TMMC. At present, the species-specificity of the pinniped poxviruses is unknown, but poxvirus infection could be transmitted from those hospitalized seals to hospitalized California sea lions during those months if the latter are susceptible. A high hospital caseload is typically associated with an increased infection pressure from horizontally transmitted diseases and, as a result, an increased incidence of hospital-acquired infections (Ayliffe et al. 1999). When the caseload of California sea lions is high, higher numbers of both susceptible sea lions and infected sea lions are likely to be present in the hospital, compared with the numbers present at other times. Also, when the caseload is high, sea lions are housed in larger groups and consequently, more direct contact between infected and noninfected individuals is possible. As a result, pathogens are more easily transmitted, which may account for the increased incidence of poxvirus infections during periods when the caseload is high. In the present study, California sea lions with a history of prior hospitalization at TMMC were more likely to develop poxvirus lesions. Two explanations may be considered for the observed association between a history of prior hospitalization and high risk of poxvirus infection. First, the incubation period of the infection may correspond to the time between initial release and restranding of the animals. Sea lions may be exposed to the poxvirus during their initial hospitalization at TMMC, but only

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76 develop clinical disease during their subsequent hospitalization. The three affected, readmitted sea lions developed poxvirus lesions 23 to 42 days after their initial release. The incubation period of poxvirus infections in seals has been suggested to be between 3 and 8 wks (Hastings et al. 1989); however, this remains to be confirmed. The incubation periods of other mammalian poxvirus infections range from 48 hours to 14 days (Kitching and Taylor 1985, Chandra et al. 1986, Di Giulio and Eckburg 2004). Alternatively, the health of sea lions that are readmitted for rehabilitation may be more severely compromised than the health of sea lions that have not been admitted before. These more severely compromised sea lions are likely to be more susceptible to secondary infections, such as poxvirus infections. The fact that sea lions readmitted to TMMC in our study were at high risk for development of poxvirus lesions suggests that those sea lions should be kept in a separate pen. In other species, transmission of poxviruses requires either direct transmission or indirect transmission via fomites. Other infection control measures to help prevent the transmission of poxviruses from those sea lions to handlers and other hospitalized animals should therefore include the strict separation of sea lions with poxvirus lesions from those without, the use of designated animal handling equipment for each pen, and the use of disposable protective gear (e.g., gloves and towels) for handling individual animals. Hospitalized California sea lions with high counts of circulating band neutrophils at the time of admission were less likely to develop poxvirus lesions than sea lions with band neutrophil counts that were within reference limits. This may suggest that animals with acute inflammatory processes are more protected against poxvirus infections because the immune system is already upregulated. In various species, it is known that

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77 the host response to an infection with one organism can activate the immune system, thereby increasing the general level of resistance to other agents (Campos et al. 1993, Crawford et al. 1994, El Tayeb and Hanson 2001, Hengel and Masihi 2003). This augmentation of immunity against infection may be initiated by a specific, adaptive immune response. However, the non-specific protective effect is mediated via nonspecific modulators of immune function (e.g., granulocyte colony-stimulating factors and interferons) and subsequently effected by nonspecific, innate immune cells such as granulocytes, mast cells, macrophages, and natural killer cells (Crawford et al. 1994, Hengel and Masihi 2003). Innate interferon-mediated antiviral mechanisms do play an important role in controlling poxvirus infections (Ruby and Ramshaw 1991, Karupiah et al. 1993); interferons limit the spread of poxviruses by inducing an antiviral state in uninfected cells and directing nonspecific immune cells to sites of viral replication. Therefore, the high counts of circulating immature granulocytes detected in California sea lions without poxvirus lesions may be an indicator of augmented innate immune activity, in which case any infecting poxvirions would be effectively combated before clinical disease develops. No signs of poxvirus disease were detected in pups and adult sea lions at TMMC. Two explanations can be considered for the lack of clinical signs in pups and adult sea lions: low frequency of sea lion pups admitted to TMMC and acquired immunity among adults. Only six sea lion pups were admitted to TMMC in 2002. Numbers of hospitalized pups are usually low, because the pups are born on islands off Southern California. The pups remain on the rookeries and do not frequent the coastline of the mainland until they are at least six months of age (Reeves et al. 2002). The fact that none

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78 of the 75 hospitalized adult sea lions developed pox lesions may suggest that adults may have previously acquired immunity against poxviruses and hence were protected against reinfection. It is possible that exposure to poxviruses in wild California sea lion populations is a common event. However, the prevalence of poxvirus infections in wild California sea lions is unknown, and results of a serologic survey for anti-poxvirus antibodies in California sea lions would help determine the reason that adult sea lions did not have clinical signs of poxvirus infection in our study.

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79 Table 5-1. Results of the univariable analysis of risk factors prior to admission associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility Risk factors prior to admission No. of cases No. of controls Crude OR 95% CI p Month of admission Dec-Feb 1 4 0.85 0.07-9.87 0.89 Mar-May 2 23 0.30 0.05-1.63 0.15 Jun-Aug 10 28 1.21 0.35-4.20 0.75 Sep-Nov 5 17 1.00 ref n.a. Origin (county) Sonoma, Marin, Alameda 1 9 0.61 0.06-6.34 0.67 San Mateo 3 8 2.06 0.38-9.94 0.40 Santa Cruz 2 15 0.73 0.12-4.60 0.37 Monterey 8 18 2.44 0.64-9.64 0.18 San Luis Obispo, Santa Barbara 4 22 1.00 ref n.a. Sex Male 11 35 1.66 0.58-4.77 0.34 Female 7 37 1.00 ref n.a. Age group Adult 0 18 n.d. n.d. n.d. Subadult 11 22 n.d. n.d. n.d. Yearling 7 30 n.d. n.d. n.d. Pup 0 2 1.00 ref n.a. Previous admission to facility Yes 3 3 4.60 0.95-22.37 0.06 No 15 69 1.00 ref n.a. OR = Odds ratio. CI = Confidence interval. n.a. = not applicable, n.d. = not determined.

