Whole Genome Sequence Analysis And Molecular Detection Techniques Of Porcine Bocavirus In Slaughter Pigs

The recently discovered porcine PBoV is a member of the Parvoviridae family,and it is potentially associated with swine disease. Thus, we developed a PCR assay totarget the PBoV NS1gene, obtained the whole viral genome by PCR amplificationfrom the PBoV positive swine sample, used purified His-NP1as coating antigen fordevelop an indirect ELISA for detection the antibody in serum to PBoV. We aimed toprovide a basis for evaluating the impact of the virus on pig production and porkquality assessment.Firstly, a survey was conducted to evaluate the prevalence of PBoV in slaughterpigs, sick pigs, asymptomatic pigs and Classic Swine Fever Virus (CSFV) eradicationplan herds in five provinces of China (Henan, Liaoning, Shandong, Hebei and Tianjin)by means of PCR targeting NS1gene of PBoV. Among the total of403tissue samples,11.41%were positive for PBoV. The positive rates of Spleen (20.75%) and inguinallymph node (27.18%) are higher than those of other organs. PCR products of twentyPBoV positive samples from slaughter pigs were sequenced for phylogenetic analysis.The result revealed that PBoV could be divided into6groups (PBoV-a~PBoV-f). AllPBoV sequenced in this study belong to PBoV-a-PBoV-d with90.1%to99%nucleotide identities. PBoV-HN2strain may be a new mutation or recombinantPBoV3. Our results exhibited significant genetic diversity of PBoV and suggested acomplex prevalence of PBoV in Chinese swine herds. Whether this diversity of PBoVhas a significance to pig production or even public health remains to be furtherstudied.Secondly,3pairs of primers were designed according to the nucleotide sequencesof PBoV published in GenBank, and3fragments covering the whole viral genomewere obtained and sequenced by PCR amplification from the PBoV positive swinesample(PBoV-HN2).The accession number of PBoV-HN2strain in GenBank isKC473563.Comparative analysis with the other14genomic sequences of PBoVrevealed that PBoV-HN2shared49～90%with the other genomic sequences of PBoV.The phylogenetic tree was generated based an complete genomic sequences, revealedPBoV-HN2had closer relationship with PBoV3、PBoV4and PBoV5in comparisonwith PBoV1and PBoV2. Comparative analysis with PBoV-HN2genome all coding regions (NS1, NP1, VP1, VP2) respectively with previously reported PBoV1andPBoV2, PBoV3PBoV4and PBoV5, PBoV-HN2nonstructural protein NS1and NP1,structure protein VP2had closer relationship with PBoV3, structural protein VP1hadcloser relationship with PBoV4.Thirdly, the pET28-NP1expression vector was constructed using pET28a vector,the recombinant protein (His-P25) with his-tag was expressed in E coli BL21(DE3).The recombinant proteins were analyzed by Western blot, results showed that therecombinant proteins could specifically react with sera from PBoV infected pigs,indicating that the recombinant proteins shared good immunogenicity. An indirectELISA was developed for detection of antibody in serum to PBoV, using purifiedHis-NP1as coating antigen.The creation of the PCR method targeting NS1gene of PBoV and the indirectELISA for detection of antibody in serum to PBoV provided a basis for PBoVepidemiological investigations and pork quality assessment.