Regulation of intestinal viral infection

by the microbiota

Megan Baldridge, MD, PhDDivision of Infectious Diseases, Department of Medicine

Edison Family Center for Genome Sciences & Systems Biology

Washington University School of Medicine, St. Louis, MO

baldridgelab.wustl.edu

Disclosure

Consultant, Orphanon Inc.

Microbiota-virus interactions in

the gut

VirusesBacteria

Infection

InflammationProducts and

metabolites

Overall goal: identify specific actors and mechanisms by which microbiota regulates infection.

Human norovirus

(+)ssRNA, non-enveloped

member of the Caliciviridae

family

Responsible for ~20 million

illnesses per year in the US

and 685 million cases

worldwide

Causes gastroenteritis or

“stomach flu”, with severe

vomiting and diarrhea in

acute phase of infection

Katpally, et al., J. Virol. 2008

Adapted from Saito et al., Clin Infect Dis. 2014

Duration of Norovirus excretion (days)

0 15050 100

GI

GII.4

GII.6

GII.17

Norovirus

genotypes

Variation in responses to

norovirus infection

Patient 1

Patient 2

Patient 3

Weeks after

first sampling

GE Illness

PCR (+)

GE Illness

PCR (+)

GE Illness

PCR (+)

1 2 3 4 5 6 7 8 9 1011121314151617 * 26

Death

Adapted from Siebenga et al. J Infect Dis. 2008

Murine norovirus (MNoV)

Murine norovirus (MNoV) is a tractable model system

Both acute and persistent strains

Nice, et al., J. Virol. 2013Karst & Wobus, et al., Science. 2003

Two Stories

Regulation of persistent MNoV by commensal

bacterial control of permissive cells

Regulation of persistent MNoV by other

persistent elements of the virome via IFN-l

Two Stories

Regulation of persistent MNoV by commensal

bacterial control of permissive cells

Regulation of persistent MNoV by other

persistent elements of the virome via IFN-l

Virus transmitted by fecal-oral

route – regulated by microbiota?

Commensal bacteria facilitate

persistent MNoV infection

Baldridge et al., Science. 2015

Vancomycin

Neomycin

Ampicillin

Metronidazole

(Abx)

-14 0 14

Persistent

MNoV Harvest

Day

Day 14

Microbiota-virus interactions in

the gut

Bacteria

Regulation of

critical cell types?

Persistent MNoV infects rare

intestinal epithelial cells

Lee, et al., Cell Host Microbe, 2017

NS1/2

Restricted expression of MNoV

receptor CD300LF on tuft cells

Orchard, et al., Science, 2016 Wilen et al., Science. 2018

MNoV persistent reservoir is in tuft cells,

which are regulated by microbiota

PBS VNAM

Wilen et al., Science. 2018

Rare chemosensory cells in the epithelial layer

of the intestine expressing taste receptors

Microbiota-virus interactions in

the gut

Bacteria

Regulation of

tuft cells CD300LF

Bacteroides sp. are sufficient to

rescue persistent MNoV

Elizabeth Kennedy & Forrest Walker

Bacteroides sp. regulate succinate

to promote tuft cells

➢ B. ovatus GF mouse

colonization: 11.9-14.2

μmol/g succinate in cecum

➢ B. theta GF colonization:

0.7-2.0

➢ Succinate is a microbial

metabolite, regulated by

protists and helminths,

which promotes tuft cells

via SUCNR1

Faith et al., Sci Trans Med. 2014

Nadjsombati et al., Immunity. 2018

Schneider et al., Cell. 2018

Lei et al., PNAS. 2018

The bacteriome promotes persistent MNoV infection by promoting tuft cells, the reservoir

for persistence, potentially by maintaining levels of metabolite succinate in the gut.

No a

bx

VA o

nly

VA, +

B. o

vatu

s

VA, +

KO

B. o

vatu

s

VA, +

L. murin

us

VA, +

L. muri

nus +s

uccin

ate

3

4

5

6

7

Lo

g10 M

No

V c

op

ies/f

ecal p

ellet

No a

bx

VA o

nly

VA, +

succ

inat

e

VA, +

monom

ethyl

succ

inat

e

4

6

8

Lo

g10 M

No

V c

op

ies/f

ecal p

ellet

Microbiota-virus interactions in

the gut

B. ovatus,

others?

