Virology 266, 26–32 (2000) doi:10.1006/viro.1999.0068, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Comparison of Nucleotide Sequences between Northern and Southern Philippine Isolates of Rice Grassy Stunt Indicates Occurrence of Natural Genetic Reassortment

Gilda J. Miranda,* Ossmat Azzam,† and Yukio Shirako,*,‡,1

*Graduate School of Agricultural Life Science and ‡Asian Center for Bioresources and Environmental Sciences (ANESC), University of Tokyo, Bunkyo-ku, Tokyo 113-8657, ; and †International Rice Research Institute, MCPO Box 3127, 1271 Makati City, The Received March 5, 1999; returned to author for revision March 31, 1999; accepted October 25, 1999

Rice grassy stunt virus is a member of the genus , is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia (similar to North and South America). We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5Ј untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors. © 2000 Academic Press

INTRODUCTION 337-kDa, polymerase protein on its cRNA (Toriyama et al., 1994). RNAs 2–4 are ambisense, coding for a single (RGSV) is a member of the polypeptide at the 5Ј terminal region of both virus-sense genus Tenuivirus, which includes rice stripe virus (RSV) RNA (vRNA) and cRNA; RNA 2 (3.5 kb) codes for a 23-kDa as the type species, rice hoja blanca virus (RHBV), protein on vRNA and a 94-kDa, putative membrane pro- stripe virus, Echinochloa hoja blanca virus, and Urochloa tein on cRNA (Takahashi et al., 1993; Estabrook et al., hoja blanca virus (Falk, 1998; Ramirez and Haenni, 1994; 1996), RNA 3 (2.5 kb) codes for a 24-kDa protein on vRNA Toriyama and Tomaru, 1995). Characteristics of tenuivi- and a 35-kDa capsid protein on cRNA (Kakutani et al., ruses include long, thread-like ribonucleoprotein (RNP) 1991; Zhu et al., 1991), and RNA 4 (2.2 kb) codes for a particles consisting of four to six negative-sense or am- 21-kDa major noncapsid protein (NCP) on vRNA and a bisense RNA segments and a single species of capsid 32-kDa protein on cRNA (Kakutani et al., 1990; Zhu et al., protein of about 36 kDa. A polymerase protein of about 1992). RHBV (de Miranda et al., 1994, 1996d, 1997; 340 kDa is also a minor component of RNP particles and Ramirez et al., 1993) also has four RNA segments with has an amino acid sequence homology with those of the same genome organization as RSV. Maize stripe Bunyaviridae (Toriyama et al., 1998). are virus (Huiet et al., 1991, 1992, 1993; Estabrook et al., 1996) transmitted by planthoppers in a persistent manner. RGSV infects rice plants (Oryza sativa L.), causing ex- and Echinochloa hoja blanca virus (de Miranda et al., cess tillering and stunting of the plants in South, South- 1996a–c) have an additional 1.3-kb, monocistronic nega- east, and East Asian countries, and is transmitted by the tive-sense RNA 5. brown planthopper Nilaparvata lugens (Stal) (Hibino et Recently, the complete nucleotide sequence of an IRRI al., 1985; Hibino, 1996). (International Rice Research Institute) isolate of RGSV The genome of RSV, the type Tenuivirus, is composed from a northern part of the Philippines (Laguna Province) of four RNA segments. RNA 1 (9.0 kb) encodes a single was determined (Toriyama et al., 1997, 1998). RGSV ge- nome consists of six ambisense RNA segments with two additional RNAs between RNA 2 and RNA 3 of RSV in

1 size. RNA 1 (9.8 kb) codes for a 19-kDa protein on vRNA To whom reprint requests should be addressed at Asian Center for and a 339-kDa polymerase protein on cRNA. RGSV RNAs Bioresources and Environmental Sciences (ANESC), University of To- kyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Fax: 81-3-5841- 2 (4.1 kb), 5 (2.7 kb), and 6 (2.6 kb) are equivalent to RSV 8036. E-mail: [email protected]. RNAs 2, 3, and 4, whereas RGSV RNAs 3 (3.1 kb) and 4

0042-6822/00 $35.00 Copyright © 2000 by Academic Press 26 All rights of reproduction in any form reserved. NORTHERN AND SOUTHERN PHILIPPINE ISOLATES OF RGSV 27

TABLE 1 RGSV Isolates and Variant

Isolation

Designation Symptoms Location Year Passage length Reference

South Cotabato Moderate South Cotabato 1989 6 years Miranda et al. (1991) Laguna Moderate Laguna early 1980s ϳ10 years Miranda et al. (1991) Toriyama et al. (1997, 1998) Laguna.M Mild IRRI 1989 7 years Miranda et al. (1991)

Note. Individual isolates were passaged in rice plants using brown planthoppers with 3- to 4-month intervals for indicated periods until virus purification. The Laguna.M variant was isolated during serial passages of the Laguna isolate at the International Rice Research Institute (Miranda et al., 1991).