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80 Table 5-2. Results of the univariable analysis of risk factors at admission associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility. See Table 5-1 for remainder of key Risk factors at admission No. of cases No. of controls Crude OR 95% CI p Primary clinical finding Trauma or entanglement 4 27 0.59 0.09-3.89 0.58 Domoic acid intoxication 0 13 n.d. n.d. n.d. Infectious disease 10 20 2.00 0.36-11.24 0.42 Malnutrition 2 4 2.00 0.19-21.04 0.51 Neoplasia or other problem 2 8 1.00 ref n.a. Body condition Moderate to emaciated 13 39 2.33 0.59-9.12 0.21 Mildly underweight 2 12 1.17 0.17-8.19 0.87 Normal for size 3 21 1.00 ref n.a. Hospital caseload 32 to 44 9 19 2.96 0.79-11.1 0.10 18 to 31 5 28 1.12 0.27-4.62 0.88 3 to 17 4 25 1.00 ref n.a. Sea surface temperature (C) 14.8-17.3 5 23 1.52 0.37-6.33 0.56 12.6-14.7 9 21 3.00 0.81-9.98 0.09 10.0-12.5 4 28 1.00 ref n.a. Total protein (g/dL) High (10.8-10.86) 0 1 n.d. n.d. n.d. Low (4.8-7.6) 5 29 0.41 0.13-1.30 0.12 Within ref range (7.7-10.7) 13 31 1.00 ref n.a. Leukocytes (10 3 cells/L) High (22.8-44.6) 3 11 0.80 0.19-3.28 0.75 Low (4.8-9.77) 1 8 0.37 0.04-3.19 0.35 Within ref range (9.78-22.8) 14 41 1.00 ref n.a. Neutrophils (10 3 cells/L) High (16.69-39.25) 0 13 n.d. n.d. n.d. Low (3.02-5.13) 0 5 n.d. n.d. n.d. Within ref range (5.14-16.68) 18 42 1.00 ref n.a. Band neutrophils (10 3 cells/L) High (0.69-4.14) 2 18 0.28 0.06-1.37 0.10 Within ref range (0-0.68) 16 41 1.00 ref n.a. Eosinophils (10 3 cells/L) High (1.48-5.02) 2 7 0.95 0.18-5.02 0.95 Within ref range (0-1.47) 16 53 1.00 ref n.a. Lymphocytes (10 3 cells/L) Low (0.13-1.17) 3 16 0.54 0.14-2.11 0.37 Within ref range (1.18-8.38) 15 43 1.00 ref n.a.

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81 Table 5-3. Results of the univariable analysis of risk factors during hospitalization associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility. See Table 5-1 for remainder of key. Risk factors during hospitalization No. of cases No. of controls Crude OR 95% CI p Quarantine Yes 6 29 1.10 0.35-3.57 0.85 No 8 43 1.00 ref n.a. Length of hospitalization (days) 46-174 7 9 18.67 2.00-99.77 0.01 27-45 3 18 4.00 0.38-41.70 0.21 5-26 3 21 3.43 0.33-35.51 0.28 1-4 1 24 1.00 ref n.a. Housed in pen B Yes 8 21 3.24 1.03-11.16 0.04 No 6 51 1.00 ref n.a. Housed in pen C Yes 10 26 4.42 1.34-14.63 0.01 No 4 46 1.00 ref n.a. Housed in pen D Yes 11 42 2.62 0.69-9.93 0.15 No 3 30 1.00 ref n.a. Housed in pen E Yes 1 2 2.69 0.69-9.93 0.15 No 13 70 1.00 ref n.a. Housed in pen G Yes 10 60 2.12 0.59-7.69 0.25 No 4 12 1.00 ref n.a. Housed in pen H Yes 4 13 1.71 0.47-6.25 0.41 No 10 59 1.00 ref n.a. Housed in pen I Yes 4 17 1.29 0.36-4.68 0.69 No 10 55 1.00 ref n.a. Anorexia Yes 6 40 0.60 0.19-1.91 0.38 No 8 32 1.00 ref n.a. Tube feeding Yes 8 27 2.22 0.70-7.02 0.17 No 6 45 1.00 ref n.a. Administration of NSAIDs Yes 8 7 4.31 1.38-13.49 0.01 No 6 55 1.00 ref n.a.

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82 Table 5-3. Continued Risk factors during hospitalization No. of cases No. of controls Crude OR 95% CI p Administration of corticosteroids Yes 4 24 0.80 0.23-2.83 0.73 No 10 48 1.00 ref n.a. Administration of phenobarbital Yes 5 13 2.52 0.74-8.60 0.13 No 9 59 1.00 ref n.a. Anesthesia Yes 6 18 2.25 0.70-7.27 0.17 No 8 54 1.00 ref n.a. Ultrasonography Yes 1 8 0.62 0.07-5.32 0.65 No 13 64 1.00 ref n.a. Radiography Yes 5 14 2.30 0.68-7.83 0.85 No 9 58 1.00 ref n.a. Surgery Yes 1 5 1.00 0.11-9.69 0.97 No 13 67 1.00 ref n.a. Final diagnosis Trauma or entanglement 5 24 1.15 0.19-7.00 0.88 Domoic acid intoxication 2 15 0.73 0.09-6.23 0.77 Infectious disease 6 19 1.70 0.30-9.24 0.54 Malnutrition 3 3 5.50 0.64-9.47 0.15 Neoplasia or other problem 2 11 1.00 ref n.a. Discharge status Released alive 14 39 2.96 0.91-9.59 0.07 Died during hospitalization 4 33 1.00 ref n.a.

