Investigation Of Porcine Viruses Co-infection In Some Areas Of Anhui Province And Establishment Of PCV2 Detection Method

The growing pig industry in China is facing more and more challenges,and porcine viral infections are threatening the development of the pig industry.In view of the co-infection of porcine viruses in Anhui,in this study,a total of 76 swine fecal samples were collected in Hefei,Huaibei,Bozhou,Bengbu and Luan cities of Anhui Province,of which 29samples were from diarrhea pigs and 47 samples were from healthy pigs,and these 76samples were tested for porcine virus metagenomic.The results showed that 19 porcine viruses were detected in the 76 samples collected,among which the porcine circovirus(PCV)had the highest prevalence(31.58%),with PCV detected in 24 samples,16 from diarrheic pigs and 8 from healthy pigs.A total of 18samples were detected with mixed infections of two or more porcine viruses,with a rate of14.47%(11/76)for duplex mixed infections,1.32%(1/76)for both triple and quadruple mixed infections,and 6.58%(5/76)for quadruple or more mixed infections.Ten mixed infections of porcine viruses,including PCV,porcine bocavirus(PBo V),porcine astrovirus(PAst V),porcine parvovirus(PPV),porcine epidemic diarrhea virus,porcine circovirus-like virus,porcine teschovirus virus,sapovirus,porcine serum-associated circovirus,and porcine associated smacovirus,which had the highest number of virus species infections.The genome sequences of four viruses with the highest infection rate were spliced,and 8 strains of PCV,5 strains of PBo V,4 strains of porcine kobuvirus(PKo V)and a torque teno sus virus(TTSu V)were successfully obtained and uploaded to Genebank to obtain the accession numbers.Nucleotide homology analysis and genetic evolution tree analysis were performed on the complete genome sequences of these four viruses to explore their evolutionary status.The results of nucleotide homology analysis showed that the whole genome homology among the eight PCV strains ranged from 94.8%to 99.9%,and from 91.1%to 99.7%with other reference strains;the whole genome homology among the five PBo V strains ranged from46.1%to 93.3%,and from 46.7%to 99.1%with other reference strains;the whole genome homology among the four PKo V strains ranged from 88.3%to 90.8%and from 86.9%to91.3%with other reference strains;the whole genome homology among the one TTSu V strain ranged from 42.2%to 85.6%with other reference strains.The genetic evolutionary tree results showed that the PCV genome-wide genetic evolutionary tree formed one major branch and five minor branches,and all eight PCV strains belonged to PCV2,among which one belonged to PCV2a,three belonged to PCV2b,and four belonged to PCV2d;the PBo V genome-wide evolutionary tree formed three branches,of which two strains belonged to PBo VG1,one belonged to PBo VG2 and two belonged to PBo VG3.The PKo V genome-wide genetic evolutionary tree was divided into two major branches,and three strains existed with the same branch;the TTSu V genome-wide genetic evolutionary tree formed two major branches,among which TTSu V1 formed two minor branches,and the TTSu V strain in this study belonged to TTSu V1b.Since PCV infection rate is the highest,and PCV2 is the most pathogenic compared to PCV1,PCV3 and PCV4,this study designed specific primers based on the conserved gene Rep of PCV2 and established a real-time quantitative fluorescent PCR(q PCR)assay based on SYBR Green I which facilitates future studies of PCV2 in our laboratory.The establishment of PCV2 assay showed that the minimum detection limits of q PCR and conventional PCR were 2.79×10~1copies/μL and 2.79×10~3copies/μL,respectively,and the sensitivity was 100 times of that of conventional PCR.In the specificity experiment,the method showed good specificity and did not react with PBo V,PKo V,TTSu V,PAst V and PPV.In the repeatability experiment,the intra-and inter-assay coefficient of variation was0.19%～0.25%and 0.27%～0.39%,indicating good repeatability.In the detection of clinical samples,the detection rates of q PCR and conventional PCR were 28.9%and19.7%respectively,and the accuracy of q PCR was higher.In conclusion,the co-infection of porcine viruses in some areas of Anhui Province and the evolutionary status of some porcine viruses investigated in this study provide a reference for disease prevention in the pig industry in Anhui,and the q PCR rapid detection method established for PCV2 can also provide effective technical support in clinical testing.