(2.9 kb) are unique in the genus Tenuivirus; RNA 3 codes clones), 2.3 kb (19 clones), 2.5 kb (34 clones), and 2.6 kb for 23-kDa and 31-kDa proteins and RNA 4 codes for (27 clones) were constructed (Fig. 1), which were iden- 19-kDa and 60-kDa proteins on their vRNA and cRNA, tified to be originated from RNAs 2, 4, 5, and 6, respec- respectively. Between the equivalent proteins of RSV and tively, by nucleotide sequence and translated amino acid RGSV, amino acid sequence similarities were less than sequence comparisons with those of other tenuiviruses 30%, leading Toriyama et al. (1998) to propose that RGSV and the Laguna isolate of RGSV. The 5Ј terminal and 3Ј be placed in a genus other than Tenuivirus. terminal nucleotide sequences were determined after In this study, we determined the complete nucleotide RT-PCR cloning of G-tailed cDNA. sequence from another IRRI isolate from South Cotabato, a There was only two and three cDNA clones that derived southern part of the Philippines, and compared it with that from RNA 1 and RNA 3, respectively, in the library. There- of the Laguna isolate (Table 1). South Cotabato in Mind- fore, the entire nucleotide sequences of RNA 1 and RNA 3 anao Island and Laguna in Luzon Island are about 1000 km were determined not only from cDNA clones but also from apart from south to north. The result showed that the two clones derived after RT-PCR using internal primer pairs (Fig. isolates are distinct in RNAs 2, 3, and 6 but nearly identical 1). The 5Ј terminal and 3Ј terminal regions were cloned in RNAs 1, 4, and 5, indicating that there was genetic after RT-PCR of G-tailed cDNA and sequenced. reassortment in the two geographical isolates. This is the first report on the possible natural occurrence of genetic Nucleotide and amino acid differences between the reassortment of a with a segmented RNA ge- northern and southern Philippine isolates of RGSV nome as deduced from sequence analyses. The total numbers of nucleotide of RNAs 1–6 of the RESULTS South Cotabato isolate were 9760, 4069, 3127, 2909, 2704, and 2590, respectively. Nucleotide sequence determination Table 2 shows comparisons of nucleotide sequences of RNP particles were purified by differential centrifuga- RNAs 1–6 between the South Cotabato and Laguna iso- tion, followed by a sucrose density gradient centrifuga- lates. Overall differences at the nucleotide level were 0.08% tion, which resulted in two distinct light-scattering bands in RNA 1, 2.19% in RNA 2, 8.38% in RNA 3, 0.58% in RNA 4, in the upper half of the gradient. RNA extracted from a 0.26% in RNA 5, and 3.63% in RNA 6. Between the two gradient-purified RNP contained five distinct double- isolates, the numbers of nucleotides were identical in two stranded RNAs (dsRNAs) ranging from 4.1 to 2.6 kb with open reading frames (ORFs) of all six RNAs. The nucleotide diffused minor bands migrating faster than the dsRNA numbers in the 5Ј and 3Ј untranslated regions of all six bands in a 0.8% agarose gel probably corresponding to RNAs were also identical between the two isolates except single-stranded forms of five RNA species (data not for the 3Ј end of vRNA 4; the South Cotabato segment was shown). DsRNA corresponding to intact 9.8-kb RNA 1 7 nucleotides shorter than the Laguna isolate in this region was not detectable, probably due to degradation during due to a deletion of one of four tandem repeats of AA- storage and shipment of the infected materials or in virus GAAAA, which may have occurred in a single event during purification process. replication. On the contrary, nucleotide sequences in the From a random cDNA library prepared to the total RNA intergenic region differed more than the other regions by preparation from gradient-purified RNP particles, we ini- single and multiple substitutions and deletion/insertions, tially obtained 118 independent cDNA clones with an particularly in RNAs 2, 3, and 6. In the intergenic regions, average insert size of 1.5 kb. Four nucleotide sequence the two isolates differed by 9.12% in RNA 2, 11.83% in RNA assemblages with the sizes of 4.0 kb (consisting of 12 3, and 6.86% in RNA 6, whereas they differed by only 28 MIRANDA, AZZAM, AND SHIRAKO

