Molecular Epidemiology, Genome Sequence Analysis And Expression Of VP1Gene Of Porcine Kobuvirus In Henan Provence And Development Of An Indirect Elisa For Detecting Antibody Against Kobuvirus

Porcine kobuvirus is classified into the genus Kobuvirus in the familyPicornaviridae,. Porcine kobuvirus was first identified in Hungary in2008. Since October2010, alarge-scale continuing outbreak of diarrhea has been observed in swine farms in China. Somestudies have indicated that porcine kobuvirus infection is certainly involved in diarrhea disease.But some studies showed that porcine kobuvirus was widerspread in healthy pig herds. Therefore,the pathogenic role of the virus has to be further investigated. This study was designed toinvestigate the porcine kobuvirus infection in Henan province and the molecular and evolutionarycharacteristics of the complete genome. A preliminary study on biological function of VP1genelay the foundation for futher studies of porcine kobuvirus and screening monoclonal antibody.Molecular and Phylogenetic Analysis of Porcine Kobuvirus VP1Region in Henan Province，CHINATo evaluate the infection of porcine kobuvirus in pig herds in Henan province, China, onehundred and fifty fecal samples from pigs with diarrhea and23fecal samples from clinical healthypigs were collected from84pig farms during2010to2012, and porcine kobuvirus in thesesamples was detected by using RT-PCR. The result revealed that142(82.1%) samples and69(82.1%) farms were positive for porcine kobuvirus, which indicated that porcine kobuvirusinfections in pig herds are endemic in Henan province. The positive rate was80.0%(120/150) and95.7%(22/23) in porcine samples collected from pigs with diarrhea and clinical healthy pigs,respectively. Statistical analysis of the porcine kobuvirus infection status with respect to clinicalsigns (diarrhea) indicates that PKV infection has no significant correlation with the occurrence ofdiarrhea in pigs (P>0.05) by Pearson’s chi-square test. VP1gene sequences of8porcine kobuvirusisolates were cloned successfully for molecular and phylogenetic analysis, The nucleotideidentities between the8strains and other reported strains isolated from different parts of Chinaranged from80.7%to99.9%，and the identities were83.9%to99.6%at the amino acid level.Phylogenetic analysis of VP1gene suggested that all porcine kobuvirus strains used in this studywere divided into4branches and the8isolates in Henan province were respectively distributed inthree of them.Molecular characterization of a porcine kobuvirus srain XX in ChinaTo investigate the molecular characterization of a porcine kobuvirus srain XX and find therecombination events in genome, ten pairs of primens were synthesized and RT-PCR method was used for amplification of the complete genome. The predicted cleavage sites of XX wereconsistent with reference strain S-1-HUN and Y-1-CHI. A phylogenetic tree based on the entiregenome sequence of representative kobuvirus strains showed that XX was more closely related toporcine kobuvirus than to other kobuviruses. The genome sequence of XX shared87.9%-89.1%identity at the nucleotide level to the other porcine kobuvirus strains. The highest rates ofnucleotide identity to strain S-1-HUN and Y-1-CHI were seen in the3’-UTR(98.2%and95.2%),and the lowest rates of nucleotide identity to strain S-1-HUN and Y-1-CHI were seen in VP0(81.3%and82.4%). Compared with the strain S-1-HUN, the highest and the lowest rates of aminoacid identity were seen in3B (100%) and VP1(88.2%), and compared with the strain Y-1-CHI,they were seen in2C (100%) and VP1(87.4%). Recombination analysis showed that there wererecombination signal at1466-2155nt in VP0. This study further demonstrated the diversity of viralgenome of the porcine kobuvirus, and indicated the homologous recombination and site mutationsmight promote the evolutionary process.Expression and immunogenicity analysis of VP1gene of porcine kobuvirusBased on the published capsid protein gene(VP1) of porcine kobuvirus strain, a pair of primerswere designed and synthesized. The VP1gene sequence was amplified by RT-PCR and inserted inexpression plasmid carrier pET-32a(+) in accordance with predetermined reading flame afterdouble digesting by BamH I and Xho I,building recombination expression vector pET32a-VP1.The vector were restructured into host bacterium BL21for expression of induction. The result ofSDS-PAGE showed that the optimal induction concentration of IPTG was1.0mmol/L, and optimalinduction time was5h. Our fusion protein which was40.6kDa has specific biological activities bywestern blotting identification, provided a preferred antigen for preparation of a monoclonalantibody and developing fast and specific immunological detection method.Establishment of indirect ELISA assay for specific antibody of pocine kobuvirusThe expressed fusion soluble protein and inclusion body protein was purified by His-taggedpuridication kit respectively, then the inclusion body protein was refolded, and some porcinekobuvirus positive material were collection to prepare inactivated virus vaccines after sterilizationtreatment simultaneously, and then protein antigens and inactivated tissue vaccine was employedto immunize the Kunming mice for preparing specific antiserum against VP1protein and porcinekobuvirus. The recombinant soluble VP1protein as coating antigen was used to initiallyestablished the indirect ELISA assay for detection serum antibody of anti-porcine kobuvirus,laying the foundation of serological testing and screening of anti-porcine kobuvirus monoclonalantibody.