嗜神经病毒躲避宿主先天性免疫反应的机制及应用课件.ppt

1、 嗜神经病毒躲避宿主先天性免疫反应的机制及对策赵 凌 教授10-15-2013Rabies virusRabies virus180nm x 75nm嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Robert Hurt-USCRabies pathogenesis嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师4Rabies infection and innate immunityRabies infection and innate immunityWang et al.Journal of Virology,2005Wang et al.Journal of Virology,

2、2005嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师5Zhao et al.Journal of Virology,2009Zhao et al.Journal of Virology,2009重组狂犬病病毒构建重组狂犬病病毒构建嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师6Expression of MIP-1MIP-1 attenuated RABV pathogenicity,while expression of RANTES or IP-10 increased RABV pathogencityZhao et al.Journal of Virology,2009Z

3、hao et al.Journal of Virology,2009嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师7Control rHEP HEP-MIP1a a HEP-RANTES HEP-IP10D3D6D9 HE staining of mouse brainsHE staining of mouse brainsZhao et al.Journal of Virology,2009Zhao et al.Journal of Virology,2009嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Expression of Expression of MIP-1 MIP-

4、1 enhances VNA production and protectionenhances VNA production and protectionZhao et al.Journal of Virology,Zhao et al.Journal of Virology,20102010嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Zhao et al.Journal of Virology,2010外周过量表达外周过量表达MIP-1MIP-1会会吸引更多的树突状细胞和吸引更多的树突状细胞和B B细胞细胞嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师嗜神经病毒躲避宿主先天性

5、免疫反应的机制及应用赵凌老师 Antigenic GroupVirusHostDiseaseI229ENL63humanhumanrespiratory infectionrespiratory infection,croupTGEVpigrespiratory and enteric infectionCCVdogenteric infectionFECVcatenteric infectionFIPVcatrespiratory,enteric,hepatitis and neurological infectionIIOC43humanRespiratory and possibly e

6、nteric infectionSARS-CoVHKU1humanhumanRespiratoryRespiratoryMHVmouseRespiratory,enteric,neurologic infectionHEVpigRespiratory,enteric,neurologic infectionBCVcowEnteric infectionTCVturkeyRespiratory and enteric infection IIIIBVchickenRespiratory and enteric infection,hepatitisCoronaviruses cause dise

7、ases in humans and domestic animalsAdapted from Holmes and Lai,Fields Virology嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Why MHV?Why MHV?MHV produces a broad spectrum of disease in the mouse -pneumonia(MHV-1)-hepatitis(MHV-A59)-encephalitis(MHV-A59/JHM)-demylination(MHV-A59)It provides excellent small animal models

8、for hepatitis,for SARS,and for multiple sclerosis嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Part I:Part I:MHV ns2 interferes type I interferon MHV ns2 interferes type I interferon responsesresponses Mutation of ns2 confers attenuation of hepatitis but not CNS disease ICIC 500PFU500PFUIHIH 500PFU500PFU Roth-Cross,J.K

9、.et al,JVI,2009,83(8):3743-3753.Roth-Cross,J.K.et al,JVI,2009,83(8):3743-3753.brainbrainliverliver嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Ns2 is an organ specific virulence factor Ns2 is an organ specific virulence factor 1a1b2a4 5a5bHESEMINMHV 2H phosphodiesterase;2 predicted catalytic His-x-Thr/Ser motifs(Mazum

10、der et a.,2002;Snijder et al.,2003)ns2(Mazumbder et al.,2002Snijder et al.,2003;Roth-Cross,2009)嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Mutation of ns2 confers attenuation of replication in macrophages and microglia but not in other cell types Zhao,L.et al,JVI,2011.Oct;85(19):10058-10068.Zhao,L.et al,JVI,2011.Oct

11、;85(19):10058-10068.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师MOI 1MOI 0.01 ns2 mutants recover the ability to replicate efficiently in macrophages and microglia from IFNAR knockout miceMOI 1 Zhao,L.et al,JVI,2011.Oct;85(19):10058-10068.Zhao,L.et al,JVI,2011.Oct;85(19):10058-10068.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Type 1 i

12、nterferon induction and signaling pathwaysIFN-IFNTYK2JAK1STAT1 STAT1 orSTAT2IFNAR1IFNAR2ISREIRF9STAT1STAT2 ISGF3STAT2IRF-9ISGs:OAS,MxA,ISG15,ISG54,MDA5TLR3TLR7/8TLR9dsRNACpGssRNATIRTBK-1IKK-IRF-3IFN-NF-BATF-2RIG-ICBPPMDA5TIRTIRTRIFMyD88MyD88ISREIFN-嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师L2AstroBMMIFNAR-/-BMMns2

13、mutants are not defective in induction of IFN-/mRNABoth wt RA59 and ns2 mutants induce minimal amounts of IFN-,mRNA in L2 cells and astrocytes wt RA59 and ns2 mutants induce similar levels of IFN-,mRNA in BMM from both B6 and IFNAR-/-mice Zhao,L.et al,JVI,2011.Oct;85(19):10058-10068.Zhao,L.et al,JVI

14、,2011.Oct;85(19):10058-10068.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师BMMBMMMicroglians2 mutants are more sensitive to the antiviral effects of IFN-IFN-/than wt A59 in macrophages and microgliabut not in other cell typesL2Astro嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Viral replication and IFN sensitivity in the Viral replication

15、 and IFN sensitivity in the hepatocyteshepatocytesNo IFNNo IFNIFNIFN嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师In vivo macrophage depletionLiposomes,encapsulating the Clodronate molecules(squares),are ingested by macrophages via endocytosis.After fusion with lysosomes(L)containing phospholipases(arrowheads),the latt

