Coelectrophoresis of double-stranded RNA from maize rough dwarf and rice black-streaked dwarf viruses.

Rice black-streaked dwarf virus was shown to contain ten dsRNA segments in a genome with an approximate molecular weight of 18.96 × 10⁶ or 19.66 × 10⁶, depending on the buffer employed for electrophoresis in polyacrylamide gels. In coelectrophoreses of genomes from rice black-streaked dwarf and maize rough dwarf virus, all segments coincided electrophoretically except segments 1, 3, and 10. In an earlier study of maize rough dwarf virus that had been maintained in a greenhouse, two types of genomes were distinguished by a slight difference in the electrophoresis of segment 10. In the present study, the virus samples were collected directly from the field, and they showed the same two kinds of segment 10.     

Electrophoretic separation of all components of the double-stranded RNA of wound tumor virus

The use of a new buffer solution for electrophoresis of the dsRNA of wound tumor virus permitted the electrophoretic separation and identification of all 12 ds RNA components of WTV RNA. The molecular weight of the genome was estimated to be 15.06 × 10⁶ daltons. All 10 components of reovirus type 2 could be separated by use of the same solution. The electrophoretic patterns for the first 7 components of wound tumor virus and rice dwarf virus are very similar.     

Electrophoretic separation of dsRNA genome segments from Fiji disease and maize rough dwarf viruses

By employing two different buffer solutions for polyacrylamide gel electrophoresis, all genome segments of Fiji disease and maize rough dwarf virus were separated. Fiji disease virus contains ten genome segments with approximate genome molecular weights of 19.26 x 10(6) and 19.85 x 10(6), depending on the buffer employed for electrophoresis. Maize rough dwarf virus possesses ten dsRNA segments and according to the buffer employed for electrophoresis the approximate molecular weights of the genome were 18.91 x 10(6) and 19.61 x 10(6). In the samples of maize rough dwarf virus analysed, the evidence indicates that two types of virions were present which were distinguished by a slight difference in molecular weight for segment 10. The electrophoretic patterns for all dsRNA components of Fiji disease and maize rough dwarf viruses were very similar suggesting a close relationship between them.     

Purification and some properties of reovirus-like particles from leafhoppers and their possible involvement in wallaby ear disease of maize

Reovirus-like particles, occurring in association with viroplasms, crystalline arrays and tubules, in the cytoplasm of Cicadulina bimaculata capable of inducing wallaby ear disease in maize, were purified from the insects by differential centrifugation, treatment with the nonionic detergent, Nonidet P-40, and sucrose density gradient centrifugation. The purified particles have a double-shelled icosahedral structure about 70 nm in diameter with external projections (A spikes) about 10 nm long located at the 12 vertices. These intact particles (IPs) are morphologically similar to those of Fiji disease virus (FDV), but are more stable. Cores were produced by enzymatic digestion of IPs with alpha-chymotrypsin. The cores are icosahedra about 57 nm in diameter with projections (B spikes) located at the 12 vertices, resembling those of FDV and cytoplasmic polyhedrosis virus. Immunization of a rabbit with purified IPs resulted in the production of antibodies specific to IPs, cores, and dsRNA. Immunoelectron microscopic investigations revealed that there is no relationship between this virus and FDV, maize rough dwarf, oat sterile dwarf, pangola stunt, and rice ragged stunt viruses, all members of the genus Fijivirus in the family Reoviridae. The nucleic acid extracted from partially purified virus was resolved into 10 segments by polyacrylamide gel electrophoresis. Reovirus-like particles or viroplasms could not be detected in thin sections of maize seedlings colonized by C. bimaculata showing wallaby ear symptoms. In the light of these data the possible etiology of wallaby ear disease is discussed.     

The presence of common host sequences in different populations of substituted SV40 DNA.

