Discovery, Phylogenetic Analysis, Diagnostic Test Development, and Surveillance of the Astroviruses of Marine Mammals

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Discovery, Phylogenetic Analysis, Diagnostic Test Development, and Surveillance of the Astroviruses of Marine Mammals
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1 online resource (145 p.)
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english
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Wellehan Jr, James Francis Xavier
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University of Florida
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
Jacobson, Elliott R.
Committee Members:
Isaza, Ramiro
Flanegan, James B.
Condit, Richard C.
Nollens, Hendrik Hans
Miyamoto, Michael M.
Farmerie, William G.

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Veterinary Medicine -- Dissertations, Academic -- UF
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Electronic Thesis or Dissertation
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theses   ( marcgt )
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Abstract:
Astroviridae are a family of small nonenveloped positive stranded ribonucleic acid (RNA) viruses associated with enteritis. Knowledge of astrovirus diversity is very limited, with only six astrovirus species from mammalian hosts officially recognized, and additional human, cheetah, rat, dog, and bat astroviruses recently described. We used consensus polymerase chain reaction (PCR) techniques for initial identification of fifteen astroviruses from marine mammals; three from California sea lions (Zalophus californianus), one from a Steller sea lion (Eumetopias jubatus), nine from bottlenose dolphins (Tursiops truncatus), and two from minke whales (Balaenoptera acutorostrata). Bayesian and maximum likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Some astroviruses from marine mammals clustered within the genus Mamastrovirus, whereas others were in a clade outside of known genera. Mamastroviruses identified did not form a monophyletic group. Recombination analysis found that a relatively recent recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same host at one point. A bottlenose dolphin astrovirus sequence was also consistent with the result of a recombination event. An enzyme linked immunosorbent assay (ELISA) and a quantitative PCR (qPCR) assay were designed for a bottlenose dolphin Mamastrovirus and used to survey seroprevalence, virus prevalence, and virus load. The results showed that animals seroconvert at a young age, and virus prevalence was even higher than what has been seen with astroviruses of terrestrial animals. Virus load correlated with abnormal behavior reported by trainers. A qPCR assay was designed for an astrovirus from a bottlenose dolphin that did not cluster with known genera. This assay was used for detection of virus prevalence and load. The prevalence was similar to that found in terrestrial mammal astroviruses, but loads were very low. It is unclear whether this virus is actually infecting dolphins. There is significant diversity amongst marine mammal mamastroviruses. They have a very high prevalence. These findings, together with their similarity to terrestrial astroviruses and recombination frequency, suggest that the marine environment plays an important role in mamastroviral ecology.
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2010.
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Adviser: Jacobson, Elliott R.

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1 DISCOVERY, PHYLOGENETIC ANALYSIS, DIAGNOSTIC TEST DEVELOPMENT, AND SURVEILLANCE OF THE ASTROVIRUSES OF MARINE MAMMALS By JAMES FRANCIS XAVIER WELLEHAN JR. A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 James F.X. Wellehan Jr.

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3 To my wife, Karen, and my children, Xavier and Elseya

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4 ACKNOWLEDGMENTS I would like to thank the present and past members of the Marine Animal Disease Laboratory for their help Linda Archer, Rebecca Rivera, Jennifer Muller, Celeste Benham, Jennifer Burchell, Heather Maness, Brian Stacy and Hendrik Nollens This work could not have been done without them. Brian and Rebecca deserve additional thanks for moral support. I thank the Navy Marine Mammal Program and the Navy Marine Mammal Program Foundation for their help and support, including Stephanie VennWatson, Cynthia Smith, Eric Jensen, and Carolina Ruiz. Stephanie was essential for the epidemiological correlation. Funding from the Office of Naval Research supported much of this work financially through research grant s N00014061 0250 and N0001409 1 0252 to H.Nollens., and N6600108D 0070 to P. Yochem Additional financial support was provided by the Aquatic Animal Health program at the University of Florida College of Veterinary Medicine, and Charlie Courtney is specifically appreciated for this. Additional samples for this work wer e graciously supplied by Charlie Innis at New England Aquarium, Frances Gulland at The Marine Mammal Center, Judy St. Leger at SeaWorld, Randy Wells and Brian Balmer with the Sarasota Dolphin Research Program, and Pam Yochem with Hubbs SeaWorld Research In stitute. I thank my committee for their mentorship my chair, Elliott Jacobson, and Hendrik Nollens, Ramiro Isaza, Mike Miyamoto, Bill Farmerie, Rich Condit, and Bert Flanegan. I thank my additional colleagues in the UF Zoological Medicine Service for th eir support, Dr. Darryl Heard and the zoo med residents during my graduate studies. I thank Andy Kane for getting us the cool new building. My parents deserve special thanks for supporting me as a perpetual student. My wife Karen and children Xavier and Elseya have been incredibly supportiveI love you.

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5 Chapter 2 has already been published as Rivera, R., Nollens, H.H., VennWatson, S., Gulland, F.M.D., Wellehan, J.F.X., 2010. Characterization of phylogenetically diverse astroviruses of marine mammals. J. Gen. Virol 91, 166173. Jim Wellehan did the primer design, sequence editing and assembly, phylogenetic analysis, and recombination analysis. Jim Wellehan and Rebecca Rivera wrote the manuscript together. Stephanie VennWatson and Frances Gulland wer e both involved in sample selection and contribution. All authors were involved in revising the manuscript.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 11 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 16 CHAPTER 1 INTRODUCTION .................................................................................................... 18 Marine Mammals and Virology ............................................................................... 18 Astr oviruses ............................................................................................................ 19 History .............................................................................................................. 19 Taxonomy ......................................................................................................... 20 Structure and Genomic Organiz ation ............................................................... 20 Epidemiology .................................................................................................... 22 Pathology ......................................................................................................... 23 Future Directions .............................................................................................. 24 2 INITIAL CHARACTERIZATION OF PHYLOGENETICALLY DIVERSE ASTROVIRUSES OF MARINE MAMMALS ............................................................ 25 Introduction ............................................................................................................. 25 Materials and Methods ............................................................................................ 26 Animals and Samples ....................................................................................... 26 Negative Staining Electron Microscopy and Sample Processing ..................... 27 Degenerate Polymerase Chain Reaction (PCR) .............................................. 27 Sequence Extension ........................................................................................ 28 Phylogenetic Analysis ...................................................................................... 29 Recombination Analysis ................................................................................... 30 Results .................................................................................................................... 31 Negative Staining Electron Microscopy ............................................................ 31 Degenerate PCR .............................................................................................. 31 Phylogenetic Analysis ...................................................................................... 32 Recombination Analysis ................................................................................... 33 Discussion .............................................................................................................. 34

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7 3 USE OF A PEPTIDEBASED INDIRECT ENZYMELINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF A HUMORAL IMMUNE RESPONSE TO BOTTLENOSE DOLPHIN ASTROVIRUS 1 (BDASTV1) ............................................................................................................. 45 Introduction ............................................................................................................. 45 Materials and Methods ............................................................................................ 47 Animals and Sera ............................................................................................. 47 Astrovirus Peptide Design ................................................................................ 47 Monoclonal Antibody ........................................................................................ 48 Optimization of ELISA Parameters ................................................................... 48 Peptide Indirect ELISA ..................................................................................... 49 Initial Validation ................................................................................................ 50 Further Validation ............................................................................................. 50 Results .................................................................................................................... 51 Initial Validation ................................................................................................ 51 Optimization of ELISA Parameters ................................................................... 51 Peptide Indirect ELISA ..................................................................................... 51 Further Validation ............................................................................................. 52 Discussion .............................................................................................................. 52 4 USE OF A RECOMBINANT CAPSID PROTEININDIRECT ELISA FOR THE DETECTION OF A HUMORAL IMMUNE RESPONSE TO BOTTLENOSE DOLPHIN ASTROVIRUS 1 ..................................................................................... 56 Introduction ............................................................................................................. 56 Materials and Methods ............................................................................................ 58 Animals and Sera ............................................................................................. 58 BDAstV1 Antigen .............................................................................................. 58 Monoclonal Antibody ........................................................................................ 59 Optimization of ELISA Parameters ................................................................... 60 Recombinant Protein ELISA ............................................................................. 60 Results .................................................................................................................... 61 BDAstV1 Antigen .............................................................................................. 61 Optimization of ELISA Parameters ................................................................... 61 Recombinant Protein ELISA ............................................................................. 62 Discussion .............................................................................................................. 63 5 USE OF A QUANTITATIVE PCR ASSAY FOR THE DETECTION OF BOTTLENOSE DOLP HIN ASTROVIRUS 1 ............................................................ 76 Introduction ............................................................................................................. 76 Materials and Methods ............................................................................................ 77 Sam ples ........................................................................................................... 77 RNA Extraction ................................................................................................. 77 Quantitative PCR .............................................................................................. 78 Confirmatory Hemin ested PCR ........................................................................ 79

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8 Results .................................................................................................................... 80 Quantitative PCR .............................................................................................. 80 Confirmatory Hemines ted PCR ........................................................................ 81 Discussion .............................................................................................................. 82 6 FURTHER INVESTIGATION OF ASTROVIRUS DIVERSITY IN CETACEAN FECAL SAMPLES .................................................................................................. 92 Introduction ............................................................................................................. 92 Materials and Methods ............................................................................................ 93 Samples ........................................................................................................... 93 RNA Extraction ................................................................................................. 93 Consensus PCR ............................................................................................... 93 Sequence Extension ........................................................................................ 94 Phylogenetic Analysis ...................................................................................... 94 Results .................................................................................................................... 95 Consensus PCR ............................................................................................... 95 Sequence Extension ........................................................................................ 96 Phylogenetic Analysis ...................................................................................... 96 Discussion .............................................................................................................. 98 7 USE OF A QUANTITATIVE PCR ASSAY FOR THE DETECTION OF BOTTLENOSE DOLPHIN ASTROVIRUS 6 .......................................................... 109 Introduction ........................................................................................................... 109 Materials and M ethods .......................................................................................... 110 Samples ......................................................................................................... 110 RNA Extraction ............................................................................................... 110 Quantitative PCR ............................................................................................ 111 Confirmatory Heminested PCR ...................................................................... 112 Results .................................................................................................................. 112 Quantitative PCR ............................................................................................ 112 Confirmatory Heminested PCR ...................................................................... 113 Discussion ............................................................................................................ 113 8 CONCLUSIONS ................................................................................................... 118 APPENDIX A MUSCLE ALIGNMENT OF PARTIAL ASTROVIRAL RIBONUCLEIC ACID (RNA) DEPENDENT RNA POLYMERASE AMINO ACID SEQUENCES ............. 121 B T COFFEE ALIGNMENT OF PARTIAL ASTROVIRAL CAPSID AMINO ACID SEQUENCES ....................................................................................................... 124

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9 C OPTICAL DENSITY AT 405 NANOMETERS (OD405) VALUES FOR 46 DOLPHIN SERUM SAMPLES USING A BOTTLENOSE DOLPHIN ASTROVIRUS 1 (BDASTV1) PEPTIDE ENZYMELINKED IMMUNOSORBENT ASSAY (ELISA) AGAINST SELECTED PEPTIDE PAIRS .................................... 126 D OD405 VALUES FOR 146 DOLPHIN SERUM SAMPLES USING A BDASTV1 PEPTIDE ELISA AGAINST ALL FOUR PEPTIDES ( TT322, TT399, TT455, TT616) .................................................................................................................. 128 LIST OF REFERENCES ............................................................................................. 135 BIOGRAPHICAL SKETCH .......................................................................................... 145

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10 LIST OF TABLES Table page 2 1 Primers used to sequence California sea lion Astroviruses 13 (CSLAstV1 to V 3), Steller sea lion astrovirus 1 (SSLAstV1) and bottlenose dolphin astrovirus 1 (B DAstV1) ....................................................................................... 39 4 1 Summary of OD405 control values for groups of dolphins ................................. 67 5 1 BDAstV1 copies detected by quantitative polymerase chain reaction ( qPCR) f rom the managed openwater collection ............................................................ 87 5 2 Comparisons of mean BDAstV1 fecal load by animal appetite, behavior, reason for sampling among a population of common bottlenose dolphins ( T ursiops truncatus ) ............................................................................................ 88 5 3 Comparisons of mean chloride, potassium, and sodium by presence or absence of BDAstV1 in feces among a population of common bottlenose dolphins ( Tursiops truncatus ) ............................................................................. 88 5 4 BDAstV1 copies detected by qPCR from wild cetaceans. .................................. 89 6 1 Additional primers used for 3 rapid amplif i cation of complimentary deoxyribonucleic acid ends ( RACE) of novel astroviruses from cetacean feces ................................................................................................................. 103 6 2 Results of astroviral consensus polymerase chain reaction ( PCR) and sequencing of bottlenose dolphin fecal samples. ............................................. 104 6 3 Results of astroviral consensus PCR and sequencing of other cetacean samples. ........................................................................................................... 105 7 1 BDAstV6 copies detected by qPCR from the managed openwater collect ion. 116

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11 LIST OF FIGURES Figure page 2 1 Negative Staining Electron Microscopy ............................................................. 40 2 2 Hu man Ast rovirus 1 genome compared to marine mammal astrovirus genome segments. ............................................................................................. 41 2 3 Bayesian phylogenetic tree of predicted 237254 amino acid partial astroviral ribonucleic acid ( RNA) dependent RNA polymerase sequences based on MUSCLE alignment. ........................................................................................... 42 2 4 Bayesian phylogenetic tree of predicted 186207 amino acid partial astroviral capsid sequences based on TCoffee alignment. ............................................... 43 2 5 Bootscanning analysis of MUSCLE alignment of astroviral sequences produced using the RDP3 suite. ......................................................................... 44 3 1 MUSCLE alignment of Human Astrovirus 1 and Bottlenose dolphin astrovirus 1 ( BDAstV1 ) capsid amino acid sequences. ....................................................... 54 3 2 Correlation of optical density at 405 nanometers ( OD405) values between different pairs of peptides. .................................................................................. 55 4 1 MUSCLE alignment of Human Astrovirus 1 and BDAstV1 capsid amino acid sequences. ......................................................................................................... 68 4 2 Amino acid sequence of cloned BDAstV1 antigen. ............................................. 69 4 3 Coomassie blue stained sodium dodecyl sulfate polyacrylamide gel e lectrophoresis ( SDSPAGE) gel of the expressed BDAstV1 fragment. ............. 70 4 4 Western blot using an anti polyhistidine antibody. .............................................. 71 4 5 Enzyme linked Immunosorbent Assay ( ELISA) values (OD405 control) of openwater collection mature dolphins over time. ............................................... 72 4 6 ELISA values (OD405 control) of openwate r collection calves over time. ......... 73 4 7 ELISA values (OD405 control) vs. days in age among common bottlenose dolphin ( Tursiops truncatus ) calves, including all ten calves. ............................. 74 4 8 ELISA values (OD405 control) vs. days in age among selected individual bottlenose dolphin ( Tursiops t runcatus ) calves with significantly increasing titers by days in age. ........................................................................................... 75 5 1 The standard curve for the BDAstV1 quantitative polymerase chain reaction ( qPCR) ............................................................................................................. 90

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12 5 2 BDAstV1 copies detected in from the managed openwater collection plotted on a logarithmic scale on the vertical axis against date on the horizontal axis. .. 91 6 1 MUSCLE alignment of sequence from animal 27 and reference BDAstV1. ..... 106 6 2 Bayesian phylogenetic tree of predicted 100133 amino acid partial astroviral RNAdependent RNA polymerase sequences based on MUSCLE alignment. 107 6 3 Bayesian phylogenetic tree of predicted 388429 amino acid partial astroviral capsid sequences based on TCoffee alignment. ............................................. 108 7 1 The standard curve for the BDAstV6 qPCR. .................................................... 117

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13 LIST OF ABBREVIATIONS AMV avian myeloblastosis virus AIDS acquired immune deficiency syndrome BD Bottlenose Dolphin Bp base pairs BDAstV1 Bottlenose Dolphin Astrovirus 1 BDAstV2 Bottlenose Dolphin Astrovirus 2 BDAstV3 Bottlenose Dolphin Astrovirus 3 BDAstV5 Bottlenose Dolphin Astrovirus 5 BDAstV6 Bottlenose Dolphin Astrovirus 6 BDAstV7 Bottlenose Dolphin Astrovirus 7 BDAstV8 Bottlenose Dolphin Astrovirus 8 BDAstV9 Bottlenose Dolphin Astrovirus 9 BSA Bovine Serum Albumin DMSO dimethyl sulfoxide cDNA complementary DNA CSLAstV1 California sea lion astrovirus 1 CSLAstV2 California sea lion astrovirus 2 CSLAstV3 California sea lion astrovirus 3 DNA Deoxyribonucleic acid dNTP deoxynucleotide triphosphates E. coli Escherichia coli ELISA Enzyme linked immunosorbent assay FMOC 9 fluorenylmethoxycarbonyl HAstV1 Human astrovirus 1

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14 HAstV2 Human astrovirus 2 HAstV3 Human astrovirus 3 HAstV5 Human astrovirus 5 HPLC High performance liquid chromatography IACUC Institutional Animal Care and Use Committee ICTV International Committee of Taxonomy of Viruses IgG Immunoglobulin G mAb monoclonal antibody MgCl2 Magnesium c hloride ML Maximum likelihood MLB1 Human Astrovirus MLB1 MMLV Moloney Murine Leukemia Virus MWAstV1 Minke Whale Astrovirus 1 MWAstV2 Minke Whale Astrovirus 2 NaCl Sodium c hloride NEM negativestaining electron microscopy OD405 optical density at 405nm OoAstV1 Orca Astrovirus 1 ORF open reading frame PBS Phosphate Buffered Saline PCR polymerase chain r eaction PNPP P Nitrophenyl Phosphate qPCR quantitative polymerase chain reaction (a.k.a. real time PCR) RACE Rapid Amplification of cDNA Ends RdRp RNAdependent RNA polymerase

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15 RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid SARS Severe acut e respiratory syndrome SDSPAGE sodium dodecyl sulfatepolyacrylamide gel electophoresis SSLAstV1 St eller sea lion astrovirus 1

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16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillmen t of the Requirements for the Degree of Doctor of Philosophy DISCOVERY, PHYLOGENETIC ANALYSIS, DIAGNOSTIC TEST DEVELOPMENT, AND SURVEILLANCE OF THE ASTROVIRUSES OF MARINE MAMMALS By James Francis Xavier Wellehan Jr. December 2010 Chair: Elliott Jacobson Major: Veterinary Medical Sciences Astroviridae are a family of small nonenveloped positive stranded ribonucleic acid (RNA) viruses associated with enteritis. Knowledge of astrovirus diversity is very limited, with only six astrovirus species from mammal ian hosts officially recognized, and additional human, cheetah, rat, dog, and bat astroviruses recently described. We used consensus polymerase chain reaction (PCR) techniques for initial identification of fifteen astroviruses from marine mammals; three from California sea lions ( Zalophus californianus ), one from a Steller sea lion ( Eumetopias jubatus ), nine from bottlenose dolphins ( Tursiops truncatus ) and two from minke whales ( Balaenoptera acutorostrata) Bayesian and maximum likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Some astroviruses from marine mammals clustered within the genus Mamastrovirus whereas others were in a clade outside of known genera. Mamastroviruses id entified d id not form a monophyletic group. Recombination analysis found that a relatively recent recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same

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17 host at one point. A bottlenose dolphin astrovirus sequence was also consistent with the result of a recombination event. An enzyme linked immunosorbent assay (ELISA) and a quantitative PCR (qPCR) assay were designed for a bottlenose dolphin Mama strovirus and used to survey seroprevalence, virus prevalence, and virus load. The results showed that animals seroconvert at a young age, and virus prevalence was even higher than what has been seen with astroviruses of terrestrial animals. Virus load c orrelated with abnormal behavior reported by trainers. A qPCR assay was designed for an astrovirus from a bottlenose dolphin that did not cluster with known genera. This assay was used for detection of virus prevalence and load. The prevalence was similar to that found in terrestrial mammal astroviruses, but loads were very low. It is unclear whether this virus is actually infecting dolphins. There is significant diversity amongst marine mammal mamastroviruses. They have a very high prevalence. These findings, together with their similarity to terrestrial astroviruses and recombination frequency, suggest that the marine environment plays an important role in mamastroviral ecology.

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18 CHAPTER 1 INTRODUCTION Marine Mammals and Virology The marine environment has amazing diversity. Cellular life first evolved in the oceans, and it is probable that viruses evolved shortly thereafter The abundance of viruses in marine environment s is approximately 15fold that of bacteria and archaea (Suttle, 2007) Marine mammals are charismatic animals with a strong appeal to humans. Humans are having a major impact on marine environments, with negative impacts on marine mammal populations (Bejder et al., 2006). Relatively little is understood about the viruses of marine mammals. Diseases such as dolphin morbillivirus have demonstrated the potential to cause significant morbidity and mortality in marine mammal populations (Schulman et al. 1997). The known diversity of marine mammal viruses has just begun to expand rapi dly (Nollens et al., 2010, Colegrove et al., 2010, Wellehan et al., 2008). A greater understanding of dolphin virology may positively impact health management of both wild and captive marine mammals. Additionally, there may be unknown potential viral zoonoses present in the marine environment, presenting risks to human populations. Emerging disease is frequently associated with host switches. One recent metaanalysis of human diseases found that 816 of 1407 (58%) are zoonotic, and of human diseases, zoonotic diseases are significantly more likely to be emerging (Woolhouse and GowtageSequeria, 2005). Most recent emerging diseases have been associated with host switches, including severe acute respiratory syndrome (SARS) coronavirus, H5N1 avian influenza, Hendra virus, Nipah virus, and acquired immunodeficiency syndrome (AIDS). The

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19 aforementioned study also found that viral diseases were much more likely to be emerging, especially ribonucleic acid (RNA) viruses (Woolhouse and GowtageSequeria, 2005). Most emerging diseases have zoonotic origins, and viruses in the marine environment are poorly understood. Understanding of the marine ecosystem is necessary to comprehend viral ecology, and has great significance for human health. Marine mammals are useful s entinels for marine viruses, allowing earlier identification of zoonotic agents and development of diagnostic and epidemiologic strategies. San Miguel Sea Lion Virus has been shown to infect humans and is associated with disease (Smith et al., 1998). Medic ine has traditionally waited for viruses to cause epidemics or epizootics before significant surveillance occurs. With our increased understanding of virus ecology and evolution, it becomes more feasible to identify probable candidates for future novel di sease outbreaks, and increase surveillance. An understanding of diverse viruses in wildlife may enable more appropriate epidemiologic responses to new virus infections. Astroviruses History Astroviruses were discovered relatively recently having first been reported in 1975 from human diarrhea cases (Madeley and Cosgrove, 1975). Once recognized, they were soon associated with childhood enteritis; an early study of a human astrovirus in Oxford, England found that 7% of 6 to 12 month old children had a posi tive titer, whereas 75% of 5 to 10 year old children were positive (Kurtz and Lee, 1978). Studies of experimental infections in volunteers established them as an etiology of gastroenteritis, although the very high prevalence of antibody titers in adults, indicating

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20 the presence of an acquired immune response, made reliable experimental infection difficult (Kurtz et al., 1979). The first sequence was obtained from human Astroviruses in 1992 (Major et al., 1992, Willcocks and Carter, 1992). Taxonomy The f amily Astroviridae is divided into two genera, Avastrovirus found in avian hosts, and Mamastrovirus found in mammal hosts (Monroe et al. 2005). Species are defined by the International Committee on the Taxonomy of Viruses (ICTV) on the basis of the host species (Monroe et al. 2005) In the genus Avastrovirus recognized species include Chicken Astrovirus Duck Astrovirus, and Turkey Astrovirus Recognized species in the genus Mamastrovirus include Bovine astrovirus Feline astrovirus Human astrovirus Mink Astrovirus Ovine astrovirus and Porcine astrovirus (Monroe et al. 2005) There has been significant recent discovery of additional mamastroviruses, including viruses from cheetahs (Atkins et al., 2009), Asian bat species ( Chu et al., 2008, Zhu et al., 2009) humans (Finkbeiner et al., 2008, Finkbeiner et al., 2009a, Finkbeiner et al., 2009b, Kapoor et al., 2009), and rats (Chu et al., 2010). Recently, a divergent astrovirus open reading frame (ORF) 1a sequence has been identified from bat guano f ound under a mixedspecies roost in North America (Li et al., 2010). Phylogenetic analysis found that it was weakly supported as basal to other mamastroviruses in a neighbor joining tree. Structure and Genomic Organization Astroviruses are small nonenvelo ped viruses with a positive stranded RNA genome and a distinct star like surface morphology seen on electron microscopy Virus particles have been reported on electron microscopy as 2834 nanometers in diameter with a round unbroken edge, a six pointed star with a white center, and triangular

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21 surface hollows (Madeley, 1979). However, astroviruses resemble other small round viruses somewhat morphologically, and a significant rate of misidentification using negativestaining electron microscopy of feces has been reported (Oliver and Phillips, 1988). Astrovirus genomes are range between 6.87.9 kb and contain three open reading frames. ORF1a encodes nsp1a, a 101 kiloDalton ( kDa ) nonstructural polyprotein, which is cleaved by a viral serine protease and host pr oteases (Kiang and Matsui, 2002, Geigenmller et al., 2002). After processing, the amino terminal fragment has a mass of approximately 20kDa ( Mndez et al., 2003), and may be a helicase since it shares limited motifs with the pestiviral helicase (AlMutai ry et al., 2005). The next fragment, containi ng the serine protease, is a 27kDa protein (Geigenmller et al., 2002, Mndez et al., 2003). This segment is the best structurally characterized astroviral protein, and is currently the only region where a detailed crystallographic structure is available (Speroni et al., 2009). Following the serine protease fragment, the carboxy terminal fragment of nsp1a contains a hypervariable region that colocalizes with viral RNA and endoplasmic reticulum. Changes in this hypervariable region have significant effects on viral replication (Guix et al., 2005). It has been proposed, based on sequence analysis, that this region may represent a genome linked viral protein, homologous to VPg in other plus sense singlestranded R NA viruses (AlMutairy et al., 2005). ORFs 1a and 1b are linked by a translational frameshift. They encode polyprotein nsp1ab, w ith a mass of 145kDa, which is cleaved to a 57kDa protein encoding an RNA dependent RNA polymerase ( Mndez et al., 2003).

