Comparative Analysis Of Effection On Interferon Signaling Of Rabies Virus BD06 And SRV9

Rabies remains a severe threat of public health especially in developing areas and countries. Due to the limit of knowledge about the molecular mechanism of rabies pathogenesis and lack of efficient treatment, rabies present a clinical fatality of almost 100% with over 2000 rabies-related deaths in China per annum. The major etiological agent of rabies is rabies virus(RABV), which consists of a negative single strand RNA genome and five encoding structural proteins(nucleoprotein, N; phosphoprotein, P; matrix protein, M; glycoptotein, G; and the large protein, L). To observe the RABV pathogenesis, recent focuses are turned to the interaction between viral components and host biological processes, such as influence on innate immune response by RABV structural proteins, with the expectation of some advances in rabies therapy. Excitingly, great and increasing progress has been made during the past tens of years. Examples include the findings that RABV P presents as an interferon(IFN) β antagonist, which affects the virus pathogenicity, and that specific amino acid substitution in N switches the RABV lethality in a manner similar to P by changing its suppression activity of IFN induction. Nevertheless, many uncertainties still require additional clarification, including, not least, the correlation between virus pathogenicity and its IFN antagonism, potential effect on IFN-related signaling of M and G besides of P and N, and roles of IFN antagonism in virus infection. With the attempt to unravel some mysteries, we designed this project.We chose an attenuated RABV SRV9 stain, extensively used for vaccine production worldwide, and a pathogenic BD06 strain, widely prevalent in China, for the comparative analysis. The IFN response in vivo and expression profile in vitro after infection of either virus were compared, and also, the IFN antagonist activities of the two viruses were comparatively analyzed. In this manner, we clarified the correlation between virus IFN antagonist activity and pathogenicity. Details are presented as below.BD06 culture with high titer was firstly screened and identified for challenge use. Equivalent amount(104ffu/30μl) of RABV or DMEM(mock)were inoculated into adult Balb/c mice following an intracerebral(i.c.) route. Mice infected with SRV9 underwent only transient weight losses, while those infected with BD06 developed rabies at day 5 and died at day 6 or 7. This differential infection outcome may stem into the distinctive IFN response induced by RABV, for IFN mediated innate immunity is the exclusive countermeasure especially in the early phase for mice infected i.c. with RABV. Therefore, we investigated the IFN responses by quantitatively analyzing the IFNβ and IFNβ induced OAS-1G and Mx1 expression levels in mouse brains at day 0, 2, 4 and 6 post infection of either virus or mock. Results suggest a significant difference between the kinetics of the IFN responses induced by the two viruses. SRV9 infected mice raised an IFN response with a high level at day 2 which peaked at day 4 but decreased at day 6, whereas, BD06 elicited a sustained increase of IFN response despite a relatively lower level at the very early phase.These results indicate that sole IFN response is not sufficient for virus elimination.We next analyzed the expression profile of 293 AD cells infection with either virus at the early stage. RNA was sequenced using an Ion proton platform. Over 80% of the screened clean reads(approximately 12M) could be aligned to the reference genome, indicative of an excellent sequencing. The expressed genes were then quantitatively analyzed after calculating their RPKM values by aligning to the reference gene hg19. We screened 2317(SRV9 against mock), 3331(BD06 against mock) and 2760(BD06 against SRV9) differential expressed genes(DEGs), and analyzed them using GO function and KEGG pathway enrichment methods. Results showed that no significant enrichment of DEGs of neither SRV9 nor BD06 against control was found, but DEGs of BD06 against SRV9 significantly enriched to the term “cytokines”. KEGG pathway enrichment analysis revealed that enrichment of DEGs between BDO6 and SRV9 focused on pathways related to antiviral response. These data together suggest that BD06 induces stronger virus-host interaction than SRV9 dose.Given that the RABV proteins dedicated to suppress the innate immune response, we nest compared the IFN antagonisms of SRV9 and BD06. To mimic the RABV infection, plasmid bearing human RIG-I-CARDs was used to activate the IFN response, RABV proteins were co-expressed to suppress this process. In this manner, we checked and compared the IFN antagonist activities of N, P, M and G form either BD06 or SRV9 by analyzing the induction of IFNβ and IFNβ stimulated PKR and OAS. Results showed that RABV P played a major role in antagonizing IFN activation. Despite that RABV N was reported to affect the IFN induction, in our experiment, RABV couldnot inhibit the IFN activation significantly. For RABV M and G, no IFN pathway antagonism was observed despite the ability of analogous proteins of other viruses to suppress IFN induction. More over, ability of SRV9 P to counterpart the IFN induction is stronger than that of BD06 P, while this ability of SRV9 N dose not vary significantly with BD06 N.Collectively, we demonstrated:(1) Kinetics of IFN response induced by SRV9 and BD06 differ significantly. Virus elimination cannot be enacted solely by IFN responses.(2) BD06 induced up-regulation of gene expression mainly focuses on virus related response, while SRV9 appears to induce a non-polar gene expression change.(3) In view of the weaker ability of P, and similar ability of other proteins in IFN antagonisms, the BD06 shows a limited ability to suppress the IFN induction compared with SRV9.(4) The data together clearly suggest a lack of positive correlation between virus IFN antagonism and pathgenicity.