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83 Table 5-4. Results of the multivariable analysis of risk factors associated with development of poxvirus lesions in 90 California sea lions in a rehabilitation facility. See Table 5-1 for remainder of key. Risk factor No. of cases No. of controls Adjusted OR 95% CI p Age group Pup, yearling 7 32 2.09 0.28-15.75 0.11 Subadult 11 40 1.00 ref n.a. Month of admission Sep-Nov 5 17 1.09 0.02-61.05 0.96 Jun-Aug 10 28 0.37 0.00-29.19 0.66 Mar-May 2 23 0.05 0.004.33 0.19 Dec-Feb 1 4 1.00 ref n.a. Previous admission to facility Yes 3 3 43.48 1.67-1,129 0.02 No 15 69 1.00 ref n.a. Hospital caseload 32-44 9 19 16.49 0.81-336.01 0.07 18-31 5 28 6.74 0.4697.67 0.16 3-17 4 25 1.00 ref n.a. Band neutrophils (10 3 cells/L) High (0.69-4.14) 2 18 0.05 0.00-0.66 0.02 Within ref range (0-0.68) 16 41 1.00 ref n.a. Duration of hospitalization (days) 46-174 7 9 8.96 1.53-52.44 0.01 1-45 7 63 1.00 ref n.a.

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CHAPTER 6 THERAPEUTIC POTENTIAL OF CIDOFOVIR FOR THE TREATMENT OF PARAPOXVIRUS INFECTIONS OF PINNIPEDS Introduction The recognized members of the genus Parapoxvirus currently consist of orf virus, bovine papular stomatitis virus, pseudocowpoxvirus and Parapoxvirus of red deer of New Zealand (Moss 1996). The natural hosts of these viruses are small and large ruminants, but orf virus, bovine papular stomatitis virus and pseudocowpoxvirus are known to infect man as well (Mercer et al. 1997). Other tentative parapoxviruses include camel contagious ecthyma virus, chamois contagious ecthyma virus (Moss 1996) and the various parapoxviruses of pinnipeds, the group of animals consisting of all seals and sea lions (Simpson et al. 1994, Osterhaus et al. 1994, Nettleton et al. 1995, Sentsui et al. 1999, Becher et al. 2002, Tryland et al. 2005). In pinnipeds, the typical clinical manifestation of Parapoxvirus infections consists of proliferative lesions, 2-3 cm in diameter, of the skin or the mucosal surface of the mouth and nasal passages (Nollens et al. In press, Becher et al. 2002). Pox lesions are sometimes observed in free-ranging seals and sea lions, but they are especially common as a complication in the treatment of debilitated, stranded pinnipeds in specialized marine mammal rehabilitation centers. The pinniped parapoxviruses are considered zoonotic agents as well, as several handlers have acquired clinical “sealpox” lesions from contact with captive, lesion-bearing pinnipeds (Hicks and Worthy 1987, Clark et al. 2005). 84

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85 In humans, infections with the different parapoxviruses are clinically indistinguishable. Lesions typically develop on the hands and fingers and usually resolve spontaneously within 6-8 weeks (Shelley and Shelley 1983, Hicks and Worthy 1987, Clark et al. 2005). Complications can occur and these include prolonged resolution time, ulceration, secondary bacterial infection, lymphangitis, lymphadenitis and bullous pemphigoid (Yirrell et al. 1991, Murphy and Ralfs 1996, Reid 1998). More extensive and recurring parapoxvirus infections can occur, especially in immunosuppressed patients (Geerinck et al. 2001, Toutous-Trellu et al. 2004). These more complicated orf virus, bovine papular stomatitis virus and pseudocowpoxvirus infections of humans have been successfully treated via cryotherapy (Degraeve et al. 1999), excision of the lesions (Shelley and Shelley 1983) and, in one case, amputation of the affected finger (Reid 1998). More recently, the ability of cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine) to reduce the in vitro replication of three orf virus strains was reported (Nettleton et al. 2000). Since the discovery of the effectiveness of the anti-parapoxviral effects of cidofovir, infections of humans with these parapoxviruses have been effectively eliminated via local, intranasal or intravenous administration of this drug (Geerinck et al. 2001, De Clercq 2002, McCabe et al. 2003, Toutous-Trellu et al. 2004). Cidofovir, including several lipid variants for oral administration, is now under clinical investigation for a variety of potential therapeutic applications (Quenelle et al. 2004, Buller et al. 2004). Other compounds that are known to interfere with the effective replication of a number of DNA viruses include isatin-beta-thiosemicarbazone (IBT), rifampicin, phosphonoacetic acid (foscarnet) and the nucleotide analogue acyclovir (9-(2

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86 hydroxyethoxymethyl)guanine). IBT inhibits viral transcription termination during vaccinia infection (Prins et al. 2004) and inhibits in vitro growth of vaccinia virus and cowpox virus (Condit et al. 1991,Pirrung et al. 2005). Rifampicin interferes with the function of a structural protein of vaccinia virus and consequently inhibits the maturation of virions (Szajner et al. 2005). Phosphonoacetic acid (PAA) is a pyrophosphate analogue with activity against herpes viruses, vaccinia virus (Sridhar and Condit 1983), human immunodeficiency virus, and other RNA and DNA viruses. PAA and its analogues achieve their antiviral effects via inhibition of viral polymerases (Crumpacker 1992). The molecule blocks the cleavage of the pyrophosphate moiety from deoxynucleotide triphosphates (dNTPs), which in turn blocks the DNA chain elongation. Acyclovir and cidofovir are respectively guanine and cytosine nucleoside analogues that are approved by the Food and Drug Administration for the medicinal treatment of human herpesviral infections. Cidofovir is converted entirely via cellular enzymes to its pharmacologically active diphosphate form, whereas acyclovir and it derivatives require viral nucleoside (thymidine) kinases to be phosphorylated into its active form (Elion 1983, Morfin and Thouvenot 2003). The phosphorylated active forms of acyclovir and cidofovir stop viral DNA replication via incorporation into and termination of the DNA chain, and via inactivation of the DNA polymerase (Elion 1983, Magee et al. 2005). While the efficacy of cidofovir against some parapoxviruses has been reported (Nettleton et al. 2000) and while rifampicin, PAA and acyclovir are known to have a varying degree of antiviral activity against molluscum contagiosum and various orthopoxviruses, the antiviral activity of these compounds against the parapoxviruses of pinnipeds has not yet been determined. In this study a parapoxvirus (Sea lion poxvirus 1;

