关键词: 猪繁殖与呼吸综合征病毒, 分离鉴定, 重组, 遗传演化分析

Abstract:

【Objective】This study aimed to provide basic data for prevention and control of swine diseases in pig farms through analysis about molecular characterization of PRRSV isolated from the lungs of diseased piglets in pig farm in Tianjin. 【Method】Lung tissues of diseased piglets were collected, and PRRSV nucleic acid positive samples were inoculated into porcine alveolar macrophages (PAMs) for virus isolation and identification. The virus was purified by limited dilution method and identified by indirect immunofluorescence assay (IFA). Then, it was segmented amplification using overlapping primers of RT-PCR, and whole genome sequence was obtained by cloning. The nucleotide and amino acid sequences of N1 strain were analyzed for homology, genetic evolution, and recombination events using biological analysis software. 【Result】The PRRSV strain, named N1, was successfully isolated with the whole genome length of 15016 bp, excluding polyA tail. The homology analysis based on whole genome sequences showed that the homologous of N1 strain was 85.0%-90.0% to other lineage PRRSV strains, with the highest homology of 90.0% to lineage 8.3, the representative strain of JXA1, and the N1 isolate shared 93.7% identity with lineage 3 (QYYZ-like) in the ORF2a, ORF2b, and 3'UTR regions, and shared 96.2% identity with in the ORF5-ORF7 regions with lineage 1.8 (NADC30-like), while the rest of the regions had a high degree of homology with sublineage 8.3 (JXA1-like). It had similar 131 (111+1+19aa) discontinuous amino acid deletion pattern with lineage 1.8 (NADC30-like) in Nsp2-coding region. The virulence sites and major neutralizing antigenic epitopes of GP5 protein were identical to lineage 1 (NADC34-like, NADC30-like), but three N-glycosylation sites locating at N³³, N⁴⁴ and N⁵¹ were mutated to a certain extent compared with the other representative strains. These potential glycosylation sites were likely to contribute to the differences in viral virulence and led to the occurrence of viral immune escape. According to the genetic phylogenetic tree constructed based on ORF5 and whole genome sequences, the genetic evolution tree based on the ORF5 gene showed that N1 strain was closer to lineage 1.8 (NADC30-like) and belonged to the same branch, but N1 strain was in the same branch as lineage 8.3 (JXA1-like) strain based on whole genome genetic evolution tree, suggesting the possibility of this strain recombination. Recombination events of the N1 strain were analyzed by Simplot and RDP4.0 softwares, and the results of the recombination analyses showed that the N1 was a multiple recombinant virus from NADC30-, JXA1-, and QYYZ-Like strains with 13 recombination events and recombinant fragments located at A(1-421 nt), B(422-1 735 nt), C(736-3 598 nt), D(3 599-4 439 nt), E(4 440-5 762 nt), F(5 763-6 451 nt), G(6 502-7 226 nt), H(7 227-8 295 nt), I(8 296-9 312 nt), J(9 313-11 536 nt), K(11 537-12 854 nt), L(12 855-13 859 nt), M(13 860- 15 016 nt) with recombinant breakpoints in Nsp1, Nsp2, Nsp3, Nsp4, Nsp7, Nsp9, Nsp12, ORF3, and ORF5. 【Conclusion】The PRRSV N1 strain isolated in this experiment was a three-lineage recombinant strain of PRRSV with the sublineage 8.3 (JXA1-like) strain as its main parent, it exhibited extensive and complex recombination patterns between sublineage 1.8, lineage 3, and sublineage 8.3, and the results provided the reference for the comprehensive prevention and control of porcine reproductive and respiratory syndrome in Tianjin area.

Key words: PRRSV, isolation and identification, recombination, genetic evolution analysis