Type Species

Wound tumor virus

(WTV)

Distinguishing Features

Phytoreovirus particles have icosahedral symmetry with a distinctive angular appearance and possess 12 dsRNA species. They are transmitted by cicadellid leafhoppers to susceptible plant species, replicating in both hosts and vectors.

Virion Properties

Morphology

Virions of Rice dwarf virus (RDV) are icosahedral, and appear to be double-shelled ~ 70 nm in diameter (Figs. 20 and 21). The outer layer of RDV contains 260 trimers of P8(T13) (Mr 46 10³) representing a total of 780 molecules, arranged with T = 13 l symmetry (Fig. 21). Neighboring capsomers on the particle form pentameric or hexameric rings. The inner capsid is reported to be a complete protein shell, composed of 60 dimers of P3 (Mr 114 10³), a total of 120 molecules, arranged with a suggested T = 1 icosahedral symmetry (Fig. 21). The layer appears to be comparable to the ‘subcore’ shell of the orbiviruses, which has been described by Grimes, Burroughs, Gouet, Diprose, Malby, Zeintara, Mertens and Stuart, 1998 as 120 molecules arranged as lattice with a geometrical quasi equivalence with T = 2 symmetry. The RDV particle structure appears to be comparable to that of ‘core’ or double layered particles of some other genera (Orbivirus and Rotavirus respectively). Wound tumor virus (WTV) is reported to possess three protein shells, including an outer amorphous layer, a middle layer of distinct capsomers, and a smooth core that is about 50 nm in diameter but lacking spikes.

Physicochemical and Physical Properties

The Mr of phytoreoviruses is about 75 10⁶. The virion S_20w is about 510S. The optimal stability of particles is at pH 6.6. The buoyant density of RDV is 1.39-1.42 g/cm³ and the virion is unstable losing P8 in CsCl. CCl₄ removes P2 from the RDV virion.

Nucleic Acid

Phytoreoviruses have 12 genome segments of linear dsRNA. Segment 1 to segment 12 are numbered according to their migration during PAGE. However, their relative sizes based on RNA sequence data indicate that segments 4 and 5, or segments 9 and 10, may migrate in the reverse order during AGE (Table 16). The RNA constitutes about 22% of the virion dry weight. The dsRNA Mr is in the range 0.3 to 3.0 10⁶, with characteristic sizes for each virus. For WTV these are: S1 ?bp; S2 ?bp; S3 ?bp; S4 2565 bp; S5 2613 bp; S6 1700 bp; S7 1726 bp; S8 1472 bp; S9 1182 bp; S10 1172 bp; S11 1128 bp; S12 851 bp. G+C content is 38-44% and 41-48% for WTV and RDV dsRNA respectively. The positive strand of each genome segment, of all viruses in the genus, contains the conserved sequence; 5-GG(U/C)A---UGAU-3 except for RDV S9 which has 5-GGUA---CGAU-3. These genus-specific terminal sequences are situated adjacent to inverted repeats, which are 6-14 bases long. These sequences differ for each RNA segment. Individual isolates of RDV can frequently be distinguished by electrophoretic profiles of at least one of the 12 segments in PAGE. RDV particles encapsidate the dsRNA in supercoiled form.

Proteins

Phytoreoviruses have 6 to 7 structural proteins with Mr in the range 45 to 160 10³. RDV has 6 structural proteins (P1(Pol), P2, P3, P5(Cap), P7, and P8(T13)). For WTV the seven CPs are organized in three shells consisting of an amorphous outer layer of 2 species, an inner ‘core’ shell of 2 species and a subcore of three species. Protein constitutes about 78% of the particle dry weight. Removal of the outer shell is not required for activation of the virus transcriptase and associated enzymes. Removal of RDV P2 abolishes the ability to infect vector cell monolayer but virus particles without P2 retain viral transcriptase activity and can infect vector insect by an injection method. P1(Pol) is the transcriptase/polymerase and binds to genomic dsRNA. P7 has non-specific nucleic acid binding activity. P3 binds to P3, P7, and P8(T13). P7 binds to P1 and P8(T13). P5(Cap) is probably an guanylyltransferase and has GTP, ATP and UTP binding activities. Pns 12 can be phosphorylated.

Lipids

None known.

Carbohydrates

None known.

