| PRRS is an acute and highly contagious viral infectious disease that occurred for the first time in 1987,causing infected herds to develop diseases characterized by reproductive failure and respiratory symptoms.PRRSV was first reported in China in1995,and PRRSV was first isolated from aborted fetuses in pigs by Guo’s team in 1996 and named CH-1a.In 2006,an outbreak of a disease originally known as "swine hyperpyrexia" was reported in China,affecting farms in more than a dozen provinces and causing huge economic losses,after which it was reported that this outbreak was caused by highly pathogenic blue ear disease(HP-PRRSV).After 2013,the epidemic trend of NADC30-like PRRSV gradually increased in the country.PRRSV is prone to mutation,combined with vaccine immunization is still controversial,so it is important to strengthen the continuous monitoring of PRRSV clinical epidemic strains for the diagnosis the disease.The clinical strains of PRRSV from 2020 to 2021 were investigated,and some strains were sequenced and analyzed.1.Detection and sequencing analysis of PRRSV in clinical samples3536 samples were collected from affected pig farms in 24 provinces,municipalities and autonomous regions of China and 1141 samples were collected from slaughterhouses in some areas of Hubei Province from 2020 to 2021,including lung,lymph nodes,brain,stillbirth,spleen and blood samples.The ORF5 and Nsp2 genes of the PRRSV-positive samples were tested by RT-PCR and the full length of the ORF5 gene of the 257 positive samples was sequenced.The results showed that 974 samples from farms tested positive for PRRSV and 28 samples from slaughterhouses tested positive for PRRSV,with positive detection rates of 32.8% and 2.4%,respectively.Among all samples,257 positive samples were randomly selected to sequence the ORF5 gene,and genetic evolution analysis was performed based on this gene,and the results showed that: Lineage 1(NADC30-like)and Lineage 8.7(JXA1-Like/CH-1a-like)were the main epidemic strains,with detection rates of 37.74% and 29.96%,respectively.2.Isolation and identification of PRRSVIn this study,26 PRRSV strains were isolated from 1002 positive samples.Sequencing analysis of the ORF5 and Nsp2 genes was performed on the 26 isolated strains,and the results showed that based on the ORF5 gene,all 26 isolated strains belonged to PRRSV-2,of which 18 isolated strains belonged to Lineage 1(NADC30-like);4 isolated strains belonged to Lineage 8.7(JXA1-like/CH-1a-Like);3 isolated strains belonged to Lineage 3(QYYZ-like);and 1 isolated strain belonged to Lineage 5.1(VR2332-like).Amino acid deduced sequence analysis of the isolated strains and the reference strains(VR2332,JXA1,GM2,NADC34,CH-1a and NADC30)showed that the mutation sites of ORF5 were mainly concentrated in the signal peptide and linear epitope regions.Strains of Lineage 1(NADC30-like)have a discontinuous deletion of 131 amino acids on the Nsp2 protein,which is consistent with reports at home and abroad.Among them,Hu B-2-China-2021 had a deletion of 135 amino acids(111+5+19)at positions322-432,482-486 and 504-522.In this study,we showed that both Nsp2 and ORF5 genes of PRRSV clinical strains were greatly mutated,suggesting that the widespread use of attenuated vaccines may be responsible for the epidemic of PRRSV.3.Whole genome sequencing analysis of some PRRSV isolated strainsFive of the 26 isolated strains were subjected to whole genome sequencing,and the five strains were named SN-2-China-2021,GS-9-China-2021,GX-11-China-2021,HE-2-China-2021,and XJ-1-China-2021.The sequencing results were analyzed.The results showed that all the 5 isolated strains belonged to NADC30-like strains;in addition,all the 5 PRRSV strains had recombination phenomenon,and the main parental strains were NADC30;the analysis of each gene and its amino acid sequence of the whole genome showed that the largest mutation regions of each gene were Nsp2,followed by GP4. |
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