Viruses Associated with Gastroenteritis

Ghazi Jamjoom

VIRAL AGENTS CAUSINGVIRAL AGENTS CAUSING

GASTROENTERITISGASTROENTERITIS

1. Rotavirus

2. Enteric adenoviruses

3. Noroviruses :

a. Norwalk-like viruses

b. Calicivirus

c. Astrovirus

Major Viruses

Noroviruses

Norwalk-like Caliciviruses Astroviruses other

viruses viruses

Viruses associated with gastroenteritis (cont) :

Other viruses (minor):

• Coronaviruses

• Parvoviruses

• Pestiviruses

• Toroviruses

ROTAVIRUSROTAVIRUS

Family Reoviridae

Genus Rotavirus

ROTAVIRUSROTAVIRUS

• First isolated in 1973 from children with

diarrhea

• EM identification from duodenal biopsies

• Human and animal strains

RotavirusRotavirus

Rotavirus- EM StructureRotavirus- EM Structure

ROTAVIRUSROTAVIRUS

• 60-80nm in size

• Non-enveloped virus

• Double capsid

• EM appearance of a wheel with radiating spokes

• Icosahedral symmetry

• double stranded (ds) RNA in 11 segments

(double – double)

ROTAVIRUS- 3D STRUCTUREROTAVIRUS- 3D STRUCTURE

Viral Structural Proteins (VP)Viral Structural Proteins (VP)

• Outer structural proteins - VP7 and VP4

VP7VP7=glycoprotein

VP4VP4=protease-cleaved, P protein, viral

hemagglutinin, and forms spikes from the surface

• Inner core structural proteins VP 1, 2, 3, 6

• VP6 is an important antigenic determinant

• Genome is composed of 11 segments of double-stranded RNA,

six structural

coding for proteins

five nonstructural

• Seven serological groups have been identified (A-G), three of

which (groups A, B, and C) infect humans .

STRUCTURESTRUCTURE

Gene coding assignment

ClassificationClassification

•Groups, subgoups, serotypes based on viral capsid

proteins

•7 Groups (A through G)

•Group A is the most common and has 2 subgroups

•10 human serotypes based on G protein (VP 7)

•8 P protein serotypes

ClassificationClassification (contd.)

•Electropherotypes

mobility of RNA segments by PAGE

Used in epidemiologic studies

Rotavirus - PropertiesRotavirus - Properties

• Virus is stable in the environment

• Relatively resistant to handwashing agents

• Susceptible to disinfection with 95% ethanol,

‘Lysol’, formalin

PathogenesisPathogenesis

• Targeted host cells- mature enterocytes lining

the tips of intestinal villi

• Intermediate/infective sub-viral particle (ISVP)

produced through proteolysis

• Enter host cell by endocytosis

• Virus replicates in the host cell cytoplasm

ReplicationReplication

• mRNA transcription with viral RNA polymerase

• Capsid proteins formed

• mRNA segments formed, assembled into immature

capsid

• mRNA replicated to form double stranded RNA

genome

HistopathologyHistopathology

• Mature enterocytes lining the tips of intestinal

villi are affected

• Villous atrophy and blunting

• Death of the mature enterocytes

HistopathologyHistopathology

Epidemiology - WorldwideEpidemiology - Worldwide

• Millions are affected

• 600,000-850,000 deaths/year

• A major cause of diarrhea-associated

hospitalizations

• Seroprevalence studies show that antibody is

present in most by age 3y.

Rotavirus-Rotavirus-Worldwide distributionWorldwide distribution

(source- centers for disease control and prevention)

Estimated Global Distribution of The 800,000 Annual Deaths

Caused By Rotavirus Diarrhea

Epidemiology : U.S.Epidemiology : U.S.

•No. of children under 5y. affected ~ 2.7 million

•Physician visits per year ~ 500,000

•Hospitalizations per year ~ 50,000

•Deaths per year ~ 20 - 40

•% cases w/ dehydration ~ 1-2.5%

EpidemiologyEpidemiology

• Age- 4mo - 2 years

Protection of younger infants through transplacental

antibody transfer

• Asymptomatic infections are common, especially in

adults

• Nosocomial infections

• Outbreaks

EpidemiologyEpidemiology (contd.)

