Molecular Epidemiological Investigation Of Porcine Circovirus Type 2 In Partial Jiangsu Area Of China And Studies On Its Pathogenicity

Porcine circovirus(PCV)belongs to Circoviridae,Circovirus,and it has a single-stranded circular DNA genome without capsule.Now,PCV has two genotypes,which are PCV1 and PCV2.The genotype of PCV2 is divided into five subtypes,which are PCV2a,PCV2b,PCV2c,PCV2d,PCV2e.PCV2 does not cause deaths of susceptible pigs while restrain the function of immune system.PCV2 is the predominant agent of postweaning multisystemic wasting syndrome(PMWS).When it infects pigs with other pathogens,it may lead to the death of pigs.It caused a large loss to the farm,so we must pay attention to PCV2 associated diseases(PCVD).In recent years,the genotype of PCV2 is always changing which caused the complexity of epidemic situation.In this study,the clinic samples from some large-scale slaughterhouses in Yangzhou and the diseased pigs subjected to the animal hospital of Yangzhou University were collected,and the genomic fragments of Porcine circovirus type 1(PCV1)?Porcine circovirus type 2(PCV2)?Porcine pseudorabies virus(PRV)?Porcine parvovirus(PPV)?Classical swine fever virus(CSFV)and Porcine reproductive and respiratory syndrome virus(PRRSV)were detected from the collected samples by polymerase chain reaction(PCR)or reverse-transcription PCR(RT-PCR).The samples positive in PCV2 PCR detection is homogenized and inoculated into the PCVs-free Dulac cells and determine its 50%tissue culture infective dose(TCID50).At same time,part of the PCV2 isolates were subjected to genetic evolution analysis and pathogenicity evaluation in pigs,aimed to provide the epidemic information related to PCV2 and their evolution situation.In addition,two PCV2 isolates were selected and inoculated into susceptible piglets to evaluate their pathogenicity.Study 1.Epidemiological investigation of PCV2 and other common viruses in partial Jiangsu region during 2015 and 2016575 lymph samples of PMWS suspicious pigs collected from 2015 to 2016 in partial Jiangsu area of China were detected for the swine common viruses by PCR and RT-PCR.The results elucidated that 46 cases were PCV1 positive,with the positive rate 8%,287cases were PCV2 positive,with the positive rate 50%;133 cases were PPV positive,with the positive rate 23%;80 cases were PRV positive,with the positive rate 14%;34 cases were PRRSV positive,with the positive rate 6%;39 cases were CSFV positive,with the positive rate 7%,respectively.The positive rate of PCV2/PPV co-infection rate was 17.5%(70 of 575);PCV2/PCV1 mixed infection rate was 11.5%(46 of 575);PCV2/PRV mixed infection rate was 8.7%(35 of 575).To inquire the genetic variation of PCV2 in partial Jiangsu area mentioned above,twenty-eight PCV2 strains were isolated and identified by sequencing.The results showed that the PCV2 isolates with their complete genome sequence could be classified into three genotypes,PCV2b(7 of 28),PCV2a(1 strain),and PCV2d(20 of 28),with the genome length of 1767 base pairs(bp)(27 strains)and 1766 bp(1 strain).PCV2 genotypes harbored special mutation sites in their ORF2 accordingly.At the genotype level,PCV2d was the dominant cluster in above region between 2015 and 2016.Study 2.Studies on pathogenicity of PCV2 isolatesStrain 151107 YZ and strain 160924 YC both belonging to PCV2d were selected to evaluate their pathogenicity in susceptible piglets.First,the Dulac cell line was.chose to isolate and replicate virus.Genomic DNA of passages F3,F7,F10,cultures was extracted and their TCID50 was determined.We found that virus titer rise along with the virus passage.Eight pigs without PCV2 antibody are randomly assigned into three groups:mock control group(n = 2);pigs in group 2 challenged with PCV2d(151107 YZ;n = 3),pigsin group 3 challenged with PCV2d(160924 YC;n = 3).Blood samples were collected at day post-inoculation(dpi)3,7,10,14,17 and 21 to detect the presence of PCV2 antibody and copies of PCV2 DNA.The results showed that the PCV2 isolate can replicate in Dulac cells and passage.The results indirect immunofluorescence assay(IFA)indicated that the TCID50 of two PCV2 isolate reached to 106.0/mL,respectively.In the piglet challenge model,the infected animals exhibited PCV2 viremia and PCV2 viral genomic loads with the peak level at dpi 7.Pathological examination results indicated that all challenged pigs exhibited typical lesions caused by PCV2 at vary degrees compared with the mock pigs.