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87 SLPV-1) of California sea lions (Zalophus californianus) was used as a model virus for testing the in vitro efficacy of IBT, rifampicin, acyclovir, cidofovir and PAA against the parapoxviruses of pinnipeds. Materials and Methods Cell Cultures Frozen early passage California sea lion kidney cell cultures (CZC-K) and California sea lion skin cell cultures (CZC-S) were obtained from the Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, and were grown to confluency in 100 mm culture dishes in our laboratory. The culture media consisted of 85% (volume:volume) DMEM-F12 (Cellgro, Herndon, VA), 15 % (volume:volume) cosmic fetal calf serum (Hyclone, Logan, UT), 2 mM L-glutamine (Cellgro, Herndon, VA), 0.1 mM Na-pyruvate (Cellgro, Herndon, VA), 50 IU penicillin/ml and 50 g streptomycin (Cellgro, Herndon, VA). All cultures were kept at 37.0C, 7.5% CO2 and approximately 90% relative humidity. When confluent, the cells were split at 1:5 ratio. For splitting, the cells were removed from the monolayers by aspirating the culture medium and incubating with 1.0 ml of trypsin-EDTA in saline for 5 min at 37.0C, after which the cells were resuspended in culture media and seeded onto tissue culture plates. Virus A parapoxvirus of California sea lions (SLPV-1) was used as a model virus for the parapoxviruses of pinnipeds. Surface morphology of the virions on negative staining electron microscopy and partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of this poxvirus to the genus parapoxvirus (Nollens et al. In press). A purified SLPV-1 preparation was used to evaluate sensitivity to the antiviral compounds. For purification, SLPV-1 was propagated

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88 in early passage California sea lion kidney cells. When more than 90% of the monolayer showed cytopathic effect, the cells and supernatants were harvested and freeze-thawed twice and the virus was concentrated as previously described (Nollens et al. In press). Briefly, intracellular mature virions (IMV) were purified from the infected cell lysates by high-speed centrifugation through a 40% sucrose cushion followed by banding of the IMV on a 25-50% Na-diatrizoate density gradient. The virus was resuspended in 10 mM Tris (pH 8.2) and stored at -80C until analysis. The titer of the purified SLPV-1 stock was determined via tissue culture infectious dose 50 (TCID 50 ) assay. For the TCID 50 assay, CZC-K cell suspensions were grown to confluency in 24-well tissue culture plates (1 ml per well; Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) under the culture conditions described above. An aliquot of the SLPV-1 stock was serially diluted six times 1:10 in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Ten wells were infected with 50 l of each virus dilution and adsorbed for 90 min at 37.0C. Non-infected control wells were mock infected with 50 l PBS alone. The inoculum was then removed and replaced with 1 ml of culture medium. After five days of incubation at 37.0C all cultures were inspected for cytopathic effect (CPE) by light microscope and the presence or absence of CPE in each well was recorded. CPE was defined as rounding and clumping of the cells and, eventually, their detachment. SLPV-1 does not induce plaque formation in infected monolayer. TCID 50 is defined as that dilution of virus that results in infection in 50% of replicate inoculations (50% endpoint). The virus titer of the concentrated SLPV-1 stock was determined as previously described(Reed and Muench 1932) and was calculated at 10 5.5 TCID 50 /ml.

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89 Compounds and Concentrations The following compounds were evaluated for their efficacy and cytotoxic effects: isatin-beta-thiosemicarbazone (IBT; Pfaltz and Bauer, Inc., Waterbury, CT), rifampicin (Sigma, St. Louis, MO), phosphonoacetic acid (PPA, Alfaproducts, Danvers, MA) and 9-(2-hydroxyethoxymethyl)guanine (Acyclovir, Calbiochem, La Jolla, CA). Cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine) was kindly provided by Chimerix (La Jolla, CA). IBT was dissolved in acetone at a concentration of 5 mg/ml and then diluted with four volumes of 0.25 M NaOH immediately before use. Rifampicin, acyclovir and cidofovir were dissolved in ddH 2 O. NaOH or HCl were added to adjust to a pH of 7.0. All compound solutions were diluted in ddH 2 O to the desired concentrations and filter sterilized through a 200 m pore filter. The concentrations used in this study were selected based on minimum inhibitory concentrations of the compounds against other DNA viruses in vitro and, when available, on in vivo peak serum titers and therapeutic dosages for humans (Sridhar and Condit 1983, Baldick, Jr. and Moss 1987, Terry et al. 1988, Nettleton et al. 2000,Tsankov and Angelova 2003). CPE Reduction Assays The antiviral activity against SLPV-1 of the compounds was assayed via a CPE reduction assay. CZC-K and CZC-S cell suspensions were grown to confluency in 24-well tissue culture plates under the culture conditions described above. All wells were infected with 50 l of SLPV-1 virus stock diluted 1:500 in PBS (approximately 32 TCID 50 per well or MOI = 10 -3 TCID 50 ) and adsorbed for 90 min at 37.0C. The inoculum was then removed and replaced with 1 ml of culture medium. Triplicate wells of CZC-K were incubated for five days in the presence of culture media containing the antiviral compounds at the concentrations indicated in Table 6-1 and in the presence of

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90 culture media alone (positive control). After five days of incubation at 37.0C all cell cultures were inspected by light microscope and the presence or absence of CPE was recorded. Compounds that appeared to be able to inhibit virus-induced CPE were included in a replicate CPE reduction assay. This second assay was performed following the exact same protocol but using CZC-S cell cultures (Table 6-1). Antiviral activity was expressed as minimum antiviral concentration (IC 50 ). The IC 50 was defined as the compound concentration needed to reduce the CPE to 50% of the drug-free control (i.e. 1 CPE positive of three wells). Virus Yield Assay Compounds that were able to reduce the frequency of CPE to the CZC-S monolayers to below 50% of the control (i.e. 1 positive of three wells) were included in the virus yield assay to evaluate the effect of the compounds at the observed IC 50 on virus yield. The monolayers of one drug-free control (0 g/ml; Figure 6-1) and of one well incubated at each drug concentration were scraped and harvested. Cells were lysed by three freeze-thaw cycles and the infected cell lysates were subsequently diluted for virus titer determination in a TCID 50 assay on CZC-K cells as described above. Briefly, an aliquot of each well was serially diluted six times 1:10 in PBS. Five wells were infected with 50 l of each virus dilution and adsorbed for 90 min at 37.0C. Non-infected control wells were mock infected with 50 l PBS alone. The inoculum was then removed and replaced with 1 ml of culture medium. After five days of incubation at 37.0C all cultures were inspected for CPE by light microscope and the presence or absence of CPE in each well was recorded. The virus yield of each well was expressed as TCID 50 /ml.

