A divergent strain of melon chlorotic spot virus isolated from black medic (Medicago lupulina) in Austria.

A tenuivirus, referred to here as JKI 29327, was isolated from a black medic (Medicago lupulina) plant collected in Austria. The virus was mechanically transmitted to Nicotiana benthamiana, M. lupulina, M. sativa, Pisum sativum and Vicia faba. The complete genome was determined by high throughput sequencing. The genome of JKI 29327 consists of eight RNA segments closely related to those of melon chlorotic spot virus (MeCSV) isolate E11-018 from France. Since segments RNA 7 and 8 of JKI 29327 are shorter, its genome is slightly smaller (by 247 nts) than that of E11-018. Pairwise comparisons between the predicted virus proteins of JKI 29327 and their homologues in E11-018 showed aa identities ranging from 80.6 to 97.2%. Plants infected with E11-081 gave intermediate DAS-ELISA reactions with polyclonal antibodies to JKI 29327. Since JKI 29327 and E11-018 appear to be closely related both serologically and genetically, we propose to regard JKI 29327 as the black medic strain of MeCSV. To our knowledge, JKI 29327 represents the second tenuivirus identified from a dicotyledonous plant. Serological and molecular diagnostic methods were developed for future detection.

Immune function of an angiotensin-converting enzyme against Rice stripe virus infection in a vector insect.

Angiotensin-converting enzyme (ACE) plays diverse roles in the animal kingdom. However, whether ACE plays an immune function against viral infection in vector insects is unclear. In this study, an ACE gene (LsACE) from the small brown planthopper was found to respond to Rice stripe virus (RSV) infection. The enzymatic activities of LsACE were characterized at different pH and temperature. Twenty planthopper proteins were found to interact with LsACE. RSV infection significantly upregulated LsACE expression in the testicle and fat body. When the expression of LsACE in viruliferous planthoppers was inhibited, the RNA level of the RSV SP gene was upregulated 2-fold in planthoppers, and all RSV genes showed higher RNA levels in the rice plants consumed by these planthoppers, leading to a higher viral infection rate and disease rating index. These results indicate that LsACE plays a role in the immune response against RSV transmission by planthoppers.

Development of a colloidal gold-based immunochromatographic strip for rapid detection of Rice stripe virus.

Rice stripe virus (RSV) causes dramatic losses in rice production worldwide. In this study, two monoclonal antibodies (MAbs) 16E6 and 11C1 against RSV and a colloidal gold-based immunochromatographic strip were developed for specific, sensitive, and rapid detection of RSV in rice plant and planthopper samples. The MAb 16E6 was conjugated with colloidal gold and the MAb 11C1 was coated on the test line of the nitrocellulose membrane of the test strip. The specificity of the test strip was confirmed by a positive reaction to RSV-infected rice plants and small brown planthopper (SBPH), and negative reactions to five other rice viruses, healthy rice plants, four other vectors of five rice viruses, and non-viruliferous SBPH. Sensitivity analyses showed that the test strip could detect the virus in RSV-infected rice plant tissue crude extracts diluted to 1:20 480 (w/v, g/mL), and in individual viruliferous SBPH homogenate diluted to 1:2560 (individual SPBH/µL). The validity of the developed strip was further confirmed by tests using field-collected rice and SBPH samples. This newly developed test strip is a low-cost, fast, and easy-to-use tool for on-site detection of RSV infection during field epidemiological studies and paddy field surveys, and thus can benefit decision-making for RSV management in the field.

Characterization of an endonuclease in rice stripe tenuivirus Pc1 in vitro.

Rice stripe tenuivirus (RSV) initiates its mRNA transcription by using the cap-snatching mechanism during which an endonuclease activity is required for the cleavage of host mRNA. In this study, we aim to characterize the endonuclease in RSV. Sequence alignment revealed the presence of a cap-snatching endonuclease domain in RSV Pc1. Expression and in vitro enzymatic activity assay demonstrated that this domain indeed had a manganese-dependent endonuclease activity. The enzyme could efficiently degrade ssRNA with preference for unstructured ssRNA, but not DNA. Mutations in the endonuclease domain allowed the identification of four key residues (D547, D567, E585 and K604). The endonuclease of RSV was similar but not identical to other known viral endonucleases, suggesting that RSV endonuclease may have some distinct catalytic characteristics.

Characterization of the first tenuivirus naturally infecting dicotyledonous plants.

A mechanically transmissible virus tentatively named "melon chlorotic spot virus" (MeCSV) was isolated in southeastern France from a melon plant showing chlorotic spots and yellowing of the older leaves. Its complete sequence was obtained by Illumina and Sanger sequencing. The genome comprises eight RNAs for a total size of 20,079 nt and is distantly related to Ramu stunt virus and maize yellow stunt virus, two tentative tenuiviruses. MeCSV differs from other tenuiviruses by its number of genomic fragments, by being readily mechanically transmissible, and by infecting only dicotyledonous hosts. MeCSV should thus be considered a member of a tentative new species related to tenuiviruses.

