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A multiplex Taqman-based real-time reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to identify potential severe strains of Citrus tristeza virus (CTV) and separate genotypes that react with the monoclonal antibody MCA13. Three strain-specific probes were developed using intergene sequences between the major and minor coat protein genes (CPi) in a multiplex reaction. Probe CPi-VT3 was designed for VT and T3 genotypes; probe CPi-T36 for T36 genotypes; and probe CPi-T36-NS to identify isolates in an outgroup clade of T36-like genotypes mild in California. Total nucleic acids extracted by chromatography on silica particles, sodium dodecyl sulfate-potassium acetate, and CTV virion immunocapture all yielded high quality templates for real-time PCR detection of CTV. These assays successfully differentiated CTV isolates from California, Florida, and a large panel of CTV isolates from an international collection maintained in Beltsville, MD. The utility of the assay was validated using field isolates collected in California and Florida.

When Culicoides variipennis (Coquillett) ingested a bluetongue virus (BTV)-defibrinated sheep blood suspension, BTV adsorbed to sheep red blood cells (RBCs) within 2 h. The virus had entered RBCs by 6 h and was still seen in RBCs 2 d after ingestion of the blood meal, even though the RBCs had been dehydrated. The peritrophic membrane began to form on day 1, and it contained breaks by day 3. The peritrophic membrane did not prevent infection of the midgut epithelium. Viral replication occurred in the midgut cells; virions matured through the basolateral extracellular membrane into the extracellular spaces between the plasma membrane and the basal lamina. The greatest number of virions was seen on day 3. The virus did not accumulate but rather exited the cells when mature. No cytopathology was observed in virus-infected cells, and midgut cells became vacuolated and sloughed off into the midgut lumen by day 3 in both control and virus-infected cells.