Investigation Of Porcine Reproductive And Respiratory Syndrome Virus Infection And Analysis Of Recombinant Evolution Of Epidemic Strains

Porcine reproductive and respiratory syndrome(PRRS)is caused by Porcine reproductive and respiratory syndrome virus(PRRSV).It is commonly known as blue ear disease and its characteristics are mainly pregnant mothers.Pigs suffer from reproductive problems such as premature birth,miscarriage,stillbirths,and mummified fetuses.Pigs and fattening pigs have severe respiratory diseases and are virulent viral infections.Since the first report in China,the disease has experienced more than 20 years of epidemic and evolution in China,especially the outbreak of highly pathogenic blue ear disease(HP-PRRS)in China in 2006,which brought huge economic losses to the pig industry.The NADC30-like strains with the same deletion characteristics as the NADC30 variant strains isolated in the United States in 2008 have appeared successively in 13 provinces and regions in China,and have recombined with domestic epidemic strains,and the types of recombination are complex and diverse.PRRSV is highly variable,leading to significant genetic diversity.There are certain differences in the pathogenicity and antigenicity of different strains,as well as the development of vaccines that cannot keep up with the speed of variation,preventing and controlling PRRS epidemics.Bring great difficulties.The aim of this study was to investigate the molecular epidemiology of PRRSV in Shandong province,the isolation and identification of three new variant strains,genome-wide sequence analysis,recombination analysis and genetic evolution analysis,which provided a reference for prevention and control of PRRS in this area.1.Molecular epidemiology of PRRSV in Shandong province from 2014 to 2017In this study,we collected a total of 347 clinical samples from 13 pig farms in Shandong province from June 2014 to April 2017.PRRSVs were detected by RT-PCR,and 243 samples were PRRSV-positive.The results of sequence analysis showed that all the strains belonged to North American type,and the proportion of NADC30 strain was 50.62%,higher than the classical PRRSV(6.67%)or HP-PRRSV(42.80%).2.Isolation and identification of 3 PRRSV recombinant strainsTo understand the prevalence of PRRSV in Shandong Province,PRRSV-positive samples were inoculated into Marc-145 cell lines or primary PAM cells and passed blindly for three generations.The virus was detected by RT-PCR and indirect immunofluorescence.Identification,the results of the separation of three strains of new strains of PRRSV laid the foundation for further study of PRRSV pathogenicity and genetic evolution and other aspects.3.Whole genome sequencing and analysis of 3 new strains of PRRSVIn order to further understand the genetic variation of SDhz1512,SDlz1601 and SDYG1606 in Shandong province,12 pairs of primers were designed according to the genome-wide sequence of the representative virulent strains which were close relatives in GenBank.The whole genome was amplified,sequenced,assembled and sequenced.The recombinant viruses were analyzed by RDP and Simplot recombination software.The genetic evolution was analyzed by RaxML and BEAST bioinformatics software.The results showed that the genetic variations of SDlz1601 and SDYG1606 shared by Shandong strain and HP-PRRSV were far from the whole genome and closely related to NADC30,but SDhz1512 strain was associated with HP-PRRSV.The close relationship between NADC30 and NADC3 0-like in some genes such as ORF7 showed that the three isolates were newly emerged mutants in China.Our results suggested that the importance of recombinant strains in Shandong,China,might be due to varying degrees of recombination between classical,HP-PRRSV or NADC30 strains.Therefore,it was indicated that not only the new variant strains undergo recombination and the recombination was more and more diversified,the strain might be highly recombined by adapting to the living environment of PRRS in our country rapidly,which should be given high priority..4.Multiple TaqMan real-time fluorescent PCR assays to identify classical PRRSV strains,highly pathogenic variants and NADC30-like strains and their clinical application.To establish a rapid diagnostic method for porcine reproductive and respiratory syndrome virus(PRRSV),to design Nsp2 as a target gene to design classic type,highly pathogenic variant strains,and PRRSV-specific primers and specific probes for NADC-like 303 types,targeting ORF7.Gene Design American-type universal primers and universal probes,through the optimization of the fluorescence PCR reaction conditions and reaction system,the establishment of multiple real-time fluorescence PCR method.The method is highly specific,sensitive,and has a detection limit of 10 virus copies.The use of multiple real-time fluorescent PCR detection methods for clinical samples,compared with the sequencing results are consistent,indicating that the method is accurate,especially for different PRRSV strains mixed infection of the sample compared to PCR sequencing,the advantages are more prominent,Closed pipe operation,no pollution.The establishment of this method provides an effective technique for the rapid identification and epidemiological investigation of PRRSV.