Circovirus Infection in Cattle
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Mohammad Yahya Halami Circovirus Infection in Cattle Inaugural-Dissertation Leipzig, 2014 Halami: Circovirus Infection in Cattle 2014 From the Institute of Virology, Faculty of Veterinary Medicine, University of Leipzig Circovirus Infection in Cattle Inaugural-Dissertation to obtain the degree of a Doctor medicinae veterinariae (Dr. med. vet.) from the Faculty of Veterinary Medicine University of Leipzig submitted by Mohammad Yahya Halami From Deirazzor/Syria Leipzig, 2014 Mit Genehmigung der Veterinärmedizinischen Fakultät der Universität Leipzig Dekan: Prof. Dr. Manfred Coenen Betreuer: Prof. Dr. Hermann Müller und Prof. Dr. Dr. Thomas Vahlenkamp Gutachter: Prof. Dr. Hermann Müller und Prof. Dr. Dr. Thomas Vahlenkamp Institut für Virologie, An den Tierkliniken 29, 04103 Leipzig Prof. Dr. Annette Mankertz Robert Koch-Institut, Nordufer 20, 13353 Berlin Tag der Verteidigung: 04.11.2014 DEDICATED TO MY PARENTS AND MY WIFE NADA CONTENTS CONTENTS List of abbreviations...……………………………………………………………………..III List of figures..……………………………………………………………………………..V List of tables..………………………………………………………………………………V 1 INTRODUCTION .......................................................................................................... 1 1.1 Study objectives ......................................................................................................... 2 1.2 Thesis organization .................................................................................................... 4 2 LITERATURE REVIEW ............................................................................................... 5 2.1 Circoviruses ................................................................................................................ 6 2.1.1 Taxonomy ............................................................................................................... 6 2.1.2 Viral structure and genome organization ............................................................... 7 2.1.3 Entry and replication of circoviruses in pigs ........................................................ 10 2.1.4 Clinical manifestation of porcine circovirus-2 (PCV2) infection in pigs ............ 11 2.1.5 Pathogenesis of PCV2 .......................................................................................... 12 2.1.6 Diagnosis of PCV2 ............................................................................................... 13 2.1.6.1 Polymerase chain reaction (PCR) ................................................................ 13 2.1.6.2 In situ hybridization (ISH) ........................................................................... 14 2.1.6.3 Enzyme-linked immunosorbent assay (ELISA) ........................................... 14 2.1.7 PCV2 in non-porcine hosts .................................................................................. 16 2.1.7.1 Cattle ............................................................................................................ 16 2.1.7.2 Rodents ......................................................................................................... 16 2.1.7.3 Humans ......................................................................................................... 17 2.2 Bovine neonatal pancytopenia (BNP) ...................................................................... 18 I CONTENTS 3 AIM OF THE WORK .................................................................................................. 20 4 RESULTS ..................................................................................................................... 22 4.1 PUBLICATION 1: Bone marrow aplasia with haemorrhagic disease in calves in Germany: characterization of the disease and preliminary investigations on its aetiology . 24 4.2 PUBLICATION 2: Whole genome sequence of two strains of porcine circovirus 2 isolated from calves in Germany ......................................................................................... 36 4.3 PUBLICATION 3: Susceptibility of calves to porcine circovirus-2 (PCV2) .......... 42 5 GENERAL DISCUSSION ........................................................................................... 63 5.1 BNP syndrome and detection of circoviruses .......................................................... 65 5.2 Complete genome sequencing of PCV2 ................................................................... 69 5.3 Pathogenicity of PCV2 in cattle ............................................................................... 70 5.4 Potential risk of PCV in humans .............................................................................. 74 6 SUMMARY ................................................................................................................. 