This document provides information about Caliciviruses/Calciviruses including their classification, morphology, genome organization, replication cycle, pathogenesis, clinical manifestations, epidemiology, and methods of diagnosis, prevention and control. It discusses that Caliciviruses have a non-enveloped structure, a positive-sense RNA genome, and cause acute gastroenteritis in humans. Noroviruses are the most common cause of outbreaks worldwide and spread primarily through fecal-oral transmission. Diagnosis can be done through electron microscopy, ELISA/RIA, or RT-PCR methods. Prevention focuses on hand hygiene, disinfection, and isolation of infected individuals.

1. Calicivirus/Calcivirus
By- Sanju Sah
St. Xavier’s College, Maitighar, Kathmandu
Department of Microbiology

2. Classification and morphology
2

3. Genera (five) within the family Caliciviridae
Norovirus
Nebovirus
Lagovirus
Sapovirus
Vesivirus
1. Feline calicivirus (species)
2. Vesicular Exanthema of Swine (species)*
• Swine isolates 1932-1956 (13 Serotypes)
• San Miguel Sea-lion virus 1972-present (17 serotypes)
• Cetacean, bovine, reptile, primate, rabbit, walrus, human,
skunk etc. 1978-present (>10)
* Colloquially grouped as Marine Caliciviruses/Vesiviruses
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Laboratory for Calicivirus Studies

4. Classification and morphology
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5. 5
• The prototypic Norwalk virus described as non-enveloped,
round, 27-nm particles with a ‘ragged’ outer edge but lacking
a definite surface structure.
• Many other Noroviruses have - in common a buoyant density
of 1.33–1.41 g/cm3, an inability to propagate in vitro.
• Noroviruses possess a single capsid protein.
• The genome is a non-segmented, single-stranded, positive
polarity RNA genome.
• The RNA genome is present in a 27-nm naked (non-
enveloped) capsid consisting of 60,000-Da capsid protein.
• The genome does not contain any virion polymerase.
• Ten prominent spikes and 32 cup-shaped depressions can be
seen on the virion by microscopy.

6. • The structure of the capsid protein is organized into two
domains joined by a flexible hinge.
• The inner shell (S) domain is composed of the N-terminal 225
residues and is involved in the formation of the icosahedral
capsid shell.
• The shell domain also shows the classic eight-stranded barrel
structure typical of many viral capsid proteins.
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• The protruding (P) domain forms prominent structures
extending from the surface of the shell, and is formed from
the C-terminal half of the protein.
• The P domain is further organized into two subdomains (P1
and P2), and it has been suggested that these structures may
be involved in binding to cellular receptors and may also be
the determinants of strain specificity.

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Genome organization
• Phylogenetic analyses of the RNA polymerase and capsid
regions of the genome indicate that human Noroviruses are
divided into two genetic groups.
• The genome of group I viruses is slightly larger (7.7 kb) than
the group II genome (7.5 kb), although the reading frame
usage of these two genetic groups is very similar.
• The genomes of both group I and group II Noroviruses are
characterized by a large 50 open reading frame (ORF) encoding
a nonstructural polyprotein of approximately 1700 amino
acids, followed by a smaller ORF at 3’ end encoding the capsid
structural protein and small basic protein.

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• This genome organization clearly distinguishes Noroviruses
from other positive-strand RNA viruses, such as the
picornaviruses.
• The smaller size of the group II Norovirus genome is
attributable to its smaller ORF1.
• A characteristic feature of the animal calicivirus genomic and
subgenomic RNAs (smaller sections of the original transcribed
template strand) is that the 5’ termini are highly conserved.

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• The 5’ terminal sequence of ORF1 is repeated around the 5’
terminal region of ORF2, suggesting that human enteric
caliciviruses, like their animal counterparts, also produce a
subgenomic RNA.
• The 5’ genomic and subgenomic termini of both groups I and
II Noroviruses begin with the sequence GU.
• The conserved GU residues at the 5’ genomic termini appears
to be a common feature of the Caliciviridae.
• The number of serotypes are not known.

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Polyprotein processing
• In common with picornaviruses, caliciviral
synthesized as a large polyprotein precursor which
proteins are
is
subsequently processed in a proteolytic cascade by the viral
3C-like protease.
• In vitro transcription and translation of genomic clones does
not appear to generate the intact polyprotein, suggesting that
proteolytic processing probably occurs cotranslationally.

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Viral replication
• The Norwalk virus replicates in the cytoplasm with release of
viral particles on cell destruction.
• The virus is presumed to replicate in a manner similar to that
of picornaviruses.

