Effects On Biological Charactersof Rabies Virusby M Gene Codon-pair Deoptimization

Rabies is caused by the rabies virus?RABV?which leads to fatal and highly neurotropic zoonotic infectious diseases.There is no effective therapy for this disease and prophylactic immunization is the primary countermeasure.Therefore,it is critical to explore the replication and pathogenesis of the RABV,and develop the affordable and efficient vaccines.The preference of codon-pair effects on the translation process via the variation of the available t RNAs amount which thus influences the protein expression.In recent years,few studies on the virus codon optimization have been reported.Codon-pair optimization deoptimization approach has been used successfully to affect replication and pathogenicity of vesicular stomatitis virus?VSV?,poliovirus?PV?,human respiratory syncytial virus?RSV?,influenza A virus?AIV?and HIV type 1.But research of the M gene optimization of RABV is absent.This study will explore the replication,transcription,pathogenicity and immunogenicity of RABV virusby M gene codon-pair deoptimization.This studyon the established reverse genetic operating platform in laboratory,the M gene of the recombinant virus rHEP-dG with double G genes isdeoptimization.The amount of dinucleotide of the viral genes CpG and Up A was increased to construct the recombined full-length c DNA plasmid pHEP-dG-M^min,successfully rescuing the recombined virus rHEP-dG-M^min.The results of the biological characters study of the recombined virus rHEP-dG-M^min indicated that the recombined virus rHEP-dG-M^min could stably proliferate in N A cells.At MOI of 3,the virus titer of recombined strain r HEP-dG-M^min was higher than that of the strain rHEP-dG.The transcription level and virus replication rate of rHEP-dG-M^min were both higher than those of the parent strain rHEP-dG at 24h post-infection.The expression of G protein in rHEP-dG-M^min was significantly higher than that in parent strain rHEP-dG after48h post-infection,at the same time,there was no significant difference in the expression of other structural proteins.At MOI of 0.01,the virus titer of the recombined strain rHEP-dG-M^min was lower than that o f strain r HEP-dG before 48h post-infection,but higher after 48h,in consistent with the spread experiment results at the low MOI.The genomic RNA level of r HEP-dG-M^min was lower than that of the parent strain rHEP-dG within 72hpost-infection.The transcription level of each gene was lower than that of the parent strain in the early stage of infection but progressively increased after48hpost-infection.At the early stage of the infection,the level of protein expression was lower.The expression of M prote in in r HEP-dG-M^min strain was lower than that in rHEP-dG strain within 72 h,while no significant difference was found between other structural proteins after 48h post-infection.The results of the pathogenicity study indicated that there was no significant difference in body weight variation between the adult mice taking intramuscular injection of the recombined virus strains by IE inoculationof r HEP-dG-M^min and the parent rHEP-dG,indicating no significant pathogenicity difference.The results of immunoge nicity indicated that both the recombined virus strains,successfully induced the production of the rabies antibody in mice.The qualified antibody level was monitored on 10d,and no significant difference was found in rabies antibody levels between the two strains over the 50 d of monitoring.The results of challenge experiment indicated that the level of rHEP-dG-M^min-induced antibody against CVS-24 in micewas no significant difference with rHEP-dG.Therefore,the immunoprotection of r HEP-dG-M^min wasno significant difference than rHEP-dG.In summary,deoptimized M gene of RABV indicated increased virus titer and different variation replication ability,transcription level and structural protein expressionof the RABV.For the ICR mice by IE inoculation of the recombined virus strain with deoptimization,its pathogenicity,immunoprotection and immunogenicity indicated no significant difference from its parent strain.To further study the applicability of the recombined RABV rHEP-dG-M^min,other immunization routs should also be applied to the KM or BALB/C mice to explore the pathogenicity and immunogenicity of the recombined viruses.