Numerous virus pathogens are transmitted by specific arthropod vectors. Understanding the mechanism of transmission is a critical step in the epidemiology of plant viruses and is crucial for the development of effective disease control strategies. In this study, we describe the localization and distribution of Wheat dwarf virus (WDV), an economically important and widespread single-stranded DNA virus, in its leafhopper vector, Psammotettix alienus. The results suggest that WDV not only can move to the salivary glands from the anterior and middle midgut via the hemocoel but also can pass directly through the sheath of the filter chamber and be readily transmitted to healthy wheat plants within 5 min of an acquisition access period on infected plants. When a bacterial-expressed recombinant capsid protein (CP) was incubated with the internal organs of leafhoppers, CP-immunoreactive antigens were found at the anterior and middle midgut. Furthermore, when leafhoppers were fed with an antiserum raised against the CP, the accumulation of WDV in the gut cells, hemocoel, and salivary glands was significantly reduced. These data provide evidence that transmission of WDV is determined by a CP-mediated virion-vector retention mechanism.
The bird cherry-oat aphid (Rhopalosiphum padi), an important pest of cereal crops, not only directly sucks sap from plants, but also transmits a number of plant viruses, collectively the yellow dwarf viruses (YDVs). For quantifying changes in gene expression in vector aphids, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a touchstone method, but the selection and validation of housekeeping genes (HKGs) as reference genes to normalize the expression level of endogenous genes of the vector and for exogenous genes of the virus in the aphids is critical to obtaining valid results. Such an assessment has not been done, however, for R. padi and YDVs. Here, we tested three algorithms (GeNorm, NormFinder and BestKeeper) to assess the suitability of candidate reference genes (EF-1α, ACT1, GAPDH, 18S rRNA) in 6 combinations of YDV and vector aphid morph. EF-1α and ACT1 together or in combination with GAPDH or with GAPDH and 18S rRNA could confidently be used to normalize virus titre and expression levels of endogenous genes in winged or wingless R. padi infected with Barley yellow dwarf virus isolates (BYDV)-PAV and BYDV-GAV. The use of only one reference gene, whether the most stably expressed (EF-1α) or the least stably expressed (18S rRNA), was not adequate for obtaining valid relative expression data from the RT-qPCR. Because of discrepancies among values for changes in relative expression obtained using 3 regions of the same gene, different regions of an endogenous aphid gene, including each terminus and the middle, should be analyzed at the same time with RT-qPCR. Our results highlight the necessity of choosing the best reference genes to obtain valid experimental data and provide several HKGs for relative quantification of virus titre in YDV-viruliferous aphids.
The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteranSpodoptera frugiperdaare widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations ofS. frugiperdain the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells ofSpodoptera exigua,Heliothis subflexa, andBombyx mori. Feeding studies demonstrated the ability ofS. frugiperdalarvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCEInsect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish thatSpodoptera frugiperdarhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.
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