| Japanese encephalitis(JE)is an acute mosquito born zoonotic infectious viral disease caused by Japanese encephalitis virus(JEV).JEV can break through the blood brain barrier(BBB)and damage to the central nervous system.Thus JEV can lead to human being dead or leave nervous sequelae.Also JEV can cause sow abortion and boar orchitis.The commercial avirulent vaccine may mutant to virulent virus.The wildlife zoo animals and dogs are usually not vaccinated by the JE avirulent vaccine.Currently,the epidemiology of JEV in these animals is not clear.And the molecular pathogenic mechanism of how JEV breakthrough the blood brain barrier(BBB)is not clear and there is no effective drug can completely cure the disease.It is well known that most Flaviviruses are isolated by inoculation of suckling mice,thus mice are an ideal model for the study of JEV pathogenic mechanism.Interferon y(IFNG,IFN-y,Gamma interferon)plays an important role in regulation of innate immunity,cellular immunity,blood brain barrier and inflammatory reaction.But the role of IFNG in the molecular pathogenesis of JE is unknown.MicroRNA(miRNA)is a non encoding RNA,produced by the host or some viruses,plays an important role in the regulation of post transcription.Therefore,this study carried out the epidemiological survey of JEV in serum of Kunming and Shanghai wildlife zoo animals and dogs in several provinces in China.And we screened the affinity peptides binding to JEV particles by phage display peptides.Then,we established a model of wild type suckling mice and IFNGR(interferon gamma receptor)deficient suckling mice with JEV infection.And the mRNA and miRNA transcriptome of brains of JEV infected or mock infected suckling mice were studied by high throughput sequencing.And also the results were analyzed and validated.Detail information should be seen in the following sections:1.JEV epidemiological surveyMany kinds of mammalian and avian animals can be infected with JEV by mosquitoes bitten,and they can be as an intermediate host to make the spread and diffusion of JEV.But the epidemiology mechanism of JEV between various susceptible animals and mosquitoes is not clear.Therefore,this research collected the serum of dogs in several provinces of China and the serum of animals in Kunming and Shanghai wildlife zoo.The HI test(Hemagglutination inhibition test),PRNT(Plaque reduction neutralization test)and JEV EdIII(Envelope domain III)indirect ELISA(Enzyme linked immuno sorbent assay)were used tot detect the JEV antibodies in the serum.And JEV antigen in the serum was tested by the differential diagnosis of JEV genotype I and III antigen using qRT-PCR(Quantitative realtime Reverse Transcription PCR).Results:The JEV HI antibody positive rate of wildlife was 36.8%;the positive rate of dogs was 55.6%;the serum antibody positive rate of wild animals mammals is higher than the avian animals;the dog serum positive rate in several provinces is higher than the others;test results of PRNT,indirect ELISA are consistent with the HI test results;The detection results of qRT-PCR for JEV antigen shows that JEV genotype III is the main antigen.Conclusion:there are JEV antibody and antigens in the serum of wildlife zoo animals and dogs.There are urgent needs to establish scientific serum epidemiological surveillance of JEV in these animals to better prevention and control of JE.2.Screening the affinity peptides binding to JEV by phage display technologyIn order to find the JEV receptor and the neutralizing epitope sequence to JEV.In this study,we carried out the screening of affinity peptides binding to JEV amplified by C6/36 cell line using phage display technology.And the phage clones showing affinity binding to JEV were acquired.And 23 peptide sequences were obtained through affinity verification and DNA sequencing.The antiviral experiment of these peptides showed that they can prevent the entry of JEV into BHK cells.And these peptides can neutralize JEV.For the three peptides of good antiviral effects,we made these peptides labeled with HRP and FITC to carry out the sandwich ELISA and immunological fluorescence tests.And the results indicated that all the three peptides can bind to the JEV antigen.Conclusion:we successfully screened the affinity peptides binding to JEV;and this research can provide valuable suggestions to find the receptor of JEV,to develop antiviral drugs and to differential characterize the JEV antigen.3.Establishment of JE infection model on wild type and IFNGR deficient