Development Of RT-LAMP Methods For Different Sub-Genotypes Of PRRSV And Pathogensis And Immunology Of Chimeric Virus Strans

Porcine reproductive and respiratory syndrome virus(PRRSV)has been recognized as one of the most important swine pathogen that causes tremendous economic losses in the world.The causative agent,characterized by reproductive failure and respiratory disorders in pigs,with higher genetic mutation and recombination characteristics which lead to the emergence of genetically and antigenically heterogeneous population.Since the first reported in china,PRRS gradually spread throughout the country,such as the highly pathogenic PRRSV that broke out in 2006,and the NADC30-like PRRSV strains and QYYZ strains that have been popular in recent years.At present,there are few research for identifying different subtypes on PRRSV.Based on the above background,this research studied the following aspects:1.Establishment and Application of RT-LAMP detection methods for different genotypes and subtypes of PRRSVDNAstar software was used to compare the two genotypes PRRSV strains,and specific primers were designed for the conserved regions on ORF7,then the method for identification of European and American-type PRRS V were successfully established.Each set of primers contains the outer primer F3/B3,the inner primer FIP/BIP and the loop primer Loop F/Loop B.According to the biological characteristics of different strains which prevalent in china,it can be roughly divided into three subtypes:classical strain,HP-PRRSV and NADC30-like strain.Based on the background,three reaction tubes according the deletion characteristics of Nsp2 gene was designed for detection and differentiation the three subtypes PRRSV strains.The specificity and sensitivity of the above methods were evaluated.The results showed that the above methods had no cross-reactivity with CSFV,PCV2,PRV and other pathogens,and was consistent with the sensitivity of Real-Time PCR.Finally,the assay is proved to be simple,sensitive and can be used for rapid detection of PRRSV.2.Development of the American Type PRRSV Freeze-Dry Diagnostic KitThe LAMP method does not require special equipment.The results can be directly judged by the naked eye,and is particularly suitable for the early diagnosis at the grassroots.Based on the PRRSV RT-LAMP method,the loop-mediated isothermal amplification reagent was freeze-dried,and the three freezes without any effect on the reaction were obtained.Finally,the optimized lyoprotectant formulation was 5%trehalose+1.25%mannitol+1.25%bovine serum albumin.The components of the PRRSV freeze-dried diagnostic kit are:isothermal amplification reagent lyophilized powder,betaine solution,chromogenic solution,positive control solution and negative control solution.The detection limit of the kit was 102 copies/μL.In addition,the kit is valid for 12 months at 4℃.The PRRSV isothermal amplification lyophilization kit established in the assay provided technical support for diagnosis and monitoring of epidemic dynamics with the advantages of high sensitivity,rapid and practicality.3.Pathogenicity and immune efficacy of the chimeric strain of Porcine Reproductive and Respiratory Syndrome virusIn this assay,three chimeric strains rTd111,rTd111/F2-7,rTd111/F5-6 constructed in the laboratory were used for the in-vitro culture experiments of porcine alveolar macrophages(PAMs).The results showed that the virus titer of the three chimeric strains were similar to those of the female TJM-F92.The levels of inflammatory cytokines induced by rTd111/F5-6 were similar to those of rTd111,slightly higher than TJM-F92,but the levels of rTd111/F2-7 were significantly higher than those of rTd111,rTd111/F5-6 and TJM-F92.When inoculated by the three chimeric strains,piglets can produce antibody against PRRSV without obvious abnormal clinical symptoms,the virulence of rTd111/F5-6 was similar to that of TJM-F92,which was significantly lower than that of FJ1402,and the virulence of rTd111/F2-7 was higher than that of rTd111/F5-6,indicating that rTd111/F5-6 has potential as a candidate for vaccine.To further validate the immunoprotective efficacy of the chimeric vaccine,piglets were attacked by HP-PRRSV BB0907 and NADC30-like strain FJ1402 respectively.It was found that rTd111/F5-6 could provide effective protection against FJ1402 and BB0907,which lays an important foundation for the development of new vaccines.