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22 ORF 2 encodes the capsid precursor protein. In human astroviruses, the 90kDa membrane bound capsid precursor is formed into viral particles that are subsequently released from the cell membrane by caspase cleavage to form a 70kDa protein ( Mndez et al., 2004) Trypsin cleavage further cuts the capsid protein into an early 38.541 kDa section forming the main capsid, and a later 2529 kDa section forming spikes, giving the virus its star shape ( Mndez et al., 2002). This trypsin cleavage markedly increases infectivity. Neutralizing epitopes have been found on the 2529 kDa protein ( Bass and Upadhyayula, 1997). Recently, an ORF (ORFX) overlapping the 5 end of ORF2 in a different frame has been identified (Firth and Atkins, 2010). ORFX is conserved amongst Mamastrovirus but not Avastrovirus It is unknown whether ORFX is expressed, and if so, what function is serves. Epidemiology Most epidemiological data have examined Human Astrovirus As a prevalent enteric disease in children, exposure to Human Astrovir us at a young age is typical. In Oxford, England, 75% of 5 to 10 year olds were positive (Kurtz and Lee, 1978). A study in London found that over 50% of children between 5 and 12 months of age had antibody responses to Human astrovirus 1 (HAstV1), and seroprevalence was 90% by the age of 5 (Kriston et al., 1996). In the Netherlands, seroprevalence of HAstV1 was 100% by 5 years of age, and although overall seroprevalence was lower for human astroviruses 24, the age of conversion was similar (Koopmans et al., 1998). However, Koopmans et al. (1998) found a later age of onset for HAst V 5 A study in Virginia found that t he seroprevalence of HAstV1 was 94% at 6 to 9 years of age ( Mitchell et al.,

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23 1999). A study in Japan found that by age 3, seropositivity t o HAstV1 and HAstV3 approached 100% (Kobayashi et al., 1999). Human astrovirus quantitative polymerase chain reaction (qPCR) assays have found the prevalence in diarrheic human feces to range from 6% 9% (Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in press). One study of fecal electron microscopy of cats found that astroviruses were the most common virus particles seen in cats with diarrhea (Marshall et al., 1987). Human astrovirus data clearly indicates a cosmopolitan distribution. Ther e is also evidence that Feline astrovirus may be cosmopolitan; astroviruses have been documented in cats from Australia (Marshall et al., 1987), England (Harbour et al., 1987), Germany (Herbst and Krauss, 1988), New Zealand (Rice et al., 1993), and the United States (Hoshino et al., 1981). A human astrovirus qPCR assay was used for a study of clinical correlation of virus load to clinical features. A tendency for longer duration of diarrhea with higher copy numbers was seen. Lower copy numbers were associated with rotavirus coinfection (Zhang et al., 2006). Pathology Astroviruses are strongly associated with enteric disease. Unlike other characterized viral causes of enteritis, but m uch like Vibrio cholerae, astroviruses cause a secretory diarrhea without m uch of a histologic footprint on enterocytes on light microscopy ( Koci et al., 2003, Moser et al., 2007, Nighot et al., 2010). Turkey astrovirus 2 (TAstV2) does not increase apoptosis in the intestine ( Koci et al., 2003). Human a strovirus 1 capsid protein interacts with apical enterocyte membranes, increasing permeability independent of viral replication. I ncreased permeability correlates with

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24 disruption of the tight junction protein occludin and a reduction in actin stress fibers in infected cells, suggest ing that tight junctions between enterocytes may become leaky ( Moser et al., 2007). With TAstV2 infections, actin rearrangement was also seen, resulting in ultrastructural changes that were not visible on light microscopy ( Nighot et al., 2010) R edistrib ution of the sodium/hydrogen exchanger 3 from the membrane to the cytoplasm was seen, which was likely the cause of the decreased sodium absorption that was found (Nighot et al., 2010). Following infection of turkey poults TAstV 2 can be recovered from mul tiple tissues, and animals are viremic ( Koci et al., 2003). While diarrhea is the most common clinical sign seen with astroviral infection, a Mamastrovirus has been found in a human encephalitis case (Quan et al., 2010), and Avastrovirus disease may also be renal or hepatic ( Imada et al., 2000, Fu et al., 2009). There is a paucity of information on the pathology and tissue tropisms of astroviruses in sites other than enterocytes. Future Directions Astroviruses are known to cause disease in mammals. As sm all RNA viruses, they are capable of rapid evolution, which is advantageous when invading new habitats such as novel host species. One recent study scored the viruses infecting mammals for biological properties that were considered advantageous to host swi tching, and found that Astr oviridae scored very highly (Pulliam, 2008). Astroviruses are very stable in aquatic environments (Espinosa et al ., 2008) T he marine environment is central in the ecology of caliciviruses, a better studied group of small nonenv eloped positive stranded RNA vir al human pathogens (Smith et al ., 1998). The astroviruses of wildlife merit further surveillance, and marine mammals represent an important potential reservoir.

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25 CHAPTER 2 INITIAL CHARACTERIZATION OF PHYLOGENETICALLY DIVER SE ASTROVIRUSES OF MARINE MAMMALS Introduction Astroviruses are small nonenveloped viruses with a positive stranded RNA genome and a distinct star like surface morphology They were relatively recently discovered and first reported in 1975 (Madeley & Cosgrove, 1975). Based mainly on the host of the virus and the genome structure, t he family Astroviridae is divided into two genera. Members of the genus Avastrovirus are found in avian hosts, whereas the genus Mamastrovirus is found in mamm al hosts (Monroe et al ., 2005). Known astrovirus diversity is very limited, with only three astrovirus species recognized from avian hosts by the International Committee on the Taxonomy of Viruses and six recognized astrovirus species from mammalian hosts ( Bovine astrovirus, Feline astrovirus, Human astrovirus (serotypes 18) Mink Astrovirus, Ovine astrovirus, and Porcine astrovirus ) (Monroe et al ., 2005). Recently, a divergent human astrovirus from a child with diarrhea (Finkbeiner et al ., 2008), and a number of astroviruses from vespertillonid and rhinolophid bats have been described (Chu et al ., 2008). The mamastroviruses have a small, positive sense, singlestranded RNA genome of less than 7,000 base pairs that codes for three open reading frames (ORF) named ORF1a, OR F1b and ORF2. A frame shift between ORF1a and ORF1b allows ORF1 to encode both a protease and an RNAdependent RNA polymerase (RdRp). ORF2 encodes the viral capsid protein. Cloning of ORF2 in expression vectors has allowed for the invitro assembly of vi rus like particles (Caballero et al ., 2004). While avastroviruses can cause intestinal or renal disease, mamastroviruses predominantly establish infections in the gastrointestinal tract of their hosts. Human

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26 astrovirus es are a frequent cause of enteric di sease in neonatal, elderly and immunocompromised humans (Dennehy et al., 2001, Gallimore et al., 2005) and one study of fecal electron microscopy of cats found that astroviruses were the most common virus particles observed in cats with diarrhea (Marshall et al ., 1987). The capsid protein plays a unique role in the pathogenesis of the diarrhea. Astrovirus capsid protein interacts with apical enterocyte membranes, increasing permeability independent of viral replication. Much like Vibrio cholerae, astroviru ses cause a secretory diarrhea without much of a histologic footprint (Moser et al ., 2007). Histopathology is therefore an insensitive test for diagnosis of astroviral diarrhea, potentially leading to underdiagnosis of astrovirus infections. Only very rec ently has the presence of astroviruses in wildlife hosts been reported. Previous reports in nondomestic hosts include identification of astroviruses in cheetahs and multiple species of bats (Atkins et al ., 2009)(Chu et al ., 2008). Here, we report on the f irst detection of five genetically distinct astroviruses from three marine mammal host species. Materials and Methods Animals and Samples Fecal samples were collected as part of routine health surveillance from two clinically healthy California sea lions ( Zalophus californianus ) and one bottlenose dolphin ( Tursiops truncatus ) housed in open ocean enclosures at the U.S. Navy Marine Mammal Program in San Diego, California (CA). In addition, fecal samples were collected from one stranded, freeranging California sea lion pup with diarrhea and one stranded, freeranging Steller sea lion ( Eum e topias jubatus ) pup without outward signs of diarrhea, both housed at The Marine Mammal Center in Sausalito, CA. All samples

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27 were collected between December 2006 and October 2008. Fecal samples were stored in sterile vials and frozen at 80 C elsius (C) until laboratory analysis. Negative Staining Electron Microscopy and Sample Processing Upon arrival to the laboratory, each fecal sample was divided into three fractions The first fraction was sent to the Florida State Diagnostic Lab for negativestaining electron microscopy (NEM). The second frac tion was stored at 80 for future analysis. The third fraction was suspended at a 1:10 ratio in 0.89% sodium chloride ( NaCl) and centrifuged at 4000 x G for 20 minutes at 4C The clarified supernatant was collected using a sterile syringe and consecutively passed through 0.8 micrometers ( m ) 0.4 5 m and 0.22 m syringe filters to eliminate cellular and bacterial particles. The final filtrate was transferred to a M icrosep concentrator column (Pall Life Sciences) and centrifuged at 1500 x G for 25 to 45 minutes at 4 C. A 140 microliter ( l ) aliquot of the concentrated filtrate w as used for RNA extraction using a Viral RNA Mini Kit (Qiagen) following the manufacturer s instructions. Degenerate Polymerase Chain Reaction ( PCR) Degenerate primers designed based on conserved a strovir al sequences (Atkins et al ., 2009) were used in a nested or a semi nested format to amplify conserved regions of the ORF1b ( RdRp ) and ORF2 ( capsid) For amplification of the partial RdRp gene, primers Astr4380F (5'GAYTGGRCNCGNTWYGATGGNACIAT 3') and Astr4811R (5'GGYTTNACCCACATNCCAAA3) (round #1), and primer s Ast r4574F (5 GGNAAYCCMTCWGGICA3 ) and Ast r4722R (5'ARNCKRTCATCNCCATA 3') (round #2) were used on all five isolates. For individual viruses, additional degenerate primer combinations were used to obtain more astrovirus RdRp gene sequence (see table 2-

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28 1). For amplification of t he partial capsid gene primer s Astr4811F ( forward, 5'TTTGGNATGTGGGTNAARCC3 ') and Astr5819R (5'TCATTNGTGTYNGTNANCCACCA3) ( round #1), and primers Astr5159F (5'TGGAGGGGMGGACCAAAG3) and Astr5819R (round #2) were used on all five isolates. For the first round of the PCR assays, fecal RNA was reverse transcribed using a OneStep RTPCR Kit (Qiagen) at 50 minutes and then denatured at 94 minutes followed by 36 cycles of denaturation at 94 seconds; annealing at 45seconds, and extension at 72seconds, with a final elongation step at 72 minutes Three l of product from the first round was used as template in a 20l nested or semi nested second round. The second round amplifications conditions using Platinum Taq DNA Polyme rase (Invitrogen) were as follows: 5 minutes denaturation at 94 seconds) annealing at 45 seconds) extension at 72 seconds) with a final elongation step at 72minutes PCR products from both rounds were run in 1% agarose gels and the DNA bands were visualized under UV light after ethidium bromide staining. Bands of interest were cut from the gel and their DNA extracted using the Qiaquick gel extraction kit (Qiagen). Direct sequenci ng was performed using the BigDye Terminator Kit (Perkin Elmer) and ABI automated sequencers. All amplicons were sequenced at least twice in both directions, and primer sequences were edited out prior to constructing contiguous sequences. Sequence Extensi on For each isolate, the gap between the upstream ORF1b and downstream ORF2 sequence segment was amplified using specific forward and reverse primers (see Table

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29 2 1) that were designed based on the sequences obtained via degenerate PCR. Again, a ll amplico ns were sequenced at least twice in both directions A ttempt s were made to sequence the remaining 5 and 3 sections of the viral genome using a GeneRacer kit ( Invitrogen). Specific rapid amplification of complimentary deoxyribonucleic acid ends (RACE) p rimers were designed, and viral genomic RNA was amplified using the manufacturers instructions Briefly, for 3 RACE, RNA was reverse transcribed using AMV reverse transcriptase and amplified with a forward gene specific primer and the GeneRacer 3 Primer For 5 RACE, RNA was treated with calf intestinal phosphatase, treated with tobacco acid pyrophosphatase to remove the 5 cap structure, and ligated to the GeneRacer RNA oligo. The dephosphorylated, uncapped and ligated RNA was then reverse transcribed and subsequently amplified using a specific reverse gene specific primer (see table 21) and the GeneRacer 3 p rimer. PCR products were run in a 0.7% agarose gel, and bands of interest were sequenced as previously described. Phylogenetic Analysis Sequences were compared to those in GenBank (National Center for Biotechnology Information, Bethesda, Maryland), European Molecular Biology Laboratory ( EMBL ) (Cambridge, United Kingdom), and Data Bank of Japan (Mishima, Shizuoka, Japan) databases using TBLASTX (Altschul et al ., 1997). The p redicted homologous 237254 amino acid sequences of astroviral RdRp and 186207 amino acid sequences of astroviral capsid protein were aligned using the following three methods: ClustalW2 (Larkin et al ., 2007), TCoffee (Not redame et al ., 2007), and MUSCLE (Edgar, 2004). Bayesian analyses of each alignment were performed using MrBayes 3.1 (Ronquist & Huelsenbeck, 2003) with gamma distributed rate variation and a proportion

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30 of invariant sites. M ixed amino acid substitution models were used. The first 10% of 1,000,000 iterations were discarded as a burn in. Maximum likelihood (ML) analyses of each alignment were performed using PHYLIP (Phylogeny Inference Package, Version 3.66) (Felsenstein, 1989), running each alignment usi ng the program ProML with amino acid substitution models JTT (Jones et al ., 1992), PMB (Veerassamy et al ., 2003), and PAM (Kosiol & Goldman, 2005) further set with global rearrangements, five replications of random input order, gamma plus invariant rate di stributions, and unrooted. The values for the gamma distribution were taken from the Bayesian analysis. Avian nephritis virus 1 (GenBank accession number AB033998) was designated as the outgroup. The alignment producing the most likely tree was then used to create data subsets for bootstrap analysis to test the strength of the tree topology (200 resamplings) (Felsenstein, 1985), which was analyzed using the amino acid substitution model producing the most likely tree in that alignment. Recombination Analy sis A nucleotide alignment was created using MUSCLE on the sequence between primers Astr4380F and Astr5819R of 13 mamastroviruses: the five marine mammal astroviruses from this study, Ovine astrovirus (GenBank accession # NC002469), Bat astrovirus AFCD337 (EU847155), Mink astrovirus (AY179509), MLB1 astrovirus (FJ222451), Human astrovirus 1 (AY720892), Human astrovirus 3 (AF141381), Human astrovirus 4 (DQ070852), and Human astrovirus 5 (DQ028633). Potential recombination patterns were screened using RDP (M artin & Rybicki, 2000), Geneconv (Padidam et al ., 1999), MaxChi (Maynard, 1992), Chimaera (Posada & Crandall, 2001), and 3Seq (Boni et al ., 2007) in the RDP3 suite (Martin et al ., 2005b) using the step

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31 down correction for multiple comparisons and a P value cutoff of 0.05. Regions of potential recombinant interest were also checked with LARD (Holmes et al ., 1999), Recscan (Martin et al ., 2005a), and SiScan (Gibbs et al ., 2000). Results Negative S taining Electron Microscopy Viral particles were detected in all five fecal samples. The 5 isolates are henceforth referred to as California sea lion astroviruses 13 (CSLAstV1, CSLAstV2, CSLAstV3), Steller sea lion astrovirus 1 (SSLAstV1) and bottlenose dolphin astrovirus 1 (BDAstV1). Individual capsids were 3035 nm in diameter. In all samples, the nonenveloped icosahedral virus particles had distinct star like surface projections and were consistent in size and morphology with members of the astrovirus family (Figure 21). Degenerate PCR The primer combination Astr4380F/Astr4811R yielded a band of 431 base pairs ( bp) on isolates CSLAstV1 CSLAstV3 SSLAstV1 and BDAstV1 The primers Astr4574F/Astr4722R yielded a band of 148bp on isolates CSLAstV1 CSLAstV2 CSLAstV3 and SSLAstV1 The primer combination 5159F/5819R yielded a band of 660bp on all five isolates. Seq uence extension via specific PCR yielded final contiguous molecules of 1,340 bp (CSLAstV3) and 1,348 bp (SSLAstV1). Additional 3 RACE yielded final contiguous molecules of 3,174 bp (CSLAstV1), 3,505 b p (CSLAstV2), and 3,985 bp (BDAstV1). The contiguous molecules corresponded to the partial capsid gene (ORF1b) and the fulllength RdRp gene of reference astroviruses (Fig ure 2 2 ). The contiguous sequences were submitted to GenBank under accession numbers FJ890351 ( CSLAstV1 ),

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32 FJ890352 ( CSLAstV2 ), FJ890353 ( CSLAstV3 ), FJ890354 ( SSLAstV1 ) and FJ890355 ( BDAstV1 ). Comparison with other sequences in GenBank revealed that all five contiguous molecules represented novel astroviruses. TBLASTX results for bottlenose dolphin astrovirus 1 showed the highest identity score with Human astrovirus 3 (GenBank accession # AF141381) at the RdRp and Human Astrovirus 7 (GenBank accession # Y08632) for the capsid precursor. TBLASTX results for California sea lion astrovirus 1 showed the highest score with Ovine astrovirus (GenBank accession # AF141381) at the RdRp and Mink astrovirus (GenBank accession # AY179509) for the capsid precursor. TBLASTX results for California sea lion astrovirus 2 showed the highest score with Human astrovirus 2 (GenBank accession # L13745) at the RdRp and Porcine astrovirus (GenBank accession # AB037272) for the capsid precursor. TBLASTX results for California sea lion astrovirus 3 showed the highest score with Human astrovirus 3 (GenBank accession # AF141381) at both the RdRp and the capsid precursor. TBLASTX results for Steller sea lion astrovirus 1 showed the highest score with Mink astrovirus (GenBank accession # AY179509) at both the RdRp and the capsid precursor. Phylogenetic Analysis Bayesian phylogenetic analysis showed the greatest harmonic mean of estimated marginal likelihoods using the MUSCLE alignment for the RdRp ( Appendix A ) and the T Coffee alignment for the capsid gene ( Appendix B ). For the RdRp, the W AG model of amino acid substituti on was found to be most probable with a posterior probability of 1.000 (Whelan & Goldman, 2001). For the capsid precursor protein, the W AG model was also most probable with a posterior probability of 0.993, and a posterior probability

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33 of 0.007 for the JTT model. Bayesian trees using the MUSCLE alignment for the RdRp ( Figure 23 ) and the T Coffee alignment for the capsid gene are shown ( Figure 24 ). ML analysis found the most likely tree from the MUSCLE alignment and the PMB model of amino acid substituti on for the RdRp, and the TCoffee alignment and the JTT model of amino acid substitution for the capsid precursor. These parameters were used for bootstrap analysis. Bootstrap values from ML analysis are shown on the trees ( Figures 23 and 24 ). Recombination Analysis The MUSCLE nucleotide alignment was 1479 nucleotides in length. Recombination analysis identified a probable recombination event in CSLAstV3 from parents Human Astrovirus 4 and CSLAstV2 with a P value after corrections for multiple compari sons of 4.911 x106. It was supported by RDP (P = 2.197x104), Recscan (P = 4.359 x103), MaxChi (P = 7.943 x104), Chimaera (P = 4.911 x106), SiScan (P = 3.089 x105), LARD (P = 2.339 x106), and 3Seq (P = 2.634 x103). Geneconv did not support this ev ent. This event started at nucleotide 832 of the alignment with a P value for the beginning breakpoint of 8.637 x103 (Fig. 27). This initial breakpoint falls early in the coding region for ORF2, at the 15th amino acid. Prior to the breakpoint, CSLAstV 3 showed greater homology with Human Astrovirus 4, and greater homology with CSLAstV2 after the breakpoint. The endpoint was not clear although predicted for nucleotide 1062 of the alignment, the P value was only 0.600. A bootscanning diagram for this e vent is shown in Figure 25. Differences in branching patterns between the capsid and polymerase in the Mink Astrovirus / CSLAstV1 / SSLAstV1 clade were not reconciled by identification of a recombination event in the initial analysis, so other sequences in the alignment were

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34 masked for additional analysis. Weak support was found for a recombination event between nucleotides 727 and 847, without correction for multiple comparisons. This region covers the end of ORF1b and the start of ORF2. This event was s upported by RDP (P = 0.02957), Recscan (P = 0.04462), SiScan (P = 7.198 x104), LARD (P = 2.405 x104), and 3Seq (P= 0.01088) but not Geneconv (P = 0.074), MaxChi (P = 0.1178), or Chimaera (P= 0.2422). Discussion T his report documents the first identificat ion of astroviruses in marine mammals. Surprising diversity was identified in these hosts. With the exception of MLB1 (Finkbeiner et al ., 2008), the astroviruses of humans, the host species that has been investigated most heavily, constitute a single spec ies with eight serotypes. In contrast, the evolutionary distance between the first three astroviruses found in California sea lions is comparable to that seen between recognized species within the Astroviridae. BDAstV1 and SSLAstV1 also appear to be disti nct from other astroviruses at a distance consistent with species differentiation. T he genetic distances between these novel viruses was generally slightly greater in the capsid region than in the polymerase region (Fig. 3 and 4) even though we examined t he capsid region which is expected to be most conserved. This is consistent with other studies. Capsids are typically under strong positive selective pressure from the host immune system, and one analysis found that most positively selected sites in astro viruses are present in the capsid (Van Hemert et al ., 2007). The phylogenetic topology determined in this study is largely in agreement with previous analyses of astrovirus phylogeny (Jonasee et al ., 2001)(Lukashov & Goudsmit, 2002), although we do not fi nd support for the findings of Chu et al (2008) (Chu et al.,

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35 2008) that bat astroviruses AFCD11 and AFCD57 are not monophyletic with the other chiropteran mamastroviruses. Of the six mamastrovirus species recognized by the ICTV, five are from hosts from o ne mammalian superorder, Laurasiatheria. The other superorder of placental mammals, Euarchontoglires, has only one host (humans) from which an astrovirus is recognized, human. The greater diversity of astroviruses within laurasiatherian hosts may imply a longer host virus relationship. Bats, from whom additional astroviruses have recently been described, are also members of Laurasiatheria. The astroviruses of bats mostly appear to form a distinct monophyletic group, unlike those of marine mammals, which are distributed across the tree of the known mamastroviruses. E vidence of bat astroviruses outside of the clade of the viruses found by Chu et al was recently published (Zhu et al ., 2009). Astroviruses are very stable in aquatic environments (Espinosa et al ., 2008). Surveys have found a Human astrovirus prevalence of up to 61% in some marine shellfish populations, which are good particle concentrators (Elamri et al ., 2006). The wide diversity seen in astroviruses that were identified from marine mammals implies that the marine environment may play a large role in astroviral ecology. Similarly, t he marine environment is central in the ecology of caliciviruses, a better studied group of small nonenveloped positive stranded RNA viruses (Smith et al ., 1998). An understanding of diverse astroviruses in wildlife may enable more appropriate epidemiologic responses to new astrovirus infections in humans. Our data suggest that a relatively recent recombination event may have occurred between a human and a marine mammal astrovirus isolate, resulting in CSLAstV3

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36 Recombination is common in other nonenveloped positive stranded RNA viruses such as picornaviruses and caliciviruses, and as a result, evolution of structural and nonstructural regions of the genome may appear semi independent (Simmonds, 2006). Previous studies have found evidence for recombination amongst Human astrovirus serotypes, as well as turkey astroviruses (PantinJackwood et al ., 2006)(Simmonds, 2006). The lineage of the region of CSLAstV3 after the area of strong homology with CSLAstV2 is not clear. This may represent a separate recombination event with an as yet unidentified clade of astroviruses. An analysis of the capsid region of a cheetah astrovirus found that it clustered relatively more closely with human astroviruses than was found in the analysis of the polymerase (Atkins et al ., 2009). If due to a recombination event, this is an apparent opposite event from that seen in CSLAstV3 which has a Human astrovirus like polymerase and a CSLAstV2 like capsid, at least in the 5 end of ORF2. The evidence for recombination in the Mink Astrovirus / CSLAstV1 / SSLAstV1 clade is less clear. Data from additional viruses in thi s clade would be needed to clarify whether a recombination event in these v iruses is probable. Recombination is an important mechanism for rapid evolution of a virus, allowing rapid acquisition of sequence that is less likely to be deleterious than random mutations. The most common mechanism of recombination among nonenveloped positive stranded RNA viruses is switching of the polymerase complex from copying one template to another (Jarvis et al ., 1992). Nonreplicative RNA recombination has also been shown in picornaviruses, but also requires unencapsulated RNA cotransfected into the same cell (Gmyl et al ., 1999). Therefore, a recombinant of a human astrovirus and a sea lion astrovirus implies that both viruses infected a sea lion, a human, or a

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37 third host species at the same point. Emerging disease is frequently associated with host switches. One recent metaanalysis of human diseases found that 816 of 1407 (58%) are zoonotic, and of human diseases, zoonotic diseases are significantly more likely to be emerging (Woolhouse & GowtageSequeria, 2005). Most recent emerging human diseases have been associated with host switches, including SARS, Hendra virus, Nipah virus, and AIDS. The aforementioned study also found that viral diseases were much more likely to be emerging, especially RNA viruses (Woolhouse & GowtageSequeria, 2005) The apparent ability of some of these viruses to infect disparate hosts suggests further study of the ecology and host range of astroviruses may be relevant to human health. We were unable to clarify t he clinical significance of astro viruses for marine mammals from our dataset Only one sea lion case ( CSLAstV3 from a stranded, freeranging pup) had clinical diarrhea. However, bacterial culture of the diarrheal sample yielded a Salmonella sp. culture. Given the clinical signs associated with other mama stroviruses, it would be reasonable to hypothesize that these viruses may cause a secretory diarrhea which is most likely to be clinically important in neonates and weanlings Further studies are underway to determine the clinical relevance of these virus es to their marine mammal hosts In conclusion, we have identified five novel astroviruses from marine mammal hosts. These viruses are diverse and all appear to be consistent with novel species. These viruses are situated across the mamastrovirus tree, and do not form a monophyletic group. There is evidence of recombination between human and marine

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38 mammal astroviruses. Further study of these viruses and their clinical significance in marine mammal populations are indicated.

