| Rabies virus (RABV) is a strictly neurotropic virus that can result in neurodegeneration in central nervous system (CNS). The mortality of rabies stays at a high level, there is an estimated 70,000 human annually dying from rabies worldwide, which is a severe public healthy problem. Only pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) can efficiently prevent the transmission of rabies virus at present, besides strengthen the immunization of mankind, we must also enhance the administration of domestic and widelife animals simultaneously.Rabies virus genome composes of five open reading frames (ORF), encoding N, P, M, G and L protein, respectively. M protein is correlated with budding and release of virus particles, and plays a role in balance of virus transcription and replication. Based on the previous studies, in this study, selected the street rabies virus GX01 isolated from Guangxi Province was selected as the research object, for further exploring the effect of rabies virus M protein on its biological characteristics. GX01 isolate is high pathogenic rabies virus that 100% lead adult mice to death after intracerebral inoculation (i.c). The full-length genome infectious cDNA clone of RC-HL strain used for production of animal rabies vaccine in Japan was selected as the background to generate rabies virus mutants. RC-HL strain is a laboratory-fixed attenuated virus that causes nonlethal infection in adult mice after i.c. We replaced the 44 and/or 46 residues of rRC-HL M gene with the corresponding residues of GX01 M gene, utilized reverse genetic technique to rescue 3 mutant viruses named rRC-HL (M44), rRC-HL (M46) and rRC-HL (M44/46) strain that contains F44L, S46G and F44L/S46G mutations of M protein, respectively.The parent virus rRC-HL strain, recombinant rabies vims mutants rRC-HL (GX01M), rRC-HL (M44), rRC-HL (M46) and rRC-HL (M44/46) strain, and pathogenic GX01 were further used to explore the different biological characteristics. The results showed that the virus titers of rRC-HL (GX01M) and rRC-HL (M44/46) are both significantly lower than that of rRC-HL in BSR/T7-9 cells, while higher than GX01. The plaque assay showed that the cell-to-cell spread capacity of rRC-HL strain in BSR/T7-9 cells is higher than the other mutants, rRC-HL (GX01M), rRC-HL (M44), rRC-HL (M46) and rRC-HL (M44/46),1.8,1.5,1.3 and 2.6 folds lower than that of parent rRC-HL strain, respectively. The transmission capacity of street strain GX01 is the lowest, 15.6 folds lower than rRC-HL strain, indicated that F44L/S46G substitutions of M protein could significantly decrease the cell-to-cell spread capacity of rabies virus. Both of rRC-HL strain and rRC-HL (M46) strain induce severe cytopathic effect (CPE) in BSR7T7-9 cells at 48 hpi, the CPE induced by rRC-HL (M44) are less than rRC-HL strain, rRC-HL (GX01M), rRC-HL (M44/46) and GX01 induce no CPE as well as the control. These data suggest that F44L/S46G substitutions of M protein dramatically decrease CPE induced by rabies virus.Parental rRC-HL strain and recombinant rRC-HL (GX01M) strain don’t kill adult mice, consistence with the results of previous study. Mutants rRC-HL (M44), rRC-HL (M46) and rRC-HL (M44/46) only cause mice nonlethal infection that just lose weight between 4-8 days, then recover to normal level. However, the GXO 1 leads all mice to death in 7 day. The result indicated that M protein of GXO 1 strain is not the main virulent factor, F44L/S46G substitutions of M protein also do not increase pathogenecity. The expression of N and M gene mRNA in BSR/T7-9 cells infected with rRC-HL (M46) strain at an MOI of 0.1 are similar to rRC-HL strain, which are higher than the other mutant viruses. The mRNA levels of N and M gene of rRC-HL (M44/46) decrease obviously, only followed by street virus GX01. The expression of N and M protein are corresponding with the mRNA level, the expression of N and M protein of rRC-HL strain and rRC-HL (M46) are both high, followed by rRC-HL (GX01M) and rRC-HL (M44), the expression of N and M protein of rRC-HL (M44/46) are both significantly lower than rRC-HL strain, similar to the previous result of rRC-HL (GX01M2) strain. The expression of N and M protein of the GX01 are the lowest among all of the rabies viruses.All of the atteunated rabies viruses rRC-HL, rRC-HL (GX01M), rRC-HL (M44), rRC-HL (M46) and rRC-HL (M44/46) could propagate in RAW264.7 cells and TLR3 deleted RAW264.7 cells (RAW264.7 TLR3-/- cells), while GX01 strain are highly limitted in RAW264.7 cells. Deleting TLR3 gene results in decreasing virus titer of all rabies viruses, however, virus titer in macrophages is significantly lower than that in BSR/T7-9 cells. Both of mRNA and protein expressed in RAW264.7 cells and RAW264.7 TLR3-/- cells infected with rabies virus are corresponding with those expressed in BSR/T7-9 cells. We determined the concentration of type I IFN induced by each rabies virus through ELISA, and found that just rRC-HL (M44/46) could induce high level of IFN-α/β in RAW264.7 cells at 12 hpi, all of rabies viruses could induce high level of IFN-α/β in RAW264.7 cells at 24 hpi except the GX01. However, the level of IFN-α/β induced by rRC-HL (M44/46) is the highest among all of rabies viruses, the concentration of IFN-a and IFN-β is 2212.17 and 2036.66 pg/mL respectively. In RAW264.7 TLR3-/- cells, rRC-HL (GX01M), rRC-HL (M44) and rRC-HL (M44/46) could induce high level of IFN-a/p at 12 hpi, the concentration of IFN-α reach 532.5,2070.5 and 2168.6 pg/mL, respectively. The expression level of IFN-α/β induced by all of rabies viruses except the GX01 is nearly the same. These data demonstrate that the GX01 M gene can induce high level of IFN-α/β, morever, F44L/S46G mutations of the M protein plays key role in the induction of IFN-α/β. Both of the normal RAW264.7 cells and RAW264.7 TLR3-/- cells express IFN-α/β induced by rabies virus, which indicate that TLR3 is not essential for inducing IFN-α/β by rabies virus.The production of antiviral protein Viperin in RAW264.7 cells and RAW264.7 TLR3-/- cells infected with rabies virus is positive correlated with the expression level of IFN-α/β, this showed that Viperin that render the cell enter into antiviral status could be induced by IFN-α/β. In addition, the protein level of IRF3 after infection with rabies virus is consistent with IFN-α/β and Viperin, indicated that IRF3 modulate the induction of IFN-α/β. NF-kB is up-regulated in RAW264.7 cells after infection with rabies virus, whereas, it is irregular in RAW264.7 TLR3-/- cells, showed that NF-kB is dispensable in the induction of IFN-α/β. We need to further detect the effect of NF-κB on the induction of IFN-α/β. The MyD88 do not change significantly after infection with rabies virus, demonstrated that it might not modulate the production of IFN-α/β. |
PDF Full Text Request