Development and Application of an Enzyme-Linked Immunosorbent Assay for the Detection of Rift Valley Fever Viral Antigen in Mosquitoes

Abstract

An indirect double-antibody sandwich ELISA was developed for detection of RVF viral antigen in mosquitoes. The assay was optimized by using mosquito-propagated preparations of the ZH501 strain of RVF virus, and the ELISA was capable of detecting < 7 ng/ml of total protein in a purified RVF virus preparation. The ELISA detected high levels of RVF viral antigen in inoculated adult female Cx. pipiens mosquitoes, and reliably detected a single RVF virus-infected mosquito in the presence of 99 uninfected mosquitoes. Recovery of RVF viral antigen from Cx. pipiens larval pools was also successful, and the ELISA detected antigen in some of these pools even in the absence of infectious virus by plaque assay. The ELISA detected viral antigen from all 9 mosquito species tested including specimens infected with 4 different strains of RVF virus. While the ELISA detected antigen in all samples stored at 4 different temperatures (-70°C, -20°C, 4°C, and 22°C), plaque assay in Vero cells failed to detect virus in samples stored at 22°C for 21 days, and a major reduction of viral titers was observed in samples stored at -20°C for 3 or more days. There was no effect of 3 freeze-thaws on the recovery of RVF viral antigen from adult female Cx. pipiens mosquito pools by ELISA. In contrast, a major reduction of viral titers in pool sizes of 50 and 100 mosquitoes each was observed after 3 freeze-thaw cycles. An attempt to increase sensitivity of the ELISA for detection of RVF viral antigen was investigated by using different capture antibodies. Antibodies prepared i against the surface glycoproteins (G1 and G2) showed little reactivity when used individually as the capture antibody. In contrast, antibody species directed against the nucleocapsid protein (N) detected RVF viral antigen at levels comparable to the standard capture antibody (monoclonal blend). The distribution of RVF virus and antigen in dissected mosquito tissues of orally infected adult Cx. pipiens was also studied.