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Hepatitis A Virus _HAV_

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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:

								
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