, three , and four 
primary translation products; (4) one or two additional very small gene products encoded by a polycistronic genome segment; (5) all members of the second two subgroups induce syncytium formation.
|
Type Species |
(MRV) |
Orthoreoviruses infect only vertebrates and are spread by the respiratory or oral-fecal routes. All members of the genus have: (1) a well defined capsid structure as observed by negative staining; (2) 10 dsRNA segments with three large (L), three medium (M), and four small (S) size-class RNA genome segments; (3) a characteristic protein profile with three , three , and four primary translation products; (4) one or two additional very small gene products encoded by a polycistronic genome segment; (5) all members of the second two subgroups induce syncytium formation.
Virions are icosahedral with a roughly spherical appearance and possess a double-layered protein capsid discernible by negative staining (Fig.1A). High resolution images have been obtained by cryoelectron microscopy and image reconstruction of various MRV isolates. A similar morphological examination of Avian orthoreovirus (ARV) virions reveals minor differences in the particle morphology of the two species. The virion consists of a central compartment (about 48 nm in diameter) containing the dsRNA genome segments, surrounded by an inner capsid (60 nm diameter) and an outer capsid (85 nm diameter) which has T = 13 (laevo) icosahedral symmetry. The surface of virions is covered by 600 finger-like projections arranged in 60 hexameric and 60 tetrameric clusters that surround solvent channels which extend radially into the outer capsid layer (Fig.1B). Intact virions also contain large, open depressions with a flower-shaped structure at the 5-fold axes, resulting in an angular capsid profile when viewed in the 3-fold orientation (Fig.1A, 1B). Intermediate subviral particles (ISVPs), generated by partial removal of the outer CPs (Fig.1C), are approximately 80 nm in diameter. The flower-shaped structures at the 5-fold axes of the ISVPs may contain an extended form of the 1 cell attachment protein, which protrudes as a 40 nm spike from the vertices. Core particles, generated by more extensive removal of the outer CPs (Fig.1D), have 150 ellipsoidal nodules on their surface and distinctive “turrets” located at the 5-fold axes. These projections, which are altered conformations of the flower-shaped structures, are about 10 nm in length and possess central channels 5-8 nm in diameter extending into the central compartment.
Physicochemical and Physical Properties
The virion Mr is about 13 
107 with a buoyant density in CsCl of 1.36 g/cm3 (1.38 g/cm3 for ISVPs, 1.43 g/cm3 for core particles). The virion, ISVP, and core S2Ow values are about 730S, 630S and 470S, respectively. Virions are remarkably stable and withstand extremes in ionic conditions, temperatures up to 55°C, pH values between 2 and 9, lipid solvents, and detergents. Exposure to UV irradiation reduces infectivity.
All orthoreoviruses have 10 linear dsRNA segments that range from 0.6 to 2.6 106 Mr. The total Mr of the MRV-3 genome is about 1.5 107 (23,549 bp) and constitutes approximately 11.5% of the virion mass. Based on their resolution by gel electrophoresis, the genomic dsRNAs are grouped into 3 size classes commonly referred to as large (L1-L3, about 3.9-3.8 kbp), medium (M1-M3, about 2.3-2.2 kbp) and small (S1-S4, about 1.6-0.9 kbp). The gel mobilities of certain genome segments are indicative of the three distinct subgroups of orthoreoviruses. In comparison to subgroup 1 (MRV), subgroup 2 [which includes avian isolates (ARV) and Nelson Bay orthoreovirus (NBV)] displays a retarded S1 segment migration, while subgroup 3 (BRV) has a severely truncated S1 genome segment (887 bp) which migrates as the S4 segment by SDS-PAGE.
Complete virus particles contain numerous oligonucleotides (2 to 20 residues in length) representing approximately 25% of the total RNA content. Three quarters of these are abortive reiterative 5
-terminal transcripts, produced by the reovirus core-associated transcriptase and capping enzymes, while the remainder are oligoadenylates. The 5-terminus of the plus-sense RNA strand of each genome segment contains dimethylated cap 1 structures (m7GpppGm2). The genomic RNAs lack polyA tails and do not contain covalently linked proteins. The complete genomic sequence of the type species (MRV-3, strain Dearing) has been determined from cloned cDNAs, along with numerous genome segments of other MRV isolates. The four S class genome segments have been sequenced from two ARV isolates, as well as from NBV and from Baboon orthoreovirus (BRV). The S1 and S3 genome segments have also been sequenced from several additional ARV isolates. Genomic dsRNA segments contain 5- and 3-terminal sequences of four or five bp which are conserved in all ten genome segments within a particular virus species. The 3-terminal consensus sequence (UCAUC-3) is also conserved between the three subgroups of orthoreoviruses, at least for the four S class genome segments. The 5-terminal conserved sequences vary and may be useful for assigning new isolates to one of the three subgroups (Table 1).