16、er disrupt the bilayers of the liposomes.The more concentric bilayers are disrupted,the greater is the Clodronate release within the cell.The cells are killed by Clodronate through apoptosis.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Liposome ClodronateA59ns2-H126RA59ns2-H126RPBSLiposome ClodronateH&E stainingN prot

17、ein stainingns2 mutants replicate and induce hepatitis in macrophage depleted mice500 fold vs 10 fold嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师 Model:Kupffer cells provide a barrier to the liver parenchyma to viruses=rA59LSECKCHepatocytesinusoidparenchyma=ns2-H126A嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Part I:conclusionsPart I:

18、conclusionslns2 is an organ specific virulence factor and antagonizes IFN signalinglns2 is required for replication in macrophages;depletion of macrophages in vivo promotes ns2 mutant virus replicationlwe suggest that MHV has to replicate in Kupffer cells in the liver sinusoids in order to reach the

19、 liver parenchyma and induce hepatitis 嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Type 1 interferon induction and signaling pathwaysIFN-IFNTYK2JAK1STAT1 STAT1 orSTAT2IFNAR1IFNAR2ISREIRF9STAT1STAT2 ISGF3STAT2IRF-9ISGs:OAS,MxA,ISG15,ISG54,MDA5TLR3TLR7/8TLR9dsRNACpGssRNATIRTBK-1IKK-IRF-3IFN-NF-BATF-2RIG-ICBPPMDA5TIRTIR

20、TRIFMyD88MyD88ISREIFN-嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师pCAGGS-IFNpCAGGS-IFNNS2a-IFNNS2a-IFNSV5V-IFNSV5V-IFNNDV-bioassay in Vero cellspCAGGS-no IFNpCAGGS-no IFNTansfection of pCAGGSTansfection of pCAGGSor pCAGGS-ns2 or pCAGGS-SV5Vor pCAGGS-ns2 or pCAGGS-SV5Vin Vero cellsin Vero cellsTreatment with or w/oTre

21、atment with or w/o1000U/ml for 16h1000U/ml for 16hInfection of NDV-GFPInfection of NDV-GFP24h24h12h12hGFP GFP 嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师ISG screening in KO BMMISG15ISG15IFIT1IFIT1IFIT2IFIT2PKRPKRRNase LRNase L嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Part II:Part II:MHV ns2 antagonizes OAS-RNase L pathway MHV ns2 a

22、ntagonizes OAS-RNase L pathwayInterferon signaling modelInterferon signaling modelViral dsRNAMDA5*,RIG-I*IFNAntiviral ISGsOAS*2-5ARNase LCellular and viral RNASmall RNAs2-PDEns2?Main pathwayOAS-RNase L pathwayOAS=2,5-oliogoadenylate synthetase2-PDE=2 phosphodiesterase2-5A=2,5oligoadenylate嗜神经病毒躲避宿主先

23、天性免疫反应的机制及应用赵凌老师Wild type A59 replication in BMM was not affected by Wild type A59 replication in BMM was not affected by the OAS-RNase L systemthe OAS-RNase L system嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Defective replication of ns2 mutant is restored in Defective replication of ns2 mutant is restored in RNase

24、L-/-BMMRNase L-/-BMMBut not in PKR-/-BMMZhao,L et al.Cell Host&Microbe,2012,(11)607616.Zhao,L et al.Cell Host&Microbe,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师ns2 expression in 293T cellsns2 expression in 293T cellspCAGGS-ns2pCAGGS-ns2pCAGGSpCAGGSpCAGGS-ns2-pCAGGS-ns2-H126RH126RZhao,L et al.Cell Ho

25、st&Microbe,2012,(11)607616Zhao,L et al.Cell Host&Microbe,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师ns2 prevents rRNA cleavage and 2-5A production in BMMZhao,L et al.Cell Host&Microbe,2012,(11)607616.Zhao,L et al.Cell Host&Microbe,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Overexpression of ns2 in 293

26、T cells prevents rRNA cleavage and 2-5A production induced by poly I:CZhao,L et al.Cell Host&Microbe,2012,(11)607616.Zhao,L et al.Cell Host&Microbe,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Ns2 cleaves 2-5A into ATP and AMPZhao,L et al.Cell Host&Microbe,2012,(11)607616.Zhao,L et al.Cell Host&Microbe

27、,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Ns2 enhances virus replication in the liverZhao,L et al.Cell Host&Microbe,2012,(11)607616.Zhao,L et al.Cell Host&Microbe,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师Ns2 facilitates the induction of viral hepatitisZhao,L et al.Cell Host&Microbe,2012,(11)607616

28、.Zhao,L et al.Cell Host&Microbe,2012,(11)607616.嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师lMouse hepatitis virus ns2 protein inhibits the IFN-induced OAS-RNase L pathway lns2 reduces the intracellular level of 2,5-oligoadenylate(2-5A)lns2 has a 2,5-phosphodiesterase to directly cleave 2-5A lns2 mutant virus replica

29、tion and hepatitis-induction are rescued in RNase L deficient micePart II:conclusionsPart II:conclusions嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师1.狂犬病病毒如何逃逸中枢神经系统先天性免疫反应？2.狂犬病病毒如何限制血脑屏障的打开？3.如何打开血脑屏障清除狂犬病病毒？研究方向研究方向嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师41谢 谢！嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师此课件下载可自行编辑修改，供参考！感谢您的支持，我们努力做得更好！嗜神经病毒躲避宿主先天性免疫反应的机制及应用赵凌老师