Ribonucleic acids (RNAs) of turnip mosaic (TurMV), tobacco etch (TEV), and maize dwarf mosaic viruses (MDMV) have molecular weights of 3.31 × 10⁶, 3.14 × 10⁶, and 2.89 × 10⁶ determined by polyacrylamide gel electrophoresis, and of 3.07 × 10⁶, 3.33 × 10⁶, and 2.94 × 10⁶ determined by sedimentation analysis in linear log sucrose density gradients. These correspond to S values of 39.0, 40.6 and 38.2. When denatured by formaldehyde, molecular weights are TurMV, 3.07 × 10⁶; TEV, 3.04 × 10⁶; and MDMV, 2.90 × 10⁶ determined by gel electrophoresis. Sedimentation coefficients after denaturation are TurMV, 24.7 S; TEV, 24.9 S; and MDMV, 24.2 S, corresponding to molecular weights of 3.16 × 10⁶, 3.32 × 10⁶, and 3.06 × 10⁶ for TurMV, TEV, and MDMV, respectively. The reduction in electrophoretic mobility and sedimentation coefficient after denaturation, along with hyperchromicity and T_m measurements, suggests that all three virus RNAs have a similar degree of secondary structure.     

Double-stranded ribonucleic acid from carnation cryptic virus

The nucleic acid of carnation cryptic virus has been identified as double-stranded RNA on the basis of (i) susceptibility to RNase in 0.1 x SSC (SSC = 0.15 M NaCl-0.015 M sodium citrate buffer, pH 7.0) and resistance to RNase in 2 x SSC and to DNase, (ii) reaction with an antiserum containing antibodies to dsRNA, and (iii) electron microscopic appearance in nondenaturing media similar to that of the dsRNA of maize rough dwarf virus. Upon electrophoresis in 5% polyacrylamide gels carnation cryptic virus RNA separated into three major segments plus a fourth minor component, and had a total molecular weight of about 4 x 10(6).     

A black-streaked dwarf disease on rice in China is caused by a novel fijivirus

An isolate of a plant reovirus causing severe stunting and dark leaf symptoms on rice from Guangdong, China, was similar in virion morphology and serologically related to rice black-streaked dwarf virus (RBSDV). The electrophoretic profiles of genome segments of the two viruses in agarose or polyacrylamide gel were indistinguishable. The four genome segments of the new isolate corresponding to RBSDV S7–S10 were amplified by ligation RT-PCR and sequenced. The size and organization of each genome segment was very similar to its counterparts in RBSDV, maize rough dwarf virus (MRDV), and mal de Rio Cuarto virus (MRCV). Sequence identity was greatest to RBSDV and MRDV (ranging from about 60–85% depending on the protein), but identities were always much lower than those between RBSDV and MRDV. These comparisons and phylogenetic analyses suggested that the virus represents a new species in genus Fijivirus group 2, tentatively named Rice black-streaked dwarf virus-2.     

Evidence of recombination and genetic diversity in southern rice black-streaked dwarf virus

Southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in China and Vietnam. Sequence identities of S10, encoding the major capsid protein, were 98.0 %-100 % and 98.3 %-100 % at the nucleotide and amino acid level, respectively. Our results suggest that the codon at position 550 of S10 is under positive selection, while most of the other codons are under neutral evolution. Putative recombination events were identified in genomic RNA segments S1, 2, 4, 5, 6 and 10, which are rare in plant-infecting dsRNA viruses. This study reveals the current state of SRBSDV evolution.     

Phylogenetic Analysis Reveals that a Dwarfing Disease on Different Cereal Crops in China is due to Rice Black Streaked Dwarf Virus (RBSDV)

A viral disease with dwarfing symptoms is associated with severe damage of different cereal crops including rice, maize, wheat and sorghum grown in China. It is believed that the pathogenic agent of the disease on rice and sorghum is rice black streaked dwarf virus (RBSDV), however, the cause of maize dwarf disease in China is still inconclusive. In this report, dsRNA was isolated from virus particles obtained from the diseased plants of rice, maize, wheat and sorghum from two Chinese provinces. Full-length cDNAs of genome segments 9 (S9) and 10 (S10) were obtained through a RT-PCR approach. Sequence analysis showed that the S9 sequences of Chinese isolates and Japanese RBSDV isolate were very similar to each other (89.1–89.6% identity at the nucleotide level, 92.3–92.9% and 95.8–98.6% identity at the amino acid level for ORF1 and ORF2, respectively). In addition, the S10 sequences of Chinese isolates and Japanese RBSDV were very similar to each other (93.0–95.4% identical nucleotides and 96.2–97.0% identical amino acids, respectively). However, there were lower similarities for S9 and S10 sequences between Chinese isolates and an Italian Maize Rough Dwarf Virus (MRDV) isolate. Phylogenetic analysis indicates that Chinese viral isolates found to infect rice, maize, wheat and sorghum and leading to similar cereal dwarfing manifestations could be grouped to the same virus species, RBSDV.     