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39 Table 2 1. Primers used to sequence California sea lion Astroviruses 1 3 ( CSLAstV1 to V 3), Steller sea lion astrovirus 1 ( SSLAstV1 ) and bottlenose dolphin astrovirus 1 ( BDAstV1 ) Primer 5 3 Sequence Primer Type Virus Isolate Astr4574R TGNCCWGAKGGRTTICC Degenerate CSLAstV1 Astr45 42F TRCCMWSNGGTGARRTCAC Degenerate BDAstV1 Astr4811F TTTGGNATGTGGGTNAARCC Degenerate BDAstV1 CSLAstro4805F AGAGTGGTGGATATGTATAAGG Specific CSLAstV1 & BDAstV1 CSLAstro5167R GCTGCTTGGTTGGCGTGAGCCAT Specific CSLAstV1 CSLAstro777F TGGTGGNTNACNRAYACHAATGA Specific CSLAstV1 CSLAstro1121R CNSCNKYRTYNAVNAYHTGCCA Specific CSLAstV1 CSLAstro4588F CAACCACCAGCAGCCCAGTGGGG Specific CSLAstV1 CSLAstro4934R GCGGAGTAGTTGGTGAACTCCCA Specific CSLAstV1 CSLAstro5067F TCCTTGAAATGACACTGCCGA Specific CSLAstV1 CSLAstro6227 R TGWWGRARKTKTCMSCYMAGGCA Specific CSLAstV1 CSLAstroGSP F GCGTCCTTGAAATGACACTGCCGAAGGA 3RACE CSLAstV1 CSL0686GSPseq1 TAAGCCTGACGGCACAACTCACT 3RACE CSLAstV1 ZcAstrV 1GSPR2 CACACCGTTCCCAGGCATCACAGA 5RACE CSLAstV1 TtAstroGSPF CCCCTTTGATCGGACCCTCAGCAATC A 3RACE BDAstV1 TtAstroGSPR GCCGGAGGCTTTAACCTCAACGCTAACA 5RACE BDAstV1 TtAstVGSPR3 TGGTCTGCTCTGTCACTTCACCCG 5RACE BDAstV1 ZcAstrV 2GSPF CGGCTCAAGCCAAGAGACCTCGCTGG 3RACE CSLAstV2 ZcAstrV 2GSPF2 CACCACCACCAACACCACACTTTCA 3RACE CSLAstV2 ZcAstrV 2GSP R GGGAGAACAACTTTACGGTGGACGAG 5RACE CSLAstV2 AFEFfor CTGCAAGCCTTCGAGTTTG Specific CSLAstV3 VEVKRev CCATTGGACTTGACCTCAACA Specific CSLAstV3 SSLF1 TGCATCCGTGCAAGACTCTA Specific SSLAstV1 SSLR1 TGAAGACTGGGAAAGGGTTG Specific SSLAstV1 SSLF2 GTCCACAGTCCGTTTC GTCT Specific SSLAstV1 SSLR2 AGTTGCAGACACACGGACAG Specific SSLAstV1

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40 A B Figure 2 1. Negative Staining Electron Microscopy Negative staining revealed clusters of viral particles were detected in fecal samples from three California sea lions ( Zalo phus californianus ), one Steller sea lion ( Eumetopias jubatus ) and one bottlenose dolphin ( Tursiops truncatus ). Individual capsids were 3035 nm wide. The nonenveloped icosahedral virus particles had distinct star like surface projections consistent with astroviruses. Only isolates CSLAstV1 (A) and BDAstV1 (B) are shown.

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41 Figure 22. HuAstV1 genome compared to marine mammal astrovirus genome segments. Diagram representing the alignment of the 3,174bp CSLAstV1 genome segment, 3,505bp CSLAstV2 genome segment, 1,340bp CSLAstV3 genome segment, 1,348bp SSLAstV1 genome segment and the 3,985bp BDAstV1 genome segment compared to the full length genome sequence of Human Astrovirus 1 (6697bp, GenBank accession # AY720892). The relative positions of the ORFs encoding the nonstructural and structural proteins are also represented. Partial gene segments of the polymerase and capsid genes used for phylogenetic analysis are indicated.

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42 Figure 23. Ba yesian phylogenetic tree of predicted 237254 amino acid partial astroviral RNAdependent RNA polymerase sequences based on MUSCLE alignment. Bayesian posterior probabilities of branchings as percentages are in bold, and ML bootstrap values for branchings based on 200 resamplings are given to the right. Avian nephrit is virus 1 (GenBank accession number NP_620617) was designated as the outgroup. Virus genera are delineated by brackets. Marine mammal astroviruses are bolded. Sequences retrieved from GenBank include Human astrovirus 1 (GenBank accession # AAW51881 ), Hu man astrovirus 3 ( AAD28539 ), Human astrovirus 4 ( AAY84778 ), Human astrovirus 5 ( AAY46273 ), Human astrovirus 8 ( AAF85963 ), Human astrovirus MLB1 ( YP002290967), Miniopterus magnater bat astrovirus WCF90 (ACF75856), Miniopterus magnater bat astrovirus AFCD57 (ACF75852), Miniopterus pusillus bat astrovirus AFCD337 (ACF75864), Miniopterus pusillus bat astrovirus WCF214 (ACF75862) Pipistrellus abramus bat astrovirus AFCD11 (ACF75853), Ovine astrovirus ( NP_059945), Mink astrovirus ( AAO32082), Turkey astrovirus 1 ( CAB95006 ), Turkey astrovirus 2 ( NP_987087), and Avian Nephritis Virus 1 ( NP_620617)

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43 Figure 24. Bayesian phylogenetic tree of predicted 186207 amino acid partial astroviral capsid sequences based on TCoffee alignment. Bayesian posterior probabilit ies of branchings as percentages are in bold, and ML bootstrap values for branchings based on 200 resamplings are given to the right. Avian nephritis virus 1 (GenBank accession number NP_620618) was designated as the outgroup. Virus genera are delineated by brackets. Marine mammal astroviruses are bolded. Sequences retrieved from GenBank include Human astrovirus 1 (GenBank accession # BAE97460), Human astrovirus 2 ( AAA62427 ), Human astrovirus 4 ( BAA93440 ), Human astrovirus 5 ( AAY46274 ), Human astrovirus 8 ( AAF85964), Human astrovirus MLB1 ( YP 002290968), Feline astrovirus ( AAC13556), Cheetah astrovirus 1 ( ACD13861 ), Porcine astrovirus ( CAB95000 ), Miniopterus pusillus bat astrovirus AFCD337 (ACF75865), Ovine astrovirus ( NP_059944), Mink astrovirus ( NP_795336 ), Turkey astrovirus 1 ( CAB95007 ), Turkey astrovirus 2 ( NP_987088), and Avian Nephritis Virus 1 ( NP_620618)

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44 Figure 25. Bootscanning analysis of MUSCLE alignment of astroviral sequences produced using the RDP3 suite. The thick line indicates bootstr ap support for monophyly of Human astrovirus 4 and CSLAstV3, with a 200bp window size and a step size of 20, with 200 bootstrap replicates. The dotted line indicates support for monophyly of CSLAstV2 and CSLAstV3. The second dotted line indicates bootstrap support for monophyly of Human astrovirus 4 and CSLAstV2. The grey area is the identified possible recombinant region. In addition to the marine mammal viruses found in this study, sequences used in the MUSCLE nucleotide alignment include Human astrovi rus 1 (GenBank accession # AY720892), Human astrovirus 3 ( AF141381), Human astrovirus 4 ( DQ070852), Human astrovirus 5 ( DQ028633), Human astrovirus MLB1 ( FJ222451), Mink astrovirus ( AY179509), Ovine astrovirus (NC_002469), and Miniopterus pusillus bat astr ovirus AFCD3 37 (EU847155).

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45 CHAPTER 3 USE OF A PEPTIDEBASED INDIRECT ENZYMELINKED IMMUNOSORBENT ASSAY ( ELISA) FOR THE DETECTION OF A HUMORAL IMMUNE RESPONSE TO BOTTLENOSE DOLPHIN ASTROVIRUS 1 Introduction Astroviruses are small round nonenveloped viruses with a positive stranded RNA genome. They were relatively recently discovered, and were first reported in 1975 (Madeley and Cosgrove, 1975). The family Astroviridae is divided into two genera, Avastrovirus found in avian hosts, and Mamastrovirus found in mammal hosts (Monroe et al., 2005). Human astrovirus is a significant cause of enteric disease in human children (Dennehy et al., 2001). We have recently reported the discovery of diverse astroviruses in marine mammals, including Bottlenose Dolphin As trovirus 1 (BDAstV1) (Rivera et al., 2010). As a prevalent enteric disease in children, seroconversion to H uman Astrovirus at a young age is typical. An early study of a human astrovirus in Oxford, England found that 7% of 6 to 12 month olds had a positiv e titer, whereas 75% of 5 to 10 year olds were positive (Kurtz and Lee, 1978). A study in London found that over 50% of children between 5 and 12 months of age had antibody responses to Human astrovirus 1 (HAstV1), and seroprevalence was 90% by the age of 5 (Kriston et al., 1996). In the Netherlands, age seroprevalence of 7 different human astrovirus serotypes was examined; seroprevalence of HAstV1 was 100% by 5 years of age, and although overall seroprevalence was lower for human astroviruses 24, the age of conversion was similar (Koopmans et al., 1998). However, Koopmans et al. found a later age of onset for HAst V 5 A study in Virginia found that t he seroprevalence of HAstV1 decreased from 67% in infants <3 months of age to 7% by 6 to 8 months of age, consistent with

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46 loss of transplacental antibodies, followed by a marked increase to 94% at 6 to 9 years of age ( Mitchell et al., 1999). Mitchell et al. found a similar age distribution but lower seroprevalence for HAstV3. A study in Japan found that by age 3, seropositivity to HAstV1 and HAstV3 approached 100% (Kobayashi et al., 1999). Attempts at culture of BDAstV1 were unsuccessful, so alternate means of obtaining antigen for serological testing were needed. The capsid (ORF2) contains the characterized neutralizing antibody epitopes in astroviruses ( Snchez Fauquier et al., 1994, Bass and Upadhyayula, 1997). There are experimental data from human astroviruses suggesting that protease cleavage cuts the capsid protein into an early section forming the m ain capsid (called VP32 or VP34, VP38.5, or VP41 at different stages of processing by different investigators) and a later section forming the projections (known as VP25, VP26, VP27, VP28 or VP29 at different stages of processing by different investigator s). Trypsin cleavage is important in processing of the projections ( Mndez et al., 2002). Caspase cleavage of the carboxy terminal part of the protein is probably involved with release of the virus from the cell ( Mndez et al., 2004), and conserved caspas e sites suggest this is also likely for other astroviruses. This region is likely cleaved off before exit from the cell, and is not likely to be a target for antibodies. The projections are likely to be external and most exposed to antibodies. Matsui et al. (1993) found that a clone of the region immediately carboxy terminal to the trypsin cleavage site is recognized by antibody es from infected individuals. Synthetic oligopeptides have been used as antigens in successful immunoassays to detect antibodies in the host (Kwang and Torres, 1994, Aizaki et al., 1995, Nilsen et

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47 al., 2003). We hypothesized that as similar approach may work with marine mammal astroviruses. In this study, the utility of a synthetic oligotide ELISA assay for detecting anti BDAstV1 antibodies was assessed. Materials and Methods Animals and Sera Serum samples were collected from 61 bottlenose dolphins from a managed openwater collection, 31 animals from three different captive closedwater collections, and 64 wild dolphins. Multipl e time points were taken from 7 dolphins from the managed openwater collection. B lood samples were collected from the ventral tail vein, the ventral fluke vein, or if taken at post mortem examination, via cardiocentesis Samples were typically collected using a 20 or 21 gauge 1.5 inch Vacutainer needle (Becton Dickinson VACUTAINER Systems, Rutherford, New Jersey 07070) and blood was collected into a Vacutainer serum separator tube or a Vacutainer without anticoagulant. The blood samples were centrifuged at 3,000 r evolutions per minute (rpm) at 21C for 10 minutes. Fibrin clots were removed and serum was transferred to cryovials. Archived sera were stored at 80C. Astrovirus Peptide Design Peptides were selected from the predicted capsid protein of B DAstV1. BDAstV1 was aligned with Human Astrovirus 1 using MUSCLE (Edgar, 2004) t o determine homologous conserved regions (figure 31) P ossible antigenic peptides within likely antigenic regions were designed using an antigenic site prediction program ( ht tp://immunax.dfci.harvard.edu/Tools/antigenic.html ) based on the methods of Kolaskar and Tongaonkar (1990), as well as hydrophilicity analysis using ProtScale (http://expasy.org/tools/protscale.html) based on the methods of Kyte and Doolittle

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48 (1982). The s elected peptides were Tt 322 ( KASEVIVQVVDA), Tt399 ( TLAWQQMNKPN), Tt455 ( ALAPYWQSLELW ), and Tt 616 ( DMVLLISWV). T wo of these peptides (Tt 399 and Tt455) wer e targeted in the area homologous to the antigenic region identified by Matsui et al. (1993) Peptides were synthesized using 9fluorenyl methoxycarbonyl (FMOC) chemistry and analyzed through HPLC and mass s pectrophotometry at the University of Florida Interdisciplinary Center for Biotechnology Research. Crude peptides were lyophilized. Peptides were recons tituted in water to 1mg/ml and stored at 80C To improve solubility, Tt 455 was reconstituted with 5% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO) and Tt 616 was reconstituted with 3% DMSO. Monoclonal Antibody The biotinylated monoclonal antibody (mAb ) HL1 912, chain of T. truncatus IgG was used as a secondary antibody The derivation, evaluation, and validation of HL1912 specificity for T. truncatus IgG has been described in detail (Nollens et al., 2007). Optimization of ELISA P arameters The positive serum sample used was from the index dolphin case 13 days after fecal shedding was detected (28 February 2007). N egative control serum samples were collected from the index animal one week prior to fecal virus shedding (8 February 2007) as well as an immunologically naive neonate bottlenose dolphin that had not yet nursed, and noserum negative controls were used to optimize the assay signal. All assay parameters were varied (working volume: 50 and 100 l; peptide concentration: 0.3, 0.5, 1, 5, and 10 g/ml ; serum dilution: 1:50, 1:100, 1:250, and 1:500; HL1 912 mAb concentration: 5 g/ml and 7.5 g/ml; streptavidinalkaline phosphatase ( AP) dilution:

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49 1:500 and 1:1,000; developing time: 15, 30, 45, and 60 min), and the assay condi tions with the highest ratio of optical density at 405 nanometers (OD405) of the sample to OD405 of the negative control were chosen. Peptide Indirect ELISA Serum samples from 146 dolphins were assayed with the ELISA using peptide antigens. Wells of high protein binding microplate (Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) were coated with 50 l of a mixture of peptides Tt 322, Tt 399, Tt 455, and Tt 616 in PBS, each at 1 g/ml and left to adsorb overnight at 4C. After this and each subsequent step, all wells were washed three times with phosphate buffered saline ( PBS) with 0.05% Tween 20 using an automated ELx405 microplate washer (Biotek Instruments, Winooski, VT) Each subsequent step of the ELISA was incubated with gentle agitation (Nutator; Adam s, Fisher Scientific, Pittsburgh, PA) for 1 hr at room temperature. After washing, all wells were blocked with 300 l of Superblock blocking buffer (Pierce, Rockford, IL) in PBS and incubated D olphin sera were applied in triplicate (1: 50 in 1% bovine s erum albumin [BSA] in PBS) and incubated A no serum negative control and one positive control serum were included on each plate. B iotinylated HL1912 monoclonal antibody (mAb) was added at a concentration of 5 g/ml (in 1% BSA in PBS) as the reagent for t he detection of bound antibodies and incubated. StreptavidinAP was used as the secondary detection reagent (1: 5 00 in 1% BSA) and incubated Finally, 1.0 mg.ml1 P Nitrophenyl Phosphate (PNPP; Sigma, St. Louis, MO) substrate was added and the absorbance a t 405 nM (OD405) was recorded after 60 minutes using a Synergy HT m icroplate r eader (BioTek, Winooski, VT) Tri plicate OD405 readings for each sample were averaged. For analysis, the average

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50 OD405 of the 1% BSA negative control was subtracted from the average OD405 of all other samples. Initial Validation Correlation of ELISA results for 46 bottlenose dolphin serum samples using peptides Tt 322 and Tt 616 as antigen versus ELISA results for the same data set using peptides Tt 399 and Tt 455 as antigen was ex amined. The samples were run as described above and the OD405 values from the Tt322/Tt 616 ELISA and Tt 399/Tt 455 ELISA were compared by Spearmans nonparametric correlation test using inStat (GraphPad Software, San Diego, CA). Further V alidation Due to rel atively low OD405 values and potential concerns about sufficiency of these small peptides as antigens, further validation of the peptide ELISA was pursued. As controls, the amino acid composition was maintained to keep hydrophobicity/mass the same, but the amino acid order was scrambled to remove conformational recognition. The control peptides were: Tt 322scr ( VVEVVSKAIQAD), Tt399scr ( KNNMQALTPQW ), Tt455scr ( LSLWPYLWAAEQ ), and Tt616scr ( LVWSILDMV). Peptides were synthesized using FMOC chemistry and analyz ed through HPLC and mass spectrophotometry at the University of Florida Interdisciplinary Center for Biotechnology Research. Crude peptides were lyophilized. Peptides were reconstituted in water to 1mg/ml and stored at 80C For consistency, Tt 455scr wa s reconstituted with 5% DMSO to improve solubility as Tt 455 had been, and Tt 616scr was reconstituted with 3% DMSO as Tt 616 had been. Correlation of ELISA results for the same 46 bottlenose dolphin serum samples used in the initial validation was examined, using peptides Tt 322 and Tt 616 as antigen

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51 versus ELISA results for the same data set using peptides Tt 399 and Tt 455 as antigen was examined. The samples were run as described above. The OD405 values from the Tt 399/Tt 455 ELISA vs. Tt 399scr/Tt 455scr i ELISA, and the Tt322scr/Tt 616 scr ELISA vs. Tt 399/Tt 455 ELISA were compared by Spearmans nonparametric correlation test using inStat. Results Initial Validation OD405 values for the 46 samples using the Tt322/Tt 616 ELISA and Tt 399/Tt 455 ELISA are displayed in blue on figure 32 and in Appendix C. The r value for the Spearmans nonparametric correlation of the Tt322/Tt 616 ELISA and Tt 399/Tt 455 ELISA was 0.6537, with a 95% confidence interval of 0.4414 to 0.7967. The twotailed P value was <0.0001, indicating that these values correlate strongly. This correlation is expected if antigenic response to these two pairs of peptides represent immune responses to the same agent. Optimization of ELISA P arameters The highest ratio of the OD405 of sample to negative c ontrol was seen with a working volume of 50 l; peptide concentration of 1 g/ml; serum dilution of 1:50; HL1 912 mAb concentration of 5 g/ml, a streptavidinAP dilution of 1:500, and a developing time of 60 min. Peptide Indirect ELISA Results of the pepti de indirect ELISA on 146 dolphin sera are presented in Appendix D. Time series were run on 7 selected dolphins. Dolphins were selected because they had multiple time points available.

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52 Further V alidation OD405 values for the 46 samples using the Tt 399scr/T t 455 scr ELISA are displayed in Appendix C. The corresponding OD405 values between the ELISA assay using peptides Tt399scr/Tt455scr and the ELISA assay using peptides Tt399/Tt455 are shown in red on figure 32. The corresponding OD405 values between the ELI SA assay using peptides Tt322scr/Tt616scr and the ELISA assay using peptides Tt399/Tt455 are shown in yellow on figure 32. The r value for the Spearmans nonparametric correlation of the Tt 399 /Tt 455 ELISA and Tt399scr/Tt455scr ELISA was 0.4815, with a 95% confidence interval of 0.2138 to 0.6820. The twotailed P value was 0.0007, indicating that these values correlate strongly. The r value for the Spearmans nonparametric correlation of the Tt 3 22/Tt616 ELISA and Tt322scr/Tt616scr ELISA was 0.7431, with a 95% confidence interval of 0.5713 to 0.8525. The twotailed P value was < 0.0001, indicating that these values correlate strongly. The r value for the Spearmans nonparametric correlation of the Tt 3 22/Tt616 ELISA and Tt399scr/Tt455scr ELISA was 0.6289, w ith a 95% confidence interval of 0.4068 to 0.7808. The twotailed P value was < 0.0001, indicating that these values correlate strongly. The r value for the Spearmans nonparametric correlation of the Tt399/Tt455 ELISA and Tt322scr/Tt616scr ELISA was 0.5371, with a 95% confidence interval of 0.2842 to 0.7201. The twotailed P value was 0.0001, indicating that these values correlate strongly. Discussion The relatively low OD405 values seen in this assay raised doubts about assay validity. The correlation of the results of the scrambled peptide assays with the specific peptide assays is expected if antigenic response is nonspecific. One plausible explanation for this is that we failed to identify suitable antigenic epitopes for the virus.

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53 There is little comparative structural data for astroviruses, and none exists for BDAstV1. The studies on antibody epitopes of astroviruses are very limited, and while two of the peptides designed were within a region homologous to a 93 amino acid segment shown to be an antibody epitope (Matsui et al., 1993) the antigenic portion may have been missed, or the BD dolphin may not have homologous epitopes. Alternatively, the short oligopeptides may not have developed the appropriate conformation that enabled antibody recognition of the longer native BDAstV1 capsid protein, as has been seen with other oligopeptide ELISA assays (Plagemann, 2001). In conclusion, this oligopeptide ELISA assay does not appear to be useful for detecting humoral immune response of bottlenose dolph ins to BDAstV1.

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54 HumAst1_BA MASKSNKQVT VEVSNNGRNR SKSRARSQSR GRDKSVKITV NSKNR--TRR TtAstVcaps MANDRSKDVS VEVKASGSQR SKSRSRSRSR GRTPAVKVTV NSKAKRFTRR QSGRGKHQSS QRVRNIVNKQ LRKQGVTGPK PAICQRATAT LGTVGSNTSG TTEIEACILL PSGRSFRAKN NSVKQQVRNQ LKKQGLTGPA PAVVQTATAT LGTIGPNTGN DAEREISFYL NPVLVKDATG STQFGPVQAL GAQYSMWKLK YLNVKLTSMV GASAVNGTVL RVSLNPTSTP NPALTKENTG SNAFGPVQAL AAQYSMWRCS RAEIRFTPLI GPSAISGTAY RCSLNMAGTP SSTSWSGLGA RKHLDVTVGK NATFNLKPSD LGGPRDGWWL TNTKDNASDT LGPSMEIHTL SQTSWSGLGS RKHKDMHIGK SGSFKLTKKE LSGPKETWWL TNTNEEGGQT LGPAVEIHSI GRTMSSYEN--EQFTGGLF LVELASEWCF TGYAANPNLV NLVKSTDKQV SVTFEGSAGS GKTVRVFTSQ TGQTYDGPVF LVELRATWEF ANFSANPGLV ALEKGEDTA RINFSGNIGE PLIMNVPEGS HFARTVLARS TTPTTLARAG ERTTSDTVWQ VLNTAVSAAE LVTPPPFNWL PLVMKVTGSS DFHARMMRVM GDDATYTRTG EI KASEVIVQ VVDA GTDIIS STVPGFGWL VKGGWWFVKL IAGRTR TGTR SFYVYPSYQD ALSNKPALCT GSTPGGMRTR NPVTTTLQFT IKAGWFFIKK LAGLSR NGDG EYAVYASYAD AQNNRPCI---LPSTVTDV TPKPT TLAWQ QMNQPSLGHG EAPAAFGRSI PTPGEEFKV ILTFG-------------APMSPNANNK QMNKPN LGLE TGSYAMSRSM PVPVEGSYKA ILQLDNYAQM IHQLQADYPR P ALAPYWQSL QNWVNKPLDA PSGHYNVKIA K--DVDHYLT MQGFTSIASV DWYTTDFQPS EAPAPIQGLQ ELW VGKGNDY GGGQHADRIT QVYKVNRGVF LNQF-----------YDQV MQPEPTLGYS VLVNSS--KK ADVYAVKQFV TAQTNNKHQV T------TLF LVKVTTGFQV NNY--LSYF IFSNTSHGKI GEVLGFQSYH MPGPTESGAE SLGPVAFNVY LGRITLSSKW SVQYKDTAYF YRASASGDAT TNLLVRGDTY TAG---ISFT Q-----------------VGWYLLTNTS PRASDEWGKS AAVMMKDWY GLGPPKPSYT TDGNGVVKPP RFDPD D MVLL ISWV RFSEGR IVDGALPPG WIWNNVELKT NTA-------------------YHMDKG LIHLIMPLPE NTD--LPVDK WCNTAMDYTY NVTVSGRKLA RDGQVRVPAG VPYWYYQDVQ TINGSDPVVQ STQMCYEMLT SIPRSRAAGY GYE----SDN TEYLDAPDF ADQLREDIET DTHIETTEDE NRESIFEFET EVPVQRSALL SLKKSAPSRA VKYDEEEEVY YTTLPKQGPP TAPWRMVEDD DDEADRF-------DIIDT SDEEDE NETD RVTLLSTLVN QGMTMTRATR IARRAFPTLT DHDSDSSYWD NDMSDDDFES SEEEELQDVD VDVLANTLEN SGFTRKEA----------DRIKRGVYMD LLVSGASPNS AWSHACEEAR KAAGEINPCT SGSRGHAE ------------------R AYAQAARDAV LKDGPTAKTV KFSDAPQE Figure 31 MUSCL E alignment of Human Astrovirus 1 and BDAstV1 capsid amino acid sequences Yellow = potential RNA binding domain (inside of capsid, likely not antigenic) Green = Casp ase cleavage motif Red = potential trypsin cleavage site, separating VP34 analogue, Ita l ics = antigenic region cloned by Matsui et al.(1993), Bold = selected peptides

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55 Figure 32. Correlation of OD405 values between different pairs of peptides. Blue dots represent corresponding OD405 values between ELISA assay using peptides Tt322/Tt616 and ELISA assay using peptides Tt399/Tt455. Red dots represent corresponding OD405 values between the ELISA assay using peptides Tt399scr/Tt455scr and the ELISA assay using peptides Tt399/Tt455. Yellow dots represent corresponding OD405 values between the ELISA assay using peptides Tt322scr/Tt616scr and the ELISA assay using peptides Tt399/Tt455.