Genome Organization and Replication

The coding strand of each dsRNA has a single ORF, except for segments 11 and 12 of RDV, segment 9 of RGDV and segment 9 of WTV. RDV-S11 has two in-frame initiation codons, and thus two ORFs. RDV-S12, RGDV-S9 and WTV-S9 possess a second, small out-of-frame and overlapping ORF, downstream within the major ORF. No evidence has been obtained for the expression of this second ORF. Five structural CP and five NS proteins of WTV have been assigned to their respective genome segments. RDV S1 encodes the putative transcriptase. Genus-specific and segment-specific sequence motifs appear to be necessary for replication, translation and encapsidation. Laboratory strains having internal deletions in some segments, but intact termini, replicate and compete favorably with wild-type virus, although the proteins expressed are aberrant, and the ability of the viruses to be transmitted by vectors may be lost. Virus replication occurs in the cytoplasm of infected cells in association with viroplasms. WTV and RGDV are confined to phloem tissues of the plant host, whereas RDV can also multiply elsewhere.

Antigenic Properties

The three recognized phytoreoviruses are antigenically distinct. Epitopes in the outer surface are unrelated to each other, while the inner surface epitopes of the capsid of RDV and RGDV will cross react.

Biological Properties

Plant hosts are either dicotyledons (WTV), or in the family Graminae (RDV and RGDV). WTV was originally identified in northeastern USA in the leafhopper Agalliopsis novella and was recently rediscovered in New Jersey USA in a single periwinkle (Catharanthus roseus) plant set out as bait for phytoplasmas in a blueberry (Vaccinium) field. The experimental plant host range of WTV is wide and encompasses many dicotyledons. The name of this virus derives from the fact that infected plants develop phloem-derived galls (tumors) at wound sites, notably at the emergence of lateral roots.

RDV and RGDV have narrow and overlapping host ranges in the Graminae. RDV causes severe disease in rice in south-east Asia, China, Japan and Korea, Nepal and the Philippines, although RGDV has only been reported in Thailand. RDV induces white flecks and streaks on leaves, with stunting and overproduction of side shoots. RDV is the only plant reovirus that is not limited to the phloem. Plants infected with RDV are stunted and fail to bear seed. Since the virus is widespread in rice in southern China and other Asian countries, it is considered likely to cause a significant reduction in rice production. RDV does not provoke enlargement or division of infected cells and does not induce galls, enations, or tumors. RGDV induces stunting, shoot proliferation, dark green color and enations in rice.

Phytoreoviruses induce no marked disease in the insect vectors. Virus replication occurs in the cytoplasm of infected cells in association with viroplasms. In the vector, there are no particular tissue tropisms. However, RDV induces abnormalities in fat body cells and mycetocytes. They are all transmitted propagatively by cicadellid leafhoppers (Hemiptera, Cicadellidae, e.g., Agallia, Agalliopsis, Nephotettix, and Recilia). Virus is acquired from plants shortly after feeding. The latent period in leafhoppers is about 10-20 days. Thereafter, infected insects have a lifelong ability to transmit virus to plants. Phytoreoviruses are also transmitted transovarially in their insect vectors. Phytoreoviruses have not been shown to be mechanically transmissible from plant to plant. No seed transmission has been reported.

List of Species Demarcation Criteria in the Genus

In common with the other genera within the family Reoviridae, it has been agreed that the prime determinant for inclusion of virus isolates within a single phytoreovirus species will be “ability to exchange (reassort) genome segments during co-infection, thereby exchanging genetic information and generating viable and novel progeny virus strains”. Data providing direct evidence of segment reassortment between isolates is very limited. Other techniques are therefore normally used to examine the level of similarity that exists between different isolates thus providing polythetic species parameters.

These include:

1.

The ability to exchange genetic material by genome segment reassortment during dual infections, thereby producing viable progeny virus strains. RDV isolates, from Japan, China, and Philippines can exchange genomic segments. Exchange ability between RDV, WTV, RGDV has not been tested.

2.

Nucleotide sequence homology. So far, nucleotide sequence identities among RDV isolates including those from different countries, are more than 90%. Some isolates have deletions [Chinese strain of RDV S11 (U36568) compared to Japanese RDV-Akita S11 (D10249)] or duplications [RDV-P S12]. The most extensive comparisons of S12 of 12 RDV isolates from five countries shows identitiy of 94.7 to 98.7% (Fig. 22). Sequence comparisons indicate that conservation between different species is at a much lower level.

3.

Protein sequence homology. Protein sequence homology between species is less than 50% and within species is more than 80%. This data indicates that high levels of serological cross-reaction would be detected within species by ELISA complement fixation (CF), or agar gel immunodiffusion (AGID), using either polyclonal sera, or monoclonal antibodies against conserved antigens. Distinct species may show significantly lower levels of cross-reaction. However, such serological methods are not routinely used to compare these viruses.

4.

Conserved terminal oligonucleotide sequences. The tetranucleotides at both ends of the genome segments are highly conserved within the genus. Two additional nucleotides flanking the 5-terminal tetranucleotide on the plus-strand are common within species.

5.

Cross-hybridization (Northern or dot blots, with probes made from viral RNA or cDNA using conditions designed to detect >80% homology). RNA-RNA hybridization detects all the segments within a species.

