Reduced Recovery of Rift Valley Fever Virus Associated with Assay of Mosquito (Diptera: Culicidae) Larval Pools1

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Abstract
Experiments were conducted to determine if the number of uninfected mosquitoes per pool affected recovery of Rift Valley fever (RVF) virus. While the inclusion of as many as 100 uninfected adult male or female mosquitoes per pool did not affect recovery of virus by plaque assay employing Vero cells, the presence of as few as 5 uninfected larvae in a pool significantly reduced the infectivity titer of RVF virus recovered. This phenomenon was observed for larvae of both species tested (Culex pipiens and Aedes aegypti), and for pools containing either an experimentally infected (inoculated) larva or an added constant amount of RVF virus. By inoculation of larval pools into hamsters, virus could be detected in some pools that were negative by plaque assay, but inoculation failed to detect virus from some of the pools to which 102.9 plaque-forming units (PFU)/ml had been added. An enzyme-linked immunosorbent assay (ELISA) was not affected by pool size but failed to detect antigen from pools to which less than 103 PFU/ml had been added. Inoculation of Toxorhynchites amboinensis was the most sensitive assay system for detection of virus in pools containing 10 or more larvae to which a RVF viral suspension had been added (RVF virus was recovered from pools that were negative by plaque assay, hamster inoculation, and ELISA). However, both ELISA and mosquito inoculation were equally efficient systems for detecting virus in pools that contained an infected larva. These findings may be important in the analysis of larvae to detect transovarial transmission.