MicrobiologyBytes: Virology: Picornaviruses Updated: September 11, 2007 Search

Picornaviruses

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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'.

CoverMolecular 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
Parechoviruses: Minireview

Human parechovirus

Vertebrates

Erbovirus Equine rhinitis B virus Vertebrates
Kobuvirus Aichi virus Vertebrates
Teschovirus Porcine teschovirus Vertebrates

Classification of Picornaviruses

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:

Picornavirus genome

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:

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.

Picornavirus replication

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, 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:

Picornavirus particles

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:

Receptor binding

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 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!):Picornavirus genome expression

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:

Genome replication

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:
Poliovirus assembly

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.

PleconarilRecently, 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:

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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.

Bodian's classic diagram of polio pathogenesis 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:

UK Department of Health current vaccination guidelines     |    Global Polio Eradication Initiative

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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:

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

MMWR: Enterovirus Surveillance - United States, 1970--2005

(Human) Rhinoviruses (HRV)

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 cells
Uninfected primary human fibroblasts
HRV-infected cells
Primary human fibroblasts infected with human rhinovirus 16

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 ElderPliny 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:

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.

 

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Aphthoviruses

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.

 

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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:

Clinically indistinguishable from:

Very similar to:

Affects:


© Russell Kightley Media

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)

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.

The UK was finally declared oficially free from FMDV again in January 2002, 11 months after the beginning of the outbreak.

 

Cardioviruses

The genus currently comprises of two virus species:

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.