• Seasonality

Winter months (Nov. through May in US)

Gradual spread W to E

Year-round in the tropics

• Incubation period - thought to be <4 days

Epidemiology : U.S.Epidemiology : U.S.(Source- centers for disease control and prevention)

EpidemiologyEpidemiology (transmission)

• Mainly person to person via fecal-oral route

• Fomites

• Food and water-borne spread is possible

• Spread via respiratory route is speculated

EpidemiologyEpidemiology (spread)

• Contagious from before onset of diarrhea to a few

days after end of diarrhea

• Large amounts of viral particles are shed in diarrheal stools

• Infective dose is only 10-100 pfu

EPIDEMIOLOGYEPIDEMIOLOGYDifferences in Groups

• Group A infections are most common

• Group B has been associated with outbreaks in

adults in China

• Group C is responsible for sporadic cases of

diarrhea in infants around the world

Clinical FeaturesClinical Features

• Incubation period - thought to be <4 days

• Fever- can be high grade (>102F in 30%)

• Vomiting, nausea precede diarrhea

• Diarrhea

- usually watery (no blood or leukocytes)

- lasts 3-9 days

- longer in malnourished and immune deficient indiv.

- NEC and hemorrhagic GE seen in neonates

Mechanism of diarrheaMechanism of diarrhea

• Watery diarrhea due to net secretion of intestinal fluid

• Activation of the enteric nervous system -possible role of enterotoxin

Clinical FeaturesClinical Features (contd.)(contd.)

• Dehydration is the main contributor to mortality.

• Secondary malabsorption of lactose and fat, and chronic diarrhea are possible

• Recovery is usually complete.

• However, severe diarrhea without fluid and electrolyte

replacement may result in dehydration and death .

Immunological Aspects

• Immunoglobulin (Ig) A , in the lumen of the gut immunity

to infection .• Actively or passively acquired antibodies (including antibodies

in

colostrum and mothers milk) lessen the severity of disease

but does not consistently prevent reinfection .• Absence of antibody small amounts of virus infection

and diarrhea .

DiagnosisDiagnosis

• Antigen detection in stool by ELISA, Latex

Agglutination (for Group A rotavirus)

• EM- non-Group A viruses also

• Culture- Group A rotaviruses can be cultured in

monkey kidney cells

• Serology for epidemiologic studies

Treatment and PreventionTreatment and Prevention

• Treatment- Treatment-

Supportive - oral, IV rehydration

• PreventionPrevention-

Handwashing and disinfection of surfaces

VaccineVaccine• Live tetravalent rhesus-human reassortant vaccine

(Rotashield)

• Licensed for use in August 1998

• Removed from the market in October 1999 due to

risk of intussusception

• Cases were seen 3-20 days after vaccination

• Approx. 15 cases/1.5 million doses

• New vaccine from bovine rotavirus under trial

Production of rhesus rotavirus (RRV), human rotavirus (HRV) x rhesus rotavirus (RRV)reassortant quadrivalent vaccine wit VP7 serotype 1, 2, 3, and 4 specificity

GASTROENTERITIS GASTROENTERITIS DUE TO ENTERIC DUE TO ENTERIC

ADENOVIRUSADENOVIRUS

GASTROENTERITIS DUE GASTROENTERITIS DUE TO ADENOVIRUSTO ADENOVIRUS

• Types 40, 41

• Belong to serogroup F

• Some cases due to types 31, 3, 7

Diarrhea due to Diarrhea due to Enteric AdenovirusEnteric Adenovirus

• Age <4 years

• Year round

• Spread via fecal-oral route

Clinical features of Enteric Clinical features of Enteric Adenovirus gastroenteritisAdenovirus gastroenteritis

• Incubation period 3 -10 days

• Diarrhea lasts for 10 -14 days

• Can also cause intussusception, mesenteric

adenitis, appendicitis

Diagnosis- Enteric adenovirusesDiagnosis- Enteric adenoviruses

• Isolation requires special media-Graham 293

• ELISA for rapid detection is available

HUMAN HUMAN CALICIVIRUSESCALICIVIRUSES

HUMAN CALICIVIRUSESHUMAN CALICIVIRUSES(HuCV)(HuCV)

•Belong to Family Caliciviridae

•Non-enveloped RNA viruses

with ss RNA

•27-35 nm in size

•Contain a single capsid protein

HUMAN CALICIVIRUSESHUMAN CALICIVIRUSES

•Genomic analysis divides it into 4 groups

•Human caliciviruses belong to 2 genera

CLASSIFICATION OF HuCVCLASSIFICATION OF HuCV

NLV (Norovirus)Norwalk virus

Hawaii virus

Snow Mountain virus

Montgomery county virus

Taunton (England)

SLV (Sapovirus)

Sapporo virus

Manchester virus

Houston/86

London/92

Morphology of HuCV- typicalMorphology of HuCV- typical

• Typical morphology

• 32 cup-like depressions

• EM appearance of “Star of David”

