Effect Of Gene Substitution On Biological Character Of Rabies Virus And Transcriptional Profiles Of Mice Brain Infected With Rabies Virus

As anancient zoonosis,rabies still cause 60,000 deaths each year.Although great progress has been made in rabies virus(RABV)research in recent years,the pathogenic mechanism of virulent RABV remain to be further investigated.However,a thoroughunderstanding ofRABV pathogenesis is critical for exploring rabies treatment program,understanding the epidemic characteristics of rabies and eliminating rabies.A number of studies,including the previous work of our group,show that insertion of exogenous immune factors and rearrangement of the viral genome can affect the biological characteristics of the virus.However,these studies do not elucidate the differences in biological characteristics between virluent and attenuated strains.In view of the fact that gene replacement is widely used to study the differences between viruses of different virulence,in order to study the effect of viral gene on the biological characteristics of virulent strainGD-SH-01,our group had successfully rescued a series of chimeric RABV by replacing the single gene in the backbone of HEP-Flury with that of GD-SH-01,and were namedas rHEP-shN,rHEP-shP,rHEP-shM,rHEP-shGand rHEP-shL,respectively.Our previous study found that N and L genes of wild-type(wt)strains can significantly affect the proliferation of the virus,G and M gene palyed a major role in the pathogenicity of the virus,replacement of the P gene significantly reduced viral virulence,however,the underlying mechanism is not clear.This study was designed toelucidate possible mechanisms.After the replacement of GD-SH-01 N gene,the intracellular viral genomic RNA increased significantly,while after the replacement of GD-SH-01 L gene,the viral cell-to-cell spread ability significantly increased;although two different strategies wereadopted,both of themacquired an enhanced proliferative capacityin vitro.After replacement of GD-SH-01 G gene,the viral load and innate immune-related molecules in the mice brain were significantly upregualted 7dayspost infection,while the adaptive immune-related molecules were not significantly up-regulated,and the viral neutralizing antibodies(VNA)in the peripheral blood did not reach the protective level(less than 0.5IU/mL),combined with previous reports of high levels of innate immune responsecontributes to RABV pathogenicity,a variety of factors account for rHEP-shG's increased pathogenicity in mice;when intracranially infected with 2FFU,the mortality rate of rHEP-shM-infected mice was significantly higher than that of HEP-Flury,suggesting that M gene could affect the viral virulence.By analyzing the immunological molecules in the brain of rHEP-shP-infected mice,we found that the adaptive immune molecules in the rHEP-shP group were significantly higher than those in the HEP-Flury group,and the levels of VNA in the peripheral blood were also significantly higher than those in the HEP-Flury group,which may explain to some extent the weakened mechanism of rHEP-shP.At the same time,we found that though mice infected with wt strains had a high level of VNA in the peripheral blood before dying,they died of rabies eventually,andanother study perfomed in our group showed that wt strain GD-SH-01 viaintranasal route can open the blood-brain barrier at the late stage of infection,therefore VNA is not enough to clear the large amount of virus inthe brain,which can not avoid fatal outcome.We found that wild-type strain GD-SH-01 had higher neurotropism than HEP-Flury by studying the one-step and multi-step growth curve of recombinant virus.Another previous studies in this study showed that GD-SH-01 was able to stimulate NA cells to produce more pronounced apoptosis than HEP-Flury did.This is not consistent with previous studiesthat wtRABV do not induce apoptosis,so we further validate this phenomenon and explore its mechanism.The results of this study show that,in addition to M and G genes,P gene also played an important role in GD-SH-01-inducedapoptosis.It was found that the recombinant virus rHEP-shP induced more apoptosis than HEP-Flury,mainly involving the activation of intrinsic apoptotic pathway: rHEP-shP infection inhibited the expression of pro-apoptotic protein bax and downregulated anti-apoptotic protein bcl-2 expression,resulting in thedecrease of mitochondrial membrane potential,and then cytochrome c were released into the cytoplasm,cytochrome c combined with Apaf-1 and pro-Caspase-9,which then further activated Caspase-3,and the lattercleaved the substrate PRAP,eventually leading to the occurrence of apoptosis.In addition,the differences in the pathogenicity of GD-SH-01 and HEP-Flury,and the functional mechanisms of GD-SH-01 G gene and M gene were analyzed systematically.Balb/c mice were infected with the parent strain GD-SH-01 and HEP-Flury,as well as the recombinant virus rHEP-shM and rHEP-shG,and the brains of mice infected with different virus at indicated time points were sequenced and compared by analyzing gene expression in different infection groups,we found that a wide range of host gene were upregulated induced by RABV infection,in order to counteractinvading pathogens.Through the short time series trend analysis(STEM),we obtained a significant profile of genes that showed the same expression trend in differernt virus infected groups,then send the gene cluster for functional enrichment and cluster analysis,analyze immune response related GO terms.Under these GO terms,differentially expressed genes which were significantly up-regulated or down-regualted in GD-SH-01-,rHEP-shM-and rHEP-shG-infected groups compared to HEP-Flury-infected groups were screenedout to participate in specific response process,which may be related to the correspondingviral gene.By analysis,we found that four virus-infected groups had two common significant trends(profile 6 and profile 8),but only profile 8 contained the expected pathways.By analyzing the GO function enrichment of profile 8,we found that the number of terms enriched into biological process was 113 for rHEP-shM-infected group,88 for rHEP-shG-infected group,112 for GD-SH-01-infected group,and 107 for HEP-Flury-infected group.According to further in-depth analysis of this trend,most of the termsrelated tohost's antiviral immunity.By drawing venn charts of four virus-infected groups,the common gene set Aand the gene set B and C,whose functions wererelated to GD-SH-01 M and G,respectively,were found.A total of 36 candidate genes were screened out in different RABV-infected groups and validated by RT-qPCR.Screening and identification of these genes will provide valuable clues and data for the further research of pathogenesis of pathogenic RABV.In this study,we further studied the function of each virus gene of wtRABV GD-SH-01 in the biological characteristics of the recombinant viruses.We first discovered and proved that P gene played an important role in RABV-induced apoptosis.At the same time,transcriptome was used to analyze the host genes with different biological characteristics.This study has enriched our understanding of wtRABV,and provided new ideas and comprehensive informationfor the mechanism ofthe pathogenicity and immunogenicity of RABV.