viral dirrhoea gastroentrities dirrhoea

1. Viruses causing Diarrhea
Guide: Dr. Deshpande
Dr. Sshrutkirti Gupta

2. • Introduction
• Etiological agents
• Clinical features
• Laboratory Diagnosis
• Treatment
• Prophylaxis

3. Introduction
• A variety of viruses are found in the human
gastrointestinal tract
• Including nonpathogenic bacteriophages &
viruses that use GIT as portal for entry( entero,
hepato & some Adenoviruses)
• Worldwide, acute gastroenteritis almost 500
million children annually.
• In developing countries, acute gastroenteritis,
including viral gastroenteritis -- leading cause of
death of children < 4 years

4. % of cases Community -
based
% of cases Hospital-
based
Rotavirus 5-40 25-65
Calicivirus 10-25 20-30
Adenovirus 40/41 10-15 5-12
Astrovirus 10-25 5-10
Coronavirus 1-3 1-2
Breadavirus ? <1
Torovirus ? 0-3.5
Picobirnavirus ? <1
Relative contributions of viral enteropathogens to
childhood gastroenteritis

5. Etiological agents
1)Rotavirus
2)Caliciviruses
3)Astroviruses
4)Enteric Adenovirus
5)Enterovirus
6) Coronavirus, Toroviruses, Aichiviruses-?
7) Nipah & Hendra viruses

6. • Clinical Features:
• Watery diarrhea, vomiting, anorexia,
abdominal pain, & fever
• Greenish-yellow or pale, without blood or
mucus
• Self-limited, recovery in 5-10 days
• Shedding for several weeks
• No effective antivirals,
• Symptomatic relief.
• Rehydration therapy- important

7. • Bacterial Diarrhea
• Inflammatory,
noninflammatory
• Abdominal pain, bloody
stools,
• leukocytosis,
• Prolonged diarrhea are
more likely- responding to
antimicrobials
• Viral Diarrhea
• noninflammatory
diarrhea
• Watery diarrhea, no
blood, no mucus
• Leucocytosis – absent
• Self limiting diarrhea- 5-
7days

8. Laboratory Diagnosis

9. • Collection, transport, & Storage of
Specimens
• Stool:
• Rotaviruses, Norwalk virus, enteric Adenovirus 40 &
41, enteroviruses
• Within 48hrs- preferable, although shedding occurs
for several weeks
• Bedpans- transferred to container/ test tube
• Diaper(5-10ml freshly passed), wooden tongue
depressor
• Wrap part of diaper with plastic to capture watery
stool
• Rectal swabs- discouraged
• Used for enteroviruses- suspected aseptic
meningitis

10. Preparation of Specimens:
• Formed stool: Make suspension – 1g stool in 4ml
Hanks BSS- centrifuge- add antibiotics to
supernatent fluid
• A cotton swab rotated in stool- vigorously agitate
in Hanks BSS
• Rectal swab- use this swab directly in Hanks BSS
Decontamination methods:
1) Antibiotics incorporated in tissue culture fluids-
simplest, practicable
2) Seitz/Selas filter- used- last resort- viruses may
adsorb to filter
3) Ultracentrifugation

11. • Storage:
• 5 days- 40C
• More than 6 dyas -20/-70oC
• Fluid samples- should be diluted in VTM(1:2 to 1:5)- & stored
• For multiple tests/ multiple pathogen testing- aliquots made
• Infectivity is lost during prolonged storage- especially
enveloped viruses
• Transport:
• Synthetic swabs- Dacron & Rayon are acceptable, should be
emulsified in Viral transport medium- can stay for 1hr at RT
• VTM- small fluid volumes, tissues, scrapings & swabs
• Fetal calf containing serum should be used if at all serum is
required
• Stuarts, Amie’s, Leibovitz-Emory, Hanks Balanced salt solution,
Eagles tissue culture medium
• Ice/ dry ice

12. • Serum samples for Ab:
• Serum should be separated as soon as
possible
• Hours- 4oC
• Weeks/months- -20oC
• Testing done on
• Acute sera
• Convalescent sera– 2-3 weeks later
• Long term- -20/-70oC in aliquots

