Gastroenteritis Viruses Prof. Mary K. Estes · Gastroenteritis Viruses Prof. Mary K. Estes The screen versions of these slides have full details of copyright and acknowledgements

Gastroenteritis Viruses

Prof. Mary K. Estes

The screen versions of these slides have full details of copyright and acknowledgements 1

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Gastroenteritis VirusesGastroenteritis VirusesGastroenteritis VirusesGastroenteritis Viruses

Mary K. Estes, Ph.D.Professor of Molecular Virology and Microbiology

and Medicine-GI, BCM

Director, Texas Medical Center DDC

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Outline

• Gastrointestinal viruses

–Rotaviruses

–Noroviruses

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• The amount of diarrheal water

equals the amount of water

over Victoria Falls in 1 min

• Diarrhea is caused

by many infectious agents,

including viruses

• It has been estimated

that in any given 24 hr period,

200 million people on Earth

have gastroenteritis

Diarrhea is a health problem worldwide


Prof. Mary K. Estes


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Viruses causing diarrheamajor pathogens in humans

• Rotaviruses

• Norwalk virus/Norovirus (Calicivirus)

• Sapovirus (Calicivirus)

• Astroviruses

• Adenoviruses (serotypes 40 and 41)

• Aichi virus

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Rotavirus

Dehydrating diarrhea

in young children

Viral gastroenteritis

Almost every child

(< 5 years) gets infected

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Epidemiology

World wide infection and disease

111 million (M) episodes of gastroenteritis

25 M clinic visits, 2 M hospitalizations

~ 440,000 deaths in children < 5 years

(Parashar et al., 2003, Emerg Infect Dis 9: 565-72)


Prof. Mary K. Estes


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Rotavirus pathogenesis

• Disease is age-restricted:

– Virus replication in all ages

– Generally asymptomatic infection in adults

– Symptomatic infection occurs

in the young children 3 months to 2 years of age;

< 2 week old mice and rats

• Main tissue tropism – enterocytes

• Diarrhea caused by multiple factors:

– Malabsorption

– NSP4 enterotoxin

– Enteric nervous system

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Rotaviruses infect mature enterocytes of villus epithelium of the small intestine

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RV-gnotiobiotic piglets

Slide 7

mke1 mestes, 5/22/2007


Prof. Mary K. Estes


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Rotavirus infection in calvesVillus blunting & loss of absorptive capacity

4 dpiUninfected

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Rotavirus infection

Time:

Virus replication:

Diarrhea:

0

0

0

Histopathology: None

7 dpi

0

0

None

Rotavirus pathogenesis

24 – 72 hpi

High titersduo<j ej = ileum

Villus atrophy and blunting,inflammation, mononuclearinfiltration of lamina propria,

v acuolation of epithelial cells

4+

ENS

12 hpi

Patchy v iral antigenduo<j ej <ileum

None

4+

Enterotoxin

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dsRNA

segment

1

23

4

Protein

5

6

789

10

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VP1VP2VP3

VP4

NSP1

VP6

NSP3

NSP2VP7

NSP4 - enterotoxi n

NSP5, NSP6

antigen

VP2

VP6Subgroup

SubcoreVP1, VP3

VP4

antigen

VP7

antigen

Neutralization

Neutralization

Rotavirus genes and proteins


Prof. Mary K. Estes


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Rotavirus replication

• Occurs in the cell cytoplasm

• Viral RNA always is inside a protein shell

• Many aspects not well understood

• A unique process occurs

during the assembly of newly made particles

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αααα 2 ββββ 1 αααα v ββββ 3αααα x ββββ 2

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Participating proteins:

VP4, VP7, and NSP4

Transient lipid

bilayer formation

Following dissolution,

mature triple layered

particles are formed

Unique particle assembly by budding into the ER

• Is Ca2+-dependent ( requires ↑[Ca2+]i )

• Is NSP4-dependent


Prof. Mary K. Estes


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Lumen

Cytoplasm

COOH

NH2

CHO

CHO

L25

ER membrane

175

47

67 85

140 ?

112AAH

Toxin

93

Cleavage133

NSP4: an intracellular receptorand enterotoxin

H2

H1

H3

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NSP4-inoculated mouse

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Prof. Mary K. Estes


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Properties of Rotavirus enterotoxin

• First described viral enterotoxin (NSP4)

• A novel toxin;

No sequence similarity to other know n toxins

• Induces age-dependent diarrhea

• Mobilizes intracellular Ca2+ – not age-dependent

• Does not induce histologic changes in the intestine

• Induces age-dependent chloride secretion

in intestinal mucosa of mice

• May be a target for treatments or vaccines

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How does the Rotavirus enterotoxin NSP4 act?

• NSP4 is released from virus-infected cells

• Cells have a receptor for NSP4

• Binding to this receptor causes cell signaling

that leads to chloride secretion and diarrhea

• Antibody to NSP4 will partially protect against disease

• Some avirulent rotaviruses have mutated NSP4 genes

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Effects of Rotavirus infection of polarized epithelial cells

• Disrupts

– Tight junctions

– Microvillar, microfilament network

• Perturbs protein targeting

– Sucrase-isomaltase

• Induces chemokine responses

• Evokes intestinal secretion

– Enterotoxin

– Enteric nervous system


Prof. Mary K. Estes


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Enterocytes

Nucleus

NSP4

Cl -

Ca 2+

Cl -

Cl -

?

