Fig. 5.

Deletion of Asp483 in WNV NS3 decreases fitness in chickens and mosquitoes. (A) Day-old chickens were inoculated s.c. in the cervical region with 2 × 10³ PFU of WNV containing equal proportions of each test virus (NS3 and WT) and a genetically marked reference virus that has been previously described. Serum was withdrawn by brachial venipuncture at the time of peak viremia (48 h p.i.) and RNA extracted according to standard methods. The proportions of test and reference viruses were estimated by quantitative sequencing. (B and C) To assess fitness in mosquitoes, colonized C. quinquefasciatus mosquitoes were offered an infectious blood meal containing 10⁷ PFU of equal proportions of test and reference viruses and held for 7 (B) or 14 (C) days of extrinsic incubation (EI). After EI, mosquitoes were sacrificed and homogenized in RNA lysis buffer (RNEasy) according to standard methods. Proportions of test and reference viruses were determined as described above. Dotted lines indicate approximate linear ranges of quantitative sequencing. For chicks, the mean fitness levels of NS3 and WT viruses were compared by a one-sample t test using the observed WT virus-reference virus proportion as a predicted mean. For mosquitoes, fitness of NS3 and that of the WT were compared using a Mann-Whitney test and a Gaussian approximation.