13. 1)Microscopy:
A) Electron Microscope:
Liquid stool- centrifuged
Solid stool- 10-20% suspension in D/W or 1%
ammonium acetate- EM grid
negatively stained with phosphotungstic acid –
electron dense substance-contrast
Alternatives:
1) ammonium molybdate
2) uranylAcetate

14. • Stool Centrifuged at 3000rpm for 10min
• Supernatent is centrifuged at 7000rpm- 30min
• Supernatent is 3-5ml is ultracentrifuged at
50,000 rpm for 1hour
• Sediment in 0.2ml d/w
• Drop to Formavar-carbon coated grid(300-400
mesh)-dry
• Dipped 3 times in d/w
• Dipped in 2% potassium phosphotungstate,
pH-6- blot dry- examined (critical period)

15. Symmetry/geometrical category:
a) Icosahedral
b) Helical
c) Combined/comple
d) Enveloped- compound symmetry,
e) Non-enveloped- simple symmetry

16. Simple Icosahedral symmetry
1) Rotaviruses
2) Norwalk viruses
3) Adenoviruses
Simple spherical symmetry
1) Calicivirus
2)Astrovirus
Enveloped virus
Corona virus

17. For low amounts of virus in sample-
1) Ultracentrifugation
2) stool suspension in agar block diffusion method
Spherical structures:
• Adenovirus, Rotavirus- spherical structures- easily seen
• Aichivirus- smooth surface indistinguishable from picornavirus
Distinctive surface
structure
Amorphous (Small round structured
viruses)SRSV
Astrovirus- distinct 5 or
6 point star
Caliciviruses
• Sapovirus- rigid surface, with cup like
depression of a typical “Star of David”
appearance
• Norovirus- less rigid, feathery outline

18. • Nonspherical structures:
Coronavirus- pleomorphic, dumbell shaped
peplomers/spikes- dark halo
Torovirus- less well defined peplomers, kidney
shaped, dark staining regions in center
Other structures:
Bacteriophages- confused with small viruses- if
without tails
Cell membrane blebs, cell wall components
Disadvantages of EM
requires at least 107 particles/ml
High cost, maintenance

19. Rotavirus
Sapporovirus Norwalkvirus
Astrovirus
Adenovirus

20. B) Immune Electron Microscope:
• Concentrates the viruses in sample- as low as 106
viral particles/ml can be detected
• Virus- antibody aggregates- easy to identify
• IgG & IgM antibodies in sample
Performed on
• patients‘ stool sample mixed antibody
• convalescent sera are mixed with a virus
• Modifications:
1) Antibody labelled with colloidal gold
2) Precoating the grid with Ab- captures the virus

21. Cryo-electron microscopy:
• Uses computer analysis of digitalized images of hydrated virus
particles to generate an electron density map & a 3D figure

22. Astrovirus: Smooth but rippled capsid surface- but
this feature is usually not prominent

23. 2] Antigen detection :
Highly sensitive & specific, but disadvantages;
a)unavailability of reagents for many viruses
b)Genetic and antigenic variation

24. A) ELISAs
Monoclonal/ Polyclonal hyperimmune antibodies
ELISA kits-96- well plates
Stool suspension(10-20%) in PBS, pH 7.4 or
Na carbonate buffer, pH 9.5
EIA for Astrovirus:
• Monoclonal ab against group antigen- in virus
grown in cell culture, stool sample
• Modified form using biotinylated detector Ab

25. • Antigen detection for Rotavirus: widely used
• Large amounts very high in diarrhea- widely
used
1) Direct & indirect solid phase ELISA-
quantification can be done
Ex: Ridascreen
2) membrane based ELISA
More sensitive than Latex agglutination
3) Latex agglutination

26. • Antigen assay for Astrovirus:
• For Human Astrovirus-1
• Antigen detection in Sapovirus
• Solid phase EIA using hyperimmune guinea-pig serum
• Antigen detection in Norovirus:
• EIA- Hyperimmune antisera prepared against
recombinant Norovirus capsid antigens
• Modified: 1st commercially available
Mixture of polyclonal sera- antigen capture
monoclonal sera- for detection of genogroupI
& genogroup II strains

27. B) Latex agglutination:
Latex beads coated with specific antibodies
Negative control latex beads coated with
nonspecific antibodies
A 10% stool suspension in PBS- centrifugation-
supernatent mixed with latex reagent
Visible clumping- positive
Simple, inexpensive, shorter duration but less
sensitive than ELISA

28. 3) Antibody detection:
Rotavirus antibody detection:
1) VP6- nonneutralizing Mab to it
2) VP7- 2nd most common ag(mannose residues)
3) VP4- immnogenic (children and adults)-
neutralizing
• A fourfold rise in antibody titre is required
• Rotavirus IgG and IgM antibodies --sera
• Secretary IgA (sIgA)Coproantibodies-- sera and
stools.