suckling miceIn order to study the pathogenesis mechanism of JEV breakthrough the blood brain barrier(BBB)of wild type and IFNGR deficient suckling mice,we used Aedes albopictus cell line C6/36 to amplify the NJ2008 JEV strain and to establish an infection model on 9 day old suckling mice by intrperitoneal injection with JEV.The Evan’s blue injected by intraperitoneal was used to observe the effect of JEV on mouse BBB and the damage situation after JEV infection each day;quantitative real time reverse transcription PCR(qRT-PCR)method was used to detect changes of JEV titer in brains of suckling mice after infection;and the distribution of JEV in mouse brain was observed by immunohistochemical(IHC)analysis using anti JEV envelope protein domain III(Ed III)monoclonal antibody;finally the changes of ultra structure and virus particle distributions in the suckling mice cerebral cortex were analyzed by transmission electron microscope(TEM)after four days post infection.Results:wild type mice began to show neurological symptoms in the fourth day post infection and began to die in the fifth day.While the IFNGR deficient suckling mice began to show neurological symptoms in the fifth day post infection and began to die in the fifth or sixth day.The time of BBB destroyed by JEV in wild type mice was in the fourth day,while in the IFNGR deficient suckling mice was in the fifth day.JEV titers in brains of both group mice after JEV infection in the third and fifth days were not significantly different(P>0.05);while in the fourth day there was a extremely significant difference(P<0.01).And there were more JEV in brains of wild type suckling mice than IFNGR deficient mice;IHC analysis showed that JEV infection on the fourth day of wild type mouse significantly more serious than IFNGR deficient mice;TEM observation of the fourth day of wild type mice showed that BBB damage is serious,the infected cell was death,rupture,dissolved and JEV particles can be observed in the cerebral;For the IFNGR deficient mice in the fourth day after infection showed that the condition of the damaged BBB is not serious.Conclusion:JEV infection on 9 day old wild type and IFNGR deficient suckling mice can cause death;the BBB of wild type mice was damaged early than IFNGR deficient mice;JEV infected wild type mice died earlier than IFNGR deficient mice.4.Study of the mRNA transcriptome of brains of wild type and IFNGR deficient suckling mice infected with JEVIn order to further study of the molecular pathogenesis mechanism of blood brain barrier of IFNGR deficient and wild type suckling mice infected with JEV,this research takes the full brain of IFNGR deficient and wild type suckling mice in four days post infection with JEV to extract total RNAs.The RNA sequencing library was build using construction kits.After the libraries were quality inspection qualified,the Illumina Hi-seq 2000 technology was used to sequence the library.The whole genome mRNA transcriptome sequencing was successfully completed;and the data was analyzed and validated.Results:a total of four groups of ten sequencing libraries were constructed by brains of JEV infected and mocked infected wild type and IFNGR deficient suckling mice;the sequencing data set a good correlation within group,and there were significant differences between groups;the corrected p value(padj,q)<0.05 was set as the standard.The differential genes between JEV infected and mock infected wild type mice(WT_T vs WT_C)and IFNGR deficient mice(KO_T vs KO_C)were 2502 and 628,respectively;the differential genes between mock infected IFNGR deficient and wild type mice(WT_C vs KO_C)were 59;the differential genes between JEV infected IFNGR deficient and wild type mice(WT_T vs KO_T)were 1181.Gene ontology(GO)analysis found that:WT_T vs WT_C,KO_T vs KO_C,WT_T vs KO_T,mainly clustered in such biological processes,immune,antiviral defense,the cellular component and molecular function;The signaling pathways(KEGG)clustering analysis found that:WT_T vs WT_C,KO_T vs KO_C,WT_T vs KO_T,mainly in innate immunology and inflammatory signaling pathways,such as cytokines and its receptor interactions,tumor necrosis factor,toll-like receptors,NF-kappa B,osteoclast differentiation,Jak-STAT and so on.WT_C vs KO_,C clustered without any significant enrichment of signaling pathways.Finally we focused on the Venn diagram of KO_T vs WT_T and KO_C vs WT_C,and selected the differential genes(Set A)response to JEV infection since IFNGR deficient.Then qRT-PCR method was