Viruses Associated with Gastroenteritis Ghazi Jamjoom

VIRAL AGENTS CAUSING GASTROENTERITIS

VIRAL AGENTS CAUSING GASTROENTERITIS Major Viruses Rotavirus Enteric adenoviruses Noroviruses : a. Norwalk-like viruses b. Calicivirus c. Astrovirus

Noroviruses Norwalk-like Caliciviruses Astroviruses other viruses viruses

Viruses associated with gastroenteritis (cont) : Other viruses (minor): Coronaviruses Parvoviruses Pestiviruses Toroviruses

Family Reoviridae Genus Rotavirus

ROTAVIRUS First isolated in 1973 from children with diarrhea EM identification from duodenal biopsies Human and animal strains

Rotavirus

Rotavirus- EM Structure

ROTAVIRUS 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)

STRUCTURE Double capsid (outer and inner capsid) Core with genome Capsid is cleaved by trypsin to form ISVP- infective sub-viral particle

ROTAVIRUS- 3D STRUCTURE

ROTAVIRUS- ultrastructure

Rotavirus- details of EM structure

Viral Structural Proteins (VP) Outer structural proteins - VP7 and VP4 VP7=glycoprotein VP4=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

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

STRUCTURE

Gene coding assignment

Classification 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

Classification (contd.) Electropherotypes mobility of RNA segments by PAGE Used in epidemiologic studies

Rotavirus - Properties Virus is stable in the environment Relatively resistant to handwashing agents Susceptible to disinfection with 95% ethanol, ‘Lysol’, formalin

Pathogenesis 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

Replication mRNA transcription with viral RNA polymerase Capsid proteins formed mRNA segments formed, assembled into immature capsid mRNA replicated to form double stranded RNA genome

Histopathology Mature enterocytes lining the tips of intestinal villi are affected Villous atrophy and blunting Death of the mature enterocytes

Histopathology Infiltration of lamina propria with mononuclear cells Repopulation of the villous tips with immature secretory cells [crypt hyperplasia]

Histopathology

Epidemiology - 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- Worldwide distribution (source- centers for disease control and prevention)

Estimated Global Distribution of The 800,000 Annual Deaths Caused By Rotavirus Diarrhea

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%

Epidemiology Age- 4mo - 2 years Protection of younger infants through transplacental antibody transfer Asymptomatic infections are common, especially in adults Nosocomial infections Outbreaks

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

Spread in the U.S. (Source- centers for disease control and prevention)

Epidemiology (transmission) Mainly person to person via fecal-oral route Fomites Food and water-borne spread is possible Spread via respiratory route is speculated

Epidemiology (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

EPIDEMIOLOGY Differences 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 Features Incubation period - thought to be <4 days Fever- can be high grade (>102F 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 diarrhea Watery diarrhea due to net secretion of intestinal fluid Activation of the enteric nervous system -possible role of enterotoxin

Clinical Features (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 .

Diagnosis 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 Prevention Supportive - oral, IV rehydration Prevention- Handwashing and disinfection of surfaces

Vaccine 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 DUE TO ENTERIC ADENOVIRUS

GASTROENTERITIS DUE TO ADENOVIRUS Types 40, 41 Belong to serogroup F Some cases due to types 31, 3, 7

Diarrhea due to Enteric Adenovirus Age <4 years Year round Spread via fecal-oral route

Clinical features of Enteric Adenovirus gastroenteritis Incubation period 3 -10 days Diarrhea lasts for 10 -14 days Can also cause intussusception, mesenteric adenitis, appendicitis

Diagnosis- Enteric adenoviruses Isolation requires special media-Graham 293 ELISA for rapid detection is available

HUMAN CALICIVIRUSES

HUMAN CALICIVIRUSES (HuCV) Belong to Family Caliciviridae Non-enveloped RNA viruses with ss RNA 27-35 nm in size Contain a single capsid protein

HUMAN CALICIVIRUSES Genomic analysis divides it into 4 groups Human caliciviruses belong to 2 genera

CLASSIFICATION 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- typical Typical morphology 32 cup-like depressions EM appearance of “Star of David” E.g.- Sapporo-like viruses

HUMAN CALICIVIRUSES - SLV

Morphology of HuCV- atypical Atypical morphology Smooth surface Small Round Structured viruses E.g.- Norwalk-like viruses

SRSV- NORWALK VIRUS

CLINICAL 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-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 virus Person-to-person Fecal-oral spread (stool/vomitus) Fecal contamination of food or water Spread through fomites?

Epidemiology-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 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 Caliciviruses Specimen- stool , vomitus, environmental swabs, [not yet on foods] Immune EM RT-PCR in state public health labs. Serology for epidemiologic purposes

HUMAN ASTROVIRUS

ASTROVIRUS 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- 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 STRUCTURE

ASTROVIRUS - 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

ASTROVIRUS - 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

ASTROVIRUS - 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

Endemic worldwide (represents > 95% of 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 cases of diarrhea in children in many countries. 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 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

Affects approximately 130 million infants and children worldwide. 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.

When do people get infected ? In the United States, rotavirus causes outbreaks of diarrhea during the winter months. In temperate areas, it occurs primarily in the winter. In the tropics it occurs throughout the year.

Who catch the infection ? 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.