Regulation of

tuft cells CD300LF

Succinate

SUCNR1

Commensal bacteria promote

infection by many enteric viruses

Robinson, Curr Opin Virol. 2019

Two Stories

Regulation of persistent MNoV by commensal

bacterial control of permissive cells

Regulation of persistent MNoV by other

persistent elements of the virome via IFN-l

Harshad Ingle & Sanghyun Lee

Unusual finding in

immunocompromised mice

Rag1-/- – lack B and T cells

Rag2-/-Il2rg-/- – lack B, T, NK

cells, and innate lymphoid

cells

Ingle & Lee et al., Nat Micro. 2019

Wild-type Rag2-/-Il2rg-/- Rag1-/- Rag2-/- Il2rg-/-

+ +

Cohousing

Transfer of resistance with

filtered fecal transplantation

Wild-type

Rag2-/-Il2rg-/- Rag1-/-

Rag1-/-

Filtered through0.2um filter

What are characteristics of

transferable factor?

Only effectively

maintained/active in

immunocompromised host

Smaller than 0.2 microns

Sensitive to heat,

proteinase K, and UV

Transferable factor – murine

astrovirus?

Kang, Hunter's Tropical Medicine and

Emerging Infectious Disease, 2013

Toh, J Virology, 2016

(+)ssRNA, non-enveloped

member of the Astroviridae

family

Novel murine astrovirus

Can we prevent transfer of

resistance by blocking astrovirus?

Rag2-/-Il2rg-/-

Rag1-/-

Purified polyclonal

antibody againstmuAstV capsid

Isotypecontrol

or

FFT

Rag

1-/-

+ PBS

Rag

1-/- +

Rag

2-/- Il2

rg-/- F

FT

Rag

1-/- +

FFT +

Isoty

pe Ab

Rag

1-/- +

FFT +

anti-

muA

stV A

b

2

3

4

5

6

Lo

g10 M

No

V c

op

ies/f

ecal p

ellet ***

***

ns

Ingle & Lee et al., Nat Micro. 2019

Viral interference via IFN?

What is driving antiviral activity in Rag2-/-Il2rg-/- mice?

Lazear, et al., Immunity, 2015

IFN-λ is upregulated in naïve

immunocompromised mice

IFN-λ is responsible for resistance

If we cure astrovirus, do mice

become sensitive to norovirus?

Wild-type

Rag2-/-Il2rg-/-

bulk splenocytes

14 days MNoVinfection

STL5 MNoVIfnl2/3

Ingle & Lee et al., Nat Micro. 2019

Microbiota-virus interactions in

the gut

B. ovatus,

others?

Regulation of

tuft cells CD300LF

Succinate

SUCNR1

Astrovirus

IFN-λ

T cells?

Next steps

Antibiotics treated and germ-free mice are tractable for

mechanistic studies of the influence of microbiota

factors on enteric viral infections

Permissive cells and innate immunity

Adaptive immune responses to vaccines

Next stages: colonization of germ-free mice with human

microbiota samples, administration of factors to enteroid

cultures, high-throughput CRISPR screening to identify

host factors of import

Baldridge Lab

Lara Crock

Hongju Deng

Bria Dunlap

Ebrahim Hassan

Mike Hogarty

Harshad Ingle

Elizabeth Kennedy

Andrew Kim

Dylan Lawrence

Sanghyun Lee

Efia Nuako

Rachel Rodgers

Larry Schriefer

Meagan Sullender

Forrest Walker

Alumni

Stefan Peterson

Acknowledgments

CGSSB

Bonnie Dee

Kathleen Matheny

Keith Page

Virgin Lab

Rob Orchard (UTSW)

Tim Nice (OHSU)

Craig Wilen (Yale)

Darren Kreamalmeyer

Jonathan Miner

Stacey Schultz-Cherry