using wild mice to tackle genetic control of resistance to ... · controlling resistance to...

Using wild mice to tackle genetic control of resistance to infectious diseases (West-Nile, Plague, Rift Valley Fever)

Xavier Montagutelli, DVM, PhD

Fondation Mérieux Oct 12, 2011

Outline

3 – Analyzing wild-derived mouse strains can be key to success (3 examples).

1 – Two complementary approaches to identify genes controlling resistance to infectious diseases relevant for the human and animal species.

2 – Classical laboratory inbred strains lack genetic and phenotypic variation.

4 – The Collaborative Cross as the genetic reference population for this decade (and more…).

Reverse genetic approach

Genetic difference Phenotypic difference

resistant Random mutations

Targeted mutations

susceptible

Identify mutation

Bruce Beutler Nobel 2011

Forward genetic approach

Phenotypic difference Genetic difference

resistant

susceptible Causal polymorphism

C R

T S

+ genotyping

Forward genetic approach

Vidal et al. 2008 Ann. Rev. Immunol. 26:81:132

Origin of laboratory mice

M.Y.

0

0.2

3

0.3

5

M.m. musculus

M.m. castaneus

M.m. domesticus

M.m. molossinus

92%

7%

1%

B6

Yang et al., Nat. Genet., 2007

Laboratory strains

0

10

20

30

40

50

60

70

80

90

100

Days after inoculation

7 8 9 10 11 12 13 14 15 0 6

% s

urv

ivors

BALB/c C57BL/6

West Nile virus

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9 10 11 12 13 14

Days after inoculation

% s

urv

ivors

Yersinia pestis

B6/J

BALB/CByJ

C3H/HeN

DBA/2J

FVB/N

Wild-derived inbred strains

M.m. musculus

M.m. castaneus

M.m. domesticus

M. spretus

M.Y.

0

0.2

3

0.3

5

1.1

0

M.m. molossinus

WLA/Pas, WMP/Pas WSB/Ei

MBT/Pas, PWK/Pas, PWD/Ph

MOLF/Ei

CAST/Ei

SEG/Pas, STF/Pas, SPRET/Ei

Flaviruses are positive-sense, single-stranded RNA viruses

Phylogenetic relationships of flaviviruses

Meningo-encephalitis syndromes

West Nile virus

2003

• 515 annual cases in USA (2009) CDC, Atlanta. 278 (54%) West Nile meningitis or encephalitis (neuroinvasive disease), 222 (43%) West Nile fever (milder disease)

WN virus spreading in the USA

1999

2001

Dead-end hosts

Mosquito vectors Culex

Transovarian sexual routes

Vertebrate reservoirs

WN virus cycle(s)

Mild illness (West Nile fever) ~ 20% among infected individuals

Clinical severity < 1% of infected individuals develop severe neurological disease

Case-fatality rate ~ 5-20 % among hospitalized patients during 2002-2003 outbreaks ~ 10% among patients with meningo-encephalitis

This suggests the existence of genetics factors controlling the susceptibility of the host

WN Neuroinvasive Disease (WNND)

BC1

F1

pare

nta

l

Dominant resistance allele

WNV susceptibility in mice

Mashimo et al., PNAS, 2002

203 [(MBT/Pas x BALB/c)F1 x BALB/c]BC1 progeny

The mapping was done using only the 74 BC1 mice that died.

