| Rabies is a zoonotic disease caused by rabies virus (RV), and almost all warm-blooded animals are susceptible to RV. Due to its high neuroinvasiveness, the fatality rate of rabies ranks at the top of the infectious disease cases; once onset occurs, the death rate is almost 100%. Because of this, it is a serious threat to human and animal health; however, no one has yet developed effective methods of treatment to rabies. More than 55,000 people die from canine rabies every year, over 95% of which exist in the developing countries of Asia, Africa and Latin America regions, with most (84%) of these cases occurring in rural areas. Moreover, most of human rabies cases arise from animals. So control of rabies in animals (especially dogs) plays a key role in preventing rabies in humans. Currently inactivated rabies vaccines are mainly used for preventing animal rabies in mainland China, while glycoprotein (GP) determines the effectiveness of inactivated vaccine to a large extent. GP is the the main antigen to inducing virus neutralizing antibodies (VNA), and is related to the neurotropism of the virus. The main feature of RV is the neural invasive, that is, after infection, it will invade the central nervous system from peripheral sites in a unique way by reverse axonal transport. GP is also associated with the pathogenicity of RV. Recombinant rabies virus containing two, even three G genes in genome have been constructed by inserting G gene before N gene, between N, P genes, and between G, L genes respectively, and comparing it to parent viruses. These recombinant viruses performed as pathogenicity reduced, expression of G protein in infected cells improved, and immunogenicity enhanced. However, recombinant viruses produced by inserting an extra G gene between P, M genes has not yet reported. In this study, we constructed a recombinant rabies virus Flury LEP that has an additional G gene between P and M genes (we name it as rLEP-PGM) via negative-strand RNA virus reverse genetics technology. Then we studied the biological characteristics of the recombinant virus and its pathogenicity on mice compared to its parental strain LEP. The result showed that the in vitro growth characteristic of rLEP-PGM was similar to the LEP strain, the growth titer on BHK-21 cell of LEP and rLEP-PGM were 4 x 10~6 FFU/mL and 2.5 x 10~6 FFU/mL respectively, and on NA cells were 4 x 10~7 FFU/mL and 2.5 x 10~7 FFU/mL respectively. Western blot analysis of GP expression showed the GP expression level of rLEP-PGM to be 1.5 times higher than LEP. The LD50 of rLEP-PGM and LEP were 3 FFU and 1 FFU by intracerebral injection, which indicated no difference, but the LD50 of intramuscular injection were 4.6 Lg FFU and 5.5 Lg FFU respectively. Thus, the insertion of an additional G gene between P and M genes can significantly raise the expression level of GP and enhance its ability to invade the central nervous system from peripheral sites. |
