Arbovirus Detection in Mosquitoes

Pre- and post-feeding blood samples and inoculum at the beginning and end of injection period were inoculated as 10-fold dilutions in the wells of a 96-well microtiter plate seeded with C6/36 cells. Plates were incubated at 28 °C for 7 days before being fixed with 20 % acetone and stored at -20 °C. The bodies and legs + wings from all experiments, and females and males collected in pools from the vertical transmission experiment were homogenized in a QIAGEN TissueLyser II (Qiagen, Hilden, Germany), centrifuged at 14,000 g before being filtered through a 0.2 μm filter (Pall Corporation, Ann Arbor, MI). The body filtrate and filtered saliva expectorates were inoculated as 10-fold dilutions in the wells of a 96-well microtiter plate containing confluent monolayers of C6/36 cells. Legs + wings and mosquito pools were inoculated in quadruplicate onto C6/36 cell monolayers within a 96-well microtiter plate. Plates were incubated, fixed and stored as described above. Presence of virus in fixed plates was determined by fixed cell enzyme immunoassay using specific monoclonal antibodies 3D6 for PCV [4 (link)] and 4G2 for WNV [16 (link)].