MBT/BALB

BALB/BALB

2.7 cM, 5.2 Mb

Mapping of WNV resistance gene

The critical interval was further refined to 0.4 cM by genotyping additional recombinants


20 genes /ESTs :

D5Mit408 D5Mit242

FLN29, gene with ligase activity Oligo-adenylate synthetase genes: Rbm19, RNA binding motif Oas 1a to Oas1h, eight genes RPL6, ribosomal protein L6 Oas2, Oas3 PTN11,Tyrosine phosphatase Deltex Rabphilin 3A SDS, Serine dehydratase Rasal1, RAS protein activator like 1 LHX5, LIM homeobox protein Tpcn1, calcium ion transport

Gene map of the critical region


BALB/c

Stop codon

Laboratory mice (susceptible)

129/Sv

DDK/Pas

C3H/HeJ

DBA/2J

GGGCTTCTGAACCGT

GGGCTTCTGAACCGT

GGGCTTCTGAACCGT

GGGCTTCTGAACCGT

NZW/Ola

NZB/Ola

C57BL/6 GGGCTTCTGAACCGT

Mus m domesticus

MBT/Pas

Arginine

GGGCTTCCGAACCGTC

Wild mice (resistant)

MAI/Pas

WMP/Pas

PWK/Pas

GGGCTTCCGAACCGTC

SEG/Pas

STF/Pas

Mus m musculus

Mus spretus

PL/J

Laboratory mice (resistant)

GGGCTTCTGAACCGT

Non-sense mutation in Oas1b gene


GGGCTTCTGAACCGT

GGGCTTCTGAACCGT

GGGCTTCCGAACCGTC

GGGCTTCCGAACCGTC

GGGCTTCCGAACCGTC

GGGCTTCCGAACCGTC

GGGCTTCCGAACCGTC

CAG Oas1b cDNA pA SV40

CGA

CMV/chicken b-actin promoter BALB/c

genetic background

Transgenic complementation

Survival curve

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

day

+/+

tg/+

Perc

ent

surv

ival

Days after infection

BALB/c Tg/+

BALB/c +/+

***

Simon et al. 2011 Virology, in press

Relevance for other species

Yersinia pestis and plague

Yersinia pestis

• Gram-negative, enterobacteria

• infects wild rodents (>200 species)

Alexandre Yersin in front of his "laboratory"

Alexandre Yersin (1863-1943) identified the bacteria responsible for plague, during the Hong-Kong epidemics (1894)

Paul-Louis Simond (1858-1947)

demonstrated the transmission of the

bacteria by rat fleas (1897)

• transmitted through flea bites

• causes plague epidemics in humans

The plague disease

Bubonic form

• inflammation of the lymph node draining the bite site: "bubo"

• intense replication of bacteria

• evolves to suppuration and dissemination to other lymph nodes

Septicaemic form

• following massive or IV infection

• usual signs of septicaemia with septic shock

Pneumonic form

• the most dangerous and fatal form

• secondary to septicaemic or by inhalation

• fulminant haemorrhagic pneumonia

Human plague

Black death

• spread from Asia to Europe ~1350

• 20 M people died (1/3 of Europe)

• Paris, Venice, etc… : > 50% died

• 200 M in human history

• >2,000 cases reported each year

(99% in Africa) – 200 deaths

Plague epidemics

• coincide with foci of infected rodents

Y. pestis : evidence for host genetic control

"Not all of them died, but all were struck"

Cats, rats, mice, rabbits, camels, turkeys, and monkeys are highly susceptible to plague.

Dogs and cows are resistant.

In humans, not all exposed individuals become sick or die.

Mouse models of plague

• Laboratory strains are highly susceptible to fully virulent Y. pestis infection.

• Differences in susceptibility can be observed only when using Y.p. strains lacking virulence factors.

SEG/Pas mice are exceptionnaly resistant to Y.p.

100 cfu, strain C092, s.c., 12-15 wks

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9 10 11 12 13 14

days post-infection

% s

urv

ivors

B6/J (n=16)

SEG/Pas (n=16)

BALB/CByJ (n=11)

C3H/HeN (n=12)

DBA/2J (n=12)

FVB/N (n=12)

CAST/EiJ (n=10)

Blanchet et al., Genes and Immunity, 2010

QTL mapping of resistance loci

Y.p. challenge : 100 cfu, strain C092, s.c., 12 wks

SEG male B6 female

F1 female B6 male

>300 backcross females

High-density SNP genotyping (>700 markers)

