Interplay of Rice Stripe Virus and Rice Black Streaked Dwarf Virus during Their Acquisition and Accumulation in Insect Vector.

Plant viruses transmitted by hemipteran vectors commonly cause losses to crop production. Rice stripe virus (RSV) and rice black streaked dwarf virus (RBSDV) are transmitted to rice plants by the same vector, the small brown planthopper (SBPH), Laodelphax striatellus Fallén, in a persistent propagative manner. However, rarely do the respective diseases they cause occur simultaneously in a field. Here, we determined the acquisition efficiency of RSV and RBSDV when acquired in succession or simultaneously by SBPH. When RBSDV was acquired first, RSV acquisition efficiency was significantly lower than when only acquiring RSV. However, RBSDV acquisition efficiency from insects that acquired RSV first was not significantly different between the insects only acquiring RBSDV. Immunofluorescence assays showed that the acquisition of RBSDV first might inhibit RSV entry into midgut epithelial cells, but RSV did not affect RBSDV entry. SBPHs were more likely to acquire RBSDV when they were feeding on plants coinfected with the two viruses. When RBSDV was acquired before RSV, RBSDV titer was significantly higher and RSV titer first declined, then increased compared to when only acquiring RBSDV or RSV. Only 5% of the SBPHs acquired both viruses when feeding on plants coinfected with RSV and RBSDV. These results provide a better understanding of the interaction between two persistent viruses when present in the same vector insect and explain why RSV and RBSDV occur in intermittent epidemics.

A Real-Time Loop-Mediated Isothermal Amplification for Detection of the Wheat Dwarf Virus in Wheat and the Insect Vector Psammotettix alienus.

Wheat dwarf virus (WDV; genus Mastrevirus, family Geminiviridae) is an economically important and widespread pathogen of cereal crops. It causes huge yield loss in wheat because of the unavailability of resistant varieties and rapid transmission by the vector leafhopper, Psammotettix alienus (Dahlb). To monitor and forecast this viral disease, an early diagnosis method is required for WDV detection in both infected plants and the virus vectors. In this study, we developed a real-time loop-mediated isothermal amplification (LAMP) assay for WDV detection. The positive sample could be detected within 28 to 32 min by following a simple, cost-effective procedure. The real-time LAMP assay showed a sensitivity of 2.7 × 105-6 copies/µl for detection and a high specificity for WDV amplification, with a similar accuracy to quantitative PCR. Furthermore, a closed-tube dye method facilitates the inspection of the LAMP reaction and avoids cross-contamination in the detection of the virus. This valuable detection assay could serve as an important tool for diagnosis and forecasting wheat dwarf disease intensity in the field.

Rescue of an Infectious cDNA Clone of Barley Yellow Dwarf Virus-GAV.

Barley yellow dwarf virus-GAV (BYDV-GAV) is one of the most prevalent viruses causing yellow dwarf disease in wheat in China. The biology and pathology of BYDV-GAV are well studied; however, gene functions and molecular mechanisms of BYDV-GAV disease development are unclear because of the lack of a reverse genetics system. In this study, a full-length complementary DNA (cDNA) clone of BYDV-GAV was constructed and expressed via Agrobacterium-mediated inoculation of Nicotiana benthamiana. Virions produced by BYDV-GAV in N. benthamiana were transmitted to wheat by an aphid vector after acquisition via a sandwich feeding method. Infectivity of the cDNA clone in wheat was verified via reverse transcription PCR and western blot assays, and the recombinant virus elicited typical reddening symptoms in oats and was transmitted between wheat plants. These results confirm the production of biologically active transmissible virions. Using the BYDV-GAV infectious clone, we demonstrate that viral protein P4 was involved in cell-to-cell movement and stunting symptoms in wheat. This is the first report describing the development of an infectious full-length cDNA clone of BYDV-GAV and provides a useful tool for virus-host-vector interaction studies.

Ribavirin targets sugar transporter 6 to suppress acquisition and transmission of rice stripe tenuivirus by its vector Laodelphax striatellus.