The orthoreovirus structural proteins are designated in terms of their relative sizes and size csses (1, 2, 3; 1, 2; 1, 2, 3: in ARV these proteins are also referred to as A, B, C; A, B; A, B, C) (Table 2). The following discussion refers to the nomenclature scheme for the prototype MRV (as used for strain MRV-3). The stabilizing lattice of the outer capsid is composed of 200 interlocking trimers of the cleaved Mr 76 103 1 protein (72 103 1C and 4 103 1N) (Fig. 1). The 1 subunits also interact with monomers of the 3 protein, which represents the finger-like projections on the surface of the virion. Pentameric subunits of the 2 protein make up the flower-like structures and turrets at the vertices of virus particles and cores. The 2 structures interact with subunits of the tetrameric 3 clusters and with the 1C lattice and represent essential structural components of the outer capsid. This essentially outer CP remains associated with the core particles, unlike the other outer CPs. The fourth component of the outer capsid, the 1 protein, exists as 12 homotrimers associated with the vertices of virions and ISVPs and may assume either a condensed or extended conformation. The 1 (120 copies) and 2 proteins (120 copies) represent the major structural proteins of the inner capsid and both may contact the genome since both display some degree of dsRNA-binding activity. The final two structural proteins of the virus, 3 and 2, are present at 12 copies per virion located on the inside of the inner capsid. The 3 protein forms 7 nm projections that extend toward the interior of the core, underlying the 12 vertices of the capsid. The 2 protein may be associated with these 3 structures.
Mature virions lack a lipid envelope. The major outer capsid lattice protein, 1, and its 1N cleavage product are N-terminally myristoylated.
Whether orthoreovirus proteins are glycosylated remains controversial. Detection of glycoproteins by labeling with radiolabeled glucosamine, or via staining by periodic acid oxidation suggests that all of the MRV and ARV proteins may be glycosylated, except the major inner CP [2 of MRV, 1 (A) of ARV].
Genome Organization and Replication
The genome consists of ten segments of dsRNA, which are packaged in equimolar ratios (one copy of each within each virion). The segments possess terminal NTRs which are shorter at the 5-terminus (12-32 bp for MRV-3) than at the 3-terminus (35-85 bp). Most segments possess a major ORF, which varies in length from 353 to 1,298 codons. One genome segment is polycistronic, containing more than one functional ORF. The MRV S1 segment is bicistronic, encoding the Mr 49 103 1 protein and the Mr 14 103 1s protein from a second overlapping ORF (Fig. 2). The S1 genome segment of ARV and NBV appears to be tricistronic, containing the cell attachment protein ORF (3/C), a potential ORF (P17) that may be equivalent to the MRV 1s ORF (although there is no sequence homology with MRV) and a unique ORF encoding the small, transmembrane, fusion-inducing protein P10. The truncated S1 genome segment-equivalent of BRV (S4, 887 bp), contains two sequential 140 codon ORFs, the first of which encodes a unique fusion-inducing P15 protein. The second ORF on the BRV S4 genome segment may encode the cell attachment protein (P16) but it exhibits no sequence or sequence-predicted structural similarity to the cell attachment proteins of the other subgroups.
The overall course of infection involves adsorption, low pH-dependent penetration and uncoating to core particles, asymmetric transcription of capped, non-polyadenylated mRNAs via a fully conservative mechanism (the nascent strand is displaced), translation, assembly of plus strands into progeny subviral particles, conversion of plus strands to dsRNA, and further rounds of mRNA transcription and translation. The efficiency of translation of the various orthoreovirus mRNA species varies over a 100-fold range while the proportions of the mRNA species found in infected cells vary inversely to their proportionate size. The final stage of the replication cycle involves the assembly of the outer capsid on progeny subviral particles, to form infectious progeny virions. These progeny particles accumulate in paracrystalline arrays in the perinuclear region of the cell cytoplasm and are released when infected cells lyse late in the replication cycle. The exception to the above generalized replication cycle involves the formation of multinucleated syncytia by members of subgroups 2 and 3 (ARV, BRV and NBV). Syncytia formation commences 10-12 hr post-infection resulting in a more rapid lytic response and enhanced kinetics of virus release.
