Taxonomic Structure of the Family
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Virions are somewhat pleomorphic, generally either brick-shaped (220-450 nm long 140-260 nm wide 140-260 nm thick) with a lipoprotein surface membrane displaying tubular or globular units (10-40 nm). They can also be ovoid (250-300 nm long 160-190 nm diameter) with a surface membrane possessing a regular spiral filament (10-20 nm in diameter) (Fig. 1).
Negative contrast images show that the surface membrane encloses a biconcave or cylindrical core that contains the genome DNA and proteins organized in a nucleoprotein complex (Fig. 1). One or two lateral bodies appear to be present in the concave region between the core membrane and the surface membrane. The initial intracellular mature virions (IMV) are enveloped by a membrane. Some IMV virions acquire a Golgi-derived envelope which contains additional virus-specific proteins. Some vertebrate viruses (e.g. Cowpox virus) may also be sequestered within inclusion bodies. Others (e.g., entomopoxviruses) may be occluded.
Physicochemical and Physical Properties
Particle weight is about 5 10-15 g. S20w is about 5000S. Buoyant density of virions is subject to osmotic influences: in dilute buffers it is about 1.16 g/cm3, in sucrose about 1.25 g/cm3, in CsCl and potassium tartrate about 1.30 g/cm3. Virions tend to aggregate in high salt solution. Infectivity of some members is resistant to trypsin. Some members are insensitive to ether. Generally, virions are sensitive to common detergents, formaldehyde, oxidizing agents, and temperatures greater than 40°C. The virion surface membrane is removed by nonionic detergents and sulfhydryl reagents. Virions are relatively stable in dry conditions at room temperature; they can be lyophilized with little loss of infectivity.
Nucleic acids constitute about 3% of the particle weight. The genome is a single, linear molecule of covalently-closed, dsDNA, 130-375 kbp in length.
Proteins constitute about 90% of the particle weight. Genomes encode 150-300 proteins depending on the species; about 100 proteins are present in virions. Virus particles contain many enzymes involved in DNA transcription or modification of proteins or nucleic acids. Enveloped virions have viral encoded polypeptides in the lipid bilayer, which surrounds the particle. Entomopoxviruses may be occluded by a virus-coded, major structural protein, spheroidin. Similarly, orthopoxviruses may be within inclusion bodies again consisting of a single protein.
Lipids constitute about 4% of the particle weight. Enveloped virions contain lipids, including glycolipids that may be modified cellular lipids.
Carbohydrates constitute about 3% of the particle weight. Certain viral proteins, e.g., hemagglutinin in the envelope of orthopoxviruses, have N- and C-linked glycans.
Genome Organization and Replication
The poxvirus genome comprises a linear molecule of dsDNA with covalently closed termini; terminal hairpins constitute two isomeric, imperfectly paired, “flip-flop” DNA forms consisting of inverted complementary sequences. Variably sized, tandem repeat sequence arrays may or may not be present near the ends (Fig. 2). Replication takes place predominately if not exclusively within the cytoplasm (Fig. 3). Entry into cells of intracellular virus (IMV) and extracellular enveloped virus (EEV) is suggested to be via different pathways. After virion adsorption, IMV entry into the host cell is by fusion between the plasma membrane after which cores are released into the cytoplasm and uncoated further. EEV entry, unlike IMV, may necessitate fusion with endosomal membranes to release the core.
Polyadenylated, capped primary mRNA transcripts, representing about 50% of the genome, are initially synthesized from both DNA strands by enzymes within the core, including a virus-encoded multisubunit RNA polymerase; transcripts are extruded from the core for translation by host ribosomes. During synthesis of early proteins, host macromolecular synthesis is inhibited. Virus reproduction ensues in the host cell cytoplasm, producing basophilic (B-type) inclusions termed “viroplasms” or “virus factories”. The genome contains closely spaced ORFs, lacking introns, some of which may partially overlap preceded by virus-specific promoters that temporally regulate transcription of three classes of genes. One class, the early genes, are expressed from partially uncoated virions prior to DNA replication (these encode many non-structural proteins, including enzymes involved in replicating the genome and modifying DNA, RNA, and proteins designed to neutralize the host response). Early genes also encode intermediate transcription factors. Intermediate genes (which encode late transcription factors), are expressed during the period of DNA replication and are required for subsequent late gene transcription. Finally, late genes are expressed during the post-replicative phase (these mainly encode virion structural proteins but also early transcription factors). Despite a cytoplasmic site of replication, there is mounting evidence for the requirement of host nuclear proteins in post-replicative transcription. The mRNAs are capped, polyadenylated at the 3 termini, but not spliced. Many intermediate, late and some early mRNAs have 5-poly(A) tracts, which precede the encoded mRNA. Early protein synthesis is generally decreased during the transition to late gene expression, but some genes are expressed from promoters with both early and late activity. Certain proteins are modified post-translationally (e.g., by proteolytic cleavage, phosphorylation, glycosylation, ribosylation, sulfation, acylation, palmitylation and myristylation). Proteolytic cleavage of late proteins is required for virion morphogenesis.
