Studies On Mechanisms Of Japanese Encephalitis Virus Infection In Human Neural Cells

Backgrounds and Objectives:Japanese encephalitis virus(JEV),the pathogen of Japanese encephalitis,belongs to mosquito-borne virus and is one of the main causes of viral encephalitis.Japanese encephalitis is a natural focus disease of humans and animals,mainly transmitted by mosquitoes.Vertebrates such as pigs,horses and waterfowls can be infected with Japanese encephalitis virus,and the main amplification hosts,while humans are the terminal host.Japanese encephalitis virus is mainly distributed in about 24 countries in Southeast Asia,Northern Oceania and Western Pacific.At present,based on the WHO reports,there are nearly ten thousand patients with Japanese encephalitis each year.The major symptoms of severe patients are high fever,headache,coma,convulsion and so on.The mortality rate of the patients with severe disease symptoms can be as high as 30%.About30%-50%of survivors with severe disease symptoms are accompanied by paralysis,mental disorders and other neurological sequelae.However,due to the increasing number of tourists in epidemic areas,the increase of imported cases in non-epidemic areas,the spread of mosquito vectors caused by climate change and the occurrence of virus mutation,there are outbreaks and epidemics of Japanese encephalitis in some countries or regions.The current situation of Japanese encephalitis still can not be ignored.However,there are no specific antiviral drugs for JEV,and the relationship between virus and the host is not clear.Therefore,Japanese encephalitis is still an important international public health problem,and its pathogenesis still needs to be further studied,which is conducive to the development of specific antiviral drugs and effective treatment measures.The vaccine is the most effective measure to prevent virus infection.The live attenuated vaccine SA14-14-2 of Japanese encephalitis virus is the most widely used vaccine in China.However,the mechanism of attenuated live vaccine SA14-14-2 has not been fully understood.Construction of the infectious clone of attenuated live vaccine SA14-14-2through reverse genetics will help to analyze the gene function,pathogenic mechanism and life cycle of the virus,even for the development of chimeric vaccine.However,the genome of flavivirus has genetic instability when it replicates in E.coli.Insertion,deletion or mutatation often occurred in the geneome sequence spontaneously.At the same time,the c DNA itself or the expressed protein has certain toxicity to E.coli,which limits the construction of full-length infectious clone of the virus.Therefore,it is necessary to construct the infectious clone of attenuated live vaccine SA14-14-2 of Japanese encephalitis virus.To explore the attenuated mechanism of the caccine is of great significance for us to study the mechanisms of neuroinvasion and neuropathogenicity of JEV,which helps for the development of JEV therapeutic drugs targeting central nervous system infection.JEV is neurotropic virus,which can break through the blood-brain barrier and invade the central nervous system(CNS)to cause encephalitis,resulting in serious central nervous system infection and complications.However,the molecular mechanism of JEV infection in nervous system is not clear.JEV infection of host cells is a complicated process,including the invasion,replication,assembly and release of virus in host cells.In these processes,JEV infection often causes changes in the environment of host cells,resulting in the activation or inhibition of a large number of host genes,involving many signal pathways and host cell molecules.On the one hand,some changed genes are playing important roles in resisting virus infection.On the other hand,some genes can also be used and hijacked by the virus,so as to promote the replication of the virus.Due to the lack of secondary lymphoid tissue in the central nervous system,adaptive immune response can not be activated,so the natural immune response in neural cells is very important to limit JEV infection.Guanylate binding protein 5(GBP5)belongs to interferon stimulate gene(ISG).The mechanisms of GBP5 is not clear in neurotropic virus.Therefore,to explore the role of GBP5 in JEV-infected neural cells is helpful to understand the neuropathogenicity of JEV.Ubiquitin proteasome system and autophagy lysosome system are two main ways to regulate protein catabolism.Neural precursor cell expressed,developmentally down-regulated 4(Nedd4),belonging to the E3 ubiquitin-protein ligase,plays an important role in the relationship between ubiquitin proteasome system and autophagy lysosome system.The role of Nedd4 in the pathogenesis of JEV remains unclear.Therefore,to explore the role of Nedd4 will help to understand the role of host factors in JEV infected-neural cells,and provide new targets for antiviral drugs.Part Ⅰ:Construction of infectious clone of Japanese encephalitis virus vaccine strain SA14-14-2Materials&methods:The full genomic sequence of live attenuated vaccine strain SA14-14-2 was synthesized from two segments.The synonymous mutations of A356G and T359C were introduced into the segment,and the introns were introduced into the positions of nt356 and nt2217 to stabilize the genomic sequence.The two segments were linked to the vector p ACNR by the method of enzyme cutting and connection,and the full-length c DNA plasmid of p ACNR-SA14-14-2 was constructed.Using c DNA as the template,infectious viral RNA was synthesized by transcription in vitro,and transferred into BHK-21 cells by electrotransfection to rescue the virus.The effect of cytopathy was observed,and the rescued virus was identified by plaque formation test and immunofluorescence test.The growth curves of parental virus and rescued virus were compared,and the neurovirulence and invasiveness of rescued virus were detected in mice.Results:The genome of JEV could not be stabilized at 25℃,but the stable clone could be obtained by introducing intron sequence into the capsid protein gene region(nt356)and envelope protein gene region(nt2217).The SA14-14-2 full-length c DNA plasmid template was successfully constructed and