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56 CHAPTER 4 USE OF A RECOMBINANT CAPSID PROTEININDIRECT ELISA FOR THE DETECTION OF A HUMORAL IMMUNE RESPONSE TO BOTTLENOSE DOLPHIN ASTROVIRUS 1 Introduction Ast roviruses are small round nonenveloped viruses with a positive stranded RNA genome. They were relatively recently discovered, and were first reported in 1975 (Madeley and Cosgrove, 1975). The family Astroviridae is divided into two genera, Avastrovirus found in avian hosts, and Mamastrovirus found in mammal hosts (Monroe et al., 2005). Human astrovirus is a significant cause of enteric disease in human children (Dennehy et al., 2001). We have recently reported the discovery of diverse astroviruses in marine mammals, including Bottlenose Dolphin Astrovirus 1 (BDAstV1) (Rivera et al., 2010). As a prevalent enteric disease in children, seroconversion to H uman Astrovirus at a young age is typical. An early study of a human astrovirus in Oxford, England found that 7% of 6 to 12 month olds had a positive titer, whereas 75% of 5 to 10 year olds were positive (Kurtz and Lee, 1978). A study in London found that over 50% of children between 5 and 12 months of age had antibody responses to Human astrovirus 1 (HAst V1), and seroprevalence was 90% by the age of 5 (Kriston et al., 1996). In the Netherlands, age seroprevalence of 7 different human astrovirus serotypes was examined; seroprevalence of HAstV1 was 100% by 5 years of age, and although overall seroprevalence was lower for human astroviruses 24, the age of conversion was similar (Koopmans et al., 1998). However, Koopmans et al.found a later age of onset for HAst V 5 A study in Virginia found that t he seroprevalence of HAstV1 decreased from 67% in infants <3 months of age to 7% by 6 to 8 months of age, consistent with

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57 loss of transplacental antibodies, followed by a marked increase to 94% at 6 to 9 years of age ( Mitchell et al., 1999). Mitchell et al. found a similar age distribution but lower seroprevalence for HAstV3. A study in Japan found that by age 3, seropositivity to HAstV1 and HAstV3 approached 100% (Kobayashi et al., 1999). Attempts at culture of BDAstV1 were unsuccessful, so alternate means of obtaining antigen for serological testing were needed. The capsid (ORF2) contains the characterized neutralizing antibody epitopes in astroviruses. There are experimental data from human astroviruses suggesting that protease cleavage cuts the capsid protein into an early section forming the main capsid (called VP32 or VP34, VP38.5, or VP41 at different stages of processing by different investigators, hereafter called main capsid) and a later section forming the projections (known as VP25, VP26, VP27, VP28 or VP29 at different stages of processing by different investigators, hereafter called arm), giving the star shape. Trypsin cleavage is important in processing of the arm ( Mndez et al., 2002). Casp ase cleavage of the carboxy terminal part of the protein is probably involved with release of the virus from the cell ( Mndez et al., 2004), and conserved caspase sites suggest this is also likely for other astroviruses. This region is likely cleaved off before exit from the cell, and is not likely to be a target for antibodies. The arm projections are likely to be external and most exposed to antibodies. Matsui et al. (1993) found that a clone of the region early after the trypsin cleavage site is recognized by antibodies from infected individuals. In two different studies, the VP26 proteins of HAstV1 and HAst V2 have been found to have neutralizing epitopes ( Snchez Fauquier et al., 1994, Bass and Upadhyayula, 1997). The BDAstV1 arm region is therefore a good candidate antigen for serodiagnostics.

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58 Materials and Methods Animals and S era Serum samples were taken from 60 bottlenose dolphins from a managed openwater collection in California, 35 animals from three different captive closedwater collections, and 67 wild dolphins from Florida (60 healthy captures and 7 strandings). Multiple time points were taken from 7 dolphins from the managed openwater collect ion (177 total samples), and multiple time points were examined from an additional 10 calves from the openwater collection (55 total samples). B lood samples were collected from the ventral tail vein, the ventral fluke vein, or if taken at post mortem examination, via cardiocentesis Samples were typically collected using a 20 or 21 gauge 1.5 inch Vacutainer needle (Becton Dickinson VACUTAINER Systems, Rutherford, New Jersey 07070) and blood was collected into a Vacutainer serum separator tube or a Vacu tainer without anticoagulant. The blood samples were centrifuged at 3,000 rpm at 21C for 10 minutes. Fibrin clots were removed and serum was transferred to cryovials. Archived sera were stored at 80C. BDAstV1 Antigen The antigen for expression was selected from the predicted capsid protein of BDAstV1. Primers TtAst1spkF1 (ACTGTCCCAGGATTTGGATG) and TtAst1spkR1 (CCACATCCTGCAGTTCCTCT) were designed to amplify the region of BDAstV1 expected to form the outer projections, between the predicted trypsin cleavage site and the predicted caspase cleavage site ( Figure 4 1). A 20 L reaction w as used that included 1L eac h of forward and reverse primers at 20M, 2 L 10x Buffer, 0.8 l 50mM MgCl2, 0.4 l 10mM dNTP, 0.5 units polymerase (Platinum Taq DNA polymeras e, Invitrogen, Carlsbad, CA, U nited States of America [U SA] ), 11.7 L sterile

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59 water, and 3 l of complementary deoxyribonucleic acid (cDNA) template from 3RACE amplifications of BDAstV1 as template (Rivera et al., 2010). The mixture was amplified in a thermal cycler (PCR Sprint, Thermo Hybaid Franklin, MA ) with an initial denaturation step at 9 5 C for 5 minutes, followed by 45 cycles of denaturation at 95 C for 30 s seconds annealing at 54C for 30 s econds and extension at 72C for 90 seconds, and a final extension at 72C for 10 min. The PCR product was resolved in 1% agarose. The band was excised and purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) To confirm product identity, Sanger sequencing was performed directly on an ali quot using the BigDye Terminator Kit (Applied Biosystems, Foster City, CA) and analyzed on an A BI 3130 automated DNA sequencer at the University of Florida Interdisciplinary Center for Biotechnology Research Sequencing Facilities to confirm identity The PCR product was ligated into pDrive plasmid (Qiagen) and cloned into EZ Competent Cells (Qiagen). Individual clones were selected and sequenced as above to confirm identity. The protein was expressed and purified by Genscript Corp., Piscataway New Jerse y, USA. Briefly, the insert had sequence encoding a polyhistidine tag (predicted amino acid sequence MGSSHHHHHHSSGLVPRGSHM ) added to the 5 end and was then cloned into an E. coli expression vector. The full amino acid sequence is given in figure 42. The protein was then expressed and purified. The protein was then run on an SDS polyacrylamide gel and stained with Coomassie blue to assess purification (Figure 43), and further confirmed with western blotting using an anti polyhistidinetag antibody (Fi gure 44). Monoclonal Antibody The biotinylated monoclonal antibody (mAb) HL1 912, chain of T. truncatus IgG was used as a secondary antibody The derivation,

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60 evaluation, and validation of HL1912 specificity for T. truncatus IgG has been described in detail (Nollens et al., 2007). Optimization of ELISA P arameters The positive serum sample used was from the index dolphin case (see chapter 2) 13 days after fecal shedding was detected (28 February 2007). N egative control serum samples were collected from the index animal one week prior to fecal virus shedding (8 F ebruary 2007) as well as an immunologically naive neonate bottlenose dolphin that had not yet nursed, and noserum negative controls were used to optimize the assay signal. Assay parameters were varied (protein concentration: 0.1, 0.25, 0.5, 1, 5 and 10 g/ml; developing time: 10, 15, 20, 30, 45, and 60 min), and the assay conditions with the highest ratio of the OD405 of sample to negative control were chosen. Recombinant Protein ELISA Serum samples from 146 dolphins were assayed with the recombinant protein ELISA. Wells of high protein binding microplates (Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) we re coated with 50 l of the cloned protein antigen at 1 g/ml in PBS and left to adsorb overnight at 4C. After this and each subsequent step, all wells were washed three times with PBS with 0.05% Tween20 using an automated ELx405 microplate washer (Biotek Instruments, Winooski, VT) Each subsequent step of the ELISA was incubated with gentle agitation (Nutator; Adams, Fisher Scientific, Pittsburgh, PA) for 1 hr at room temperature. After washing, all wells were blocked with 300 l of Superblock blocking buffer (Pierce, Rockford, IL) in phosphate buffered saline ( PBS) and incubated D olphin sera were applied in triplicate (1: 50 in 1% BSA in PBS) a nd incubated A no serum negative control and one positive control serum were included on each plate. B iotinylated HL1912 mAb was added at a concentration of 5

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61 g/ml (in 1% BSA in PBS) as the reagent for the detection of bound antibodies and incubated. S treptavidinAP was used as the secondary detection reagent (1: 5 00 in 1% BSA) and incubated Finally, 1.0 mg.ml1 P Nitrophenyl Phosphate (PNPP; Sigma, St. Louis, MO) substrate was added and the absorbance at 405 nM (OD405) was recorded after 15 minutes usi ng a Synergy HT m icroplate r eader (BioTek, Winooski, VT) Tri plicate OD405 readings for each sample were averaged. For analysis, the average OD405 of the 1% BSA negative control was subtracted from the average OD405 of all other samples. Comparisons between populations were analyzed using inStat (GraphPad Software, San Diego, CA). For statistical comparison of populations, average values were used for animals with more than one time point. Correlation with age was analyzed using SAS ( SAS Institute Inc., Cary, NC) Results BDAstV1 Antigen Of four PCR amplicon clones selected that were sequenced, one was 100% identical with the reference sequence and was selected for expression. The other three had 1 or 2 nucleotide substitutions resulting in 0 to 2 predi cted amino acid changes. No substitutions were shared by any of the clones. The predicted molecular mass of the protein was 45176.7 daltons, and the purified expressed protein was consistent with this (Figures 4 3 and 44). Optimization of ELISA P arameters The highest ratio of the optical density at 405nm (OD405) of sample to negative control was seen with a protein concentration of 1 g/ml. A developing time of 15 minutes was chosen because of the high ratio of OD405 of sample to negative control

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62 and overloading of the limits of reader detection for some samples at longer times. At these parameters, the post shedding index case OD405 was 4.8 times that of the preshedding index case OD405, and 114.5 times that of the prenursing neonatal dolphin. Recombi nant Protein ELISA The values of subpopulations (67 wild dolphins, 60 mature animals from the openwater managed collection, 10 calves from the openwater managed collection, and 35 animals from three different captive closedwater collections) were analyz ed for normality using the Kolmogorov Smirnov test ( Dallal and Wilkinson, 1986). All populations except calves were found to have nonGaussian distributions. The mean, median, range, and quartiles of OD405 values of the groups of animals are given in table 4 1. The Kruskal Wallis test with Dunns post test was used to compare groups. The calves (median value 0.39) were significantly lower than all other groups, with P<0.001. No differences were identified (defined by P>0.05) between wild animals (median value 1.27) and openwater mature animals (median value 1.05). The values from calves (median value 0.38) were significantly lower than openwater mature animals (P<0.01 ), wild animals ( P<0.0 01), and closed system animals (median value 0.94, P<0.05). The values from closed system animals were also lower than those from wild dolphins (P<0.05). Of wild animals examined, the only two animals with values less than 0.5 were both calves. Time series of mature openwater collection animals and of calves are gi ven in figures 45 and 4 6 Animals 1 and 20 show significant increases in titer at the same time in Winter 2007. There were no significant linear associations with age in mature animals (R2=0.10). Amongst calves, t here was a weak linear association with increasing age (R2=0.28, P < .0001) (Figure 47) Animal 168 also shows a significant

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63 increase in titer in Winter 2007.There were several individual calves with strong associations between age and BDAstV1 antibody levels (Figure 48 ). Discussion Our resu lts suggest that, similar to HAstV1 in humans, BDAstV1 exposure in bottlenose dolphins is near ubiquitous and cosmopolitan. Wild dolphins from the Atlantic oceans and openwater collection animals from the Pacific had significantly higher titers than calv es. Titers in closedwater collection animals were lower than wild animals but still significantly higher titers than calves, despite smaller populations in which to maintain virus and the use of ozone and UV sterilization to maintain excellent water qual ity. Astroviruses are very stable in aquatic environments (Espinosa et al., 2008). Surveys have found a Human astrovirus prevalence of up to 61% in some marine shellfish populations, which are good particle concentrators (Elamri et al., 2006). W ide diver sity has been seen in marine mammal astroviruses implying that the marine environment may play a large role in astroviral ecology (Rivera et al., 2010), and the finding of high prevalence further supports that. The marine environment is central in the ec ology of caliciviruses, a better studied group of small nonenveloped positive stranded RNA viruses (Smith et al., 1998). Clinical diagnosis of astroviral disease is challenging. Astrovirus capsid protein interacts with apical enterocyte membranes, increas ing permeability independent of viral replication. Much like Vibrio cholerae, astroviruses cause a secretory diarrhea without much of a histologic footprint on enterocytes on light microscopy ( Koci et al., 2003, Moser et al., 2007, Nighot et al., 2010). As trovirus culture is also challenging, with few cell lines capable of supporting Human astrovirus and a high requirement for trypsin (Taylor et al., 1997). Attempts at culture of marine mammal astroviruses to date have

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64 been unsuccessful. Astroviruses resemble other small round viruses somewhat morphologically, and a significant rate of misidentification using negativestaining electron microscopy of feces has been reported (Oliver and Phillips, 1988). N egativestaining electron microscopy of feces for viru s detection has also been shown to be comparatively insensitive (van Niewstadt et al., 1988). Histopathology culture, and negativestaining electron microscopy are theref ore insensitive test s for diagnosis of a stroviral diarrhea. More sensitive techniques such as PCR are not yet widely applied to marine mammals, leading to probable underdiagnosis of astrovirus infections. Clinical assessment of diarrhea in dolphins is also challenging. Dolphins normally have poorly formed stool that is excreted in water making it is difficult to observe and to assess. A secretory diarrhea would not be observed as reliably as it would in a terrestrial animal. Previous immune exposure may have significant effects on astroviral disease. Human astrovirus is a neonatal dis ease and has an even lower age of median infection amongst hospitalized children than rotavirus (Dennehy et al., 2001). One study found that 41 of 603 young children with diarrhea (6.8%) had human astrovirus in their feces as determined by antigen ELISA, whereas none of 141 control children did (Dennehy et al., 2001). In a study of nave turkey poults, 100% of naive turkey poults inoculated with Turkey astrovirus 2 animals exhibited diarrhea by 3 days post infection. However, experimental infections in v olunteer human adults found that only one of eight who ingested Human astrovirus developed diarrhea and vomiting, with a second volunteer developing abdominal discomfort (Kurtz et al., 1979) A second experiment in the same report found that five of nine volunteers ingesting fecal filtrate from the earlier volunteer

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65 developed abdominal discomfort and an increase in antibody titer. Volunteers with no preexisting anti Human astrovirus antibodies developed more severe symptoms and were more likely to shed virus (Kurtz et al., 1979). Another study found that none of 17 adult volunteers ingesting a smaller amount of HAstV5 developed clinical signs, and one of 2 volunteers ingesting a larger amount developed vomiting and diarrhea (Midthun et al., 1993). The volunteer developing clinical signs did not have a detectable antibody titer prior to the experiment and had a very high titer following the experiment. Eight of the 19 volunteers showed a fourfold or greater increase in antibody titer. The lower rate of dis ease in adults combined with the very high seroprevalence in age groups beyond early childhood suggest that acquired immune responses are a significant complicating factor in deciphering the clinical significance of astrovirus infections. While several of the calves showed strong associations between age and BDAstV1 antibody levels, two of the calves (170 and 171) showed an initial decrease in antibody followed by an increase. This may represent loss of maternal antibodies followed by exposure, as has been seen with HAstV1 in humans by Mitchell et al.(1999) Examination of the mature animal time series shows that the trends of two of the animals (9 and 31) largely parallel each other, although these animals were housed on different piers. Dolphin 55 shows a moderate titer followed by a persistent elevation for over two years, from December 2005 until April 2008. This elevation coincides temporally with viral shedding by this animal as detected by qPCR (see BDAstV1 qPCR chapter), suggesting persistent infection in this animal. This contrasts with the briefer

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66 elevation seen in animal 1. Persistent infection has been seen with astrovirus infection in humans; persistent gastroenteritis was seen with 8.5% of astrovirus infections in children in Spain (Caballero et al., 2003). A study of children in Bangladesh found that Human astrovirus was more commonly associated with persistent diarrhea than acute diarrhea (Unicomb et al., 1998). Animal 55 may have served as a source of infection for other animals. Animal 1 showed a marked increase in titer between 15 February 2007 and 20 February 2007. Animal 20 showed a marked increase in titer between 25 October 2006 and 23 Mar ch 2007. Calf 168 showed a marked increase in titer between 3 January 2007 and 16 March 2007. All three show marked increases in antibody titer in late winter of 2007, indicating a possible common source of infection. Animal 55 was moved into closer contact with animal 20 and calf 168 in late December 2006. Identification of persistently shedding animals may enable management to keep them separate from young calves, where the risk of disease is likely most significant. In conclusion, this assay is useful for determining humoral immune response of bottlenose dolphins to BDAstV1. Prevalence of expos ure appears to be very high and is geographically widespread, similar to Human astrovirus in humans. Both transient and persistent infections with BDAstV1 appear to occur in dolphins. Based on the observed parallels to human astrovirus infection, BDAstV1 is more likely to be clinically significant in calves than other age groups, and this is where management and future investigations should focus.

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67 Table 4 1. Summary of OD405 control values for groups of dolphins Mature openwater collection Calves openwater collection Wild Closed water collection Mean 1.19 0.48 1.43 1.03 Median 1.05 0.38 1.27 0.94 Range 0.26 2.82 0.15 1.05 0.07 2.94 0.41 2.26 1st quartile 0.7425 0.29 0.995 0.685 3rd quartile 1.4975 0.6475 1.735 1.18

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68 HumAst1_BA MASKSNKQVT VEVSNNGRNR SKSRARSQSR GRDKSVKITV NSKNR--TRR BDAstV1cap MANDRSKDVS VEVKASGSQR SKSRSRSRSR GRTPAVKVTV NSKAKRFTRR QSGRGKHQSS QRVRNIVNKQ LRKQGVTGPK PAICQRATAT LGTVGSNTSG TTEIEACILL PSGRSFRAKN NSVKQQVRNQ LKKQGLTGPA PAVVQTATAT LGTIGPNTGN DAEREISFYL NPVLVKDATG STQFGPVQAL GAQYSMWKLK YLNVKLTSMV GASAVNGTVL RVSLNPTSTP NPALTKENTG SNAFGPVQAL AAQYSMWRCS RAEIRFTPLI GPSAISGTAY RCSLNMAGTP SSTSWSGLGA RKHLDVTVGK NATFNLKPSD LGGPRDGWWL TNTKDNASDT LGPSMEIHTL SQTSWSGLGS RKHKDMHIGK SGSFKLTKKE LSGPKETWWL TNTNEEGGQT LGPAVEIHSI GRTMSSYEN--EQFTGGLF LVELASEWCF TGYAANPNLV NLVKSTDKQV SVTFEGSAGS GKTVRVFTSQ TGQTYDGPVF LVELRATWEF ANFSANPGLV ALEKGEDTA RINFSGNIGE PLIMNVPEGS HFARTVLARS TTPTTLARAG ERTTSDTVWQ VLNTAVSAAE LVTPPPFNWL PLVMKVTGSS DFHARMMRVM GDDATYTRTG EIKASEVIVQ VVDAGTDIIS STVPGFGWL VKGGWWFVKL IAGRTR TGTR SFYVYPSYQD ALSNKPALCT GSTPGGMRTR NPVTTTLQFT IKAGWFFIKK LAGLSR NGDG EYAVYASYAD AQNNRPCI---LPSTVTDV TPKPTTLAWQ QMNQPSLGHG EAPAAFGRSI PTPGEEFKV ILTFG-------------APMSPNANNK QMNKPNLGLE TGSYAMSRSM PVPVEGS YKA ILQLDNYAQM IHQLQADYPR PALAPYWQSL QNWVNKPLDA PSGHYNVKIA K--DVDHYLT MQGFTSIASV DWYTTDFQPS EAPAPIQGLQ ELWVGKGNDY GGGQHADRIT QVYKVNRGVF LNQF ----------YDQV MQPEPTLGYS VLVNSS--KK ADVYAVKQFV TAQTNNKHQV T------TLF LVKVTTGFQV NNY--LSYF IFSNTSHGKI GE VLGFQSYH MPGPTESGAE SLGPVAFNVY LGRITLSSKW SVQYKDTAYF YRASASGDAT TNLLVRGDTY TAG---ISFT Q-----------------VGWYLLTNTS PRASDEWGKS AAVMMK DWY GLGPPKPSYT TDGNGVVKPP RF DPDD MVLL ISWVRFSEGR IVDGALPPG WIWNNVELKT NTA-------------------YHMDKG LIHLIMPLPE NTD -LPVDK WCNTAMDYTY NVTVSGRKLA RDGQVRVPAG VPYWYYQDVQ TINGSDPVVQ STQMCYEMLT SIPRSRAAGY GYE----SDN TEYLDAPDF ADQLREDIET DTHIETTEDE NRESIFEFET EVPVQRSALL SLKKSAPSRA VKYDEEEEVY YTTLPKQGPP TAPWRM VEDD DDEADRF-------DIIDT SDEEDE NETD RVTLLSTLVN QGMTMTRATR IARRAFPTLT DHDSDSSYWD NDMSDDDFES SEEEELQDVD VDVLANTLEN SGFTRKEA----------DRIKRGVYMD LLVSGASPNS AWSHACEEAR KAAGEINPCT SGSRGHAE ------------------R AYAQAARDAV LKDGPTAKTV KFSDAPQE Figure 41 MUSCLE alignment of Human Astrovirus 1 and BDAst V1 capsid amino acid sequences Yellow = potential RNA binding domain (inside of capsid, likely not antigenic) ; Green = Caspase cleavage motif ; Red = potential trypsin cleavage site; separating VP34 analogue; Ital ics = antigenic region cloned by Matsui et al.(1993); Bold = cloned protein

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69 1 MGSSHHHHHH SSGLVPRGSH MRNGDGEYAV YASYADAQNN RPCILPSTVT DVTPKPTTLA 61 WQQMNKPNLG LETGSYAMSR SMPVPVEGSY KAILQLDNYA QMIHQLQADY PRPALAPYWQ 121 SLELWVGKGN DYGGGQHADR ITQVYKVNRG VFLNQFYDQV MQPEPTLGYS IFSNTSHGKI 181 GEVLGFQSYH MPGPTESGAE SLGPVAFNVY LGRITLSSKW SVQYKDTAYF PRASDEWGKS 241 AAVMMKDWYG LGPPKPSYTT DGNGVVKPPR FDPDDMVLLI SWVRFSEGRN TDLPVDKWCN 301 TAMDYTYNVT VSGRKLARDG QVRVPAGVPY WYYQDVQTIN GSDPVVQNRE SIFEFETEVP 361 VQRSALLSLK KSAPSRAVKY DEEEEVYYTT LPKQGPPTAP WRM Figure 4 2 A mino acid sequence of cloned BDAstV1 antigen. Added histidine tag is in bold.

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70 Figure 4 3 Coomassie blue stained SDS PAGE gel of the expressed BDAstV1 fragment. Markers are given in kilodaltons.

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71 Figure 4 4. Western blot using an anti polyhist idine antibody. Markers are given in kilodaltons.

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72 Figure 4 5. ELISA values (OD405 control) of openwater collection mature dolphins over time. Animal ID numbers are given on the right

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73 Figure 46. ELISA values (OD405 control) of openwater col lection calves over time. Animal ID numbers are given on the right

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74 Figure 47. ELISA values (OD405 control) vs. days in age among common bottlenose dolphin ( Tursiops truncatus ) calves, including all ten calves.

