Picornaviruses
MicrobiologyBytes: Latest Updates
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)
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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:
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 image 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: |
# Serotypes: |
Receptor: |
Description: |
| Human Rhinovirus |
91 |
ICAM-1 (Intracellular Adhesion Molecule 1, CD54) |
Immunoglobulin-like molecule; 5 domains |
| Human Rhinovirus |
10 |
LDLR (Low Density Lipoprotein Receptor) |
| Poliovirus |
3 |
CD155 |
Immunoglobulin-like molecule; 3 domains |
| Coxsackie A |
3 |
ICAM-1 |
| Echo |
2 |
VLA-2 |
Integrin-like molecule |
| Echo |
6 |
DAF (Decay Accelerating Factor, CD55)
Also used by: CAV21, EV70 |
Regulation of complement activation |
| EMCV |
1 |
VCAM-1 (Vascular Cell Adhesion Molecule, CD106) |
Adhesion molecule |
Rossmann
MG, et al. (2002) Picornavirus-receptor interactions. Trends Microbiol. 10: 324-331
Imaging
Poliovirus Entry in Live Cells. 2007 PLoS Biol. 5 (7): e183
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:
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 Infection: |
Event: |
| ~1-2h |
Sharp decrease in cellular macromolecular synthesis; margination of chromatin
(loss of homogeneous appearance of nucleus) |
| ~2.5-3h |
Start of viral protein synthesis; vaculoation of cytoplasm, 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:
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Species:
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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 |
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Total:
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89 serotypes
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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:
MicrobiologyBytes: Latest Updates
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.
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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.
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~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:
UK
Department of Health current vaccination guidelines | Global
Polio Eradication Initiative
Latest:
Coxsackieviruses:
- Algonquin indian name of village in N.Y. where first isolated (Daldorf and
Sickles/suckling mice/1948). Two groups, based on pathology in suckling mice:
- Group A: Cause acute myositis (muscular inflammation) with inflammation
and necrosis. 24 serotypes.
- Group B: Cause degenerative 'plaques' in brain, muscle and pancreas
(model for induced diabetes in mice). 6 serotypes.
- Coxsackievirus B4 (CB4) damages tissue, which causes the release of sequestered
islet antigen. This in turn stimulates autoreactive T cells and results in
the initiation of insulin-dependent diabetes mellitus.
(Horwitz
MS, et al. Presented antigen from damaged pancreatic beta cells activates autoreactive
T cells in virus-mediated autoimmune diabetes. J Clin Invest 2002, 109: 79-87)
- In man, these viruses show a seasonal, epidemic pattern of infection (mostly
sub-clinical), associated with meningitis, paralysis (usually less severe
than acute poliomyelitis), myocarditis, etc. These are common infections world-wide
(inc. UK) - no effective treatment/prophylaxis! Coxsackie A16 (CAV16) causes
the common childhood infection hand-foot-mouth
disease (no, not foot and mouth: hand-foot-mouth). CAV21 appears to be
a recombinant between a rhinovirus (P1) and poliovirus (P2-P3).
Echoviruses:
Enteric Cytopathic Human Orphan
viruses; not linked to any human disease (hence 'orphan'). 32 serotypes (echo
10 = reovirus 1; echo
28= HRV1A). Common cause of enteric infections.
'New' Enteroviruses:
Since 1969, 'new' Enteroviruses have been assigned numbers, not names:
Cause of 'the common cold' (but not the only one!). ~105
serotypes (hence repeated infections). Relatively fragile viruses (c.f. Enteroviruses),
with
optimum growth temperature of 33°C (URT infection). Extensive human volunteer
studies show no evidence for susceptibility when exposed to cold/wet conditions
(!) although general immune status is probably important. Relatively little
c.p.e., although this varise from strain to strain:
Uninfected primary human fibroblasts
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Primary human fibroblasts infected with human rhinovirus
16
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Many HRV types grow very poorly in vitro. Replicate in ferrets
- other animal reservoirs? Symptoms are due to damage
to ciliated epithelium in the URT, of little consequence in itself, but
predisposes to secondary bacterial infections - a major problem in infants and
elderly: 5-10% of viral upper respiratory tract infections progress to bacterial
sinusitis.
In addition, rhinoviruses are a major economic pest world-wide
(lost working days). There is little or no cross-protection between serotypes.
Protection relies on levels of secreted Ab in URT - may be relatively short-lived
(e.g. a few years rather than lifelong).
Pliny
the Elder (Gaius Plinius Secundus, 23-79 AD) recommended 'kissing the hairy
muzzle of a mule as a cure for colds (Natural History, Book 30). OTOH, Pliny
also recommended:
- placing the right foot of a hyaena on a woman during childbirth
- powdered sow's dung to relieve labour pains, or goose semen
mixed with water or "the liquids that flow from a weasel's uterus through
its genitals"
so judge for yourselves...
