The Rapid Detection Method For Prrsv And Construction Of GP4 N-glycosylation Mutants

Porcine reproductive and respiratory syndrome（PRRS）is still one of the diseases affecting the healthy development of the pig industry.As a highly epidemic virus,the outbreak of RRSV has brought serious economic losses.After PRRSV was introduced into our country,the high heritability and recombination rate led to the continuous change of the circulating strains in our country,causing more difficulties in PRRSV prevention and control,and producing more obstacles to the development of vaccines.At present,there are no effective vaccines on the market to control PRRSV invasion and infection.In this study,two methods for rapid detection of PRRSV were established based on recombinase aided amplification（RAA）combined with fluorescence quantification and immunolayer test strips.Specific primers with probes were designed in the PRRSV ORF7 region.The pathogen can be detected by a constant temperature reaction in a short time.Three different lineages of PRRSV p MD18T-ORF7 eukaryotic expression vectors were successfully constructed.The sensitivity of the PRRSV RAA method was verified by 10-fold serial dilution of the copy number of the PRRSV plasmid.The results showed that the PRRSV RAA test paper method could be detected at9.9×10¹⁰-10¹copies/μl;the PRRSV RAA fluorescence method could be detected at 9.9×10⁹-10⁶copies/μl.The specificity of the PRRSV RAA method is proved by comparison with porcine related pathogens,and PRRSV strains of different lineages can be detected.157 clinical samples were collected from different pig farms in Hainan Province,and the PCR method was used to compare and verify that the sensitivity and specificity of the RAA fluorescence method were 94.0%and100.0%.In addition,the sensitivity and specificity of the RAA test paper method were 100%and97.8%.Several strains of PRRSV was isolated from the positive samples detected by the RAA method,and selected two typical strains for sequencing and named them as HNHK1-2021 and HNLG1-2021in our laboratory.Through genome analysis of PRRSV ORF5 and construction of phylogenetic tree,it was proved that both PRRSV HNHK1-2021 and HNLG1-2021 belong to North American strains,PRRSV HNHK1-2021 belongs to lineage 3（QYYZ-like PRRSV）,and PRRSV HNLG1-2021 belongs to lineage 5（VR-2332-like PRRSV）.In order to further study the mechanism of PRRSV function,the N-glycosylation mutant of PRRSV ORF4 was constructed to study whether the glycosylation mutation affects the effect of PRRSV on the CD163 receptor.The eukaryotic expression vectors of p EGFP-ORF4 and p RFP-CD163 were successfully constructed.Confocal microscopy successfully demonstrated that all PRRSV ORF4 N-glycosylation mutants could spatially colocalize with the CD163 receptor,and both of them were expressed in the cytoplasm.Therefore,our study provide a theoretical foundation for studying the invasion mechanism of PRRSV.