76 7 ZUSAMMENFASSUNG ............................................................................................. 79 8 REFERENCES ............................................................................................................. 82 9 APPENDIX ................................................................................................................ 101 10 ACKNOWLEDGEMENTS ....................................................................................... 106 II LIST OF ABBREVIATIONS LIST OF ABBREVIATIONS aa amino acid AvGyV2 avian gyrovirus 2 BaCV barbel circovirus BNP bovine neonatal pancytopenia BTV blue tongue virus BVDV bovine viral diarrhea virus CaCV canary circovirus cap capsid (gene) Cap capsid (protein) CAV chicken anemia virus CfCV cat fish circovirus CsaCV chimpanzee circovirus CyCV cyclovirus DCVC s-(1,2-Dichlorovinyl)-L-cysteine DfCyV dragonfly cyclovirus DNA deoxyribonucleic acid DuCV duck circovirus ELISA enzyme-linked immunosorbent assay FiCV finch circovirus ICTV international committee on taxonomy of viruses GAGs glycosaminoglycans GoCV goose circovirus GuCV gull circovirus HGyV1 human gyrovirus 1 IFA immunofluorescence assay III LIST OF ABBREVIATIONS IgG immunoglobulin G IPMA immunoperoxidase monolayer assay ISH in situ hybridization kb kilo base MHC major histocompatibility complex nt nucleotide ORF open reading frame PBMC peripheral blood mononuclear cells PCR polymerase chain reaction PCV1 porcine circovirus type 1 PCV2 porcine circovirus type 2 PCVAD porcine circovirus associated disease PDNS porcine dermatitis and nephropathy syndrome PK15 porcine kidney 15 PiCV pigeon circovirus PMWS post-weaning multisystemic wasting syndrome PNP proliferative and necrotizing pneumonia PRDC porcine respiratory disease complex RaCV raven circovirus RCR rolling circle replication rep replication (gene) Rep replication (protein) ssDNA single stranded circular deoxyribonucleic acid StCV starling circovirus SwCV swan circovirus IV LIST OF FIGURES AND TABLES LIST OF FIGURES Figure Title Page 1 Circovirus particles 7 2 Genome organization of PCV2 9 3 Life cycle of PCV 11 4 Phylogenetic analysis of the members of the family Circoviridae 73 5 Phylogenetic analysis of PCV2 genomes 74 6 Possible interspecies transmission of PCV 75 LIST OF TABLES Table Title Page 1 The members of the family Circoviridae 8 2 Proposed terminology for porcine circovirus diseases 15 V 1 INTRODUCTION 1 INTRODUCTION 1 INTRODUCTION 1.1 Study objectives Circoviruses are small, spherical viruses with a circular single-stranded DNA genome of 1.7 to 2.4 kb in size. The genome contains two major open reading frames (ORFs), encoding capsid (Cap) and replication (Rep) proteins (TODD et al. 2000). Porcine circovirus (PCV) was first identified as a cell culture contamination (TISCHER et al. 1974). Subsequently, research studies have shown that PCV was widespread in the pig population (TISCHER et al. 1986; ALLAN et al. 1995; TISCHER et al. 1995). The findings of JACOBSEN et al. (2009) showed that PCV2 has been present in the pig population in northern Germany since 1962. In 1991, a severe wasting syndrome in pigs was observed in Canada (HARDING and CLARK 1997), the United States of America (USA) and shortly thereafter in Europe (DAFT et al. 1996; LECANN et al. 1997). A PCV was found to be associated with the new syndrome (ELLIS et al. 1998a). Compared to the previously described circovirus in pigs the virus showed less than 80% sequence homology, indicating a new genotype (MEEHAN et al. 1998). Today, two distinct PCV genotypes are known: The non-pathogenic PK-15 cell line associated PCV1 and the pathogenic disease causing PCV2 (MEEHAN et al. 1998). PCV2- associated microscopic lesions were identified in archived tissues as early as 1985 in Spain (RODRÍGUEZ-ARRIOJA et al. 2003). Based on the sequence of capsid gene, the PCV2 can be further subdivided into PCV2a, PCV2b and PCV2c (OLVERA et al. 2007; GRAU-ROMA et al 2008; SEGALÉS et al. 2008), and is now present in all major pork producing countries. The first detection of antibodies against PCV in sera of cattle, humans and mice was reported in 1995 (TISCHER et al. 1995). Studies performed in Canada also detected circovirus DNA in lung tissue samples of bovine respiratory disease and aborted foetuses (NAYAR et al. 1999). In two other studies, however, no antibodies to PCV2 were detected in 2 INTRODUCTION sera from cattle (n=405), sheep (n=120), horse (n=100) and humans (n=120) (ALLAN et al. 2000a; ELLIS et al. 2001). Since the early 1990s, PCV2 has been associated with a number of disease manifestations including post-weaning multisystemic wasting syndrome (PMWS), respiratory disease, porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, enteritis, and neuropathy (CHAE 2005).