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75 A B C D Figure 48. ELISA values (OD405 control) vs. days in age among selected individual bottlenose dolphin ( Tursiops truncatus ) calves with significantly increasing titers by days in age. A) calf 165, B) calf 168, C) calf 169, D) calf 172

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76 CHAPTER 5 USE OF A QUANTITATIVE PCR ASSAY FOR THE DETECTION OF BOTTLENOSE DOLPHIN ASTROVIRUS 1 Introduction Astroviruses are small round nonenveloped viruses with a positive stranded RNA genome. They were relatively recently disc overed, and were first reported in 1975 (Madeley and Cosgrove, 1975). The family Astroviridae is divided into two genera, Avastrovirus found in avian hosts, and Mamastrovirus found in mammal hosts (Monroe et al., 2005). Human astrovirus is a significant cause of enteric disease in human children (Dennehy et al., 2001). We have recently reported the discovery of diverse astroviruses in marine mammals, including Bottlenose Dolphin Astrovirus 1 (BDAstV1) (Rivera et al., 2010). Seroconversion to a strovirus at a young age is typical, and a number of studies have shown that most humans have seroconverted by 5 years of age (Kriston et al., 1996, Kobayashi et al., 1999). Our serological survey of BDAstV1 found that most bottlenose dolphins seroconvert at a young age as well. Given the complications this introduces to serodiagnosis of BDAstV1 infection in populations other than young calves, methods for direct detection of BDAstV1 rather than antibody response have merit. Quantitative PCR (qPCR, a.k.a. real time PCR) has been used previously for detection of human astroviruses (Grimm et al., 2004, Royuela et al., 2006, Zhang et al., 2006, Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in press) and Turkey astrovirus 2 (Spackman et al., 2005) in fecal samples. Quantitative PCR human astrovirus assays have been shown to be significantly more sensitive than culture by more than two orders of magnitude (Royuela et al., 2006). The human qPCR assays

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77 have found prevalences in diarrheic human feces from 6% 9% (Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in press). A human astrovirus qPCR assay was used for a study of clinical correlation of virus load to clinical features; a tendency for longer duration of diarrhea with higher copy numbers was seen, and lower copy numbers were associated with rotavirus coinfection (Zhang et al., 2006). Materials and Methods Samples A total of 62 fecal samples were collected from 38 bottlenose dolphins ( Tursiops truncatus ) from a managed openwater collectio n in California This was the same managed openwater collection used in the ELISA survey in chapter 3, although samples were not available from all animals surveyed by ELISA. A total of 22 lower gastrointestinal samples were taken from 13 stranded wild c etaceans in New England, including 2 bottlenose dolphins (feces and mesenteric lymph node of each), 2 minke whales ( Balaenoptera acutorostrata) (colon, duodenum, and feces of one and feces of another), 2 pygmy sperm whales ( Kogia breviceps ) (colon, small i ntestine, and feces of one and feces of another), 3 short beaked common dolphins ( Delphinus delphis ) (feces and mesenteric lymph node of one and mesenteric lymph node of the other two), 2 harbor porpoises ( Phocoena phocoena) (mesenteric lymph nodes), and 2 Atlantic w hite sided d olphin s ( Lagenorhynchus acutus ) (colon, small intestine, and mesenteric lymph node of one and mesenteric lymph node of another). Samples were stored after collection at 80C. RNA E xtraction Samples were maintained on ice at all points during RNA extraction. 0.10g of feces were measured and 900 0.9% NaCl was added to suspend the sample by

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78 vortexing. Samples were spun at 3000 x G for 30 minutes at 4 collected and filtered serially through 800nm, 450nm, and 200nm filters (Millipore, Billerca, MA). Filtrate was concentrated with Microsep concentrator columns (Pall Life Sciences) and centrifuged at 1500 x G for 30 minutes at 4 T he concentrated filtrate was used for RNA extraction using a High Pure Viral RNA Kit (Roche, Indianapolis, IN) following the manufacturers instructions. Tissue samples were cut to 0.10g portions and extracted using RNeasy Mini Kit (Qiagen) following the manufacturers instructions. Quantitative PCR cDNA was synthesized using a MMLV r everse t ranscriptase kit (Advantage RTfor PCR, Clontech Mountain View, CA) using primer TtAstR ( CCTGCCATATTCAGGGAACAA) for initial strand synthesis. A BDAst V1 PCR amplicon of the capsid from the index BDAstV1 case amplified using consensus primers Ast r5159F and Astr5819R (Atkins et al., 2009) was used as a positive control for the standard curve, and RNasefree water was used as a negative control The positive control was quantified by both comparison to a mass ladder standard (Low mass ladder DNA st andard, Invitrogen, Carlsbad, CA ) on gel electrophoresis as well as spectrophotometry ( NanoDrop 8000, Thermo Scientific, Wilmington, DE). The standard curve, run on each plate, used 10fold serial dilutions, ranging from 106 to 10 copies. Quantitative P CR was performed using forward primer TtAst F ( TTGATCGGACCCTCAGCAAT ) reverse primer TtAstR and probe TtAst V probe (6FAM AGTGGGACAGCGTATC MGBNFQ ) targeting the Bottlenose dolphin astrovirus 1 ( BDAst V1) capsid gene. All samples were run in triplicate and a mean Ct value was calculated.

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79 (TaqMan Fast Universal PCR Master Mix 2X, Applied Biosystems). A 7500 Fast Real Time PCR System (A pplied Biosystems) was used to amplify the reactions with cycling conditions as follows: initial denaturation at 95 C for 20 seconds; 50 cycles of 95 C for 3 seconds followed by 60 C for 30 seconds. Comparison of years was analyzed using inStat (GraphPa d Software, San Diego, CA). Correlation with clinical values, sex, and age was analyzed using SAS ( SAS Institute Inc., Cary, NC ) Confirmatory H eminested PCR Samples with an appropriate curve were confirmed using a BDAStV1 specific heminested PCR and sequencing of PCR products. This confirmatory PCR used forward primer TtAst1SpecF1 ( ACCAAATACTGGCAATGATGC) and TtAstR as a first round, and TtAst1SpecF2 ( GTTTGGTCCTGTGCAAGCATT) and TtAstR as a second round. For the first round, 3.2 l of fecal RNA was reverse transcribed using a OneStep RT PCR Kit (Qiagen, Valencia, CA) at 50 minutes and then denatured at 95 for 10 minutes, followed by 45 cycles of denaturation at 95seconds; annealing at 55 seconds, and extension at 72 seconds, with a final elongation step at 72 n Two l of product from the first round was used as template in the 20l second round. The second round amplification used Platinum Taq DNA Polymerase (Invitrogen) and conditions were as follows: 5 minutes denaturation at 95 denaturation at 95 seconds, annealing at 55 for 30 seconds, and extension at 72 seconds, with a final elongation step at 72 PCR products were resolved in 1% agarose gels, excised, and puri using the QIAquick gel extraction kit (Qiagen). Sanger sequencing was performed directly using the BigDye Terminator Kit (Applied Biosystems, Foster City, California)

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80 and analyzed on ABI 3130 automated DNA sequencers. Results from the qPCR that were not confirmed by the heminested PCR were considered negative and given as 0. Results Quantitative PCR The BDAstV1 qPCR assay accurately detected 10 to 106 cDNA copies. The standard curve for the BDAstV1 qPCR assay had a slope of 3.33 and a correlation coefficient (R2) of 0.997 (Figure 5 1). Results of the qPCR for the managed openwater collection are given in table 51. From the managed openwater collection, 31 of 62 fecal samples (50%) were positive, representing 25 of 38 animals (66%). The mean and median BDA st V 1 detected copy numbers were 114 and 1.5 (range 05180). There were no significant differences in mean BDAst V 1 levels when comparing sex (P = 0.4) or linear associations with age (R2=0.04). The BDAstV1 detected copy numbers found in managed openwater collection samples fr om 2007 (n=23) and those from 2008 (n=39) were analyzed for normal distribution using the Kolmogorov Smirnov test ( Dallal and Wilkinson, 1986). Both populations were found to have nonGaussian distributions. For 2007, the mean detected copy number was 303, the median was 54, and the range was 05180. For 2008, the mean detected copy number was 3, the median was 0, and the range was 044. The MannWhitney test was used to compare medians of the different years for significance. The 2007 values were signi ficantly higher than those from 2008, with a two tailed P<0. 0 001. A plot of detected viral copies detected vs. date is shown (Figure 5 2). Forty two fecal samples from 32 animals had paired clinical observations and hematological data. The fecal sample w ith 5,180 copies detected was a routine sample

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81 collected from a young male dolphin with no abnormal clinical signs identified Because this count was so high compared to other reported levels (next highest level was 445), this value was not included in comparative analyses involving mean fecal viral levels and clinical parameters Animals with abnormal behavior were more likely to have higher mean BDAst V 1 copies detected compared to animals with reported normal behavior (Table 5 2). There were no significant differences in mean BDAst V 1 copies detected when comparing animals with normal or abnormal appetite or reason for blood sampling (routine or clinical). There were no significant differences in mean serum sodium, potassium, or chloride levels when compar ing animals with or without BDAst V 1 in their feces (Table 5 3). Results of the qPCR for the wild cetaceans are given in table 54. In total, BDAstV1 was found in 7 of 13 (54%) of stranded animals examined. One of two stranded bottlenose dolphins was posit ive in both the feces and mesenteric lymph node. Both minke whales were positive, as were both pygmy sperm whales. One of 3 s hort beaked common dolphins was positive. Neither of the harbor porpoises or the Atlantic white sided dolphins were positive on qPCR. The mean and median BDA st V 1 numbers of detected copies detected in the 7 wild cetacean fecal samples were 9 and 6 (range 026). Confirmatory H eminested PCR The heminested PCR resulted in a product of 135 bp, as expected. All qPCR positives with m ore than 10 copies present were confirmed as positive by heminested PCR. Of the 19 positive samples found by qPCR to have less than 10 copies detected, 4 were not confirmed by hemi nested PCR and were considered negative. No

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82 differences from the reference sequence were seen in any of the 15 positive sequences. Samples with less than 10 detected copies that were confirmed as positive averaged 4.8 detected copies (range 28), whereas those that were rejected averaged 1.9 detected copies (range 13). Discussion The qPCR results further confirm the high prevalence of BDAstV1 in bottlenose dolphins, as serological data had indicated. Fifty percent of fecal samples from the managed collection, many of which were from clinically normal animals, were found to be positive, and 86% of fecal samples from stranded cetaceans were positive. This is higher than values seen with qPCR surveys of human diarrheic samples, where 6% 9% prevalence was found (Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in pres s). Differences in assay sensitivity are one possible explanation for this discrepancy. An additional possibility is that the prevalence of astroviruses may be higher in the marine environment. Astroviruses are very stable in aquatic environments (Espino sa et al., 2008). Surveys have found a Human astrovirus prevalence of up to 61% in some marine shellfish populations, which are good particle concentrators (Elamri et al., 2006). W ide diversity has been seen in marine mammal astroviruses implying that th e marine environment may play a large role in astroviral ecology (Rivera et al., 2010), and the finding of high prevalence in dolphins by qPCR further supports that. T he marine environment is central in the ecology of caliciviruses, a better studied group of small nonenveloped positive stranded RNA viruses (Smith et al., 1998). We found BDAstV1 in three odontocete species (bottlenose dolphin, pygmy sperm whale, s hort beaked common dolphin) and one mysticete species (minke whale), implying low host fidelit y. As small RNA viruses, astroviruses are evolutionarily

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83 fascinating. Due to lack of proofreading by their polymerases, RNA viruses have the fastest mutating genomes found in nature, and m any RNA viruses accumulate one mutation per copy, which is thought to be the limit before accumulation of deleterious mutations would lead to extinction (Moya et al., 2004). This provides the ability to adapt rapidly to niches such as novel hosts Most recent emerging diseases have been associated with host switches, and in humans, RNA viral diseases a re much more likely to be emergent (Woolhouse and GowtageSequeria, 2005). We have found evidence of marine mammals playing a role in human astroviral ecology (Rivera et al., 2010). Further understanding of host range and limiting factors of astroviruses is needed. Virus was found in both Atlantic and Pacific cetacean populations, in agreement with the serological data. With a high prevalence in social species that have large geographic ranges, stability in a marine environment, and known presence in two oceans, it is probable that BDAstV1 is distributed throughout the worlds oceans. Virus prevalence differed significantly between the two years surveyed in the managed openwater collection, indicating that there are dynam ics to BDAstV1 epidemiology. The qPCR was more useful than serological methods for distinguishing this. Further work is needed to understand the epidemiology of BDAstV1 and the factors impacting it. It should be noted that our qPCR values are for copies detected, which may be expected to differ from actual virus numbers present. We were unable to cultivate virus (data not shown), and hence were unable to spike control samples directly with virus. We used dilutions of known copy numbers of BDAst V1 PCR amp licon as a standard curve. This control is a DNA template and does not reflect loss during extraction or reverse transcription. The presence of PCR inhibitors or nucleases may result in falsely

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84 low readings. These are common in feces, so t his is of special concern for fecal samples Control 18S rRNA amplification was variable in fecal samples ansd so equivalency is based solely on use of an equivalent amount of feces used for initial extraction. Further, extraction methods differed between tissue samples and fecal samples, so caution should be used to avoid over interpretation when comparing different sample types and quantitative data should not be used for this purpose. Kochs postulates have not been fulfilled for BDAstV1, and the barriers to doing so m ake this unlikely to happen in the near future, including inability to culture this virus, societal concerns about doing experimental infections in dolphins, and the large expense that would be required to acquire and house specific pathogenfree calves for this. The evidence is fairly strong that this virus infects bottlenose dolphins and is not merely passing through with ingesta; dolphins seroconvert to BDAstV1, some animals shed significant amounts in feces, and BDAstV1 was found in a lymph node of one animal. Clinical diagnosis of astroviral infection is challenging. Astrovirus culture is challenging, with few cell lines capable of supporting Human astrovirus and a high requirement for trypsin (Taylor et al., 1997). Attempts at culture of marine mammal astroviruses to date have been unsuccessful. Astroviruses resemble other small round viruses somewhat morphologically, and a significant rate of misidentification using negativestaining electron microscopy of feces has been reported (Oliver and Phillips, 1988). N egativestaining electron microscopy of feces for virus detection has also been shown to be comparatively insensitive (Logan et al., 2007, van Nieuwstadt et al., 1988). Culture, and negativestaining electron microscopy are theref ore insensitive test s for

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85 diagnosis of astroviral infection. Nucleic acid amplification diagnostic techniques appear to be the best current option, and development of this assay provides this option for BDAstV1. Diagnosis of astroviral disease is also challenging. Much like Vibrio cholerae, astroviruses cause a secretory diarrhea without much of a histologic footprint on enterocytes on light microscopy ( Koci et al., 2003). Nighot et al.(2010) found that Turkey astrovirus 2 causes sodium malabsorption. Our data did not show a correlation between BDAstV1 copies detected and electrolyte values. One possible explanation is that the presence of an acquired immune response attenuated disease manifestation. Volunteers experimentally infected with human astrovirus who had no pre existing anti human astrovirus antibodies developed more severe symptoms and were more likely to shed virus (Kurtz et al., 1979). The only parameter we found to correlate with BDAstV1 copies detected were trainer reports of abnormal behavior. This sit uation may differ in previously unexposed calves. We did not identify a correlation with age and BDAstV1 shedding. Although human astroviral disease prevalence is much higher in young children, there is conflicting data on whether infection prevalence dif fers by age. Similar virus prevalence was found in adults and children in China (Dai et al., 2010), but higher virus prevalence was found in children than in adults in Europe (van Maarseveen et al., in press). Dolphin 55 was found to shed virus at two t ime points. Serological data from dolphin 55 showed a moderate titer followed by a persistent elevation for two years. This elevation coincides temporally with viral shedding by this animal, suggesting persistent infection in this animal. Persistent infection has been seen with astrovirus

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86 infection in humans; persistent gastroenteritis was seen with 8.5% of astrovirus infections in children in Spain (Caballero et al., 2003). A study of children in Bangladesh found that Human astrovirus was more commonly associated with persistent diarrhea than acute diarrhea (Unicomb et al., 1998). In conclusion, this qPCR assay is useful for detecting and quantitating BDAstV1 in clinical samples from bottlenose dolphins. Prevalence of infection appears to be very high and is geographically widespread, similar to Human astrovirus in humans. Viral load correlates with abnormal behavior. Based on the observed parallels to human astrovirus infection, BDAstV1 is more likely to be clinically significant in calves than other age groups, and this is where management and future investigations should focus.

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87 Table 5 1 BDAstV1 copies detected by qPCR from the managed openwater collection Animal ID Date BDAstV1 copies detected Animal ID Date BDAstV1 copies detected 1 6/18/08 0 40 1/25/07 90 1 10/28/08 5 42 5/29/08 0 1 11/3/08 4 42 7/17/08 0 2 12/20/07 31 46 8/15/08 0 3 3/27/08 0 47 2/14/07 80 5 9/5/07 0 47 2/18/08 0 6 3/2/07 29 52 5/28/08 0 6 9/21/07 0 52 6/25/08 12 7 6/25/08 0 52 8/27/08 0 8 2/14/07 445 55 2 /7/07 70 8 9/11/08 5 55 6/21/07 60 9 1/24/07 3 56 2/14/08 0 9 3/20/08 0 56 3/20/08 0 12 1/12/07 16 59 9/17/07 56 14 2/21/07 310 60 5/29/08 0 14 7/29/08 0 60 9/24/08 5 15 5/21/08 0 60 10/28/08 0 15 6/26/08 0 163 5/22/08 0 17 2/8/07 74 163 7/30/08 0 22 2/21/07 5180 168 7/8/08 0 22 12/12/07 11 168 9/24/08 5 23 9/5/07 15 173 1/24/07 54 24 2/1/07 58 174 7/7/07 4 26 8/2/07 46 175 5/28/08 0 27 5/21/08 0 176 1/31/07 8 27 8/27/08 0 176 6/19/08 44 30 8/10/07 336 177 3/12/08 0 30 12/ 3/08 0 177 3/20/08 0 31 4/16/08 0 178 12/4/08 12 35 1/23/08 0 39 7/8/08 0 39 9/24/08 5 39 10/31/08 22

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88 Table 5 2 Comparisons of mean BDAst V 1 fecal load by animal appetite, behavior, reason for sampling among a population of common bottlenose dolphins ( Tursiops truncatus ) Health Variable Sample number Mean fecal BDAstV1 copies detected P value Reason for sample Initial or follow up Routine 21 21 29 58 0.53 Appetite Abnormal Normal 4 38 86 38 0.14 Behavior Abnormal Normal 9 33 81 32 0.05 Table 5 3. Comparisons of mean chloride, potassium, and sodium by presence or absence of BDAst V 1 in feces among a population of common bottlenose dolphins ( Tursiops truncatus ) Serum Variable Mean serum value P value BDA stV1 negative feces n=26 BDAstV1 positive feces n=16 Chloride (mEq/L) 119 120 0.55 Potassium (mEq/L) 3.8 3.7 0.58 Sodium (mEq/L) 153 154 0.41

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89 Table 54. BDAstV1 copies detected by qPCR from wild cetaceans. Animal ID Species Sample Copies detected 179 Minke Whale Colon 0 179 Feces 13 179 Duodenum 0 180 Minke Whale Feces 6 181 Pygmy sperm whale Feces 26 182 Pygmy sperm whale Feces 6 182 Colon 0 182 Small Intestine 0 183 Short beaked common dolphin Mesenteric LN 0 184 Short beaked commo n dolphin Feces 6 184 Mesenteric LN 0 185 Short beaked common dolphin Mesenteric LN 0 186 Harbor Porpoise Mesenteric LN 0 187 Harbor Porpoise Mesenteric LN 0 188 Atlantic white sided dolphin Small Intestine 0 188 Large Intestine 0 188 Mesenteric LN 0 189 Atlantic white sided dolphin Mesenteric LN 0 190 Bottlenose dolphin Feces 0 190 Mesenteric LN 0 191 Bottlenose dolphin Feces 3 191 Mesenteric LN 2

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90 Figure 5 1 The standard curve for the BDAstV1 qPCR. Ct values are plotted on the vert ical axis against log10 of the cDNA copy number of the standard curve on the horizontal axis

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91 Figure 5 2 BDAstV1 copies detected in from the managed openwater collection plotted on a logarithmic scale on the vertical axis against date on the horizont al axis

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92 CHAPTER 6 FURTHER INVESTIGATION OF ASTROVIRUS DIVERSITY IN CETACEAN FECAL SAMPLES Introduction Astroviruses are small round nonenveloped viruses with a positive stranded RNA genome. They were relatively recently discovered, and were first reported in 1975 (Madeley and Cosgrove, 1975). Human astrovirus is a significant cause of enteric disease in human children (Dennehy et al., 2001). The family Astroviridae is divided into two genera, Avastrovirus found in avian hosts, and Mamastrovirus fou nd in mammal hosts (Monroe et al., 2005). Recognized species in the genus Mamastrovirus include Bovine astrovirus, Feline astrovirus, Human astrovirus, Mink Astrovirus, Ovine astrovirus, and Porcine astrovirus (Monroe et al., 2005). There has been signific ant recent discovery of additional mamastroviruses, including viruses from cheetahs (Atkins et al., 2009), Asian bat species ( Chu et al., 2008, Zhu et al., 2009) humans (Finkbeiner et al., 2008, Finkbeiner et al., 2009a, Finkbeiner et al., 2009b, Kapoor et al., 2009), and rats (Chu et al., 2010). We have recently reported the discovery of diverse astroviruses in marine mammals, including Bottlenose Dolphin Astrovirus 1 (BDAstV1) the first cetacean astrovirus (Rivera et al., 2010). The cetacea are composed of two clades, the odontocetes (toothed whales) and the mysticetes (baleen whales) (McGowen et al., 2009). There are limited data on cetacean viruses. Most attention has focused on viruses of odontocetes, which are smaller and have been kept in captivit y.

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93 Materials and Methods Samples A total of 7 2 fecal samples were collected from 41 bottlenose dolphins ( Tursiops truncatus ) from a managed openwater collection in California. Three fecal samples were taken from 3 stranded wild cetaceans in New England, including 2 minke whales ( Balaenoptera acutorostrata) and a pygmy sperm whale ( Kogia breviceps ) Two fecal samples were taken from two orca ( Orcinus orca) from a captive closedwater collection. Samples were stored after collection at 80C. RNA E xtra ction Samples were maintained on ice at all points during RNA extraction. 0.10g of vortexing. Samples were spun at 3000 x G for 30 minutes at 4 collected and filtered serially through 800nm, 450nm, and 200nm filters (Millipore, Billerca, MA). Filtrate was concentrated with Microsep concentrator columns (Pall Life Sciences) and centrifuged at 1500 x G for 30 minutes at 4 was used f or RNA extraction using a High Pure Viral RNA Kit (Roche, Indianapolis, IN) following the manufacturers instructions. Consensus PCR Samples were tested for the presence of astroviruses using BDAStV1specific PCRs and sequencing of PCR products. The first nested PCR targeted the RdRp and used forward primer Astr4380F and reverse primer Astr4811R in the first round, and Astr4 574F and reverse primer Astr4722R in the sec ond round (Atkins et al., 2009). The second heminested PCR targeted the capsid and used f orward primer Astr4811F and reverse primer Astr5819R in the first round, and Astr 5159F and reverse primer

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94 Astr 5819R in the second round (Atkins et al., 2009) PCR products were resolved in 1% agarose gels excised, and puri QIAquick gel extract ion kit (Qiagen, Valencia, CA). Sanger sequencing was performed directly using the BigDye Terminator Kit (Applied Biosystems, Foster City, California) and analyzed on ABI 3130 automated DNA sequencers. PCR products that resulted in mixed sequences using consensus primers were either resequenced using specific primers or cloned using a TA plasmid ligation kit into an E. coli vector (Invitrogen, Carlsbad, CA), extracted using a QIAprep Spin Miniprep Kit (Qiagen) and sequenced as above. Sequence E xtension For each novel virus, a ttempts were made to obtain the remaining 3 sections of the viral genome by 3 r apid a mplification of cDNA e nds (3RACE) using a kit (GeneRacer, Invitrogen) according to manufacturers instructions. Briefly RNA was reverse transcribed using AMV reverse transcriptase and amplified with a forward gene specific primer (Table 61 ) and the Gene Racer 3 Primer. PCR products were run in a 0.7% agarose gel, and bands of interest were sequenced as previously described. Phylogenetic A nalysis Sequences were compared to those in GenBank (National Center for Biotechnology Information, Bethesda, Maryland), EMBL (Cambridge, United Kingdom), and Data Bank of Japan (Mishima, Shizuoka, Japan) databases using BLASTX (Altschul et al., 1997). The predi cted homologous 100133 amino acid sequences of astroviral RdRp and 388429 amino acid sequences of astroviral capsid protein were aligned using the following three methods: ClustalW2 (Larkin et al., 2007), TCoffee (Notredame et al., 2007), and MUSCLE (Edgar, 2004).