BSB : (B6 x SEG)F1 x B6

Three QTLs increase survival by 20%

57 Mb

Chr 3

0

20

40

60

80

100

2 4 6 8 10 12 14

% s

urv

ivors

B/B (n=142)

B/S (n=179)

Chr 3 : Yprl1

p = 0.00024

Days post-infection

2 4 6 8 10 12 14

B/B (n=146)

B/S (n=176)

0

20

40

60

80

100

p = 0.00026

Days post-infection

% s

urv

ivors

Chr 4 : Yprl2

p = 0.00026

2 4 6 8 10 12 14

Days post-infection

0

20

40

60

80

100

% s

urv

ivors

B/B (n=148)

B/S (n=174)

Chr 6 : Yprl3

69 Mb 87 Mb

Chr 4 Chr 6

Blanchet et al., Genes and Immunity, 2010

Y.p. resistance in SEG : a model

0

10

20

30

40

50

60

70

80

90

100

Surv

ival (%

)

B6 SEG 1 QTL

2 QTLs

3 QTLs

??

Y.p. resistance in congenic mice

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Days post infection

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Surv

ival ra

te

B6

B6 + Chr 4+6

Zoonosis in Africa

Spread to Yemen and Saudi Arabia in 2000

Infectious disease due to an enveloped virus

transmitted mainly by mosquitoes

(Phlebovirus) Isolation, 1930

1987, 1998

1977, 1993 2000

1990-1991 2006-2007

1951, 1975

1997,-1998

Human and animal health problem

No safe vaccine for protection nor antiviral agents

for therapy

Arbovirus transmitted by many species of

mosquitoes

Potential bioterrorism agent

L

M

S

Gn

Gc

RNP

L

90 à 100 nm Aedes

Culex

5’

5’

5’

L: RNA-dependent RNApolymerase

3’ Segment L (6404 nt)

3’

Gc Gn NSm

Segment M ( 3885 nt)

3’ NSs

N

Segment S (1690 nt)

Rift Valley fever virus

Reasons to study RVF virus

The different outcomes both in animals and humans suggest the existence of genetic factors controlling the susceptibility of the host

Hepatic syndrome, vasculitis and necrosis of the liver due to RVF

Macroscopically foetuses often show

haemorrhages and haemothorax

RVF in animals

Abortions

High fatalities in young animals

Necrotic hepatitis with or without

haemorrhagic state

Indigen breeds of African cattle and sheep are resistant

RVF in humans

Usually uncomplicated acute febrile illness but

o hepatocellular failure o acute renal failure o hemorrhagic manifestations o meningoencephalitis and retinitis o death associated with hepatorenal failure, shock, severa anemia

occur in 1% of humans that become infected with Rift valley fever virus

Rift Valley fever virus-induced diseases

♀ BALB/c

late susceptible

♀ MBT/Pas

early susceptible

♀ C57BL/6

intermediate

Rift Valley fever virus in mice

Survival of various inbred strains of mice following infection by 102 PFU RVF ZH548 i.p.

BALB/cByJ

100 PFU Rift Valley fever virus

D0

MBT/Pas

100 PFU Rift Valley fever virus

D3

4.2 ± 0.5

7.7±0.3

D1

3.1±0.3

0

D4

All mice were already dead

All mice were still alive

Viral load in BALB/c and MBT infected mice

log(PFU/ml)

log(PFU/ml)

Earlier and higher viral production in MBT mice than in BALB/c mice.

MBT cells produce higher virus titers than BALB/c cells, at various MOI,

suggesting that BALB/c cells are able to limit viral production.

6

7

8

9

log P

FU

/10

6 c

ells

15 hours post-infection

MOI 1 MOI 5 MOI 10 MOI 1 MOI 5 MOI 10

20 hours post-infection

***

***

** ***

***

**

BALB/c and MBT-derived MEFs infected at MOI of 1, 5 and 10

BALB/cByJ

MBT/Pas

Viral production by MEFs

Zaverucha do Valle et al., J. Immunol., 2010

Infection differentially regulates more cellular genes in MBT cells than in BALB/c cells.