Whole genome deep sequencing revealed host impact on population structure, variation and evolution of Rice stripe virus.

High-throughput deep sequencing and variant detection showed that variations of Rice stripe virus (RSV) populations obtained from small brown planthopper-transmitted rice plants and sap-inoculated N. benthamiana plants were single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels). The SNPs were more uniform across RSV genome, but InDels occurred mainly in the intergenic regions (IRs) and in the 5' or 3' noncoding regions. There were no clear patterns of InDels, although the inserted sequences were all from virus itself. Six, one, and one non-synonymous substitutions were respectively observed in the RdRP ORF, IR and the movement protein ORF. These non-synonymous substitutions were found to be stable, resulting in new consensus sequences in the NBL11 RSV population. Furthermore, the numbers of SNPs and InDels in RSV genome from N. benthamiana plants were much higher than that from O. sativa plants. These differences are likely caused by selection pressures generated by different host plants.

Genomic variations in the 3'-termini of Rice stripe virus in the rotation between vector insect and host plant.

A large number of plant RNA viruses circulate between plants and insects. For RNA viruses, host alternations may impose a differential selective pressure on viral populations and induce variations in viral genomes. Here, we report the variations in the 3'-terminal regions of the multiple-segment RNA virus Rice stripe virus (RSV) that were discovered through de novo assembly of the genome using RNA sequencing data from infected host plants and vector insects. The newly assembled RSV genome contained 16- and 15-nt extensions at the 3'-termini of two genome segments compared with the published reference RSV genome. Our study demonstrated that these extensional sequences were consistently observed in two RSV isolates belonging to distinct genetic subtypes in RSV-infected rice, wheat and tobacco. Moreover, the de novo assembled genome of Southern rice black-streaked dwarf virus also contained 3'-terminal extensions in five RNA segments compared with the reference genome. Time course experiments confirmed that the 3'-terminal extensions of RSV were enriched in the vector insects, were gradually eliminated in the host plant and potentially affected viral replication. These findings indicate that variations in the 3'-termini of viral genomes may be different adaptive strategies for plant RNA viruses in insects and plants.

Bacterial microbiota in small brown planthopper populations with different rice viruses.

The small brown planthopper (SBPH) is an important virus vector, transmitting Rice stripe virus (RSV), and Rice black-streaked dwarf virus (RBSDV). Insect symbionts play an essential role in the insect fitness, however, it is still unclear about their contributions to viral transmission by SBPH. Here, we investigated endosymbiont communities in non-viruliferous, RSV-infected, and RBSDV-infected SBPH populations using Illumina 16S rRNA gene MiSeq sequencing. In total, 281,803 effective sequences of the 16S rRNA gene were generated from different samples. Sequence analysis revealed the percentages of these bacterial groups in different SBPH populations on several taxonomic levels ranging from phyla to genera. The extremely consistent bacterial diversity and abundance indicated that RSV or RBSDV infection did not affect the composition and abundance of symbionts in SBPH. It was notable that Wolbachia was dominant in all populations. The symbiosis between Wolbachia and SBPH might be potentially studied and utilized to control pest SBPH in the future.

Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén).

BACKGROUND: The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples. RESULTS: We established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation. CONCLUSIONS: A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.

Artificial feeding Rice stripe virus enables efficient virus infection of Laodelphax striatellus.

Rice stripe virus (RSV), the causative agent of rice stripe disease, is transmitted by Laodelphax striatellus in a persistent-propagative manner. Efficient virus acquisition is primary for studies of virus transmission and virus-insect vector interactions. However, under greenhouse conditions, less than 30% of the L. striatellus population, on average, become viruliferous during feeding on RSV-infected plants. Here, we explored a method for efficient RSV acquisition by feeding the insects with a virus-containing artificial diet. Virus particles were partially purified from frozen infected rice leaves. A series of RSV concentrations in a 5% sucrose solution were tested in the feed of L. striatellus nymphs. The percentage of infected insects increased along with the increasing viral concentration, and the highest infection percentage 96% was achieved using a 1200ngµL(-1) crude RSV suspension after 48h feeding. RSV particles acquired in this manner were able to spread to L. striatellus salivary glands. This improved method of obtaining viruliferous insects should assist the study of RSV transmission mechanisms in L. striatellus.

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

BACKGROUND: Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. METHODS: The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. RESULTS: UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. CONCLUSIONS: We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

A simplified method for simultaneous detection of Rice stripe virus and Rice black-streaked dwarf virus in insect vector.

