INTRACELLULAR IMMUNIZATION OF MOSQUITO CELLS TO LACROSSE VIRUS USING A RECOMBINANT SINDBIS VIRUS VECTOR

A cDNA of the small RNA genome segment of La Crosse (LAC) virus was in serted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated geno mic RNA that was electrotransfected into BHK-21 cells to produce virus . Northern blot analysis of RNA isolated from infected Aedes albopictu s (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgen omic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, T E/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ ANTI-S virus yielded at least 4 log(10) T CID50/ml less LAC virus than cells infected with either TE/3'2J or AR3 39 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of ge nes or sequences of interest in arthropod cells, and for evaluating st rategies for intracellular immunization against arboviruses.