FIG. 1. Locations of individual random cDNA clones and RT-PCR amplified regions aligned to each RNA segment. Solid lines indicate nonchimeric cDNA clones; dashed line, cDNA was derived from other RNA fragments or an unidentified origin; and lines with filled circles at the both ends indicate that the regions were amplified by RT-PCR before cloning into a plasmid vector.

0.78% in RNA 4 and 0.34% in RNA 5. In RNA 1, nucleotide protein (0.38%). In RNA 5, there was only one amino acid differences were found only in the ORF 2. Differences in difference in the 22-kDa protein (0.52%), whereas both nucleotide sequences between the two isolates were par- two nucleotide differences in the 36-kDa capsid protein ticularly distinct in the intergenic region as shown in Fig. 2. gene were silent. In RNA 2, there were four nucleotide differences caus- ing two conservative amino acid substitutions in the Nucleotide sequence variations within the two 23-kDa ORF (1.00% difference at the amino acid level), isolates in the intergenic regions of RNA 5 and 6 whereas 11 of 28 nucleotide differences caused amino acid changes in the 94-kDa ORF (1.34%). It was peculiar To examine the extent of heterogeneity at the nucleotide that the 5Ј terminal 0.7-kb region of the 94-kDa gene was level within individual isolates, we examined nucleotide identical between the two isolates. sequence variations in the intergenic region of RNAs 5 and RNA 3 was most diverged among six RGSV segments 6 of the South Cotabato isolate and a mild isolate derived between the two isolates. There were 7 amino acid from the Laguna isolate (designated Laguna.M) (Table 1). differences in the 22-kDa ORF (3.59%) and 48 amino acid The intergenic region was amplified by RT-PCR and cloned differences in the 31-kDa ORF (17.65%) (Fig. 3). into pCR2.1 plasmid (InVitrogen, San Diego, CA). Nucleotide On the contrary, the high rate of nucleotide differences in sequence was determined from both ends of the insert RNA 6 between the two isolates did not cause much diver- from four independent clones. As a result, nucleotide se- gence in amino acid sequence. All 10 nucleotide differ- quence variations in the intergenic regions of RNA 5 and 6 ences in the 21-kDa NCP gene and 19 of 21 nucleotide of the South Cotabato isolate were 0.11 Ϯ 0.07% and 0.38 Ϯ differences in the 36-kDa protein on cRNA did not cause 0.29%, respectively. With the Laguna.M isolate, nucleotide amino acid differences, likely due to strong constraint for sequence variations in the intergenic regions of RNA 5 and their functions of the translated products. Two amino acid RNA 6 were 0.65 Ϯ 0.22% and 0.33 Ϯ 0.13%, respectively. differences in the 36-kDa protein were both conservative. Therefore, there was no significant differences in nucleo- On the contrary, proteins coded on RNAs 1, 4, and 5 tide variations between RNA 5 and RNA 6 as shown in both were mostly identical between the two isolates. In RNA 1, isolates. On the other hand, the Laguna isolate and the there were only three amino acid differences in the Laguna.M isolate differed by only 0.23% in the intergenic 339-kDa protein (0.10%). In RNA 4, there were only one region of RNA 5 and by 0.66% in that of RNA 6, indicating conservative amino acid difference in the 19-kDa protein that the mild isolate was closely related to the parental (0.61%) and two amino acid differences in the 60-kDa Laguna isolate even after 7 years of passage after isolation. NORTHERN AND SOUTHERN PHILIPPINE ISOLATES OF RGSV 29

TABLE 2 Comparison of Six RGSV RNA Segments between the South Cotabato and Laguna Isolates at the Nucleotide Level