identified through the enzyme digestion and sequencing.The rescued virus was successfully obtained.There are no intron sequences found in the rescued virus identified by sequencing.The expression of E protein,NS1 protein and NS3protein of the rescued virus was detected by immunofluorescence test.The plaque size and morphology of the rescued virus and the parental virus are similar,and they have similar proliferation characteristics.The results of mice infection showed that the rescued virus and the parental virus had no neurovirulence and invasiveness to mice under the amount of 2×10~2PFU and 5×10~4PFU,respectively.Conclusion:The infectious clone of JEV attenuated live vaccine strain SA14-14-2 has been constructed,and the rescued virus can be used for the study of the pathogenic mechanism and attenuated mechanism of JEV.Part Ⅱ:The inhibition role of GBP5 in JEV-infected neural cellsMaterials&methods:In SH-SY5Y cells,GBP5 expression was down-regulated by si RNA of GBP5 or up-regulated by plasmid overexpressing GBP5 to detect the effect of GBP5 on JEV infection.Using virus bingding test and virus internalization test to detect the impact of GBP5 on the virus entry process.The effect of GBP5 on JEV replication was detected by transfection of JEV replicon RNA.The role of GBP5 in JEV infection was analyzed by using functional mutant plasmid of GBP5 and truncated plasmid.Subsequently,the effect of GBP5 on Furin protein and the effect of Furin protein on JEV infection were examined.Results:JEV infection can up-regulate the expression of GBP5 in HUVEC cells and Huh7 cells,and the up-regulation was in an infection dose dependent manner.In SH-SY5Y cells and SK-N-SH cells,JEV infection can cause the down-regulation of GBP5,and the decrease was along with the increase of infection doses.Up-regulation of GBP5 in SH-SY5Y cells can inhibit JEV infection,and down-regulation of GBP5 can promote JEV infection.GBP5 does not influence the entry process of JEV infection in SH-SY5Y cells,but affect the process of replication and the production of infectious virions.The GBP5 functional mutant plasmid with C583A mutation without isoprenylation lost the anti-JEV effect.Further detection showed that GBP5 could down-regulate the expression of Furin,and affect the infectivity of JEV in SH-SY5Y cells through Furin protein.Conclusion:GBP5 is of a significant effect in inhibiting JEV.The antiviral effect on JEV depends on the isoprenylation of GBP5,and affects JEV maturation through the Furin protein.The down-regulation of GBP5 in JEV-infected neural cells may be related to the hijacking of antiviral mechanism of the host for effective replication.The changes of GBP5in neural cells provides a new way to elucidate the neuropathogenic mechanism of JEV.Part Ⅲ:Promotion of Nedd4 on JEV replication in human neural cellsMaterials&methods:In SK-N-SH cells,the influence of Nedd4 on JEV infection was detected by down-regulating Nedd4 with si RNA of Nedd4 protein or up-regulating Nedd4 with plasmid over-expressing Nedd4.The influence of Nedd4 on viral entry process was detected by virus binding test and virus internalization test.The effect of Nedd4 on JEV replication was detected by transfection JEV replicon RNA.Then,the transformation of LC3I/II protein and the level of autophagy in JEV infected SK-N-SH cells were detected by Western blotting and m Tag RFP-m Wasabi-LC3,respectively.The function of autophagy in JEV infected SK-N-SH cells was verified by the si RNA of Beclin1.Finally,the role of Nedd4 in Beclin1 and the autophagic pathway during JEV infection was tested.Results:JEV infection can induce the expression of Nedd4 in SK-N-SH cells and the up-regulation was along with the progress of infection.Down-regulation of Nedd4 in SK-N-SH cells can significantly inhibit JEV infection and up-regulation of Nedd4 can promote JEV infection in SK-N-SH cells.Down-regulation of Nedd4 did not participate in the entry process of JEV,but significantly inhibit the replication of JEV.JEV infection can activate autophagy in SK-N-SH cells,while inhibition of autophagy in SK-N-SH cells can promote JEV replication.In JEV infection,down-regulation of Nedd4 can induce the expression of Beclin1 and inhibit JEV replication through promoting autophagy.In non-neural cells,down-regulation of Nedd4 did not affect JEV infectivity.Conclusion:Nedd4 plays an important role in the process of JEV infection in neural cells.JEV infection can promote the expression of Nedd4 in neural cells.Down-regulation of Nedd4 can promote the expression of Beclin1 protein and enhance autophagy,thus inhibiting JEV replication.The results showed that JEV infection in neural cells can hijack Nedd4 and promote JEV infection in neural cells by inhibiting autophagy.The role of Nedd4and autophagy in neural cells is helpful to understand the neuropathogenicity of JEV and provide potential targets for the development of antiviral drugs.SummaryAt present,there are few researches on the neuropathogenicity of JEV.The roles of structural and non-structural proteins in the JEV-infected neural cells are not clear.The molecules in host cell essential for JEV to infect neural cells and mechanism of molecules involved in regulating JEV still need to be clarified.In this study,we successfully constructed the infectious clone of JEV attenuated live vaccine strain SA14-14-2,and successfully rescued the virus,which provided a basis for the study of the neuropathogenic mechanism of JEV.The establishment of effective infection depends not only on the pathogenicity of the virus,but also on the resistance of the host.On the one hand,JEV can inhibit the expression of antiviral protein GBP5 and interfere with its antiviral effect,so as to promote the virus infection in neural cells.On the other hand,JEV infection can also promote the expression of Nedd4 in neural cells,which is beneficial to JEV infection in neural cells by inhibiting autophagy.The above results are helpful to understand the biological characteristics and neuropathogenicity of JEV,which is of great significance to clarify the neuropathogenic mechanism of JEV,and to provide potential targets for the development of antiviral drugs.