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 inserted, 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 genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/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 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.
Powers
A M
AM
Department of Microbiology, Colorado State University, Fort Collins 80523.
Olson
K E
KE
Higgs
S
S
Carlson
J O
JO
Beaty
B J
BJ
D000330:Aedes; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D016373:DNA, Antisense / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D007114:Immunization; D018062:La Crosse virus / Q000235:genetics / Q000502:physiology*; D008647:Mesocricetus; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D012845:Sindbis Virus / Q000235:genetics*; D014158:Transcription, Genetic; D014762:Viral Interference; D014779:Virus Replication
D016373:DNA, Antisense; D018076:DNA, Complementary; D012367:RNA, Viral