Studies on toroviruses of humans and cattle Lynn Marie Duckmanton A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Graduate Department of Molecular and Medical Genetics, University of Toronto Copyright by Lynn M. Duckmanton, 1999 National Library Bibliotheque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395, rue Wellington OttawaON KIA ON4 OttawaON KlAON4 Canada Canada The author has granted a non- L'auteur a accorde une licence non exclusive licence allowing the exclusive pennettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, loan, distribute or sell reprodwe, preter, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la foxme de microfiche/film, de reproduction sur papier ou sur format Bectronique. The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protege cette ththe. thesis nor substantial extracts fiorn it Ni la these ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent &re imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Studies on toroviruses of humans and cattle, Degree of Doctor of Philosophy, 1999, Lynn Marie Duckmanton, Graduate Department of Molecular and Medical Genetics, University of Toronto. Abstract Human torovirus (HTV) was purified from patient stool specimens and characterized. By negative contrast electron microscopy (EM) and thin-section EM, torovirus-like particles were found to be morphologically similar to Berne virus (BEV) and Breda virus (BRV). HTV was shown to react with BRV antiserum and human convalescent serum from torovirus-positive patient stools, by hemagglutination inhibition (HI), immunoelectron microscopy (IEM), and immunoblotting. RNA extracted from HTV preparations was amplified by RT- PCR using primers bracketing a 219-base region at the 3' end of the BEV genome. Sequence analysis of amplicons from five HTV specimens showed a high degree of sequence similarity among the human viruses as well as with BEV. Using EM and RT-PCR, the incidence of torovirus excretion in calves with diarrhea from farms in Southern Ontario was investigated and compared to the excretion of other enteric pathogens. By RT-PCR, torovirus RNA was detected in 36% of diarrheic specimens. The incidence of torovirus in asymptomatic control specimens by RT-PCR was only 12%, thereby allowing us to make a significant correlation between disease and virus shedding. Nucleotide sequence analysis of 5 of the arnplicons from EM- and RT-PCR- positive samples revealed that the 3' end of the genome of bovine torovirus found in Southern Ontario manifested 96%-97% sequence identity to that of the BRV-1 strain found in Iowa. The nucleotide sequence of 7.5kb in the 3' region of the BRV-1 genome was derived from an arnplicon of BRV-f RNA amplified by long RT-PCR. Sequence analysis revealed the presence of 4 open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, as well as an ORF for a novel I .2kb gene located between the M and N genes. This gene was 99% identical in nucleotide sequence to the hernagglutinin- esterase (HE) gene of BRV-2. Using primers designed from the BRV-1 HE gene, an amplicon was obtained from HTV RNA, and on sequencing was shown to have 75% sequence identity with the BRV-I HE gene. The BRV-1 N gene, and the HE genes of BRV-1 and HTV were cloned and expressed. Guinea pig antisera to the N and HE recombinant proteins were shown to be reactive with bovine and human toroviruses by immunoblot, IEM, and dot blot analyses. iii Acknowledgments The work described in this thesis took approximately five years to complete, and there are a number of people whom I would like to thank for their help and support during this period. First, Dr. Paul Widden at Concordia University is responsible for introducing me to the field of scientific research, and encouraging me to pursue graduate studies at the University of Toronto. The encouragement and guidance provided by my supervisor, Dr. Martin Petric, aided me to complete my research and to pursue a career in industry. I am certainly grateful for his support. I would also like to thank Dr. Raymond Tellier for his advice and direction throughout the final years of this project. The wonderful staff in the Virology laboratory at the Hospital for Sick Children also aided me by providing valuable technical assistance and ideas, as well as motivation and friendship. I would like to thank John, Sumita, Antonietta, Willy, Maria, Anna, Rose, Fran, Gloria, Lily, and my office mate, Karen. I am grateful to my parents, John and Ckline, for their constant love and support. Their enthusiasm and encouragement were indispensable