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• Norwalk virus enter the body predominantly via the oral
route.
• Virions are acid stable, consistent with an ability to survive
passage through the stomach.
• The virion causes infection first by binding to the cell receptor
on the cell membrane and enter the cell.
• Receptor binding triggers conformational change which
results in release of viral RNA into cell cytoplasm.
• VPg is removed from the viral RNA.
– VPg (viral protein genome-linked) is a protein that is covalently
attached to the 5′ end of positive strand viral RNA and acts as a
primer during RNA synthesis in a variety of virus families

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• Positive stranded RNA serves both as genomic and mRNA for
these viruses and is translated into a large polypeptide
known as non capsid viral protein.
• Subsequently the viral protein is utilized by the viral enzymes
protease to form capsid protein of the progeny as well as
several noncapsid protein including the RNA polymerase.
• RNA polymerase initiates the synthesis of progeny RNA
genomes.

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• The infecting viral RNA is copied and the complementary
strand serves as template for the synthesis of new plus
strands.
• The subgenomic RNA serves as a template for translation of
both the capsid and terminal ORF proteins.
• Replication is followed by packaging of plus strands into
virions and maturation involves the several cleavage events.
• The progeny virion assembly occurs by coating of genomic
RNA with the capsid protein in the cell cytoplasm called as
encapsidation.
• The release of progeny virions occurs by the lysis of the cell.

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 Incubation Period
• Based on volunteer studies with the Norwalk virus, the
incubation period ranges from 10 to 51 hours, with a mean
of 24 hours.
• Norwalk viruses are highly contagious.
• 10–100 infectious particles may be needed to initiate
infection.
• Human caliciviruses enter the body predominantly via the
oral route.
Pathogenesis and pathology

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20. 20
• Virions are acid stable, consistent with an ability to survive
passage through the stomach.
• Indirect evidence from epidemiologic studies suggests that
viruses may enter also via aerosols, such as in those
generated from the explosive vomiting that often occurs
during illness .
• The site of primary replication for the human caliciviruses
has not been established, but it is assumed that they
replicate in the upper intestinal tract.

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• Biopsies of the jejunum of volunteers who develop
gastrointestinal illness following oral administration of the
Norwalk or Hawaii virus exhibit histopathologic lesions .
• Partial flattening and broadening of villi with disorganization
of the mucosal epithelium.
• Lamina propria infiltrated with mononuclear cells and
vacuolization of mucosal epithelium.
• Crypt cell hyperplasia is common.
• Dilatation of the rough and smooth ER with an increase in
multivesiculate bodies in mucosal epithelial cells.

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• In general, Norovirus infections seem to cause mild atrophy
(waste away, especially as a result of the degeneration of
cells) of the villi of the small intestine, assumed to arise from
limited virus replication that damages the mucosal cells.
• The appearance of mucosal lesions was paralleled by a
decrease in brush border (the microvilli-covered surface of
simple cuboidal epithelium and simple columnar epithelium
cells) enzymes, which returned to normal values during
convalescence.

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Host immunity
• Norwalk virus infection confers a brief and short immunity.
• Recurrent infection occurs throughout life, because of the
absence of long-term immunity, lack of cross-strain immunity,
and because of the diversity of Norwalk virus strains.

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Clinical manifestations
• Norwalk viruses cause gastroenteritis in adults.
• The illness in symptomatic cases typically begins after an
incubation period of 24–48 hours.
• The illness is characterized by sudden onset of nausea,
vomiting, which can be projectile and severe.
• Low grade fever and diarrhea usually occur, the latter being
relatively mild.

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• In contrast to bacterial gastroenteritis, diarrheal stools do not
contain blood, mucus, or white cells.
• Fecal leukocytes are absent.
• Other symptoms- mild abdominal pain , malaise and
headache.
• Vomiting may arise from a decrease in gastric motility, giving
rise to a reflux action into the stomach.
• Norwalk virus gastroenteritis is short-lived and typically lasts
for 24–48 hours.

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• In general, Norovirus infections are self-limiting and affected
patients rarely need to be hospitalized.
• There have, however, been occasional reports of severe
dehydration that required the administration of intravenous
fluids.
• Deaths associated with Norovirus infections are exceptionally
rare and have not been directly attributed to this group of
viruses.
• Gastric emptying is delayed, and malabsorption of fat, D-xylose,
and lactose has been observed.

