Genetic and mechanistic Determinants of Rotavirus Host Range Restriction

Transcription

1 Genetic and mechanistic Determinants of Rotavirus Host Range Restriction Harry Greenberg MD and the Greenberg Lab Stanford University School of Medicine Eleventh International Rotavirus Symposium New Delhi, India 9/04/14

2 Three LID investigators how spent their careers focused on host range restricted pathogens

3 PFU equivalent/g intestinal tissue Replication of murine and simian RV in the suckling mouse intestine THE REPLICAION DIFFERENCE IS SUBSTATIAL (>10 4 )!! EW (murine RV ) (simian RV ) RV Infected enterocytes 10,000,000,000 1,000,000, ,000,000 10,000,000 1,000, ,000 10,000 1, EW Days post infection

4 First Host Range Restricted Rotavirus Vaccine (Rotashield TM ) A quadravalant set of Simian x Human monoreassortants A Modified Jennerian Vaccine att: AZ Kapikian VP4 Neutralization antigen (SIMIAN) VP6 Group Antigen (SIMIAN) VP7 Neutralization antigen (HUMAN) THE Simian x Human Rotavirus Vaccine Was Attenuated In Children Figure courtesy MK Estes MODIFIED

5 The Merck Pentavenlant Reassortant Vaccine RotaTeq- a second Modified Jennerian Vaccine G2 G3 G1 P[8] G4 The Bovine-Human Reassortant Vaccine is Also Attenuated in Children

6 Host Range Restriction of Local vs. Systemic Infection in Mice EC = Murine RV; = simian RV Quantities of rotavirus (-)RNA and antigen in suckling mice innoculated with EC or rotavirus Rotavirus (-)RNA Rotavirus antigen sample inoculum Geometric mean* Geometric mean** Feces EC 6.8 (+0.6) 4.5 (+1.0) 6.2 (+0.9) 1.3 (+2.3) Small Int. EC 5.0 (+1.0) 0.3 (+0.8) 4.7 (+1.8) 1.2 (+2.4) MLN EC 3.9 (+1.7) 3.0 (+ 1.9) 3.0 (+2.5) 2.4 (+3.3) Blood EC 1.0 (+1.1) 0.4 (+0.8) 1.6 (+2.2) 0.7 (+1.8) Liver EC 2.2 (+1.0) 0.3 (+0.8) 0.3 (+1.0) 2.1(+2.4) Lung EC 0.9 (+0.9) 1.6 (+2.0) (+2.7) Kidney EC 1.8 (+1.2) 0 NA NA * The log value of the geometric mean on 3 through 7 dpi -RNA copy numbers/microgram ** The log value of the geometric mean of PFU/ml equivelant values of ELISA titer on 3 through NA= not available Enteric HRR is not Present at Systemic Sites Fenaux, M. et al, J Virol; 2006

7 Rotavirus replication in the epithelial cells of murine biliary tract is viral strain specific but not host range restricted UK (bovine) (simian) EW (murine) VP6, K19, Nucleus Feng et al. J Virol 2010

8 1) Does the Host Range Restriction Phenotype Occur In Vitro and What Is Its Basis? 2) What is the Genetic and Mechanistic Basis of the Host Range Restricted Phenotype In Vivo?

9 Rotavirus Replication and the Role of Interferons in Regulating Growth in Mouse Embryo Fibroblasts (MEFs) 1.E+08 1.E sv IFNR KO STAT1 KO 1.E+06 1.E+05 1.E+04 1.E+03 Bovine Porcine Murine Simian Heterologous Homologous IFN signaling restricts bovine and porcine RV replication Murine and simian replication is not restricted by IFN Lack of restriction based on the ability of the virus to degrade IRF3 or NFkB (data not shown) Feng N. et al, J Virol,2009

