Epidemiological Investigation Of Porcine Reproductive And Respiratory Syndrome Virus In Shandong Province From 2021-2023

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).It can cause sow abortion,premature delivery,stillbirth,deformed fetus and respiratory symptoms of pigs.Since the discovery of PRRSV in the United States in 1987,PRRSV has spread to all countries in the world.China isolated PRRSV in 1996,broke out highly pathogenic PRRSV in 2006,In 2014,the NADC30-like strain became the main epidemic strain in some regions of China,and in 2017,a new NADC34-like strain appeared,and the detection rate in some regions was rising.The PRRSV genome continues to undergo different degrees of mutation and recombination,which makes the prevention and control of PPRSV more difficult,causing serious economic losses to China’s pig industry.In order to understand the prevalence and genetic variation of PRRSV in Shandong Province,this study collected 2973 lung,lymph nodes and whole blood samples and 4360 serum samples from some pig farms in Shandong Province from 2021 to 2023,Using the detection primer designed according to PRRSV N gene for RT-PCR detection,a total of 543 positive samples were detected by RT-PCR using the detection primer designed according to the PRRSV N gene,with a positive rate of 18.26%.Among them,the detection rate of PRRSV in the lung was 20.83%,which was higher than that in lymph nodes and whole blood.1090 pig sera were tested for PRRSV antibody.The overall antibody positive rate was 77.7%,while the lowest PRRSV antibody positive rate in Yantai was 43.1%,and the vaccine protection rate was low.The positive samples detected by RT-PCR were isolated,identified,amplified and sequenced,and 78 PRRSV ORF5 gene sequences,37 PRRSV NSP2 gene sequences and 2PRRSV genome sequences were obtained.According to the genetic evolution tree constructed by ORF5 gene,the prevalent PRRRSV in Shandong Province is type II,of which 47 strains belong to Sublineage1.8(NADC30-like),19 strains belong to Sublineage8.7(JXA1-like),8strains belong to Sublineage1.5(NADC34-like),3 strains belong to Sublineage8.1(CH-1a-like),1 strain belongs to Lineage5(VR-2332-like),and NADC30-like strain is the main epidemic strain in Shandong Province,with a detection rate of 60.26%,Moreover,we found that the detection rate of NADC34-like virus strain is higher than before,and it may also be widely prevalent in Shandong Province in the future.This is what we need to pay attention to.The nucleotide and amino acid homology of ORF5 gene and NSP2 gene were analyzed respectively.The highest nucleotide and amino acid homology of ORF5 gene and CH-1a strain were83.6% ～ 98.8% and 81.1% ～ 99.0%,respectively,and the lowest nucleotide and amino acid homology with QYYZ strain were 82.6% ～ 85.6% and 80.0% ～ 85.6%,respectively,The highest nucleotide and amino acid homology of the isolated NSP2 gene and JXA1 strain were 57.7%～97.5% and 40.2%～95.9%,respectively.It was found that the NSP2 gene was more variable than the ORF5 gene.The GP5 protein encoded by ORF5 gene was analyzed,and R13 and R151 were the expression of virulent strains.The virulent strains in Lineage1 were not found,but were common in Lineage8.The 137 amino acid can distinguish vaccine strain(A137)and wild strain(S137).Only one strain in Lineage5 is A137,and the rest are S137.The strain in Lineage1 has a specific mutation in the amino acid at position 47,from isoleucine(L)to leucine(I).In addition,there are also antigenic epitopes in GP5 protein,of which the antigenic epitopes at positions 37 to 45(S37H38F/L39Q40L41I42Y43N44L45)are conservative and can be recognized by the corresponding lymphocytes in the host’s serum and induce the body to produce a specific immune response.Mutation of these amino acids may lead to the failure of the body to produce a corresponding immune response and reduce the immune protection rate of the vaccine.The analysis showed that there were mutations in different branches,The 39 th amino acid of WF-3isolate in Sublineage1.8 mutated to valine(V),and the 41 st amino acid of YT-10 mutated to serine(S);The 39 th amino acid of WF-3 isolate in Sublineage1.8 mutated to valine(V),and the 41 st amino acid of YT-10 mutated to serine(S);The 41 st amino acid of the isolates LY-1,TA-2 and JINAN-2 in Sublineage1.5 also mutated to serine(S);The 37 th amino acid of isolate YT-4 in Sublineage8.7mutated to phenylalanine(F),and the 39 th amino acid of the strain in this branch is isoleucine(I);The 38 th amino acid of the strain in Sublineage 8.1basically showed glutamine(Q),and the 39 th amino acid specific mutation was phenylalanine(F).A better understanding of the epitopes of PRRSV is conducive to the development of new vaccines.The deduced amino acid of NSP2 gene found that compared with the reference strain VR-2332,20 isolates in the subcliniage1.8 branch had discontinuous 131(111+1+19)amino acid deletions(322-432 aa,483aa,504-522aa),while 7 isolates in this branch had additional amino acid deletions.Among them,YT-6,YT-7 and QD-1 have additional 11 amino acid deletions at464-474 aa,isolate JINAN-3 has additional 5 amino acid deletions at 460-464 aa,isolate YT-10 has additional 4 amino acid deletions at 474-477 aa,isolate RZ-1 and BZ-2 have additional 3amino acid deletions at 523-525 aa.seven isolates in the subcliniage8.7 branch had discontinuous 30(1+29)amino acid deletions(481aa,533-561aa),two isolates in the subcliniage8.1 branch had no deletion of amino acids;one solates in the subcliniage1.5 branch had deletion of 100 continuous amino acid.According to the construction of genetic evolution tree based on the whole genome of the isolate and reference strain,it was found that ZB-1 belongs to Sublineage8.7 branch,and HZ-2 belongs to Sublineage1.8 branch.The nucleotide homology of ZB-1 is the highest with reference strain JXA1(99.2%)and the lowest with reference strain JS2021NADC34(83.1%);HZ-2 has the highest nucleotide homology with reference strain NADC30(88.6%)and the lowest nucleotide homology with reference strain QYYZ(82.2%).Through recombination analysis,it was found that ZB-1 does not have recombination phenomenon,HZ-2 has recombination phenomenon.and NADC30 is parent strain,which is formed by the recombination of NADC30,JXA1 and JS2021NADC34.In summary,PRRSV was prevalent in various regions of Shandong Province from 2021 to 2023,with NADC30-like strain being the main epidemic strain,followed by HP-PRRSSV.NADC34-like strain was also beginning to become prevalent in some regions of Shandong Province.The detection rate of PRRSV in lung is high.By analyzing the NSP2 and ORF5 genes of the virus,it was found that the PRRSV gene was easily mutated to different degrees,mainly mutations,deletions,and recombination.According to the different deletion of NSP2,the branch of the strain can be preliminarily determined.By analyzing the whole genome sequence of the virus,it was found that the recombination phenomenon would also occur between different branch virus strains.These mutations can complicate the prevalence of PRRSV and lead to low protection rate after vaccination.Therefore,investigating and analyzing the variation and prevalence of PRRSV strains in Shandong Province can increase the epidemiological data of PRRSSV in Shandong Province and provide information for vaccine development.