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Hepatitis A Virus

HAV
▸ HAV, first identified in 1973, is:

non-enveloped

spherical
◦ positive stranded RNA virus

genus hepatovirus

picornavirus family
▸ HAV strains recovered from widely separated
regions of the world are antigenically similar.
In humans, a single serotype of HAV exists.
HAV
▸ HAV is known to produce disease in humans
and non-human primates.
▸ In vitro, the wild type virus is generally

difficult to grow and no cytopathic effect is


observed.
▸ Attenuated HAV strains adapted to cell

culture have been used to develop vaccines.


▸ HAV infection induces lifelong protection

against re-infection.
Morphology
▸ HAV is among the smallest and structurally
simplest of the RNA animal viruses.
▸ The virion is non-enveloped and, with a

diameter of 27-32 nm, it is composed


entirely of viral protein and RNA.
▸ Empty capsids, abundant in faeces collected

during early infection, band at 1.20 and 1.29


- 1.31 g/cm3, with sedimentation
coefficients ranging from 50 S to 90 S,
predominantly 70 S.
Morphology
▸ Electron microscopy (EM) analyses show
particles with icosahedral symmetry although
no structural details could be discerned.
▸ Morphologically, HAV particles are

indistinguishable from other picornaviruses.


Genome and proteins
▸ The hepatitis A genome consists of a linear, single
stranded, positive-sense RNA of approximately 7.5
kb containing a 5'-nontranslated region with
complex secondary and tertiary structure.
▸ The 5'-end represents a non-coding region (NCR)
extending over 10% of the genome, it is uncapped
and covalently linked to the viral protein VPg (2.5
kD).
▸ The 3'-end terminates with a poly(A)tail of 40 - 80
nucleotides.
Genome and proteins
▸ HAV genomic replication occurs exclusively in the
cytoplasm of the infected hepatocyte by a
mechanism involving an RNA-dependent RNA
polymerase.
▸ Sequences for known human HAV isolates are
highly similar even when geographic and temporal
origins are widely separated, yet seven distinct
genotypes have been identified to date.
Antigenicity
▸ HAV has only one known serotype, and one
neutralization site is immunodominant. Different
viral strains show similar reactivity to monoclonal
anti-HAV antibodies.
▸ HAV is neutralized by both IgG and IgM.
▸ No serologic or hybridizing cross-reactivity
between HAV and other viral hepatitis agents,
including hepatitis E virus (HEV), has been
observed.
▸ The nonstructural proteins of HAV are also
immunogenic during natural and experimental
infections.
Antigenicity
▸ Antigens of the intact virion are conformational and
different from those of isolated proteins.
▸ Antibodies to purified capsid proteins or to
synthetic peptides have weak or no detectable
neutralizing activity.
▸ Hepatitis A capsids contain 60 copies of VP1 (30 to
33 kD), VP2 (24 to 30 kD) and VP3 (21 to 28 kD).
▸ Exposed parts of VP1 (residues Ser102 and Ser114)
and of VP3 (residue Asp70) on the capsid surface
define the conformational immunodominant
antigenic site of HAV.
Stability
▸ HAV has no lipid envelope and is stable when
excreted from the infected liver to the bile to enter
the gastrointestinal tract.
▸ It has been found to survive in experimentally
contaminated fresh water, seawater, wastewater,
soils, marine sediment, live oysters, and creme-
filled cookies.
▸ HAV is extremely resistant to degradation by
environmental conditions, a property that allows its
maintenance and spread within populations.
Stability
▸ HAV is resistant to:

Storage at -20°C for years

Thermal denaturation (survives at 70°C up to 10
min)

Acid treatment (pH 1 for 2 h at room temperature),
20% ether, chloroform, dichlorodifluoromethane,
and trichlorotrifluoroethane

Perchloracetic acid (300 mg/l for 15 min at 20°C)
◦ detergent inactivation (survives at 37°C for 30 min
in 1% SDS)
Stability
▸ HAV is inactivated by:
◦ heating to 85°C for 1min

autoclaving (121°C for 20 min)
◦ ultraviolet radiation (1.1 W at a depth of 0.9 cm
for 1 min)

formalin (8% for 1 min at 25°C)

ß-propriolactone (0.03% for 72 h at 4°C)

potassium permanganate (30 mg/l for 5 min)

iodine (3 mg/l for 5 min)
Stability

HAV is also inactivated by

chlorine (free residual chlorine concentration of 2.0 to
2.5 mg/l for 15 min) and

chlorine-containing compounds (3 to 10 mg/l sodium
hypochlorite at 20°C for 5 to 15 min)
▸ Shellfish from contaminated areas should
be heated to 90°C for 4 min or steamed for
90 sec
Hepatitis E Virus
HEV
▸ HEV, first identified in 1973, is:

non-enveloped

spherical
◦ positive stranded RNA virus
▸ Although originally classified within the
family of caliciviruses, they are now
unclassified.
▸ All HEV strains studied appear to comprise a

