Genetic characterisation and phylogenetic analysis of bovine astroviruses and kobuviruses
Calf diarrhoea remains the most important cause of economic loss to both dairy and beef cattle industries worldwide, with approximately 50% of deaths among weaning calves resulting from diarrhoeal disease. Complex etiopathogenesis involving the infection of one or multiple pathogens, as well as other non-infectious factors such as the environment and nutrition, contributes to its devastating effects. Astroviruses (AstVs) and Kobuviruses (KoVs) are two single-stranded, positive-sense RNA viruses previously detected in healthy and diarrhoeic calves. AstV was identified in healthy and diarrhoeic calves in similar proportions, while KoV was predominantly associated with diarrhoeic individuals. In order to investigate the KoV strains found in diarrhoeic calves, the full genome of KoV from a diarrhoeic calf was sequenced. This KoV was then compared with the bovine U-1, porcine KoV and Aichi virus strains. Specifically-designed PCRs were used to target the full KoV genome of positive samples, and amplicons were cloned to allow the internal sequencing of one single KoV detected among possible mixed infections. Upon assembly of the genome sequences, some animals were found to be co-infected with multiple KoVs. The main region of diversity, the VP1 (capsid) region, was amplified from multiple samples to determine the diversity of KoV in Scotland. The genome sequenced in this study will be used to produce an infectious clone for future challenge studies to establish the potential role of bovine KoV in calf diarrhoea. Another aspect of the study was to explore the diversity and epidemiology of AstV in diarrhoeic and healthy calves by capsid gene analysis. As the AstV capsid protein is the major target of host antibody production, a serological test for AstV infection could then be developed. Following amplification of AstV capsid genes by PCR, phylogenetic analysis identified 4 lineages from which capsids from 2 of these lineages were successfully cloned ready for use in the baculovirus expression system. This information and the expression plasmids containing representative AstV capsid genes can then be used to develop serological tests for AstV, enabling estimation of the prevalence of AstV in the British cattle population.