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viral gastroenteritis
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What causes viral gastroenteritis?
RotavirusesCalicivirusesCalicivirusesAstrovirusesSRV (Small Round Viruses)SRV (Small Round Viruses)TorovirusesAdenoviruses 40 41Adenoviruses 40 , 41
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Diarrhea Causing Agents in World
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ROTAVIRUSROTAVIRUS
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Genus Segments Host VectorFamily Reoviridae
Genus Segments Host VectorOrthoreovirus 10 Mammals None
Orbivirus 11 Mammals Mosquitoes, fliesOrbivirus 11 Mammals Mosquitoes, fliesRotavirus 11 Mammals NoneColtivirus 12 Mammals Ticks
Seadornavirus 12 Mammals TicksAquareovirus 11 Fish NoneIdnoreovirus 10 Mammals None Cypovirus 10 Insect NoneFijivirus 10 Plant Planthopper
Phytoreovirus 12 Plant LeafhopperO i 10 Pl t Pl thOryzavirus 10 Plant Planthopper
Mycoreovirus 11 or 12 Fungi None?
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REOVIRUSREO: respiratory enteric orphan,
early recognition that the viruses caused respiratoryearly recognition that the viruses caused respiratory and enteric infections
incorrect belief they were not associated with disease, hence they were considered "orphan " viruses
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ROTAVIRUSROTAVIRUS‐‐ PROPERTIESPROPERTIES
Virus is stable in the environment (months)
Relatively resistant to hand washing agents
Susceptible to disinfection with 95% ethanol, ‘Lysol’, p 95 , y ,formalin
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STRUCTURAL FEATURES OF ROTAVIRUSSTRUCTURAL FEATURES OF ROTAVIRUS
60‐80nm in size
Non‐enveloped virusNon enveloped virus
EM appearance of a wheel with radiating spokes
Icosahedral symmetry
Double capsidp
Double stranded (ds) RNA in 11 segments
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Rotavirus structureRotavirus structure
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The rotavirus genome consists of 11 segments of double-stranded RNA, which code for 6 structural viral proteins, VP1, VP2, VP3, VP4, VP6 and VP7 and 6 non-structural proteins, NSP1-NSP6 , where gene segment 11 encodes both NSP5 and 6.
Genome is encompassed by an inner core consisting of VP2, VP1 and VP3 proteins. Intermediate layer or inner capsid is made of VP6 determining group and subgroup
ifi i ispecificities.
The outer capsid layer is composed of two proteins, VP7 and VP4 eliciting neutralizing antibody responses.
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CLASSIFICATIONCLASSIFICATION‐‐ GroupsGroupsCLASSIFICATIONCLASSIFICATION‐‐ GroupsGroups7 Groups (A through G) and Within group A, four different7 Groups (A through G) and Within group A, four different
subgroups (SG); SGI, SGII, SGI and II, and nonI/nonII, have been
di ti i h d th b i f VP6 di it hi h ibldistinguished on the basis of VP6 diversity, among which possibly
SGI or SGII are the only humans strains
Group A is the most common
Group B (outbreaks in China)p ( )
Group C (worldwide)
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CLASSIFICATIONCLASSIFICATION SerotypesSerotypesCLASSIFICATION CLASSIFICATION ‐‐ SerotypesSerotypesSerotypes based on viral capsid proteins inducing neutralizing Ab
15 G serotypes based on VP7 (G serotypes) differencesyp
5 predominant strains in U.S. (G1-G4, G9) account for 90% of isolates
Strain G1 accounts for 73% of infectionsStrain G1 accounts for 73% of infections
25 P serotypes based on VP4 (P serotypes) with P4/P8 predominance
Common PG combinations are: P8G1, P8G2, P4G2, P8G4
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Rotavirus gene function
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Rotavirus PathogenesisRotavirus PathogenesisEntry through mouth
Targeted host cells - mature enterocytes lining the tips of
intestinal villi
Intermediate/infective sub-viral particle (ISVP) produced
th h t l ithrough proteolysis
Enter host cell by endocytosis
Replication in epithelium of small intestine
Replication outside intestine and viremia uncommonReplication outside intestine and viremia uncommon
Infection leads to isotonic diarrhea
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CLINICAL FEATURESCLINICAL FEATURESCLINICAL FEATURESCLINICAL FEATURES
Incubation period ‐ thought to be <4 days
Fever‐ can be high grade (>102°F in 30%)g g ( )
Vomiting, nausea precede diarrhea
Di hDiarrhea
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MECHANISM OF DIARRHEAMECHANISM OF DIARRHEA
Watery diarrhea due to net secretion of intestinal fluid and loss of absorptive surfacep
Activation of the enteric nervous systemRole of NSP4 peptide regions as an enterotoxinRole of NSP4 peptide regions as an enterotoxinDehydration is the main contributor to mortality Secondary malabsorption of lactose and fat andSecondary malabsorption of lactose and fat, and chronic diarrhea are possible
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EPIDEMIOLOGYEPIDEMIOLOGYEPIDEMIOLOGYEPIDEMIOLOGYA hild 4 2Age‐ children 4mo - 2 yearsAsymptomatic infections are common, especially in adults