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91 Cytotoxicity Assays The cytotoxicity of the compounds for CZC-K and CZC-S cells was determined both on confluent monolayers and on exponentially growing cells. A cytotoxic effect of the antiviral compounds to confluent monolayers can be falsely interpreted as virus-induced CPE. For evaluation of this cytotoxic effect, CZC-K and CZC-S cell suspensions were grown to confluency in 24-well tissue culture plates under the culture conditions described above. Triplicate confluent monolayers of CZC-K and CZC-S were incubated for five days in the presence of culture media containing the antiviral compounds at the concentrations indicated in Table 6-1, in parallel with the virus-infected cell cultures. Triplicate confluent monolayers of CZC-K and CZC-S were used as controls (drug-free cells) and were incubated in the presence of culture media alone. After five days of incubation at 37.0C all cell cultures were inspected by light microscope and the presence or absence of cytotoxicity, defined as apparent CPE, was recorded. Cytotoxicity to confluent monolayers was expressed as the 50% cytotoxic concentration (CCc 50 ). The CCc 50 was defined as the concentration required to cause a detectable cytotoxic effect to 50% of the monolayers. The inhibitory effect of the compounds on the growth of CZC-K and CZC-S cells was studied in parallel with the CPE reduction assays described above. Cells were removed from confluent monolayers using trypsin-EDTA as described above, and counted. All cell counts were made manually using a hemocytometer. The CZC-K and CZC-S cells were seeded into 24-well plates in a volume of 1.0 ml at a concentration of 34.3 5.6 cells/mm 3 and 28.6 6.3 cells/mm 3 for CZC-K and CZC-S respectively. All cells were allowed to proliferate under the conditions described above in the presence of culture media containing the antiviral compounds at the concentrations indicated in

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92 Figure 6-2 and Figure 6-3 (four wells per concentration per compound), in parallel with the virus-infected cell cultures. After the cells were allowed to grow for two days, all cells of one of each set of four wells were harvested and counted. For harvesting, the culture medium of each well was aspirated and the monolayer was incubated with 150 l of trypsin-EDTA in saline for 5 min at 37.0C, after which the trypsin was inactivated by resuspending the cell/trypsin suspension in 850 l culture media to a total volume of 1.0 ml. The remaining three of each of the four wells were allowed to proliferate for four days after which they were harvested and counted. For analysis, all individual cell counts were expressed as mean proportion (%) of cell counts ( SD) for each drug concentration relative to the mean cell count in the drug-free cell controls as determined on day 4. Mean proportions were compared using a Student’s t-test for comparison of means. Cytotoxic effect on cell growth was expressed as the 50% cytotoxic concentration (CCg 50 ). The CCg 50 was defined as the concentration that reduces the proliferation of cells to 50% of drug-free control cells. Results CPE Reduction Assays After five days incubation with SLPV-1, all three drug-free CZC-K control wells were CPE positive (Table 6-1). All wells infected in the presence of IBT, rifampicin or acyclovir showed CPE at all concentrations tested. One and two infected wells incubated in the presence of 5 g/ml and 20 g/ml cidofovir respectively were CPE negative. Additionally, one infected well incubated in the presence of PAA at 200 g/ml was CPE negative. All other wells showed CPE. Based on these results, the antiviral activity of cidofovir and PAA against SLPV-1 was assessed using CZC-S cell cultures. After five days incubation with SLPV-1, all three drug-free CZC-S control wells were CPE positive

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93 (Table 6-1). A reduction in the frequency of CPE was observed using cidofovir at concentrations 2 g/ml and when using PAA at concentrations 200 g/ml. At the concentrations tested, PAA was unable to reduce the frequency of CPE to below 50% of the controls (i.e. 1 CPE positive well per three wells). The IC 50 for CPE of cidofovir was between 5 and 20 g/ml and between 2 and 5 g/ml when using California sea lion kidney and skin cell cultures respectively. Virus Yield Assay The calculated virus yield of CZC-S cells infected with SLPV-1 in the absence of cidofovir (0 g/ml) was 10 6.8 TCID 50 /ml. A gradual decrease in virus yield was observed with increasing concentrations of cidofovir (Figure 6-1). The SLPV-1 yield of infected cells incubated in the presence of 0.2, 0.5 g/ml of cidofovir for five days was 10 6.8 , 10 6.6 TCID50/ml respectively. The SLPV-1 yield of infected cells incubated in the presence of 2, 5 and 20 g/ml of cidofovir was reduced to 10 6.0 , 10 5.1 and 10 4.1 TCID 50 /ml respectively, or 13.4%, 2.0% and 0.1% of the virus yield of the drug-free control. Cytotoxicity Assays After five days incubation no signs of cytotoxicity were observed in the drug-free CZC-K and CZC-S control monolayers (Table 6-1). All monolayers incubated in the presence of IBT, acyclovir and PAA remained intact. Rifampicin, especially when used at a concentration of 50 g/ml and higher was toxic to the CZC-K cells. Additionally, one of three CZC-K and CZC-S monolayers incubated in the presence of cidofovir at a concentration of 20 g/ml showed cytotoxicity. All other monolayers remained intact. The observed CCc 50 of rifampicin to confluent CZC-K monolayers was 50 g/ml. Cidofovir did not evoke a cytotoxic effect to more than 50% of the CZC-K and CZC-S monolayers (i.e. cytotoxicity in 2 per three monolayers).