The functions and properties of specific viral proteins influence various stages of the MRV replication cycle (Table 2). The MRV 1 cell attachment protein determines the cell and tissue tropism of the virus strain and has hemagglutinin activity. The 1 protein is N-terminally myristoylated and forms a complex with 3 in solution which triggers cleavage of 1 to 1N and 1C. The 1 protein is further proteolytically cleaved into 
and polypeptides during virus entry to cells and is responsible for membrane interactions. The 1 protein also influences strain-specific differences in capsid stability, transcriptase activation, and neurovirulence. In addition to complexing with 1 and forming the outer protective layer of the virion, the 3 protein is involved in translation regulation. The 2 (Cap) core spike is the guanylyl transferase involved in mRNA capping while the 1 and 2 major inner CPs both bind dsRNA. The 1 (Hel) protein may also function as a helicase and a RNA triphosphatase. The minor inner CP 3 (Pol) is the viral polymerase while the second minor inner CP 2, along with the major inner CP 1, are involved in the NTPase activity associated with core particles. There are also at least three nonstructural proteins encoded by the virus genome. The NS and NS proteins are produced in high abundance during an infection and, together with 3, associate with mRNA to form virus mRNA-containing complexes, presumed precursors of progeny virus assembly. The NS protein also associates with the cytoskeleton. The 1s protein is a small, basic protein expressed in cells infected by all three MRV serotypes. This protein appears to be dispensible for growth in cell culture but may be involved in virus-induced cytopathic effects and the inhibition of DNA synthesis.
Recent analysis of members of subgroups 2 and 3 (the avian/Nelson Bay and baboon isolates) has revealed a similar situation to that described for MRV, with some notable exceptions. The truncated cell attachment protein of ARV and NBV, 3 or C (Mr 35 103), exists as a multimer with a coiled-coil domain similar to that of MRV but possesses no hemagglutinin activity. The S class genome segments of BRV, encode no homologue of the ARV or MRV cell attachment proteins. The dsRNA-binding domain of the MRV 3 protein is not conserved in the homologous 2 proteins of ARV, NBV, or BRV. Viruses in the avian and baboon subgroups encode an additional protein responsible for syncytium formation. The P10 fusion proteins of ARV and NBV, show sequence (35%) and structural similarities but are unrelated to the P15 fusion protein of BRV. All of these fusion proteins are small, basic transmembrane proteins and induce fusion in transfected cells in the absence of other viral proteins.
The serotype specific antigen of the orthoreoviruses is protein l (3/C of the avian species), which reacts with neutralizing antibodies. Antigenic recognition of this protein is the basis for three serotypes of MRV and 5-11 serotypes of ARV. The MRV l and 1s proteins elicit strain-specific and cross-reactive cytotoxic T-cell activities. The MRV proteins 2 and 3 are group-specific antigens, similar to the 2/B and 2/B proteins of ARV (Fig. 3). The considerable sequence homology that exists between different isolates in the same species, but not between species, is reflected by limited antigenic cross reactions between species. The most extensive antigenic similarity occurs between ARV and NBV, which agrees with the increased amino acid identity between these species and their assignment to one subgroup.
Transmission is by the enteric or respiratory route, no arthropod vectors are involved, and infection is restricted to a variety of vertebrate species (baboons, bats, birds, cattle, humans, monkeys, sheep, snakes, and swine). Orthoreovirus distribution is ubiquitous and worldwide. Human orthoreoviruses are generally benign but may cause upper respiratory tract illness or enteritis in infants and children (albeit rare). In mice, orthoreovirus disease can cause diarrhea, runting, oily hair syndrome, hepatitis, jaundice, mycocarditis, myositis, pneumonitis, encephalitis, and neurologic symptoms. A variety of symptoms may be associated with orthoreovirus infection of domestic animals including upper and lower respiratory illnesses and diarrhea. In monkeys, orthoreoviruses cause hepatitis, extrahepatic biliary atresia, meningitis, and necrosis of ependymal and choroid plexus epithelial cells. The BRV isolate was obtained from baboons suffering from meningoencephalomyelitis and the isolates from snakes were obtained from animals displaying neurological symptoms. Avian orthoreoviruses do not infect mammalian species. The outcome of infection of birds may range from inapparent to lethal and depends on the virus strain and the age of the host bird. Systemic infection results in virus isolation from numerous tissues. Disease presentations in chickens include feathering abnormalities, gastroenteritis, hepatitis, malabsorption, mortality, myocarditis, paling, pneumonia, stunted growth, and weight loss. In turkeys, avian orthoreoviruses cause an infectious enteritis. Birds that survive an acute systemic infection may develop obvious joint and tendon disorders (tenosynovitis) that resemble the pathology of rheumatoid arthritis in humans.
List of Species Demarcation Criteria in the Genus
The orthoreoviruses include four species and two unassigned viruses. Conclusive species classification requires the direct demonstration of exchange of genetic material via reassortment of genome segments. To date, there is no evidence of reassortment between the four species identified which reflects the extensive sequence divergence between species.
