Isolation And Identification Of European-type Porcine Reproductive And Respiratory Syndrome Virus Strains And Sequencing And Analysis Of Whole Genomes

Porcine Reproductive and Respiratory Syndrome(PRRS)is an acute contact infectious disease of pigs caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)infection.It is characterized by reproductive disorders in pregnant sows and respiratory symptoms in piglets.PRRSV can be divided into European type(EU-type)and American type(NA-type)based on its genetic differences.Both genotype strains are prevalent in China.Among them,the American PRRSV has a wider epidemic range and is more harmful to the pig industry.However,the detection rate of European-type PRRSV in pig herds is increasing year by year,and the impact on the pig industry is also gradually increasing.Because domestic epidemiological survey data and research on European-type PRRSV are far less than American-type PRRSV,Therefore,tracking and testing the clinical prevalence of European PRRSV is of great significance to disease prevention and control.In this study,a dual TaqMan fluorescent quantitative RT-PCR method for distinguishing American and European PRRSV was established,and it was used to detect clinical samples of suspected PRRSV infection collected in Henan and surrounding areas from 2018 to 2020,Therefore,tracking and testing the clinical prevalence of European PRRSV is of great significance to disease prevention and control.1.Establishment and preliminary application of dual TaqMan fluorescence quantitative RT-PCR identification method for American and European PRRSVIn order to distinguish between European and American PRRSV strains more quickly and accurately,in this experiment,based on the ORF7 gene sequence characteristics of GenBank Central American and European PRRSV strains,two pairs of specific primers and two TaqMan probes with different fluorescent signals were designed.After optimizing the concentration of primers and probes in the reaction body and the reaction conditions,a dual TaqMan fluorescence quantitative RT-PCR identification method for American and European PRRSV was established.This method can specifically distinguish American and European PRRSV,and has no cross-reactivity with other common swine viruses;The detection limits of the American and European PRRSV recombinant plasmids are 10 copies/?L and 100 copies/?L,respectively;The coefficient of variation in the stability test is less than 2.00%,which proves that this method has good specificity,sensitivity and stability.This method was used to test 240 clinical samples from 35 pig farms in Henan and surrounding areas.110 were PRRSV positive samples,with a positive rate of 45.8%,of which 105 were American PRRSV and 5 were European PRRSV.This indicates that there are European PRRSV strain infections in Henan and surrounding areas,and the surveillance,prevention and control of European PRRSV should be strengthened.2.Isolation and identification of the European PRRSV strainTissue grinding was performed on 5 clinical samples tested as European type PRRSV,and PAM cells were inoculated with the tissue grinding fluid.Among them,4 samples showed cytopathic changes after inoculation with PAM cells.Fluorescence quantitative RT-PCR and indirect immunofluorescence(IFA)identification were performed on the harvested 5 cell fluids.The results showed that 4 of the 5 cell fluids were positive for European PRRS V,which proved that 4 European PRRSV strains were successfully classified.3.Determination and analysis of the whole genome sequence of the European PRRSV strainIn order to further study the characteristics of the genome sequence of European PRRSV,this study determined the complete genome sequence of the virus in 4 isolated European PRRSV strains and 1 piece of disease material slurry.The homology and genetic evolution of the 5 European-type sequences obtained in this study and the one European-type sequence obtained by our laboratory in 2015 were analyzed.The results showed that the nucleotide sequence similarity between the six European-type sequences and the American-type reference strain VR2332 was 60.0-60.4%,and the nucleotide sequence similarity with the European-type reference strain Lelystad virus was 85.0-89.8%.Compare the deduced amino acid sequences of NSP2,ORF3,and ORF4 with the Lelystad virus strains with large variation,the results showed that six European PRRSV strains had different numbers of amino acid deletions,and four of them showed new deletion characteristics.These deletions increased the genetic diversity of European PRRSV,but the biological characteristics caused by them are still unclear.Perform genetic evolution analysis on the whole genome and ORF5 gene sequence,It shows that all 6 European PRRSVs belong to Subtype I(Global),Among them,two strains are in the same evolutionary branch as BJEU06-1 strain,one is in the same evolutionary branch as NMEU09-1,one is in the same branch as the Amervac vaccine strain,and two strains form new branches.Recombination analysis showed that there was no recombination in the 6 European PRRSV strains.This study provides a reference for studying the variation and genetic evolution of European PRRSV in China.