Influence Of Phosphoprotein Of Virulent RABV Strain GD-Sh-01 On Permeability Of Blood-brain Barrier

Rabies is an acute infectious disease caused by rabies virus(RABV),and the lethality reaches about 100% once clinical symptoms appear.As a worldwide zoonotic disease,rabies leads over 55,000 human deaths each year.Up to now,vaccination is the main way to prevent rabies infection.Therefore,better understanding of RABV structural protein facilitates the development of effective vaccine.Blood-brain barrier(BBB)serves as an organic barrier between peripheral vessels and central nerves system(CNS),which helps maintain the innate stability by prohibiting the entry of exotic substances.Normally,BBB is integrated by tight junction(TJ)protein to form an intensive barrier,however,under some condition,microbes may lead breakdown of BBB resulting in enhancement of BBB permeability.Thus,in the view of clinical medicine,BBB may protect the CNS against exotic substances,on the other hand,it may keep therapeutic substances out so that makes it difficult to cure neurological diseases.There is no report about influence of each gene of RABV on BBB yet.In this study,we infected mice with virulent strain GD-SH-01,lab-attenuated strain HEP-Flury,or recombinant strain r HEP-SH-N,rHEP-SH-P,rHEP-SH-M,r HEP-SH-G or rHEP-SH-L respectively,and then the BBB permeability of infected mice was detected.We found that only the BBB permeability of mice infected with r HEP-SH-P kept relative integrated post-infection,and rare clinical symptoms appeared,and other strains of RABV enhanced the BBB permeability and led symptoms appear in varying extent.We found rHEP-SH-P did not decrease the expression of TJ protein using western blotting,however,the parent strains will depress its expression.In addition,we tested transcription level of P gene and N gene of GD-SH-01,HEP-Flury or r HEP-SH-P,and found that P gene expression of r HEP-SH-P was depressed compared with GD-SH-01,which results in the depression o f N gene.The result demonstrated that the replication capacity of rHEP-SH-P has been decreased.Then,we found the viral load of r HEP-SH-P was higher than parent strains in vivo using qRT-PCR and fewer T cells infiltrated into CNS in mice infected with rHEP-SH-P by immunological histological chemistry,which suggested that the relatively integrated BBB hampered T cells infiltration into CNS,so that the virus was not eliminated.Finally,some innate immune molecules were determined in the brain of infected mice,and expression of cytokines in mice infected with r HEP-SH-P was lower than parent strain GD-SH-01.In conclusion,P gene expression of GD-SH-01 was depressed in genome background of HEP-Flury,so that the viral replication was lowered and the ability to induce immune response was decreased,which kept relative BBB integrated.This study demonstrated that the replication capability was the key factor of inducing immune re sponse.