Rice stripe virus (RSV) and Rice black-streaked dwarf virus (RBSDV) are transmitted by their common vector small brown planthopper (SBPH) that cause serious crop losses in China. A simple reverse transcription-PCR method was developed for the simultaneous detection of RSV and RBSDV in single SBPH. Three primers targeted to RSV-RNA4 and RBSDV-S2 segments were designed to amplify respectively 1114-bp and 414-bp fragments in a reaction. The method is reliable, rapid and inexpensive for detecting the two viruses in vector, which could facilitate better forecasting and control of the virus diseases. Using this method, it was found that SBPH could carry RSV and RBSDV simultaneously.

Infection route of rice grassy stunt virus, a tenuivirus, in the body of its brown planthopper vector, Nilaparvata lugens (Hemiptera: Delphacidae) after ingestion of virus.

Rice grassy stunt virus (RGSV), a tenuivirus, is transmitted by the brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), in a persistent-propagative manner. In this study, immunofluorescence microscopy was used to investigate the infection route of RGSV in the internal organs of BPH after acquiring the virus by feeding on RGSV-infected rice plants. The sequential infection study revealed that RGSV initially infected the midgut epithelium, then crossed the basal lamina into the midgut visceral muscles, from where RGSV apparently spread into the hemolymph, then into the salivary glands of its BPH vector. The mechanism underlying this infection route of RGSV in its BPH vector may confer an advantage for the direct spread of RGSV from the initially infected epithelium to the salivary glands in BPH, contributing to efficient transmission of RGSV by its insect vector.

Investigation on subcellular localization of Rice stripe virus in its vector small brown planthopper by electron microscopy.

BACKGROUND: Rice stripe virus (RSV), which is transmitted by small brown planthopper (Laodelphax striatellus Fallén, SBPH), has been reported to be epidemic and cause severe rice stripe disease in rice fields in many East Asian countries, including China. Investigation on viral localization in the vector is very important for elucidating transmission mechanisms of RSV by SBPH. In this study, transmission electron microscopy and immuno-gold labeling technique were used to investigate the subcellular localization of the ribonucleoproteins (RNPs) of RSV in the digestive tract, muscles, ovary and testes of SBPH. RESULTS: A lot of amorphous RSV inclusion bodies with high electron density were observed in the cytoplasmic matrix and vacuoles of follicular cells of ovarioles in viruliferous SBPH, which were very similar to viral inclusions formed in rice cells. After magnified, it was found that sand-like or parallel filamentary structures were constructed inside the electron-dense inclusions. A large numbers of RSV RNPs distributed diffusely throughout the eggshell surface and interior of ovum, midgut lumen and epithelial cells, while the amount of the virus in muscles was far less than that in the ovary and midgut tissues. Besides RSV, numerous endogenous microorganisms were also observed in SBPH body, including yeast-like endosymbiotes (YLES), endosymbiotic bacteria and insect virus. CONCLUSIONS: According to the results of the virus localization, a potential mechanism of RSV transovarial transmission was proposed that RSV might replicate and accumulate initially in the inclusions of follicular cells, then exploit the pathway of the nutrition transportation to pass through the eggshell and spread into the oocytes along with the nutrition. Moreover, RSV might exploit muscles for its spread in vector body with a lower efficiency.

One-step multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of three rice viruses.

Rice stripe virus (RSV), Rice black-streaked dwarf virus (RBSDV), and Rice dwarf virus (RDV) are major rice-infecting viruses in Korea that can cause serious crop losses. A one-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for the simultaneous detection of these rice viruses. Three sets of specific primers targeted to the capsid protein coding genes of RSV, RBSDV, and RDV were used to amplify fragments that were 703 bp, 485 bp, and 252 bp, respectively. The one-step mRT-PCR assay proved to be a sensitive and rapid method for detecting the three rice viruses. This method could be used to facilitate better control of rice viruses.

Strong resistance against Rice grassy stunt virus is induced in transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or movement proteins as targets for RNA interference.

Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes significant economic losses in rice production in South, Southeast, and East Asian countries. Growing resistant varieties is the most efficient method to control RGSV; however, suitable resistance genes have not yet been found in natural rice resources. One of the most promising methods to confer resistance against RGSV is the use of RNA interference (RNAi). It is important to target viral genes that play important roles in viral infection and proliferation at an early stage of viral replication. Our recent findings obtained from an RNAi experiment with Rice stripe virus (RSV), a tenuivirus, revealed that the genes for nucleocapsid and movement proteins were appropriate targets for RNAi to confer resistance against RSV. In this study, we transformed rice plants by introducing an RNAi construct of the RGSV genes for the nucelocapsid protein pC5 or movement protein pC6. All progenies from self-fertilized transgenic plants had strong resistance against RGSV infection and did not allow the proliferation of RGSV. Thus, our strategy to target genes for nucleocapsid and movement proteins for conferring viral resistance might be applicable to the plant viruses in the genus Tenuivirus.