Regions in RNA genomea RNA segments 5Ј UTR ORF 1 IGR ORF 2 3Ј UTR Total

1 Numbers of total ntb 165/165 501/501 108/108 8778/8778 208/208 9760/9760 Numbers of different ntc None None None 8 None 8 % of different ntd None None None 0.09 None 0.08 2 Numbers of total nt 209/209 603/603 581/568 2469/2469 207/207 4069/4056 Numbers of different nt 3 4 53 28 1 89 % of different nt 1.43 0.66 9.12 1.13 0.48 2.19 3 Numbers of total nt 159/159 588/588 1386/1382 819/819 175/175 3127/3123 Numbers of different nt 6 23 164 63 6 262 % of different nt 3.77 3.91 11.83 7.69 3.34 8.38 4 Numbers of total nt 227/227 498/498 511/510 1578/1578 95/102 2909/2915 Numbers of different nt None 1 4 5 7 17 % of different nt None 0.20 0.78 0.32 7.30 0.58 5 Numbers of total nt 103/103 576/576 878/878 978/978 169/169 2704/2704 Numbers of different nt None 2 3 2 None 7 % of different nt None 0.35 0.34 0.20 None 0.26 6 Numbers of total nt 74/74 540/540 919/913 978/978 79/79 2590/2584 Numbers of different nt None 10 63 21 None 94 % of different nt None 1.85 6.86 2.15 None 3.63

a Genome sense is as described by Toriyama et al. (1998). UTR, untranslated region; ORF 1, the 5Ј proximal ORF on the genome strand; IGR, intergenic region; ORF 2, the 5Ј proximal ORF on the complementary strand. b South Cotabato isolate/Laguna isolate. c Each nucleotide substitution, deletion and insertion between the two isolates was counted as 1. d The number of different nt was divided by the number of total nt of the South Cotabato isolate in each region.

DISCUSSION from more than three independent cDNA clones or RT- PCR-derived clones covering the entire genome. The In this study, we determined the complete nucleotide analysis of the intergenic regions of the two isolates from sequences of the six RNA segments of the South Cota- Laguna also supports that accumulation rates of muta- bato isolate of RGSV from a southern part of the Philip- tions among individual RNA segments are not signifi- pines and compared them with those of the Laguna isolate from a northern part of the Philippines. The re- cantly different. As shown in Table 2 and Fig. 2, con- sults showed that there were significant differences in straints to genetic divergence are stronger for coding the accumulation of mutations between the two isolates regions than for the noncoding intergenic regions. depending on RNA segments. Among different tenuiviruses, RNA 3 (equivalent to RNAs 1, 4, and 5 are nearly identical throughout the RGSV RNA 5) is generally more diverged than RNA 4 segments, with 0.08%, 0.58%, and 0.26% differences, re- (equivalent to RGSV RNA 6) (Toriyama et al., 1997). RNA spectively, whereas RNAs 2, 3, and 6 differed by 2.19%, 3 codes for a capsid protein on cRNA, which is ex- 8.38%, and 3.63% as a whole and by 9.12%, 11.83%, and pressed both in host plants and in vector insects, 6.86% in the intergenic region, respectively. In RNA 3, the whereas RNA 4 codes for a major noncapsid protein, two isolates differed considerably not only in the inter- which is detected in abundance in host plants but not genic region but also in a coding region, resulting in a detectable in viruliferous insects (Falk et al., 1987; 17.65% difference at the amino acid level of the 31-kDa Miranda and Koganezawa, 1995). RNA 3 may replicate protein. These results indicate that there was genetic more than RNA 4 during circulation between host plants reassortment in the two geographical isolates; RNAs 1, 4, and insects, and during the course of evolution, RNA 3 and 5 of the two isolates came from a common ancestor, may have diverged more rapidly than RNA 4 among whereas RNAs 2, 3, and 6 were from two different an- different tenuiviruses. Considering that RNA 3 is more cestors. diverged than RNA 4 among tenuiviruses, near identities A possibility that some RNA segments accumulate in RGSV RNAs 1, 4, and 5 and significant divergence in mutations more rapidly than the others within an isolate RGSV RNAs 2, 3, and 6 between the two RGSV isolates during a natural replication process can be excluded may support the natural occurrence of genetic reassort- because there were no significant differences in nucle- ment. otide variations among six RNA segments as determined Besides this study, there have been two reports on 30 MIRANDA, AZZAM, AND SHIRAKO

