Optimization Of Fluorescent Antibody Virus Neutralization Test For Rabies Virus

Rabies is a zoonosis caused by rabies virus(RABV)infection with an extremely high mortality.Current,there is no effective treatment for rabies,and vaccination is the most effective way to prevent and control rabies by inducing the virus neutralizing antibody(VNA).Fluorescent Antibody Virus Neutralization(FAVN)test is a widely accepted method and recommended by OIE to determine the VNA level for RABV.Currently,two imported reagents,standard serum and FITC-labeled anti-RABV-N fluorescent antibody(FITC-RABV-N for short),are required during the detection process of FAVN.However,these two reagents are expensive and often have the problem of being unable to be carried out due to the unstable supply.Therefore,to solve the shortage of these reagents,and further optimize the detection method and reduce the detection cost as well,a monoclonal antibody(MAb)with neutralizing activity against RABV G protein was screened to replace the standard serum.Moreover,a recombinant RABV expressing e GFP was constructed to omit the step of detecting unneutralized RABV by FITC-RABV-N.These strategies not only save the detection cost but also shorten the detection time,which would optimize the FAVN method.The results are as follows:1.Screening of monoclonal antibodies against RABV with neutralization activity to replace the standard serum.Glycoprotein(G)is the only exposed protein on the surface of RABV virions,which is also the main antigen for VNA production.A recombinant Canine distemper virus(CDV)expressing RABV G protein constructed previously in our lab The purified RABV virons were used as immunogen to screen MAb against RABV G protein.The preparation and monoclonal of hybridoma cells were then carried out,and the supernatant of monoclonal hybridoma cells was then screened to find out those could recognize the CDV-RABV-G infected cells by indirect immunofluorescence assay(IFA).A total of 9MAbs against RABV-G were screened out and named as 1E11,2B10,5E9,6G10,7A3,8A5,9A7,9F7 and 9F10,respectively.It was found that the RABV neutralization activity of MAb 7A3 was the highest among all the screened Mabs.Furthermore,7A3 was double diluted,and the VNA titers of 7A3 at different dilutions were determined by FAVN test.The results showed that the VNA titer of 7A3 would be 0.5 IU/m Lwhen its concentration was 6.5 ug/m L,which is the VNA titer of standard serum used in FAVN test,suggesting that 7A3 at concentration of 6.5 ug/m L could replace the standard serum as the reference for FAVN test.2.Construction of recombinant RABV expressing e GFP to replace the FITC labeled anti-RABV-N Mab.Conventionally,the CVS-11 strain was used to incubate with serum samples,and the unneutralized CVS-11 virus was then detected by FITC labeled anti-RABV-N antibody.The VNA titers were calculated by comparing the numbers of RABV positive fluorescent foci between testing samples and standard sample.To solve the reagent shortage of FITC-RABV-N and simplify the FAVN test as well,we plan to construct a recombinant RABV with autofluorescence to omit the use of FITC-RABV-N and simplify the FAVN test.Therefore,a recombinant RABV expressing e GFP was constructed in this study based on CVS-B2 c strain,whose genome sequence similarity of which is higher than99.9% with CVS-11,by inserting e GFP gene between G and L genes of RABV genome which was,therefore,named as r B2c-G/e GFP/L.To further enhance the expression level of e GFP and its fluorescence intensity,the e GFP gene with nuclear localization sequence(NLS)was inserted between N and P genes of CVS-B2 c genome,and the recombinant RABV expressing NLS-e GFP was successfully rescued and named as r B2c-N/NSL-e GFP/P.Comparing the two recombinant r RABVs,we found that the m RNA level of e GFP was higher in r B2c-N/NSL-e GFP/P infected cells than that in r B2c-G/e GFP/L infected cells,which indicated that expression of e GFP could be improved by advancing the insertion site in RABV genome.In addition,the e GFP was partially expressed in the nucleus of r B2c-N/NSL-e GFP/P infected cells through NLS,which would make the fluorescence more concentrated than that of r B2c-G/e GFP/L.All these improvements will make it easier to observe the fluorescence and improve the accuracy of FAVN.Together,FAVN method was optimized by replacing the standard serum with 7A3 and introducing the recombinant RABV r B2c-N/NSL-e GFP/P into FAVN test,which would shorten the detection time and save the detection cost as well.