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The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

Vertical transmission of Homalodisca vitripennis reovirus (HoVRV) from glassy-winged sharpshooter (GWSS, Homalodisca vitripennis (Germar)) females to progeny occurred in laboratory assays at frequencies too low (2.6%–15.4%) to account for HoVRV incidence (90–100%) in field populations resident in citrus. Because citrus is immune to HoVRV and no plant host is known, horizontal transmission of HoVRV from insect-to-insect was evaluated. Exposure of colony-reared, virus-free test nymphs to HoVRV-infected source adults held in the same cage for 10 days on virus-immune cowpea resulted in HoVRV transmission (13.3%–30.7%) to test nymphs. HoVRV was not transmitted when exposure was indirect and required passive movement of virions through the xylem of immune citrus seedlings. Collectively, these results demonstrate direct insect-to-insect horizontal transmission of HoVRV, providing a plausible explanation for high incidence of HoVRV in GWSS field populations in the absence of efficient vertical transmission or a plant host.

A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris (Ehrh.) and sequenced. This virus, named Thelephora terrestris virus 1 (TtV1), contains two reading frames in different frames but with the possibility that ORF2 could be translated as a fusion polyprotein after ribosomal -1 frameshifting. Picornavirus 2A-like motif, nudix hydrolase, phytoreovirus S7, and RdRp domains were found in a unique arrangement on the polyprotein. A new genus named Phlegivirus and containing TtV1, PgLV1, RfV1 and LeV is therefore proposed. Twenty species of oribatid mites were identified in soil material in the vicinity of T. terrestris. TtV1 was detected in large amounts in Steganacarus (Tropacarus) carinatus (C.L. Koch, 1841) and in much smaller amounts in Nothrus silvestris (Nicolet). This is the first description of mycovirus presence in oribatid mites.

The minor outer capsid protein P2 of Rice dwarf virus (RDV), a member of the genus Phytoreovirus in the family Reoviridae, is essential for viral cell entry. Here, we clarified the structure of P2 and the interactions to host insect cells. Negative stain electron microscopy (EM) showed that P2 proteins are monomeric and flexible L-shaped filamentous structures of ∼20 nm in length. Cryo-EM structure revealed the spatial arrangement of P2 in the capsid, which was prescribed by the characteristic virion structure. The P2 proteins were visualized as partial rod-shaped structures of ∼10 nm in length in the cryo-EM map and accommodated in crevasses on the viral surface around icosahedral 5-fold axes with hydrophobic interactions. The remaining disordered region of P2 assumed to be extended to the radial direction towards exterior. Electron tomography clearly showed that RDV particles were away from the cellular membrane at a uniform distance and several spike-like densities, probably corresponding to P2, connecting a viral particle to the host cellular membrane during cell entry. By combining the in vitro and in vivo structural information, we could gain new insights into the detailed mechanism of the cell entry of RDV.

Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae , induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis . Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis . Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis . For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors. IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral passage through the apical plasmalemma into salivary cavities. This mechanism could be extensively exploited by other persistent viruses to overcome salivary gland release barriers in insect vectors, opening new perspectives for viral control.

In the double-shelled capsid of Phytoreovirus, the outer capsid attaches firmly to the 3-fold axes of the T=1 core. It then forms a T=13 lattice via lateral interactions among the P8 trimers (Wu et al., 2000, Virology 271, 18-25). Purified P8 molecules also assemble into hexagonal monolayers as well as tubular crystals. To explore the mechanisms of formation of these structures, the configurations of P8 trimers were compared and verified in particles of Rice dwarf virus and in tubular crystals (tubes) whose structure was determined by cryoelectron microscopy using helical reconstruction technique. Remarkable variations in intertrimer contacts were observed in the tubes and in the surface lattice of Rice dwarf virus capsid. Superposition of the atomic structure of P8 trimers in the structures analyzed by cryoelectron microscopy allowed us to identify groups of specific and stable interactions, some of which were preserved in the tubes and the quasi-equivalent T=13 icosahedral lattice of the virion's shell. The flexible nature of the binding between P8 trimers, created via electrostatic interactions that hold radially inward, appears to allow the outer-capsid P8 trimers to envelop the ragged surface of the core, forming the double shell of an intact viral particle.