Nosocomial infections
Outbreaks
Severe Disease young, immunocompromised Se e e sease y g, p
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R t i I itRotavirus Immunity
Antibody against VP7 and VP4 partially protective
First infection usually does not lead to permanent immunity
Reinfection can occur at any age
Subsequent infections generally less severe
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Rotavirus detection and strainRotavirus detection and strain characterization
Laboratory procedures for diagnosis of rotavirus include :
electron microscopy (EM)electron microscopy (EM),
passive latex agglutination assays (LA),
electropherotyping using polyacylamide gel
electrophoresis (PAGE),p ( ),
enzyme-linked immunosorbent assays (ELISA)
reverse transcription-polymerase chain reaction (RT-PCR)
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TREATMENT AND PREVENTIONTREATMENT AND PREVENTION
Treatment Treatment Treatment Treatment Supportive‐ oral, IV rehydration
PreventionPreventionHand hygiene and disinfection of surfacesHand hygiene and disinfection of surfaces
VaccineVaccine
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Calicivirus
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HUMAN CALICIVIRUSESHUMAN CALICIVIRUSES((HuCVHuCV))
• Family Caliciviridae
Non enveloped RNA viruses• Non-enveloped RNA viruses
with ss [+] sense RNA
• 27-35 nm in size
• Contains single capsid protein g p p
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CLASSIFICATIONCLASSIFICATION
Caliciviruses can be divided into:Caliciviruses can be divided into:Norwalk and "Norwalk-like" viruses (NLV)
"Sapporo-like" viruses (SLV)
Vesiviruses
LagovirusesLagoviruses
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CLASSIFICATIONCLASSIFICATIONNLV (Norovirus) SLV (Sapovirus)NLV (Norovirus)Norwalk virusHawaii virus
S (Sapov us)Sapporo virusManchester virus
Snow Mountain virusMontgomery county virus
Houston/86London/92
Taunton (England)
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Norwalk virus: Clinical Features• 24 hour incubation period
• Vomiting prominent
• Headache, myalgia, fever
• Diarrhea 1-3 days, less severe than rotavirus
• Affects all ages• Affects all ages
• Treatment symptomatic
• rehydration, antidiarrheals
• Complications rare
• immunocompromised
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SPREADSPREAD
Person-to-person fecal-oral spread (stool/ vomitus)p p ( )
Fecal contamination of food or water
F it ( t l/ it )Fomites (stool/ vomitus)
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EPIDEMIOLOGYEPIDEMIOLOGY
Worldwide distribution
>23 million cases/year in the U.S.
Major cause of food-borne outbreaks of Major cause of foodا(50%<) borne outbreaks of ( 50%)
Most people have had infections by age 4 years (by l di )sero-prevalence studies)
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DIAGNOSISDIAGNOSIS
diagnosed by electron microscopy only, often difficult to diagnose because of small size
Specimen: stool, vomitus, food, environmentalSpecimen: stool, vomitus, food, environmental swabs (during outbreak investigations)
i bli h l h l bRT-PCR in state public health labs.
Serology for epidemiologic purposes
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Astrovirus Particles
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Classification of AstrovirusClassification of Astrovirus
Genus Astrovirus
Family Astroviridae
Human serotypes: HuAstV 1-8
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ASTROVIRUSASTROVIRUS‐‐ structurestructureASTROVIRUSASTROVIRUS‐‐ structurestructure
Small ss RNA virusSmall ss RNA virus Non-enveloped 27-32nm in sizeContain 3 structural proteinsGenome has been sequenced
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ASTROVIRUSASTROVIRUS li i lASTROVIRUSASTROVIRUS ‐ Clinical Features
Infants and children are most often affected
Eld l d i i d lElderly and immunecompromised persons also
Short incubation period 1-4 daysy
Nausea, vomiting, abdominal cramping and watery
di hdiarrhea
Constitutional symptoms-fever, malaise, headachey p
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ASTROVIRUSASTROVIRUS id i lASTROVIRUSASTROVIRUS ‐ Epidemiology
Endemic worldwide
Mainly in children <7 years of ageMainly in children <7 years of age
Transmission- person-to-person [fecal-oral]
Outbreaks due to fecal contamination of sea-food or
waterwater
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ASTROVIRUSASTROVIRUS ‐ DiagnosisASTROVIRUSASTROVIRUS Diagnosis
EM (virus shed in stool in great numbers)
EIA
RT-PCR
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TorovirusesFamily Coronaviridae
Genus TorovirusGenus Torovirus
Human and animal pathogen
Pl hi t d ( ) RNA iPleomorphic, coated ss (+) RNA virus
Watery diarrhea in 2 – 12 months old
Diagnosis: EM
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Adenovirus Particle
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Enteric AdenovirusesEnteric AdenovirusesNaked DNA viruses, 75 nm in diameter.
