Isolation And Identification,Sequence And Pathogenicity Analysis Of Analysis Of Different PRRSV Strains

Porcine reproductive and respiratory syndrome(PRRS)is an infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),and characterized by reproductive failure in sows,respiratory problems affecting pigs of all ages and increased morbidity and mortality in piglets.The disease was first found in 1987 in the United States and became one of the main infectious disease of pigs.Nowadays the virus circulates in most parts of the world.In China,since the first PRRSV strain was isolated in 1996,PRRSV has already experienced prevalence and evolution over twenty years.At present,there co-exist the classic PRRSV,the highly pathogenic PRRSV(HP-PRRSV)and the NADC30-like PRRSV strains which were recently found in 2012 and easily recombined with other PRRSV strains.However,the existing PRRSV vaccines,including the classic vaccine and the highly pathogenic one,cannot protect the host from the harm caused by NADC30-like strains effectively.What’s more,some attenuated HP-PRRSV vaccine has some risk of virulence return,increasing the difficulty on the prevention and control of the disease.In this study,the PRRSV strains were isolated and analyzed based on the NSP2 genes,ORF5 genes and complete genome to reveal the variation characteristics,the genetic variation and the recombination situation of PRRSV,as well as to revealed the latest molecular epidemiology and genetic variation of PRRSV in the fields.Moreover,the pathogenicity comparison of different PRRSV strains were made to provide reference for outbreak alert,clinical diagnosis and vaccine development of PRRSV.1.Isolation and identification of different PRRSV strainsTo understand the characteristics of PRRSV strains in the fields,217 samples of suspected PRRSV infected pigs collected from pig farms during 2015～2016 in different regions of Henan province were detected by RT-PCR.The results showed that 54 samples were PRRSV positive,and the positive rate was 24.88%.The PRRSV positive specimens were passed through a 0.22-μm filter and inoculated onto MARC-145 cells,and the viruses harvested from the supernatant were identified by RT-PCR and indirect immunofluorescence assay(IFA).The results revealed that 10 PRRSV strains were isolated from 54 positive specimens,including HP-PRRSV strains,HP-PRRSV vaccine revertant strains,NADC30-like strains and their recombinants.These isolates were designated as HENZK-1,HENZMD-9,HENXC-4,HENZZ-8,HENXY-2,HENXY-3,HENLY-6,HENNY-4,HENPY-2 and HENHB-8,and viral titers of their fifth viral passage were determined as 106.8TCID50/mL,106.7TCID50/mL,106.33TCID50/mL,106 75TCID50/mL,106TCID50/mL,106.15TCID50/mL,106 75TCID50/mL,106.53TCID50/mL,106.6TCID50/mL and 106.35TCID50/mL,respectively.This assay provided reliable reference materials for the research about pathogenicity and evolution of PRRSV.This assay provided reliable reference materials for the research about evolution and pathogenicity of PRRSV.2.Sequence determination and analysis of NSP2 gene,ORF5 gene and complete genome of different PRRSV isolatesNSP2 and ORF5 genes of the PRRSV isolates were amplified,sequenced and analyzed.The results showed that 5 of the 10 isolates,including HENZK-1,HENZZ-8,HENLY-6,HENNY-4 and HENPY-2,were highly homologous with HP-PRRSV JXA1,and the rest isolates were highly homologous with NADC30 containing a discontinuous 131-amino acids deletion in Nsp2 protein.Two isolates of either group were chose to be sequenced and analyzed for the complete genome.The results showed that the isolates HENZK-1,HENZZ-8,HENLY-6,HENNY-4 and HENPY-2 were 15319bp,15320bp,15017bp and 15023bp in length,respectively,excluding the poly(A)tail.The results of the nucleotide sequence similarity,genetic evolution and recombination analysis showed that HENZZ-8 was a HP-PRRSV,HENZK-1 was a revertant of JXA1-R vaccine,HENXC-4 was a NADC30-like strain while HENZMD-9 was a recombinant of HP-PRRSV and NADC30-like strains."Thus,the four isolates were considered to belong to four different subtypes of PRRSV strains.This assay provided scientific reference data not only for the study on the prevalence,genomic evolution and virulence of HP-PRRSV strains,but also for the development and use of vaccine of PRRS.3.Pathogenicity study of different PRRSV isolatesTo understand the pathogenicity of different isolates,four different subtypes of PRRSV isolates(HENZK-1,HENZMD-9,HENXC-4 and JXA1-R vaccine revertant HENPDS-2)were tested on animal experiment.The results revealed that the pathogenicity of four isolates from strong to weak were HENXC-4,HENZK-1 HENZMD-9 and HENPDS-2 in order.JXA1-R vaccine group showed no obvious clinical symptoms but some pathological symptoms.Compared with JXA1-R,HENZK-1 showed strong pathogenicity,speculated that the major cause of the endemic situation of the herds was related to this isolate,but the cause of its pathogenicity still need further research.This assay provided reliable reference to determine the pathogenicity of different PRRSV strains as well as the scientific use of PRRSV vaccines.