Courtesy of A. Einerhand

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New unexpected information about Rotavirus replication

Rotavirus infections are not limited to intestinal enterocytes

Rotavirus can be found in the serum of infected animals

and children (viremia)

Extraintestinal rotavirus can be seen

in the absence of diarrhea

Does extraintestinal Rotavirus infect other tissues

and cause non-diarrheal disease?

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Treatment and vaccines


Prof. Mary K. Estes


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Treatment?

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Oral rehydration therapy

Per liter of clean water

• 90 mEq sodium

• 20 mEq potassium

• 80 mEq of chloride

• 30 mEq of citrate

• 20 g glucose

• ¾ T. table salt

• 1 T. baking soda

• 1 c. orange juice or 2 bananas

• 4 T. sugarOR

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Prof. Mary K. Estes


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Vaccines?

• Live-attenuated reassortant vaccines

– Three currently approved and others being tested

– Not yet clear if these vaccines will protect

in developing countries where needed most

• Inactivated virus

• Virus-like particles

Need to be tested

as backup plan

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Conclusions

• Rotaviruses cause diarrhea by several mechanisms -

Enterotoxin, Malabsorption, ENS

• Why is disease age-restricted?

– Age-dependent chloride channel, Immunity

• Rotaviruses cause extraintestinal infections-

do they cause other diseases?

• Live, attenuated vaccines are safe and their effectiveness

in developing countries needs to be established

• New, effective vaccines and therapies may target

the rotavirus enterotoxin

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First identified human Calicivirus

Prototype strain of genetically related Noroviruses

23 million cases of gastroenteritis per year in USA

• Cause illness in babies, children, adults and elderly

• Cause outbreaks in daycare centers, schools,

nursing homes, hospitals, military

• Cause >96% of all outbreaks of nonbacterial gastroenteritis

Norwalk virus (NV)

Noroviruses


Prof. Mary K. Estes


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• Highly stable

• Highly infectious (1-10 virions)

Norovirus gastroenteritis

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• Many asymptomatic infections

and prolonged post-symptomatic shedding

• Some people resistant to infection (20%)

– Possibly due to genetic factor?

• Persistent infections

– Transplant patients, immunosuppressive therapy

• Unusual clinical presentation

– Disseminated intravascular coagulation

• Spread by contaminated food,

water and environmental surfaces

Norovirus background

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Norovirus pathogenesis

• Incubation period 1 – 2 days

• Produces shortening of microvilli in small intestine

with infiltration of mononuclear cells

• Transient malabsorption

and delayed gastric emptying may occur

• Cause of vomiting unknown

• Immune responses poorly characterized


Prof. Mary K. Estes


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Clinical responses of 2 adult volunteers given NV

Dolin et al., JID 123: 307-312 (1971)

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1970s NV studies

12 volunteers6 ill

NV

Immunity due to previous exposure?

Repeated susceptibilityRepeated resistance

6 not ill

6 ill

6 not ill

NV> 2 yrs

Genetic factor may determine susceptibility or resistance to NV

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Challenges studying Norwalk virus (NV)

• Infection and disease only in humans-

no small animal model

• No cell culture –

no in vitro system for infection and replication

• Low concentration of virions in stool


Prof. Mary K. Estes


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Virus-like particles (VLPs)

• Expressed capsid proteins VLPs

- Virus surrogate

- Structurally and antigenically similar

to Norw alk virion capsids

- Model for Norw alk virus-cell binding

50 nm

(Prasad et al., Science 1999)

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Norwalk VLPs hemagglutinate

human red blood cells

Discovered the Norwalk VLPhemagglutination receptor is the H antigen

Model for Norwalk virus-host cell binding:

Hutson et al., J Virol (2003)

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What is the H antigen?

• Carbohydrate histo-blood group antigenon the cell surface and on secreted mucins

• Presence controlled by genetically determined

expression of a carbohydrate modifying enzyme, a fucosyltransferase, FUT2

FUT2-/- = 20% of population

FUT2+/+ = secretor

Henry et al., Vox Sang (1995)


Prof. Mary K. Estes


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The H antigen is a host susceptibility factorfor Norwalk virus infection

Non-infected Infected

FUT2-/- 8 0

FUT2+/-, FUT2+/+ 1 42

n = 51

• FUT2-/- individuals are resistant to NV infection(0/8 infected, p < 0.0001)

Hutson et al. (J. Med. Virol. 77: 116; 2005)

FUT2 genotype versus NV challenge outcome

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Is host susceptibility similar for all Noroviruses ?

NO

• Hemagglutination patterns and CHO binding patterns

are strain specific

• Secretor status does not influence susceptibility

to Snow Mountain virus infection

• All people will be susceptible to some norov irus!

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Summary: Noroviruses

Clinical significance

is increasing –

numbers and settings

H histo-blood group

CHO antigen is host

susceptibility factor

Knowledge of NV-CHO

binding site (P domain)

or the structure of other

viral proteins may lead

to antivirals to help

prevent transmission

NN

CC

H 329H 329

W 375W 375N 331N 331

NN

CC

H 329H 329

W 375W 375N 331N 331


Prof. Mary K. Estes


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Gastroenteritis Viruses Prof. Mary K. Estes · Gastroenteritis Viruses Prof. Mary K. Estes The screen versions of these slides have full details of copyright and acknowledgements