10 Intestinal replication of mouse x simian reassortants in Wt / STAT1 KO mice Reassortants Rotavirus Genes Wild type STAT1 KO (nsp3) 8 (nsp2) 9 (VP7) pfu/g pfu/g B7/2 R R E R R R R R R R R 30 (0/4) 179,055 (6/6) A15/1 R R R R R R E E E R R 30 (0/7) 16,502 (4/4) B2/1 R E E R R R R R E R E 30 (0/6) 51,667 (5/5) B4/1 R R E R R R E E E R R 30 (0/7) 37,251 (7/7) B6/1 R E E R R R R R E R E 30 (0/4) 160,707 (8/8) E14/2 E E E R R E E R E E R 30 (0/9) 118,166 (7/7) H5/1 E E E R R E R E E E R 52 (1/7) 40,907 (6/6) E9/1 E E E R R E E E E E R 77 (1/4) 155,672 (6/6) D4/3 E E E R R E E E E E E 70 (2/8) 26,889 (6/6) D10/2 R R R R E R R R R R R 900 (3/4) 18,736 (12/12) E4/1 R R E R E R R E E R R 224 (4/7) 7,167 (7/7) A/11 R R R R E R E R R R R 194 (5/10) 130,731 (7/7) EA R R E R E R E R R R R 253 (6/9) 44,344 (4/4) DEA R R E R E R R R R R R 6,162 (6/6) 39,719 (4/4) DEA R R R R E R E E R R R 4,531 (8/8) 138,792 (3/3) D6/2 E E E R E E E E E E E 127,487 (11/11) 134,217 (5/5) D1/5 R E E R E R E E R R E 1,274,569 (10/10) 11,687,256 (5/5) C3/2 R E E R E R E E R R E 185,580 (4/4) 4,365,849 (6/6) EA R R E R E R E E R R R 74,944 (9/9) 862,379 (4/4) BE R E E R E R E E E R R 26,923 (4/4) 41,918 (4/4) R R R R R R R R R R R 39 (1/9) 90,828 (9/9) EW E E E E E E E E E E E 13,774,839* (5/5) 28,786,099* (5/5) NSP1 and IFN primarily responsible for host range restriction Feng et al. J Virol. 2013

11 PFU equivalent/g intestinal tissue Replication of wild type EW, and mono VP4 reassortant (D6/2) in mouse intestine 10,000,000,000 1,000,000,000 * ** ** * ** 100,000,000 10,000,000 ** 1,000, ,000 10,000 1,000 EW D6/ Days post infection VP4 very modestly restricts replication in the mouse Feng, N. et al, J Virol,20013

12 Bovine VP4 restriction of RV replication in of STAT1 KO mice Reassortant Rotavirus gene STAT1 KO Proportion of pfu/g viral positive R R R U R R R R R R R 145 (30-2,333) 2/6 UK U U U U U U U U U U U 30 0/5 R R R R R R R R R R R 131,139 (46, ,000) 5/5 Bovine VP4 greatly restricts intestinal replication of in the permissive IFN defficient STAT1 KO mice Feng et al. J Virol. 2013

13 Conclusions Murine RV replication is >10,000X than simian in mouse intestine. NSP1 confers the restricted heterologous replication phenotype. EW NSP1 is necessary but not sufficient for a murine replication phenotype. VP4 efficiently mediates intestinal infection (replication reduced 10X compared to mouse VP4). Bovine VP4 reduces replication 10000X In addition to murine NSP1 and VP4, other proteins (eg.vp3, NSP2/3) are required for the full murine intestinal replication phenotype. What is the mechanism by which NSP1 affects host range in vivo?

14 Category 1-based Enterocyte Hi/Low clusters Categorical clustering of single villous epithelium cells and assessment of IFN transcriptional induction Assay Category: Non- Infected Hi Low RV + Hi Low Hi Infected (CIAs) Bystanders Low Cells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ranscripts Low Ct value Clustering data demonstrated inhibition of IFN induction in RV infected and bystander IECs % High Sen et al, PNAS, 2012

15 Bulk intestinal IFN induction by homologous and heterologous RV infection in suckling mice Relative Expression (2 -DDCt ) NSP5 NSP3 VP6(+) = simian virus EW = murine virus Murine RV replicates efficiently despite substantial IFN induction. Simian RV does not replicate well but also induces a substantial IFN transcriptional response Fold change over uninfected(/gapdh) IFN-α4 ISG20 IFI203 Sen et al, PNAS, 2012

16 RV-mediated early IFN induction occurs primarily in immune (CD45+) cells 10 7 Fold over uninfected (/GAPDH) IFNA4 IFNB1 RSAD2 IP-10 Ifit Epithelial (ESA + CD45 - ) Hematopoietic (ESA - CD45 + ) Stromal (ESA - CD45 - ) Cells sorted into epithelial (#1 = EpCam+CD45-) and hematopoietic (#2 = CD45+EpCam-) Type I IFN is induced primarily in intestinal hematopoietic cells But which CD45+ cells?