to me. Many thanks, also, to Diane, Chandan and Adrienne for their kindness and reassurance. I save a hug for Simba, whose companionship warmed my lap and my heart while writing this thesis. Finally, I would profoundly like to thank my husband, Arun, whose love and devotion gave me the will to persevere and attain my goals. His listening ear and continuous motivation made this work possible. I share all this with you. Table of Contents Page Title page i Abstract ii Acknowledgements iv Table of contents v List of abbreviations viii List of tables xi List of figures xi i Dissemination of work arising from this thesis xvi Chapter 1: Introduction i lnfectious diarrhea 2 Viral gastroenteritis 3 Nidovirales: Coronaviridae and Arteriviridae 10 Arterivirus Morphology Genetics and replication Epidemiology Coronavirus Morphology Genetics and replication Antigenic properties Pathology and pathogenesis Diagnosis Epidemiology Prevention and control Torovinrs Morphology Physicochemical properties Genome organization Replication Antigenic properties Pathology and pathogenesis Epidemiology Diagnosis Prevention and control Evolutionary relationships Summary Chapter 2: Objectives Chapter 3: Characterization of torovirus from human fecal specimens Summary Introduction Materials and methods Results Discussion Chapter 4: Detection of bovine torovirus in fecal specimens of calves with diarrhea from Ontario farms Summary lntroduction Materials and methods Results Discussion Chapter 5: Bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of Breda virus Summary Introduction Materials and methods Results Discussion Chapter 6: The novel hemagglutininesterase genes of human torovirus and Breda virus Summary lntroduction Materials and methods Results Discussion Chapter 7: Discussion References vii List of Abbreviations alc acute and convalescent AHL Animal Health Laboratory baBRV bovine anti-Breda virus baBRV-1 bovine anti-Breda virus-I baBRV-2 bovine anti-Breda virus-2 BCIP-NBT 5-bromo4thloro-3-indolyl phosphate- nitro blue tetrazolium BCV bovine coronavirus BEV Berne virus BRV Breda virus BRV-1 Breda virus-I BRV-2 Breda virus-2 Blv bovine torovirus BVDV bovine viral diarrhea virus ccv canine coronavirus CVLP coronavirus-like particle EAV equine arteritis virus ELlSA enzyrne-linked immunosorbent assay EM electron microscopy ER endoplasmic reticulum FlPV feline infectious peritonitis virus PHI guinea pig hyperimmune gpHla5RV guinea pig anti-Breda virus4 HE protein hyperimmune gpHlaHTV guinea pig anti-human torovirus HE protein hyperimmune gpHlaN guinea pig anti-Breda virus4 N protein hyperimmune 9 PPI guinea pig preimmune gpPlaBRV guinea pig anti-Breda virus-I HE protein preirnmune gpPlaHTV guinea pig anti-human torovirus HE protein preimmune gpPlaN guinea pig anti-Breda virus-I N protein preimmune HA hemagglutination HCV human respiratory coronavirus viii HE hemagglutinin-esterase HECV human enteric coronavirus HEF hemagglutinin-esterase fusion protein HI hemagglutination inhibition HTV human torovirus huA human acute huC human convalescent IBV avian infectious bronchitis virus ICW International Committee for the Taxonomy of Viruses ICV influenza C virus IEM immunoefectron microscopy IFA immunofluorescence assay LDV lactate dehydrogenase-elevating virus M viral envelope MHV murine hepatitis virus Mr molecular mass N viral nucleocapsid NS non-structural ORF open reading frame PCR polymerase chain reaction POL viral polymerase PRRSV porcine reproductive and respiratory syndrome virus PVDF polyvinylidene fluoride RaGP rabbit anti-guinea pig RIA radioimmunoassay RT-PCR reverse transcription-polymerase chain reaction S viral peplomer SDS-PAGE sodium dodecyl sulfate-polyacrytamide gel electrophoresis SHW simian hemorrhagic fever virus SRSV small round structured virus TBST Tris-buffered saline containing 0.5% Tween-20 TCV turkey bluecomb coronavirus TGEV transmissible gastroenteritis virus of swine ix TS-EM thin section-electron microscopy TVLP torovirus-li ke particle VBS veronal buffered saline X Beme virus pseudogene List of Tables Page 1. Species within the genera Coronavirus, Tomvvhrs, and Arterivims 2. Properties and functions of the toroviral structural proteins 3. EM and ELISA results for 89 stool specimens from children with diarrhea 4. Comparison of features of coronaviruses, toroviruses, and Artenviruses 5. IEM and HI results for two TVLP-positive human fecal specimens 6. Detection of torovirus by EM in symptomatic and asymptomatic patients 7. HI results showing seroconversion to human torovirus 8. EM and RT-PCR