9 hours post-infection BALB/cByJ MBT/Pas

700

600

500

400

300

200

100

0

100

200

300

1 2

Num

ber

of diffe

rentially e

xpre

ssed g

enes

Upregulated

Downregulated

152

77

614

205

Differentially regulated genes (twofold cutoff)

229 0.82 %

819 2.9 %

Number of genes : % cellular transcripts :

27,000 unique transcripts

Micro-array analysis in MEFs


229 unique genes

differentially regulated

following infection with

Rift Valley fever virus

87 genes are highly upregulated after infection in BALB/cByJ cells, but moderately or not upregulated in MBT/Pas cells



Genes highly upregulated in MEFs from BALB/c, but not from MBT/Pas mice

………………………………………………. Interferon stimulated gene 20





………………………………………………. Interferon induced gene 44

………………………………………………. Interferon induced gene 35

……………………………………… Member of the P200 family




…………………………………… ……………. p47 GTPase family

…………………………………… ……………. p47 GTPase family

………………………………………………………………………………………………………………………. MDA-5 RNA helicase

………………………………………………………………………………………………………………………………….. RIG-I RNA helicase

…………………………………………………………………………………………… dsRNA-dependent protein kinase PKR

………………………………………………………………………………………..…………………cytosolic DNA sensor DAI/ZBP1 …………………………………………………………………… Oligoadenylate synthetase (OASl1)

…………………………………………………………………… Oligoadenylate synthetase (OASl2)

…………………………………………………………………… Oligoadenylate synthetase (OAS1a)

…………………………………………………………………………………..Chemokine

…………………………………………………………………………………..Cytokine

………………………………………………………………………………. Interferon regulatory factor 9 (IRF9)

………………………………………………………………………………. Interferon regulatory factor 7 (IRF7)

