The Stability Of Virulence Of PRRSV FJ1402 Strain And Establishment Of An Indirect ELISA For Detecting The Antibody To The Viral GP5

Porcine reproductive and respiratory syndrome virus is one of the most important pathogens that can infect pigs of all ages,causing respiratory and reproductive diseases.PRRSV is an RNA virus,which is very prone to recombination and mutation to produce new virus strains.In 2006,China's outbreak of highly pathogenic PRRSV caused serious economic losses.Since 2013,a new PRRSV-like NADC30 variant strain(NL-PRRSV)has appeared in some provinces in China,and has gradually become the main epidemic strain in China.The significant changes in viral antigenicity and pathogenicity have brought new challenges to the prevention and control of PRRS in pig farms in China.At the same time,PRRSV antibody detection mainly relies on the commercial indirect ELISA antibody detection kit for detecting the N protein of PRRSV,which cannot be used to measure the immune protection level of the infected pigs.In this study,the NL-PRRSV isolated strain FJ1402 was serially passaged on Marc-145 cells to 60 generations.And it was proved that its pathogenicity to piglets gradually decreased.Meanwhile,we also established an indirect ELISA to detect the antibody against GP5 of PRRSV.And it indicated that this method has high specificity and sensitivity,and could be used to detect to antibody to PRRSV in swine.The main research contents are as follows:1.The Stability of Virulence of PRRSV NADC30-like FJ1402 strain.In this study,NL-PRRSV FJ1402 isolated strain was continuously passaged to 60 generations on Marc-145.The virus titers of F5,F30 and F60 generations were 105.5,105.5 and 106.5 TCID50/mL.Twenty healthy piglets aged 30-35 days without PRRSV and PCV2 infection were selected and randomly divided into 4 groups.Groups 1-3 were inoculated with the same dose(106 TCID50/mL)of F5,F30,and F60 virus solutions.Group 4 was inoculated with PBS as negative control.They were raised and observed for 14 days,body temperatures and clinical symptoms were measured and recorded daily.And the blood were collected regularly.At 14 days after inoculation they were killed for the pathological examination.The viremia and lung viral load were detected by fluorescence quantitative PCR.The results showed that both F5 and F30 can cause high fever in piglets,which can cause high levels of blood and lung viruses,and obvious interstitial pneumonia can be seen in lung tissue.Compared with the lower generation virus strains,F60 significantly reduced the level of viremia,clinical symptoms and pathological changes.It proved that the virus titer of the NL-PRRSV FJ1402 strain gradually increased after passage,but the pathogenicity of piglets gradually decreased,which provided basic data for the study of PRRSV vaccine.2.Prokaryotic expression of GP5 protein of PRRSV and establishment of an indirect ELISA antibody detection methodIn this study,based on the characteristics of the GP5 gene of PRRSV FJ1402 strain,the GP5/4m gene fragments were designed by deletion of the third transmembrane zone and optimization of GPS as E.coli preference codons and obtained by the gene synthesis.It was cloned into pCold-TF E.coli expression vector and constructed a recombinant plasmid pCold-TF-GP5/4m.And the soluble GP5/4m protein was expressed highly and purified by nickel column supernatant purification method,which had GP5 antigen detected by Western blot.Then the purified GP5 protein was used as coating antigen to establish indirect ELISA method.The results showed that antigen coating concentration was 1.0?2.0?g/mL,serum dilution was 1:100,enzyme-labeled antibody dilution was 1:5000,and the substrate color development time was 10 min.The cut-off value of ELISA was as follows:S/P?0.28 was considered to be positive and S/P<0.21 was considered to be negative,and others were suspicious.Sensitivity experiments showed that the method could detect 160-fold diluted positive serum.It had no cross-correlation with the serum antibodies against PRV,FMDV,CSFV,SVA and PCV2.The intra-batch and inter-batch repeatability coefficient of variation was 1.17%?6.43%.This method was used to detect antibody against PRRSV in 306 clinical pig serum samples,together with the commercial IDEXX PRRSV ELISA detection kit.The results showed that the coincidence rate of these 2 methods was 88.89%.It indicated that this method has high specificity,sensitivity and repeatability,and provides a new detection method for anti-PRRSV antibody monitoring in swine.