The nucleotide sequences of segments S1 and S12 of a Chinese isolate of Rice gall dwarf virus (RGDV) were determined. This provides the first complete sequences of these segments. The complete sequence of S1, the largest genome segment of RGDV, was 4,505 nucleotides in length and was predicted to encode a large protein of 1,458 amino acids with a calculated molecular mass of nearly 166.2 kDa. The protein was related to that encoded by S1 of Rice dwarf virus (RDV; 50% identity and 67% similarity) and (to a lesser extent) to some large proteins of other reoviruses. It appears to be an RNA-dependent RNA polymerase (RdRp) and is probably present in particles as a minor core protein. S12, the smallest genome segment of RGDV, was 853 nucleotides in length, encoding a single major protein of 206 amino acids with a calculated molecular mass of nearly 23.6 kDa. This protein, though a little larger than those of RDV S11 and Wound tumor virus (WTV) S12 in size, showed some similarity to them, especially in the conserved N-terminal region and may have RNA-binding properties. Despite having a common host plant, RDV and RGDV were not more closely related to one another than either of them was to WTV. Phylogenetic analysis of the RdRp showed that members of the genus Phytoreovirus were more closely related to those of the genus Rotavirus than to any other genus within the family Reoviridae.

Rice dwarf virus (RDV) is characterized by its unusual ability to multiply in both plants and leafhopper vector insects and by its transovarial mode of transmission. Colonies of Nephotettix cincticeps, derived originally from pairs of leafhoppers infected with an ordinary strain of RDV, were maintained for 6 years in the laboratory and were found, at the end of this time, still to harbor RDV. Moreover, the isolate of RDV, designated RDV-I, obtained from these colonies retained the ability to infect rice plants. When we raised leafhoppers separately from eggs that had been placed individually on pieces of water-soaked filter paper and reared them in the presence of healthy rice seedlings, we found that all of these leafhoppers harbored RDV. This observation suggested that RDV-I had been maintained in the leafhoppers by transovarial transmission. Two further observations, namely, the low rate of acquisition of RDV by virus-free insect nymphs on symptomless plants on which viruliferous insects had been reared, and the fact that only 2 to 5% of plants had symptoms when rice seedlings were inoculated via RDV-I-viruliferous insects, confirmed that the maintenance of RDV-I by any other mode of transmission through plants and insects was unlikely. This efficient and long-term maintenance of RDV in a population of viruliferous insects might explain the prolonged duration of rice dwarf disease in the field, once there has been a serious outbreak.

The complete nucleotide sequences of the double-stranded RNA segments S1, S4, S6, S7 and S12 of the genome of a Rice gall dwarf virus (RGDV) isolate from Thailand were determined. The segments consisted of 4505, 2622, 1648, 1652 and 853 nucleotides, encoding putative proteins of 1458, 725, 489, 511 and 206 amino acids with molecular masses of approximately 166, 80, 53, 59 and 24 kDa, respectively. Homology searches indicated that each of the putative proteins has a counterpart in isolates of Rice dwarf virus (RDV) and Wound tumor virus, two other species in the genus Phytoreovirus. However, no similarities were found to other registered sequences, including those of other viruses that belong to the family Reoviridae. The identities between homologous structural proteins of RGDV and RDV ranged from 34 to 51% and were thus higher than those between homologous non-structural proteins of RGDV and RDV (16-37%). Among the nonstructural proteins, the highest amino acid sequence identity (37%) was observed for RGDV Pns11 and RDV Pns10, a constituent of tubular inclusions. This observation suggests that a specific amino acid backbone might be required for maintaining not only the three-dimensional structure of virions but also that of inclusions. The entire sequence of the RGDV genome is now available.

