Development And Application Of A Porcine Reproductive And Respiratory Syndrome Virus(PRRSV) Stably Expressing A Gaussia Luciferase And Genomic Analysis Of A Natural Recombinant PRRSV

Porcine reproductive and respiratory syndrome virus(PRRSV),an etiological agent of porcine reproductive and respiratory syndrome(PRRS),has caused huge economic losses to the global pork industry.Due to the lack of proofreading capacity of its RNA-dependent RNA polymerase,PRRSV is a fast-evolving RNA virus with the emergence of novel strains every few years.Since first reported in China,PRRSV has been widespread across the country and become one of the most important swine diseases.In recent years,the frequency of the recombination events between different PRRSV strains has been increased significantly,especially between vaccine strains and field strains,which leads to new challenges to the prevention and control of PRRSV.PRRSV has been explored as a viral vector to express foreign genes,including fluorescent protein genes,luciferase genes,and protective antigens from other pathogens.In this study,a reverse genetics platform for the highly pathogenic PRRSV TA-12 strain was established,and the recombinant TA-12M virus containing genetic marker was successfully rescued.To construct a PRRSV reporter virus stably expressing a secreted Gaussia luciferase(Gluc),we inserted the expression cassettes of Gluc into two sites in the TA-12M virus genome,nsp12/ORF2a and ORF7/3' UTR,respectively.Through cell transfection and virus infection experiments,the PRRSV reporter virus(rTA-Gluc2)containing Gluc insertion between ORF7 and 3'UTR was successfully rescued.The results of the virus growth curve and plaque assay showed that the growth characteristics of the reporter virus were similar to that of the parental virus,although its titers were slightly lower than those of the parental virus.By the luciferase assay,Gluc was detected in the cell culture supernatant after the reporter virus infection,and the dynamic of Gluc expression was consistent with the kinetics of the reporter virus titer,indicating that the Gluc in virus supernatant can be used as an indicator of viral replication.The results of luciferase assay and viral genome sequencing showed that the reporter virus could stably express Gluc after 10 consecutive passages on MARC-145.Based on the stability of the reporter virus and the high sensitivity of Gluc detection,we applied rTA-Gluc2 to develop the Gluc readout-based PRRSV-specific antiviral drug screening assay and PRRSV neutralizing antibody detection assay.In short,a reverse genetics of HP-PRRSV was established,which laid a foundation for in-depth analysis of its pathogenic mechanism;the PRRS V reporter virus stably expressing a secreted luciferase was successfully rescued,which provided a fast and efficient technology for PRRSV-specific antiviral drug screening and neutralizing antibody detection.Currently,the majority of PRRSV strains in China belong to lineages 1,3,5,and 8 of PRRSV-2,and NADC30-like strains in lineage 1 have gradually become the dominant strains.Many studies have reported the recombination between different PRRSV strains,especially between NADC30-like strains and other field strains,in which NADC30-like strains usually served as the major parent strain.In this study,we isolated a PRRSV strain from the lung tissue of a dead newborn piglet,and this virus was designated as JS2020.The virus only infected porcine alveolar macrophages but not MARC-145.The complete genome of JS2020 was determined to be 15016 bases in length.In comparison with the prototype strain VR-2332,JS2020 contains a deletion pattern of "111+1+19" amino acids in the nsp2 hypervariable region,which is consistent with the characteristics of the NADC30-like strains.NCBI BlastN sequence alignment revealed that 15HEN1 shares the highest genetic similarity with JS2020 which is only 91.53%.The results of recombination analysis suggested that JS2020 was created through the recombination between a NADC30-like strain and a NADC34-like strain.The ORF2a?ORF6 region(11751 nucleotide?14231 nucleotide)of the JS2020 genome is derived from a NADC34-like strain,while the rest genomic regions were derived from a NADC30-like strain.Taken together,a novel recombinant PRRSV virus was isolated,which provides a basis for the understanding of current PRRSV epidemic situation in China.