Establishment Of Rabies Virus FVAN Method And Its Primary Application In Detection Of The Efficacy Of Veterinary Inactivated Rabies Vaccine

Rabies is a lethal zoonotic disease caused by rabies virus,which mainly affects the central nervous system and has a high mortality rate.There is no effective trea tment after clinical symptoms.Vaccine immunization is the most effective means of prevention and treatment of the disease,and the monit oring of serum antibody levels is the most direct and effective evaluation method for the effect of vaccine immunization.As a cell-level virus neutralization test,fluorescent antibody neutralization test（Fluorescent Antibody Virus Neutralization,FAVN）is a standard test method recommended by OIE to quantify neutralizing antibodies to rabies virus in serum.Not only a stable fluorescein-labeled specific m Ab is required but also reproducible observations are not possible in the currently used FAVN method.This experiment will establish a new type of fluorescent antibody neutralization test method,based on recombinant rabies virus which can express green fluorescent protein.Then the veterinary rabies vaccine immunization efficacy was evaluated using the established FVAN method after comparative analysis with the FAVN method recommended by OIE.The specific results are as follows.Firstly,the rabies virus CVS-11 strain and the recombinant rabies virus labeled with green fluorescent protein（r Flury-LEP-GFP）were tested for virulence.The results showed that the poison value of CVS-11 was 10^4.5 TCID₅₀/0.1m L,and the titer of r Flury-LEP-GFP was 10^5.5 TCID₅₀/0.1m L.Neutralization experiments were conducted with the above two viruses respectively,it is found that the FVAN method can see clear cell morphology under the fluorescence microscope,while the FAVN method is dominated by point-like fluorescence.Also,in the actual application process,it can be found that the flu orescence decay rate of the FVAN method is very slow,so repeated observations can be made.Differently,the fluorescence of the FAVN method decays rapidly,so the results must be observed and recorded quickly.Secondly,the titers of 15 positive sera were detected by the new FVAN method,and the correlation was compared with the conventional FAVN method.The experimental data show that the results of the FVAN and FAVN are significantly related.Meanwhile,the new FVAN show better specificity,and its specificity and reproducibility are good in subsequent experiments.Therefore,the new FVAN method constructed in this experiment can effectively replace the traditional FAVN method,and has broad application prospects in the monitoring,diagnosis,treatment effect identification and vaccine evaluation of human and animals.Thirdly,in order to evaluate the application effect of the newly established FVAN method in practice,it was applied in the efficacy evaluation of veterinary inactivated rabies vaccine in this experiment.Using 8 Inactivated rabies vaccine for veterinary use,including the reference vaccine,diluted 5 times,25 times,125 times and 625 times respectively.32groups of 160 mice were immunized,5 mice in each group.The blood of the mice was collected 14 days and 28 days after the first immunization,and the serum neutralizing antibody titer was detected by the FVAN method.The results show that serum titer at day 28were overall higher than at day 14.At the same time,we tested the efficacy of these eight batches of vaccines using the mouse efficacy test method（NIH method）for inactivated rabies vaccine specified in the"Veterinary Pharmacopoeia of the People’s Republic of China".Using statistical methods to analyze the results of the two,we found that the serum titer of the 25-fold dilution group detected by the FVAN method on the 28th day of the first vaccination was significantly correlated with the vaccine titer ob tained by the NIH method.The R² value of the slope/difference reached 0.8,showing the same trend for both.It shows that this method can be used in the evaluation of the efficacy of inactivated rabies vaccine.In summary,this experiment established the FVAN method for the detection of rabies virus neutralizing antibodies,optimized the standard detection method of rabies virus neutralizing antibodies in animal serum recommended by OIE,and evaluated the immune efficacy of veterinary rabies vaccines.As a new method,it lays the foundation for the application in the efficacy evaluation and detection of veterin ary inactivated rabies vaccines.At the same time,because this method can not only effectively reduce the amount of experimental animals,but also reduce the time required for detection due to its non-challenging characteristics.,which is more in line with the principle of animal welfare,and is the development direction of the efficacy testing of rabies inactivated vaccines.