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Epidemiology
• Noroviruses are now established as the most important cause
of epidemic nonbacterial outbreaks of gastroenteritis
worldwide.
• Norwalk virus gastroenteritis is found worldwide in adults.
• National surveillance and diagnosis by EM of outbreaks of
nonbacterial gastroenteritis in the UK have shown that
Noroviruses are a more common cause of infective
gastroenteritis than Salmonella or Campylobacter.
• The infection typically occurs in group settings, such as schools,
hospitals, nursing homes, etc.

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• It is a strict human infection, and humans are the major source
of infection.
• The virus is excreted in the vomitus and feces for several weeks
after recovery; hence vomitus and feces are important sources
of infection.
• Infection is transmitted from person to person by ingestion of
food or water contaminated with the virus.
• Subsequent surveillance and investigations of outbreaks in
many parts of the world identified the potential of Noroviruses
for causing epidemic gastroenteritis in semiclosed or
community wide populations, for example families, healthcare
institutions, holiday locations including cruise ships,
educational establishments, and the catering industry.

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• Outbreaks occur among children and adults, but rarely among
neonates or very young children.
• Although considerable data are available on the causal role of
Noroviruses in outbreaks, less is known about their role in
endemic disease, perhaps because of the relative mildness of
the illness.
• Hospitalization of individual cases is thus unusual, and
opportunities to investigate sporadic cases are scarce.

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Laboratory Diagnosis
VIRUS ISOLATION
• There are no reports of the isolation of Noroviruses in either
cell or human intestinal organ cultures.
• Tests with a wide range of animals have also failed to identify
a suitable model, although recently it was reported that
macaques could be experimentally infected with human
Noroviruses.
• Some success has been achieved with the transmission of
Norwalk virus in chimpanzees, in which serological responses
and excretion of Norwalk virus antigen in stools were
described.
• It is, however, unlikely that isolation in cell cultures will ever
be used for diagnosis, as in vitro methods are too slow and
labor-intensive.

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ELECTRON MICROSCOPY
• direct detection of Noroviruses in stools.
• Virions possess an amorphous surface structure, lacking a
defined symmetry, with a ragged outline that probably
explains the wide range of particle diameters (32– 38 nm)
reported.

32. 32
• It is essential that preparations of Noroviruses are examined
in the absence of antibody, which can mask the surface
structure and lead to incorrect identification.
• Noroviruses are commonly excreted in feces as small
aggregates and in very low numbers at 106 viruses/g ofstool.
• These concentrations are close to the limit of sensitivity of the
EM, so careful examination of preparations is necessary.

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SEROLOGICAL METHODS
• Radioimmunoassay (RIA) or ELISA are also used to detect the
virus and viral antigen in the stool.
• Both ELISA and RIA are the serodiagnostic tests frequently
used to detect specific antibodies to Norwalk virus in the
serum.
• The EIA format for detection of Noroviruses in clinical
specimens offers considerable advantages over RT-PCR.
• EIA is cheap, rapid, and simple, and thus easy to deploy in the
routine diagnostic setting.

34. 34
RT-PCR
• The characterization of the Norovirus genome has allowed the
application of molecular techniques to the diagnosis of
Norovirus infection.
• Based on RNA polymerase and capsid antigen of Norovirus.
• These techniques are not used for routine diagnosis because
of cost considerations and the need for specialized laboratory
equipment (e.g. thermal cyclers), and are thus restricted to
the research or reference laboratory.

35. Prevention and control
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36. 36
Prevention and control
• No specific treatment is available for Norwalk virus.
• No vaccine is available against the virus.
• The high infectivity of Noroviruses and the explosive
outbreaks of gastroenteritis that often occur in semiclosed
communities present a major challenge to control of infection
and require aggressive intervention.
• Measures for interrupting the various modes of transmission
must include ‘enteric’ precautions, but these alone are
ineffective and should be supplemented by measures to deal
with patients who vomit.

37. 37
• Symptomatic healthcare workers should be excluded from
contact with patients for at least 2 days after resolution of
symptoms.
• Strict personal hygiene, such as effective hand washing should
be routine
• People caring for patients should wear disposable gloves,
gowns, and masks when cleaning vomit, fecal material, or
contaminated clothing
• All potentially contaminated surfaces in toilets, bathrooms,
and rooms occupied by patients should be disinfected with a
chlorine-based product and cleaned with a hot detergent
solution.

38. 38
• Contact with other patients should be avoided
• Common-source outbreaks are well documented.
• Symptomatic food handlers have often been incriminated and
must be excluded from the workplace for at least 2 days after
resolution of symptoms.
• Decontamination of all potentially infected surfaces in the
kitchens and associated rest and toilet facilities is essential .

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• Thank you…