Genetic Determinants of Sindbis Virus Mosquito Infection Are Associated with a Highly Conserved Alphavirus and Flavivirus Envelope Sequence

Abstract

Wild-type Sindbis virus (SINV) strain MRE16 efficiently infects Aedes aegypti midgut epithelial cells (MEC), but laboratory-derived neurovirulent SINV strain TE/52J infects MEC poorly. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of MRE16 and TE/52J differ at 60 residue sites. To identify the genetic determinants of MEC infection of MRE16, the TE/52J virus genome was altered to contain either domain chimeras or more focused nucleotide substitutions of MRE16. The growth patterns of derived viruses in cell culture were determined, as were the midgut infection rates (MIR) in A. aegypti mosquitoes. The results showed that substitutions of MRE16 E2 aa 95 to 96 and 116 to 119 into the TE/52J virus increased MIR both independently and in combination with each other. In addition, a unique PPF/.GDS amino acid motif was located between these two sites that was found to be a highly conserved sequence among alphaviruses and flaviviruses but not other arboviruses.The majority of medically important mosquito-borne alphaviruses (family Togaviridae) and flaviviruses (family Flaviviridae) are vectored by two Culicinae mosquito genera, Aedes and Culex. The mosquito Aedes aegypti is a major vector in the epidemic disease cycles of the flaviviruses dengue virus (DENV) and yellow fever virus, as well as being a competent vector for a number of alphaviruses, including the prototypic alphavirus Sindbis virus (SINV). There are three major genotypes of SINV, the Paleoarctic-Ethiopian (P/E), which is found in Europe and Africa, the Oriental-Australian (O/A), which is found in Asia and Australasia, and the Southwest genotype, which is found in the southwestern region of Western Australia (28,34,40,41). SINV strain MRE16 is an O/A genotype isolated between 1966 and 1969 from a pool of Culex tritaeniorhynchus mosquitoes in Malaysia (20a). A full-length infectious clone (IC) of MRE16 has been generated, and it is currently the model O/A genotype being investigated in laboratory mosquito infection assays (24,25,29,31,43). MRE16 was previously found to have a midgut infection rate (MIR) in A. aegypti mosquitoes of 100% at 7 days postinfection (dpi) (24). In contrast, recombinant SINV strain TE/5Ј2J (P/E genotype), a double subgenomic SINV constructed from a chimeric mouse neurovirulent variant of SINV AR339 called TE12, infects less than 15% of A. aegypti mosquito midguts when analyzed similarly (31).The MRE16 E2 gene has been implicated as having genetic determinants of midgut infection (31). Sequence comparison between the SINV TE/5Ј2J and MRE16 E2 genes identified approximately 316 nucleotide (nt) (24.9%) and 60 amino acid (aa) residue (14.2%) differences (43). In this work, SINV strain TE/5Ј2J was mutated to generate viruses containing MRE16 sequences within the E2 gene (as either chimeras or point mutations) and the viruses were evaluated for the ability to productively infect the MEC of A. aegypti mosquitoes. The data demonstrated that TE/5Ј2J viruses having the specific MRE1...

Effects of Manipulating Apoptosis on Sindbis Virus Infection of Aedes aegypti Mosquitoes

“…To test this hypothesis, we utilized adult A. aegypti females of the Rexville D strain, which has been used extensively in studies with SINV (22)(23)(24). Adult female Rexville D mosquitoes were injected with 345 ng dsRNA corresponding to either Aeiap1, Aedronc, or a negative-control sequence not present in the A. aegypti genome (the bacterial cat gene) and monitored for survival.…”

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

“…MRE16 5’ds GFP and MRE16 5’ds were generated from infectious clones [23,24]. Chikungunya virus (La Reunion strain LR2006-OPY-1) and Western Equine Encephalitis virus (McMillian strain) infections were performed in biosafety level 3 [25,26].…”

Complexity of virus–vector interactions

“…Mechanisms behind these barriers have been reviewed thoroughly [17]. Surface structures of the virus particle itself may be responsible for efficient crossing of tissue barriers [18], and/or the barrier may be physically altered during virus replication [17]. …”