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94 IBT, rifampicin, PAA and acyclovir had a marked (p 0.05) inhibitory effect on the growth of uninfected CZC-K (Figure 6-2) and/or CZCS cells (Figure 6-3) at high concentrations. Cidofovir, at a concentration of 5 g/ml and higher, significantly (p 0.05) decreased the proliferation of CZC-K cells, but not of CZC-S cells. Rifampicin, at a concentration of 100 ug/ml, reduced the proliferation of CZC-K cells to 32 % of the drug-free control (CCg 50 of rifampicin on cell growth = 100 ug/ml). At no time did any of the other four compounds reduce cell growth to below 50% of the drug-free control. Discussion Our finding showed an unequivocal antiviral effect of cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine) against the sea lion parapoxvirus SLPV-1. The frequency of virus-induced cytopathic effect to cell cultures was reduced when SLPV-1 was cultured in the presence of cidofovir. Virus yield assays confirmed the ability of cidofovir to reduce SLPV-1 replication to 10 -2 that of the control cultures. This study confirms the findings of a previous study reporting the antiviral activity of cidofovir against parapoxviruses (Nettleton et al. 2000). The sensitivity of SLPV-1 to cidofovir appears to be lower than the sensitivities previously reported for parapoxviruses of cattle and sheep (strains orf-11, MN, NZ-2) and is more similar to that of vaccinia virus (strain Lister) (Nettleton et al. 2000). It was previously reported that cidofovir at 5 g/ml inhibited any detectable growth of orf-11 and MN virus after 96 hrs of incubation, and delayed and reduced yields of NZ-2. Also, orf-11, NZ-2 and MN parapoxvirus production was entirely inhibited by cidofovir at 20 g/ml. However, the growth of vaccinia virus when measured after 96 hrs was similar to the growth of SLPV-1, as it remained detectable even when grown in the presence of 20 g/ml although virus yields were markedly reduced (Nettleton et al. 2000). It should be

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95 noted here that this apparently atypical sensitivity of the SLPV-1 parapoxvirus could in part be due to limitations of the titering method used in this study. SLPV-1 fails to induce plaque formation in any of the cell types tested and, as a result, virus doses and yields were estimated using a TCID 50 assay. The TCID 50 endpoint method does measure infectivity, but importantly the unit of infectivity measured by the endpoint method may require more than one infectious particle and the number of virus particles may be underestimated. If the TCID 50 of SLPV-1 stock and inoculum is underestimated, the quantitations of antiviral activity of cidofovir reported in this study may be a conservative estimate of the actual antiviral activity as well. Cidofovir has good therapeutic potential for the treatment of human cases of sealpox. Pharmacokinetic data are available for cidofovir for a number of species, including rats (Cundy et al. 1996a), rabbits (Cundy et al. 1997), monkeys (Cundy et al. 1996b) and humans (Wachsman et al. 1996, Cundy 1999). The observed effective cidofovir concentrations for SLPV-1 did fall below peak cidofovir serum levels of all species and within the therapeutic dosage for humans. Due to its low oral bioavailability, cidofovir is administered to humans as a 1-hour intravenous (IV) infusion with saline hydration. When administered IV at 10 mg/kg dose, peak serum levels are achieved of 23.6 4.88 mg/l with a half-life of 2.6 1.9 hours (n = 25) (Cundy 1999). In comparison, cidofovir exhibited antiviral activity against SLPV-1 when used at concentrations of 2 g/ml and higher. At 2 g/ml a ten-fold reduction in SLPV-1 yield was observed, and the virus proliferation decreased exponentially when the cidofovir concentration was increased to 5 and 20 g/ml. In addition, human cases of orf virus infection have been successfully treated via topical application of 1% cidofovir cream

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96 (Geerinck et al. 2001, McCabe et al. 2003). We therefore suggest that cidofovir should be the treatment of choice for treating human cases of sealpox infections as well, being administered either intravenously or topically. Cidofovir has previously been suggested for treatment of parapoxvirus of animals as well (Nettleton et al. 2000). Whereas topical administration would make the use of cidofovir in domestic animal patients more practical, when treating wildlife, such seals and sea lions, the usefulness of even a topical cream may at times be limited due to the need for handling, the ability of animals to lick off creams and other practical constraints. For treatment of parapoxvirus infections of pinnipeds in rehabilitation centers, only topical application would be feasible and only in severely debilitated animals. For more routine use of cidofovir for the treatment of parapoxvirus infections in pinnipeds, a cidofovir formulation with good oral bioavailability would be required. Further, a pharmacokinetic study would be needed to determine appropriate dosages and administration intervals. No antiviral activity against SLPV-1 was detected when using IBT, rifampicin, PAA and acyclovir (9-(2-hydroxyethoxymethyl)guanine). Where rifampicin inhibits the maturation of vaccinia virions at concentrations in the range of 100 g/ml, we were unable to evaluate the antiviral activity of rifampicin at concentrations above 10 g/ml because of the cytotoxic effect of rifampicin at these concentrations to the monolayers (Baldick, Jr. and Moss 1987). PAA was, at the tested concentrations, unable to reduce viral CPE to below 50% of drug-free controls, although a more subtle CPE reduction to 66% (2 out of 3) was observed at 200 and 500 g/ml. The growth of vaccinia virus is typically inhibited in the presence of 100 g/ml PAA (Sridhar and Condit 1983), but the

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97 antiviral activity of PAA against SLPV-1 at concentrations greater than 500 g/ml could be further explored. The results presented in this study confirm the previously proposed therapeutic potential of cidofovir for the treatment of parapoxvirus infections (Nettleton et al. 2000). The in vitro efficacy of cidofovir against SLPV-1 further indicates a therapeutic potential of cidofovir for the treatment of parapoxvirus infections of pinnipeds and humans.