E.g.- Sapporo-like viruses

HUMAN CALICIVIRUSES - SLVHUMAN CALICIVIRUSES - SLV

Morphology of HuCV- atypicalMorphology of HuCV- atypical

• Atypical morphology

• Smooth surface

• Small Round Structured viruses

E.g.- Norwalk-like viruses

SRSV- NORWALK VIRUSSRSV- NORWALK VIRUS

CLINICAL FEATURESCLINICAL FEATURES

• Adults and Children

• Usual incubation Period is <24 hours

(ranges from 12hrs. to 4 days)

• Short duration of illness <3 days

• Nausea, vomiting, fever, headache

• Abdominal cramping

• Watery diarrhea

Epidemiology-NorovirusesEpidemiology-Noroviruses

• Worldwide distribution

• >23 million cases/year in the U.S.

• Major cause of foodborne outbreaks of GE

• Most people have had infections by age 4

years (by seroprevalence studies)

Spread of Norwalk virusSpread of Norwalk virus

A. Person-to-person Fecal-oral spread (stool/vomitus)

B. Fecal contamination of food or water

C. Spread through fomites?

Epidemiology-NorovirusesEpidemiology-Noroviruses

•Asymptomatic infections- seroconversion but asymptomatic shedding of virus

•Low infective dose •Viral excretion during convalesence

(up to 2 weeks)•Ability to survive in water chlorination at

routine levels

Epidemiology of OutbreaksEpidemiology of Outbreaks

• Cruise ships, schools, nursing homes, etc. • Can involve infants and school-age children

• Source usually is contaminated food and water (seafood-oyster and shellfish etc.)

Diagnosis- Human Diagnosis- Human CalicivirusesCaliciviruses

• Specimen- stool , vomitus, environmental swabs,

[not yet on foods]

• Immune EM

• RT-PCR in state public health labs.

• Serology for epidemiologic purposes

HUMAN HUMAN ASTROVIRUSASTROVIRUS

ASTROVIRUSASTROVIRUS

• Described in relation to an outbreak of

gastroenteritis in 1975

• Detected by EM

• Immunologically distinct from Human Caliciviruses

• Belong to family Astroviridae

• 8 human serotypes are known

ASTROVIRUS- structureASTROVIRUS- structure

• Small ss RNA virus

• Non-enveloped

• 27-32nm in size

• Round with an unbroken, smooth surface

• EM appearance of a 5 or 6 pointed star within

smooth edge

• Contain 3 structural proteins

ASTROVIRUS- EM ASTROVIRUS- EM STRUCTURESTRUCTURE

ASTROVIRUSASTROVIRUS - Epidemiology

• Worldwide

• Mainly in children <7 years of age.

• Transmission person-to-person via

fecal-oral route

• Outbreaks due to fecal contamination of

sea-food or water

ASTROVIRUSASTROVIRUS - Clinical Features

• Infants and children are most often affected

• Short incubation period 1-4 days

• Nausea, vomiting, abdominal cramping and watery

diarrhea

• Constitutional symptoms-fever, malaise, headache

ASTROVIRUSASTROVIRUS - Diagnosis

• EM (virus shed in stool in great numbers)

• EIA

• RT-PCR

Rotaviruses Ghazi Jamjoom

• Twenty-five years ago, little was known about the causes of diarrhea, which kills an estimated 3 million infants and children worldwide every year.

• Scientists knew that bacteria and parasites were implicated in only approximately 10 to 20 % of all cases of diarrhea..

• In 1973,, researchers in Australia discovered a virus in infants with severe diarrhea and named it “rotavirus” for its wheel-like shape.

• One year later, NIAID researchers were the first to identify rotavirus in the United States.

Rotavirus Biology

•Rotaviruses belong to the family Reoviridae ,

genus Rotavirus.

• They have a characteristic wheel-like appearance when viewed by electron microscopy.

•Nonenveloped, double-shelled

Group A rotaviruses

• Endemic worldwide (represents >

95% of

currently identified strains in humans

)

• The leading cause of severe diarrhea among infants and children . • Accounts for about half of the cases requiring hospitalization.

Group B rotavirus,

• Also called adult diarrhea rotavirus or

ADRV

• Has caused major epidemics of severe

diarrhea affecting thousands of persons

of all ages in China.

Group C rotavirus•

• Has been associated with rare and sporadic

cases of diarrhea in children in many

countries.

• First outbreaks were reported from Japan

and England

• Subgroups classification based upon

neutralization epitopes of the outer capsid proteins,

VP4 and VP7

Antigenic specificity of VP7 G serotypes

Antigenic specificity of VP4 P serotypes

Fourteen G serotypes and twenty one P serotypes have been detected in humans.