……………………………………………………………………………………………………..STAT1

……………………………………………………………………………………………………..STAT2



BALB/c cells display a strong innate immune response to infection with the RVFV

Upregulated in BALB/cByJ

Unchanged in BALB/cByJ

Not present in the microarray

NSs NSs viral protein

IFNαs

IFNβ

5’ triphosphate

ssRNA dsDNA dsRNA

IRF3 IRF3 IFNβ

IFNβ

ISG

TBK-1

MAVS

eIF2α

IKKα/β IKKε

NFκB

TRAF

IFNAR1 IFNAR2

ISG IFNα

IFNβ

JAK1 TYK2

ISRE

IRF3

SOCS1

PIAS1

dsRNA

Early signaling events Late signaling events

NSs

NSs NSs

NSs

NSs

NSs

ISG15

IFIT2

MDA5 LGP2

IFIT1

RIG-I

OASL1

IFI204

IFIT3

IIGP1

MDA-5

IP-10

ISG12

OAS1a

IFI202b

IFI203

IFI35

ISG20

LGP2

OASL2

PKR

GVIN1

IFI205

IIGP2

DAI

IFI44

RIG-I DAI

TRIM25

PKR

STAT1 STAT2

IRF9

IRF7

ISGF3

IFN

IFN

Type I interferon antiviral innate response

Gene activation in BALB/c

5’ triphosphate

ssRNA dsDNA dsRNA

IRF3 IRF3 IFNβ

IFNβ

ISG

TBK-1

MAVS

eIF2α

IKKα/β IKKε

NFκB

TRAF

IFNAR1 IFNAR2

ISG IFNα

IFNβ

JAK1 TYK2

ISRE

IRF3

SOCS1

PIAS1

dsRNA

Early signaling events Late signaling events

NSs

NSs NSs

NSs

NSs

NSs

Upregulated in BALB/cByJ and MBT/Pas

Unchanged in BALB/cByJ and MBT/Pas

Not present in the microarray

Upregulated in BALB/cByJ, but unchanged in MBT/Pas

ISG15

IFIT2

MDA5 LGP2

IFIT1

RIG-I

OASL1

IFI204

IFIT3

IIGP1

MDA-5

IP-10

ISG12

OAS1a

IFI202b

IFI203

IFI35

ISG20

LGP2

OASL2

PKR

GVIN1

IFI205

IIGP2

DAI

IFI44

NSs NSs viral protein

IFNαs

IFNβ

RIG-I DAI

TRIM25

PKR

STAT1 STAT2

IRF9

IRF7

ISGF3

IFN

IFN

Weak gene activation in MBT

Type I interferon antiviral innate response

MBT cells display a weak innate immune response to infection with the RVFV

Kinetic study by gRT-PCR

Group 1 Late higher expression in MBT/Pas cells

**

*

Ifit3

0 5

10 15 20 25 30 35 40 45 50

0 3 6 9 Hours after infection

Rela

tive e

xpre

ssio

n

*

Ifna4

Hours after infection

* Ifnb1

0

200

400

600

800

1000

1200


BALB/cByJ

MBT/Pas

0

5

10

15

20

25

30

0 3 6 9

IFN-4 IFN-

Expressed in a Stat2- dependent manner

Group 2 Delayed induction in MBT/Pas cells

*

Ifit1

0

10

20

30

40

50

60

70


BALB/cByJ

MBT/Pas

***

***

Rig-I

0

1

2

3

4

5

6

7


*

*

Stat2

0

2

4

6

8

10

12

14

16


Rela

tive e

xpre

ssio

n

Direct target of IRF3 Known sensor of RVFV dsRNA IFNAR activation pathway

Group 3 Low or no induction in MBT/Pas cells

*** *** Isg15

Hours after infection

***

* *

*** Oasl2

0

5

10

15

20

25

30

35

40


BALB/cByJ

MBT/Pas

*** ***

***

*** Irf7

0

1

2

3

4

5

6

7

8


Rela

tive e

xpre

ssio

n

0

2

4

6

8

10

12

0 3 6 9

Required for IFN-4 expression Ubiquitin-like protein that modifies > 150 proteins by ISGylation

Oligoadenylate synthetase

Gene expression study by RT-PCR

Do the genes whose expression in MBT/Pas cells is low or delayed play a role in limiting viral production ?

• Inhibition of RVFV-induced genes by specific siRNA

• Effects on the inhibition on viral production

Isg15 and Oasl2 play functional role in the inhibition of viral production

Irf7

0

20

40

60

80

100

120

mR

NA

rela

tive

expre

ssio

n (

%)

**

*

Mock

-inf

RVFV

Inf. siR1 siR2 siR3

Isg15

0

20

40

60

80

100

120

*** *** *** ***

Mock

-inf

RVFV

Inf. siR1 siR2 siR3

Rig-I

0

20

40

60

80

100

120

***

*** ***

***

Mock

-inf

RVFV

Inf. siR1 siR2 siR3

Oasl2

0

20

40

60

80

100

120

*

**

mR

NA

rela

tive

expre

ssio

n (

%)

Mock

-inf

RVFV

Inf. siR1 siR2 siR3

7,8

7,9

8,0

8,1

8,2

8,3

Scrambled

siRNA Irf7 Isg15 Oasl2 Rig-I

log

10 P

FU

/ 10

6 c

ells

***

*

Gene knock-down experiments


0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8

Days following infection

Surv

ivin

g m

ice

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8

♀ BALB/c

♂ MBT/Pas

♀ MBT/Pas

♂ BALB/c

♂ (BALB/c x MBT/Pas)F2

♀ (BALB/c x MBT/Pas)F2

F2 mice are intermediate between MBT/Pas and BALB/c mice

Mapping resistance loci

576 F2 mice were challenged

with 102 PFU RVF virus.