29. • Ab detection for Astrovirus:
• Baculovirus expressed antigen- serotype
specific Ab is detected
• Recombinant antigen

30. • Indirect fluorescent-antibody test CF
counterimmunoelectrophoresis hemagglutination
inhibition
• immune adherence hemagglutination
• Reverse passive hemagglutination inhibition
neutralization
• dot-immunobinding assay a
• biotin-amplified immunobinding assay (299), an avidin-
• biotin RIA, and several dot hybridization techniques

31. • Immunofluorescence:
• Can be done on samples
• Tissue cultures- rapid identification even before CPE is seen
Direct:- FITC labelled antibody added- to homologous virus on
slide
• Used when large quantities of virus is present in sample
Indirect: 1st an unlabelled antibody- then FITc stained antiglobin
Ab
• Increases senstivity, used when small qty is present
• Major problems:
1) Nonspecific fluorescence
2) Low specificity of reagents
3) Maintenance of Fluorescent microscope

32. 4] Nucleic acid Detection:
RNA/DNA
Electrophoresis
Hybridization
Amplification-PCR/RT-PCR
Selection of method depends on:
DNA/RNA, single/double stranded,
single/segmented genome, rapid diagnosis,
typing, research purpose

33. A) Restriction Enzyme(RE) Digestion
SmaI - RE– Adenovirus 40 & 41
B) Electropherotyping
RNA extracted from stool- electrophorosed on agar gel-
stained by ethidium bromide/silver stains
As sensitive as EM
Ex. dsRNA, segmented Rotavirus
C) Dot Blot Hybridization
Require longer hybridization time
Combined with EM- improved sensitivity & specificity
Ex. Torovirus(esp), Rota, Calici, Astro,Norovirus

34. D) PCR & RT PCR
Most sensitive
1) Serotyping of Rotavirus to distinguish G & P
serotypes- primers against VP7 & VP4
2) Genotyping, Genetic variation
Internal nucleic acid controls- Inhibitors in stool
Guanidium thiocyanate(RNAzol, TRIzol)- extraction
Phenol chloroform for recovery of extracted NA
Ethanol precipitation/silicon particles/membrane

35. 5) Virus isolation
Cell culture:
Cultivation is difficult and not reliable,
However, certain specific cell lines used are:
1) Monkey Kidney cells- broad host range-
poliovirus, adenovirus
2) Hela lines- poliovirus, Adenovirus
Suckling mice & adult mice
Embryonated eggs
• Identification:
• Site of formation- cytoplasm/nucleus

36. • When a CPE is seen-
1) 0.2ml tissue culture fluid is subpassed- to
check agent is transmissible
2) Bacterial & fungal contamination- turbid
• Followed by Neutralization test for
identification
• Disadvantage: only viral activity, but does not
differentiate between all viruses- similar CPE
• CPE can be quantitated

37. • Those not producing CPE- detected by indirect
methods
• Hemadsorption
• Haemagglutination tests
• If no CPE in 7 days--Passage the cell cultures at
least once before reporting as negative
• Polychrome dyes used to stain- infected tissue
culture cells-
Inclusion bodies(cytoplasmic/nuclear)- ancillary not
pathognomic-
Intranuclear basophilic inclusion- Adenovirus

38. • For Rotavirus cultivation
• monkey kidney cell lines (MA-104 and CV-1) (Wa
strain) in the presence of trypsin
• Roller cultures of MA-104 cells, a line of fetal
rhesus monkey kidney cells( passage2-7)
• Human intestinal cell lines (CaCo-2 cells) grown
on permeable filter membranes(apical cells)-
slower
• LLC-MK2 cells (a continuous line of rhesus
monkey kidney) and human embryonic
fibroblasts, using nonspecial techniques.