Neutralization assays measure reactivity

predominantly to VP7 proteins.

Distribution of Rotavirus Strains From A Global Collection of 2,748 Strains.

Transmission

• Rotavirus infection is very

contagious .

• Viral particles pass in the stool of infected persons before and after they have symptoms of the illness .

• Spread is by the oral-fecal route

• The virus can survive for long periods on hard surfaces (e.g.

toys and tables) and is not killed by standard

disinfectants... children forget to wash their hands often

enough, especially before eating and after using the toilet.

toilet Get infected

• Infected food handlers may contaminate foods that require

handling and no further cooking, such as salads, fruits.

• The infective dose is presumed to be 10-100 infectious viral

particles.

Because a person with rotavirus diarrhea often excretes large

numbers of the virus (108-1010 infectious particles/ml of

feces), infection doses can be readily acquired .

• Asymptomatic rotavirus excretion has been well

documented may play a role in perpetuating

endemic disease .

N.B some have reported low titers of virus in respiratory

tract secretions and other body fluids..

Epidemiology

• Rotavirus is the single most important cause of life-threatening diarrhea in children younger than 2 years.

• Affects approximately 130 million infants and children worldwide.

• In the United States alone, rotavirus causes more than 3 million cases of childhood diarrhea each year, leading to an estimated 55,000 to 100,000 hospitalizations and 20 to 100 deaths.

• Humans of all ages are susceptible to rotavirus infection,

although Children , premature infants, the elderly,

and the immunocompromised are prone to more severe

symptoms caused by infection with group A rotavirus.

Who catch the infection ?

• An infant’s first bout of diarrhea from rotavirus is the most severe , subsequent reinfections decrease in severity. These findings indicated that infants gradually develop partial immunity to the virus and that a vaccine might prevent the disease.

• Temporary lactose intolerance may occur.

• NSP4 protein may act in a toxin-like manner

Calcium ion influx into enterocytes

Release of neuronal activators

Neuronal alteration in

water absorption

• Loss of the ability to absorb water net secretion

of water and loss of ions watery diarrhea

Watery diarrhea dehydration (most commonly

isotonic) and may lead to metabolic acidosis and

death .

• The incubation period ranges from 1-3 days .

• Symptoms often start with vomiting followed by 4-8 days of

diarrhea.

Symptoms and Signs

• Some may have a slight rise in temperature .

Immunological Aspects

• Immunoglobulin (Ig) A , in the lumen of the gut immunity

to infection .• Actively or passively acquired antibodies (including antibodies

in

colostrum and mothers milk) lessen the severity of disease

but does not consistently prevent reinfection .• Absence of antibody small amounts of virus infection

and diarrhea .

• Infection in infants and small children is generally symptomatic .

• In adults infection is usually asymptomatic .

• Asymptomatic rotavirus infections are common in neonates

because of passively acquired maternal immunity , breast

feeding , and possible infection with less virulent strains

(EIA) 1) Identification of the viral antigen in the patient's stool latex

agglutination most widely used screening test for clinical specimens .

2) Electron microscopy (EM)

3) polyacrylamide gel electrophoresis (PAGE) is used in some laboratories

for RNA typing.

4) A reverse transcription-polymerase chain reaction (RT-PCR) has been

developed to detect and identify all three groups of human rotaviruses.

Diagnosis

• Serotypes can be identified using monoclonal antibodies

against VP7 and VP4.

• Neutralization is detected on tissue cultures as a CPE

Antigenic specificity of VP7 G serotypes

Antigenic specificity of VP4 P serotypes

• Treatment is nonspecific and consists of : 1) Oral rehydration therapy to prevent dehydration.

2) About one in 40 children with rotavirus gastroenteritis will

require hospitalization for intravenous fluids .

• For persons with healthy immune systems, rotavirus gastroenteritis is a self-limited illness, lasting for only a few days.

Treatment

• Total prevention of the spread of rotavirus is virtually impossible.

prevention

• In hospitals health officials control rotavirus outbreaks by isolating

infected patients and by ordering strict hand-washing procedures.

• Even in the cleanest environments with the best hygiene, most children

still become infected with rotavirus before age 4 or 5.

Rotavirus

Vaccine

Rotavirus vaccine

• Scientists knew that although many strains of rotavirus exist, only four

cause the majority of diarrhea cases in young children in the United

States.

• Aiming for prevention : NIAID researchers developed a vaccine

(RRV-TV) designed to protect against the four strains of rotavirus .

