Alamdary Ashkan, Gholami Alireza, Azizi Mohammad, Noormohammadi Zahra
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Viral Vaccines Production Unit, Pasteur Institute of Iran Production and Research Complex, Karaj, Iran.
Adv Biomed Res. 2025 Jul 31;14:75. doi: 10.4103/abr.abr_480_24. eCollection 2025.
The most effective approach to control and prevent rabies is by proper pre- and post-exposure immunization. Glycoprotein (G) is a structural protein of the rabies virus that plays a crucial function in host protection against the virus. This study assessed the effects of integrating an extra copy of the glycoprotein gene into the rabies virus genome using reverse genetics on the immunogenicity and titer of the recombinant virus.
The expression of the recombinant viral glycoprotein was compared to the PV strain using qRT-PCR and western blot techniques. The virus titers were studied using a fluorescent antibody test (FAT). The inactivated recombinant virus was administered to BALB/c mice as a vaccine, and the immunogenicity was assessed using the rapid fluorescent focus inhibition test (RFFIT).
Glycoprotein overexpression was observed using qRT-PCR (2.47-fold increase) and confirmed by western blot analysis (3.4-fold increase). The double G virus showed significantly higher virus titers than the PV strain. The immunogenicity of the double G virus was also considerably increased. The VNA titers induced by the double G virus were approximately two and three times higher at 14 and 21 days post-inoculation, respectively.
Based on our findings, the PV strain's G gene duplication showed greater viral titers, better VNA induction, and higher G expression levels. Both the development of rabies vaccines and the investigation of various cellular processes involved in the virus's life cycle may be advanced by characterizing the recombinant double G strain.
控制和预防狂犬病最有效的方法是进行适当的暴露前和暴露后免疫接种。糖蛋白（G）是狂犬病病毒的一种结构蛋白，在宿主抵御该病毒的过程中发挥着关键作用。本研究评估了利用反向遗传学将糖蛋白基因的额外拷贝整合到狂犬病病毒基因组中对重组病毒免疫原性和滴度的影响。
使用qRT-PCR和蛋白质免疫印迹技术将重组病毒糖蛋白的表达与PV株进行比较。使用荧光抗体试验（FAT）研究病毒滴度。将灭活的重组病毒作为疫苗接种给BALB/c小鼠，并使用快速荧光灶抑制试验（RFFIT）评估免疫原性。
使用qRT-PCR观察到糖蛋白过表达（增加2.47倍），并通过蛋白质免疫印迹分析得到证实（增加3.4倍）。双G病毒的病毒滴度显著高于PV株。双G病毒的免疫原性也显著增强。双G病毒诱导的病毒中和抗体（VNA）滴度在接种后14天和21天分别高出约两倍和三倍。
根据我们的研究结果，PV株的G基因复制显示出更高的病毒滴度、更好的VNA诱导以及更高的G表达水平。对重组双G株进行特性分析可能会推动狂犬病疫苗的研发以及对病毒生命周期中各种细胞过程的研究。
