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Hepatitis A Virus (HAV) HAV causes 'infectious/epidemic hepatitis'. Known for centuries & (wrongly) believed to be spread by aerosols. Spread by faecal-oral route - outbreaks frequently associated with consumption of shellfish. HAV is the commonest cause of acute viral hepatitis - probably something like half of all cases are due to this virus: Clinically, HAV infection is very variable: >90% childhood infections asymptomatic, 25-50% adult infections (as usual, the older you get, the worse it is). Incubation period from 10-50 days, fever, jaundice are main symptoms. 99% cases recover completely, a few cases experience permanent liver damage, fatalities ~0.1%. The virus was first isolated by Purcell in 1973. In vitro, grows in a variety of cell lines, but rather poorly. HAV is a Picornavirus, formerly classified in the genus Enterovirus. Genome studies (sequence homology) showed that it did not belong in this genus and it has been reclassified in a genus of its own: Hepatovirus Family Genus Type Species (Subfamily) Picornaviridae Enterovirus Poliovirus Rhinovirus Human rhinovirus A Hepatovirus Hepatitis A virus Cardiovirus Encephalomyocarditis virus Aphtovirus Foot-and-mouth disease virus O Parechovirus Human parechovirus Erbovirus Equine rhinitis B virus Kobuvirus Aichi virus Teschovirus Porcine teschovirus Both inactivated and attenuated vaccines are available, the inactivated form being more widely used. The availability of assays for and vaccines against HAV means that the incidence is likely to decrease in future. A combined hepatitis A and B vaccine (Twinrix® - GlaxoSmithKline Biologicals) is now licenced for use in persons aged 18 years. This consists of the antigenic components used in Havrix (HAV) and Engerix-B (HBV) vaccines. Picornaviruses Introduction: Picornaviruses are among the most diverse (more than 200 serotypes) and 'oldest' known viruses (temple record from Egypt ca. 1400 B.C.). FMDV was one of the first viruses to be recognized - Loeffler and Frosch 1898. Poliomyelitis as a viral disease was first recognized by Landsteiner and Popper, 1909 (though the virus was not isolated until the 1930's. Name: 'Pico (Greek = very small) RNA Viruses'. Molecular Biology of Picornaviruses The field of picornavirus research has exploded over the past decade, placing picornaviruses at the forefront of discovery in molecular virology and yielding a wealth of information on nearly all aspects of picornavirus biology and disease. Molecular Biology of the Picornaviruses offers an up-to-date, in-depth analysis of all major aspects of picornavirus research, providing a summary of the many significant accomplishments in picornavirus research as well as a road map of the path to future discoveries. (Amazon.co.UK) Classification: Originally based on physical properties (particle density & pH-sensitivity) & serological relatedness, more recently based on nucleotide sequence. The most recent revision of virus taxonomy has recognized nine genera within the family: Group IV: (+)sense RNA Viruses Family Genus Type Species Hosts Picornaviridae Enterovirus Poliovirus Vertebrates Rhinovirus Human rhinovirus A Vertebrates Hepatovirus Hepatitis A virus Vertebrates Cardiovirus Encephalomyocarditis virus Vertebrates Aphthovirus Foot-and-mouth disease virus O Vertebrates Parechovirus Human parechovirus Vertebrates Parechoviruses: Minireview Erbovirus Equine rhinitis B virus Vertebrates Kobuvirus Aichi virus Vertebrates Teschovirus Porcine teschovirus Vertebrates Genome: The genome consists of one s/s (+)sense RNA molecule of between 7.2kb (HRV14) to 8.5kb (FMDV). A number of features are conserved in all Picornaviruses: Genomic RNA is infectious (~1x106-fold less infectious than intact particles, although infectivity is increased if the RNA is introduced into cells by transfection) - CHARACTERISTIC OF (+)SENSE RNA VIRUSES !!! There is a long (600-1200 base) untranslated region at the 5' end (important in translation, virulence and possibly encapsidation and a shorter 3' untranslated region (50-100 bases) - important in (-)strand synthesis. The 5' UTR contains a 'clover-leaf' secondary structure known as the IRES: Internal Ribosome Entry Site (see below). The rest of the genome encodes a single 'polyprotein' of between 2100- 2400 aa's. Both ends of the genome are modified, the 5' end by a covalently attached small, basic protein VPg (~23 AA's), the 3' end by polyadenylation (polyadenylic acid sequences are not genetically coded, there is a 'polyadenylation signal' upstream of the 3' end as in eukaryotic mRNAs): Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. (Cello J, Paul AV, Wimmer E. Science 2002 297: 1016-18) Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling oligonucleotides of plus and minus strand polarity. The synthetic poliovirus cDNA was transcribed by RNA polymerase into viral RNA, which translated and replicated in a cell-free extract, resulting in the de novo synthesis of infectious poliovirus. Experiments in tissue culture using neutralizing antibodies and CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice confirmed that the synthetic virus had biochemical and pathogenic characteristics of poliovirus. Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence. Structure: The capsid consists of a densely-packed icosahedral arrangement of 60 protomers, each consisting of 4 polypeptides, VP1, 2, 3 and 4 - all derived from cleavage of the original protomer VP0, with (pseudo) T=3 packing. The particle is 27-30nm in diameter (depending on type and degree of desiccation), while the length of the genome (stretched-out) is ~2,500nm therefore the genome is tightly packed into the capsid, together with sodium or potassium ions or polyamines (in rhinoviruses) to counteract the negative charges on the phosphate groups. An electron micrograph of negatively-stained picornavirus particles. A computer generated animation of a picornavirus capsid. This image is based on the real atomic co-ordinates of rhinovirus 16 and shows a view inside the capsid. In this video: VP1: is in blue VP2: is in green VP3: is in red VP4: is in yellow (only visible on the inside of the particle) Replication: We know a great deal about Picornavirus replication due to single-step growth curve type experiments performed at high multiplicity of infection. Replication occurs entirely in the cytoplasm - it can occur even in enucleated cells and is not inhibited by actinomycin D. Receptors: The cellular receptors for several different groups of picornaviruses have been identified using a number of different techniques over the last few years: Binding competition between different viruses MAbs which block virus binding Fluorescently labelled virus (Echovirus) # Virus: Receptor: Description: Serotypes: Human ICAM-1 (Intracellular Adhesion Immunoglobulin-like 91 Rhinovirus Molecule 1, CD54) molecule; 5 domains Human 10 LDLR (Low Density Lipoprotein Receptor) Rhinovirus Immunoglobulin-like Poliovirus 3 CD155 molecule; 3 domains Coxsackie A 3 ICAM-1 Echo 2 VLA-2 Integrin-like molecule DAF (Decay Accelerating Regulation of complement Echo 6 Factor, CD55) activation Also used by: CAV21, EV70 VCAM-1 (Vascular Cell Adhesion EMCV 1 Adhesion molecule Molecule, CD106) Rossmann MG, et al. (2002) Picornavirus-receptor interactions. Trends Microbiol. 10: 324-331 The atomic structure of poliovirus-receptor complex has been described: Belnap DM et al (Hogle). Three-dimensional structure of poliovirus receptor bound to poliovirus. PNSA USA 97, 73-78 (2000); He Y et al (Rossman). Interaction of the poliovirus receptor with poliovirus. PNAS USA 97, 79-84 (2000); Rossmann, M.G. et al (2000) Cell Recognition and Entry by Rhino- and Enteroviruses. Virology 269: 239-247 The structure of serotype 1 poliovirus bound to CD155 was compared with the structure of rhinovirus bound to its cellular receptor, ICAM-1. In both cases the receptor molecule is a long molecule, sticking out from of the surface of the cell and binding to a "canyon" on the virus particle. However, in the case of the rhinovirus, ICAM-1 is a long molecule and sticks straight into the canyon, whereas CD155 lies on the surface of the virus particle along the canyon: Uncoating: After adherence to the receptor, the virus can be eluted again, but if this happens, the particle undergoes conformational changes due to the loss of VP4 and infectivity is lost - this is also the first stage in uncoating: Picornavirus-receptor interactions. Trends Microbiol. 2002 10:324-331. Translation: The kinetics of Picornavirus replication are rapid, the cycle being completed in from 5-10 (typically 8) hours. Genomic RNA is translated directly by polysomes, but ~30 min after infection, cellular protein synthesis declines sharply, almost to zero, this is called 'SHUTOFF' - the primary cause of c.p.e: Time after Event: Infection: Sharp decrease in cellular macromolecular synthesis; ~1-2h margination of chromatin (loss of homogeneous appearance of nucleus) Start of viral protein synthesis; vaculoation of cytoplasm, ~2.5-3h beginning close to nucleus & spreading outwards ~3-4h Permeabilization of plasma membrane ~4-6h Virus assembly in cytoplasm (crystals sometimes visible) ~6-10h Cell lysis; release of virus particles Shutoff of host cell translation is due to cleavage of the cellular protein eIF-4G, a component of the 220kD 'cap-binding complex' (CBC or CBP). This cleavage is carried out by enterovirus & rhinovirus 2A proteinases and the aphthovirus L proteinase. CBC is binds the m7G cap structure at the 5' end of all eukaryotic mRNAs and subsequently binds the small ribosomal subunit / tRNAmet complex during initiation of translation. The 43S initiation complex then 'scans' the 5' UTR until the first initiating AUG codon is encountered. Cleavage of eIF-4G prevents the complex binding the cap structure and the 43S complex. However: The long picornavirus 5' UTR contains an IRES: Internal Ribosome Entry Site or 'landing pad'. Normally, translation is initiated when ribosomes bind to the 5' methylated cap then scan along the mRNA to find the first AUG initiation codon. The IRES forms an elaborate secondary structure which can bind ribosomes and deliver them directly to the polyprotein initiation AUG without scanning upstream sequences - hence in a m7G cap independent mode. In picornavirus-infected cells, cleavage of eIF-4G knocks out ("shuts-off") the normal cap-dependent mode of translation of cellular genes, but does not affect picornavirus IRES-driven translation (cap independent mode). In this manner the virus shuts-off the host cell translation but leaves its own translation unaffected - a method whereby the virus can sequester the host-cells resources for its own purposes. The extent of host cell shutoff varies for