Cloning And Sequence Analysis Of The Complete Genome Of Porcine Kobuvirus CH441and Porcine Bocavirus CH437

Diarrhea is a cause of morbidity and death among pigs worldwide, leading to hugeeconomic losses directly or indirectly. The virus is one of the pathogens that cause diarrhea. Inrecent years, new pathogenesis has been detected in the herd of swine, including porcinekobuvirus and porcine bocavirus. Though there are a lot of PKoV and PBoV complete sequencesavailable in GenBank, a little report about the sequence of Gansu samples. The study on Gansusamples is helpful for an inside look at the evolutionary relationship among different strains. Theresearch contents are as follows:1. The complete sequence and phylogenetic analysis of the strain CH44110sets of primers were designed to cover the genome, according to the sequences depositedin GenBank. We determined the complete genome sequence and genetic evolution of thekobuvirus strain CH441using the RT-PCR and RACE methods, with the software DNAStar andMEGA5.0. The viral genome is8149nt long, including a29-Poly (A) tail of the3′end,90ntshorter in the2B-coding region than other kobuvirus strains in GenBank. The sequence encodesa single open reading frame (ORF) with2488amino acids (aa), including a leader (L) protein,structural proteins P1(VP0, VP3, and VP1), non-structural proteins P2(2A,2B,2C) and P3(3A,3B,3C,3D). Nucleotide sequence analysis showed that CH441strain shared a lowest level ofnucleotide identity with K-4/2012/CH (88.3%), and the highest with CH/HNXX-4(90.8%); thelowest level of amino acid similarity with Y-1-CHI (94.3%), the highest with CH/HNXX-4(97.7%). Phylogenetic analysis found strain CH441is in different branch with those detected InHungary. It was most closely related to porcine kobuvirus detected in China but it was not in thesame branch with the strains detected in Gansu province, indicating that porcine kobuvirus mayhave geographic and host differences in evolution.2. The complete sequence and phylogenetic analysis of the strain CH4378sets of primers were designed to cover the genome, according to the sequences depositedin GenBank. We determined the complete genome sequence and genetic evolution of thebocavirus strain CH437using the PCR method, with the software DNAStar and MEGA5.0. Thecomplete genome of CH437was5275nt in length, contains of three ORFs, ORF1encodes NS1(2004nt,667aa), ORF3encodes NP1(681nt,226aa), ORF2encodes VP1(2049nt,682aa)and VP2(1641nt,546aa). Analysis of nucleotide sequences demonstrated that CH437sharedthe lowest nucleotide sequence identity with strain HEN0922-5400(37.0%), the highest withstrain SH17N-2(86.9%), the NS1gene shared24.2%to88.6%nucleotide sequence identity, theNP1shared21.3%to89.9%, According to the species demarcation criteria for members of the genus Bocavirus stipulated by the International Committee on Taxonomy of Viruses (ICTV), aconclusion was given that CH437should be classified as a novel PBoV species.