Complete Genome Analysis And Molecular Epidemiology Of The Porcine Kobuvirus Using Infected Pigs From Jiangsu Province, China

Porcine kobuviruses are small, non-enveloped viruses with single-stranded, positive-sense genomic RNA within the family Picornaviridae, genus kobuvirus. The virus was first detected in 2008 in fecal samples of domestic pigs in Hungary, and then the virus was detected in many countries. Since December 2010, outbreaks characterized by watery diarrhea, dehydration, and vomiting have occurred in suckling piglets in 10 provinces in China with a morbidity of 80% to 100% and 50% to 90%mortality. Outbreaks have caused severe economic losses and consequent social effects and porcine kobuvirus were detected in most of these diarrhea pigs. PKVs have detected in many counties and some reports suggested that the virus may relate to porcine diarrheal.At present, PKVs can’t be cultured in vitro; the pathogenicity of PKVs and its role in pig diarrheal disease is not well understood. Most reports about PKVs were focused on molecular characterization of this virus. The 3D gene was usually used to detect the PKVs.3D gene is the most conservative region of PKVs, its coding protein in animal virus proliferation in the body and infection in cell cultures found a virus specificity of non-structural protein, known as a viral infection related proteins. The VP1 gene of the PKVs is the most variable region in the capsid gene of the virus among PKVs, the genetic diversity in different pigs may have a close relationship with pig diarrhea. In this study, to increase the awareness of the situation and to stimulate further molecular epidemiological and clinical studies about porcine kobuvirus, we analyzed the genetic characterization of 3D gene and VP1 gene of PKVs and two strains PKVs were sequenced the complete genome using infected pigs from Jiangsu Province, China. We hope this study can enrich the epidemiological survey data of PKVs and help for learning the relationship between PKVs and pigs diarrhea.1. Development of RT-PCR for detection of Porcine KobuvirusIn this study, a pair of primers was designed based on the 3D gene conserved region of PKVs; a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method for the detection of PKVs was developed. Using the RT-PCR assay, the specific fragment of 217bp was amplified from feces sample of the PKVs only, not from PEDV, TGEV, GARV, PRRSV, CSFV, PCV and Escherichia coli. The sensitivity of the RT-PCR was 0.1263pg for the PKVs nucleic acids. The newly developed method was used to detect PKVs from clinical specimens of pigs in Jiangsu Province, China.2. Genetic characterization of porcine kobuvirus 3D gene and VP1 gene and detection of coinfecting pathogens in diarrheic pigs in Jiangsu Province, ChinaIn this study,396 samples from diarrheic pigs on 46 pig farms in Jiangsu Province, China, were analyzed by RT-PCR. One-hundred eighty-one pigs from 37 farms tested positive for PKVs. Phylogenetic analysis of the 3D gene from 19 isolates showed sequence homology of 88.0%～100% and 69.4%～100% for nucleotides and amino acids, respectively, while similarity to isolates of other kobuviruses was 69.6%～78.8% and 27.8%～56.9%, respectively. One-hundred eighty-five samples contained two or more pathogens, and 31/68 PKVs positive samples tested positive for other diarrheic pathogens, confirming the existence of PKVs infection and coinfection. In another study,91 samples from diarrheic pigs and 126 samples from healthy pigs were analyzed by RT-PCR,64.8%(59/91) diarrheic piglets and 19.8% (25/126) normal piglets are positive for PKVs.28 positive samples were randomly selected and analyzed the VP1 gene, the nucleotides and amino acids homology showed 79.6%-100% and 80.3%-100%, respectively. Phylogenetic analysis revealed that all PKVs strains were divided into 4 clusters.15 strains collected from those suffering diarrhea pigs segregated into different branches, all the VP1 sequences detected in clinically healthy pigs clustered into a big group. The results demonstrated that the VP1 gene showed genetic diversity in pigs those with diarrhea but conservative among clinic healthy pigs in Jiangsu province.3. Complete genome sequence of the porcine kobuvirus in Jiangsu provinceBased on the sequences of porcine kobuvirus reference strains, twelve pairs of oligonucleotide primers were designed to amplify the different regions of the porcine kobuvirus genome. In this study, two porcine kobuvirus strains, JS-01-CHN and JS-02a-CHN which were detected in diarrheal and asymptomatic piglets, respectively. The sequence of the two strains was analyzed using the bioinformatics software package. The full-length genome of JS-02a-CHN, which was detected in healthy piglets, is 8121 nucleotides (nt) long without the polyA tail. There was a 30-amino-acid deletion in 2B-coding region of JS-02a-CHN, as it is the first report that a 30-amino-acid deletion in porcine kobuvirus of asymptomatic piglets, indicating that porcine kobuvirus may have geographic and host differences in evolution. Recombination events were found in strain JS-01-CHN isolated from piglets with diarrhea. Therefore, we suggest that recombination may contribute to the evolution of PKV virulence. To address these limitations in future studies, virus cultured in vitro and more complete genome sequences of PKV field isolates should be used for recombination and epidemiological analyses of PKV.