Genotyping with 315 polymorphic

markers, 240 SNPs and 75

microsatellites distributed along

the whole genome.

Mapping resistance loci

c/c c/m m/m

Genotype at 168.21

6

5

5.5

4.5

Chr. 2

c/c c/m m/m

Genotype at D5Mit168

6

5

5.5

4.5

Chr. 5

c/c c/m m/m

Genotype at D11Mit214

5

5.5

4.5

Chr. 11

QTL mapping

Chr.2 Chr.5 Chr.11

Three QTLs affect time to death

Do differentially regulated genes in BALB/c and MBT/Pas MEFs map to the QTLs on chromosome 2, 5 and 11 ?

Chr 11 Oasl2

Chr 2

QTL mapping and gene expression

Towards an ideal mouse population ?

Inbred strains provide information dependent on their genotype.

Outbred stocks are not a valuable alternative (lack of genetic variation, irreproducibility, poor genetic characterization).

Testing several inbred strains is an optimal design but common lab strains are closely related.

??

Towards an ideal mouse population ?

Include genetic variation present across M. musculus subspecies in a novel genetic reference population with ideal features :

• Maximize genetic variation across a collection of strains

• Optimize genetic resolution to facilitate gene identification

• Balanced contribution from each progenitor

• Detailed characterization of the genotype of each strain

M.Y.

0

0.2

3

0.3

5

M.m. musculus

M.m. castaneus

M.m. domesticus

M.m. molossinus

Collaborative Cross design 129S1/S

vIm

J

A/J

NO

D/L

tJ

C57BL/6

J

NZO

/H1LtJ

CAST/E

i

WSB/E

iJ

PW

D/P

hJ

Mitochondrial DNA

Inbred Collaborative Cross strain

Developed at : - University of North Carolina (Chapel Hill), - Tel-Aviv University, - Perth (Australia).

300-400 strains at the completion of the program.

First 70 strains to be released beginning of 2012.

These 8 strains capture 89% of the mouse genetic variation (2x that observed across human populations).

Collaborative Cross

PWK/PhJ NOD/LtJ C57BL/6J A/J

129S1/SvImJ NZO/H1LtJ CAST/EiJ WSB/EiJ

Data on 184 lines

Collaborative Cross : first data

Collaborative Cross

European Cooperation in the field of Scientific and Technical Research

www.cost.esf.org

SYSGENET: European systems genetics network for the study of complex genetic human diseases using mouse genetic reference populations

Action BM0901 2009 - 2013

Participating countries: AT, CH, CZ, DE, DK, EE, ES, FI, FR, GR, IL, IT, LU, NL, PL, UK Chair: Klaus Schughart, DE, [email protected]

Working Groups :

1) GRP Resources

2) Phenotyping and genotyping

3) Bioinformatics

4) International outreach

5) Training and mobility

Plans to import at least 75 strains in Europe. To be distributed by TAU.

Conclusions

Wild-derived inbred strains provide much larger genetic and phenotypic variation than laboratory strains.

There is tremendous variation across mouse strains in susceptibility to infectious diseases.

These natural variations are often controlled by multiple genes with moderate effects.

The Collaborative Cross will provide unprecedented opportunity to discover new mouse models, in all biological fields.

Acknowledgements

Mouse functional genetics unit

Laurent Guillemot

Isabelle Lanctin

Charlène Blanchet

Lucie Chevallier

Tania Zaverucha do Valle

Dominique Simon

Jean Jaubert

Jean-Jacques Panthier

Yersinia unit

Christian Demeure

Elisabeth Carniel

Molecular Genetics of Bunyaviruses unit

Agnès Billecocq

Michèle Bouloy

Molecular Interactions Flaviviruses-Host

Marie-Pascale Frenkel

Philippe Desprès

Klaus Schughart

Rudi Alberts

using wild mice to tackle genetic control of resistance to ... · controlling resistance to...

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