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

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

RRV (simian RV )EW (murine RV )

PFU

eq

uiv

ale

nt/

g in

test

inal

tis

sue

Days post infection

Replication of murine and simian RV in the suckling mouse intestineTHE REPLICAION DIFFERENCE IS SUBSTATIAL (>104)!!

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

1,000,000,000

10,000,000,000

2 4 6 8 10 12 14

EW

RRV

RV Infected enterocytes

First Host Range Restricted Rotavirus Vaccine (RotashieldTM)A quadravalant set of Simian x Human monoreassortants

“A Modified Jennerian Vaccine” att: AZ Kapikian

VP4

Neutralizationantigen

(SIMIAN)

VP6 GroupAntigen

(SIMIAN)

VP7Neutralization

antigen(HUMAN)

Figure courtesy MK Estes

MODIFIED THE Simian x Human Rotavirus Vaccine Was Attenuated In Children

RotaTeq- a second “Modified Jennerian Vaccine”

G3G2

G1

G4P[8]

The Bovine-Human Reassortant Vaccine is Also Attenuated in Children

The Merck Pentavenlant Reassortant Vaccine

Quantities of rotavirus (-)RNA and antigen in suckling mice innoculated with EC or RRV rotavirus

sample inoculum

Feces EC

RRV

Small Int. EC

RRV

MLN EC

RRV

Blood EC

RRV

Liver EC

RRV

Lung EC

RRV

Kidney EC

RRV

* 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 7 dpi

NA= not available

Rotavirus (-)RNA

Quantities of rotavirus (-)RNA and antigen in suckling mice innoculated with EC or RRV rotavirus

Geometric mean*

6.8 (+0.6)

4.5 (+1.0)

5.0 (+1.0)

0.3 (+0.8)

3.9 (+1.7)

3.0 (+ 1.9)

1.0 (+1.1)

0.4 (+0.8)

2.2 (+1.0)

0.3 (+0.8)

0.9 (+0.9)

1.6 (+2.0)

1.8 (+1.2)

0

* 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 7 dpi

Rotavirus (-)RNA Rotavirus antigen

Quantities of rotavirus (-)RNA and antigen in suckling mice innoculated with EC or RRV rotavirus

Geometric mean**

6.2 (+0.9)

1.3 (+2.3)

4.7 (+1.8)

1.2 (+2.4)

3.0 (+2.5)

2.4 (+3.3)

1.6 (+2.2)

0.7 (+1.8)

0.3 (+1.0)

2.1(+2.4)

0

1.7 (+2.7)

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 7 dpi

Rotavirus antigen

Host Range Restriction of Local vs. Systemic Infection in Mice

EC = Murine RV; RRV = simian RV

Fenaux, M. et al, J Virol; 2006 Enteric HRR is not Present at Systemic Sites

RRV (simian)UK (bovine)

Rotavirus replication in the epithelial cells of murine biliary

tract is viral strain specific but not host range restricted

EW (murine)

RRV

VP6, K19, Nucleus

Feng et al. J Virol 2010

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?

Rotavirus Replication and the Role of Interferons in Regulating Growth in Mouse Embryo Fibroblasts (MEFs)

• IFN signaling restricts bovine and porcine RV replication

• Murine and simian RRV replication is not restricted by IFN

• Lack of restriction based on the ability of the virus to degrade IRF3 or NFkB

(data not shown)

Heterologous Homologous RRV

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

Bovine Porcine Murine Simian

129sv

IFNR KO

STAT1 KO

Feng N. et al, J Virol,2009

Reassortants Rotavirus Genes Wild type STAT1 KO

1 2 3 4 5 6

7

(nsp3)

8

(nsp2)

9

(VP7) 10 11 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 11-1-3 R R E R E R E R R R R 253 (6/9) 44,344 (4/4)

DEA 24-2-1 R R E R E R R R R R R 6,162 (6/6) 39,719 (4/4)

DEA 3-1-2 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 4-1-2 R R E R E R E E R R R 74,944 (9/9) 862,379 (4/4)

BE 1-1-3 R E E R E R E E E R R 26,923 (4/4) 41,918 (4/4)

RRV 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)

Intestinal replication of mouse x simian reassortants in Wt / STAT1 KO mice

Feng et al. J Virol. 2013NSP1 and IFN primarily responsible for host range restriction

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

1,000,000,000

10,000,000,000

0 2 4 6 8 10 12 14 16

EW

D6/2

RRV

**

*

**

PF

U e

qu

iva

len

t/g

in

testin

al tissu

e

Days post infection

Replication of wild type EW, RRV and mono RRV VP4

reassortant (D6/2) in mouse intestine

*

**

**

Feng, N. et al, J Virol,20013RRV VP4 very modestly restricts replication in the mouse

Bovine VP4 restriction of RV replication in of STAT1 KO mice

Bovine VP4 greatly restricts intestinal replication

of RRV in the permissive IFN defficient STAT1 KO

mice

Reassortant Rotavirus gene STAT1 KO Proportion of

1 2 3 4 5 6 7 8 9 10 11 pfu/g viral positive

19-1-2 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

RRV R R R R R R R R R R R 131,139 (46,667 - 500,000) 5/5

Feng et al. J Virol. 2013

Murine RV replication is >10,000X than simian RRV in mouse intestine.

RRV NSP1 confers the restricted heterologous replication phenotype.

EW NSP1 is necessary but not sufficient for a murine replication phenotype.

RRV 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?