single serotype.
HEV
▸ Replication in cell culture was first reported in
1993; yields of virus are generally very low.
▸ In natural infections, the virus replicates in

hepatocytes.
▸ In vivo infected macaque hepatocytes support

HEV replication after isolation and placement


into tissue culture.
Morphology
▸ HEV is a small and structurally simple RNA
animal virus.
▸ The virion is non-enveloped and, with a

diameter of 27-34 nm, is composed entirely


of viral protein and RNA.
▸ Full virions have a buoyant density of 1.29 g/

cm3 in potassium tartrate/glycerol gradients


and a sedimentation coefficient of 183 S in
neutral sucrose gradients, empty capsids of
165 S under the same conditions.
Morphology
▸ Electron microscopy (EM) analyses show
spherical particles of possible icosahedral
symmetry, with indefinite surface
substructure, resembling the caliciviruses.
▸ Morphologically, HEV is similar to Norwalk

virus, a member of the calicivirus family,


although the sequence of HEV most closely
resembles the sequence of rubella virus, a
togavirus, and beet necrotic yellow vein virus,
a plant furovirus.
Genome and Proteins
▸ The hepatitis E genome consists of a linear, single-
stranded, positive-sense RNA (that is, mRNA) of
approximately 7.5 kb containing a 3' poly(A) tail
and short 5' and 3' non-coding (NC) regions.
▸ Three overlapping open reading frames (ORFs)
exist, and all three coding frames are used to
express different proteins.
▸ The genomes of several HEV strains from different
parts of the world have been sequenced and
compared. Overall, they appear to fall into four
major genetic groups:
Genome and Proteins
1.South-East Asian (Burmese, some Indian strains),
North and Central Asian (strains from China, Pakistan,
Kyrgyzstan, and a few from India), and North African
strains form one somewhat heterogeneous genotype,
2.the single North American (Mexico) isolate
comprises a second,
3.the US and swine isolates comprise a third and
4.a subset of isolates from China and most isolates
from Taiwan comprise a newly described fourth
group.
Genome and Proteins
▸ ORF1 (5 kb) is located towards the 5' end of the genome and
encodes a polyprotein of about 1690 amino acids that probably
undergoes post-translational cleavage into multiple nonstructural
proteins required for virus replication, including a methyltransferase,
a putative papain-like cystein protease, an RNA helicase and an
RNA-dependent RNA polymerase.

▸ ORF2 does not overlap with ORF1; it is located at the 3'-end of the
genome and encodes the principal and probably only structural
protein. It is a capsid protein of 660 amino acids (71 kDa).

▸ ORF3 begins with the last nucleotide of ORF1; it overlaps extensively


with ORF2 and is the shortest of the open reading frames, encoding
a small immunogenic 123 amino acid phosphoprotein (14.5 kDa)
which associates with the cytoskeleton, suggesting a possible role in
the assembly of virus particles.
Genome and Proteins
▸ Genetically heterogeneous isolates from several European countries
have been designated new genotypes, but should probably be
grouped with the US isolates into a large, heterogeneous group.
▸ Two novel isolates of HEV have recently been described in Argentina.
Distinct from all previously described isolates, they represent two
diverse subtypes of a new genotype of HEV.
▸ The genome of swine HEV, an animal strain of HEV, has recently
been identified and characterized.
◦ The putative capsid gene (ORF2) of swine HEV shares about 80%
sequence identity at the nucleotide level and about 92% identity at
the amino acid level with that of human HEV strains.
◦ The small ORF3 of swine HEV has about 84% nucleotide sequence
identity and about 80% amino acid identity with human HEV
strains.
Antigenicity
▸ All HEV strains studied appear to comprise a single
serotype.
▸ Western blot data indicate that type-specific (virus-
specific) epitopes exist and can be used to
differentiate serologically different isolates.
▸ Antibodies to swine HEV cross-react with capsid
antigens from strains of human HEV.
▸ No serologic or hybridizing cross-reactivity
between HEV and other viral hepatitis agents,
including hepatitis A virus (HAV), has been
observed.
Stability
▸ HEV is extremely sensitive to high salt
concentrations (CsCl).
▸ The virus is sensitive to degradation by proteolytic
enzymes.
▸ Virions remain unaltered after exposure to
trifluorotrichloroethane.
▸ HEV should be stored as cold as possible, although
it is rapidly degraded when freeze-thawed.
▸ For transportation, specimens containing HEV
should be kept frozen in dry ice (solid CO2, -70°C),
or preferably in liquid N2 (-120°C).
Stability
▸ HEV is excreted from the liver via the common bile
duct into the duodenum of the small intestine.
Survival in the gastrointestinal tract suggests
relative stability to acid and mild alkaline
conditions.
▸ The amount of infectious virions shed in the faeces
during infection is low, consistent with the low
rates of secondary spread during epidemics.
▸ Outbreaks of HEV have been successfully controlled
by chlorination of water supplies. Iodinated
disinfectants or autoclaving destroys the virus.
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