17 Relative Viral Load (/GAPDH) Homologous and heterologous RV replication in the intestine are differentially regulated by type I, II, and III IFNs RV gene ~25x ~250x ~20x 10 5 EW IFN receptor/stat-1-mediated signaling substantially restricts simian but not murine RV replication in the intestine The primary restriction of simian (250X) and murine EW(<25X) is due to IFN I and II Type III IFN appears to play a role in restricting but not EW RV Sen, et al; PNAS 2012

18 pfu equivalent/100 mg of fecal samples What is the specific role of IFNλ in regulating homologous and heterologous rotavirus replication in suckling mice 1.E P > E E P < E E E E E Wild type IFNλR KO Wild type, EW IFNλR KO, EW Replication of simian RV is restricted by IFNλ. Murine RV replication is not significantly restricted by IFNλ

19 pfu equivalent per gram of stool Murine RV replication in the intestine of suckling and adult mice 10,000,000,000 1,000,000, ,000,000 10,000,000 1,000, ,000 10,000 1, , Adult STAT1, Adult 129, Pup STAT, Pup Days post infection In adult mice, interferon inhibits murine RV replication more efficiently than in suckling mice

20 Amplification Phase of Interferon Signaling IFN induction IFN signaling Exogenous IFN-α, β, γ, or λ Virus Sensor Y IFN signal IRF3 NF-kB IFN-α/β SB1A STAT1 p(y701) Antiviral State IFN Secretion of type I IFN In HT29 cells: porcine SB1A degrades β-trcp; simian degrades IRF3 Experimental Design HT29 cells infected with or SB1A, stimulated with IFN and stained for RV (VP6+) and py701-stat1 by flow cytometry

21 FITC-VP6 Unstimulated +IFN-β RV regulation of STAT1 activation at 16hpi Mock SB1A Mock - IFN-β + IFN-β Bystanders RV + SB1A Arcsinh ratio APC-pSTAT1 (Y701) and SB1A infected cells effectively inhibit IFN-mediated STAT1-pY701 SB1A also effectively inhibits IFN-mediated STAT1-pY701 in bystander cells Sen et al, J Virol, 2013

22 Acknowledgements Ningguo Feng Adrish Sen Nitya Nair Cathy Cruz Sergie Kotenko Susan Weiss Gourab Mukherjee Xiaosong He Funded by grants from NIH, NIAID and the VA

23 Select examples of IFN responses from intestinal DCs during RV infection Fold over uninfected population (/18SRNA) Key: P = Peyers Patch Key: P = Peyers Patch M= Mesenteric Lymph Node L = Lamina Propria DC+ = WT DC- = BDCA Origin: P P M M L L pdc: Phenotype: SP (CD11b-CD103+) Type I IFN was induced in single positive DCs (CD11b-CD103+) from the PP Across intestinal DC subsets, considerable heterogeneity in innate responses (location and whether pdc-dependent) was observed Fold over uninfected population (/18SRNA) M= Mesenteric Lymph Node L = Lamina Propria DC+ = WT DC- = BDCA Origin: P P M M L L L M M M pdc: Phenotype: SP (CD11b-CD103+) DP (CD11b+CD103+) WT/SP/PP/16hpi BDCA2/SP/PP/16hpi WT/SP/mLN/40hpi BDCA2/SP/mLN/40hpi Dendritic subsets (CD11b/CD103) BDCA2/SP/LP/16hpi WT/DP/LP/16hpi BDCA2/DP/LP/16hpi WT/DP/mLN/40hpi BDCA2/DP/mLN/40hpi BDCA2/DP/mLN/16hpi IFN-a4 Dendritic IFN-b subsets (C NLRP12 ILTIF

24 RV Host Range Restriction/Virulence Background 1) VP3, VP4, VP7, NSP1, NSP4 all associated with regulating RV Virulence/DD50 in the pig gut. 2) VP4 or VP4 and NSP1 associated with regulating RV Virulence and/or Replication in the mouse biliary tract 3) In Vitro studies of Replication in MEFs and mouse cholangiocytes implicated RV VP4 and NSP1 in regulating Replication and this restriction was linked to both RV cell entry and innate immunity mechanisms 4) Limited analysis of genetic or mechanistic basis of host range restriction of Replication in the GI tract What is the basis of host range restriction of rotavirus replication in the GI tract?