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98 Table 6-1. Number of infected (CPE) and control (cytotoxicity) CZC-K and CZC-S wells displaying apparent CPE in the presence of the antiviral compounds at concentrations indicated N wells displaying CPE N wells displaying cytotoxicity Antiviral compound Concentration CZC-K CZC-S CZC-K CZC-S No drug n.a. 3 3 0 0 IBT (M) 90.0 3 0 45.0 3 0 22.5 3 0 Rifampicin (g/ml) 100 3 3 50 3 3 10 3 0 5 3 1 1 3 0 Acyclovir (g/ml) 20 3 0 5.0 3 0 2.0 3 0 1.0 3 0 0.5 3 0 0.2 3 0 0.1 3 0 0.05 3 0 0.02 3 0 0.01 3 0 Cidofovir (g/ml) 20 1 1 1 1 5 2 1 0 0 2 3 2 0 0 0.5 3 3 0 0 0.2 3 3 0 0 PAA (g/ml) 500 3 2 0 0 200 2 2 0 0 100 3 3 0 0 50 3 3 0 0 25 3 3 0 0

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99 Figure 6-1. Sea lion parapoxvirus (SLPV-1) yield of infected CZC-S cells and incubated in the presence of 0.2, 2, 5 or 20 g/ml of cidofovir for five days. Virus yields were determined via a TCID 50 assay on CZC-K cells. The drug-free control is indicated on the graph as 0 g/ml. Units of Y-axis are 10x TCID 50 /ml.

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100 A B C D E Figure 6-2. Effect of isatin-beta-thiosemicarbazone (A), rifampicin (B), acyclovir (C), cidofovir (D) and phosphonoacetic acid (E) on growth of early passage California sea lion kidney cells. Cell counts were determined in triplicate cultures incubated in the presence of IBT, rifampicin acyclovir, cidofovir and phosphonoacetic acid at concentrations indicated for four days. Values are mean cell counts ( SE) on Day 0, 2 or 4. Full lines indicate counts that were significantly different at day 4 from the drug-free control (p < 0.05).

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101 A B Figure 6-3. Effect of cidofovir (A) and phosphonoacetic acid (B) on growth of early passage California sea lion skin cells. Cell counts were determined in triplicate cultures incubated in the presence of cidofovir and phosphonoacetic acid at concentrations indicated for four days. Depicted values are mean cell counts ( SE) for each drug concentration on Day 0, 2 or 4 respectively. Full lines indicate cell counts that were significantly different at day 4 from the drug-free control (p < 0.05) and dotted lines indicate differences that were not significant (p > 0.05). Error bars of cell counts that were not significantly different from the control are not depicted.

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CHAPTER 7 CONCLUSIONS Each year, several hundred pinnipeds, including California sea lions, northern elephant seals and harbor seals, are admitted to specialized marine mammal rehabilitation centers in the United States, and suspected poxvirus infections are observed in these animals. These infections are especially common among juvenile pinnipeds that already have other health problems, and appear to be hospital-acquired. This potential for hospital acquisition prompted this study into the etiology and epidemiology of poxvirus infections in pinnipeds. The ultimate objective of this project was to gain the information needed to develop useful and practical recommendations to prevent and manage poxvirus epizootics in marine mammal rehabilitation centers and to determine the threat these animals pose to the free-ranging pinniped populations. As a result, the exposure of rehabilitation workers to this zoonotic agent would be reduced, and rehabilitation practices would be improved. Most of the information we acquired is from California sea lions as this was the species most readily accessible for study. However, we recommend that the information collected for California sea lions, except for the seroprevalence data presented as part of Chapter 4, be used to guide rehabilitation practices pertinent to poxviruses for other pinniped species until more information is available for these species. Based on the work presented in this dissertation, information on pinniped poxviruses and recommendations for the prevention and management of poxvirus epizootics in marine mammal rehabilitation centers include the following: 102

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103 Not one, but several poxvirus strains circulate among the various North American pinniped populations. At present, all poxviruses identified in North American pinnipeds belong to the genus Parapoxvirus. However, poxviruses belonging to other poxvirus genera may circulate as well. Parapoxviruses from Atlantic pinnipeds are phylogenetically distant from those of Pacific pinnipeds. Quarantine procedures should therefore be put in place to avoid the introduction of novel poxvirus strains into nave wild populations when pinnipeds, such as animals unsuitable for release, are transferred between facilities on the Atlantic and Pacific coast. It appears that parapoxviruses are not commonly transmitted between free-ranging phocids and otariids. However, at least some pinniped parapoxvirus strains are capable of infecting multiple species within a phylogenetic family and possibly species belonging to different phylogenetic families. A parapoxvirus of California sea lions was isolated and can be used in future studies as a model virus for pinniped parapoxviruses. Previous exposure to a parapoxvirus does not correlate with protection against re-infection. A prevention plan for poxvirus epizootics that is based on (1) determining of serologic status of newly admitted animals, (2) quarantining newly admitted animals until serologic status is known, followed by (3) isolation of seronegative animals, is therefore not likely to be a successful strategy. The lack of protection of California sea lions following exposure to parapoxviruses suggests that the usefulness of a vaccine may be as ambiguous as orf virus vaccines are in sheep. Some poxvirus infections that are observed in rehabilitation centers are acquired while in the rehabilitation center. It may therefore be possible to reduce the incidence of poxvirus infections in pinnipeds in rehabilitation centers using prophylactic measures. Parapoxviruses can survive in the environment for months to years and are typically transmitted directly although indirect routes are possible. Restriction of animal movement is therefore advised. Parapoxviruses can resist dehydration, but are very susceptible to bleach. Effective cleaning practices should consist of thorough scrubbing of pens to remove all organic material, followed by washing with bleach or detergents with known virucidal activity. Leaving pens empty and dry for extended periods of time without previous scrubbing and bleaching is not likely to inactivate the pinniped parapoxviruses.