nucleotide level (our analysis), with multiple deletion/ insertions in the intergenic region in both RNAs. The Chinese isolate was more distantly related from either of the two Japanese isolates, indicating no proof of recent genetic reassortment. On the other hand, the two iso- lates of RHBV differed by 1.36% in RNA 3, with mostly a single nucleotide substitutions, and by 4.90% in RNA 4, including multiple deletion/insertions in the intergenic region, suggesting a possibility of reassortment (de Miranda et al., 1997). Genetic reassortment of segmented RNA such as Orthomyxoviridae (Murphy and Webster, 1996), Reoviridae (Ramig and Ward, 1991; Ramig, 1997; Uyeda et al., 1995), and Bunyaviridae (Henderson et al., 1995; Rodriguez et al., 1998; Qiu et al., 1998) is well documented as a source of naturally occurring vari- ants as well as an experimental tool to map a biolog- ical phenotype to a specific RNA segment. In plant viruses with segmented RNA genomes, although there have been reports on the generation of reassortants after coinoculation or coinjection of two related but distinguishable viruses in a greenhouse or a labora- tory (Uyeda et al., 1995; Qiu et al., 1998; Masuta et al., 1998), natural cases of reassortment of segmented plant RNA viruses appear to be rare (Fraile et al., 1997; FIG. 2. Positions of nucleotide and amino acid differences between Hu and Ghabrial, 1998). This is the first report of the Laguna and South Cotabato isolates of RGSV. Rectangular boxes indi- possible natural occurrence of reassortment of plant cate ORFs. Vertical bars below the horizontal line and rectangular segmented RNA virus demonstrated with the entire boxes indicate positions of nucleotide differences, whereas those genome at the nucleotide level. above the boxes indicate positions of amino acid differences. Except for the 31-kDa protein coded on cRNA, amino acid differences are indicated above the vertical bars as the amino acid in Laguna isolate MATERIALS AND METHODS and the position from the amino terminus to the amino acid in South Virus isolates and variants Cotabato isolate. A South Cotabato isolate was collected from the prov- ince of South Cotabato, a southern part of the Philip- sequence comparisons of different geographical isolates pines, in 1989 and passaged for 6 years before use in of tenuiviruses, RSV and RHBV. In case of RSV, RNAs 3 this study. An isolate from Laguna and characterized by and 4 of two Japanese and one Chinese isolates were Toriyama et al. (1997, 1998) was originally collected from compared (Qu et al., 1997). The two Japanese isolates the province of Laguna, a northern part of the Philippines differed by 3.23% in RNA 3 and 5.33% in RNA 4 at the in early 1980s, and passaged for about 10 years (Miranda

