Poxviruses

Introduction:

The largest and most complex viruses known! Have been known about for centuries - the characteristic "pocks" produced by variola virus (Smallpox) giving their name to all forms of infectious disease "a dose of the pox". Smallpox first appeared in China and the Far East at least 2000 years ago.

The Pharaoh Ramses V (left) died of smallpox in 1157 B.C. The disease reached Europe in 710 A.D. and was transferred to America by Hernando Cortez in 1520 - 3.5 million Aztecs died in the next 2 years. In the cities of 18th century Europe, smallpox reached plague proportions and was a feared scourge - highly infectious. Five reigning European monarchs died from smallpox during the 18th century.

Smallpox has now been eradicated - the last naturally occurring outbreak of smallpox was in Somalia on 26th October 1977.

Taxonomy:

Subfamily: Genus: Members:
Chordopoxvirinae Avipoxvirus fowlpox virus
Capripoxvirus sheeppox virus
Leporipoxvirus myxoma virus
Molluscipoxvirus Molluscum contagiosum
Orthopoxvirus vaccinia virus
Parapoxvirus orf virus
Suipoxvirus swinepox virus
Yatapoxvirus Yaba monkey tumor virus
Entomopoxvirinae Entomopoxvirus A Melolontha melolontha entomopoxvirus
Entomopoxvirus B Amsacta moorei entomopoxvirus
Entomopoxvirus C Chironomus luridus entomopoxvirus

Morphology:

 Oval or "brick-shaped" particles 200-400nm long - can be visualized by the best light microscopes (just). The external surface is ridged in parallel rows, sometimes arranged helically. The particles are extremely complex, containing many proteins (more than 100) and detailed structure is not known.
The extracellular forms contain 2 membranes (EEV - extracellular enveloped virions), intracellular particles only have an inner membrane (IMV - intracellular mature virions).


Thin sections in E.M. reveal that the outer surface is composed of lipid and protein which surrounds the core, which is biconcave (dumbbell-shaped), with 2 "lateral bodies" (function unknown). The core is composed of a tightly compressed nucleoprotein. To view an electron micrograph of a negatively-stained poxvirus particle click here.
Antigenically, poxviruses are very complex, inducing both specific and cross-reacting antibodies - hence ability to vaccinate against one disease with another virus (below). There are at least 10 enzymes present in the particle, mostly concerned with nucleic acid metabolism/genome replication.

Genome:

Linear, d/s DNA of 130-300kbp. Ends of genome consist of a terminal hairpin loop (no free ends) with several tandem (i.e. direct) repeat sequences (this arrangement is found at the ends of chromosomes from a number of different organisms). The ends of the genome form direct repeats called inverted terminal repeats (ITRs). Several poxvirus genomes have been sequenced. Most of the essential genes are located in the central part of the genome, while non-essential (in tissue culture) genes are located at the ends. There are ~200 genes in the genome.

Replication:

 Occurs in the cytoplasm - the virus is sufficiently complex to have acquired all the functions necessary for genome replication (c.f. Herpesviruses). There is some contribution from the cell but it is not clear what this is - poxvirus gene expression and genome replication occur in enucleated cells, but maturation is blocked.

Receptors are not known, but probably >1 on different cell types. For Vaccinia, one of these is probably the EGF receptor (epidermal growth factor).
Penetration is complex and may also involve >1 mechanism.
Uncoating occurs in two stages, removal of the outer membrane as the particle enters the cell and in the cytoplasm, the particle (minus its outer membrane) is further uncoated and the core passes into the cytoplasm.
Gene expression is carried out (exclusively?) by viral enzymes associated with the core and is divided into 2 phases:
Early genes: ~50% genome, expressed before genome replication
Late genes: expressed after genome replication; late promoters are dependent on DNA replication for activity.
Poxvirus gene expression has been studied in detail because of the interest in the use of Vaccinia virus as a vector for expression of heterologous genes.
Genome replication is believed to involve self-priming, leading to the formation of high m.w. concatemers (isolated from infected cells) which are subsequently cleaved and repaired to make virus genomes. The many virus-encoded enzymes involved in replication (e.g. thymidine kinase - tk) offer potential targets for chemotheraputic agents.
Assembly occurs in the cytoskeleton; the events involved in putting together such a complex particle are not understood, but probably involve interactions with the cytoskeleton (e.g. actin-binding proteins). Inclusions are formed in the cytoplasm which mature into virus particles. Actin 'comet tails' form which shoot IEV through the cytoplasm to the cell surface, and possibly into adjacent cells - these have been timed moving at 3µm/min. This may provide an alternative mechanism for cell to cell spread (c.f. EEV).
Overall, replication of this large, complex virus is rather quick ~12h.

Vaccinia virus shows considerable resistance to the antiviral effects of interferons. One of the early genes of this virus, K3L, encodes a protein which is homologous to eIF-2a which inhibits the action of PKR. In addition, the E3L protein also binds dsRNA & inhibits PKR activation. As well as these, other poxvirus-encoded proteins interfere with the actions of complement, IL-1 and TNFs. Vaccinia infection of cells can confer "protection" from IFN on other viruses, e.g. Picornaviruses.

Variola and Vaccinia:

At least 9 different poxviruses cause disease in humans, but variola virus (VV) and vaccinia are the best known. VV strains are divided into variola major (25-30% fatalities) and variola minor (same symptoms but less than 1% death rate). "Variolation" = the administration of material from known smallpox cases (hopefully variola minor!!!) to protect recipients - practiced for at least 1000 years (Chinese) but risky - Jenner was nearly killed by variolation in 1756!

The precise origins of vaccinia virus are uncertain:
Edward Jenner, 14th May 1796, used cowpox (Sarah Nemes!) to "vaccinate" 8 year old James Phipps, who he later challenged with VV (ethical?) and showed that he was protected. For more than 100 years, the "vaccine strains" (many origins) were propagated from arm-to-arm, but for at least the last 50 years, Vaccinia has been a distinct virus from cowpox - origin? (In molecular terms, vaccinia is most similar to buffalopox - did the two viruses somehow become mixed at some stage?).

Vaccination was almost universally adopted worldwide around 1800, but it took a major commitment from the WHO in 1965 to achieve eradication.
Eradication of smallpox was possible for 3 reasons:

1) There is no other reservoir for VV but man (including primates) (c.f. Arboviruses)
2) VV causes only acute infections, from which the infected person either:
a) dies
b) recovers with life-long immunity (c.f. Herpesviruses)
3) Vaccinia virus is an effective immunogen.
Infection with both viruses occurs naturally by the respiratory route (high transmission rate!!!) and is systemic, producing a variety of symptoms, but most notably with VV characteristic pustules and scarring of the skin. Replication of vaccinia is more localized. Administration of vaccinia to immunocompromised hosts results in systemic infection with neurological damage or death.

Online tutorial on smallpox

Unfortunately, monkeypox shows every sign of an emerging virus disesae about to replace smallpox.

Expression Vectors:


Makes use of homologous recombination in infected cells to introduce foreign DNA coupled to VV promoter (e.g. tk) into virus genome. Numerous variants have now been made, some which include indicator systems such as beta-galactosidase to give recombinant plaques a blue colour (c.f. M13).
Problems for human vaccines:

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© AJC 1997