Guangxi Bovine Astrovirus, Molecular Epidemiology And Complete Genome Cloning And Sequences Analysis

Astroviruses are small non enveloped single stranded RNA viruses first identified by electron microscopy in1975from children suffering from diarrhea in England. Later, astrovirus was detected in both terrestrial and aquatics animals including mammals and birds. As of date, novel astroviruses in animals and humans are continually being discovered. Their species diversity and gene recombination event imply possible inter-species cross-transmission and subsequently virus adaptation to classical or new hosts and/or to the co-infection with different viral or non viral pathogens with potential risks of emergence of zoonotic astroviruses. In addition, astrovirus has been associated with neurologic diseases in humans and diverse animals. Astroviruses are cited as food and water pollutants being involved in food and water borne diseases. In this study, we focused more on the bovine astrovirus which was one of the earliest astroviruses to be discovered (1978) but poorly studied in the last3decades.(1) An epidemiology investigation of the bovine astroviruses circulating in cattle and water buffalo population in Guangxi province of China were conducted. A total of211rectal swabs collected from calves aged from0to6months with mild to severe diarrhea in5industrial farms (3dairy and2water buffalo farms) were involved in this study. Detection by RT-PCR resulted in:92/211(43.6%) samples tested positive for bovine astrovirus. Positive rate was46.10%(71/154) in cattle and36.84%(21/57) in water buffalo. These results indicate that astrovirus is widely spread in the cattle and water buffalo population in Guangxi Province of China.(2) In order to understand the co-infection status of astrovirus and most known bovine enteric viruses,32samples from this study stock were screened for BEV, BVDV, BRV, BCoV and BToV by RT-PCR. As result, astrovirus was detected along with one or more of these viruses in87.5%of the cases. Interestingly,4/32(12.5%) of the samples tested positive only for bovine astrovirus and negative for other viruses screened. These results lined out that astrovirus could be associated with diarrhea independently or may be associated with non viral pathogens (bacteria, parasites) or other predisposing facts such as hygiene and feed to induce diarrhea in calves.(3) Phylogenetic analysis based on both partial and full-length ORF2amino acid divided the Guangxi cattle and water buffalo astrovirus isolates into5sub-groups under the genus Mamastrovirus, genotype MAstV33previously comprised bovine and deer astroviruses (BAstV-B18/HK, BAstV-B76-2/HK, CcAstV-1/DNK/2010and CcAstV-2/DNK/2010). Astrovirus isolates from water buffalo belonged to this genotype although they appeared to constitute new sub-groups and suggested that water buffalo was also a host of the genus Mamastrovirus genotype MAstV33.(4) In this study, the two obtained nearly complete genomes (BAstV-GX7/CHN/2014and BAstV-GX27/CHN/2014) of bovine astrovirus were6233bp long and had96.7%nucleotide identities. Compared with NCBI existing bovine complete genome sequences, they had67.6%-87.3%nucleotide identities. The two isolates showed high similarities with the BAstV B76-2/HK—the bovine astrovirus isolates from Hong Kong in2011. However, both partial and complete genome comparison of the Guangxi bovine astrovirus show that they were highly divergent (46%-48.6%) with BAstV NeuroSl/USA—the divergent bovine astrovirus which was associated with neurologic disease in USA indicating that the Guangxi isolates may not be associated with an eventual neurologic diseases in calves. Further analysis of the two nearly complete genome sequences exhibited the3putative ORFs and2UTRs characteristic of astroviruses. The ORF1a was2397bp long deduced in798aa coding for the nspla. Using TMHMM Server v.2.0and NCOILLS version1.0predicted5transmembrane helices and2coiled-coils of the28aa indicating that the nspla was complete. The ORFlb was1579bp long in which the coding region was1509bp deduced in502aa related with the RNA-dependant RNA polymerase (RdRp). The ORF2was2316bp long deduced in771aa coding for the viral capsid protein. The ORF1a/ORF1b overlap was116nucleotides; near the3’-end of ORF1a the ribosomal frameshift signal (A6C) was identified at (2387-2393nt). The ORFlb/ORF2overlap was56nucleotides. Upstream ORF2start codon identified a highly conserved sequence motif predicted to be the signal of the putative promoter for sgRNA synthesis.In summary, this study conducted a molecular epidemiology of bovine astrovirus in Guangxi and reported the first detection of bovine astrovirus in diarrheic water buffalo calves. In addition,25partial sequences and2nearly complete genome sequences of bovine astroviruses were analyzed and submitted to the NCBI GenBank, therefore, this study is of interest both at the clinical level in future monitoring of the disease and at the molecular level by supplying useful references for further molecular study of astroviruses.