Pathogenicity and immunogenicity of recombinant rabies viruses expressing the Lagos bat virus matrix and glycoprotein genes

Abstract

Lagos bat virus (LBV) is a phylogroup II lyssavirus exclusively found in Africa. Previous studies have shown that this virus is lethal to mice after intracranial (i.c.) and intramuscular (i.m.) inoculation. Pathogenicity determinants of LBV are yet to be determined. The antigenic composition of LBV differs substantially from that of the rabies virus (RABV) and current rabies vaccines do not provide cross protection against LBV and other phylogroup II lyssaviruses. LBV is associated with Pteropodidae bat species and although no human infections have been reported to date, fatal spill-over into dogs, cats and a mongoose have been reported. To investigate the potential role of the LBV matrix (M) protein and glycoprotein (G) in the pathogenesis, reverse genetics technology was used to construct recombinant viruses. The genes encoding the G protein or the M and G protein of the attenuated RABV strain SPBN were replaced with those of LBV (LBVAFR1999) resulting in SPBN-LBVG and SPBN-LBVM-LBVG, respectively. In addition, to evaluate the immunogenicity of the LBV G, the recombinant RABV SPBNGAS-LBVG-GAS was constructed that contained the LBV G inserted between two mutated RABV G genes (termed GAS). Multi-step growth curves showed that SPBN, SPBNGAS-GAS-GAS (a recombinant RABV containing three G protein genes) had the highest growth rate followed by SPBN-LBVG and SPBNGAS-LBVG-GAS. While there was no statistically significant difference between the growth rate of these viruses (p>0.05), the growth rate of SPBN-LBVM-LBVG was lower than that of the other viruses, including LBVAFR1999. The single-step growth curves yielded similar results, with SPBNGAS-GAS-GAS, SPBNGAS-LBVG-GAS and SPBN producing the highest titres and SPBN-LBVM-LBVG and LBVAFR1999 again producing the lowest titres. The results from both growth curves indicated that both the M and G protein of LBV control the growth rate of the virus and thereby playing a role in pathogenicity. All the viruses − with a single exception, viz. SPBNGAS-GAS-GAS − were lethal to mice after i.c. inoculation, although the pathogenicity of SPBNGAS-LBVG-GAS was lower compared to the other recombinant viruses. Mice inoculated with LBV and SPBN-LBVM-LBVG had the highest percentage mortality (100%) and the shortest mean incubation period, while those inoculated with SPBNGAS-LBVG-GAS had the lowest percentage mortality (20%) and the highest incubation period. Following i.m. inoculation, only LBVAFR1999 and SPBN-LBVM-LBVG were lethal to mice, indicating that both the M and G protein of LBV play a role in the pathogenesis of LBV. Serum from mice inoculated with SPBNGAS-GAS-GAS and RABISIN (a commercial rabies vaccine used for dogs) cross-neutralised RABV and DUVV, while no detectable VNA were observed for LBV and MOKV. These findings emphasise the already known concept that vaccines derived from RABV cross-neutralise against DUVV, but not against LBV and MOKV. Most interestingly, serum collected from mice inoculated i.m. with SPBNGAS-LBVG-GAS cross-neutralised phylogroup I and II [RABV, LBV, Duvenhage virus (DUVV) and Mokola virus (MOKV)] lyssaviruses, indicating that this recombinant virus has a potential to be used for the development of a pan-lyssavirus vaccine.