Molecular Epidemiology Investigation Of PRRSV In Different Regions Of China From 2016 To 2018

At present,PRRS has frequently spread out in the world and has the characteristics of widespread epidemics,high incidence,complicated infection,and difficulty in prevention and control.It has seriously restricted the healthy development of pig industry.As a big pig-raising country in the world,China is also facing the huge threat to pig health caused by PRRSV.In order to investigate the prevalence and the genetic evolution of the PRRSV in China from 2016 to 2018,112 suspected PRRS infection samples collected from Northeast,Central,East and North China were detected by RT-PCR,and the Nsp2,ORF5(ORF5a)genes of positive samples were cloned and analyzed by molecular epidemiological methods.The results showed that the total number of PRRSV positive samples was 57,and the total positive rate was 50.9%.56 Nsp2 gene sequences and 39 ORF5(ORF5a)gene sequences were cloned.The phylogenetic tree analysis of Nsp2 and ORF5 gene construction showed that the identified PRRSV strains in this study were North American(NA)type and belonged to lineage 1,3 and 8,among which the PRRSV identification strains accounted for the highest proportion in lineage 8 and accounted for the lowest proportion in lineage 3.The nucleotide and amino acid homology analysis showed that the nucleotide homology of the Nsp2 gene of 56 identified PRRSV strains was 53.0%~99.8%,and the amino acid homology was 46.8%~99.7%.The nucleotide homology of the ORF5 gene of 39 identified PRRSV strains was 82.4%~100.0%,and the amino acid homology was 79.6%~100.0%.The nucleotide homology of the ORF5 a gene of 39 identified PRRSV strains was 84.0%~100.0%,and the amino acid homology was 80.8%~100.0%.The results of the Nsp2 protein sequence alignment of the identified strains revealed that there were 7 new insertion and deletion mutation patterns;the amino acid mutation sites of the GP5 protein sequence of the identified strains are mainly concentrated in the signal peptide and epitope region;ORF5a protein sequence of the identified strains is 46 aa,and the amino acid mutation sites are mainly concentrated in the transmembrane and intramembrane regions.In addition,the sublineage 8.7 identified strains contained 2 conserved amino acid positions,V8 and S28,respectively;and the identified strains in lineage 1 contained 4 conserved amino acid sites: G12,F28,P41,T43.The results of molecular characteristics analysis of PRRSV showed that:(1)Recombination analysis indicated that 13 potential recombinant strains were recombined in the Nsp2 gene;4 potential recombinant strains with the same origin were recombined in the ORF5 gene.(2)Selection stress analysis indicated that 10 codon sites in the ORF5 gene of the identified strains were positively selected and 31 codons were under negative selection pressure;while codons under positive selection pressure were mainly distributed in GP5 protein signal peptide,N-glycosylation site;codons under negative selection pressure are mainly concentrated in the transmembrane region.Only 1 codon site in the ORF5 a gene of the identified strains was subjected to positive selection pressure,and 8 codon sites were subjected to negative selection pressure;codons under positive selection pressure were distributed in the intramembrane region of ORF5 a protein,Codons under negative selection pressure are mainly concentrated in the transmembrane region.(3)The predicted results of GP5 protein signal peptide indicated that the signal peptide cleavage site of sublineage 8.1 identified strain HLJ/2017/1127 c was between amino acid A26/V26~A27/V27;but the signal peptide cleavage site of lineage 1 and sublineage 8.7 identified strains were both the same,all between A31~S32/N32.(4)The potential N-glycosylation site analysis of GP5 protein showed that the number of Nglycosylation sites of 39 identified strains was 2~5,and the identified strains containing 4 Nglycosylation sites were the most;the identified strains containing 2 N-glycosylation sites were the least.In addition,the 44 and 51 N-glycosylation sites were the most conserved.The results showed that PRRSV was prevalent in many regions of China from 2016 to 2018,and there are mixed infections of different lineage strains in some areas;PRRSV epidemic strains in sublineage 8.7(HP-PRRSV)are still domestic dominant strains,and PRRSV epidemic strains in lineage 1 also account for a high proportion.In addition,compared with the classical strains of various lineages at home and abroad,different types of PRRSV strains in China have showed different patterns and different degrees of genetic variation,suggesting that the monitoring of PRRSV epidemics and genetic variation should be further strengthened.