Development of a Chimeric Sindbis Virus with Enhancedper OsInfection ofAedes aegypti

Abstract

The TE/3'2J double subgenomic Sindbis (dsSIN) viruses have been used to stably express genes in Aedes aegypti nerve and salivary gland tissues. However, because these viruses inefficiently infect Ae. aegypti when administered by the per os route, TE/3'2J viruses must be intrathoracically inoculated into the mosquitoes to infect these tissues. A Malaysian Sindbis (SIN) virus isolate (MRE16) does efficiently infect Ae. aegypti midgut tissues after ingestion, and approximately 95% of these mosquitoes also develop disseminated infections within 14 days. We have sequenced the entire 26S RNA of MRE16 virus and have developed a chimeric SIN cDNA infectious clone, designated MRE1001, which contains sequence elements of TE/3'2J and MRE16 virus. MRE1001 virus efficiently infects midgut cells, and greater than 90% of infected mosquitoes develop disseminated infections after 14 days extrinsic incubation. The chimeric MRE1001 cDNA clone should allow identification of viral determinants of midgut infection and dissemination and lead to the development of new SIN virus expression systems.

Deletions in the Putative Cell Receptor-Binding Domain of Sindbis Virus Strain MRE16 E2 Glycoprotein Reduce Midgut Infectivity in Aedes aegypti

“…Sequencing of MRE16 virus genome and MRE16sp structural genes. Primers for reverse transcriptase PCR (RT-PCR) were designed from a consensus sequence generated from six previously published SIN virus or SIN virus-like genome sequences and a previously published MRE16 sequence (31). Genomic RNA was isolated from MRE16 virus seed by using a QIAamp viral RNA mini kit as recommended by the manufacturer (Qiagen, Valencia, Calif.).…”

“…The 3Ј-terminal sequence had been previously reported (31). New sequencing primers efficiently spaced to span the entire genome were designed on the basis of MRE16 sequence and previously published data (31). Genomic RNA was isolated from MRE16sp virus seed stock as described previously.…”

“…A SIN virus strain based on the mouse neurovirulent TE12 virus (22) also shows restricted midgut infection in A. aegypti, with less than 20% dissemina-tion at 14 days p.i. (31). However, a Malaysian SIN virus strain, MRE16, readily infects midguts of A. aegypti and disseminates in nearly 100% of the mosquitoes by 14 days postfeeding (31).…”

“…(31). However, a Malaysian SIN virus strain, MRE16, readily infects midguts of A. aegypti and disseminates in nearly 100% of the mosquitoes by 14 days postfeeding (31). Nucleotide sequence analyses of MRE16 genomic RNA show a close genetic relationship between MRE16 and other SIN viruses within the Oriental/Australian genetic group (29,31).…”

“…However, a Malaysian SIN virus strain, MRE16, readily infects midguts of A. aegypti and disseminates in nearly 100% of the mosquitoes by 14 days postfeeding (31). Nucleotide sequence analyses of MRE16 genomic RNA show a close genetic relationship between MRE16 and other SIN viruses within the Oriental/Australian genetic group (29,31). It has been previously shown that the determinants for this enhanced infection phenotype in A. aegypti resides in the structural genes of MRE16 virus (24,31), and it has been further shown that determinants for enhanced oral infection reside in the E2 glycoprotein (28).…”