Establishment Of Indirect ELISA Method And Development Of Inactivated Vaccine For Tilapia Lake Virus

Tilapia Lake Virus Disease(Ti LVD)is caused by a novel orthomyxo-like RNA virus:Tilapia Lake virus(Ti LV).Since 2009,Ti LV has caused severe economic losses and posed severe challenges to the global Tilapia-farming industry.At present,there is no effective control method for Ti LVD outbreaks.and early diagnosis and vaccine immunization are the most effective measures for the prevention and control of viral diseases.Therefore,the content of this research mainly includes two aspects.One is the establishment of an indirect ELISA method for the detection of Ti LV specific antibody by using the protein encoded in segment 8 of the Ti LV gene(VP20)as the envelope antigen,which provides a new serological diagnostic method for the detection of Ti LV,as well as the effect evaluation of the prepared Ti LVD vaccine.The other is the development of an inactivated Ti LV vaccine that can provide effective protection to tilapia,providing a new option and product stock for the prevention and control of tilapia lake virus disease.The research contents and results are as follows:1.Establishment of Ti LV indirect ELISAPrevious studies have shown that the encoding protein of Ti LV gene segment 8(S8)is a structural protein with high immunogenicity,with a size of about 20 k D,tentatively named VP20.Currently,there is no serological diagnostic method for Ti LV due to the lack of Ti LV immunogenic antigen and readily available monoclonal antibody(m Ab)against tilapia Ig M.In this study,the recombinant protein VP20 expressed in the E.coli expression system was used as the enveloped-antigen,and the newly developed tilapia Ig M monoclonal antibody(m Abs)was used as the secondary antibody.The concentration of envelope-antigen,serum and secondary antibody dilution ratio were optimized by chessboard titration,and an indirect enzyme-linked immune-sorbent assay(i ELISA)method was established for the detection of Ti LV-specific Ig M antibody levels in tilapia serum.By using the established i ELISA method,50 Ti LV negative samples of Ti LV-negative serum detected by IFA were tested,and the cut-off value was defined to be 0.258 OD units.The i ELISA also showed high analytic specificity and was able to distinguish between antibodies against Ti LV and other pathogens.It was more sensitive than the current IFA technique for detecting Ti LV.In addition,for detection of artificial infection samples,the results showed that the diagnostic sensitivity andspecificity of the i ELISA compared with IFA was 100% and 92.6%,respectively;while the diagnostic sensitivity and specificity of the i ELISA versus semi-nested PCR was 80.8% and95.6%,respectively.A serological survey was performed using the i ELISA with tilapia sera samples from the field compared with an unknown status regarding Ti LV,13/73 serum samples tested positive by i ELISA,indicating a 17.8% Ti LV antibody positives.In conclusion,our newly developed i ELISA was specific and sensitive,and it may be useful for large-scale investigations of Ti LV infections and monitoring trials of future vaccination protocols.2.Development of Ti LV inactivated vaccineIn this study,the newly isolated strong Ti LV-GD1710 strain was taken as the research object.Firstly,Ti B cells were used to amplify the virus,Then,the Ti LV was inactivated with formaldehyde and propanolactone,respectively,and the inactivation time,temperature and inactivation agent concentration were optimized The inactivated Ti LV was respectively combined with Montanide IMS 1312 VG and Freemix to prepare the inactivated Ti LV vaccine and inoculated tilapia with intramuscular injection.After immunization,the serum specific antibody and neutralizing antibody levels of tilapia were detected,and the relative expression levels of 7 immune-related genes,including IFN-γ,TNFα,MHC-Iα,MHC-IIβ,Ig M,IL-1βand My D88,were measured in the spleen and kidney,then the viral load of the fish in the spleen and kidney and the relative percentage survival were measured,after challenged with thevirulent strain Ti LV-GD1710,to evaluate the immune effect of the inactivated vaccine.The results showed that the inactivation of the virus with the final concentration of 0.2%formaldehyde at 37℃ for 48 h and the final concentration of 0.1% propiolactone at 4℃ for72 h could achieve a relatively ideal inactivation effect.After immunization,the serum specific antibody level and neutralizing antibody titer of each group were significantly increased compared with the control group.The serum specific Ig M antibody level of OD450 nm was between 0.42~1.04,and the serum neutralizing antibody titer was between 46~253,among which the highest were the serums of the BPL +Montanide IMS 1312 VG group.The relative expression levels of 7 immune-related genes in the spleen and kidney of tilapia were all up-regulated,among which MHC-Iα was the most significantly up-regulated one in both spleen and kidney,reaching a maximum of 61.6 in the spleen and 56.8 in the kidney.In addition,the viral load of the spleen and kidney tissues of tilapia in the immune group afterthe challenge was significantly lower than that of the non-immune control group,and the lowest group was BPL +Montanide IMS 1312 VG.Challenge infection test showed that all the vaccine immunization groups had certain immune protection effect,among wihch the BPL+Montanide IMS 1312 VG inactivated vaccine group had the best immunization effect and could provide 82.1% relative protection rate for tilapia against TiLV.