Taxonomic Structure of the Family
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The virions of viruses within the family Reoviridae have icosahedral symmetry but may appear spherical in shape. They each have a capsid, which is made up of concentric protein layers organized as one, two or three distinct capsid shells, which have an overall diameter of 60-80 nm.
It is possible to divide the nine genera into two groups. One group contains those viruses in which intact virus particles or cores have relatively large ‘spikes’ or ‘turrets’ situated at the 12 vertices of the icosahedron (including Orthoreovirus, Cypovirus, Aquareovirus, Fijivirus and Oryzavirus, as well as most of the unclassified or unassigned viruses from invertebrates). The second group includes those genera with relatively smooth or almost spherical virus particles and cores without large surface projections at their five fold axes (including Orbivirus, Rotavirus, Coltivirus and Phytoreovirus).
It is important to note that the nomenclature used to describe viral particles with different numbers of intact capsid layers varies between the different genera, although the current nomenclature will be explained in each case. The transcriptionally active core particle of the ‘spiked’ viruses appear to contain only a single complete capsid layer with T = 2 symmetry, to which the spikes are attached. In intact virus particles, the core is usually surrounded by an incomplete protein layer with T = 13 symmetry that is penetrated by the spikes, and which forms the outer capsid coat. The virus particles are therefore usually regarded as double shelled. One exception is the cypoviruses, which are distinct in having virions with only a single capsid shell, equivalent to the “core” particle of viruses from the other genera. In the genus Cypovirus the transcriptionally active single shelled particle is the intact virion, although characteristically the particles of some cypoviruses may be occluded either singly or multiply within the matrix of proteinaceous crystals called polyhedra, that are composed primarily (>90%) of the viral “polyhedrin” protein. In contrast the smooth cored viruses contain a subcore, which may be relatively fragile, with a complete T = 2 protein shell that is reinforced in the transcriptionally active core particles by a complete T = 13 core-surfaced layer. These double layered core particles do not have surface spikes and in the intact virions are surrounded by an outer capsid shell, giving rise to three layered virus particles. The innermost protein shell of the virus capsid has an internal diameter of approximately 50-60 nm, and surrounds the 10, 11, or 12 linear dsRNA genome segments. In the smooth cored genera, the enzymatically active minor proteins of the virion are also situated within this central space, attached to the inner surface at the 5 fold axes of symmetry (these include RNA-dependent RNA polymerase (transcriptase and replicase), NTPase, helicase, capping and transmethylase enzymes). In ‘spiked’ genera some of these enzyme proteins form the turrets on the surface of the core. These hollow projections appear to act as conduits for the exit of nascent mRNA synthesized by the core-associated enzymes.
Particles of some genera can leave infected cells by budding (Orbivirus) or can bud into the endoplasmic reticulum (Rotavirus), acquiring a membrane envelope derived from the cellular membranes, although in most cases this envelope appears to be transient. In some genera the outer capsid proteins (CP) can be modified by proteases (such as trypsin or chymotrypsin) forming ‘infectious’ or ‘intermediate subviral particles’ (ISVP). This may happen during, may be essential for, or may alter the particle characteristics for cell attachment and entry.
Physicochemical and Physical Properties
The virion Mr is about 12 107. The buoyant density in CsCl is 1.36-1.39 g/cm3. Virus infectivity is moderately resistant to heat, organic solvents (e.g., ether) and to non-ionic detergents (variable, depending on both virus strain and detergent). The pH stability of virions varies among the genera.
Virions contain 10, 11 or 12 segments of linear dsRNA depending on the genus. The individual Mr of these RNAs range from 0.2 to 3.0 106. The total Mr of the genome is 12 to 20 106. The RNA constitutes about 15-20% of the virion dry weight. The positive strands of each duplex have 5-terminal caps (type 1 structure). There are data to suggest that negative strands may have phosphorylated 5-termini. However, in some cases (e.g. BTV, Orbivirus) the negative strand has also been shown to be poorly labelled by treatment with polynucleotide kinase and [-32P] ATP, suggesting that like the positive strand it may also have a “blocked” 5-structure. Both RNA strands have a 3OH and viral mRNAs lack 3-poly(A) tracts. The viral dsRNA species are present within virus particles in equimolar proportions, representing one copy of each segment per virion. Intact virions of some genera also contain significant amounts of short ssRNA oligonucleotides.
At least 3 internal virion structural proteins constitute the RNA polymerase and associated enzymes that are involved in the processes of mRNA synthesis and capping [including a fully conservative dsRNA-dependent ssRNA polymerase (transcriptase), nucleotide phosphohydrolase, guanylyltransferase, two distinct transmethylase activities and dsRNA template-unwinding (helicase) activities]. Some of the minor proteins may also play a structurally significant role as components of the virion structure together with at least 3 major CPs. The proteins range in size from Mr 15-155 103. The proteins constitute about 80-85% of the dry weight of virions.
