Effect Of Rabies Virus Infection On TLRs And Identification Of Host Factors Related To Viral Replication

Rabies virus（RABV）is a typical neurotropic virus which can cause lethal zoonotic-rabies.So far,rabies has no effective treatment means worldwide,and it remains a public health threat in most part of the world.For a long time,the only way of rabies prevention is vaccination.Despite the use of RABV vaccine,more than 59,000 people died of rabies each year.The main reason is that effective therapeutic drugs are difficult to develop due to the pathogenesis of RABV was not well understood.Therefore,it is in urgent needed to explore the pathogenic mechanism of RABV and find the key host factors that mediate the infection and replication of rabies virus.This kind of research would provide novel antiviral targets and theoretical evidence for rabies treatments.In the process of infection,RABV needs to break through the body’s first line of defense against pathogens,which is the host’s innate immunity system.There are few studies about the expression and roles of Toll like receptors（TLRs）,an important family members of innate immune system,in RABV infection.In this study,we used three RABV strains with different virulence（fixed strain CVS-11,street strain GDMM and attenuated strain SRV9）to build infection mice model via intracranial injection.Subsequently,RT-q PCR and WB were performed to examine the alteration of TLRs,downstream effector molecules（IRF3,Irgm1）,as well as TLRs related cytokines（IL-1β,IFN-α,IFN-β,and IFN-γ）in mice brains.The mice of CVS-11 group and GDMM group died at 7 dpi.and 12 dpi.（days post infection）,respectively,and the copy number of RABV in mice brains increased with the extension of infection time.The expression of TLR2,TLR8,TLR9,Irgm1,IFN-α,IFN-γin these two groups were significantly up-regulated.All mice in SRV9 group were survived and viral copy numbers in their brains reached the peak value at 5dpi.,then decreased.The changes of TLR7,8,9 and IFN-γin SRV9 group were consistent with the tendency of viral copies.The results indicated that TLR2,7,8,and 9 were involved in the immune regulation during RABV infection.TLR2,8,9 may played antiviral roles by inducing inflammatory response in early stage of virus infection,but it was hard to clear large amounts of the virus in the late stage of infection,which promoted the occurrence of encephalomyelitis.In addition,TLR7 showed different trends in virulent and attenuated strains about significantly up-regulated in attenuated SRV9 infection,but slightly changed in virulent CVS-11 and GDMM.This result suggested that TLR7 played an antiviral effect in RABV infection.According to the expression and function of TLRs in RABV infection,we thought that TLRs would not be the best target to block the infection of RABV.Then,we screened key host proteins that mediate the infection and replication of RABV at gene level based on CRISPR’s genome-wide screening strategy.The mouse genome-wide CRISPR knock-out（GeCKO）library was amplified and packaged into lentivirus after quality control.The lentivirus was then used to infect RABV susceptible cell line BHK-21 cells,and a random gene-deletion cell library BHK-21^KOwas obtained by puromycin resistance filter.Subsequently,BHK-21^KO cells were infected with SRV9-e GFP（Enhanced green fluorescent proteins）recombinant virus which was constructed by reverse genetic system.Partial BHK-21^KO cells which were not infected with SRV9-e GFP were isolated by flow sorting（three rounds of sorting to minimize the false-negative cells）.Uninfected cell clones were confirmed by DFA,RT-q PCR and Western blot,and the candidate proteins were determined by sequencing.Meanwhile,the genomic DNA was extracted for high-throughput sequencing to enrich high abundance sg RNA.The data demonstrated that leukotriene B4 receptor1（Ltb4r1）had the highest sg RNA enrichment,which was consistant with the results of sequencing in the uninfected cell clones.The results indicated that knocked out of Ltb4r1 could significant anti-RABV infection and replication,which may be a potential therapeutic target after RABV exposure.Taken together,this study comprehensively revealed the expression changes of TLRs in RABV infection mice models for the first time and confirmed that many TLRs involved in the infection and replication of RABV.Importantly,we found an important host factor Ltb4r1 by CRISPR whole genome knockout library screening.This study enriched pathogenic mechanism research of RABV and may provide new targets and theoretical foundation for the research and development of preventive and therapeutic drugs.