FIG. 3. Alignment of amino acid sequences of the 31-kDa protein coded on cRNA 3 of the two RGSV isolates. For the South Cotabato protein, amino acids identical to those of the Laguna protein are indicated by dots. NORTHERN AND SOUTHERN PHILIPPINE ISOLATES OF RGSV 31 et al., 1991); this is referred to as the Laguna isolate in with an EcoRI linker, CGGAATTCCG, followed by diges- the present report. A mild isolate was obtained from the tion with EcoRI and purification with a spin column (Qia- Laguna isolate by serial passages by a vector brown gen) and LMP gel electrophoresis. The linker-ligated planthopper in rice plants (O. sativa var. Taichung Native double-stranded cDNA was ligated into EcoRI-digested 1) and is referred as the Laguna.M isolate. Two geo- pGEM3Z (Promega) and transformed into E. coli MC1061 graphical isolates and the mild isolate were confined in strain. Recombinant cDNA clones were screened for the meshed cages and separately maintained at the Inter- presence of appropriate-size inserts by restriction anal- national Rice Research Institute. Virus passage by vector ysis of miniprep DNA. insects was done as described previously (Miranda et al., 1991). Nucleotide sequence determination Inserts of all cDNA clones were sequenced from each Virus purification and RNA extraction end by using SP6 or T7 primers up to nearly 800 nucle- RGSV-infected rice plant materials were shipped fro- otides from the 5Ј terminus of the primer using the dye zen with dry ice under the quarantine permit from the terminator cycle sequencing kit (ABI) and an ABI 377 Ministry of Agriculture, Forestry and Fishery of Japan. automated DNA sequencer. From each sequence, highly Fifty grams of rice leaves and stems at 6 weeks after ambiguous regions very close to or too far from the feeding by viruliferous brown planthoppers were cut into primer-annealing region were removed with the use of small pieces and homogenized in 5 volumes of 0.1 M Factura software (ABI), followed by assembling of all sodium citrate, pH 6.5, containing 0.5% 2-mercaptoetha- sequences in batch with the use of AutoAssembler soft- nol and 10 mM diethyldithiocarbamate. The homogenate ware (ABI). After the first assembling, remaining vector was filtered through two layers of muslin cloth and cen- sequences and ambiguous sequences were removed trifuged at 10,000 rpm for 10 min in a Beckman JA-12 manually and batch assembling were repeated. Finally, rotor. The supernatant was clarified by the addition of internal sequences were edited to obtain consensus Triton X-100 to 2%, layered onto 20% sucrose, and spun sequences. at 45,000 rpm for1hinaBeckman Ti50.2 rotor. The Nucleotide and amino acid sequences were analysed pellets were suspended in water. The suspension was with the use of DNASIS (Hitachi) and DNA Strider soft- frozen at Ϫ20°C overnight, thawed without stirring, and ware (Marck, 1988). Homology analyses of nucleotide spun for 3 min in a microfuge. The supernatant was and amino acid sequences were performed with the use layered onto a 10–40% sucrose density gradient pre- of FASTA at the Website of the National Institute of pared in 0.1 M sodium citrate, pH 6.5, which was centri- Genetics, Japan. fuged at 38,000 rpm for1hinaBeckman SW41 rotor. For cloning and sequencing of the 5Ј terminal regions, Light-scattering zones containing RNP particles were individual dsRNAs fragments were purified from LMP collected, pelleted by centrifugation at 38,000 rpm for 2 h gel, heat-denatured in water, and used as templates for in SW41, and suspended in water. The purified RNP cDNA synthesis with a primer annealing to a 0.5- to suspension was treated in 2% SDS, 0.1 M NaCl, 10 mM 0.8-kb region downstream from the 5Ј terminus of vRNA Tris–HCl, pH 7.5, and 1 mM EDTA at 70°C for 5 min. or cRNA. The 3Ј end of cDNA was tailed with oligo(G) Protein was removed by phenol–chloroform extraction, using dGTP and terminal deoxynucleotidyl transferase and RNA was precipitated in ethanol. The final pellet was (Takara). G-tailed cDNA was amplified by Taq DNA poly- suspended in water. merase (Type Ex, Takara) using either d(C)12 or Ј d(C)12ACACA, in which ACACA is conserved in all 5 Construction of a random cDNA library termini of tenuivirus RNA, and an internal primer used for cDNA synthesis. PCR products were cloned directly into Approximately 2 ␮g of unfractionated RNA and 200 pCR2.1 (Invitrogen) or into pGEM3Z after digestion at a pmol of random