of Rice Stripe Virus

Lin HanxinLin QitianWu ZujianLin QiyingXie Lianhui

(Institute of Plant Virology of Fujian Agricultural University, Key Laboratory of

Plant Virology of Fujian Province, Fuzhou350002)

Abstract An isolate of rice stripe virus (designated as RSV-YL) was purified. The particles showed to be pleomorphisms under electron microscope, mainly branched filaments of about 80-250 nm in length and about 8 nm in width. There are also some open circular filaments of 3 nm and 8 nm in width, and some filaments of 13 nm in width and 130-190 nm in length. The basic morphism of RSV particles should be filaments of 3 nm in width and various length. By SDS-PAGE analysis, the molecular weight of disease-specific protein (SP) encoded by vRNA4 was 19.9 kDa and that of coat protein (CP) encoded by vcRNA3 was 33.6 kDa. When nucleic acid extracted from the purified RSV was electrophoresed under nondenaturing condition, the size of four dsRNAs (designated as dsRNA1-4 in order of decreasing size) was 4.9×10⁶,2.7×10⁶,2.0×10⁶ and 1.7×10⁶ Da, respectively, and that of four ssRNAs (designated as ssRNA1-4 in order of decreasing size) was 3.0×10⁶,1.2×10⁶,0.9×10⁶ and 0.8×10⁶ Da, respectively. A fifth segment with a size of 0.58×10⁶ Da identified as ssRNA5 associated with the purified virus sometimes. The antiserum against the coat protein further purified by preparative electrophoresis was raised and used to investigate the serological relationships between RSV-CP and RSV-SP, CP and SP of rice grassy stunt virus (RGSV) which is also a member of Tenuivirus. The results showed that RSV-CP had no serological reaction with SP of RSV and PGSV, but could weakly react with antiserum of RGSV-CP, which confirmed that there is distantly evolutionary relationship between RGSV and RSV.

Key wordsRice stripe virus, Protein, Nucleic acid, Serology

摘要提纯的水稻条纹病毒(云南宜良分离物)在电镜下的形态为多型性，但主要是宽8-10 nm，长80-250的分枝丝状体，有些为直径3 nm或8 nm的开环环状体，有些为13 nm宽，130-190 nm长的丝状体，但其基本结构应是直径3 nm、长度不等的丝状体。经聚丙烯酰胺凝胶电泳分析，vRNA4编码的病害特异蛋白(SP)分子量为19.9 kDa，而vcRNA3编码的外壳蛋白(CP)约为33.6 kDa。在非变性条件下，RSV的4条ssRNAs大小分别为3.0×10⁶(ssRNA1)、1.2×10⁶(ssRNA2)、0.9×10⁶(ssRNA3)和0.8×10⁶ Da(ssRNA4)，有时出现一条大小为0.58×10⁶ Da的单链RNA(ssRNA5)；而4条dsRNAs的分子量分别为4.9×10⁶(dsRNA1)、2.8×10⁶(dsRNA2)、2.0×10⁶(dsRNA3)和1.7×10⁶ Da(dsRNA4)。利用制备电泳分离提纯的外壳蛋白免疫家兔，得到了高特异性的抗血清。A蛋白夹心ELISA检测结果表明，RSV-CP与水稻草状矮化病毒(RGSV)CP抗血清有微弱的反应，但与RSV、RGSV的SP抗血清没有反应，而RSV-CP抗血清与RSV-SP及RGSV的SP、CP都无血清学关系，这个结果表明RGSV与RSV之间在进化上具有一定的亲缘关系。

关键词水稻条纹病毒，蛋白，核酸，血清学

Rice stripe virus (RSV) is the typical member of Tenuivirus. The other members are rice grassy stunt virus (RGSV), maize stripe virus (MStV), rice hoja blanca virus (RHBV). RSV causes severe damage to rice production in China, Japan, Korea and former USSR^［1］.

After several cycles of sucrose density gradient centrifugation, RSV can be separated into three components, M (comprising two subcomponents, M1 and M2), B and NB, which contain circular filaments or filamentous particles with various lengths, four ssRNAs and four dsRNAs were found in association with RSV nucleic acid^［2，3］. In infected plant tissues, a large amount of viral -encoded disease-specific protein (SP) accumulate, which are related to the developing of symptoms^［4，5］. In Japan, the genome of T isolate has been completely sequenced. RSV genome has an ambisense nature. The SP is encoded by viral-sense RNA4 (vRNA4) and one large open reading frame (ORF) on the viral complementary-sense RNA3 (vcRNA3) encodes coat protein^［6－9］. However, the chemical and serological properties of RSV are not clearly characterized in China yet, even though some molecular biological characteristics of RSV has also been studied^{［10－13］}. Here, we report the properties of proteins and nucleic acid of RSV YL isolate in China and the serological relationships among CP, SP of RSV and RGSV.

1Materials and Methods

1.1Virus and plant materials

RSV isolated from Yiliang County, Yunnan Province, China was maintained in a japonica rice variety (Hexi 28) by transmission via the viruliferous smaller brown planthopper, Laodelphax striaterllus. Infected rice leaves were stored in -70℃ and used for purification.

1.2Purification of RSV

RSV was purified following the method of Ishikawa et al (1989)^［2］, except that infected leaf tissues were firstly grounded in liquid nitrogen and that a RPS 65-T rotor (Hitachi) was used for sucrose density gradient centrifugation which was performed for 2.5 h at 101 700 g at 4℃. Virus bands and the zones between the bands were separately collected. Each fraction was diluted with 0.1 mol/L PBS and centrifuged for 1.5 h at 150 000g. The pellet was finally suspended in 0.01 mol/L PBS (containing 0.1% DEPC).

1.3Electron microscopy

Purified RSV was mounted on grids coated with a collodion film for 5 min, then stained with 2% phosphotungastic acid for 5 min, and examined under a JEM-1200 EX electron microscope (JEOL, Japan).

1.4Preparation and electrophoresis of RSV-RNAs

RNAs were extracted as described by Toriyama (1986)^［14］, and then analyzed in native 1.5% agrose gels (Bio-Red Chemical). Nucleic acid was also released from the purified RSV by heating for 5-10 min at 55℃ in a solution containing 2% sodium dodecyl sulfate (SDS), 4% glycerol and 1% bromephenol blue just prior to electrophoresis. After electrophoresis, the gels were soaked in distilled water for 10 min and then stained with ethidium bromide.

ssRNA marker used here was 6 583, 4 981, 2 604, 1 908, 1 383, 955, 623 and 283 bp (Promega Chemical), RDV dsRNAs of 3.29×10⁶, 2.65×10⁶, 2.39×10⁶,2.01×10⁶,1.91×10⁶,1.22×10⁶,1.20×10⁶,0.912×10⁶，0.827×10⁶，0.795×10⁶，0.553×10⁶ and 0.553×10⁶ Da were also used as molecular weight standards.

To determine the properties of RNAs, RNase A (Sigma) treatments in high- and low-salt buffers were carried out according to the method described by Ishikawa et al (1989)^［2］.

The photograph of the gels and the calculation of molecular weight were all perfomed on IS-1000 Digital Imaging System (Alpha Innotech Corporation).

1.5SDS-polyacrylamide gel electrophoresis (PAGE) of proteins

SP was purified following the method of Lin et al (1999)^［15］. Purified SP or RSV sample (1 μL) was added with 3 μL loading buffer (1% SDS, 0.5% 2-mer-captoethanol, 2 mmol/L EDTA, 4% glycerol and 0.5% bromephenol blue), then heated at 100 ℃ for 3 min and electrophoresed. After electrophoresis, the gels were stained with coomassie b