An analysis, using microarrays, of gene expression in rice plants infected with Rice dwarf virus revealed significant decreases in levels of expression of genes that are involved in the formation of cell walls, reflecting the stunted growth of diseased plants. The expression of plastid-related genes also was suppressed, as anticipated from the white chlorotic appearance of infected leaves. By contrast, the expression of defense- and stress-related genes was enhanced after viral infection. These results suggest that virus-infected rice plants attempt to survive viral infection and replication by raising the levels of expression of defense- and stress-related genes while suppressing the expression of genes required for the elongation of cells and photosynthesis.

The salivary glands of the Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar 1821, (syn. H. coagulata, Hemiptera: Cicadellidae) were collected and used to produce a cDNA library. Examination by BLASTX analyses identified 2 viral sequences, one a 610-base pair fragment and a second 839-base pair fragment, both of which had significant homology to viruses within the genus Phytoreovirus. Resequencing of the fragments confirmed sequence validities. These sequences were used for in silico protein translation and BLASTP analysis confirming the established homology. While the GWSS is the primary vector of Pierce’s disease of grapes, this is the first report that GWSS may be a vector of a phytoreoviruses. Phylogenetic and homology comparisons with BLASTX, BLASTP, and PAUP analyses indicated that the viral sequences isolated from GWSS were closely related to the viruses in the Family Reoviridae, Genus Phytoreovirus, specifically Rice Dwarf Phytoreovirus (RDV). RDV is the only plant reoviru...

The family Reoviridae separates two subfamilies and consists of 15 genera. Fourteen viruses in three genera (Phytoreovirus, Oryzavirus, and Fijivirus) infect plants. The outbreaks of the plant-infecting reoviruses cause sometime the serious yield loss of rice and maize, and are a menace to safe and efficient food production in the Southeast Asia. The plant-infecting reoviruses are double-shelled icosahedral particles, from 50 to 80nm in diameter, and include from 10 to 12 segmented double-stranded genomic RNAs depending on the viruses. These viruses are transmitted in a persistent manner by the vector insects and replicated in both plants and in their vectors. This review provides a brief overview of the plant-infecting reoviruses and their recent research progresses including the strategy for viral controls using transgenic rice plants.

Between 2005 and 2008 the prices of rice, wheat and maize were more than doubled (von Braun, 2008). The sudden surge in cereal prices led or was led by major uncertainties to secure staple food supplies in many developing countries, especially in Asia. Several economic and social factors collectively appear to have triggered the unstable supplies of cereal crops during the recent food crisis (Headey and Fan, 2008). Besides, lower cereal crop productivities due to unfavorable environments and outbreaks of pests and diseases may have been associated, at least in part, with the unstable supplies of cereals during the period. In fact, massive outbreaks of brown planthoppers (BPH) transmitting rice viruses were reported in many areas of Indochina Peninsula during 2006 and 2008, which resulted in a significant decrease in international trade of rice (Ta et al., 2013). This research topic consists of four original research and seven review articles covering various aspects of cereal viruses and their vector insects that are considered as current or potential threats to stable production of cereal crops. We also hope that this e-book can provide the readership an update on the recent studies on a variety of cereal viruses.

Transencapsidation of the Rice gall dwarf virus (RGDV) inner core by the Rice dwarf virus (RDV) outer capsid P8 protein was examined in vitro and in planta. When RGDV core particles were incubated with an extract from RDV P8-transgenic rice leaf tissue, RDV P8 encapsidated the RGDV core particles to form double-shelled virus-like particles in vitro. In contrast, when RDV P8-transgenic rice plants were inoculated with RGDV, progeny RGDV particles contained RGDV P8 but RDV P8 was not detectable in the virions. No significant differences were found in acquisition by the vector insects and subsequent transmission rates between RGDV infecting nontransgenic rice plants and those infecting RDV P8-transgenic rice plants. These results indicate that mechanisms of and/or requirements for interactions between P8 and the inner core particles of phytoreoviruses differ between in vitro and in planta.

Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of members of the family Reoviridae. Immunocytochemical and biochemical analyses were carried out to characterize the poorly understood viroplasms of the phytoreovirus Rice dwarf virus (RDV). Within 6 h of inoculation of cells, viroplasms, namely discrete cytoplasmic inclusions, were formed that contained the non-structural proteins Pns6, Pns11 and Pns12 of RDV, which appeared to be the constituents of the inclusions. Formation of similar inclusions in non-host insect cells upon expression of Pns12 in a baculovirus system and the association of molecules of Pns12 in vitro suggested that the inclusions observed in RDV-infected cells were composed basically of Pns12. Core proteins P1, P3, P5 and P7 and core virus particles were identified in the interior region of the inclusions. In contrast, accumulation of the outer capsid proteins P2, P8 and P9 and of intact virus particles was evident in the peripheral regions of the inclusions. These observations suggest that core particles were constructed inside the inclusions, whereas outer capsid proteins were assembled at the periphery of the inclusions. Viral inclusions were shown to be the sites of viral RNA synthesis by labelling infected cells with 5-bromouridine 5′-triphosphate. The number of viroplasms decreased with time post-inoculation as their sizes increased, suggesting that inclusions might fuse with one another during the virus-propagation process. Our results are consistent with a model, proposed for vertebrate reoviruses, in which viroplasms play a pivotal role in virus assembly.

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.

The viral and virus-related structures of Rice dwarf virus have been visualized by cryo-electron microscopy and tomography revealing the viral infection and replication mechanisms., It is well known that viruses utilize the host cellular systems for their infection and replication processes. However, the molecular mechanisms underlying these processes are poorly understood for most viruses. To understand these molecular mechanisms, it is essential to observe the viral and virus-related structures and analyse their molecular interactions within a cellular context. Cryo-electron microscopy and tomography offer the potential to observe macromolecular structures and to analyse their molecular interactions within the cell. Here, using cryo-electron microscopy and tomography, the structures of Rice dwarf virus are reported within fully hydrated insect vector cells grown on electron microscopy grids towards revealing the viral infection and replication mechanisms.

Rice dwarf virus (RDV) is a member of the genus Phytoreovirus , which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either β-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana . Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum , a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana . A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum . By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.

The nucleotide sequences of segments S1 to S6 of a Chinese isolate of Rice black-streaked dwarf virus (RBSDV) were determined. This provides the first complete sequence of a plant pathogenic member of the genus Fijivirus. The complete ten-segment genome has 29,141 nucleotides, making it the largest reovirus genome so far reported. Each of the segments S1-S6 is predicted to encode a single major protein. Protein comparisons indicated that S1 encoded an RNA dependent RNA polymerase, with similarities to that encoded by S1 of Nilaparvata lugens reovirus (NLRV). S2 and S3 appeared to be homologous to S3 and S4 respectively of both Fiji disease virus (FDV) and NLRV. The protein encoded on S4 showed some similarity to that of NLRV S2. The proteins encoded on S5 and S6, though similar in size to those of NLRV S5 and S6, had no detectable homologies to them or to any other known protein.

The structures of the double-shelled rice dwarf virus and of its single-shell core have been determined by cryoelectron microscopy and image reconstruction. The core carries a prominent density located at each of the icosahedral faces of its T = 1 lattice. These protrusions are formed by outer shell trimers, tightly inserted at the threefold positions of the core. Such configuration of the core may guide the assembly of the outer shell, aided by lateral interactions between its subunits, into a T = 13 lattice. The organization of the phytoreovirus capsid elucidates for the first time a general model for assembling two unique T numbers of quasi-equivalence.

A hammerhead ribozyme (Rz) with long hybridizing arms targeting the mRNA of rice dwarf virus (RDV) segment 5 and a mutated nonfunctional ribozyme (mRz) were constructed. As predicted, Rz transcribed in vitro cleaved the target mRNA of RDV segment 5 into two fragments of 138 and 238 nucleotides in length. The Rz and mRz genes were each placed under the control of the CaMV 35S promoter and used to transform Japonica rice variety 'Tongling No.1' via Agrobacterium tumefaciens. A total of 32 independent lines containing Rz or mRz was obtained as demonstrated by Southern blot analysis. Challenge inoculation with RDV viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plants containing the Rz transgene displayed high resistance or delayed and attenuated viral symptoms. In contrast, transgenic lines expressing mRz showed severe symptoms similar to the control plants transformed with the vector alone. These results suggest that Rz confers RDV resistance in transgenic rice. Genomic DNA PCR analysis confirmed that all of the examined T6 progeny plants contained the Rz transgene. However, accumulation of the Rz transcripts was detectable by RT-PCR only in the plants that were resistant to RDV. This suggested that loss of RDV resistance in progeny plants containing the Rz transgene may result from silencing of the Rz transgene.

