DNA Virus Infections - Poxviruses

Introduction: Poxciruses 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 two "lateral bodies" (function unknown). The core is composed of a tightly compressed nucleoprotein.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 ~250 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). McFadden G. Poxvirus tropism. Nat Rev Microbiol. 3: 201-213, 2005

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-2α which inhibits the action of PKR. In addition, the E3L protein also binds dsRNA and 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.

Members of the poxvirus family were the first DNA viruses shown to encode secreted versions of cellular cytokine receptors. Virus cytokine binding proteins are generally detected as either secreted glycoproteins or as transmembrane cell surface proteins. Virus cytokine receptor mimics (known as viroceptors) are soluble or membrane-bound proteins that may share significant homology to the ligand binding domains of cellular cytokine receptors and function by sequestering cytokine ligands from interacting to their cognate cellular receptors. Although many virus cytokine binding proteins were previously identified by their sequence relationship to cellular proteins, some virus-encoded soluble proteins have been identified which bind to and inhibit cytokines with high affinity but do not share any sequence identity to known cellular cytokine or immunomodulatory receptors.

Virus:	Open Reading Frame:	Function:
Myxoma virus	M-T1	secreted CC-chemokine binding protein
Myxoma virus	M-T7	secreted IFN-gamma receptor homologue and CC-,CXC-, C-chemokine binding protein
Myxoma virus	M-T2	secreted TNF receptor homologue
Vaccinia virus	B15R	secreted IL-1b receptor homologue
Vaccinia virus	B18R	secreted IFNalpha/beta binding protein
Tanapox virus	???	secreted IL-2/IL-5/IFN-gamma binding protein

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?

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 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. The vaccine is contraindicated in:

• Eczema, atopic dermatitis or other acute, chronic, or exfoliative skin conditions
• Diseases or treatments which cause immunodeficiency or immunosuppression
• Moderate or severe acute illnesses
• Previous allergic reaction to smallpox vaccine or any of the vaccine components
• Pregnancy
• Breastfeeding mothers
• Infants and children under 12 months of age

Aside for the development of new vaccines, future responses to poxvirus infection are likely to include immune-based therapies such as humanized antibodies or fragments of antibodies produced in animals, and antiviral drugs such as Cidofovir, a DNA polymerase inhibitor developed for Cytomegalovirus retinitis, which has been found to be active in preventing Variola infection in cultured cells and protects mice against lethal virus challenge.