f tidi t i d i t 40 d 41 i t d ithfastidious enteric adenovirus types 40 and 41 are associated with gastroenteritis
associated with cases of endemic gastroenteritis, usually in young childrenassociated with cases of endemic gastroenteritis, usually in young children and neonates. Can cause occasional outbreaks.
possibly the second most common viral cause of gastroenteritis (7-15% of all endemic cases)
similar disease to rotaviruses
most people have antibodies against enteric adenoviruses by the age of three
diagnosed by EM or by the detection of adenovirus antigens in faeces bydiagnosed by EM or by the detection of adenovirus antigens in faeces by
ELISA or other assays.
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Diagnosis of viral diseases
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Gastrointestinal Viral InfectionsCulture:rotaviruses, enteric adenoviruses (especially, serotypes 40and 41) noroviruses sapoviruses astroviruses and possiblyand 41), noroviruses, sapoviruses, astroviruses, and possiblytoroviruses that is not readily cultivable.
Electron Microscopy:Negative staining with uranyl acetate or phosphotungstic acidcan be performed directly on stool specimens.p y pespecially the noroviruses and sapoviruses, are bettervisualized using immune electron microscopy
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Gastrointestinal Viral InfectionsAntigen Detection:Many commercial assays that use for rotavirus detection, and at least one commercial adenovirus assay is available. yAntigen detection assays for astrovirus have been used in investigations
Nucleic Acid Amplification:In rotaviruses antigen detection assays better than RT-PCR because of the adequate sensitivity and greater simplicitybecause of the adequate sensitivity and greater simplicity.RT-PCR has been shown to be more sensitive than EIA for detection of astroviruses.RT PCR is the method of choice for detection of norovirusesRT-PCR is the method of choice for detection of noroviruses.
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T pes of Cell C lt reTypes of Cell CulturePrimary cell cultures;d i d di tl f th i l i l d i k kid llderived directly from the source animal. include primary monkey kidney cells and primary rabbit kidney cells.
Diploid or semicontinuous cells;are capable of a limited number of passages before undergoing senescence. Include human fibroblast cell cultures such as MRC-5 and WI-38 cells.
Continuous or transformed cell lines;are immortalized cells that can be passaged without limit. include HEp-2,are immortalized cells that can be passaged without limit. include HEp 2, HeLa, A549, and Madin-Darby canine kidney cells.
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Electron MicroscopElectron Microscopyfor the direct visualization of viral particles in specimen.
Advantages include speed, lack of requirement for viral viability, and that many different kinds of viral particles can potentially be seen (nature of the particle).
Disadvantages include the cost and complexity of i t i i l t i th d fmaintaining an electron microscopy, the need for a
skilled operator, and limited sensitivity related to the relatively high concentration of viral particlesrelatively high concentration of viral particles.
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1 Di t S i E i ti (EM)1. Direct Specimen Examination (EM)diagnostic virology is the evaluation of stool specimens g gy pfrom patients with suspected viral gastroenteritis.rotaviruses, enteric strains of adenovirus, noroviruses, and astroviruses, are not readily cultivable. All be seen by negatively stained using phosphotungstic acid or uranyl acetate. Rotaviruses and adenoviruses are easily seen. Noroviruses and sapoviruses are better visualized using immune electron microscopy
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2. Examination of Fixed Tissue (EM)
3. Examination of Infected Cell Cultures(EM)
h th t b id tifi d b th th dwhen they cannot be identified by other methods.
Although identification to the species level is not possible, identification of a virus family based on morphology can provide a starting point for moremorphology can provide a starting point for more detailed identification.
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Electronmicrographs
RotavirusAdenovirus
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Antigen DetectionAntigen Detection
passive latex agglutination assays (LA),
Enzyme linked immunabsorbant assay(ELISA)
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Enzyme linked immunabsorbantEnzyme linked immunabsorbantassay(ELISA)
Sample to be tested Enzyme -> colo
virus Detecting anvirus Detecting an
Capturing antibody
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Molecular MethodsRT‐PCRMethods based on the detection of viralgenome are also commonly known asgenome are also commonly known asmolecular methods. It is often said thatmolecular methods is the future direction ofviral diagnosis, and it is certain that the role ofmolecular methods will increase rapidly in the
fnear future
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Other Possible Diarrhoeal VirusesOther Possible Diarrhoeal VirusesCoronaviruses
RNA viruses with a crown-like appearance
Not convincing associated with gastroenteritis at present
Small Round Virusesll i lik i l i h h f 22 28 ismall virus-like particles with a smooth surface, 22-28nm in
diameter
may possibly be parvoviruses, enteroviruses, or cubic y p y p , ,bacteriophages
occasionally seen in the faeces of endemic or epidemic cases of t t itigastroenteritis