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.

Rotavirus can survive in the acidic environment , in a buffered Pathogenesis Rotavirus can survive in the acidic environment , in a buffered stomach , or in a stomach after a meal. Virus adsorption to columnar epithelial cells viral replication shortening and blunting of the microvilli and mononuclear cell infiltration into the lamina propria

NSP4 protein may act in a toxin-like manner Temporary lactose intolerance may occur. NSP4 protein may act in a toxin-like manner Neuronal alteration in water absorption Release of neuronal activators Calcium ion influx into enterocytes 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 .

Some may have a slight rise in temperature . Symptoms and Signs The incubation period ranges from 1-3 days . Symptoms often start with vomiting followed by 4-8 days of diarrhea. 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

Diagnosis (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.

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 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.

prevention Even in the cleanest environments with the best hygiene, most children still become infected with rotavirus before age 4 or 5. Total prevention of the spread of rotavirus is virtually impossible. In hospitals health officials control rotavirus outbreaks by isolating infected patients and by ordering strict hand-washing procedures.

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.

Why? 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.

Medline Search Oct. 15, a total of 102 confirmed and presumptive cases of intussusception following receipt of << rotavirus vaccine>> were reported to the Vaccine Adverse Events Reporting System (VAERS). 57 had onset within seven days of receipt of the vaccine and 29 underwent surgery; seven required bowel resection. One fatal case was reported in a 5-month-old, according to John Livengood, MD, director of the Epidemiology and Surveillance Division, National Immunization Program, CDC.

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).

These data indicate that the risk of intussusception is increased by 80% after rotavirus vaccination in an ever vs. never comparison. For dose one, the risk of intussusception increased 25-fold in the first three to seven days following vaccination and 7.1-fold in eight to 14 days after vaccination. For dose two, the risk of intussusception increase 13.4-fold in three to seven days after rotavirus vaccination.

problems do not appear to be at risk now.   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.

Vaccine adverse reactions

Percentage of children with adverse reactions during 5 days of surveillance following each of three doses of tetravalent rhesus-rotavirus vaccine (RRV-TV) or placebo. percentage with fever (as measured by axillary temperatures).*On day 4, 2.2% of RRV-TV recipients vs. 0.2% of placebo recipients became febrile (p = 0.02). b) percentage with diarrhea. c) percentage with vomiting. Adapted from (43).

following receipt of doses 1, 2, or 3 of RRV-TV following receipt of doses 1, 2, or 3 of RRV-TV. The number of children who have received RRV-TV is unknown, however, the observed rate of intussusception among vaccine recipients during the first 3 weeks after immunization appears to be greater than expected, with the highest rate during the first week following vaccination. These initial data suggest that intussusception occurs at a younger age in vaccine recipients than in unvaccinated children.

Prevention: In 1998, the U.S. Food and Drug Administration approved a live virus vaccine (Rotashield) for use in children. However, the Advisory Committee on Immunization Practices (ACIP) recommended that Rotashield no longer be recommended for infants in the United States because of data that indicated a strong association between Rotashield and intussusception (bowel obstruction) among some infants during the first 1-2 weeks following vaccination.  More information about rotavirus vaccine is available from the National Immunization Program Sanitary measures adequate for bacteria and parasites seem to be ineffective in endemic control of rotavirus, as similar incidence of rotavirus infection is observed in countries with both high and low health standards

Rotavirus Infection at KAAUH ?

A 1293 fecal samples were collected from patients of different age groups with gastroenteritis over a period of 6 years & 8 months (November 1995 – July 2002) in Jeddah.

708 males (54.76%) 1293 585 females ( 45.24%) All were tested using enzyme linked immunosorbent assay (ELISA).

Number of infected patients 136 73 males (53.68%) 63 females (46.32%) 5.65% males % of infected patients 10.52 4.87% females

% of infected males = 10.31 % % of infected females = 10.77 %

Statistics

N=127 (total number of patients between 0 years - 40 years) Mean = 2.69 years Std.Error= .4590

Descriptive Statistics N=127(total number of patients between 0 years-40 years) Mean = 2.69 years Std.Error= .4590

Distribution of Age of Patients 5.7 (years)

Numbers of Infected Males to Females in Different Age Groups 0-28 28d-1y 1-3 3-6 6-18 >18

Distribution of Age of Patients Excluding The Out-Liers (years)

Infected Saudies : non Saudies

Infections Among Males : Females (saudies : non saudies)

Infections in( Males : Females ) in Different Seasons

Infections of males to females in different seasons

Infections in Males in Different Seasons : Infections in Females in Different Seasons

Duration of Illness (days)

Numbers of patients from different Age Groups ( Males to Females )

Number of Patients in Different Seasons (Males : Females )

Number of Patients in Different Seasons (Males : Females )

Number of Infected Males & Females in Each Nationality

Infections Among Different Nationalities in Each Season

Distribution of Different Durations of Illness (days)

Means of Infections Among Different Age Groups

conclusion Rotavirus was found to be most prevalent among infants and young children . There is no significant difference in incidence depending on the sex. Most of the cases were presented to the hospital in the winter. The mean duration of illness is 3.3 days, with the majority having symptoms of gastroenteritis for 3-5 days.

Saudi &non

Saudi & non T-test