Post on 19-Aug-2021
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
n-
Infe
cte
d
RV
+B
ys
tan
de
rs
Infe
cte
d (
CIA
s)
Ce
lls
HighLow
Ct value
Transcripts
Ca
tego
ry 1
-ba
sed
Ente
rocyte
Hi/L
ow c
luste
rs
Hi
Low
Hi
Low
Hi
Low
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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?