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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 recognised - Loeffler and Frosch 1898. Poliomyelitis as a viral disease was first recognised by Landsteiner and Popper, 1909 (though the virus was not isolated until the 1930's.
Name: 'Pico (Greek very small ) RNA Viruses'.

Classification:

Originally based on physical properties (particle density & pH-sensitivity) & serological relatedness, more recently based on nucleotide sequence. There are 5 genera:

Aphthovirus 7 serotypes
Cardiovirus 2 serotypes
Enterovirus 111 serotypes
Hepatovirus 2 serotypes (1 human, 1 simian)
Rhinovirus 105 serotypes
Unassigned 3 serotypes
Total: ~230 viruses

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:

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. To view an electron micrograph of negatively-stained picornavirus particles, click here.

To view a computer generated animation of a picornavirus capsid, click here. This image is based on the real atomic co-ordinates of rhinovirus 16 and shows a view inside the capsid. In this video:

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:

Virus: # Serotypes: Receptor: Description:
Human Rhinovirus 91 ICAM-1 (Intracellular Adhesion Molecule 1) 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) ???
EMCV 1 VCAM-1 (Vascular Cell Adhesion Molecule) ???

Uncoating:

After adherence to the receptor, the virus can be eluted again, but if this happens, the particle undergos 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 appears to be due to cleavage of the 220kD 'cap-binding complex' (CBC) involved in binding the m7G cap structure at the 5' end of all eukaryotic mRNAs during initiation of translation. This is carried out by poliovirus protein 2A.
The 5' UTR contains the IRES: Internal Ribosome Entry Site or 'landing pad'. Normally, translation is initated when ribosomes bind to the 5' methylated cap then scan along the mRNA to find the first AUG initiation codon. The IRES overcomes this & allows picornavirus RNAs to continue to be translated after degradation of CBC.
The polyprotein is initially cleaved by P2A into P1 & P2P3. Further cleavage events are carried out by 3C - the main picornavirus protease. All of these cleavages are highly specific (drug target!):

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). In vitro transcription studies have suggested 2 possible models by which genome replication might occur:

The (-)ve sense cRNA serves as a template for multiple (+)ve sense strands, some of which are translated, others which form vRNA.

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:

Polioviruses 3 serotypes
Coxsackieviruses group A 23 serotypes
Coxsackieviruses group B 6 serotypes
Echoviruses 31 serotypes
Enteroviruses 38 serotypes
Total: 111 serotypes

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 - lymphoid/ epithelial cell in the gut and neurons in the CNS - different receptors, etc.

Replication of the virus in the CNS occurs in the 'grey matter', particularly motor neurons 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 this goal. The WHO reports that global eradication of poliomyelitis is still on course for the year 2000.

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.
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. Common infections worldwide (inc. UK) - no effective treatment/prophylaxis!

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 asymptomatic (?) enteric infections.

'New' Enteroviruses:

Since 1969, 'new' Enteroviruses have been assigned numbers, not names:

Type: Illness:
68 Pneumonia
69 None (?)
70 Acute haemorrhagic conjunctivitis (1969-1974 pandemic)
71 Meningitis
72 Hepatitis A virus (now a separate genus: Hepatovirus)

Rhinoviruses

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. Little c.p.e. Many types grow very poorly in vitro. Replicate in ferrets - other animal reservoirs? Symptoms due to damage to cilliated epithelium in URT. Little consequence in itself, but predisposes to secondary bacterial infections - a major problem in infants and elderly. In addition, a major economic pest worldwide (lost working days).
No effective prophylaxis or treatment; (Pliny the Younger recommended 'kissing the hairy muzzle of a mouse') - in spite of extensive molecular knowledge. 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 life-long).
To view a computer generated animation of a rhinovirus particle, click here. This image is based on the real atomic co-ordinates of rhinovirus 14. The antigenic sites on the surface of this particle are highlighted in purple. Note how the distribution of the antigenic sites on the capsid emphasises its icosahedral symmetry.
Medscape Article: What's New With Common Colds? Complications and Management.

Apthoviruses

This is the group of viruses responsible for foot-and-mouth disease (FMD) - a major economic pest worldwide, especially in S.America and Australasia. Controlled largely by vaccination (inactivated vaccine - occasional vaccine-linked outbreaks) or slaughter of infected animals. They are physically quite distinct from other Picornaviruses: To view a high resolution computer-generated image reconstruction of a FMDV particle, click here.

Cardioviruses

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...). Genome size ~7.8kb; 5' non-translated region contains poly-C tract of ~100-170nt (like Apthoviruses).

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