Mature virions lack a lipid envelope. Depending on the genus, a myristyl residue may be covalently attached to one of the virion proteins. For coltiviruses, rotaviruses and orbiviruses, an intermediate in virus morphogenesis or release from the cell, may have a lipid envelope that is subsequently lost or removed.
In some genera one of the outer virion proteins can be glycosylated with high mannose glycans, or O-linked N-acetylglucosamine. A small nonstructural viral protein may also be glycosylated.
Genome Organization and Replication
The viral RNA species are mostly monocistronic, although some segments have second functional in frame initiation codons, or other ORFs. Proteins are encoded on one strand only of each duplex (the mRNA species). The mode of entry of viruses into cells varies between genera but usually results in the loss of components of the outer capsid. Transcriptionally active, parental virus-derived particles (cores) are released into the cell cytoplasm. Repetitive asymmetric transcription of full-length mRNA species from each dsRNA segment occurs within these particles throughout the infection course. The mRNA products, which are produced in larger copy numbers from the smaller segments, are extruded from the icosahedral apices of the particles. Structures, termed viroplasms or virus inclusion bodies (VIB), occur in localized areas of the cytoplasm. They appear to be the sites of replication and progeny virus particle assembly. VIB have a granular and moderately electron dense appearance when viewed by electron microscopy and may contain nascent subviral particles. Outer capsid components appear to be added at the periphery of the VIB.
The mechanism of genome assembly and synthesis remains uncharacterised. Evidence has been obtained for orthoreoviruses that sets of capped mRNAs and certain NS proteins are incorporated into “assortment complexes” that are considered to be the precursors of progeny virus particles. The mRNAs are then used as template for a single round of minus strand synthesis, thereby reforming dsRNA segments. The different species of mRNAs in the cell cytoplasm are present in non equimolar ratios (see transcription of mRNA above). However, the dsRNA genome segments are usually packaged in exactly equimolar ratios (one copy of each genome segment per particle). The selection of the viral mRNAs for packaging is therefore thought to be highly specific, involving recognition signals on each mRNA species. The RNA segments have conserved terminal sequences at both ends, which may be involved as recognition signals for the RNA transcriptase and/or replicase. These sequences may also be essential for selection and incorporation of the RNAs into the nascent progeny particles. At least in some genera, the RNA within completed particles is packaged as a series of concentric and highly organized shells which also have elements of icosahedral symmetry.
In addition to the parental virus derived subviral particles (cores), progeny core particles also synthesize mRNA, providing an amplification step in replication. Depending on the genus, some NS proteins are involved in the translocation of virus particles within cells and in virus egress. Many cypoviruses also form polyhedra, which are large crystalline protein matrices that occlude virus particles and which may be involved in transmission between individual insect hosts. The steps involved in virion morphogenesis and virus egress from cells vary according to the genus. Genome segment reassortment occurs readily in cells co-infected with viruses of the same species.
The viruses that infect vertebrate hosts generally possess ‘group’ or ‘serogroup’ (species) specific antigens and, within each serogroup, more variable serotype-specific antigens. Viruses that infect plants and insects only, may show a greater uniformity and less antigenic variation in their different proteins, possibly due to the absence of antibody selective pressure on ‘neutralization antigens’. No antigenic relationship has been found between the viruses in different genera. Some viruses hemagglutinate red blood cells.
The biological properties of the viruses vary according to the genus. Some viruses replicate only in certain vertebrate species and are transmitted between hosts by respiratory or fecal/oral routes. Other vertebrate viruses replicate both in arthropod vectors (e.g., gnats, mosquitoes, or ticks, etc., - orbiviruses, coltiviruses) and vertebrate hosts. Plant viruses replicate both in plants and arthropod vectors (leafhoppers). Viruses that are pathogens of insects (cypoviruses) are transmitted by contact, or fecal oral routes.
Species Demarcation Criteria in the Family
Within the family Reoviridae, the prime determinant for inclusion of virus isolates within a virus species is an ability to exchange (reassort) genome segments during co-infection, thereby exchanging genetic information and generating viable and novel progeny virus strains. However, direct evidence of genome segment reassortment between different virus strains is limited and other methods have been used to analyse relationships, similarities and differences between viruses. These data, which allow a prediction of the “compatibility” of strains for reassortment, include: identification of vector and/or host species, clinical signs, serological comparisons, comparisons of RNA/protein sequences, cross-hybridization analysis of RNA or cDNA, analysis of conserved terminal regions, identification of the virus ‘serotype’ (neutralization type) with one that is already classified within a species, analysis of the electrophoretic migration patterns of the genome segments (electropherotype), amplification of conserved genome segments or regions by PCR (can be coupled with cross-hybridization, sequence analyses or restriction fragment analysis or the products).
Migration of dsRNA genome segments during PAGE is significantly affected by their primary sequence, as well as by their Mr. However their migration rate during agarose gel electrophoresis (AGE) appears to depend only on Mr. AGE may therefore be more suitable for comparison and identification of different virus species by electropherotyping, while PAGE is more sensitive for detection of relatively small differences between strains within a single virus species.
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