Pathogen Detection Of PRRSV In Parts Of Sichuan And Genome Sequence And Pathogenicity Analysis Of SCcd2020 Strain

Porcine reproductive and respiratory syndrome（PRRS）has been prevalent worldwide for decades,but it has not been effectively controlled so far.The reason for this is that Porcine reproductive and respiratory syndrome virus（PRRSV）,in addition to has the strong clinical infectious,and its genome characterized by rapid mutation and frequent recombination.Since the discovery of PRRSV in China in 1995,several variants of PRRSV have evolved to date.These PRRSV variants not only showed genetic diversity,but also showed differences in the pathogenicity of piglets.The emergence of variant strains complicates the PRRSV epidemic situation and increases the difficulty of PRRS prevention and control in China.The purpose of this study is to keep abreast of the epidemiological situation and evolutionary characteristics of PRRSV in Sichuan Province.A total of 210 samples of suspected PRRSV infection materials were collected from the infected pig farms in the region from 2020-2021,and 57 samples were positive,with a positive rate of 27.1%.18 strains of ORF5 genes were amplified by ORF5 gene sequencing in the positive samples.Among which 3 strains were CH-1a-like PRRSV（16.67%）,5strains were HP-PRRSV（27.78%）,3 strains were VR2332-like PRRSV（16.67%）,4 strains were NADC30-like PRRSV（22.22%）,3 strains were NADC34-like PRRSV（16.67%）.The pathogen detection results showed that NADC30-like PRRSV and HP-PRRSV were the two types of strains with high clinical detection rates in Sichuan,while the imported NADC34-like PRRSV from North America was the first detection in the region in 2020,which should be allowed to be alerted.In order to analyze the genetic characteristics of NADC34-like PRRSV in Sichuan,a strain of NADC34-like PRRSV,named SCcd2020,was successfully isolated by PAMs in this study.The isolation of SCcd2020 strain was confirmed by cytopathic effect（CPE）observation and identification by RT-PCR and indirect immunofluorescence（IFA）,followed by whole genome sequence analysis of the isolated SCcd2020 strain.The results showed that the full-length sequence of SCcd2020 strain was 15020 bp,with 79.8%～89.2%nucleotide homology with the whole genome sequence of 12 PRRSV-2 representative strains,and the highest homology with NADC30 strain,the nucleotide homology of ORF5 gene was 84.4%～97.0%,and the amino acid homology was84.1%～96.5%,and the highest homology with IA/2014/NADC34 strain with the highest homology.The deduced sequence of amino acids showed that the deletion characteristic of 111+1+29 AA in Nsp2 protein of SCcd2020 strain was the same as NADC30-like PRRSV.N34,N44,N51 and N57are the four glycosylation sites present in GP5 protein.Genetic evolution showed that the ORF5 gene of SCcd2020 strain clustered with NADC34-like PRRSV;the whole genome clustered with NADC30-like PRRSV,indicates there were differences in genetic evolution.The Bayesian differentiation time showed that the earliest divergence of NADC34-like PRRSV in China was during 2013-2014,coinciding with the outbreak of this strain in the U.S.It is speculated that domestic NADC34-like PRRSV has been introduced into my country during the outbreak of this strain in the U.S.Recombination analysis showed that the SCcd2020 strain was a recombinant strain formed by NADC30-like PRRSV,HP-PRRSV and NADC34-like PRRSV,and the recombination breakpoints were located at Nsp2（3866 nt）,Nsp2（4388 nt）,Nsp3（5211 nt）,Nsp9（7957 nt）and ORF2b（11975 nt）.The recombination event between NADC34-like PRRSV and native HP-PRRSV was reported for the first time in China,which provided a new reference for the epidemiological investigation of PRRSV in my country.To further analyze the pathogenic characteristics of the recombinant variant of SCcd2020,the SCcd2020 strain was attacked on four-week-old piglets at 1×10^9.19 TCID₅₀/m L（2 m L intramuscular injection+2 m L nasal drip）.The pathogenicity of SCcd2020 strain in piglets was evaluated by the clinical manifestations,serum viral load,antibody level and pulmonary pathological changes of piglets after challenge.The results of animal experiments showed that SCcd2020 strain infection can cause severe clinical symptoms in piglets,including high fever（>41℃）,dyspnea,weight loss,diarrhea,depression,etc.The incidence rate is 100%（5/5）,and the mortality rate is as high as 60%（3/5）.The pathological changes in the lungs on autopsy were severe,and the lung tissue was accompanied by hemorrhagic,congested and fibrotic lesions.The viral serum load peaked at approximately（9×10⁸copies/m L）on the 10th day.The antibody test turned positive on the 7th day.Animal results showed that recombinant variant SCcd2020 strain was similarly pathogenic to HP-PRRSV in piglets,significantly higher than the mind strain HLJDZD32-1902（NADC34-like PRRSV）.In conclusion,the complex recombination has occurred between NADC34-like PRRSV and domestic indigenous HP-PRRSV,and recombinant variant SCcd2020 strain is highly pathogenic to piglets.The results of this study will help China to better understand the genetic evolution characteristics of NADC34-like PRRSV and provide data to support the scientific control of this strain in China.