Comparison Of Different Immune Response Between Attenuated Rabies Virus And Street Virus In Incubation Period

Rabies is a zoonosis caused by rabies virus with typical symptom of acute progressive encephalitis.Rabies presents a public health problem worldwide,causing more than 60,000 human deaths annually,mainly in developing countries.RABV infects a board range of hosts,nearly all warm blooded animals,such as human,dogs,cats,bats and foxes.One of the characteristics of RABV is its capacity to invade the central nervous system(CNS).CNS infection with wild-type(wt)RABV is always fatal in humans.The epidemic rabies virus,which is prevalent in animal populations,is called street virus,the street virus infection is usually fatal to animals.Wt-RABV presents the ability of neurotropism,wherever the site is when the infection occurred.WtRABV will finally invade periphery neuron by local infection site,and enters CNS through retrograde axon transmission,causing severely encephalitis.Intriguingly,RABV do not directly invade CNS immediately after infection.It needs several days,maybe couples of months or years to lurk,named eclipse phase,then induces the irreversible fatal infection.Before the wt-RABV infection generated in CNS,there is no difference between infected animals and non-infected animals,neither obviously clinical symptoms nor serum reaction.In another word,during the latency period upon the RABV infection,it is impossible to identify these two cases from clinical signs or serological reaction.Attenuated virus is originated from the street RABV by artificial domestication or mutation showing less pathogenicity or no pathogenicity to animals.If it invades the body,even the CNS,in a short period,it will be cleared in the infected local site.The dramatic difference between the wt RABV and attenuated virus infection may relate from their distinct virulence.The key factor maybe the different responses against the pathogenetic RABV and attenuated RABV.It is necessary to elucidate the mechanism during the latency period or the ?hidden phase? in RABV infection.Identification of new biomarkers during the early infection may contribute to interfere or reverse RABV infection and support the early diagnosis and treatment after exposure.Street strain used in this study named BD06,isolated in 2006 from a rabid dog in Baoding,Hebei Province,2006.BD06 belongs to China genetype 1(appendix 1),and is responsible for the majority of rabies cases in humans and dogs in China.Laboratory strain SRV9 was attenuated after continually passage on animals or cells after isolated from a rabid dog in 1935,and is used worldwide.SRV9 has been developed as a vaccine strain nowadays.We have conduct this study,analyzing the gene expression profiles in mouse brain infected with street or attenuated RABV.We also compared the peripheral immune response raised from street rabies virus and attenuated virus.Based on the results above,we additionally analyzed the function of the differentially expressed genes by over-expression in immune-competent cells using a tool of lentiviral vector.Details are shown as followed.1.RNA-seq analysis on immunity response about RABV intracerebral infection.The isolated street rabies virus strain was cultured on BHK-21 cells until the titer reached 105.875TCID50/ml stably.Eighteen mice were randomly ranged into three groups,SRV9-infected,BD06-infected and mock-infected group.RNAs of brain tissues of mice infected intracerebrally were purified in a RNase-free condition at day 1 and 6 post infection(dpi)for the subsequent RNA-seq.Analysis based on the sequenced data shows that at 1 dpi,528 genes in SRV9,compared to BD06,infected mouse brain showed significantly high expression levels,most of which clustered into signal pathways related to intrinsic immune responses and some of them were involved in antigen-presenting processes.However,the profiles of genes responsible for innate immune responses in BD06 infected mouse brain showed a similar property with that of control infected group.At d6,441 genes in BD06 infected group expressed at a higher level than that in SRV9 infected group.There up-regulated genes mainly involved in the inflammatory response,chemokines secretion,and IFN response.Nevertheless,the adaptive response still had not been activated.The mouse lost dramatically in weight and showed clinical signs such as anorexia,depressed,limb paralysis.While,SRV9 induced responses increased overall compared to d1 with activated DC and B cells and recovered body weight growth and physiological functions.2.Development of TaqMan probe quantitative method for rabies viruses.In order to investigate the dynamic changes of virus loads in mice post RABV infection and additionally to reveal the intrinsic relation between the immune responses and viral replication in latent infection phase.We designed two groups of quantitative TaqMan probes and RABV primers based on the N gene,and selected the optimal primer group,G2.Based on G2,we constructed a qPCR method with a sensitivity of 101 copies per ?l.The linear regression coefficients of the standard curves were both over 0.99,which could satisfy the requirement for quantitation of BD06 and SRV9 virus loads.3.Differential host immune responses after infection with wild-type or labattenuated rabies viruses in mice.In the RNA-seq experiments,we investigated the traits of gene expression only during the early and late infection,lack of that during the interval.To reveal the whole process of virus latency period,we randomly ranged 72 SPF mice into six groups: two SRV9-infected,two BD06-infected and two mockinfected.All mice were intracerebrally injected.At d1,d3,d5,and eve of death(ED),blood,inguinal lymph nodes,and brains of mice in every first group were collected for RNA extraction.Mice in every second group were weighed daily.In SRV9-infected mice,immune response was activated at d1 and increased until d3 when DC and B lymphocytes were activated,the VNA level reached 0.5IU/ml,a level considered to be protective,and blood brain barrier(BBB)permeability was enhanced,which participated in inhibiting virus proliferation,as the infection progressed,the intrinsic immune response enhanced gradually,at the same time,the immune response contributed to clear the virus and subsequently began to recover.BD06 induced a weak immune response in mice similar to the mock in the whole process of the latent phase until the dramatical proliferation of virus.Then,the inflammatory responses were activated accompanied with more secretion of IFN and chemokines.However,the VNA remained in a low level until death.At this phase,no obviously pathological change was observed in mouse brain,but mice became emaciated,head curled,limb paralyzed,exhausted and dead.During peripheral infection,SRV9-infected mice induced a small amount of VNA at early phase of infection,and the VNA can reach protective levels in the middle period of infection,which helped clear the virus,inhibiting the virus to enter the CNS.While in BD06-infected mice,RABV evaded immune response in the early phase and a small amount of virus reached the brain through axon transmittion,incubated for a period of time,then a large number of virus replicated,causing the "storm" response of chemo-inflammatory factors,whereas the VNA levels were not enough to protect the mice from death.4.Research on antiviral function of IFITs.To further investigate the function of IFITs(IFIT1,IFIT2,IFIT3 and IFIT5)during the incubation period of RABV infection,IFIT genes were amplified from 293 T cell genome and cloned into lentivirus vector pFUGW-H1.Recombinant lentiviruses expressing IFIT1,IFIT2,IFIT3 and IFIT5 were rescued respectively by co-tranfection the package and envelop expressing plasmids based on the second generation of lentivirus packaging system,then were co-infected cells with RABV.We use the models to investigate the influence of IFITs on RABV infection and replication.In conclusion,we compared the differences of the immune responses in latent period in mice induced by lethal and attenuated RABV.In the infection of attenuated SRV9,the virus could be recognized by the RIG-I and TLR sensing system and activated the innate immune response which additionally waked up the adaptive immune response for the secretion of VNA and cleared the virus.However,the street virus BD06 evaded the host immune system in the latent period and induced an acute immune response in the very late infection when the virus was dramatically increased.Although the VNA was induced,it was little to no level to eliminate the RABV.The infected mice became emaciated,head curled,limb paralyzed,exhausted and dead without obvious clinical pathological changes in the brain.Overexpression of IFITs using a lentivirus vector system revealed that IFIT3 helped suppress the replication of RABV,especially SRV9,which provides a new choice for target screening in control of RABV infection.