different picornaviruses. For poliovirus, this is a vigorous process, with nearly all translation of cellular genes blocked. On the other hand, some strains of rhinovirus only block ~50% of translation of cellular genes blocked. The polyprotein produced is initially cleaved by the P2A protease into P1 & P2P3 peptides. Further cleavage events are carried out by 3C - the main picornavirus protease. All of these cleavages are highly specific (drug target!): Read: Barco, A. et al. (2000) Poliovirus Protease 3C pro Kills Cells by Apoptosis. Virology 266: 352-360. Genome Replication: One of the products made is the virus RNA-dependent RNA polymerase (3D), which copies the genomic RNA to produce a (-)sense strand. This forms the template for (+)strand (genomic) RNA synthesis, which occurs via a multi- stranded replicative intermediate complex (RI). The (-)ve sense cRNA serves as a template for multiple (+)ve sense strands, some of which are translated, others which form vRNA. In vitro transcription studies have suggested 2 possible models by which genome replication might occur: A recent paper shows that a long-range interaction between ribonucleoprotein (RNP) complexes formed at the ends of the poliovirus genome is necessary for RNA replication. Initiation of negative strand RNA synthesis requires a 3' poly(A) tail and a cloverleaf-like RNA structure located at the other end of the genome. An RNP complex formed around the 5' cloverleaf RNA structure interacts with the poly(A) binding protein bound to the 3' poly(A) tail, linking the ends of the viral RNA and effectively circularizing it. Formation of this circular RNP complex is required for initiation of negative strand RNA synthesis. RNA circularization may be a general replication mechanism for positive stranded RNA viruses. (Herold J, Andino R. Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol Cell 7: 581-591, 2001) Assembly: RNA is believed(?) to be packaged into preformed capsids, although the molecular interactions between the genome & the capsid responsible for this process are not clear. Empty capsids (defective) are common in all Picornavirus infections. The capsid is assembled by cleavage of the P1 polyprotein precursor into a protomer consisting of VP0,3,1 which join together enclosing the genome: Maturation: Maturation (& infectivity) relies on an internal autocatalytic (?) cleavage of VP0 into VP2 + VP4. Release: Release (in most cases) on the virus from the cytoplasm occurs when the cell lyses - probably a 'preprogrammed' event which occurs a set time after the cessation of 'housekeeping' macromolecular synthesis at shutoff. (Hepatitis A virus is relatively non-lytic & sets up a more persistent infection). Enteroviruses Enterovirus infections are common in humans; seasonal peak in autumn; frequently undiagnosed: Species: Serotypes: Bovine enterovirus 2 serotypes Human enterovirus A (coxsackie A viruses) 10 serotypes Human enterovirus B (coxsackie B viruses, echoviruses) 36 serotypes Human enterovirus C (coxsackie A viruses) 11 serotypes Human enterovirus D 2 serotypes Poliovirus 3 serotypes Porcine enterovirus A 1 serotype Porcine enterovirus B 2 serotypes Unassigned: 22 serotypes Total: 89 serotypes Enteroviruses account for an estimated 10-15 million symptomatic infections in the United States alone each year. Recently, a drug has been developed which has activity against enteroviruses and rhinoviruses. Pleconaril is a novel drug that inhibits viral replication by blocking viral uncoating, viral attachment to host cell receptors, and transmission of infectious virions, with broad-spectrum anti-EV and anti-RV activity and is high bioavailablity when administered orally. Polioviruses: To view a high resolution computer-generated image reconstruction of a poliovirus particle, click here. Note the icosahedral symmetry which is clearly visible in this image. These are the prototypic Picornaviruses; there are 3 distinct serotypes. They cause poliomyelitis (flaccid muscular paralysis). As with all the Enteroviruses, they are transmitted by the faecal-oral route. Primary site of infection is lymphoid tissue associated with the oropharynx and gut (GALT). Virus production at this site leads to a transient viraemia, following which the virus may infect the CNS. This is of interest because of this apparent 'dual tropism' of the virus for two distinct cell types - reflects the distribution of the poliovirus receptor CD155 on cells lymphoid/ epithelial cells in the gut and on neurons in the CNS. Replication of the virus in the CNS occurs in the 'grey matter', particularly motor neurones in the anterior horns of the spinal cord and brain stem. Distinctive 'plaques' produced in the grey matter are due to lytic replication of the virus & probably inflammation caused by an over- enthusiastic immune response. ~1% of people infected with the most virulent strains experience paralysis (99% asymptomatic infections). Death is usually due to respiratory failure by paralysis of the intercostal muscles and diaphragm. Effective polyvalent vaccines are available against polioviruses - OPV/IPV. In 1988, the World Health Assembly established the year 2000 for achieving global poliomyelitis eradication. By 1994, the Americas were certified as polio-free. All other regions are making steady progress towards the goal of global eradication, which is now scheduled for 2008:
"Hepatitis A Virus _HAV_"