used to validate the RNA sequencing results of randomly selected differential genes,and the important genes from several signaling pathways.Conclusion:we successfully got four groups of different mRNA transcriptome data;and we analyzed and validated the differential genes response to JEV infection since IFNGR deficient;these results provide foundation to further study the molecular mechanism of JEV breakthrough BBB and the role of IFNGR signaling pathway during these processes.5.Study of the miRNA transcriptome of brains of wild type and IFNGR deficient suckling mice infected with JEVIn order to find the key miRNAs of JEV infection on wild type and IFNGR deficient suckling mice brains.Based on the JEV infection model,this research took the full brains of JEV infected and mock infected wild type and IFNGR deficient mice in the fourth day post infection.The whole genome miRNA transcriptome was carried out by RNA-seq,and the results were analyzed and validated.Results:1324 known mature mouse miRNA,107 new miRNAs were received.After DGEseq analysis,for the JEV infected wild type mice compared with mock infected(W_T vs W_C),11 miRNAs were up regulated,1 miRNA was down;JEV infected IFNGR deficient mice compared with mock infected(K_T vs K_C),4 miRNAs were up regulated;mock infected wild type and IFNGR deficient mice(W_C vs K_C),2 miRNAs were down regulated;JEV infected wild type mice compared with JEV infected IFNGR deficient mice(W_T vs K_T),2 miRNAs were up regulated.We used Miranda to predict the miRNA target genes.Depend on the degree of enrichment of target genes to do the GO and KEGG pathway analysis.GO analysis shows that the miRNA mainly involved in the molecular function,regulating cell components and biological function.KEGG pathway analysis of miRNAs is mainly involved in JAK-STAT pathway,toll-like receptors,NF-Kappa B pathway and apoptosis,etc.Conclusion:we successfully got four groups of differential expressed miRNAs transcriptomic data and biological meanings of the predicted target genes;the very important differential expressed miRNAs were obtained,and this will provide valuable basis for further study the prevention and control of JE.6.Study of the relationship of differential miRNA and mRNA of brains of wild type and IFNGR deficient suckling mice infected with JEVIn order to further obtain the specific differential target genes of differential miRNAs,according to the negative correlation between miRNA and its target gene mRNA,using differential genes of the transcriptome sequencing results,we carried out the analysis as follows:the up regulated expression of miRNA and down regulated expression of target mRNA,down regulated expression of miRNA and up regulated expression of target mRNA.And we analyzed the GO and KEGG enrichment of these target genes.For the wild type suckling mice infection with JEV,there were 1739 mRNAs up regulated,763 mRNAs down regulated,while 11 miRNAs were increased,1 was down.The infection of JEV IFNGR deficient suckling mice,there were 576 mRNA up regulated,52 down regulation of mRNA expression,while 4 miRNAs were increased.Compared with JEV infection on wild type and IFNGR deficient scukling mice,there are 797 mRNAs up regulated,384 down regulated,while 2 miRNAs were increased.Compared with mock infected wild type suckling mice and IFNGR deficient mice,31 mRNAs were up regulated,28 down regulated,while 2 miRNAs were increased and 2 miRNAs were down,respectively.The results found that the GO enrichment was mainly involved in immunity,apoptosis,cytokine production and other biological process.KEGG pathways were enriched in Toll like receptor,TNF,RIG-I like receptor,cytokine and apoptosis signaling pathway,and so on.From the results of correlation analysis,we used dual luciferase report system to determine the target effects of miR-147-3p and STAT6,miR-27a-3p and IRF9,miR-203-3p and IL12B,miR-21a-3p and USP,mmu-miR-223-3p and IL6,mmu-miR-142-5p and CD28,miR-223-3p and IL11R,miR-21a-5p and STAT1,miR-203-3p and OASL2,miR-21a-3p and CD22,miR-203-3p and MYD88,miR-142b and TNFSF9,miR-155-5p and TNFSF9,miR-142-3p and IL7,miR-27a-3p and IFIT3.The results indicated that the fluorescence value of STAT6,USP,IL11R,STAT1,OASL2,CD22,MYD88,TNFSF9,IL7 and IFIT3 decreased more significantly.Conclusion:we successfully got four groups of miRNAs and their differential mRNA targets,as well as the biological meanings,and the correlation analysis of miRNAs and mRNA targets were tested by the dual luciferase report system. |
