Real time PCR is the modern molecular techniques used for detection and quantification of viral particle in the specimens. In this study Real Time PCR (RT-PCR) based method was developed and used to quantify the rabies virus PV-11 strains in the different viral harvests of tissue culture fluid in comparison with the Mouse Inoculation Test (MIT) and Fluorescent Antibody Test (FAT). The Eagle's MEM tissue culture medium with 5% fetal calf serum, 1% neomycin and 0.5% Amphotericin-B were used for the propagation of Vero cells in 150 cm 2 tissue culture flasks. The confluent monolayers of the Vero cells (90%) were infected with 0.2 MOI of PV-11 strain of rabies virus. Five individual viral harvests were collected from the infected vero cells at every 3 days interval and subjected for MIT, FAT to quantify the viral titre. The MIT titrated sample was used for the preparation of five point internal reference standard for RT-PCR technique to quantify the number of viral particles in the harvested fluid. The primers targeting 98 base pairs from 446 to 543 in the cDNA of the rabies virus nucleoprotein gene (N gene) were designed and synthesized for Real time PCR assay. The in-house RT-PCR assay was developed with SYBR green master mix. Viral particle in each harvests were quantified in comparison with internal reference standard included in the assay. The results of MIT, FAT and Real time PCR methods were compared with each other. The standardized Real time PCR method was applied for the quantification of viral titre in the different viral harvests cultured using Roller bottle and Bioreactor culturing methods using two different medium. Maximum log of 7.216 viruses per 1 ml of bioreactor viral harvest was quantified by using the real time PCR method. Dilution of the samples are required if the sample having higher viral titre which is not covered within the linear standard range of log 4.50 to log 6.50. This real time PCR based quantification of rabies virus has the advantage of greater accuracy, rapid, economical when compared to the other tests and no animal models required.

Vaccine against rabies is prepared by cell culture technology and these vaccines are free from many side effects when compared to nerve tissue vaccines. The vaccine production is a continuous process involving propagation of virus, harvesting, concentration, inactivation, purification and formulation with preservatives. The quantification of viral protein in the intermediate biological product is an in-process quality control test to reduce the product loss during various process of vaccine manufacturing. The conventional in-vivo & in-vitro tests employed for the quantification of rabies viral protein are time consuming, laborious and requires laboratory animals. In this study, we attempted to develop in-house serological methods such as sandwich ELISA, Dot Blot for the detection and quantification of rabies antigen in the intermediate biological material during vaccine preparation. The hyper immune sera was prepared by immunizing two animal models i.e. Guinea Pigs and rabbits with standard Rabies antigen. The sera samples were purified by saturated ammonium sulphate precipitation and further by G50 gel column. The antirabies antibody titre in the purified preparation was estimated using Rapid Fluorescent Focus Inhibition Test (RFFIT). National Reference Rabies Vaccine received from Central Drug Laboratory, Kasauli was used to prepare the local reference standard and it was included in the in-house serological methods to validate the assay. Our in-house tests are found to be simple, rapid and cost effective and require less time when compared to in-vivo animal challenge and cell culture based in-vivo tests.

The Vero cell is the continuous cell line used as a cell substrate for many viral vaccines manufacturing including Rabies vaccine. The Pitman and PV-11 strains of rabies virus are commonly used for production of rabies vaccines. In our study the PV - 11 strain is used as seed virus for the production of Vero cell derived inactivated Anti Rabies Vaccines (Lyophilized). The working cell bank and working virus seed lot were prepared and tested for its sterility and mycoplasma contamination. In this study the roller bottle method and micro carrier (cytodex-1) based bioreactor culturing method were standardized with MEM Eagles and RPMI 1640 medium. The outcome of the two methods were compared ,to find out the medium and culturing method which can give higher concentration of Vero cells to get higher viral titre in mass production. The quantities of Vero cells obtained from passage 144 to 148 were assessed using a haemocytometer cell count procedure. The quantity of the Vero cell obtained per roller bottle ranged from 222.82x106 cells to 236.67x106 cells in MEM Eagles medium and 227.80x106 cells to 230.66x106 cells in RPMI 1640 medium. The average quantity of Vero cells in roller bottles were 1.12 x 106 cells per ml of culture. In the bioreactor culturing method the Vero cells obtained in the concentration of 1.69 to 1.75 million cells / ml of culture by MEM Eagles medium and 1.67 to 1.70 million cells / ml by RPMI 1640 medium. In the bioreactor the average yield of vero cells ranged 1.70 x106 cells per ml of culture. It is estimated that about 35% increase in Vero cell quantity in bioreactor system than the roller bottle culturing method. 0.3 MOI (Multiplicity of Infection) of rabies virus was kept as constant for infection of confluent monolayered Vero cells in both roller bottle and bioreactor culture. Four viral harvests were collected at three day intervals and replenished with fresh culturing medium each time. The viral titres in each viral harvest were quantified using in-house standardized RT-PCR method; the roller bottle method yielded viral titre log 6.044 to 7.106 and the bioreactor culture yielded log 6.559 to 7.216 rabies viral particles. It is observed that the bioreactor culture yielded more viral titre than the roller bottle culture using both the medium.

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