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95 Bayesian analyses of each alignment were performed using MrBayes 3.1 (Ronquist & Huelsenbeck, 2003) with gamma distributed rate variation and a proportion of invariant sites, and mixed amino acid substitution models Four chains were run and statistical convergence was assessed by looking at the standard deviation of split frequencies as well as potential scale reduction factors of parameters. The first 10% of 1,000,000 iterations were discarded as a burn in. Maximum likelihood (ML) analyses of each alignment were performed using PHYLIP (Phylogeny Inference Package, Version 3.66) (Felsenstein, 1989), running each alignment using the program ProML with amino acid substitution models JTT (Jones et al., 1992), PMB (Veerassamy et al., 2003), and P AM (Kosiol & Goldman, 2005) further set with global rearrangements, five replications of random input order, gamma plus invariant rate distributions, and unrooted. The values for the gamma distribution were taken from the Bayesian analysis. Avian nephriti s virus 1 (GenBank accession number AB033998) was designated as the outgroup. The alignment producing the most likely tree was then used to create data subsets for bootstrap analysis to test the strength of the tree topology (200 resamplings) (Felsenstein, 1985), which was analyzed using the amino acid substitution model producing the most likely tree in that alignment. Results Consensus PCR Results of the consensus PCR assays on bottlenose dolphins are presented in table 62 On the RdRp assay, 18 of 72 s amples (25%) from the bottlenose dolphins resulted in astroviral sequences, representing 13 of 41 animals (32%). On the capsid assay, 6 of 72 samples (8%) from the bottlenose dolphins resulted in astroviral

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96 sequences, representing 6 of 41 animals (15%). R esults of the consensus PCR assays on other cetaceans are presented in table 63 On the RdRp assay, both minke whale samples and one orca sample resulted in astroviral sequences. The capsid assay did not result in any astroviral product. The bottlenose dolphins represented 9 distinct virus types, the minke whales represented two distinct virus types, and one orca represented another distinct virus type. Four of 72 samples (6%) had multiple astrovirus types present. A bottlenose dolphin sample from anim al 27 showed 99% homology (806/811) with the reference BDAstV1 sequence (GenBank accession # FJ890355) over the first 811 nucleotides, and only 67% homology (258/387) over the final 387 nucleotides. A MUSCLE alignment is given in Figure 61. Sequences were submitted to GenBank under accession numbers HQ668121HQ668143. Sequence E xtension Sequence extension only resulted in product extension on BDAstV3 from case 23 and MWAstV2 from case 40. Additional 3RACE yielded final contiguous molecules of 2,942 bp ( BDAstV3) and 3, 080 bp ( MWAstV2 ). The contiguous molecules corresponded to the partial RdRp gene (ORF1b) and the full length capsid gene (ORF2) and 3 UTR of reference astroviruses The contiguous sequences were submitted to GenBank under accession number s HQ668129 and HQ668143. Phylogenetic A nalysis BLASTX results for the RdRp of bottlenose dolphin astrovirus es 2 and 5 (BDAstV2, BDAstV5) showed the highest identity score with Turkey astrovirus 2 (GenBank accession # Q9ILI5 ) BLASTX results for bottlenos e dolphin astrovirus 3 (BDAstV3) showed the highest identity score with Human astrovirus 3 ( ADC53753) for the RdRp and with California sea lion astrovirus 2 (ACR54274) for the capsid. BLASTX

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97 results for the RdRp of bottlenose dolphin astrovirus 4 (BDAstV4) showed the highest identity score with a Miniopterus magnater bat astrovirus ( ACF75849) BLASTX results for the RdRp of bottlenose dolphin astrovirus 6 (BDAstV6) showed the highest identity score with Turkey astrovirus 2 ( ABX46574 ) BLASTX results for the RdRp of bottlenose dolphin astrovirus 7 (BDAstV7) showed the highest identity score with Duck hepatitis virus 3 ( ACF19905) BLASTX results for the RdRp of bottlenose dolphin astrovirus 8 (BDAstV8) showed the highest identity score with Duck astrovirus C N GB ( YP_002728002) BLASTX results for the RdRp of bottlenose dolphin astrovirus 9 (BDAstV9) showed the highest identity score with California sea lion astrovirus 1 ( ACR54271 ) BLASTX results for the RdRp of minke whale astrovirus 1 (MWAstV1) showed the highest identity score with Duck hepatitis virus 2 ( ACF19904) BLASTX results for the RdRp of minke whale astrovirus 2 (MWAstV2) showed the highest identity score with Human astrovirus 1 ( ACN78557 ) BLASTX results for the RdRp of orca astrovirus 1 (OoAstV1) showed the highest identity score with a Miniopterus magnater bat astrovirus ( ACF758 3 9 ) Bayesian phylogenetic analysis showed the greatest harmonic mean of estimated marginal likelihoods using the MUSCLE alignment for the RdRp (see Supplemental Material: R dRp alignment) and the TCoffee alignment for the capsid gene (see Supplemental Material: Capsid alignment). For the RdRp, the BLOSUM model of amino acid substitution was found to be most probable with a posterior probability of 0.995 ( Henikoff & Henikoff, 1992) followed by the W AG model of amino acid substitution with a posterior probability of 0.005 (Whelan & Goldman, 2001). For the capsid precursor protein, the W AG model was most probable with a posterior probability of 1.000.

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98 Bayesian trees using the MUSCLE alignment for the RdRp (Figure 6 2 ) and the TCoffee alignment for the capsid gene are shown (Figure 6 3 ). While BDAstV3 and MWAstV2 cluster within the genus Mamastrovirus the other viruses identified do not cluster within known genera. ML analysi s found the most likely tree from the MUSCLE alignment and the PMB model of amino acid substitution for the RdRp, and the TCoffee alignment and the JTT model of amino acid substitution for the capsid precursor. These parameters were used for bootstrap analysis. Bootstrap values from ML analysis are shown on the trees (Figures 6 2 and 6 3 ). Discussion This survey of astroviruses in cetacean fecal samples has identified significant diversity. The majority of viruses identified do not nest within the recognized astroviral genera, and form a well supported monophyletic group. Significant diversity is present in this group, and the distance between BDAstV4 and BDAstV7 is greater than that between most Mamastrovirus / Avastrovirus pairs. It is possible that thi s novel astrovirus clade may represent more than one genus. Both of the novel mamastroviruses (BDAstV3 and MWAstV2) identified here were successfully sequenced from the conserved RdRp region through to the 3 end. The phylogenetic analyses of both the RdR p and the capsid found that these viruses represent fairly early divergences within Mamastrovirus in the clade containing Human astrovirus and MLB1. Given the relationship to known mamastroviruses, it is probable that these viruses utilize mammalian host s and likely represent true infection in these cetaceans.

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99 Neither consensus capsid PCR nor 3RACE yielded any product from the samples that had resulted in nonMamastrovirus astrovirus sequences. Possible explanations for failure of amplification using 3R ACE include a distance from the known sequence to the 3 end that was too long for efficient amplification, and lack of sufficient copy numbers of intact template present. The failure of amplification using consensus primers is likely due to lack of conservation of the template sequences in the divergent viruses. The RdRp and the 3 portion of the capsid are some of the most conserved regions in astroviruses (van Hemert et al., 2007). Recently, an ORF (ORFX) overlapping the 3 end of the capsid in a diff erent frame has been identified (Firth and Atkins, 2010). ORFX is conserved among Mamastrovirus but not Avastrovirus It is unknown whether ORFX is expressed, and if so what function it has, but if it is relevant for mamastroviral biology then it may provide selective pressure for conservation of this region in mamastrovirsues and not other astroviruses. A divergent astrovirus sequence has recently been identified from bat guano found under a mixedspecies roost (Li et al., 2010). Random amplification and pyrosequencing produced a sequence homologous to a region of ORF1a that was weakly supported as basal to other mamastroviruses in a neighbor joining tree. Unfortunately, sequence from homologous regions is not available to compare this to the nonMamas trovirus astroviruses from cetacean feces. The BDAstV1 sequence from animal 27 showed very strong homology with the reference BDAstV1 sequence over the first 811 nucleotides sequenced, and only 67% homology over the final 387 nucleotides. This is strongly suggestive of recombination. The juncture between these regions is after the RdRp, and near the 5 end of the

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100 capsid/ORFX. Recombination appears to be common in astroviruses (Belliot et al., 1997, Walter et al., 2001, Pantin Jackwood et al., 2006, Strain et al., 2008, Rivera et al., 2010, Ulloa and Gutirrez, 2010). Recombination appears to be especially common near the end of RdRp/start of the capsid (Walter et al., 2001, Pantin Jackwood et al., 2006, Strain et al., 2008, Rivera et al., 2010), as was seen here. The less homologous final 387 nucleotides are still closer to the reference BDAstV1 than to other known astroviruses on a BLASTX search, and the probable parent sequence remains to be discovered. Comparison of the relationships of viruses in the RdRp and capsid regions reveals a number of discrepancies. The diversity and high rate of recombination of astroviruses is diagnostically and epidemiologically challenging. Further understanding of the diversity, host range, and biology of the astrovirus es is needed to improve diagnostic testing and assess risks. It should be noted that CSLAstV2was found in dolphin 36. This is clear evidence of host switching over an even larger phylogenetic distance than was identified with BDAstV1 between cetacean spec ies. Emerging disease is frequently associated with host switches. One recent metaanalysis of human diseases found that 816 of 1407 (58%) are zoonotic, and of human diseases, zoonotic diseases are significantly more likely to be emerging (Woolhouse & Gow tageSequeria, 2005). Most recent emerging human diseases have been associated with host switches, including SARS, Hendra virus, Nipah virus, and AIDS. The aforementioned study also found that viral diseases were much more likely to be emerging, especial ly RNA viruses (Woolhouse & Gowtage-

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101 Sequeria, 2005). The ability of astro viruses to infect disparate hosts suggests further study of their ecology and host range may be relevant to human health. MWAstV1 and MWAstV2 were found in samples from mysticetes. Very little is known of mysticete viruses. There is some serologic evidence of viral infection, but concerns about cross reactivity with unknown agents in poorly studied host species limit conclusions that can be drawn from this. Adenoviruses have been i solated from feces of a sei whale ( Balaenoptera borealis ) and a bowhead whale ( Balaena mysticetus ) that were not further identified, and serologic testing of the bowhead whale found no neutralizing antibodies, suggesting this may not have been infecting the whale (Smith and Skilling, 1979, Smith et al., 1987). An enterovirus was also isolated from feces of a gray whale ( Eschrichtius robustus ) (Smith and Skilling, 1979). Paramyxovirus like particles have been seen on electron microscopy in two fin whales ( Balaenoptera physalus ) with lesions histologically consistent with morbilliviral disease ( Jauniaux et al., 2000). The only sequence data available prior to this study was from a poxvirus in a skin lesion on a bowhead whale (Bracht et al., 2006). Further s tudy is needed to understand these viruses, especially those outside of known genera. A number of studies have recently begun to look at viral diversity in feces; the majority of eukaryotic viruses have often been viruses of food items (Zhang et al., 2006, Li et al., 2010). Study of fish or other prey species of cetaceans to look for the presence of these viruses, study of virus loads, and searching for acquired immune responses in cetaceans are indicated to delineate any potential role these viruses play in cetacean health. While most mamastrovirus infections are enteric, a mamastrovirus has been found in a human encephalitis case (Quan et al., 2010), and avastrovirus

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102 disease may also be renal or hepatic ( Imada et al., 2000, Fu et al., 2009). The search for these viruses should not be limited to the gastrointestinal tract. In conclusion, we have discovered several novel astroviruses present in cetacean feces. Phylogenetic analysis revealed that several of these did not cluster within known genera. Further study is needed to understand the ecology and clinical significance of these viruses.

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103 Table 61. Additional primers used for 3 RACE of novel astroviruses from cetacean feces 3RACE Primers 5 3 Sequence TtAst2RACE3: GTCGCGGCAATGTACGGAGAATGGA TtA st3RACE3: GACAGAAAGGTGGTAAYCCCTCCGGACA TtAst4RACE3: CCGCAACTACCAACCTGCGGAAAAAGA TtAst5RACE3: GGTTACGGCCATGTATGCGGAATGGA TtAst5RACE3.2: TCGTCCCATCATATCGTGCCCAATCA TtAst6RACE3: RGTCGCAGCAATGTATGGGGAATGGA TtAst7RACE3: GCATCGAATGCCCAATGGTGGAAA CA TtAst8RACE3: CCAGCAGAGCTCAAAGCCACCTACCG TtAst8RACE3.2: RCGTCGGACACTACCCAACACCAGCA BaAst1RACE3: TCCTCACGGCCTACGAAAACGCACAC BaAst2RACE3: AGACACGCGGGAATCCTTCTGGTCAA OoAst1RACE3: CGTTGAAGCCAAAACATGGGCCAACA Primers for closing sequence gap s of 3RACE products TtAstV3seq1 TGTCAGAGCAGCGGTATCAC TtAstV3F1720 CACCTTTTGGCTGGCTTATT TtAstV3R31 CTTATCTGCTGCAACCACCA BaAstV2seq1 AAGTCACACCCAGCTTGGAC BaAStV2F938 GCCTAAGCCAGCACTCTCAC BaAstV2capR31 ACAATCCAATGGTGGTCTGG

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104 Table 62. Results of astroviral consensus PCR and sequencing of bottlenose dolphin fecal samples. Animal ID Date RdRp PCR Capsid PCR Animal ID Date RdRp PCR Capsid PCR 1 2/15/07 BDAstV1 BDAstV1 36 6/29/08 CSLAstV2 1 6/18/08 37 4/9/08 1 10/28/08 BDAstV2 39 7/ 8/08 BDAstV6 1 11/3/08 BDAstV8 39 9/24/08 2 12/20/07 39 10/31/08 3 3/27/08 40 1/25/07 5 9/5/07 42 5/29/08 6 3/2/07 42 7/17/08 6 9/21/07 46 8/15/08 7 6/25/08 BDAstV5, BDAstV6 47 2/14/07 8 2/14/07 BDAstV1 47 2/13/08 8 9/11/08 47 2/18/08 9 1/24/07 52 5/28/08 9 3/20/08 52 6/25/08 12 1/12/07 52 8/27/08 12 12/9/08 55 2/7/07 14 2/21/07 55 6/21/07 14 7/8/08 56 2/14/08 14 7/24/08 BDAstV2 56 3/20/08 14 7/29/08 59 9/17/07 15 5/21/08 BDAstV4 60 5/29/08 15 6/26/08 60 9/24/08 BDAstV3 17 2/8/07 60 10/28/08 BDAstV6, BDAstV8 21 11/30/07 163 5/22/08 22 2/21/07 BDAstV 1 BDAstV1 163 7/30/08 22 12/12/07 163 7/1/08 23 9/5/07 BDAstV1, BDAstV1b, BDAstV3 BDAstV1, BDAstV3 168 7/8/08 24 2/1/07 168 9/24/08 24 4/9/08 173 1/24/07 26 8/2/07 174 7/7/07 BDAstV2 27 5/21/0 8 BDAstV1 (100%) Recomb. (68% ID) 175 5/28/08 27 8/27/08 BDAstV7 176 1/31/07 30 8/10/07 BDAstV1 BDAstV1 176 6/19/08 30 12/3/08 BDAstV5 177 3/12/08 31 4/16/08 177 3/20/08 BDAstV2, BDAstV6, BDAstV9 35 1/ 23/08 178 12/4/08

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105 Table 63. Results of astroviral consensus PCR and sequencing of other cetacean samples. Animal ID Species RdRp PCR Capsid PCR 179 Minke Whale MWAstV1 180 Minke Whale MWAstV2 181 Pygmy Sperm Whale 192 Orca OoAstV1 193 Orca

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106 27 CCCAACTCAACTTTTCCGCAGGATAAAGAAACTCCGGTGGAGTTTTATTAATAAAGAGCAACGTGAGTATTACTCA BDAstV1 CCCAACTCAACTTTTCCGCAGGATAAAGAAACTCCGGTGGAGTTTTATTAATAAAGAGCAACGTGAGTATTACTCA **************************************************************************** 27 CGTATGTATGAATGGTACTGCTATAACCTATTTAATAGGTATGTGCTTCTACCTTCGGGTGAAGTGACAGAGCAGA BDAstV1 CGTATGTATGAATGGTACTGCTATAACCTATTTAATAGGTATGTGCTTCTACCTTCGGGTGAAGTGACAGAGCAGA **************************************************************************** 27 CCAGAGGGAATCCTAGCGGACAATTTTCAACCACGATGGATAATAACATGGTTAATGTC TGGCTACAGGCCTTTGA BDAstV1 CCAGAGGGAATCCTAGCGGACAATTTTCAACCACGATGGATAATAACATGGTTAATGTT TGGCTACAGGCCTTTGA ********************************************************** **************** 27 ATTTGCATATTTCTTTGGCCCAGATAAAAAGAAATGGAGCAAGGTGGATGCCCTCATTTATGGGGACGACCGCCTC BDAstV1 ATTTGCATATTTCTTTGGCCCAGATAAAAAGAAATGGAGCAAGGTGGATGCCCTCATTTATGGGGACGACCGCCTC ******************************* ******************************************** 27 TCATCATGGCCAGAAATCCCCGTTAATTATGGGGAGAGAGTGGTTGAAATGTATAAGAAGGTGTTTGGGATGTGGG BDAstV1 TCATCATGGCCAGAAATCCCCGTTAATTATGGGGAGAGAGTGGTTGAAATGTATAAGAAGGTGTTTGGGATGTGGG **************************************************************************** 27 TGAAACCAGAGAAAGTTAAAGTGCAGAAC ACCCTAGTTGGCCTTTCCTTTTGCGGGTTTACGGTAGATCAGAATTA BDAstV1 TGAAACCAGAGAAAGTTAAAGTGCAGAAT ACCCTAGTTGGCCTTTCCTTTTGCGGGTTTACGGTAGATCAGAATTA **************************** *********************************************** 27 TGAACCCGTACCCAGCTCACCAGAAAAGTTG CTTGCAGGCCTTTTGACTCCAACCAAGAAAATGCCGGACCTTGAA BDAstV1 TGAACCCGTACCCAGCTCACCAGAAAAGTTA CTTGCAGGCCTTTTGACTCCAACCAAGAAAATGCCGGACCTTGAA ****************************** ********************************************* 27 TCACTCCATGGGAAACTCTTGTGCTTTCAGTTGCTCTCAGCGTTTTTGCCGGAGGATCACCCCTTCAAAAATTACG BDAstV1 TCACTCCATGGGAAACTCTTGTGCTTTCAGTTGCTCTCAGCGTTTTTGCCGGAGGATCACCCCTTCAAAAATTACG **************************************************************************** 27 TTGAGATGAGCCTAGCATCTACGGCTAAGCAGCTACCAGGAACCGCGCTACCACCGCGTTTCACTGAGGAGCAACT BDAstV1 TTGAGATGAGCCTAGCATCTACGGCTAAGCAGCTACCAGGAACCGCGCTACCACCGCGTTTCACTGAGGAGCAACT **************************************************************************** 27 GCATTGCATTTGGAGGGGAGGACCAAAAATTTGCG ATGGCTAACGGCCGTG GCAAAGATGTTAGCGTTGAGGTTAA BDAstV1 GCATTGCATTTGGAGGGGAGGACCAAAAATTTGCA ATGGCTAACGGCCGTA GCAAAGATGTTAGCGTTGAGGTTAA ********************************** *************** ************************* 27 AGCCTCCGGCTCACAGAGAAGCAAAAGTCGTTCCCGTTCAAGGTCTCGAGGG AGAAATCCCGCA GTT AAAGTCACA BDAstV1 AGCCTCCGGCTCACAGAGAAGCAAAAGTCGTTCCCGTTCAAGGTCTCGAGGA AGAACACCCGCT GTC AAAGTCACA *************************************************** **** ***** ** ********* 27 GTTAATA CCAAAC CAAAGAGA A A T GGACAGAACAGACGTGGT G GACGCC CTAATAGAAAT TTCA G T CGTAA ATCA G BDAstV1 GTTAATT CCAAAG CAAAAAGA T T TACCAGA--AGACCAAG T C GACGCT CTTTTAGAGCT AAAA A T AATAG ---T G ****** ***** **** ** **** **** ** ***** ** **** ** 27 T ACGGC G ACAGAT C A AGCGT G AACTTGA T AAACAAGGAGTCACC GGGCCGAC A CCGTC T GTGTCCCAGATTGCT AC BDAstV1 T CAAAC A ACAAGT T A GAAAT C AACTCAA G AAACAAGGTCTCACA GGGCCCGC C CCAGC G GTTGTCCAGACCGCG AC *** **** ******** **** ***** ** ** ***** ** ** 27 A GCTACTT T G GGA ACTATTAATGGGAATT C CAGCAATC A G GCAGAGAGGGAGC T GAGTT G CTTCCTT AATCCAGCA BDAstV1 G GCTACTC T T GGC ACTATTGGACCAAATA C TGGCAATG A T GCAGAGAGGGAGA T TTCCT T CTATCTA AATCCAGCT ****** ** ****** *** ***** ************ ** ** ******** 27 TTAATAAAG GAAAAT ACT GGC TCG AATGGTTTTGGA CCA GTTACA GCATTT GCT GCA CAGTAC TCGCT T TGGC GAT BDAstV1 TTAACCAAA GAAAAC ACA GGT TCA AATGCGTTTGGT CCT GTGCAAGCATTG GCA GCC CAGTAT TCAAT G TGGA G AT **** ** ***** ** ** ** **** ***** ** ** ****** ** ** ***** ** *** *** 27 GTA C G AGCCTTTCAGT T A AACT G ACCCCA T TGATA GGTTC T TCAGCAG T TTCAGGC ACC G G GTATCGCGTTTCAGT BDAstV1 GTT C C AGAGCCGAGAT A A GGTT T ACCCCT TTGATC GGACC C TCAGCAA T CAGTGGG ACA G C GTATCGTTGT TCCCT ** ** ***** ***** ** ****** ** ** ****** *** 27 A AATGCG A CAGGTTC T CCTTCT AGTGATA G C TGGAGTGGC CTTGGTGC T AGGAAA CAC A GGGATT T C CAA AT BDAstV1 G AATATG G CAGGGAC A CCTTCGCAGACATC G TGGTCCGGA CTTGGATC C AGGAAG CAT A AAGATA T G CAT AT *** **** ***** ** ** *** ** ***** ***** ** *** ** ** Figure 61. MUSCLE alignment of sequence from animal 27 and reference BDAstV1. Differences are highlighted in red.

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107 Figure 62. Bayesian phylogenetic tree of predicted 100133 amino acid partial astroviral RNAdependent RNA polymerase sequences based on MUSCLE alignment. Bayesian posterior probabilities of branchings as percentages are in bold, and ML bootstrap values for branchings based on 200 resamplings are given to the right or below Avian nephritis virus 1 (GenBank accession number NP_620617) was designated as the outgroup. Virus genera are delineated by brackets. Marine mammal astroviruses are bolded and in red (cetaceans) or purple (pinnipeds) Human astroviruses are in green. A reas of multifurcation are marked by arc s. Sequences retrieved from GenBank include Human astrovirus 1 (GenBank accession # AAW51881), Human astrovirus 3 (AAD28539), Human astrovirus 4 (AAY84778), Human astrovirus 5 (AAY46273), Human astrovirus 8 (AAF85963), Human astrovirus MLB1 (YP002290967), Human astrovirus MLB2 ( ACX69833 ), Human astrovirus VA1 ( ACR23347), Rat astrovirus (ADJ38390), Cheetah astrovirus, ( Miniopterus magnater bat astrovirus WCF90 (ACF75856), Miniopterus magnater bat astrovirus AFCD57 (ACF75852), Miniopterus pusillus bat astrovirus AFCD337 (ACF75864), Miniopterus pusillus bat astrovirus WCF214 (ACF75862), Pipistrellus abramus bat astrovirus AFCD11 (ACF75853), Ovine astrovirus (NP_059945), Mink astrovirus (AAO32082), T urkey astrovirus 1 (CAB95006), Turkey astrovirus 2 (NP_987087), and Avian Nephritis Virus 1 (NP_620617).