Conclusions

Assay Category: 1 2 3 4 5 6 7 8

No

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Infe

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RV

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Infe

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Transcripts

Ca

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Hi

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Clustering data demonstrated inhibition of IFN induction in RV infected and bystander IECs

Categorical clustering of single villous epithelium cells and assessment of IFN transcriptional induction

Sen et al, PNAS, 2012

Bulk intestinal IFN induction by homologous and heterologous RV infection in suckling mice

RRV = simian virus

EW = murine virus

NSP5

NSP3

VP6(

+)10-1

101

103

105

107

Rela

tive E

xpre

ssio

n (2

- DD

Ct )

IFN-α

4

ISG20

IFI2

03

100

101

102

103

104

Fold

change o

ver

unin

fecte

d(/

gapdh)

Murine RV replicates efficiently despite substantial IFN induction.

Simian RV does not replicate well but also induces a substantial

IFN transcriptional response

Sen et al, PNAS, 2012

• 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?

RV-mediated early IFN induction occurs primarily in immune (CD45+) cells

100

101

102

103

104

105

106

107

Fold

over unin

fecte

d (/G

AP

DH

)

IFNA4

IFNB1

RSAD2

IP-10

Ifit3

Epithelial

(ESA+CD45-)

Hematopoietic

(ESA-CD45+)

Stromal

(ESA-CD45-)

1

2

3

Homologous and heterologous RV replication in the intestine are differentially regulated by type I, II, and III IFNs

IFN receptor/STAT-1-mediated signaling substantially restricts simian but

not murine RV replication in the intestine

The primary restriction of simian RRV (250X) and murine EW(<25X) is due to

IFN I and II

Type III IFN appears to play a role in restricting RRV but not EW RV

105

106

107

108

109

1010

1011

~25x

~250x

~20x

Re

lati

ve V

iral

Lo

ad (

/GA

PD

H)

EW RRV

RV gene 11

Sen, et al; PNAS 2012

pfu

eq

uiv

alen

t/1

00

mg

of

feca

l sam

ple

s

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

1.E+11

Wild type RRV IFNλR KO RRV Wild type, EW IFNλR KO, EW

1011

1010

109

108

107

106

105

104

Replication of simian RV is restricted by IFNλ . Murine RV replication is not significantly restricted by IFNλ

P > 0.05

P < 0.05

What is the specific role of IFNλ in regulating homologous and heterologous rotavirus replication in suckling mice

1

10

100

1,000

10,000

100,000

1,000,000

10,000,000

100,000,000

1,000,000,000

10,000,000,000

3 6 9 12

129, Adult

STAT1, Adult

129, Pup

STAT, Pup

Murine RV replication in the intestine of suckling and adult micep

fue

qu

ival

en

t p

er

gram

of

sto

ol

Days post infection

In adult mice, interferon inhibits murine RV replication more efficiently than in suckling mice

Amplification Phase of Interferon Signaling

In HT29 cells: porcine SB1A degrades β-TrCP; simian RRV degrades IRF3

Experimental Design

HT29 cells infected with RRV or SB1A, stimulated with IFN and

stained for RV (VP6+) and pY701-STAT1 by flow cytometry

Sensor

Virus

IRF3 NF-kB

IFN-α/β

IFN signal

STAT1

p(Y701)

Antiviral State

Secretion of type I IFN

IFN

Y

IFN induction IFN signaling

RRVSB1A

ExogenousIFN-α,β,γ,orλ

+IFN

-β

APC-pSTAT1(Y701)

FITC

-VP6

Un

stim

ula

ted

• RRV and SB1A infected cells effectively inhibit IFN-mediated STAT1-pY701

• SB1A also effectively inhibits IFN-mediated STAT1-pY701 in bystander cells

RV regulation of STAT1 activation at 16hpi

Mock

RV+ Bystanders

- IFN-β

+ IFN-β

RRV

SB1A

Arcsinh ratio

Mock RRV SB1A

Sen et al, J Virol, 2013

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

Select examples of IFN responses from intestinal DCs during RV infection

• 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

SP(CD11b-CD103+) DP(CD11b+CD103+)

P M LMP L L M M MpDC: + - + - - + - + - -

Origin:

Phenotype:

Key:P=PeyersPatchM=MesentericLymphNodeL=LaminaPropriaDC+=WTDC-=BDCA

WT/SP/PP/16

hpi

BDCA2/SP/PP/16

hpi

WT/SP/m

LN/40

hpi

BDCA2/SP/m

LN/40

hpi

BDCA2/SP/LP

/16hp

i

WT/DP/LP

/16hp

i

BDCA2/DP/LP

/16hp

i

WT/DP/m

LN/40

hpi

BDCA2/DP/m

LN/40

hpi

BDCA2/DP/m

LN/16

hpi

10-1

100

101

102

104

106

Fo

ld o

ver u

nin

fect

ed p

op

ula

tion

(/18

SR

NA

)

Dendritic subsets (CD11b/CD103)

IFN-b

NLRP12

IFN-a4

ILTIF

SP(CD11b-CD103+) DP(CD11b+CD103+)

P M LMP L L M M MpDC: + - + - - + - + - -

Origin:

Phenotype:

Key:P=PeyersPatchM=MesentericLymphNodeL=LaminaPropriaDC+=WTDC-=BDCA

WT/S

P/PP

/16hp

i

BDCA2/SP/PP

/16hp

i

WT/S

P/mLN

/40hp

i

BDCA2/SP/mLN

/40hp

i

BDCA2/SP/

LP/16

hpi

WT/D

P/LP

/16hp

i

BDCA2/DP/LP

/16hp

i

WT/D

P/mLN

/40hp

i

BDCA2/DP/mLN

/40hp

i

BDCA2/DP/mLN

/16hp

i

10-1

100

101

102

104

106

Fo

ld o

ve

r u

nin

fec

ted

po

pu

lati

on

(/1

8S

RN

A)

Dendritic subsets (CD11b/CD103)

IFN-b

NLRP12

IFN-a4

ILTIF

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?