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.
Department of Microbiology, Colorado State University, Fort Collins 80523.
A M
AM
Powers
K E
KE
Olson
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S
Higgs
J O
JO
Carlson
B J
BJ
Beaty
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