deoxyoligonucleotide hexamers in 20 ␮l convenient internal restriction site. Nucleotide se- of water were heated at 100°C for 1 min and immediately quences were determined as described above. For clon- placed on ice. cDNA was synthesized by AMV reverse ing and sequencing of the internal and terminal regions transcriptase (Life Sciences/Nippon Gene) at 42°C for of RNA 1 and RNA 3, RNA extracted from a fraction above 1 h. cDNA was double-stranded by Escherichia coli DNA the light-scattering top band in the sucrose density gra- polymerase I in the presence of RNase H at 12°C for 1 h dient containing randomly fragmented RNPs was used and at 21°C for 1 h. Double-stranded cDNA was treated as templates for RT-PCR amplification. with T4 DNA polymerase to flush the ends, and an internal EcoRI site was methylated by EcoRI methylase in the presence of S-adenosylmethionine, followed by ACKNOWLEDGMENTS fractionation of 1.0–2.0 kb double-stranded cDNA in a The authors are grateful to Myron Brakke for reviewing the manu- low-melting-point (LMP) agarose gel. The 5Ј terminus of script. This work was supported by Grant-in-Aid for Creative Basic double-stranded cDNA was phosphorylated and ligated Research 09NP0901 from the Ministry of Education, Science, Sports 32 MIRANDA, AZZAM, AND SHIRAKO and Culture of Japan. G.J.M. was also supported by a scholarship from segment 3 of rice stripe virus: The first instance of a virus containing the Ministry of Education, Science, Sports and Culture of Japan. The two ambisense segments. J. Gen. Virol. 72, 465–468. nucleotide sequence data reported here have been submitted to DDBJ Marck, C. (1988). “DNA Strider”: A “C” program for the fast analysis of and assigned the accession numbers AB032180 (RNA 1), AB023777 DNA and protein sequences on the Apple Macintosh family of com- (RNA 2), AB029894 (RNA 3), AB023778 (RNA 4), AB023779 (RNA 5), and puters. Nucleic Acids Res. 16, 1829–1836 AB023780 (RNA 6). Masuta, C., Ueda, S., Suzuki, M., and Uyeda, I. (1998). Evolution of a quadripartite hybrid virus by interspecific exchange and recombina- REFERENCES tion between replicase components of two related tripartite RNA viruses. Proc. Natl. Acad. Sci. USA 95, 10487–10492. de Miranda, J., Hernandez, M., Hull, R., and Espinoza, A. M. (1994). Miranda, G. J., and Koganezawa, H. (1995). Identification, purification, Sequence analysis of rice hoja blanca virus RNA 3. J. Gen. Virol. 75, and serological detection of the major noncapsid protein of rice 2127–2132. grassy stunt virus. Phytopathology 85, 1530–1535. de Miranda, J. R., Munoz, M., Madriz, J., Wu, R., and Espinoza, A. M. Miranda, G. J., Koganezawa, H., and Bajet, N. B. (1991). Variants of rice (1996a). Sequence of Echinochloa hoja blanca tenuivirus RNA-3. grassy stunt virus in the Philippines. Philippine Phytopathol. 27, Virus Genes 13, 65–68. 20–25. de Miranda, J. R., Munoz, M., Wu, R., and Espinoza, A. M. (1996b). Murphy, B. R., and Webster, R. G. (1996). Orthomyxoviruses. In “Fields Sequence of Echinochloa hoja blanca tenuivirus RNA-4. Virus Genes Virology” (B. N. Fields, D. M. Knipe, P. M. Howley, and e. al., Eds.), pp. 13, 61–64. 1397–1445. Lippincott-Raven, Philadelphia. de Miranda, J. R., Munoz, M., Wu, R., and Espinoza, A. M. (1996c). Qiu, W. P., Geske, S. M., Hickey, C. M., and Moyer, J. W. (1998). Tomato Sequence of Echinochloa hoja blanca tenuivirus RNA-5. Virus Genes spotted wilt tospovirus genome reassortment and genome segment- 12, 131–134. specific adaptation. Virology 244, 186–194. de Miranda, J. R., Munoz, M., Wu, R., Hull, R., and Espinoza, A. N. Qu, Z., Liang, D., Harper, G., and Hull, R. (1997). Comparison of se- (1996d). Sequence of rice hoja blanca tenuivirus RNA-2. Virus Genes quences of RNAs 3 and 4 of rice stripe virus from with those 12, 231–238. of Japanese isolates. Virus Genes 15, 99–103. de Miranda, J. R., Ramirez, B. C., Munoz, M., Lozano, I., Wu, R., Haenni, Ramig, R. F. (1997). Genetics of the rotaviruses. Ann. Rev. Microbiol. 51, A. L., Espinoza, A. M., and Calvert, L. A. (1997). Comparison of 225–255. Colombian and Costa Rican strains of rice hoja blanca tenuivirus. Ramig, R. F., and Ward, R. L. (1991). Genomic reassortment in rotavi- Virus Genes 15, 191–193. ruses and other reoviridae. Adv. Virus Res. 39, 163–207. Estabrook, E. M., Suyenaga, K., Tsai, J. H., and Falk, B. W. (1996). Maize Ramirez, B.-C., and Haenni, A.