Journal:

Article Title: An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells

doi: 10.1128/JVI.78.1.378-388.2004

Figure Lengend Snippet: Soluble EIII inhibits DV infection to BHK21 and C6/36 cells by blocking virion binding to cells. (A) EIII blocked DEN-2 infection on BHK21 cells. (Left panel) BHK21 cells were mock treated or treated with various concentrations of BSA or EIII, as shown at the bottom of the figure, and infected with DEN-2 (16681) at an MOI of 5 PFU per cell. At 24 h p.i., the cell cultures were harvested and the virus titers were determined by plaque assays on BHK21 cells. (Right panel) BHK21 cells were treated with EIII (30 or 300 μg/ml) and infected with different viruses, at an MOI of 5 PFU per cell, as shown at the bottom of the figure. At 24 h p.i., cell cultures were harvested and the respective virus titers were determined by plaque assays on BHK21 cells. % Viral replication = (the number of plaques produced with EIII/the number of plaques produced without EIII treatment) × 100. (B) EIII blocked DV binding to BHK21 cells. BHK21 cells were treated with EIII (300 μg/ml) for 2 h and then infected with DEN-1, -2, -3, or -4, JEV, or VV at an MOI of 5 PFU per cell. Cells were immediately harvested after virus infection and used for immunoblot analyses with either MAb 4G2 against E protein (for DEN1 to DEN4) or a rabbit serum against whole virions (for JEV and VV). The blots were scanned and quantitated with Image Gauge, version 3.45, installed on a FUJIFILM LAS-1000 plus pictrography 3000 (Fuji Inc.). For JEV and VV blots, the most abundant three bands were averaged for quantitation. (C) EIII blocked DV infection on C6/36 cells. C6/36 cells were treated with EIII (30 or 300 μg/ml) as described for panel A. At 24 h p.i., cell cultures were harvested and the respective virus titers in supernatant were determined on BHK21 cells. (D) EIII blocked DV binding to C6/36 cells. C6/36 cells were treated with EIII (300 μg/ml) for 2 h and then infected at an MOI of 5 PFU per cell with DEN-1, -2, -3, or -4, JEV, or VV. Cells were immediately harvested after virus infection, and the amount of virus bound to cells was determined by immunoblot analyses as described for panel B. (E) EIII blocked infection of four low-passage DV isolates on BHK21 and C6/36 cells. BHK21 and C6/36 cells were treated with EIII (30 or 300 μg/ml) and infected with low-passage DVs. At 24 h p.i., cell cultures were harvested and the respective virus titers in supernatant were determined on BHK21 cells. (F) Binding of four low-passage DV isolates on BHK21 and C6/36 cells was blocked by EIII. BHK21 and C6/36 cells were treated with BSA or EIII (300 μg/ml) for 2 h and infected with each of the four low-passage DV isolates and harvested for immunoblot analyses as described for panel B. The low-passage DV isolates used for panels E and F are identical.

Article Snippet: The blots were scanned and quantitated with Image Gauge, version 3.45, installed on a FUJIFILM LAS-1000 plus pictrography 3000 (Fuji Inc.).

Techniques: Infection, Blocking Assay, Binding Assay, Produced, Western Blot, Quantitation Assay