Epidemic Investigation And Establishment Of Assays For Detection Of Porcine Kobuvirus In Hebei Province

Porcine kobuvirus(PKV)is a member of the kobuvirus genus of the picornaviridae family.It is one of the important pathogens newly discovered in recent years that can cause diarrhea in pigs.It mainly harms piglets under three weeks of age.Since PKV was first discovered in 2007,PKV has been reported in many countries around the world,and has a high positive rate in serum or fecal samples from healthy pigs and diarrhea pigs.However,the positive rate in piglets with diarrhea symptoms is much higher than that in healthy pigs,and usually has a high co-infection rate with other diarrhea viruses,which affects the healthy development of the pig industry.To investigate the prevalence of PKV in Hebei Province,this study collected 280 stool samples from 48 large-scale pig farms in various regions of Hebei Province and tested them by RT-PCR.The results showed that among the collected samples,89(31.8%)samples tested positive for PKV,and the 11 prefectures and cities in Hebei Province tested had different degrees of infection.Compared with other regions,Baoding has a higher positive rate,with a positive rate of 40%(18/45).The co-infection rate of PKV and PRRSV(46.1%)is higher than that of other diarrheal viruses PEDV(38.2%),PDCoV(3.4%)and PBOV(1.1%).PKV is widespread in Hebei Province and the infection rate is higher.The 12 PKV VP1 gene sequences were amplified and compared with the reference gene sequences registered on GenBank using MegAlign in DNA-Star software.The nucleotide homology is 78%～100%,amino acid deletions are detected at amino acid positions 231 and 239,and amino acid insertions are present at position 238.Using the biology software MEGA7.0 for systematic evolution analysis,the evolutionary tree shows that most of the strains in Hebei Province(10/12)are in the same branch,and each group contains a sequence isolated from pigs showing diarrhea symptoms,indicating that there is no the unique PKV sequence is associated with diarrhea.The biological software RDP4 and Simplot were used to detect the recombination sites of the VP1 gene.This study will provide a theoretical reference for the prevalence and evolution of PKV and provide a scientific basis for the prevention and control of PKV in Hebei Province.ELISA has the advantages of simple operation,excellent specificity,high sensitivity,and convenient mass serum detection,so it is often used as one of rapid serological diagnosis methods in clinical practice.In this study,PKV VPO protein was expressed as a coating antigen through a prokaryotic expression system,and Western blotting proved that the recombinant protein can react specifically with PKV positive serum to optimize the reaction conditions.The optimal reaction conditions for the PKV VPO indirect ELISA detection method are that the antigen coating concentration is 0.62 μg/mL,the serum dilution concentration is 1:200,the best enzyme-labeled secondary antibody dilution concentration is 1:5000,and 5%skimmed milk powder is used as incubate the blocking solution at 37℃ for 1 hour and develop the color for 10 minutes.When the sample OD450nm<0.225,the serum sample antibody can be determined to be negative,and OD450nm≧0.225 can determine the serum sample antibody to be positive,with high specificity and good repeatability.The established PKV VP0 protein indirect ELISA method,as a serological detection method with simple operation,high sensitivity and accurate results,provides technical support for the monitoring and prevention of PKV.The real-time fluorescent quantitative PCR method has been widely used in clinical detection of pathogens,and has the advantages of simple operation,high sensitivity,and large number of detections.In this study,in order to establish a real-time fluorescent quantitative PCR method that can detect porcine crest virus,a pair of fluorescent detection primers were designed,the reaction system and reaction conditions were optimized,and the amplification curve of the fluorescent quantitative PCR method established showed a good linear relationship.The established fluorescent quantitative PCR method is 100 times more sensitive than conventional PCR,with good specificity and high sensitivity.The pig feces samples collected through detection in Hebei Province have higher sensitivity than conventional PCR.This real-time fluorescent quantitative PCR method provides a powerful technical support for the pathogenic detection of swine crest virus and the monitoring of swine crest virus in Hebei Province.