• During the 1970s, NIAID scientists analyzed the genetic material of

rotavirus, Identified two important proteins , VP4 and VP7 produced

by the genes , and determined the function of these proteins.

• Proteins on the surface of the virus were found to be critical for triggering

an immune response in the body against rotavirus.

• NIAID researchers focused on these proteins to develop a vaccine.

Tetravalent Oral Live-Attenuated Vaccine

• The oral vaccine contains four different, live attenuated viral strains,

serotypes ( 1,2,3,4) .

• One strain (serotype 3) is an unmodified rhesus monkey rotavirus

(RRV) which does not cause disease in humans

• The other three are made by reassortment (genetic recombination)

of that monkey RRV with three human rotaviruses of serotypes

1,2,4.

• Each reassortant vaccine strain contains 10 monkey RRV genes and

the VP7 gene for one serotype of the human rotavirus envelope

proteins:

VP-7 (serotype 1) , VP-7 (serotype 2) , VP-7 (serotype 4)

• The combined vaccine provided comprehensive protection

against the four serotypes (1,2,3,4)

• Studies showed that high doses of the RRV-TV vaccine, designed to protect against four strains of rotavirus, were very effective in

preventing severe, dehydrating rotavirus disease.

• Breast-feeding did not interfere with the effectiveness of the rotavirus vaccine ensuring good nutrition in infants

• In August 1998, the first live attenuated rotavirus vaccine

(Rotashield{registered} {Wyeth Lederle Vaccines and

Pediatrics}) was approved for use in infants by the Food and

Drug Administration. The Advisory Committee on Immunization

Practices has recommended that this vaccine be given as a

three-dose schedule to infants aged 2, 4, and 6 months.

•However, on July 15, 1999, the US Centers for Disease Control and

Prevention (CDC) recommended that doctors stop giving the

rotavirus vaccine to infants.

• On October 22, 1999, the Advisory Committee on Immunization

Practices voted to stop recommending the vaccine

Why?

• Centers for Disease Control and Prevention (CDC) advisory committee

received an overwhelming amount of data all indicating a strong

association between ( rotavirus vaccine ) and bowel obstruction among

some infants during the first one to two weeks following vaccination.

• Apparently, many infants who received the rotavirus vaccine developed

Intussception of the bowel within one to three weeks after receiving

a dose or two of the vaccine.

• The risk of intussusception was increased 19-fold in the first 3 to 7

days after vaccination and almost fourfold (3.6) in the 8 to 14 days

after vaccination (P<0.0002).

 

• Children who have already received the vaccine and have not had

problems do not appear to be at risk now.

• In the meantime, research on better vaccines for rotavirus continues.

1293

585 females ( 45.24%)

All were tested using enzyme linked immunosorbent

assay (ELISA).

708 males (54.76%)

Number of infected patients 136

73 males (53.68%)

63 females (46.32%)

% of infected patients 10.52

5.65% males

4.87% females

% of infected males = 10.31 %

% of infected females = 10.77 %

AGE

adultsschoolpreschooltoddlersinfantsneonates

Count

40

30

20

10

0

SEX

male

female3

4

7

17

21

6

3

6

22

30

7

Numbers of Infected Males to Females in Different Age Groups

0-28 28d-1y 1-3 3-6 6-18 >18

4745

19

7

malefemale

sex

Bars show counts

Infections Among Males : Females (saudies : non saudies)

27

24

1210

15

12

13

10

malefemale

sex

Bars show counts

Infections in( Males : Females ) in Different Seasons

DURATION

10.08.06.04.02.00.0

80

60

40

20

0

Std. Dev = 1.77

Mean = 3.3

N = 118.0077

76

22

4

Duration of Illness

(days)

SEASON

automnsummerspringwinter

Count

30

20

10

0

SEX

male

female

10

12

10

24

13

15

12

27

Number of Patients in Different Seasons (Males : Females )

SEASON

automnsummerspringwinter

Count

30

20

10

0

SEX

male

female

10

12

10

24

13

15

12

27

Number of Patients in Different Seasons (Males : Females )

ALL NATIONALITIES

Count

50

40

30

20

10

0

SEX

male

female

Number of Infected Males & Females in Each Nationality

ALL NATIONALITIES

Count

40

30

20

10

0

SEASON

winter

spring

summer

automn

Infections Among Different Nationalities in Each Season

DURATION

10.08.06.04.02.00.0

80

60

40

20

0

Std. Dev = 1.77

Mean = 3.3

N = 118.0077

76

22

4

Distribution of Different Durations of Illness

(days)