39. • Cell culture for Astroviruses:
1)CaCo-2 from gut lines
2)T84
3)PLC/PRF/5- from hepatoma
Sapovirus-
Cannot be grown in culture, but single report
Dolphin kidney & HEK cells with trypsin
Norwalkvirus:
Cannot be grown in either cell/organ culture,
However, macaques- used, chimpanzees- infected

40. • Cytopathic effects:
• Enterovirus: Round & distorted refractile cells
with darkened margins, pyknotic nuclei, some
cluster together, loss of intercellular bridges,
complete degeneration of cell sheet rapidly
• Adenovirus: Clustering of cells and
cytoplasmic strands

41. • Typing by Neutralization test
• Titrate the virus in cell culture
• centrifuged – supernatent- 10-3 dilution
• Equal volume added to suitably diluted type
specific antiserum- Virus- antisera mixture
• Look daily for CPE along with a virus control which
is set up
• Antisera neutralises CPE- effect should stay for 4
days beyond time for a distinct CPE to show in
control tube
• Useful- Adenoviruses- 30 serotypes
•

42. • Embryonated eggs:
• Chick embryos-
poliovirus
• Animals:
• Monkeys, chimpanzees,
cyanmolgous monkeys,
Rodents-Poliovirus

43. • Virus grouping based on biologic &
bichemical properties:
1) Nucleic acid type
2) Particle size
3) Sensitivity to ether
4) Acid liability

44. 6) Histopathology(HPE):
• Rotavirus HPE:
• Duodenal biopsy- LM, immunoflorescent
staining
• Patchy epithelial involvement, shortened &
blunted villi with a cuboidal epithelium, crypt
hypertrophy, and mononuclear cell infiltration
of lamina propria

45. • Typing
1) Epidemiologic purposes
2) species within a family- different pathogenic
potential
• Serotyping, Genotyping
• Neutralization test- Rotavirus- P& G serotypes
• Hemagglutination inhibition
• Hemadsorption
• IF, ELISA,ICT

46. 1) Rotavirus:
• Epidemiology
• Discovered in 1973 in duodenal biopsy
• 5- 6,00,000 deaths of children/year- need for vaccine
• Children < 5years, especially between 6-24months
• Outbreaks in adults & older children- Adult diarrhea
rotavirus (ADRV)
• Temperate areas: winter/spring peaks
• Tropical areas: no seasonal variation
• Mortality : high in developing countries
Low in developed countries with high
disease burden

47. Taxonomy:
• Family Reoviridae- Respi-enteric orphan viruses
• nonenveloped, triple layered- Rota(wheel)-
diagnostic
• 11 segmented, dsRNA
1) Complete/double shelled: 70nm, smooth surface
2) Incomplete/single shelled: 60nm, rough surface
3) Empty particles: no RNA core
Capsid- 132 aqueous channels of
types I-III (type I- imp)

50. Classification:
Serological :
• Serogroups- A (common) B, C- humans &
animals
D, E,F,G- animals only
Group specific epitopes on Structural (VP6) and
nonstructural proteins
• A- children( subgrps I &II)
• B- all age groups{contain- adult diarrhea
rotavirus(ADRV)}
• C- mild in children & adults

51. Serotypes: by Neutralization assays:
• Antiserum with the epitopes on 2 major outer
capsid proteins
a) P (protease sensitive)serotype– antibodies
against VP4 -- (20 P serotypes)-- virulence
b) G serotype– antibodies against VP7-- (14 G
serotypes)
c) India & Africa- P6 predominates
d) Worldwide- G9- predominant G type-

52. Like in children in humans, also infect younger
ones in animals & birds
Related to viruses like:
1) Epidemic diarrhea of infant mice(EDIM)
2) Nebraska calf diarrhea
3) Simian virus SA11
Since Simian & calf viruses grow readily in cell
cultures- used as antigens for serological tests

53. Gene
segm
en
Protein Location Biological functions
1 VP1 Core RNA Polymerase
2 VP2 Core RNA binding
3 VP3 Core Guanyltransferase
4 VP4
(VP5 & VP8)
Outer capsid A) Cell attachment & penetration
B) Hemagglutination
C) Neutralization Ab( P serotype)
D)Restriction of growth in cell culture
5 NS53 RNA binding(zinc finger)
6 VP6 Inner capsid Major inner core protein; major subgroup antigen
7 NS34 RNA binding
8 or 9 VP7 Major outer
capsid
(glycoprotein)
Serotype specificity; major neutralization
determinant
10 NS28 Virus assembly; enterotoxin
11 VP9