-L. (1994). Molecular biology of tenuivi- stripe tenuivirus RNA2 transcripts in plant and insect hosts and ruses, a remarkable group of plant viruses. J. Gen. Virol. 75, 467–475. analysis of pvc2, a protein similar to the Phlebovirus virion mem- Ramirez, B. C., Lozano, I., Constantino, L. M., Haenni, A. L., and Calvert, brane glycoproteins. Virus Genes 12, 239–247. L. A. (1993). Complete nucleotide sequence and coding strategy of Falk, B. W., and Tsai, J. H. (1998). Biology and molecular biology of rice hoja blanca virus RNA4. J. Gen. Virol. 74, 2463–2468. viruses in the genus tenuiviruses. Annu. Rev. Phytopathol. 36, 139– Rodriguez, L. L., Owens, J. H., Peters, C. J., and Nichol, S. T. (1998). 163. Genetic reassortant among viruses causing hantavirus pulmonary Falk, B. W., Tsai, J. H., and Lommel, S. A. (1987). Differences in levels of syndrome. Virology 242, 99–106. detection for the maize stripe virus capsid and major non-capsid Takahashi, M., Toriyama, S., Hamamatsu, C., and Ishihama, A. (1993). proteins in plants and insect hosts. J. Gen. Virol. 68, 1801–1811. Nucleotide sequence and possible ambisense coding strategy of Fraile, A., Alonso-Prados, J. L., Aranda, M. A., Bernal, J. J., Malpica, J. M., and Garcia-Arenal, F. (1997). Genetic exchange by recombination or rice stripe virus RNA segment 2. J. Gen. Virol. 74, 769–773. reassortment is infrequent in natural populations of a tripartite RNA Toriyama, S., Kimishima, T., and Takahashi, M. (1997). The proteins plant virus. J. Virol. 71, 934–940. encoded by rice grassy stunt virus RNA5 and RNA6 are only distantly Henderson, W. W., Monroe, M. C., St. Jeor, S. C., Thayer, W. P., Rowe, related to the corresponding proteins of other members of the genus J. E., Peters, C. J., and Nichol, S. T. (1995). Naturally occurring Sin Tenuivirus. J. Gen. Virol. 78, 2355–2363. Nombre virus genetic reassortants. Virology 214, 602–610. Toriyama, S., Kimishima, T., Takahashi, M., Shimizu, T., Minaka, N., and Hibino, H. (1996). Biology and epidemiology of rice viruses. Annu. Rev. Akutsu, K. (1998). The complete nucleotide sequence of the rice Phytopathol 34, 249–274. grassy stunt virus genome and genomic comparisons with viruses of Hibino, H., Cabauatan, P. Q., Omura, T., and Tsuchizaki, T. (1985). Rice the genus Tenuivirus. J. Gen. Virol. 79, 2051–2058. grassy stunt virus strains causing tungrolike symptoms in the Phil- Toriyama, S., Takahashi, M., Sano, Y., Shimizu, T., and Ishihama, A. ippines. Plant Dis. 69, 538–541. (1994). Nucleotide sequence of RNA1, the largest genomic segment Hu, C.-C., and Ghabrial, S. A. (1998). Molecular evidence that strain of rice stripe virus, the prototype of the tenuivirus. J. Gen. Virol. 75, BV-15 of peanut stunt cucumovirus is a reassortant between sub- 3569–3579. group I and II strains. Phytopathology 88, 92–97. Toriyama, S., and Tomaru, K. (1995). “Tenuivirus”: Virus Taxonomy, Huiet, L., Klaassen, V., Tsai, J. H., and Falk, B. W. (1991). Nucleotide Classification and Nomenclature of Viruses, Sixth Report of the sequence and RNA hybridization analyses reveal an ambisense International Committee on Taxonomy of Viruses (C. M. F. F. A. coding strategy for maize stripe virus RNA3. Virology 182, 47–53. Murphy, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, Huiet, L., Tsai, J. H., and Falk, B. W. (1992). Complete sequence of maize M. A. Mayo, and M. D. Summers, Eds.) Springer-Verlag, New York. stripe virus RNA4 and mapping of its subgenomic RNAs. J. Gen. Virol. Uyeda, I., Ando, Y., Murao, K., and Kimura, I. (1995). High resolution 73, 1603–1607. genome typing and genomic reassortment events of rice dwarf Huiet, L., Tsai, J. H., and Falk, B. W. (1993). Maize stripe virus RNA5 is Phytoreovirus. Virology 212, 724–727. of negative polarity and encodes a highly basic protein. J. Gen. Virol. Zhu, Y., Hayakawa, T., and Toriyama, S. (1992). Complete nucleotide 74, 549–554. sequence of RNA4 of rice stripe virus isolate T, and comparison with Kakutani, T., Hayano, Y., Hayashi, T., and Minobe, Y. (1990). Ambisense another isolate and with maize stripe virus. J. Gen. Virol. 73, 1309– segment 4 of rice stripe virus: Possible evolutionary relationship with 1312. phleboviruses and uukuviruses (Bunyaviridae). J. Gen. Virol. 71, Zhu, Y., Hayakawa, T., Toriyama, S., and Takahashi, M. (1991). Complete 1427–1432. nucleotide sequence of RNA3 of rice stripe virus: An ambisense Kakutani, T., Hayano, Y., Hayashi, T., and Minobe, Y. (1991). Ambisense coding strategy. J. Gen. Virol. 72, 763–767.