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108 Figure 6 3 Bayesian phylogenetic tree of predicted 388429 amino acid partial astroviral capsid sequences based on TCoffee alignment. Bayesian posterior probabilities of branchings as percentages are in bold, and ML bootstrap values for branchings based on 200 resamplings are given to the right. Avian nephritis virus 1 (GenBank accession number NP_620618) was designated as the outgroup. Virus genera are delineated by brackets. Marine mammal astroviruses are bolded and in red (cetaceans) or purple (pinnipeds) Human astroviruses are in green. Sequences retrieved from GenBank include Human astrovirus 1 (GenBank accession # BAE97460), Human astrovir us 2 (AAA62427), Human astrovirus 4 (BAA93440), Human astrovirus 5 (AAY46274), Human astrovirus 8 (AAF85964), Human astrovirus MLB1 ( ACI62175), Human astrovirus VA1 ( ACR23349 ), Human astrovirus VA2 ( ACX83591 ), Rat astrovirus ( ADJ38391), Feline astrovirus ( AAC13556), Dog astrovirus ( CAR82569 ), Porcine astrovirus (CAB95000), Hipposideros armiger Bat Astrovirus LS11 ( ACN88714), Miniopterus pusillus bat astrovirus AFCD337 (ACF75865), Miniopterus magnater Bat Astrovirus AFCD57 ( ACU30844 ), Taphozous melanopogon B at Astrovirus LD71 ( ACN88712), Ovine astrovirus (NP_059944), Mink astrovirus (NP_795336), Turkey astrovirus 1 (CAB95007), Turk ey astrovirus 2 (NP_987088), Turk ey astrovirus 3 ( AAV37187 ), Avian Nephritis Virus 1 (NP_620618) Avian Nephritis Virus 2 (BAB2161 7) and Duck Astrovirus ( ACN82429 )

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109 CHAPTER 7 USE OF A QUANTITATIVE PCR ASSAY FOR THE DETECTION OF BOTTLENOSE DOLPHIN ASTROVIRUS 6 Introduction Astroviruses are small round nonenveloped viruses with a positive stranded RNA genome. They were relatively r ecently discovered, and were first reported in 1975 (Madeley and Cosgrove, 1975). The family Astroviridae is divided into two genera, Avastrovirus found in avian hosts, and Mamastrovirus found in mammal hosts (Monroe et al., 2005). Human astrovirus is a significant cause of enteric disease in human children (Dennehy et al., 2001). We have recently discovered diverse astroviruses in marine mammals, some of which do not cluster phylogenetically with known astroviral genera, including Bottlenose Dolphin Ast rovirus 6 (BDAstV6). Seroconversion to a strovirus at a young age is typical, and a number of studies have shown that most humans have seroconverted by 5 years of age (Kriston et al., 1996, Kobayashi et al., 1999). Our serological survey of BDAstV1 found that most bottlenose dolphins seroconvert at a young age as well; serological data on BDAstV6 is not available. Given the complications this introduces to diagnosis in populations other than young calves, methods for direct detection of BDAstV6 rather than antibody response have merit. Quantitative PCR (qPCR, a.k.a. real time PCR) has been used previously for detection of human astroviruses (Grimm et al., 2004, Royuela et al., 2006, Zhang et al., 2006, Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in press) and Turkey astrovirus 2 (Spackman et al., 2005) in fecal samples. Quantitative PCR human astrovirus assays have been shown to be significantly more sensitive than culture by more than two orders of magnitude (Royuela et al., 2006). The hum an qPCR assays

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110 have found prevalences in diarrheic human feces from 6% 9% (Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in press). Materials and Methods Samples A total of 62 fecal samples were collected from 38 bottlenose dolphins ( Tursi ops truncatus ) from a managed openwater collection in California and 22 lower gastrointestinal samples were taken from 13 stranded wild cetaceans in New England, including 2 bottlenose dolphins (feces and mesenteric lymph node of each), 2 minke whales ( B alaenoptera acutorostrata) (colon, duodenum, and feces of one and feces of another), 2 pygmy sperm whales ( Kogia breviceps ) (colon, small intestine, and feces of one and feces of another), 3 short beaked common dolphins ( Delphinus delphis ) (feces and mesen teric lymph node of one and mesenteric lymph node of the other two), 2 harbor porpoises ( Phocoena phocoena) (mesenteric lymph nodes), and 2 Atlantic w hite sided d olphin s ( Lagenorhynchus acutus ) (colon, small intestine, and mesenteric lymph node of one and mesenteric lymph node of another). Samples were stored after collection at 80C. This was the same sample set used in Chapter 5. RNA E xtraction Samples were maintained on ice at all points during RNA extraction. 0.10g of feces were measured and 900 0.9% NaCl was added to suspend the sample by vortexing. Samples were spun at 3000 x G for 30 minutes at 4 collected and filtered serially through 800nm, 450nm, and 200nm filters (Millipore, Billerca, MA). Filtrate was concentrated w ith Microsep concentrator columns (Pall Life Sciences) and centrifuged at 1500 x G for 30 minutes at 4 T he concentrated filtrate was used for RNA extraction using a High Pure Viral RNA Kit (Roche, Indianapolis, IN)

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111 following the manufacturers instruct ions. Tissue samples were cut to 0.10g portions and extracted using a RNeasy Mini Kit (Qiagen) following the manufacturers instructions. Quantitative PCR cDNA was synthesized using a MMLV r everse t ranscriptase kit (Advantage RTfor PCR, Clontech Mounta in View, CA) using consensus astroviral primers Astr4722R and Astr4811R (Atkins et al., 2009) for initial strand synthesis. A BDAst V 6 PCR amplicon of the polymerase from the index BDAstV1 case amplified using consensus primers Astr4380F and Astr4722R (At kins et al., 2009) was used as a positive control for the standard curve, and RNasefree water was used as a negative control The positive control was quantified by both comparison to a mass ladder standard (Low mass ladder DNA standard, Invitrogen, Carlsbad, CA ) on gel electrophoresis as well as spectrophotometry ( NanoDrop 8000, Thermo Scientific, Wilmington, DE). The standard curve, run on each plate, used 10fold serial dilutions, ranging from 106 to 10 copies. Quantitative PCR was performed using for ward primer TtAstV6qPCRF ( GTTRGTCGCAGCAATGTATG ) reverse primer TtAstV6qPCRR ( YCCCTAAGCTCGTCAAGTGT ) and probe TtAst V 6 probe (6FAM TGGATTTTTRAGAATGTCGGA MGBNFQ ) targeting the BDAstV6 polymerase gene. All samples were run in triplicate and a mean Ct value was calculated. PCR Master Mix 2X, Applied Biosystems). A 7500 Fast Real Time PCR System (Ap plied Biosystems) was used to amplify the reactions with cycling conditions as

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112 follows: initial denaturation at 95 C for 20 seconds; 50 cycles of 95 C for 3 seconds followed by 60 C for 30 seconds. Confirmatory Hemi nested PCR All samples from the managed openwater collection were confirmed using a consensus astrovirus hemi nested PCR and sequencing of PCR products protocol using forward primer Astr4380F and reverse primer Astr4811R in the first round, and Astr4380F and reverse primer Astr4722R in the second round (Atkins et al., 2009). PCR products were resolved in 1% agarose gels, excised, and puri gel extraction kit (Qiagen). Sanger sequencing was performed directly using the BigDye Terminator Kit (Applied Biosystems, Foster City California) and analyzed on ABI 3130 automated DNA sequencers. Results from the qPCR that were not confirmed by the heminested PCR were considered negative and given as 0. Results Quantitative PCR The DeHV 2 qPCR assay accurately detected 10 to 106 cDNA copies. The standard curve for the BDAstV6 qPCR assay had a slope of 3.34 and a correlation coefficient (R2) of 0.99 6 (Figure 1). Results of the qPCR for the managed openwater collection are given in table 71. From the managed openwater collection, 5 of 62 fecal samples (8%) were positive, representing 5 of 38 animals (13%). The mean and median BD A st V 6 detected copy numbers were 0.2 and 0 (range 0 4 ). None of the wild stranded cetaceans were found to have BDAstV6.

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113 Confirmatory H eminested PCR The heminested PCR resulted in a product of 400 bp after primers were edited out, as expected. All qPCR negatives were confirmed as negative by heminested PCR. Of the 10 samples found positive by qPCR, 5 were not confirmed by hemi nested PCR and were considered negative. S urprisingly, s amples that were confirmed as positive averaged 2.4 detected copies (range 24 ), whereas those that were rejected averaged 7.0 detected copies (range 4 11). Discussion The qPCR results find a much lower prevalence of BDAstV6 than BDAstV1 in bottlenose dolphins. Eight percent of fecal samples from the managed collection were found to be BDAstV6 positive, as compared to 50% for BDAstV1, and none of the fecal samples from stranded cetaceans were positive, as compared to 86% for BDAstV1. The BDAstV6 prevalence found is similar to values seen with qPCR surveys of human diarrheic samples, where 6% 9% prevalence was found (Logan et al., 2007, Dai et al., 2010, van Maarseveen et al., in press). Differences in assay sensitivity are one possible explanation for this discrepancy. It should be noted that our qPCR values are for copies detected, which may be expected to differ from actual virus numbers present. We were unable to cultivate virus (data not shown), and hence were unable t o spike control samples directly with virus. We used dilutions of known copy numbers of BDAst V 6 PCR amplicon as a standard curve. This control is a DNA template and does not reflect loss during extraction or reverse transcription. The presence of PCR inhi bitors or nucleases may result in falsely low readings. These are common in feces, so t his is of special concern for fecal

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114 samples Equivalency is based solely on use of an equivalent amount of feces used for initial extraction Kochs postulates have not been fulfilled for BDAstV6, and the barriers to doing so make this unlikely to happen in the near future, including inability to culture this virus, societal concerns about doing experimental infections in dolphins, and the large expense that would be required to acquire and house specific pathogenfree calves for this. It is possible that BDAstV6 is not actually a virus of dolphins but is actually a fish virus passing through with ingesta; BDAstV6 has only been found in feces so far, and the amounts shed are fairly small. As a very divergent astrovirus, it is difficult to predict what would be expected for host range or tissue tropism. While most mamastrovirus infections are enteric, an astrovirus has been found in a human encephalitis case (Quan et al. 2010), and avastrovirus disease may be renal or hepatic. Clinical diagnosis of astroviral infection and astroviral disease is challenging. Astrovirus culture is challenging, with few cell lines capable of supporting Human astrovirus and a high requiremen t for trypsin (Taylor et al., 1997). Attempts at culture of marine mammal astroviruses to date have been unsuccessful. Astroviruses resemble other small round viruses somewhat morphologically, and a significant rate of misidentification using negativest aining electron microscopy of feces has been reported (Oliver and Phillips, 1988). N egativestaining electron microscopy of feces for virus detection has also been shown to be comparatively insensitive (Logan et al., 2007, van Nieuwstadt et al., 1988). Muc h like Vibrio cholerae, astroviruses cause a secretory diarrhea without much of a histologic footprint on enterocytes on light microscopy ( Koci et al., 2003) and there are no pathognomonic lesions Culture, negativestaining

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115 electron microscopy and hist opathology are theref ore insensitive test s for diagnosis of a stroviral infection. Nucleic acid amplification diagnostic techniques appear to be the best current option, and development of this assay provides this option for BDAstV6. In conclusion, this qP CR assay is useful for detecting and quantitating BDAstV6 in clinical samples from bottlenose dolphins. Prevalence in dolphin feces appears to be lower than that of BDAstV1 and similar to Human astrovirus in human feces. The host range, tissue tropism, and disease implications of BDAstV6 are still unknown, and this is where future investigations should focus.

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116 Table 7 1 BDAstV6 copies detected by qPCR from the managed openwater collection. Animal ID Date Copies detected Animal ID Date Copies detected 1 18 Jun 08 0 47 14 Feb 07 0 1 28 Oct 08 0 47 18 Feb 08 0 1 3 Nov 08 2 52 28 May 08 0 2 20 Dec 07 0 52 25 Jun 08 0 3 27 Mar 08 0 52 27 Aug 08 0 5 5 Sep 07 0 55 7 Feb 07 0 6 2 Mar 07 0 55 21 Jun 07 0 6 21 Sep 07 0 56 14 Feb 08 0 7 25 Jun 0 8 2 56 20 Mar 08 0 8 14 Feb 07 0 59 17 Sep 07 0 8 11 Sep 08 0 60 29 May 08 0 9 24 Jan 07 0 60 24 Sep 08 0 9 20 Mar 08 0 60 28 Oct 08 2 12 12 Jan 07 0 163 22 May 08 0 14 21 Feb 07 0 163 30 Jul 08 0 14 29 Jul 08 0 168 8 Jul 08 0 15 21 May 08 0 168 24 Sep 08 0 15 26 Jun 08 0 173 24 Jan 07 0 17 8 Feb 07 0 174 7 Jul 07 0 22 21 Feb 07 0 175 28 May 08 0 22 12 Dec 07 0 176 31 Jan 07 0 23 5 Sep 07 0 176 19 Jun 08 0 24 1 Feb 07 0 177 12 Mar 08 0 26 2 Aug 07 0 177 20 Mar 08 2 27 21 May 08 0 178 4 Dec 08 0 27 27 Aug 08 0 30 10 Aug 07 0 30 3 Dec 08 0 31 16 Apr 08 0 35 23 Jan 08 0 39 8 Jul 08 4 39 24 Sep 08 0 39 31 Oct 08 0 40 25 Jan 07 0 42 29 May 08 0 42 17 Jul 08 0 46 15 Aug 08 0

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117 Figure 7 1 The standard curve for the BDAstV6 qPCR. Ct values are plotted on the vertical axis against log10 of the cDNA copy number of the standard curve on the horizontal axis

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118 CHAPTER 8 CONCLUSIONS This work demonstrates that astroviruses ar e prevalent in the marine environment, and specifically in marine mammals. As in humans, the seroprevalence study showed that dolphins develop antibodies to BDAstV1 at a young age. The BDAstV1 virus prevalence by qPCR suggests, however, that active infec tion and shedding are potentially even more common in dolphins than has been seen with any astrovirus in terrestrial species. Astroviruses are very stable in aquatic environments (Espinosa et al ., 2008), and this is likely to contribute to a higher pathogen prevalence. We have identified evidence of historical recombination in CSLAstV3 and in BDAstV1. Recombination appears to be common in astroviruses (Belliot et al., 1997, Walter et al., 2001, Pantin Jackwood et al., 2006, Strain et al., 2008, Rivera et al., 2010, Ulloa and Gutirrez, 2010). Throughout biology, hybridization is a factor allowing rapid nondetrimental change, allowing species to invade novel habitats (Nolte et al., 2005, Rieseberg et al., 2007). Recombination of viruses may provide a hybri d advantage for crossing host species The host fidelity of astroviruses does not appear to be stringent. BDAstV1 was identified in a variety of cetacean species, both odontocetes and mysticetes. CSLAstV2 was found able to infect both pinnipeds and cetac eans. The recombination between a stro virus es of humans and marine mammals as evidenced by analysis of CSLAstV3 indicates that a human and a marine mammal virus can infect the same host. The nomenclature of astroviruses is problematic. Astroviral s pecies are defined by the ICTV on the basis of host species of origin (Monroe et al., 2005) We have identified significant diversity of astroviruses in bottlenose dolphins, California sea lions,

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119 and minke whales. Similarly, others have recently identified sign ificant diversity in astroviruses of humans (Finkbeiner et al., 2008, Finkbeiner et al., 2009a, Finkbeiner et al., 2009b, Kapoor et al., 2009) and individual bat species ( Chu et al., 2008, Zhu et al., 2009). Kapoor et al. (2009) have suggested that astrov iruses be grouped and named based on both host species and a genetic distance criteria using either the capsid or RdRp loci. The apparently high recombination rate of astroviruses complicates this. Recombination appears to be especially common near the end of RdRp/start of the capsid (Walter et al., 2001, Pantin Jackwood et al., 2006, Strain et al., 2008, Rivera et al., 2010), as was seen here. Capsid and RdRp genes may have very different ancestries, and naming according one gene or the other may not ac curately indicate virus behavior. Use of a multiple gene nomenclature, as has been done with hemagglutinin and neuraminidase genes for influenza viruses, may be a potential solution. The data generated also shows that significant diversity of the Astrovir idae beyond Avastrovirus and Mamastrovirus exists. More study is indicated to determine the diversity, host range, and clinical significance of these viruses. The very high astrovirus prevalence seen in marine mammals, the great diversity seen in astrovi ruses from these species, the stability of astroviruses in the marine environment, and the widespread geographic distribution of marine mammal astroviruses all suggest that the marine environment plays a central role in astrovirus biology. We have shown t hat host fidelity of these viruses is limited. Given that most recent emerging diseases have been associated with host switches, and the marine environment appears to be a significant

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120 reservoir of astroviral diversity, there is a need to further understand marine astroviruses.

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121 APPENDIX A MUSCLE ALIGNMENT OF PARTIAL ASTROVIRAL RIBONUCLEIC ACID (RNA) DEPENDENT RNA POLYMERASE AMINO ACID SEQUENCES 1 50 AvNeph1 PVQLFQRMRELRKFFLTRRSRRRYGKLLDWYNAQLTDRITLLPTGEVTH Turkastr1 PPELFRRIKLMRFFLLDPKYKTPENRDRYNWYVENLIDKVVLLPTGEVCK Turkastr2 PKSLFWRIRQIRFFFLHDSHKTPKMRRLYNWYVKNLLEKIILLPTGEVCQ MLB1 PPTLLMHIKKLRFTLMGSMSR--KYENVYKWYCRNLINRFVVLPSGEVTA BdAstV1 PTQLFRRIKKLRWSFINKEQREYYSRMYEWYCYNLFNRYVLLPSGEVTE CslAstV2 PTPLLLHIKKLRWSMINEVQRKKYQSLHDWYCHNLVHRKVVLPSGEITE CslAstV3 PPALFRHIKEIRWNFINKEQREKYRHVHEWYVDNLLKRNVLLPSGEVTV Humastr3 PPALFRHIKEIRWNFINKDQREKYRHVHEWYVDNLLNRHVLLPSGEVTV Humastr5 PPSLFRHIKEIRWNFINKDQREKYRHVHEWYVDNLLNRHVLLPSGEVTL Humastr4 PPALFKHIKEIRWNFINKDQREKYRHVHEWYVDNLLNRHVLLPSGEVTL Humastr1 PPALFKHIKEIRWNFINKDQREKYRHVHEWYVDNLLNRHVLLPSGEVTL Humastr8 PPALFKHIKEIRWNFINKDQREKYRHVHEWYVDNLLNRHVLLPSGEVTL BatAstPa PPEVFFQIKDIRFGLLSPEYRTVRNRSVYKWYCENLINRDVVLPSGEITH BatAstMm2 PNQVFHKIKDIRFNFLSKEYRTKENREIYDWYCKNLTNRVVLLPSGEVTK BatAstMp PNEVFRHIKWFRFNMLDPVYKTDLNRSVYSWYVDQMLHRYVLLPSGEVTI BatAstMm PVEVFRHIKDFRFSMLDPVYKTDLNRSVYDWYVSQLVYRYVLLPSGEITI BatAstMp2 PVEVFRHIKNFRFMMLDPVYKTDMNKSIYDWYVGQLMYRYVLLPSGEITI SslAstV 1 PVEVFLAIKQVRFSFLADEYKTLENYDIYSWYCHNLVHRFVCMPSGEITL Ovineastr PSQIFKHIKNFRFSMLAKEYQTPELRNMYHWYVDNILRRYVCMPSGEITI Minkastro PREIFAKIKSFRFSCLAEEFQTDANRAMYQWYCDSLLDRYVLMPSGEVTR CslAstV 1 PREVFMHIKKFRFSCLADEYKTPELESMYDWYCNALLERYVLLPSGEVTL 51 100 AvNeph1 VKKGNPSGQFSTTVDNNLVNEWLTAFEFGYQHLENHGIIPTVRDYRANVD Turkastr1 IYGGNPSGQFSTTVDNNFVNVWLTVFELAYLFYKEHNRLPTICEIKKHTD Turkastr2 VKKGNPSGQFSTTVDNNMINVWLTTFEVSYLFFKQRGRLPTEKELQENCS MLB1 QQRGNPSGQMSTTMDNNMINYWLQAFEYKFLN-------LPEEEWMHFD BdAstV 1 QTRGNPSGQFSTTMDNNMVNVWLQAFEFAYFF ------GPDKKKWSKVD CslAstV 2 QHRGNPSGQFS TTMDNNCVNLWIQAFEFAYMI ------GPDKELWKKYD CslAstV 3 QTRGNPSGQFSTTMDNNMVNVWLQAFEFAYFN ------GPNKELWKNYD Humastr3 QTRGNPSGQFSTTMDNNMVNFWLQAFEFAYFN-------GPNKELWKTYD Humastr5 QTRGNPSGQFSTTMDNNMINFWLQAFEFAYFN-------GPNKDLWKTYD Humastr4 QTRGNPSGQFSTTMDNNMVNFWLQAFEFAYFN-------GPNKDLWKTYD Humastr1 QTRGNPSGQFSTTMDNNMVNFWLQAFEFAYFN-------GPDKDLWKTYD Humastr8 QTRGNPSGQFSTPMDNNMVNFWLQAFEFAYFN-------GPDKDLWKTYD BatAstPa QDRGNPSGQVSTTMDNNMINTFLQAFEFIYLN---NLTIETAKELWESYD BatAstMm2 QLNGNPSGQVSTTMDNNMVNTFCQAFEFMFVN---GLTIDEAKKKWVDYD BatAstMp QDRGNPSGQISTTMDNNLVNSFLQAFEFAFIH--PELDLDELTELYKQCD BatAstMm QDRGNPSGQISTTMDNNLVNTFLQAFEFAYVN--PELSLDELDVLYAQCD BatAstMp2 QDRGNPSGQISTTMDNNLVNTFLQAFEFAYMN--PELSSDELDTLYAQCD SslAstV 1 QERGNPSGQVSTTMDNNMCNVFFQAFEYAWLH -PCKTLDELHEDWE RVD Ovineastr QHKGNPSGQVSTTMDNNLVNVFLQAFEYAYLH--PEKSMDELRKDWESYD Minkastro QTKGNPSGQISTTMDNNLCNVFFQAFEYAYIH--PEKSIEELRESWDRCD CslAstV 1 QTKGNPSGQISTTMDNNLCNVFFQAFEFAYIN -PDLSMQELCDAWERCD

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122 101 150 AvNeph1 FLCYGDDRLLAFNPSFVNYDPQVTIDM----YKNIFGMWVKPENIKLFD Turkastr1 WICYGDDRLLAVDKRFINSYDTAAVIAM----YKDVFGMWVKPDNIKVFP Turkastr2 MICYGDDRLLSIRKGFVEYEPDTVIDM----YKNIFGMWVKRNNIKIQD MLB1 TLIYGDDRISTYRSIPNEYTKTIVDM----YKNVFGMWVKPEKVKVSE BdAstV1 ALIYGDDRLSSWPEIPVNYGERVVEM----YKKVFGMWVKPEKVKVQN CslAstV2 TLVYGDDRLSTTPKIVDNYEEKVIEM----YKNIFGMWVKPGKVKISE CslAstV3 TIVYGDDRLSTTPVVPDNYEERVIEM----YREIFGMWVKPGKVVCKD Humastr3 TVVYGDDRLSTTPSVPDNYEERVIAM----YRDIFGMWVKPGKVICRE Humastr5 TVVYGDDRLSTTPSVPENYEERVIDM----YRDIFGMWVKPGKVICRE Humastr4 TVVYGDDRLSTTPSVPNNYEERVITM----YRDIFGMWVKPGKVICKD Humastr1 TVVYGDDRLSTTPSVPDDYEERVITM----YRDIFGMWVKPGKVICRN Humastr8 TVVYGDDRLSTTPSVPDNYEERVITM----YRDIFGMWVKPGKVICRD BatAstPa SLVYGDDRVTSTPLVPSNYVERVVGM----YADIFGMWVKPDNVKVSN BatAstMm2 TIVYGDDRITSS PLVPPDYSDRVIRM----YKDIFGMWVKPENVKISD BatAstMp SAVYGDDRLSSWPCVPDDYVHQVVCM----YEHVFGMWVKPEKVKISD BatAstMm SLIYGDDRLSSWPVIPEDYVHKVSCM----YEHVFGMWVKPEKVKVSD BatAstMp2 SLIYGDDRLSSWPCVPEDYETRVSGM----YEHVFGMWVKPDKVKVSD SslAstV 1 SLVYGDDRLSFV PDVPSDYV D KVVAM ---YETVFGMWVKPTKVVVSD Ovineastr SLIYGDDRLTTSPSVPNDYVTRVVAM----YKDIFGMWVKPEKVKVSH Minkastro SLIYGDDRLTTFDHVPPDYVDRVVHM----YKDVFGMWVKPEKVIVSD CslAstV 1 SLIYGDDRLTTF PSIPSDYV NRVVDMYKDIYKDIFGMWVKPDKVVVQD 151 200 AvNeph1 SPTGSSFCGFTLVKPHGQWVGVVNVNKLLQSLKTPTRRLPDLESLWGK Turkastr1 SLEGVSFCGMVWTKRKGQYVGKPNVDKILSTLSDPVSRLPDIQSLWGK Turkastr2 TPEGLSFCGLTIVKSSTGAYVGVPNVNKILSTLENPVRRLPDVESLWGK MLB1 SLEGLSFCGFTYTPNG-----PVPSEPYKLMASLLKPATKLPDLIALHGK BdAstV 1 TLVGLSFCGFTVDQ ---NYEPVP SSPEKLLAGLLTPTKKMPDLESLHGK CslAstV 2 TLVGLSFCGFTVDQ ---NLEPIPTAPEKLMASLLKPSTKLPDLESLHGK CslAstV 3 TIVGLSFCGFTVNE ---DLEPVPTSPEKLMASLLKPYKVLPDLESLHGK Humastr3 SIIGLSFCGFTVNS----DLEPVPTSPEKLMASLLKPYKVLPDLESLHGK Humastr5 SIVGLSFCGFTVNA----DLEPVPTSPEKLMASLLKPYKILPDLESLHGK Humastr4 SIVGLSFCGFTVNE----NLEPVPTSPEKLMASLLKPYKILPDLESLHGK Humastr1 SIVGLSFCGFTVNE----NLEPVPTSPEKLMASLLKPYKILPDLESLHGK Humastr8 SIVGLSFCGFTVNE----NLEPVPTSPEKLMASLLKPYKILPDLESLHGK BatAstPa TVNGLSFCGFTNNLISNMYLPVPTNVNKLVASLITPVKKLQDIESLAGK BatAstMm2 TLVGLSFCGFTNIREKGMYLPVPSNCEKLVAALVRPVKKLPDIEALAGK BatAstMp TLVGLTFCGFTIFKDGDLYLPVPVDAWKFISSTLHPVKALPDFDALVGK BatAstMm TLEGLTFCGFTVIRSGGFYLPIPVDAWKFISSTICPTKQLPDFDALVGK BatAstMp2 TLEGLTFCGFTVIRSGGLYLPIPFDAWKFISSTLCPTKQLPDFDALVGK SslAstV 1 TPVGLTFCGFTVSP ---DLLPMPTNTEKLIAALVTPTRKLKDMDALYAK Ovineastr SPVGLSFCGFVITHQDGQYLPVPAEEAKLLASLLRPTKKLENMDALYGK Minkastro TPVGLSFCGFTVGP----DLMPVPTDCDKLVASLVTPTKKLQDIVALYSK CslAstV 1 TPIGLSFCGFTVNQ ---DFMPVPTECDKLIASLVTPTKKLADIYSLYSK