Molecular diversity of Rice grassy stunt virus in Vietnam.

Rice grassy stunt virus (RGSV, Tenuivirus) recently emerged on rice in Vietnam, causing high yield losses during 2006-2009. The genetic diversity of RGSV is poorly documented. In this study, the two genes encoded by each ambisense segment RNA3 and RNA5 of RGSV isolates from six provinces of South Vietnam were sequenced. P3 and Pc3 (RNA3) have unknown function, P5 (RNA5) encodes the putative silencing suppressor, and Pc5 (RNA5) encodes the nucleocapsid protein (N). The sequences of 17 Vietnamese isolates were compared with reference isolates from North and South Philippines. The average nucleotide diversity among the isolates was low. We confirmed a higher variability of RNA3 than RNA5 and Pc3 than P3. No relationships between the genetic diversity and the geographic distribution of RGSV isolates could be ascertained, likely because of the long-distance migration of the insect vector. This data will contribute to a better understanding on the RGSV epidemiology in South Vietnam, a prerequisite for further management of the disease and rice breeding for resistance.

Genetic diversity and population structure of rice stripe virus in China.

Rice stripe virus (RSV) is one of the most economically important pathogens of rice and is repeatedly epidemic in China, Japan and Korea. The most recent outbreak of RSV in eastern China in 2000 caused significant losses and raised serious concerns. In this paper, we provide a genotyping profile of RSV field isolates and describe the population structure of RSV in China, based on the nucleotide sequences of isolates collected from different geographical regions during 1997-2004. RSV isolates could be divided into two or three subtypes, depending on which gene was analysed. The genetic distances between subtypes range from 0.050 to 0.067. The population from eastern China is composed only of subtype I/IB isolates. In contrast, the population from Yunnan province (southwest China) is composed mainly of subtype II isolates, but also contains a small proportion of subtype I/IB isolates and subtype IA isolates. However, subpopulations collected from different districts in eastern China or Yunnan province are not genetically differentiated and show frequent gene flow. RSV genes were found to be under strong negative selection. Our data suggest that the most recent outbreak of RSV in eastern China was not due to the invasion of new RSV subtype(s). The evolutionary processes contributing to the observed genetic diversity and population structure are discussed.

A one-step real time RT-PCR assay for quantifying rice stripe virus in rice and in the small brown planthopper (Laodelphax striatellus Fallen).

Rice stripe virus (RSV) is an important pathogen affecting rice production in subtropical and temperate regions. One-step real time RT-PCR methods using the TaqMan probe are described for quantitative detection of RSV in rice tissues and in Laodelphax striatellus Fallen, the small brown planthopper (SBPH). Primers and probe for specific detection of RSV were designed within the conserved region identified within the coat protein (CP) gene sequence. A DNA fragment was amplified for mimicking the complementary RNA by PCR-based gene assembly, and was used for generation of standard RNA templates. A sensitivity assay showed that the detection limit of the assay was 20 copies, and the standard curve had a linear range from 20 to 2 x 10(5) copies, with good reproducibility. An internal control assay designed to target the rice ubiquitin 5 gene (UBQ5) was included in detecting RSV in different infected rice tissues. Simultaneously, a real time RT-PCR assay was used to detect the RSV CP gene in viruliferous SBPH. The results showed that the numbers of RSV CP genes in different tissues were variable. Accumulation of the RSV CP gene was greater in rice leaf and SBPH thoraco-abdominal tissue than in rice stem and SBPH head, respectively. As determined by an end-point dilution comparison, one-step real time RT-PCR was close to 10(4)-fold more sensitive than the indirect enzyme-linked immunosorbent assay (ELISA) for RSV detection. This quantitative detection assay provides a valuable tool for diagnosis and molecular studies of RSV biology.

Transmission of Rice stripe virus acquired from frozen infected leaves by the small brown planthopper (Laodelphax striatellus Fallen).

Rice stripe disease, caused by Rice stripe virus (RSV), is one of the most serious rice diseases in temperate and subtropical regions of the world. Since RSV is not transmissible mechanically, an insect transmission test was the original basis for identification of the viral population and cultivar resistance. A simple, rapid and reliable method is described by which virus-free small brown planthoppers acquired RSV from frozen infected rice leaves and transmitted the virus to healthy rice plants. Of 30 planthoppers tested, 9 insects fed on the frozen infected leaves acquired the virus as shown by an indirect-ELISA. In the transmission tests with a single insect, fed previously on frozen leaves, 5 of 30 plants (16.67%) became infected, compared to 7 of 30 plants (23.33%) became infected when a single insect fed on fresh infected leaves. All rice plants expressing stripe symptoms were identified with the virus by RT-PCR.