BACKGROUND: Rice stripe tenuivirus (RSV) is one of the most destructive pathogens of rice and other cereal crops. The virus is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a circulative-propagative manner. Thus, blocking transmission by the insect vector would provide an effective strategy to prevent epidemic outbreaks of the disease. RESULTS: In this study, we explored the effect of ribavirin on acquisition and transmission of the virus by specifically inhibiting the expression of sugar transporter 6 (LsSt-6), which was recently reported as a key vector component for RSV transmission. Ribavirin at the highest concentration tested (250 µmol L-1 ) significantly reduced RSV acquisition and transmission efficiency by SBPHs through inhibiting LsSt-6 messenger RNA (mRNA) level. Survival of the model insect Spodoptera frugiperda cell line (Sf9) was 95.0 ± 2.2 and 85.6 ± 2.1% after exposure to 250 µmol L-1 ribavirin or 8-azaguanine, respectively. Further study confirmed that 250 µmol L-1 ribavirin also significantly reduced LsSt-6 mRNA and protein levels in Sf9 cells. However, 8-azaguanine did not significantly inhibit viral infectivity and LsSt-6 mRNA levels in SBPH or the Sf9 cell line. CONCLUSION: This result provides evidence that ribavirin has the potential to disrupt LsSt-6 expression but not others like viral RNAs to prevent acquiring RSV, which leads to less viral accumulation in SBPH tissues and thereby lower transmission efficiency. © 2020 Society of Chemical Industry.

Determination of free amino acids in tea by a novel method of reversed-phase high performance liquid chromatography applying 6-Aminoquinolyl-N-Hydroxysuccinimidyl carbamate reagent.

We describe a novel analytical method for quantification of free amino acids in tea using variable mobile phase pH, elution gradient and column temperature of reversed-phase high-performance liquid chromatography (RP-HPLC). The study of mobile phase pH 5.7 was chosen to simultaneous quantification of 19 free amino acids in tea, while it improved maximum resolution of glutamine, histidine and theanine. Elution gradient was adapted for enhancing the solution of free amino acids, mainly because of adjustment of mobile phase A and B. The column temperature of 40 °C was conducive to separate free amino acids in tea. The limit of detection (LOD) and limit of quantitation (LOQ) of this method were in the range of 0.097-0.228 nmol/mL and 0.323-0.761 nmol/mL, respectively. The relative standard deviation of intraday and interday ranged in 0.099-1.909% and 3.231-7.025%, respectively, indicating that the method was reproducible and precise, while recovery ranged between 81.06-112.78%, showing that the method had an acceptable accuracy. This method was applied for the quantification of free amino acids in six types of tea. Multivariate analysis identified serine, glutamine, theanine and leucine as the most influencing factor for classify among analyzed sample.

New insights on the transmission mechanism of tenuiviruses by their vector insects.

Tenuiviruses, which cause serious diseases in rice, wheat, maize and other gramineae crops, recently have been assigned to the family Phenuiviridae in the order Bunyavirales. Transmission of tenuiviruses to host plants depends on the specific vector planthoppers. The interaction between the virus and insect offers critical points for developing an efficient management strategy. This review focuses on recent advancements in our understanding of the interactions between the virus and insect components. Vector components such as various proteins play major roles in virus replication, stability and transovarial transmission. The virus can either directly interact with these proteins or regulate expression of genes that encode them to alter the metabolism or defense mechanisms of the insect vectors. However, the vector components that are involved in virus infection and movement in midgut and salivary glands are not as well explored and are targets for further study.

iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation.

BACKGROUND: Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear. METHODS: We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes. RESULTS: As a whole, 681 (65.8% downregulated, 34.2% upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results. CONCLUSIONS: The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

Quantification of southern rice black streaked dwarf virus and rice black streaked dwarf virus in the organs of their vector and nonvector insect over time.

Southern rice black streaked dwarf virus (SRBSDV) and rice black streaked dwarf virus (RBSDV) are serious rice-infecting reoviruses, which are transmitted by different planthoppers in a persistent propagative manner. In this study, we quantitatively compared the spatial distribution of SRBSDV and RBSDV contents over time in their vector and nonvector insects using real time-PCR. Genome equivalent copies (GEC) were assessed every 2 days from 0 to 14 days after a 3-days acquisition access period (AAP) on infected plants. Results revealed 293.2±21.6 to 404.1±46.4 SRBSDV GEC/ng total RNA in whole body of white-backed planthopper (WBPH, Sogatella furcifera) at day 0 and 12 and 513.5±88.4 to 816.8±110.7 RBSDV GEC/ng total RNA in the whole body of small brown planthopper (SBPH, Laodelphax striatellus) at day 0 and 14, respectively, after 3-days AAP. Highest GEC of both viruses were found in the gut of their respective vectors. Although SRBSDV was detected in the gut of SBPH, it did not spread into the hemolymph or other organs. After an 8-day latent period, the transmission efficiency of SRBSDV and RBSDV by their respective vectors was significantly positively correlated with GEC in the salivary gland (r(2)=0.7808, P=0.0036 and r(2)=0.9351, P<0.0001, respectively, at α=0.05). Together, these results confirm that accumulation of >200 SRBSDV or RBSDV GEC/ng total RNA in the gut of vector, indicated threshold for further spread and the virus content in the salivary gland was significantly correlated with transmission efficiency by their respective vectors.