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123 201 250 AvNeph1 LVSLKIMCYHSDPEAVS--YLSNQIRRVEEYARAEGIELPEVGPDFYRK Turkastr1 LVSLRLLCENESDEVVD--YLDKQIESVSRHAKEAGIALPKIGPDFYAE Turkastr2 LVSLRILCENAPSNVKH--FLDEQISNVEEFAARENIQLPEVGPDFYSR MLB1 LLCFQLLMANDTAHPFYGYIEQCLQYTHRALSDVS--LPRRFTRRQLEY BdAstV1 LLCFQLLSAFLPEDHPFKNYVEMSLASTAKQLPGTA--LPPRFTEEQLHC CslAstV2 LLCYQLLSTFLDEEHPFKGYVEQCLARTSKQLRDSG--LPARFTEEQLRR CslAstV3 LLCYQLLAAFMAEDHPFKVYVEHCLSRTAKQLRASG--LPARLTEEQLHR Humastr3 LLCYQLLAAFMAEDHPFKVYVEHCLSRTAKQLRESG--LPARLTEEQLHR Humastr5 LLCYQLLAAFMAEDHPFKVYVEHCLSRTAKQLRDSG--LPARLTEEQLHR Humastr4 LLCYQLLAAFMAEDHPFKVYIEHCLSRTAKQLRDSG--LPARLTEEQLHR Humastr1 LLCYQLLAAFMAEDHPFKVYVEHCLSRTAKQLRDSG--LPARLTEEQLHR Humastr8 LLCYQLLAAFMAEDHPFKVYVEHCLSRTAKQLRDSG--LPARLTEEQLHR BatAstPa VLSFKVLMHNLPDDDPGKIFILNCESALRRHMDAVGQPWVNFTTSMLDF BatAstMm2 VLSYKVLTHNLPDDDPSKQFVLACELSINKHLRARGVDPITFTREMLDF BatAstMp ILSYQILTHNLPDDDPVKTWFEEAHASLTLHNRVHGGDPLPVMSRDMRDF BatAstMm ILSYQILTHNLPDDDPVKKWFEEAHSALVMHNRVSGGDPLPTITRDMRDF BatAstMp2 ILSYQILTHNLPDDDPVKTWFEEAHSALVVHNRVSGGDPLPTITRDMRDF SslAstV 1 LQCYGILGHNLPTDDEFKNYIYLALEVLARHIRAAGGEEPVRFTDRMLDA Ovineastr LLCYRILNHNLPNDNKFRNYILVALEVMARHYSSRGEEPPFYVTESMLDK Minkastro VLCYRILGHNLSDEHEFKRYVRVALEVLARHIRNLGGEEPVHVTERLLDK CslAstV 1 VLCYNILGYNLEDEHEFKNYARIALEVLARHIRNMGGEEPVHVTEKML DV 251 263 AvNeph1 IW----------Turkastr1 IW----------Turkastr2 IW----------MLB1 IWRGGPNDDYG-BdAstV 1 IWRGGPKICNG -CslAstV 2 IWRGGPKTCDG -CslAstV 3 IWRGGPKKCDG -Humastr3 IWRGGPKKCDG-Humastr5 IWRGGPKKCDG-Humastr4 IWRGGPKKCDG-Humastr1 IWRGGPKKCDG-Humastr8 IWRGGPKKCDG-BatAstPa LWSGGPN-----BatAstMm2 LWRGGPN-----BatAstMp LWSGGPKKDGRST BatAstMm LWS---------BatAstMp2 LWR---------SslAstV 1 LWRGGPK -----Ovineastr LWRGGPKFDYG-Minkastro LWRGGPK-----CslAstV 1 LWR GGPKRRDG -

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124 APPENDIX B T COFFEE ALIGNMENT OF PARTIAL ASTROVIRAL CAPSID AMINO ACID SEQUENCES 1 60 AvNeph1 MAGGATAPAG---------------------AKPKQPKQKQKKPSSQARKKPSQKQKAMTurkastr1 -------------MS--APAGKAGPKAQ-----KKCKVVTQ---KTKTVPKKTKQQKPRK Turkastr2 MAAMADKVVVKKTTT--RRRGRSNSRSR-----SRSRSRSRTKKTVKIIEKKPEKSILK cheetahcap MASKPGKEVTVEVNNTNGRSRSKSQPR-----SRSRGR--GKTVKITVNSKGGSRGR felineastr MASKPGKEVTVEVNNTNGRSRSKSQPR-----SRSRGR--GKTVKITVNSKGGSRGR Humastr1 MASKPSKQVTVEVN---NGRSRSRSRPR-----SQSRGR--DKSVKITVNSRNKGR--R Humastr2 MASKSDKQVTVEVNN--NGRNRSKSRAR-----SQSRGR--GRSVKITVNSHNKGR--R Humastr4 MASKSDKQVTVEVNN--NGRSRSKSRAR-----SQSRGR--GRSVKITVNSNNKGR--R Humastr5 MASKPSKQVTVEVN---NGRSRSRSRPR-----SQSRGR--DKSVKITVNSRNKGR--R Humastr8 MASKSDKQVTVEVNN--NGRSRSKSRAR-----SQSRGR--GRSVKITVNSHNKGR--R Porcineast MASKSGKDVTVKVENTNGRGRSRSRSR-----SRSRAR--NKNVKITINSKPGASGG Ovineastro --M AEKPQQKAVAS--AAKQLAKEVVKLDKITKSNGKQHPQKNVPARKWRPRQA---Minkastro MASANQAAKAEAKK--VIEKVAKEVI---KETKNSAQRN---QGPGKRWNSKKGRHMBdAstV1 MANGRSKDVSVEVKA--SGSQRSKSRSR-----SRSRGR--TPAVKVTVNSKAKRFTRR CslAstV1 MAHANQAAKSEAKK--EVKKVVKELVKDVAKEAKKDAQRR---SAPNRRWKGQRG---CslAstV2 MASASGKNVTVEVKNTG--SRSKSRGR-----SQSRGR--SKNVKITVNSKPNRK--Q CslAstV3 MASKSDKKVTVEVKSNGNGRSRSKSRSR-----SQGRGR--KSDVKITVNSKPRGG--G MLB1 MANASKGVTVNINN-----AKRKPRFT-----NNQRAR--STRPNFTPAPKFR----BatAstMp NNNITSAAPTADATP--SG--------VSTTTAPRAPRRRRRSRRVRFVNRPLIENDVFSslAstV 1 MAT AGQAAKAEAKK -EVKKLVKEVKKEVKQERKN NHAG ----------QGRRGRGQ 61 120 AvNeph1 --KPVKQELRKVEKQVR--VLKARTNGPKVNDTMKTTVTVGTLVGQTQSGLNRQLRVSF Turkastr1 VRLQKVE-------RQVKTL--KAKTRGPKISDTFSTVVTVGRIIGNNDDSLTRQLKVFV Turkastr2 KIDQAERRDA----KQLRRI--RKKVQGPPVNSRMTTVVTLGQITGNKDNTLERKHKCFL cheetahcap QNGRGKRQSTQRVRNIVNKQLRKQGVTGPKPAICQRATATLGTVGSNTSGTTEIEACILL felineastr QNGRGKRQSAQRVRKIVNKQLRKQGVTGPKPAICQRATATLGTVGSNTSGTTEIEACILL Humastr1 QTGRNKHQSNQRVRNIVNKQLRKQGVTGPKPAICQRATATLGTVGSNTSGTTEIEACILL Humastr2 QNGRNKYQSNQRVRKIVNKQLRKQGVTGPKPAICQRATATLGTIGTNTTGATEIEACILL Humastr4 QNGRNKYQSNQRVRKIVNKQLRKQGVTGPKPAICQTATATLGTIGSNTTGATEIEACILL Humastr5 QNGRNKHQSNQRVRNIVNKQLRKQGVTGPKPAICQRATATLGTVGSNTSGTTEIEACILL Humastr8 QNGRNKYQSNQRVRKIVNKQLRKQGVTGPKPAICQTATATLGTIGSNTTGATEIEACILL Porcineast QRRRGKPQSDKRVRSIVKQQLDKSGVTGPKPAIRQRATATLGTIGSNSSGKTELEACILT Ovineastro ------KPNNRRVTHKIKRELHKQGLEGPASRFRVTVSATIGKVGPNKEQGPELQIATFL Minkastro PKNNNNKGMKRTVDNEVKQKLKKEGLEGPRSRFSVRVSATIGKIGPNKEQGPELQIATFL BdAstV 1 PSRRSFRAKNNSVKQQVR NQLKKQGLTGPAPAVVQTATATLGTIGPNTGNDAEREISFYL CslAstV 1 -----KQTKQTVHKEANKKLRKEGLEGPRPRFSVRVSATIGKVGPNKEQGPELQIATFL CslAstV 2 RRTGPRGGSSKRVARLVKQHLDKSGATGPKPAIAQKATATLGVVGANTSGNTELEMCLMT CslAstV 3 RTGRGARQSNQRVARIVRKQLDKSGVKGPKPAVKQRATATLGTVGSNTSGNTELESCI FT MLB1 --------KRRFIPNRNRRRRQNTSTTGPKPAVSQTITATLGTVGSNLSDVVETECAVFL BatAstMp --SGRRRFPRRFITRAVKREIKREGLEGPKVSVQQKITSTFGMIGPNTTDNAELELNFFL SslAstV 1 RNNNHGRETKRTVDREVNRKMKREGLEGPKSRFTVRVSATIGRLGPNTTQGPELQLSAFM

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125 121 180 AvNeph1 NPLLMKSTEGGSTTPLSIRASMYEMWKPLSVEIFATPLSGFSSVVGSVGFMVITLNGL Turkastr1 NPLLMKNQDSGSTSSPLSIRASQYGLWKIAKLHVYFTPLAGSANVIGTVSFASLEQESTurkastr2 NPLLMKSQETGQTATPLSVRASQYNLWKLSRLHVRLIPLAGKANILGSVVFLDLEQEANcheetahcap NPVLVKDATGSTQFGPVQALGAQYSMWKLKYLNVKLTSMVGASAVNGTVVRVSLNPTSfelineastr NPVLVKDATGSTQFGPVQALGAQYAMWKLKYLNVKLTSMVGASAVNGTVVRVSLNPTSHumastr1 NPVLVKDATGSTQFGPVQALGAQYSMWKLKYLNVKLTSMVGSSAVNGTVVRVSLNPTSHumastr2 NPVLVKDATGSTQFGPVQALGAQYSMWKLKYLNVKLTSMVGASAVNGTVLRISLNPTSHumastr4 NPVLVKDATGSTQFGPVQALGAQYSMWKLKYLNVRLTSMVGASAVNGTVVRISLNPTSHumastr5 NPVLVKDATGSTQFGPVQALGAQYSMWKLKYLNVKLTSMVGSSAVNGTVVRVSLNPTSHumastr8 NPVLVKDATGSTQFGPVQALGAQYSMWKLKYLNVRLTSMVGASAVNGTVVRISLNPTSPorcineast NPILVKDNTGNNTFGPIVALGAQYSLWRIRFLRIKFTPMVGQSAVTGTVVRASLNPTAOvineastro HPSLVKEPNDGSNFGPLQAAAAQWGLWRISDLEVRFTPLVGSSAVTGSVTRASLNLTQMinkastro HPSLMKEPNDGTNFGPLQAAAAQWGLWRLSSLEVKCTPLVGSSAVTGSIYRMSLNLTQBdAstV 1 NPALTKENTGSNAFGPVQALAAQYSMWRCSRAEIRFTPLIGPSAISGTAYRCSLNM AG CslAstV 1 HPGLMKEPNDGTNFGP LQPPAAQWGMWRIASLSVRFTPLVGPSPVTGSVYRVSLNL TQ CslAstV 2 NPCLVKDNTGNNAFGPVQALGAQYTMWRIKNLTVKLTPLVGSSAIVGTVVRMSLNS TS CslAstV 3 NPCLVKDSTGSAQFGPIQALGAQYSLYKLSYLNVTLTPLVGASAVSGTVVRVSVNP TA MLB1 NPIIAKDSGASATFGPLQSLGAQYALWRLKWLEVRLQPLVGNSAVSGTVARVSLNMTTBatAstMp HPALAKEANDGTAFGPLQALAAQYSLWKIKYLTLRFTPMVGASAVSGTVVRASLNLSQSslAstV 1 HPSLMKEPNDGTNFGPLQAAAAQWGLWQLSSMTVRFTPLVGPSAVTGSCYRASLNL TQ 181 220 AvNeph1 EASADSIDTIKARRHVQMALGRPYRLKLSARELAGPREG Turkastr1 GVATAESPDTIKAKYHAEVPIGSRFVWKVPPRMLTGPREG Turkastr2 TAGPESVDTIKARPHVEVPIGSKTVWKVHPRSALGPRQG cheetahcap TPSSTSWSGLGARKHLDVTVGKNAVFKLRPXDLGGPRDG felineastr TPSSTSWSGLGARKHLDVTVGRDAVFKLRPSDLGGPRDG Humastr1 TPSSTSWSGLGARKHLDVTVGKNAVFKLKPADLGGPRDG Humastr2 TPSSTSWSGLGARKHMDVTVGRNAVFKLRPSDLGGPRDG Humastr4 TPSSTSWSGLGARKHLDVTVGKNAVFKLKPSDLGGPRDG Humastr5 TPSSTSWSGLGARKHLDVTVGKNAVFKLKPADLGGPRDG Humastr8 TPSSTSWSGLGARKHLDVTVGKNAVFKLKPSDLGGPRDG Porcineast TPSSTGWSGLGARRHIDIVVGKAATFNLKASDLSGPREG Ovineastro SPGATSWGGLGARKHLDVPTGVSKVWKLRRGDLTGPRQT Minkastro SPGNASWGGLGARKHKDIPAGKSVSWKLQRGDLAGPRQT BdAstV 1 T PSQTSWSGLGSRKHKDMHIGKSGSFKLTKKELSGPKET CslAstV 1 S PGNSSWGGLGARRHMDIPVGRQVTWKLTKGELYRPRQT CslAstV 2 T PSSTSWSGLGARLHADAVVGRSATFRLKPRDLAGPREG CslAstV 3 T PSSTS WSGLGARYHMDVMVGRKAVFKLRANQLNGPREG MLB1 GPTLNSWSGLGARIHKDVRVGSNLVWRIKQRLVSGPCET BatAstMp SPGGSNWSGLGTRLHIDMHPGQVATFHLRGDQVGGPRDG SslAstV 1 S PGNASWGGLGARKHVDISVGRTVSWKLSRGDLAGPRQT

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126 APPENDIX C OPTICAL DENSITY AT 405 NANOMETERS ( OD405) VALUES FOR 46 DOLPHIN SERUM SAMPLES USING A BOTTLENOSE DOLPHIN ASTROVIRUS 1 (BDASTV1) PEPTIDE E NZYMELINKED IMMUNOSORBENT ASSAY (ELISA) AGAINST SELECTED PEPTIDE PAIRS Animal ID Date plate1 399 455 plate2 322 616 plate3 399scr 455scr Blank 0.555 0.362 0.454 1 9 /28/06 0.155 0.375 0.427 2 7/26/06 0.217 0.361 0.555 3 8/3/06 0.061 0.418 0.559 4 8/10/06 0.111 0.298 0.456 5 11/2/06 0.149 0.280 0.474 6 8/24/06 0.896 0.904 0.785 7 8/11/06 0.102 0.256 0.480 8 8/3/06 0.044 0.391 0.905 21 8/24/06 0.747 0.682 1.341 24 8/8/06 0.046 0.916 0.677 25 1/26/05 0.027 0.263 0.472 26 7/6/06 0.292 0.446 0.748 27 10/3/06 1.099 1.247 1.317 28 9/26/06 0.154 0.188 0.309 29 9/26/06 0.278 1.031 0.524 30 9/22/06 0.054 0.443 0.729 31 9/27/06 0.179 0.410 0.327 33 2/13/06 0.084 0.015 0.061 33 12/28/06 0.025 0.058 0.090 34 8/7/06 0.390 0.760 0.502 35 7/31/06 0.216 0.213 0.501 36 8/16/06 0.655 0.514 0.637 37 7/6/06 0.186 0.183 0.541 38 8/29/06 0.267 0.588 0.528 39 4/15/06 0.098 0.188 0.088 40 8/23/06 0.381 0.291 0.331 13 6 1/9/07 0.656 0.826 0.913 137 6/25/00 0.446 0.450 0.496 138 8/11/06 0.281 0.194 0.201 139 8/23/00 0.594 0.410 0.508 140 12/18/01 0.234 0.735 0.347 141 11/9/93 1.128 1.000 0.988 142 3/2/92 0.372 0.658 0.599 143 3/4/93 1.415 1.437 1.409 144 9/23/92 0.618 0.595 0.428 145 11/19/93 0.710 0.585 0.488 146 8/9/95 0.784 0.779 1.070 147 1/18/07 0.103 0.243 0.462

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127 149 1/18/07 0.258 0.250 0.520 150 1/24/07 0.284 0.816 0.396 151 1/17/07 0.347 0.275 0.356 152 1/30/06 0.036 0.073 0.135 153 1/18/07 0.063 0. 512 0.292 154 12/28/06 0.375 0.391 0.301 155 1/10/07 0.375 0.582 0.458 156 1/17/07 0.268 0.438 0.347

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128 APPENDIX D OD405 VALUES FOR 146 DOLPHIN SERUM SAMPLES USING A BDASTV1 PEPTIDE ELISA AGAINST ALL FOUR PEPTIDES (TT322, TT399, TT455, TT616) Collectio n Animal ID Date OD 405 Managed Open Water 1 9/28/06 0.593 2 7/26/06 0.833 3 8/3/06 0.544 4 8/10/06 0.580 5 11/2/06 0.604 6 8/24/06 1.208 7 8/11/06 0.618 8 8/3/06 0.565 9 9/18/06 1.577 10 5/25/05 0.666 11 8/17/06 0.486 12 9/18/06 0.97 2 13 7/7/05 0.674 14 9/28/06 0.513 15 7/6/06 0.496 16 4/12/06 0.421 17 8/17/06 0.655 18 8/22/06 0.428 19 8/24/06 0.628 20 9/11/06 0.547 21 8/24/06 1.670 22 9/14/06 0.819 23 8/30/06 0.603 24 8/8/06 0.399 25 1/26/05 0.404 26 7/6/0 6 0.572 27 10/3/06 1.080 28 9/26/06 0.515 29 7/20/06 0.520 30 9/22/06 0.486 31 9/27/06 1.059 32 9/12/06 1.620 33 2/13/06 0.469 33 12/28/06 0.858 34 8/7/06 1.502 35 7/31/06 1.074 36 8/16/06 1.631 37 7/6/06 1.218 38 8/29/06 1.265 39 4/5/06 0.449 40 8/23/06 1.065

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129 41 9/15/06 0.724 42 8/11/06 1.324 43 4/11/06 0.397 44 9/18/06 0.959 45 8/8/06 0.518 46 9/14/06 0.787 47 8/8/06 0.801 48 9/13/06 0.818 49 9/19/06 1.003 50 8/29/06 0.564 51 11/2/06 0.718 52 11/7/06 0 .830 53 7/6/06 0.676 54 7/26/06 0.730 55 9/22/06 0.487 56 9/29/06 2.498 57 7/26/06 0.860 58 7/31/06 0.739 59 1/26/06 0.633 60 9/18/06 0.249 61 8/24/06 0.002 Wild 62 1.702 63 0.780 64 1.463 65 0.999 66 0.872 67 2.147 68 1.423 69 0.770 70 1.272 71 1.285 72 1.341 73 1.227 74 1.029 75 0.813 76 1.393 77 1.275 78 1.318 79 0.843 80 0.951 82 0.969 83 1.254 84 1.068 85 2.110 86 1.020

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130 87 0.951 88 1.627 89 1.246 9 0 0.664 91 0.793 92 1.414 93 1.238 94 0.418 95 0.701 96 0.954 97 1.035 98 1.521 99 0.488 68 0.959 100 0.909 101 0.638 102 0.698 103 8.830 104 1.602 105 0.698 106 6.850 107 0.466 108 0.675 109 0.2 41 110 0.860 111 0.838 112 0.523 113 0.772 114 0.781 115 0.371 116 0.346 117 0.320 118 2.267 119 1.195 120 3.147 121 1.903 122 0.075 123 2.242 124 1.042 125 2.018 Captive collection 1 126 5 Jun 06 1.089 127 16 Jul 06 0.788 128 3 Feb 07 0.937 129 1 Nov 07 1.087

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131 130 5 Apr 06 4.870 131 5 Sep 06 0.587 132 17 Oct 06 1.822 133 25 Oct 05 0.442 134 13 Mar 06 0.351 135 2 May 05 0.563 Captive collection 2 136 1/9/07 1.834 137 6/25/00 1.16 0 138 8/11/06 0.729 139 8/23/00 1.096 140 12/18/01 1.060 141 11/9/93 2.185 142 3/2/92 1.374 143 3/4/93 2.062 144 9/23/92 1.251 145 11/19/93 1.185 146 8/9/95 1.855 Captive collection 1 147 1/18/07 0.363 148 1/18/07 0.067 149 1/18 /07 0.334 150 1/24/07 0.335 151 1/17/07 0.209 152 1/30/06 0.356 153 1/18/07 0.222 154 12/28/06 0.509 155 1/10/07 0.156 156 1/17/07 0.263 Time series Animal Date Run1 Run2 4 28 Feb 06 1.034 4 10 May 06 1.026 4 10 Aug 06 0.6 65 4 9 Nov 06 0.815 4 18 Jan 07 1.270 4 25 Jan 07 1.249 4 28 Jan 07 0.792 4 8 Feb 07 0.994 4 15 Feb 07 1.003 4 20 Feb 07 0.953 4 28 Feb 07 0.783

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132 4 13 Mar 07 0.790 4 5 Apr 07 1.577 4 2 May 07 1.468 4 5 Jun 07 1.543 4 9 Aug 07 1.401 4 10 Oct 07 0.833 4 11 Jan 08 0.998 4 14 Feb 08 0.851 9 26 Jul 01 2.140 1.957 9 29 Jul 02 2.004 1.785 9 5 Jun 03 1.694 1.304 9 13 Jan 04 1.725 1.346 9 13 Oct 04 1.847 1.737 9 15 Feb 05 1.618 1.565 9 30 Jun 05 1.821 1.974 9 8 Dec 05 2.043 1 .696 9 28 Feb 06 1.814 1.753 9 5 Jun 06 1.927 1.735 9 5 Jul 06 1.800 1.669 9 25 Aug 06 1.813 1.592 9 10 Oct 06 1.739 1.867 9 24 Nov 06 1.514 1.889 9 23 Jan 07 1.565 1.734 9 19 Mar 07 1.018 1.211 9 26 Jul 07 1.034 1.124 9 27 Nov 07 1.341 1.599 9 12 Mar 08 1.106 1.313 21 11 Jan 06 0.922 0.937 21 9 Feb 06 0.795 0.611 21 8 Mar 06 1.096 0.729 21 6 Apr 06 1.302 0.495 21 1 Jun 06 8.110 5.463 21 10 Aug 06 2.188 1.299 21 13 Sep 06 1.700 1.077 21 19 Sep 06 1.453 0.719 21 25 Sep 06 2.796 1.731 21 23 Mar 07 1.093 0.598 21 19 Apr 07 1.232 0.671 32 26 Jul 01 1.058 32 29 Jul 02 0.549 32 5 Jun 03 1.079 32 13 Jan 04 0.530 32 13 Oct 04 0.655 32 15 Feb 05 1.208 32 30 Jun 05 1.270 32 8 Dec 05 0.327

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133 32 28 Feb 06 0.691 32 5 Jun 06 0.705 32 5 Jul 06 1.085 32 25 Aug 06 0.150 32 10 Oct 06 0.609 32 24 Nov 06 0.952 32 23 Jan 07 0.629 32 19 Mar 07 0.875 32 26 Jul 07 0.913 32 27 Nov 07 0.991 34 17 Sep 01 0.615 34 27 Sep 02 0.709 34 8 May 03 0.494 34 12 Dec 03 0.355 34 4 M ay 04 0.470 34 11 Jan 05 0.324 34 20 May 05 0.523 34 26 Jan 06 0.414 34 24 Apr 06 0.348 34 24 May 06 0.442 34 29 Jun 06 0.204 34 31 Jul 06 0.130 34 28 Sep 06 0.343 34 6 Dec 06 0.218 34 23 Aug 07 0.266 34 8 Feb 08 0.176 36 17 Jul 01 0.651 36 16 Jul 02 0.534 36 28 May 03 0.567 36 4 Nov 03 0.603 36 21 Apr 04 0.652 36 24 Nov 04 0.723 36 23 Feb 05 0.732 36 1 Sep 05 0.527 36 16 Feb 06 0.573 36 2 Jun 06 0.671 36 6 Jul 06 0.612 36 28 Jul 06 0.465 36 21 Sep 06 0.726 36 5 Oct 06 0.655 36 17 Nov 06 0.661 36 27 Feb 07 0.263 36 13 Sep 07 0.403 36 20 Mar 08 0.403 56 25 Oct 01 1.412 56 12 Sep 02 0.835

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134 56 28 May 03 1.359 56 22 Apr 04 1.330 56 26 Oct 04 1.432 56 8 Feb 05 1.307 56 21 Jul 05 1.461 56 20 Dec 05 1.293 56 7 Mar 06 1.281 56 8 Jun 06 1.680 56 1 Aug 06 1.348 56 8 Nov 06 1.401 56 22 Feb 07 1.273 56 24 May 07 1.159 56 18 Oct 07 1.587 56 8 Apr 08 0.795

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145 BIOGRAPHICAL SKETCH Jim Wellehan grew up in Auburn, Maine. He graduated from Bowdoin College in 1992 with a B.A. in biochemistry. In 2001, he graduated from the University of Minnesota as a Doctor of Veterinary Medicine, and a Master of Science in Veterinary Molecular Biosciences on Mycoplasma infections in passerine birds in Minnesota This was followed by an internship in Avian/Exotic/Wildlife Medicine and Surg ery at Ontario Veterinary College in 2001/2002, and a residency in Zoological Medicine at the University of Florida from 20022005. After becoming a diplomate of the American College of Zoological Medicine and serving as a clinical instructor at Disneys Animal Kingdom in 2005/2006, he retur ned to the University of Florida to begin Doctor of Philosophy studies and served as an attending clinician on the Zoological Medicine Service at the College of Veterinary Medicine. He became a diplomate of the American College of Veterinary Microbiologis ts in both the Virology and Bacteriology/Mycology subspecialties in 2006. He is currently a Clinical Assistant Professor at the University of Florida College of Veterinary Medicine and serves as an attending clinician for the Zoological Medicine Service, as service chief for the Clinical Microbiology Laboratory at the University of Florida Veterinary Hospitals, and as codirector of the Marine Animal Disease Laboratory. He is very happily married to Dr. Karen Schaedel, and they have two children, Xavier and Elseya.