Molecular and phylogenetic analysis of the porcine kobuvirus VP1 region using infected pigs from Sichuan Province, China

Abstract

Background: Porcine kobuvirus (PKoV) is a member of the Kobuvirus genus within the Picornaviridae family. PKoV is distributed worldwide with high prevalence in clinically healthy pigs and those with diarrhea.

Methods: Fecal and intestinal samples (n = 163) from pig farms in Sichuan Province, China were obtained to determine the presence of PKoV using reverse transcription polymerase chain reaction assays. Specific primers were used for the amplification of the gene encoding the PKoV VP1 protein sequence. Sequence and phylogenetic analyses were conducted to clarify evolutionary relationships with other PKoV strains.

Results: Approximately 53% (87/163) of pigs tested positive for PKoV. PKoV was widespread in asymptomatic pigs and those with diarrhea. A high prevalence of PKoV was observed in pigs younger than 4 weeks and in pigs with diarrhea. Phylogenetic analysis of 36 PKoV VP1 protein sequences showed that Sichuan PKoV strains formed four distinct clusters. Two pigs with diarrhea were found to be co-infected with multiple PKoV strains. Sequence and phylogenetic analyses revealed diversity within the same host and between different hosts. Significant recombination breakpoints were observed between the CHN/SC/31-A1 and CHN/SC/31-A3 strains in the VP1 region, which were isolated from the same sample.

Conclusion: PKoV was endemic in Sichuan Province regardless of whether pigs were healthy or suffering from diarrhea. Based on our statistical analyses, we suggest that PKoV was the likely causative agent of high-mortality diarrhea in China from 2010. For the first time, we provide evidence for the co-existence of multiple PKoV strains in one pig, and possible recombination events in the VP1 region. Our findings provide further insights into the molecular properties of PKoV, along with its epidemiology.

Figure 1

Phylogenetic analysis of PKoV VP1 protein sequences detected in Sichuan province. The phelogenetic tree was constructed using the neighbor-joining method, Kimura 2-parameter model by MEGA version 5.0. Bootstrap values (based on 1,000 replicates) >75% are shown. Filled quadrate (■) indicates PKoV VP1 protein sequences obtained in this study. Filled triangle (▲) represents a Sichuan strain PKoV VP1 protein sequence has been reported in our previous study. Kobuvirus pig/JY-2010a/CHN isolate group contained 10 sequences from China forming a tight cluster. The Genbank accession numbers are: GU292548-GU292553, GU292556-GU292559; PKoV Thailand/Japan isolate group including 10 sequences clustered in a monophyletic branch. Their accession numbers in Genbak are as follows: AB624490, AB624492-AB624497, AB624499, AB624501, AB624503.

Figure 2

Nucleotide similarity comparison between the putative parental strains and the possible recombination strain. CHN/SC/31-A1 and CHN/SC/31-A3 were the putative parental strains, and CHN/SC/31-A2 was the query sequence, CHN/SC/32-B2 was an outgroup. The x-axis indicates nucleotide positions, and the y-axis shows similarity.

Figure 3

Bootscan analysis of the putative recombinant sequence. The x-axis indicates nucleotide positions. The y-axis denotes bootstrap values that support the clustering of CHN/SC/31-A2 with the parental strains.