54. Nonstructural
proteins
NSP1 to 3, 5, 6- Replication
NSP4 morphogenesis
Act as chaperones to transport proteins to RNA
NSP1- highly
variable>> VP7&VP4

55. 2) Caliciviruses:
Epidemiology
• Nonbacterial diarrhea was first described in Southern USA- called
as hyperemesis hiemis/ winter vomiting disease- seasonality
• 40% nonbacterial epidemics
• All age groups
• Ability to cause explosive outbreaks- Catergory B agents by
National Institute of Allergy & Infectious Diseases(NIAID)
• Recently, ABO blood group antigens, Lewis &
secretor/nonsecretor types- receptors for Norovirus
• Raw oystersShellfish,

56. Taxonomy:Calicivirudae:
1)Norovirus- Norwalk virus
2) Sapovirus- Sapporo virus
3) Vesivirus
4) Lagovirus
(HuCVs) Caliciviruses
Sapporo like viruses(SLV) Norwalk like viruses(NLV)
Epidemiology Young children All ages
Season No seasonal variation Temperate areas
Faeco-oral Faeco-oral

57. Caliciviruses- calyx- cup
Structure:
Sapporovirus:
• 30-40nm, nonenveloped, positive sense, ssRNA
• Rigid, central stain filled cup surrounded by peripheral
cups- Star of David appearance- dark center with dark
surrounding patches
• Unlike Astroviruses that have smooth, entire edge,
caliciviruses have ragged outline-difficult for measuring
• Only EM and RT- PCR have been used for its
identification

58. Sapporovirus Norwalk virus
Clinical
features
Vomiting, diarhea Nausea, vomiting, diarrhea for
1-3 days
Epidemiology Sporadic , endemic
outbreaks
Cruise ships, nursing homes,
day care
Spread by- water, person to
person, airborne droplets of
vomitus
Structure Rigid, distinct
surface
morphology, cup
like depressions-
Star of David
appearance
Less rigid, amorphous
surface,feathery outline

59. Sapporovirus Norwalk
virus

60. 3) Astroviruses:
Epidemiology
• School age children, adults, immunosuppressed patients
rarely
• 2-10% pediatric diarrhea
• Human astrovirus- only intestinal
• Avian Astrovirus- intestinal & extraintestinal
• Taxonomy:
• Family Astroviridae- star like (astron- star in Greek)
Genera
1) Mamastrovirus
2) Avastrovirus

61. • Structure: Astrovirus
• 28-30nm, small, round,
nonenveloped virus,
star motif at center- in 10% virions
Star center not stained- white area in EM- diiferentiates
from Star of David appearance of Caliciviruses
Smooth surface(Parvo-like) or suface
spike protrusions sometimes
Genome: positive sense , ssRNA

62. 8 serotypes
• Serotype 1- most commonly detected

63. 4) Enteric Adenoviruses:
Epidemiology
5- 20% pediatric gastroenteritis
Diarrhea lasts for 5-12 days i.e longer than other
viral diarrheas
Taxonomy:
Family: Adenoviridae
Genera
1) Mastadenovirus- in mammals- share a common
complement fixing antigen
2) Aviadenovirus- in birds
Infantile diarrhea/ enteric- serotypes Ad 40 & 41
(fastidious)

64. • Structure:
• Aden- Gland(Greek)- isolated from adenoids
• 70-100nm, icosahedral, nonenveloped
• linear dsDNA core
Capsid shell proteins-
a) hexon- group antigens-- major epitopes
b) penton base & fibres- type specific antigens
Penton base in capsid & fibre with knob distally- space
vehicle
Subgroups 6-(A to F)- haemagglutination
, fibre length,DNA fragment analysis,
size, composition,
Serotypes- 51

65. 5) Enteroviruses
Pico-small , nonenveloped, ssRNA
Summer months, children<5yrs
Family- Picornaviridae
Genera:
1) Enterovirus
2) Rhinovirus
Poliovirus(
3 types)
Coxsackie Echovirus
Enterovirus(68-72
types)
Enterovirus

