Analysis Of Complete Genome Sequence Of Porcine Kobuvirus WUH1Strain And Isolation Of Porcine Kobuvirus

Kobuvirus is a member of Picornaviridae and they are small nonenveloped viruses with a genomic positive single-stranded RNA that can infect humans and animals. Now, Aichi virus(AiV) and bovine kobuvirus(BKV) are within the genus. In2007, the Hungarian researchers found the candidate member of kobuvirus--porcine kobuvirus(PKV) when they detected of porcine calicivirus. Since then, the detective rate of PKV in Europe, Asia and America was very high, but the relationship between the virus and disease is still unclear. It has been reported that AiV could be one of the causative agents of gastroenteritis in humans, and BKV might also cause diarrhoea in cattles. Some results show that the detective rate of PKV was significantly higher in diarrhea pigs than the ones without diarrhea. These suggest us PKV may cause swine diarrhea as a new pathogen.Now, a lot of PKV nucleotide partial sequences are available but there is little report about its complete genome. Complete genome sequence analysis is helpful for an insidelook at the evolutionary relationship among different srains of PKV. AiV and BKV were isolated in1991and2003, but PKV has not been isolated yet, which makes studies about PKV could not be carried out. Based on these techniques, we established a RT-PCR detection method and cloned the PKV complete genome sequences. The positive disease materials were used to isolate PKV. Specific contents are as follows:1. Establish a RT-PCR detection method and applicationIn this study, a special pair of primers were designed based on kobuvirus/swine/S-1-HUN/2007/Hungary (GeneBank accession number EU787450)3D region sequence which chould amplify a495bp DNA fragment of PKV. Our results indicated that the assay was specific, sensitive and reproducible. Using the established PCR method to investigate the prevalence of PKV in Central China, the results showed that the the positive rate was high. This indicated we should pay more attention.2. Genome sequenced and analysis of PKVNine pairs of primers were designed based on reported PKV sequence to amplify the complete genome of one strain from WuHan by fragments, and RACE was applied to amplify5’end. The complete genome sequence of WUHlwas8210bp in length, containing1open reading frame which encode2488amino acids. Compared to strain S-1-HUN/2007/Hungary, there was one base insertion exist in5’ UTR and one deletion in3’UTR of WUH1. The phylogenetic tree based on the genome sequence showed that WUH1was most closely related to the strains identified in China and was relatively far to the strains in Hungary.3. Isolation of PKVThe positive samples were cultivated by Vero cells, with6passaged viral nucleic acids were detected in each generation of cells. Viral nucleic acid could be detected in the initial cell generations, but with serial of passages, the viral nucleic acids were disappear gradually.