Members of an Orthoreovirus species may be identified by :
1. |
An ability to exchange genetic material by genome segment reassortment during dual infections, thereby producing viable progeny virus strains. |
2. |
Identification of conserved terminal genomic RNA sequences within a species (absolute conservation of the 5 |
3. |
Identification of extensive sequence identity between the proteins encoded by homologous genome segments (greater than 85% amino acid identity within a species versus less than 65% identity between species). |
4. |
Identification of extensive sequence identity between homologous genome segments (greater than 75% nucleotide sequence identity within a species, versus less than 60% between species). |
5. |
Identification of virus serotype (based on cross neutralization) with a virus type already classified within a specific Orthoreovirus species. |
6. |
Demonstration of extensive antigenic similarity in the major structural proteins within a species, as determined by ELISA or immunoprecipitation. |
7. |
Analysis of “electropherotype” by agarose gel electrophoresis but not by PAGE (some similarities can exist between closely related species). |
8. |
Similar organization of the polycistronic genome segment. |
9. |
Identification of host and clinical signs. |
The four species of orthoreoviruses can be divided into three distinct subgroups, as illustrated by a phylogenetic tree (Fig. 3), generated by comparison of the amino acid sequence of the 2 protein (major inner capsid). Very similar trees were also generated by comparison of 3 and NS (data not shown). Subgroup 1 includes all the nonfusogenic MRV isolates, representing a single species with three distinct serotypes. Numerous ARV isolates from commercial poultry flocks (including several different serotypes) and NBV (isolated only once from a flying fox) represent two distinct species in subgroup 2. These two species possess 40-60% amino acid identity in the S class gene products, which exceeds the 20-30% identity in homologous proteins detected between different subgroups but which is considerably lower than the >90% identity displayed by isolates of the same species. These two species induce syncytium formation, share more extensive antigenic similarity than other species, possess similar conserved terminal genome segment sequences, and display a similar gene organization of the polycistronic, fusion-inducing S1 genome segment (Fig. 2). In view of the sequence divergence and the absence of evidence for reassortment between the ARV and NBV isolates, these isolates are considered as two separate species in the same subgroup.
The third subgroup contains a single species, BRV. This isolate also induces syncytium formation but shares little sequence (20-30%) or antigenic similarity to the other fusogenic species. BRV also contains a truncated, fusion-inducing, polycistronic S1 genome segment-equivalent (the S4 genome segment) with a distinct gene organization (Fig. 2), a fusion protein with no sequence or sequence-predicted structural similarity to the fusion proteins of ARV or NBV, and a unique 5-terminal consensus sequence. This species clearly represents a distinct subgroup of the orthoreoviruses. In addition, there are two unclassified isolates from snakes which also induce cell fusion but which lack the characteristic electrophoretic mobility of the fusion-inducing genome segments of either the ARV or BRV subgroups. Classification of these isolates awaits sequence information. Ndelle virus (NDEV) was previously classified as an unassigned orbivirus but recent equence analysis have shown it to have high levels of sequence homology to the MRV in at least four genome segments.
Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus names, genome sequence accession numbers [ ], and assigned abbreviations ( ) are:
|
Subgroup 1 | ||
|
Mammalian orthoreovirus |
(MRV) | |
|
serotype 1 {strain Lang} |
(MRV-1) | |
|
serotype 2 {strain D5/Jones} |
(MRV-2) | |
|
serotype 3 {strain Dearing} |
[L1: M24734, L2: J03488, L3: M13139] |
(MRV-3) |
|
[M1: M27261, M2: M19408, M3: M27262] |
||
|
[S1: M10262, S2: M25780, S3: X01627, S4: K02739] |
||
|
Subgroup 2 | ||
|
Avian orthoreovirus |
(ARV) | |
|
strain S1133 |
[S1: L39002, S3: U20642, S4: U95952] |
(ARV-S1133) |
|
strain 176 |
[S2: AF059716, S3: AF059720, S4: AF059724] |
(ARV-176) |
|
strain SK138a |
[S2: AF059717, S3: AF059721, S4: AF059725] |
(ARV-138) |
|
Nelson Bay orthoreovirus |
[S2: AF059718, S3: AF059726, S4: AF059722] |
(NBV) |
|
Subgroup 3 | ||
|
Baboon orthoreovirus |
[S1: AF059719, S2: AF059723, S3: AF059727] |
(BRV) |
Tentative Species in the Genus
|
Python orthoreovirus |
(PRV) |
|
Rattlesnake orthoreovirus |
(RRV) |
|
Ndelle virus |
(NDEV) |
Phylogenetic Relationships within the Genus
|
|  |