The Differences Of Intracellular Replication And Assembly Among Rabies Virus Strains

Rabies,as a classical zoonosis,is an acute encephalomyelitis infectious disease caused by rabies virus(RABV)infecting the central nervous system.It is usually transmitted to humans and animals by biting through saliva of sick animals.Because of the clinical manifestation of hydrophobia,it is also called hydrophobia with a fatality rate of nearly 100%.RABV,a prototype of the rabies virus genus of rhabdavirus family,its genome consists of a no-segmented single-stranded negative-stranded RNA,encoding five structural proteins:nucleoprotein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G)and RNA-dependent RNA polymerase protein(L).Genomic RNA,together with N,P and L proteins,forms the ribonucleoprotein complex(RNP),which assists the replication and transcription of viral genomes.M protein locates under the envelope membrane of RABV linking to RNP and protective antigen G,while G protein envolves the binding to host cell surface receptors and participates in the entry and transmission between neurons of RABV.Great progress has made in the pathogenesis of RABV,especially the replication taking place in Negri bodies.However,more details about synethsizing and assembling sites for some structural proteins need to be elucidated.TCID₅₀ titers of the three RABV strains(challenge virus standard CVS-11,oral attenuated SRV-9,and street strain PB4)were titrated on N2a and BHK cells.At the same time,CVS-11,SRV-9 and PB4 were inoculated with the same amount of MOI in N2a and BHK cells respectively,specific antibodies against RABV structural proteins and endoplasmic reticulum(ER)or Golgi apparatus(Golgi)were used to stain the infected cells,and immunofluorescence observation were performed under laser co-focal microscopy.Co-localization of N,P,M,G protein in different RABV strain with ER or Golgi were detected,and immunofluorescence in frozen sections of mouse brain tissue was applied to confirm the immunofluorescent results.The results demonstrated that there were significant differences in the titers of challenge virus standard CVS-11,oral attenuated SRV-9,and street strain PB4 on the same cell type,and CVS-11 had a relatively higher titer than SRV-9,while PB4 only infected N2a cells and had a very low titer.By immunofluorescence,it was showed that M protein was dispersed distributed in cytoplasm and cell membrane,obvious co-localizations were demonstrated with ER and Golgi apparatuses.The cellular dispersed pattern and co-localization of G protein in all tested strains manifected the same disciplines as the M protein.N protein and P protein in the RABV strains displayed as globular,and no co-localization with the ER and Golgi were observed.The results further revealed both of M protein and G protein in CVS-11 and SRV-9 co-localize with ER or Golgi repectively,and M and G co-localization presented nearly cell membrane,which implied M and G proteins were synthesized and processed by ER-Golgi pathways,and co-transported to the membrane for budding as progeny virions.That P protein and N protein did not follow the ER-Golgi pathway was also indicated in our study.Based on above,it was clearly deduced that no differences of synthesized sites were found among the RABV strains either in neuronal or somatical derived cells,this issue had been approved by frozen sections of mouse brain tissue primarily,further enrichment was in the process.The RABV strains we chosen comprised challenge virus standard,oral attenuated,and street strain,all showed the same synthesized mode among the strains,which implied RABV might follow the same displines in structural protein systhesis and assembling of progeny virions.