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104 The temporal characteristics of the humoral immune response of California sea lions to parapoxviruses are similar to those of the humoral immune response of sheep to orf virus. Therefore, the incubation period of parapoxvirus infections in pinnipeds is likely to be approximately seven days. Subclinical parapoxvirus infections occur, but it is not known whether subclinically infected pinnipeds shed virus. Restriction of movement of animals, even if they are apparently clinically healthy, is therefore advised. Poxvirus infections are very common in California sea lions in the wild. Over 90% of all California sea lions are exposed to parapoxviruses at least once over the course of their lifetime. Since parapoxviruses are endemic in wild California sea lion populations, introduction of locally endemic poxvirus strains into the wild is not a concern. Therefore, the release of captive sea lions infected with locally endemic parapoxvirus strain into the wild should not increase the risk of an outbreak in wild populations, and the presence of pox lesions in fully rehabilitated California sea lions should not preclude their return to the wild. However, this recommendation can not be extrapolated to other pinniped species without any information on the prevalence of parapoxvirus infections in these other pinniped species. Pinnipeds admitted to rehabilitation centers without inflammatory disease processes (i.e. with low to normal band neutrophil counts at the time of admission) are at higher risk of acquiring a poxvirus infection while in a rehabilitation center than animals admitted with inflammatory processes. Pups or yearlings are at higher risk of acquiring a poxvirus infection while in a rehabilitation center than older animals. Pinnipeds that have previously been admitted to a rehabilitation center are at higher risk of acquiring a poxvirus infection than animals that are admitted for the first time. Pinnipeds that are hospitalized for longer than six weeks are more likely to acquire a poxvirus infection than animals that are hospitalized for less than six weeks. Hospital caseload at the time of admission is directly and positively correlated with the likelihood of acquiring a poxvirus infection during hospitalization. Acquiring a poxvirus infection during hospitalization is not associated with a decreased chance of survival. Clinical poxvirus lesions typically develop 3-5 weeks after admission to the rehabilitation center. Attempts to prevent poxvirus outbreaks via prolonged quarantine periods upon admission are therefore not practical (or useful).

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105 The following factors are not associated with acquiring a poxvirus infection while in a rehabilitation center: gender, county of origin, month of admission, sea surface temperature, body condition, blood parameters at the time of admission (except band neutrophil count), anorexia, any husbandry and veterinary procedures during hospitalization, use of steroids and other therapeutics during hospitalization. No practical etiologic treatment is currently available for parapoxvirus infections of pinnipeds. The antiviral compound cidofovir has in vitro antiviral activity against pinniped parapoxviruses and therefore has good therapeutic potential for the treatment of pinniped parapoxvirus infections in humans and animals. Oral formulations of cidofovir are still being developed. As a result, only topical application is currently feasible and only in severely debilitated animals. For more routine use of cidofovir for the treatment of parapoxvirus infections in pinnipeds, a cidofovir formulation with good oral bioavailability would be required. It has been suggested that parapoxviruses can cause persistent infections in pinnipeds. Some scant, unconfirmed information is available suggesting that cattle can become persistently infected with parapoxviruses, which would support this hypothesis. On the other hand, it is well established that parapoxviruses can persist even in small herds of cattle, thus creating the false impression that individual animals experience intermittently reactivating infections. Unfortunately, during this study no cases of apparently reactivating poxvirus infections occurred, so we were unable to obtain the samples that would be needed to prove or disprove this hypothesis. Based on the existing information available for other species, it does appear more likely that pinnipeds experience repeated parapoxvirus infections rather than intermittently reactivating persistent infections. However, we do recommend this topic as a focus for future research, given the impact the existence of persistent parapoxvirus infections would have on several of our conclusions.

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LIST OF REFERENCES Ayliffe GAJ, Babb JR, Taylor LJ (1999), Hospital acquired infection, in Hospital acquired infection. Principles and prevention, Butterworth Heineman, Oxford p 210 Baldick CJ Jr., Moss B (1987), Resistance of vaccinia virus to rifampicin conferred by a single nucleotide substitution near the predicted NH2 terminus of a gene encoding an Mr 62,000 polypeptide, Virology 156: 138-145 Becher P, Konig M, Muller G, Siebert U, Thiel HJ (2002), Characterization of sealpox virus, a separate member of the parapoxviruses, Arch. Virol. 147: 1133-1140 Buddle BM, Dellers RW, Schurig GG (1984), Contagious ecthyma virus-vaccination failures, Am. J. Vet. Res. 45: 263-266 Buller RM, Owens G, Schriewer J, Melman L, Beadle JR, Hostetler KY (2004), Efficacy of oral active ether lipid analogs of cidofovir in a lethal mousepox model, Virology 318: 474-481 Campos M, Godson D, Hughes H, Babiuk L, Sordillo L (1993), The role of biological response modifiers in disease control, J. Dairy Sci. 76: 2407-2417 Chandra R, Singh IP, Garg SK, Varshney KC (1986), Experimental pathogenesis of buffalo pox virus in rabbits: clinico-pathological studies, Acta Virol. 30: 390-396 Clark C, McIntyre PG, Evans A, McInnes CJ, Lewis-Jones S (2005), Human sealpox resulting from a seal bite: confirmation that sealpox virus is zoonotic, Br. J. Dermatol. 152: 791-793 Condit RC, Easterly R, Pacha RF, Fathi Z, Meis RJ (1991), A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase, Virology 185: 857-861 Crawford RM, Leiby DA, Green SJ, Nacy CA, Fortier AH, Meltzer MS (1994), Macrophage activation: a riddle of immunological resistance, Immunol. Ser. 60: 29-46 Crowther JR (2001), Validation of diagnostic tests for infectious diseases, in The ELISA Guidebook, ed. Crowther JR, Humana Press Inc, Totowa NJ p 301-346 106

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BIOGRAPHICAL SKETCH Hendrik Nollens graduated as a veterinarian from the College of Veterinary Medicine, University of Gent, Belgium, in July 1998. After approximately 2 years as a clinical resident in the Department of Large Animal Internal Medicine at the University of Gent, he enrolled in the master’s program at the University of Otago in New Zealand. He completed his Master of Science in marine sciences on the topic of “Shell lesions in New Zealand abalone” in December 2001. Hendrik enrolled in the PhD program of the College of Veterinary Medicine of the University of Florida in the summer of 2002. 113