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [32P]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodo-ptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 × 106 Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.

The outer capsid protein (P8) heterogeneity of rice dwarf virus (RDV) exists not only in purified virus particles, but also in RDV-infected rice, transgenic rice expressing P8, E. coli expression of P8 product and the in vitro translation products of S8. N-terminal amino acid sequencing revealed that P8 is a cleavage product of P8'. The cleavage occurs specifically at the residues of Asp362 and Pro363. The function of the proteolytic processing is unknown.

The function of rice dwarf virus segment 11 and the corresponding segments of other phytoreoviruses is not yet determined. The amino acid sequence of Pns11, encoded by segment 11, contains a putative zinc finger and five flanking basic regions at the C-terminus. The full-length Pns11 protein and three truncated derivatives, which lack the N-terminus, the zinc-finger, or the C-terminal five basic regions were expressed inEscherichia coliand their nucleic acid binding properties were studied. Pns11 interacts with single- and double-stranded forms of DNA and RNA in a sequence-nonspecific manner. The truncated derivative which contains both the zinc-finger and the C-terminal basic regions has the same binding properties as the full-length Pns11. However, removal of either of these domains prevents binding activity. The binding activity of Pns11 was drastically reduced when the blots were treated with a high concentration of EDTA. Moreover, Pns11 extracted from infected rice also binds to single-stranded RNA. These data suggest that RDV Pns11 binding activity is structure-dependent and it may play an important role in virus replication and/or genome assortment.

There are now over 30 viruses reported to infect rice through experimental tests and in nature. However, only 25 are of any direct economic impact to rice production. And most of them are transmitted by either plant or leaf hoppers. The rice grassy stunt tenuivirus (RGSV), rice hoja blanca tenuivirus (RHBV), rice ragged stunt phytoreovirus (RRSV), rice wilted stunt virus (RWSV) (a relative of Rice grassy stunt tenuivirus (RGSV)), rice stripe tenuivirus (RSV), rice black streaked dwarf fijivirus (RBSDV) are plant hopper borne, while rice bunchy stunt pytoreovirus (RBSV), rice dwarf phytoreovirus (RDV), rice gall dwarf phytoreovirus (RGDV), rice transitory yellowing rhabdovirus (RTYV), rice yellow dwarf mycoplasma-like organisms (RYDMLO's), rice waika machlovirus (RWV), rice orange leaf virus (ROLV), maize streak geminivirus (MSV) strain A, and rice tungro machlovirus/badnavirus (RTV) are transmitted by leafhoppers. The African specific rice yellow mottle sobemovirus (RYMV) is transmitted by the Ch...

Summary Symptom variability of tobacco leaf curl disease has been reported by numerous researchers worldwide. It has been suggested that the different types of leaf curl may be caused by more than one geminivirus strain (McClean, 1940; Osaki & Inouye, 1981), although no evidence exists to support this hypothesis. Previously, several tobacco plants exhibiting a range of leaf curl symptoms were collected from South Africa and Zimbabwe and grouped according to symptomology (Paximadis, Dusterwald, Duyver & Rey, 1997). Three classes (I, II & III) of leaf curl were distinguished, and class I leaf curl from South Africa was shown not to be associated with a geminivirus infection, but was consistently associated with 12 double-stranded RNA species, now believed to be of phytoreovirus origin. In this study, symptom descriptions of class II and III leaf curl have been reported, and, triple-antibody sandwich ELISAs, dot-blot hybridisation analysis, PCR, double-stranded RNA analysis and seed-transmission experiments were employed to screen class II and III tobacco plants for geminiviruses or other possible causal agents. Class II leaf curl symptoms were shown not to be associated with a geminivirus infection or with any double-stranded RNA species. Class II leaf curl symptoms were however transmitted through seed. Class III leaf curl plants tested negative for double-stranded RNA, but tested positive in PCR, dot-blot hybridisation and ELISAs for geminiviruses. It is suggested that ‘tobacco leaf curl’ is not a single disease but rather a wide collection of symptoms incited by different agents, and should therefore be described as a disease complex or syndrome.

