MRE16-eGFP and TR339-eGFP virus stocks were generated from SINV infectious cDNA clones with inserts of enhanced GFP (eGFP) driven by the second sub-genomic promoter, using standard methods [35 (link),46 (link),47 (link)]. MRE16-eGFP was passed once in baby hamster kidney (BHK-21) cells and twice in Aedes albopictus clone C6/36 cells. TR339 5'2J-eGFP was passed once in BHK-21 cells and three times in C6/36 cells.
For mosquito feedings, all viruses were diluted as indicated in de-fibrinated sheep blood and provided at 37°C in a water jacketed artificial feeder with parafilm membrane. An aliquot of each SINV stock was titered by viral plaque assay on Vero cells and the other aliquots were stored at -80°C and diluted prior to feeding. TR339-eGFP blood-meals contained approximately 3.3 × 108 pfu/ml and MRE16 meals contained 2.2 × 107 pfu/ml. Representative mosquitoes from each group were selected for dissection and eGFP detection with an Olympus epi-fluorescence microscope to confirm midgut infection with each batch of virus. Viral titers from individual mosquitoes were determined by plaque assays of filtered mosquito homogenates as previously described [29 (link)].
For mosquito feedings, all viruses were diluted as indicated in de-fibrinated sheep blood and provided at 37°C in a water jacketed artificial feeder with parafilm membrane. An aliquot of each SINV stock was titered by viral plaque assay on Vero cells and the other aliquots were stored at -80°C and diluted prior to feeding. TR339-eGFP blood-meals contained approximately 3.3 × 108 pfu/ml and MRE16 meals contained 2.2 × 107 pfu/ml. Representative mosquitoes from each group were selected for dissection and eGFP detection with an Olympus epi-fluorescence microscope to confirm midgut infection with each batch of virus. Viral titers from individual mosquitoes were determined by plaque assays of filtered mosquito homogenates as previously described [29 (link)].
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