The Effect Of Mouse MyD88 Gene Deficiency On The Pathogenicity Of Rabies Virus

Rabies virus(RABV)is a kind of virus that strictly invades the Central Nervous System(CNS),causing rabies in almost all warm-blooded animals,commonly known as hydrophobia.The number of rabies deaths in the world is as high as 59,000 annually.The human rabies,except for a few part of cases caused by surgery and/or by wild animals,is mainly caused by dog bites.Preventing pre-exposure and/or post-exposure is the most effective way to reduce the incidence of rabies.However,China is a country that has a lot of dogs and a high incidence area for rabies.Therefore,the relevant government departments have proposed an initiative to eliminate rabies via vaccinating dogs with rabies vaccines.The rabies virus contains a genome about 12 kb in length and five genes coding N,P,M,G and L proteins in order.The G gene is responsible for the pathogenicity and budding of the virus,including virulent and avirulent G protein.Our research group has previously carried out the comparison of pathogenicity differences between G proteins of virulent and avirulent strains,rescued the recombinant RABV of G protein,and found out that the amino acids S253,F263 and V273 of G protein are virulent determining sites.And these three amino acid sites of the mutant RABV also could induce a high level of type I interferon(I-IFN)in the mouse brain.The MyD88 gene is a downstream adaptor protein of the Toll-like receptor(TLR),which functions as a signal transduction and is an important molecule in the host innate immunity.Therefore,this study knocked out the MyD88 gene in C57BL/6 mice to study its effects on the RABV pathogenicity and innate immunity induced by RABV.Firstly,this study introduced the transcription termination sequence and resistance screening gene at the corresponding positions of exons 4 and 5 on MyD88 gene through the CRISPR-Cas9-mediated method to achieve MyD88 gene knockout.The MyD88 biallelic knockout(MyD88-/-)mice was validated by genotype,transcript and protein detection.Seven different RABV and its mutants were selected as objects in this study,including two parental strains(r RC-HL and GX074),four recombinant strains(r RC-HLΔG,r RC-HLΔG242-288,Mu11 and r RC-HLΔG/R333Q)and the standard challenge strain CVS-24,wherein r RC-HL strain is a Japanese animal vaccine strain which is highly attenuated;GX074 strain is a street strain isolated from Guangxi province of southern China;and Mu11 is the mutant with S253,F263 and V273 substitutions on G protein of r RC-HL.These different RABVs were used to infect wild type(wt)mice(C57BL/6)and MyD88-/-mice intracerebrally.By comparing pathogenicity,the virulent strains GX074,r RC-HLΔG,r RC-HLΔG242-288 and Mu11 were lethal to both types of mice,however,the dead peak of MyD88-/-mice were 1-2 days later than those of wt mice.It is worthy noting that r RC-HLΔG/R333 Q failed to cause all wt mice to die,whereas resulted in all of MyD88-/-mice to die.At the same time,the attenuated strain r RC-HL failed to kill wt mice,but kill 73.33% of MyD88-/-mice.Knocking out the MyD88 gene could also promote the transcription of RABV in the mouse brain,especially the attenuated virus.The results indicate that MyD88-/-exacerbates the pathogenicity of RABV.Further,wt and MyD88-/-mice were used to test the expressions of innate immune factors after different RABVs infection,including interferons(IFN-α/β/γ)and interleukins(IL-1β,IL-6 and IL-12),chemokine MCP-1,tumor necrosis factor(TNF-α)and antiviral factor(Viperin).The results indicated that the virulent RABVs could induce high level production of IFN-α/β in mice brains,and there was no significant difference between wt and MyD88-/-mice.However,MyD88 knockout could confer attenuated r RC-HL to maintain higher levels of IFN-α/β at the late stages of infection.IFN-γ did not increase in both types of mice at 4 d after infection,then increased at 7 d and 11 d,but the increasing amount of IFN-γ in MyD88 knockout mice was significant lower compared with wt mice.Among the interleukins,the expression level of IL-1βdid not increase in the two types of mice infected with RABVs,and the production of MyD88-/-mice was only slightly lower than that of wt mice.The expression level of IL-6 significantly increased after infecting with virulent strains,and there was no significant difference between the two types of mice;the attenuated r RC-HL produced IL-6 at 4 d after infection of wt mice,but returned to the normal level after 7 d;whereas in MyD88-/-mice,IL-6 produced at the late stage of infection and persisted.The expression trend of IL-12 in wt mice after infection with different RABVs was the same with IFN-α/β;but MyD88-/-mice infected with RABVs showed no difference from the DMEM group at 4 d after infection,while there was a slight increase at 7 d and 11 d,but overall the increasing amount of IL-12 in MyD88-/-mice were significantly lower than those in wt mice.The expression trend of MCP-1 was similar with IL-12,and the expression of MCP-1 also decreased after MyD88 gene knockout.The TNF-α production of wt mice was mainly concentrated after 7 d post infection,but its expression in MyD88-/-mice increased more than 2 times only at 11 d,and the levels at all time points were lower than those of wt mice.Viperin is a downstream molecule of INF-α/β,significantly increased after RABV challenge,and no significant difference had been displayed between the two types of mice.In summary,the MyD88 gene knockout enhances the transcription of RABV in the brain mainly by reducing IL-12,MCP-1,IFN-γ and TNF-αcompared with wt mice,accelerates the progression of rabies,and enables attenuated virus to acquire lethality on adult mice.Finally,MyD88 plays an important role in anti-rabies virus innate immune indicating that different RABV infections can lead to high level of type I interferon production.