The nucleotide sequence of the genome segment 2 (S2) of rice gall dwarf virus (RGDV), a phytoreovirus, when compared with the amino acid sequence of a component protein of the virus, showed that S2 potentially encoded a 127K minor outer capsid protein. This 127K protein designated as P2 and the 127K minor outer capsid protein (also termed P2) of rice dwarf virus (RDV) are similar in size, located in the outer capsid, and have well-conserved predicted polypeptide sequences, suggesting similar functions. Infectivity to insect vector cell monolayers of RGDV was maintained and the P2 protein was retained irrespective of carbon tetrachloride (CCl4) treatment. This is in contrast to the infectivity of RDV which is removed along with P2 protein following CCl4 treatment. RGDV with P2 was acquired by vector insects and transmitted to host plants, although RDV lacking P2 could not be transmitted to plants as previously published. These results imply that RDV and RGDV require P2 proteins for virus infectivity to vector insects.

The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants.

A high incidence (86%) of potyvirus infection was noted in tobacco plants exhibiting a form of leaf curl in South Africa. Despite leaf curl being reported in the literature to be of geminiviral aetiology, no geminiviruses were detected. Furthermore, no other virus particles were detected by virus purification, TEM and serology. Twelve species of dsRNA were consistently isolated from these tobacco plants, but were absent from other forms of leaf curl-affected and healthy tobacco. Aphid and mechanical inoculation demonstrated that the purified potyvirus(es) did not cause leaf curl symptoms, but rather mild mottle and mosaic symptoms in tobacco. Partial characterization of the potyvirus preparation showed a possible relationship to a South African strain of potato virus Y. Because potyvirus-inoculated plants did not manifest leaf curl symptoms, and because leaf curl symptoms were noted in some plants not infected with a potyvirus, it was concluded that the potyvirus is not involved in the leaf curl aetiology, but causes a latent infection, the symptoms of which are masked. The pattern of the dsRNA banding, induction of enations and lack of mechanical and seed transmission are common to plant reoviruses. The possibility of a phytoreovirus involvement in this form of leaf curl is currently being investigated. The results from this study suggest that tobacco leaf curl disease worldwide, with regard to geminiviruses, be re-evaluated.

Rice dwarf virus isolates were collected from several locations in Japan, the Philippines, China, Nepal and Korea. Genomic dsRNA segment profiles in polyacrylamide gel electrophoresis differed among the isolates. There were less differences in the profiles between isolates from Japan and Korea than in those between these two Countries and others. Nucleic acid hybridization was used to examine the extent of genomic variation. Full-length cDNAs to all genomic segments encoding non-structural proteins (S4, S6, S9, S10, S11 and S12) were synthesized from two Japanese isolates, and were used for dot-blot hybridization. Hybridizations using probes generated from the full-length cDNA clones failed to differentiate isolates from different geographical areas. However, cDNA probes covering a variable region of S12 were able to distinguish Japanese and Korean isolates from those of other countries. Phylogenetic tree analysis based on the amino acid sequence of P12 encoded by S12 grouped Japanese and Korean isolates together. The Chinese isolates from two different locations (Yunnan and Fujian) were closely related to each other, and were the most distantly related to Japanese and Korean isolates.

Analysis of the primary organizations of the phytoreoviruses wound tumor (WTV), rice dwarf (RDV), and rice gall dwarf (RGDV), shows that they are similar but distinct in molecular structure. Detailed correlations can therefore be sought between their phenotypic and genomic properties. This review focuses on the conserved nucleotide and amino acid sequences of phytoreoviruses with reference to the functions and physical properties of the proteins.