Identification of rice black-streaked dwarf fijivirus in maize with rough dwarf disease in China

Summary.  Three virus isolates from maize with rough dwarf in different provinces in China were analyzed at the molecular level. When compared to an isolate from diseased rice plants in Hubei Province, all four isolates had identical genomic RNA electrophoretic profiles, which were composed of ten double-stranded (ds) RNAs. Full-length cDNAs of segment 10 (S10) from each of the four isolates were cloned by RT-PCR and the complete sequences were determined. Analysis of the sequences revealed that each consisted of 1801 nucleotides and contained a single open reading frame (ORF) which potentially encoded a protein with 558 amino acids. Further, the sequences showed more than 97.0% and 98.0% identity atnucleotide and amino acid levels, respectively. In addition, theiridentities to rice black-streaked dwarf virus S10 were significantlyhigher than those to maize rough dwarf virus S10. Based on theseresults, it is suggested that the virus which causes this maize diseasein China is rice black-streaked dwarf virus. Received April 4, 2000 Accepted July 4, 2000     

The molecular weight of bacteriophage θ6 and its nucleocapsid

A diameter of 82 nm, a molecular weight of 99 × 10⁶, and a refractive index increment of 0.152 g^?1cm³ were obtained for θ6 bacteriophage from turbidity measurements made with an analytical ultracentrifuge and a conventional spectrophotometer. Turbidity was also used to obtain a diameter of 64 nm and a molecular weight of 40 × 10⁶ for the θ6 nucleocapsid produced when virus solutions are made 2% Triton X-100. The RNA content found for the intact virus by the orcinol method was 10%- by weight, which corresponds to 10 million for the total molecular weight of the three double-stranded RNA molecules comprising the genome. The phospholipid content of the intact virus was found to be 20% by weight. The relative amounts of individual protein species were determined by gel electrophoretic analyses of radioactive virus. The results yielded estimates of the number of copies of each protein in the virion.     

Nucleotide Sequences of Double-Stranded RNA Segments from a Hypovirulent Strain of the White Root Rot Fungus Rosellinia necatrix: Possibility of the First Member of the Reoviridae from Fungus

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41×10⁶ to 2.95×10⁶. Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5-end (5-ACAAUUU-3) and at the 3-end (5-UGCAGAC-3) were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.     

Genetic structure of rice black-streaked dwarf virus populations in China

Rice black-streaked dwarf virus (RBSDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Fijivirus in the family Reoviridae. The genome of RBSDV consists of ten dsRNA segments. Although RBSDV has caused significant economic losses to rice and maize production in the past few years in China, its molecular diversity and evolution remain largely unknown. To elucidate the factor(s) underlying the evolution of RBSDV, we determined segment 8 (S8; carrying ORF8 encoding the minor core capsid protein) sequences of 101 samples and segment 10 (S10; carrying ORF10 encoding the major capsid protein) sequences of 103 samples. The results show that both ORF8 and ORF10 are under negative selection. The S8 of three isolates and S10 of two isolates are recombinants. The RBSDV population in China can be classified into three groups according to S8 sequences or into two groups according to S10 sequences, irrespective of host or geographical origin. Of the RBSDV isolates with both S8 and S10 sequences available, 17 are between-group reassortants and 30 are between-subgroup reassortants. The RBSDV subpopulations from different geographical regions and hosts show frequent gene flow within or between subpopulations. The RBSDV population from maize is in a state of expansion. In this study, no new emergent population was detected. Taken together, the results indicate that, in addition to recombination and negative selection, reassortment and gene flow are important factors that drive evolution of RBSDV in China.     

Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode

The purified genomic dsRNA of bluetongue virus type 1 (BTV 1) was separated into 10 size classes by polyacrylamide gel electrophoresis. These genome segments were recovered individually from the gel, denatured with methylmercuric hydroxide and translated in vitro in a rabbit reticulocyte lysate system. The virus-specific proteins synthesised in vitro were compared by polyacrylamide gel electrophoresis with proteins from purified virus particles and cores and with virus-specified proteins synthesised in infected BHK 21 cells (in vivo). The identity of those BTV 1 proteins synthesized in vivo and in vitro which showed similar electrophoretic mobilities, was confirmed by electrophoretic analysis of their partial protease digests. The results of these experiments have allowed the assignment of each of the genome segments of BTV 1 to the protein(s) which it encodes and consequently to the structural and nonstructural proteins found in the infected cell.     

The morphogenesis of human cytomegalovirus. Isolation and polypeptide characterization of cytomegalovirions and dense bodies.

Nucleic acid isolated from subviral particles of Fiji disease virus (FDV) was identified as double-stranded (ds)-RNA by the following properties: (1) Positive orcinol reaction; (2) resistance to ribonuclease (RNase) in 1 × SSC (sodium chloride-sodium citrate buffer) but not in 0.1 × SSC; (3) susceptibility to RNase in 1 × SSC after thermal denaturation; (4) sharp thermal denaturation curve with a melting temperature of 76° in 0.01 × SSC; (5) buoyant density of 1.60 g/cm³ in Cs₂SO₄; and (6) no increase in ultraviolet absorption on treatment with formaldehyde at 37°. On electrophoresis in polyacrylamide gel, FDV-RNA separated into nine RNA segments with a total molecular weight of 15.3 × 10⁶.     

Molecular characterization of the genome segments S4, S6 and S7 of rice gall dwarf virus

Summary Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus within the family Reovirdae. Its genome has 12 segments of double-stranded RNA (dsRNA), of which the nucleotide sequences of segments S4, S6, and S7 were determined, providing the first complete genome sequence of RGDV. Each of the segments S4, S6, and S7 contained conserved terminal sequences conforming to the RGDV consensus, 5′-GGXA … UGAU-3′ (X = U or C). Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of 79.8, 58.6, and 53.3 kDa. These proteins appeared to be homologous to those encoded by the corresponding segments of rice dwarf virus and wound tumor virus, the other known members of the same genus, having about 20–30% amino acid identity to them. It is therefore likely that RGDV S4 and S6 encode non-structural proteins and S7 an inner core protein. Probable homologies between the segments of all known phytoreoviruses are summarized. Beyond these similarities, the RGDV proteins displayed no significant similarity to any other reported viral proteins.     

Electrophoresis of the complementary strands of the double-stranded Kemerovo virus RNAs in agarose-urea gel

Single-stranded (ss)RNAs derived from 10 double-stranded (ds)RNA segments of Kemerovo virus (KV) were separated into 13 RNA bands by agarose-urea gel electrophoresis. The complementary strands of the dsRNA segments 1, 9 and 10 displayed different electrophoretic mobility. An attempt was made to determine the origin of the ssRNA bands. The ssRNA bands originating from the dsRNA segments 1, 2, 3, 9 and 10 were identified unequivocally, while those originating from the dsRNA segments 4, 5, 6, 7 and 8 were characterized partially. The minus RNA strands of the dsRNA segments 9 and 10 exhibited higher electrophoretic mobilities as their complementary plus RNA strands.     

Some properties of carnation ringspot virus single- and double-stranded ribonucleic acid

From sodium dodecyl sulfate (SDS)-dissociated carnation ringspot virus (CRSV) two RNAs of different sizes were separated by density gradient centrifugation. Preparations of the smaller RNA-2 (0.5 × 10⁶ daltons) were not infectious; preparations of the larger RNA-1 (1.5 × 10⁶ daltons) were only moderately infectious. Mixtures of the two kinds of preparations were very infective. This enhancement of infectivity was demonstrated within and between the two RNAs from each of two strains of CRSV. The genetic information controlling irreversible virus particle aggregation and dissociation of virus particles by SDS at pH 5 was contained in RNA-1. Virus-infected tissue contained dsRNA replicative forms corresponding to RNA-1, RNA-2, and a minor ss-RNA component.