THE EXPRESSION OF CHLORAMPHENICOL ACETYLTRANSFERASE IN AEDES-ALBOPICTUS (C6 36) CELLS AND AEDES-TRISERIATUS MOSQUITOS USING A DOUBLE SUBGENOMIC RECOMBINANT SINDBIS VIRUS/

Genomic RNA was transcribed in vitro from the double subgenomic recomb inant Sindbis (SIN) virus expression vector, pTE/3'2J/CAT, and transfe cted into BHK-21 cells to generate recombinant virus stocks. TE/3'2J/C AT virus was used to infect C6/36 (Aedes albopictus) cells and adult f emale Aedes triseriatus. When C6/36 cells were infected with TE/3'2J/C AT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 x 10(5). Approximately 4. 0 log10TCID50 of the TE/3'2J/CAT virus was intrathoracically inoculate d into adult female mosquitoes. Titers greater than 6.0 log10TCID50/ml were detected within 4 days pi and declined to less than 4.0 log10TCI D50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 x 10(-5) units of CAT/mosquito or 1.4 x 10(10) CAT polypept ides), peaked at day 6 (4 x 10(-3) units of CAT/mosquito or 7.2 x 10(1 1) CAT polypeptides), and remained at peak levels to day 20. Immunoflu orescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neura l, midgut, ovarian, and salivary gland tissues. Alphavirus-based expre ssion vectors should be useful for expressing heterologous genes in mo squito cells as well as adult mosquitoes.