66. 6) Corona, Aichiviruses
Epidemiology:
Coronavirus:
usually associated with common cold, less commonly
intestinal
Torovirus:
Acute & persistent diarrhea in children
Nosocomial
Aichivirus:
Children(<4years), adults

67. 5] Coronavirus
• Family: Coronaviridae
• Order: Nidovirales-
• Enveloped, positive sense RNA
Coronaviridae Arteriviridae Roniviridae

68. • Structure:
• Pleomorphic, enveloped, solar corona
appearance
• Club/petal peplomers as fringe on surface
• positive sense RNA
• Genomic RNA- 25to 31kb- largest RNA viruses
• Nucleocapsid:
1) Nucleoprotein(N),
2) Envelope with membrane protein(M)
3) Lipid bilayer- inserted S (Spike),
HE(hemagglutinin- esterase).

69. 6] Aichivirus
• 30 nm, indistinguishable from picornavirus
• Positive sense
• 3 structural proteins:
• VP0, VP1, VP3
• VP0- reacts with convalescence phase sera

70. 7) Nipah & Hendra
• Family: Paramyxoviridae
• Larger & pleomorphic, helical nucleocapsid
• Fruit bats
• Horses, pigs
• Humans- subclinical infection
• Hendravirus- flulike, minor symptoms
• Nipah- fatal syptoms

71. • Treatment & Prophylaxis
• Rehydration: Oral, subcutaneous, intravenous
• Oral Rehydration therapy- especially for
Rotavirus diarrhea – modified from
Osmolrity(mol/l)
Component WHO ORS ORS light
Sodium 90 60
Potassium 20 20
Chloride 80 50
Citrate 10 10
Glucose 111 84
Total osmolarity 311 224

72. • Oral Immunoglobulins of bovine colostral origin-
shorten virus shedding, daily stool output,
duration
• Lactobacilli- shorten duration of diarrhea
• Broad spectrum antivirals- adenosine analogues-
inhibition of S-adenosylhomocysteinehydrolase
• Rececadotril- enkephalinase inhibitor-
antisecretory & antidiarrheal
• Early refeeding after rehydration
• Foods- carbohydrates, rice meat, potatoes, bread
& cereals, leanmeats, yogurt,fruits, vegetables

73. • Vaccines
• Rotavirus Vaccines:
Live-attenuated
1) From human & related species-(bovine, rhesus & lamb)
2) Isolates from children are attenuated by passaging,
from neonates-low virulence strains
3) Multivalent – (G1,2,3,4,& P1a)
4) Monovalent

74. • In 1998, rhesus-human reassortment (RRV-TV)
was licensed, Rotashield- 1st licensed Rotavirus
vaccine
Tetravalent , reassortment of 4 major serotypes
human-simian rhesus
But withdrawn in 1year- intussuception
• In 2005, Monovalent live-attenuated in Mexico(G1)
1st dose- 6-14wks
2nd dose- 14-24wks
• LLR(China)- attenuated strain of Lamb Rotavirus

75. FDA approved in US
3) Rotarix- monovalent live-attenuated
In phase III trial, contains- human rotavirus
G1
4) Rotareq- Live, oral, pentavalent
Bovine(WC3 strain reassorted with 5 different human
Rotaviruses (G1,2,3,4,PIa), including genes coding VP4 and
VP7 of 5 different - in 2006
a) Bovine viruses don’t replicate in intestine of infants
b) Produce lower first dose side-effects than RRV-TV

76. • Calicivirus vaccines
• Edible vaccines-
1) Transgenic plants expressing Norovirus
protein
2) Subviral vaccine- produced in Baculovirus
Major challenges in Calicivirus vaccines are
a) High genetic variability
b) No known animal model

77. • Astroviruses & Enteric Adenoviruses:
• Disease is mild & self-limiting
• Rarely requires Rehydration therapy
• Interruption of transmission- main focus,
by 1) increased personal hygienic measures
2) disinfection of contaminated environment
At present, no vaccines, antivirals available
• Coronaviruses, Toroviruses, Aichiviruses- real
significance in human gastroenteritis is not yet
established

78. • Structure:
• Photo.
• From Latin name- chalice/calyx-Calicivirus
• Norovirus- genome-
• Positive sense ssRNA
• 26-34nm, cubic symmetry, heat & acid stable
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