The Genetic Variation Analysis And The Pathogenicity Assessment Of PRRSV HBap4/2018 Strain

Porcine reproductive and respiratory syndrome(PRRS),an animal epidemic disease,has caused huge economic losses to the pork industry of our country in the past decade.PRRSV is highly contagious pathogen,which can lead to reproductive disorders in sows and severe respiratory diseases in piglets and growing pigs.PRRSV mutates easily under environmental or immune pressure,and itsgenetic characteristic and the pathogenicity vary among different isolates.Therefore,monitoring genetic variation in PRRSV isolates in a timely manner will provide a solid scientific basis for PRRSV prevention and control policy making and be also very helpfulfor the development of effective vaccines.In this study,we first tested 566 clinical samples of sick pigs and found that 246 of them were PRRSV positive.The Nsp9,Nsp2 and ORF5 genes of some positive samples were sequenced and analyzed.Phylogenetic trees were generated based on the ORF5 and Nsp2.Briefly,18 ORF5 genes were mainly clustered to lineage 3 and 8;28 Nsp2 genes were clustered to lineage 1,5 and 8;10 Nsp9 genes were clustered to lineage 1 and 8.The results of amino acid sequence analysis showed that Nsp2 have four different amino acid deletion patterns among different strains: 1+29 aa,111+1+19 aa,111+5+1+19 aa,1+29+14 aa.SHpd1/2018 Nsp2 exhibited 131 aa deletions,similar to NADC30 strain,and HBap4/2018 Nsp2 showed 5 additional aa deletions.It is noteworthy that,Nsp2 and ORF5 of HBap4/2018 belong to different gene subgroups and the same is shown inSHpd1/2018,so we speculate that genome recombinationmay happened in these two strains.In order to further analyze the genetic and biological characteristics of HBap4/2018 and SHpd1/2018 strains,we successfullyisolated these two strains from clinical samples,and found both could be specifically recognized by monoclonal antibodies targeting GP5 and N proteins of PRRSV HuN4 strain.However,Nsp2 protein of HBap4/2018 and SHpd1/2018 strains could not be recognized by monoclonal antibodiesfor HuN4 strain.These 2 stains also showed similar characteristics in proliferation and plaque morphology to HuN4 strainin Marc-145 cells.The results of recombination analysis showed that both strains were recombined from HP-PRRSV strain(main parent strain)and NADC30 strain(secondary parent strain).Furthermore,the recombination patterns of the two strains were different.The SHpd1/2018 strain had two recombination breakpoints(nt 2002 and nt 3205)and both are in the hypervariable region(HV II)of Nsp2 gene,while the HBap4/2018 strain had five recombination breakpoints(2000 nt?5105 nt?6292 nt?7412 nt and 14249 nt),which are located in the Nsp2,Nsp3,Nsp5,Nsp7 and ORF7 gene regions,respectively,indicating that HBap4/2018 is a new natural recombinant PRRSV strain.In order to understand the pathogenic characteristics of the new recombinant mutant strain HBap4/2018,30-day-old piglets were inoculated with HBap4/2018 strain,and clinical symptoms,virus loads in serum,viral shedding in nasal swabs were measured and histopathologicalstudy was conducted.The results showed that after infection with HBap4/2018,all the piglets developed clinical symptom including persistent high fever,dyspnea,anorexia and Depressed.There were 3 piglets died on days 4,15 and 28 after infection,respectively,and the final mortality rate reached 60%.The above results indicate that the novel recombinant PRRSV isolate HBap4/2018 is highly pathogenic to piglets.This study focused on genetic evolution of PRRSV epidemic strains found in some areas of China in recent years.We isolated a natural recombinant PRRSVstrains with new genetic characteristics.The PRRSV recombinant mutant retained the genetic and biological characteristics of the HP-PRRSV strain.We confirmed that new natural recombinant virus HBap4/2018 strain is highly pathogenic to piglets.This study will help us to deeply understand the genetic evolution trend and pathogenic characteristics of PRRSV epidemic strains in China,and provide a solid basis for the scientific prevention and control of PRRSV as well as the development of new vaccines.