Epidemiological Investigation Of PRRSV In Guangdong Province In 2018 And Construction Of Full-length CDNA Clone Of Genotype 1 Strains

Porcine reproductive and respiratory syndrome(PRRS)is a viral and infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV)that seriously affects the global pig industry.Based on the differences in viral genome and antigenicity,PRRSV is divided into European(genotype 1)and North American(genotype2).Since 2006,the Highly Pathogenic PRRS(HP-PRRS)has caused huge losses for the pig industry in China.In recent years,genotype 1 PRRSV has been reported in China,which has brought severe challenges to China’s PRRS on the prevention and control.Guangdong Province is one of the major pig breeding provinces in China.Epidemiological surveillance of PRRSV in Guangdong Province is necessary.To investigate the prevalence of PRRSV in Guangdong Province in 2018,5597 serum samples and 900 suspected disease materials infected with PRRSV were collected from large-scale pig farms in Guangdong Province.The results of detection for antigen and antibody show that the positive rate of antigen are 25.3%(228/900,95% CI: 22.5%-28.3%);the positive rate of antibody are 78.9%(4417/5597,95% CI: 77.8%-80.0%).At the same time,the results of detection for antigen and antibody are analyzed by months,quarters,and regions,and the results show that there are no significant correlation for the antigen positive rate and antibody positive rate among different months,different quarters,and different regions,respectively.The results of detection for the antigen show that genotype 1and genotype 2 PRRSV existes in Guangdong Province,but genotype 2 PRRSV is predominant.In order to reserch the genetic variation of PRRSV in Guangdong Province in 2018,ORF5 genes from antigen-positive samples were sequenced and analyed,and a total of 36 sequences are obtained.The results of analysis for homology show that the homology for the nucleotide and amino acid(aa)among the genotype 2 PRRSV ORF5 sequences obtained in 2018 are 79.9%-99.3% and 79.1%-99.0%,respectively.And ORF5 sequences exhibite83.3%-99.2%(81.1%-98.5%),83.4%-94.7%(82.1%-93.5%),82.3%-99.7%(80.1%-99.5%),83.1%-95.2%(84.1%-94.0%),and 81.8%-95.4%(80.1%-95.0%)nucleotide(amino acid)homology with HP-PRRSV representative strain JXA1,Chinese classic strain CH-1a,North American classic strain VR2332,NADC30 strain and GM2 strain,respectively.The results of analysis for genetic evolution show that the GM2-like and NADC30-like subgroup strains are predominant in Guangdong Province in 2018,and the JXA1-like subgroup strains also possess certain proportion.The status of the existence of multiple subgroup strains indicates that the PRRSV epidemic strains in Guangdong Province are characterized by diversity.The results of analysis for amino acid sites show that the mutations of amino acid sites for different subgroups strains have a higher uniqueness.Four genotype 1 PRRSV strains are isolated from the antigen-positive samples,but none of them could adapt to MARC-145 cell.The results of analysis for amino acid of Nsp2 gene show that compared with the Lelystad virus(LV)strain,the EU-GD-HD strain and EU-GDDG18 strain have a continuous deletion with 67-amino acid(aa 282-348).The EU-GDFS18 strain and EU-GDCZ18 strain don’t have deletion in this region,but EU-GDCZ18 strain has a continuous deletion with 6-amino acids(aa 418-423).The results of analysis for amino acid of ORF3 and ORF5 gene show that four isolated strains have corresponding amino acid deletion in hypervariable region and more amino acid substitutions in signal peptide region for ORF3 gene and ORF5 gene,respectively.The results of analysis for homology show that the homology for genome-wide nucleotide among the four isolated strains is 85.8%-91.4%,and the homology is low;four isolated strains exhibites 84.8%-85.5%,60.0%-60.4% and 82.0%-94.4% nucleotide homology with genotype 1 PRRSV representative strain LV,genotype 2 PRRSV representative strain VR2332 and genotype 1 PRRSV reference strains isolated from China,respectively.The EU-GD-HD strain and EU-GDDG18 strain share the highest nucleotide homology with FJQEU14 strain,they are 94.3% and 91.4%,respectively.Genetic evolution and recombination analysis show that EU-GDDG18 strain is recombined from genotype 1strains FJQEU14 and BJEU06-1.The results suggest that the genetic recombination still occurs among PRRSVs,which leading to the emergence of new strains.Monitoring of new strains should be strengthened.In order to provide tools for the further study on the pathogenic mechanism of genotype 1 PRRSV,the whole genome sequence of the isolated genotype 1 PRRSV EU-GD-HD strain is cloned into the modified low copy vector pOK-PA.The CMV promoter and the BGH termination signal peptide,the foreign gene,are inserted into the two ends of the genome,respectively,and a full-length cDNA clone of the virus is successfully constructed.The full-length cDNA clone plasmid of the genotype 1 PRRSV EU-GD-HD strain will provide important experimental materials for the rescue of viruses in vitro and the research on the pathogenicity and replication ability of genotype 1 PRRSV in the future.