Abstract
Purpose: Pakistan is one of the few countries where Rabies is endemic and a great threat to humans as well as live stocks. A large number of individuals died of rabies exposure due to either the limited access to rabies vaccines or the side-effects of sheep brain -originated Sample vaccine. Cell culture-derived rabies vaccines are mostly unaffordable to the needed population under rabies threat. Development of a safer and more affordable vaccine is necessary in Pakistan. Here, we developed two novel recombinant rabies virus vaccines using a local Pakistan rabies virus glycoprotein gene, and tested the efficacy of vaccines in mice. Methods: The glycoprotein gene (RVG) of vector ERAg3p and ERAg3m was substituted, respectively, with a modified Pakistani RVG. The resulting recombinant vectors were applied for reverse genetics to recover two vaccine viruses, PK-SG and PK-DG. The efficacy of PK-SG and PK-DG were tested in mice by intramuscular injection and oral delivery. Results: All mice survived the challenge after intramuscular vaccination using PK-SG, or PK-DG. In the oral vaccination groups, 80% mice with PK-SG and 90% mice with PK-DG survived the challenge. Meanwhile, 80% of the unvaccinated control mice succumbed after challenge. The mean rabies virus neutralizing antibody titers was ≥0.5 IU/ml in all vaccinated groups. Conclusion: Our results demonstrated the efficacy of PK-SG and PK-DG in rabies vaccination in mice. The two recombinant virus strains may be good vaccine candidates for the target animals and humans in Pakistan. Detailed investigations are necessary in the future.
Highlights
Rabies is an important international zoonotic disease and is the eleventh biggest cause of human death in the reportable infectious diseases [1,2,3]
Rabid dog bites attribute to about 99% of human rabies death toll, and more than 3.3 billion people are living under the threat of a potential exposure to the deadly rabies virus in the endemic regions [4]
The Semple vaccine is not always available in Pakistan, and cell culture-derived rabies vaccines are mostly unaffordable to the needed population under rabies threat
Summary
RNA extraction and amplification of a Pakistan rabies virus glycoprotein gene Total RNA was extracted from the cow brain tissue using Trizol Reagent according to the manufacturer’s instruction (Invitrogen, CA, USA). The Pakistan rabies virus glycoprotein (PKG) cDNA was transcribed by using RAVGF primer and Transcriptor Reverse Transcriptase (Roche, Germany) at 55°C for 30 minutes. The primer set RAVGF and RAVGR was employed to amplify the entire glycoprotein gene (RVG) under the PCR conditions: 94°C for 4 minutes, followed by 30 cycles of 94°C for 50 seconds, 53°C for 90 seconds, 72°C for 120 seconds, and the final extension at 72°C for 15 minutes. The amplified RVG was analyzed by electrophoresis on 1% gel, cloned into pCR®-XL-TOPO® vector (Invitrogen, CA, USA), and sequenced as described previously [22,26]. The recombinant vector PK-SGV and PK-DGV, along with other T7 helper plasmids, were co-transfected to the BSR cell lines (a clone of BHK cells) to recover rabies virus as described [28]. The antibody titers between the vaccinated and control groups were analyzed statistically by performing ANOVA and DNMRT with the help of computer-aided program DASTAT® (UPB, Perugia, Italy)
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