HAV Structure

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Hepatitis A Virus

Transcript of HAV Structure

Hepatitis A Virus

HAV

HAV, first identified in 1973, is:

HAV strains recovered from widely separated regions of the world are antigenically similar. In humans, a single serotype of HAV exists.

non-enveloped spherical positive stranded RNA virus genus hepatovirus picornavirus family

HAVHAV 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.

MorphologyHAV 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.

MorphologyElectron 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 cremefilled 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 -20C for years Thermal denaturation (survives at 70C 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 20C) detergent inactivation (survives at 37C for 30 min in 1% SDS)

Stability

HAV is inactivated by:

heating to 85C for 1min autoclaving (121C for 20 min) ultraviolet radiation (1.1 W at a depth of 0.9 cm for 1 min) formalin (8% for 1 min at 25C) -propriolactone (0.03% for 72 h at 4C) 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 20C for 5 to 15 min)

Shellfish from contaminated areas should be heated to 90C for 4 min or steamed for 90 sec

Hepatitis E Virus

HEV

HEV, first identified in 1973, is:

Although originally classified within the family of caliciviruses, they are now unclassified. All HEV strains studied appear to comprise a single serotype.

non-enveloped spherical positive stranded RNA virus

HEVReplication 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.

MorphologyHEV 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.

MorphologyElectron 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, singlestranded, 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 Proteins1.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 (virusspecific) 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, -70C), or preferably in liquid N2 (-120C).

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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