6.

Analysis of “electropherotype” by agarose gel electrophoresis but not by PAGE. (similarities can exist between closely related species).

7.

Identification of host plant. Dicotyledons (WTV), or the family Graminae (RDV and RGDV).

List of Species in the Genus

Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus names, insect vector and host names { }, genome sequence accession numbers [ ], and assigned abbreviations ( ) are:

Species in the Genus

Rice dwarf virus

S1: [D90198], S2: [D00608], S3: [D00607], S4: [U36562], S5: [D90033], S6: [M91653], S7: [D00639], S8: [D00536], S9: [D00465], S10: [D00473], S11: [D10249], S12: [D90200]

(RDV)

{Nephotettix cincticeps, N. nigropictus, Recilia dorsalis: Graminae}

Rice dwarf virus, isolate B

(RDV-B)

Rice dwarf virus, isolate S

(RDV-S)

Rice dwarf virus, isolate H

(RDV-H)

Rice dwarf virus, isolate China

(RDV-Ch)

Rice gall dwarf virus

S2 [D86439], S3 [D13774], S8 [D13410], S9 [D01047], S10 [D13411]

(RGDV)

{Nephotettix cincticeps, N. nigropictus, N. virescens, N. malayanus, Recilia dorsalis: Graminae}

Wound tumor virus

S4: [M24117], S5: [J03020], S6: [M24116], S7: [X14218], S8: [J04344], S9: [M24115], S10: [M24114], S11: [X14219], S12: [M11133]

(WTV)

{Agallia constricta, A. quadripunctata, Agalliopsis novella: many dicotyledons}

Tentative Species in the Genus

Tobacco leaf enation phytoreovirus

(TLEP)

Phylogenetic Relationships Within the Genus

See Fig. 22.

1.	The ability to exchange genetic material by genome segment reassortment during dual infections, thereby producing viable progeny virus strains. RDV isolates, from Japan, China, and Philippines can exchange genomic segments. Exchange ability between RDV, WTV, RGDV has not been tested.
2.	Nucleotide sequence homology. So far, nucleotide sequence identities among RDV isolates including those from different countries, are more than 90%. Some isolates have deletions [Chinese strain of RDV S11 (U36568) compared to Japanese RDV-Akita S11 (D10249)] or duplications [RDV-P S12]. The most extensive comparisons of S12 of 12 RDV isolates from five countries shows identitiy of 94.7 to 98.7% (Fig. 22). Sequence comparisons indicate that conservation between different species is at a much lower level.
3.	Protein sequence homology. Protein sequence homology between species is less than 50% and within species is more than 80%. This data indicates that high levels of serological cross-reaction would be detected within species by ELISA complement fixation (CF), or agar gel immunodiffusion (AGID), using either polyclonal sera, or monoclonal antibodies against conserved antigens. Distinct species may show significantly lower levels of cross-reaction. However, such serological methods are not routinely used to compare these viruses.
4.	Conserved terminal oligonucleotide sequences. The tetranucleotides at both ends of the genome segments are highly conserved within the genus. Two additional nucleotides flanking the 5-terminal tetranucleotide on the plus-strand are common within species.
5.	Cross-hybridization (Northern or dot blots, with probes made from viral RNA or cDNA using conditions designed to detect >80% homology). RNA-RNA hybridization detects all the segments within a species.
6.	Analysis of “electropherotype” by agarose gel electrophoresis but not by PAGE. (similarities can exist between closely related species).
7.	Identification of host plant. Dicotyledons (WTV), or the family Graminae (RDV and RGDV).


Rice dwarf virus	S1: [D90198], S2: [D00608], S3: [D00607], S4: [U36562], S5: [D90033], S6: [M91653], S7: [D00639], S8: [D00536], S9: [D00465], S10: [D00473], S11: [D10249], S12: [D90200]	(RDV)
{Nephotettix cincticeps, N. nigropictus, Recilia dorsalis: Graminae}
Rice dwarf virus, isolate B		(RDV-B)
Rice dwarf virus, isolate S		(RDV-S)
Rice dwarf virus, isolate H		(RDV-H)
Rice dwarf virus, isolate China		(RDV-Ch)
Rice gall dwarf virus	S2 [D86439], S3 [D13774], S8 [D13410], S9 [D01047], S10 [D13411]	(RGDV)
{Nephotettix cincticeps, N. nigropictus, N. virescens, N. malayanus, Recilia dorsalis: Graminae}
Wound tumor virus	S4: [M24117], S5: [J03020], S6: [M24116], S7: [X14218], S8: [J04344], S9: [M24115], S10: [M24114], S11: [X14219], S12: [M11133]	(WTV)
{Agallia constricta, A. quadripunctata, Agalliopsis novella: many dicotyledons}