There is little or no cross-protection between serotypes, therefore currently
no prospects for vaccines. Protection relies on levels of secreted antibody
in URT - may be relatively short-lived (e.g. years rather than life-long).
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.
This
is the group of viruses responsible for foot-and-mouth disease (FMD) - a major
economic pest world-wide. The disease is is endemic in parts of Asia, Africa,
the Middle East and South America, with sporadic outbreaks in other areas. For
the last few years, a pandemic of type O has been raging in many countries,
and in February 2001, this spread to the UK, possibly from S.Africa. Controlled
largely by vaccination or slaughter of infected animals (in E.U.).
Unfortunately, presently available (inactivated) vaccines are not entirely
effective. Vaccination blocks disease symptoms (making detection of infection
difficult) but does not always block transmission of the virus to other animals.
Sheep can harbour the virus for several months, cows for up to a year or even
longer. Occasional vaccine-linked disease outbreaks occur as a result.
Aphthoviruses are physically quite distinct from other Picornaviruses:
The virus is found in breath, saliva, faeces, urine, milk and semen of infected
animals as well as meat and by-products in which have remained above pH 6.0.
Infection causes:
- Vesicles or blisters on the tongue, gums, cheek, palate, lips, nostrils,
muzzle, teats, udder, snout and hooves.
- Pyrexia, anorexia, shivering, reduction in milk production for 2-3 days,
grinding of the teeth, drooling, lameness, stamping or kicking of the feet.
- Death of young stock.
Clinically indistinguishable from:
- Vesicular stomatitis (Rhabdovirus)
- Swine vesicular disease (Enterovirus - very similar to human coxsackievirus
B5)
- Vesicular exanthema of swine (Calicivirus)
Very similar to:
Affects:
- Bovidae (cattle, zebus, domestic buffaloes, yaks), sheep, goats, swine,
all wild ruminants and pigs.
- Camelidae (camels, dromedaries, llamas, vicunas) have low susceptibility.
- Horses may be carriers but do not become sick.
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Virus is highly infectious and can persist in contaminated fodder and the
environment for up to 1 month, depending on the temperature and pH. FMD is
one of the most contagious animal diseases. Transmission occurs by:
- Direct or indirect contact (droplets)
- Animate vectors (humans, etc)
- Inanimate vectors (vehicles, implements)
- Airborne, especially temperate zones (up to 60 km overland and 300 km
by sea - e.g. 1967 UK outbreak spread from France to Isle of Wight)
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| Can humans catch foot and mouth?
Yes - just:
Foot and mouth disease is a zoonosis, a disease
transmissible to humans, but it crosses the species barrier with difficulty
and with little effect. Given the high incidence of the disease in animals,
both in the past and in more recent outbreaks worldwide, its occurrence in
man is rare so experience of the human infection is limited. The last human
case reported in Britain occurred in 1966, during the last epidemic of foot
and mouth disease. The circumstances in which it does occur in humans are
not well defined, though all reported cases have had close contact with infected
animals. There is one report from 1834 of three veterinarians acquiring the
disease from deliberately drinking raw milk from infected cows. There is
no report of infection from pasteurised milk, and the Food Standards Agency
considers that foot and mouth disease has no implications for the human food
chain. The type of virus most often isolated in humans is type O followed
by type C and rarely A. The incubation period in humans is 2-6 days. Symptoms
have mostly been mild and self limiting, mainly uncomfortable tingling blisters
on the hands but also fever, sore throat, and blisters on the feet and in
the mouth, including the tongue. Patients have usually recovered a week after
the last blister formation. Foot and mouth disease should not be
confused with the human disease hand, foot, and mouth disease. This is an
unrelated and usually mild viral infection, principally of children, caused
by different viruses, principally coxsackie A virus. BMJ
322: 565-566, 2001.
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The UK was finally declared oficially free from FMDV again in January 2002,
11 months after the beginning of the outbreak.
The genus currently comprises of two virus species:
- Encephalomyocarditis virus. One serotype. Includes encephalomyocarditis
virus (EMCV) (model infection of mice), mengovirus, Maus-Elberfield virus,
Columbia virus - all considered to be strains of EMCV (really a mouse virus,
but can infect man, elephants, squirrels...).
- Theilovirus: Theiler's murine encephalomyelitis
virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV) & a few others.
The cardioviruses are most closley related to the aphthoviruses and equine
rhinoviruses. Genome size ~7.8kb; 5' non-translated region contains poly-C tract
of ~100-170nt (like Aphthoviruses).
© MicrobiologyBytes 2007.