The replication of the DNA genome appears to be mainly through the action of viral enzymes. DNA replication appears to involve a self-priming, unidirectional, strand displacement mechanism in which concatemeric replicative intermediates are generated and subsequently resolved via specific cleavages into unit length DNAs that are ultimately covalently closed. Genetic recombination within genera has been shown, and may occur between daughter molecules during replication. Non-genetic genome reactivation generating infectious virus has been shown within and between genera of the Chordopoxvirinae.
Virus morphogenesis begins following DNA replication and expression of early, intermediate and late genes. Particle assembly is initiated with the formation of crescent-shaped membrane structures. Replicated, concatameric DNA is resolved into unit genomes and packaged into immature virion particles, which further mature to form intracellular mature virions (IMVs) which are fully infectious if liberated from cells. A portion of the IMVs are further enveloped by modified Golgi membranes, transported to the periphery of the cell where fusion of the wrapped virions with the plasma membrane ultimately results in release of extracellular enveloped virions (EEVs) by an as yet incompletely understood process. Enveloped virions thereby acquire host cell lipids and additional virus-specific proteins, including the virus hemagglutinin protein. The envelope is closely positioned to the surface membrane. While both IMVs and EEVs are infectious, the external antigens on the two virus forms are different and upon infection, the two forms of virus bind to different cellular receptors and are likely uncoated by different mechanisms. Virus DNA and several proteins are organized as a nucleoprotein complex within the core of all infectious virions. The IMVs contain an encompassing surface membrane, lateral bodies, and the nucleoprotein core complex (see Fig. 1). For Vaccinia virus, the core has a 9 nm thick membrane with a regular subunit structure. Within the vaccinia virion, negative stain indicates that the core assumes a biconcave shape (Fig. 1) apparently due to the large lateral bodies, although some evidence suggests the shape may represent an artifact of sample preparation. The lipoprotein surface membrane surrounding the Vaccinia virus core and lateral bodies is about 12 nm thick and contains irregularly shaped surface tubules composed of small globular subunits. During natural infections, the virus is likely spread by that population of virus released from the cells (EEV). Although the internal structure of Vaccinia virus is revealed in thin sections, the detailed internal structure of parapoxvirus particles is less evident (Fig. 1). In negatively stained preparations of parapoxviruses, superimposition of dorsal and ventral views of the surface filament sometimes produces a distinctive “criss-cross” surface appearance.
Within each genus of the subfamily Chordopoxvirinae there is considerable serologic cross-protection and cross-reactivity. Neutralizing antibodies are genus-specific. The nucleoprotein antigen, obtained by treatment of virus suspensions with 0.04 M NaOH and 56°C treatment of virus suspensions, is highly cross-reactive among members. Orthopoxviruses have hemagglutinin antigens, although this is rare in other genera.
Transmission of various member viruses of the subfamily Chordopoxvirinae occurs by (1) aerosol, (2) direct contact, (3) arthropods (via mechanical means), or (4) indirect contact via fomites; transmission of member viruses of the subfamily Entomopoxvirinae occurs between arthropods by mechanical means. Host range may be broad in laboratory animals and in tissue culture; however, in nature it is generally narrow. Many poxviruses of vertebrates produce dermal maculopapular, vesicular rashes after systemic or localized infections. Poxviruses infecting humans are zoonotic except for Molluscum contagiosum virus and the orthopoxvirus Variola virus (smallpox, now eradicated). Members may or may not be occluded within proteinaceous inclusions (Chordopoxvirinae: acidophilic (A-type) inclusion bodies, or Entomopoxvirinae: occlusions or spheroids). Occlusions may protect such poxviruses in environments where transmission possibilities are limited.
Neutralizing antibodies and cell-mediated immunity play a major role in clearance of vertebrate poxvirus infections. Reinfection rates are generally low and usually less severe. Molluscum contagiosum infections may recur, especially by autoinoculation of other areas of the skin with virus derived from the original lesions (e.g., by scratching).
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