THE EXPRESSION OF CHLORAMPHENICOL ACETYLTRANSFERASE IN MOSQUITOS AND MOSQUITO CELLS USING A PACKAGED SINDBIS REPLICON SYSTEM

Sindbis (SIN) replicon virus was used to express chloramphenicol acety ltransferase (CAT) in Aedes albopictus (C6/36) cells and Aedes triseri atus mosquitoes. RNA transcribed in vitro from a SIN replicon plasmid (pSINrep5/CAT) and from SIN virus helper constructs (pDH-EB or pDH(26S )5'SIN) was coelectroporated into BHK-21 cells to generate replicon vi ruses, designated rep5/CAT/EB and rep5/CAT/ 26S. C6/36 cells infected with rep5/CAT/EB or rep5/CAT/26S virus at a multiplicity of infection of 3, expressed 3.8 x 10(6) and 6.0 x 10(6) CAT trimers per cell, resp ectively, at 2 days postinfection (pi). Both viruses attained peak tit ers by Day 2 pi. Adult female A. a triseriatus mosquitoes were intrath oracically inoculated with 7 x 10(4) IFU rep5/CAT/EB or 1 x 10(5) IFU rep5/CAT/26S virus. Virus titers remained at approximately 10(5) IEU/m l through Day 2 pi and decreased roughly 1 log by Day 10 pi. CAT enzym e activity was detected 2 days pi (rep5/CAT/EB, 1.49 x 10(-4) units CA T/10 mu g protein; rep5/CAT/26S, 2.03 x 10(-5) units CAT/10 mu g prote in) and remained near these levels through Day 10 pi. CAT was detected in the head, salivary glands, midgut, and ovaries of inoculated mosqu itoes by indirect immunofluorescence or CAT activity assays. These res ults suggest that packaged replicon viruses can be useful for expressi on of heterologous genes in mosquito cells and whole mosquitoes. (C) 1 995 Academic Press, Inc.