Deletions in the putative cell receptor-binding domain of Sindbis virus strain MRE16 E2 glycoprotein reduce midgut infectivity in Aedes aegypti

Abstract

The Sindbis virus (Alphavirus; Togaviridae) strain MRE16 efficiently infects Aedes aegypti mosquitoes that ingest a blood meal containing 8 to 9 log(10) PFU of virus/ml. However, a small-plaque variant of this virus, MRE16sp, poorly infects mosquitoes after oral infection with an equivalent titer. To determine the genetic differences between MRE16 and MRE16sp viruses, we have sequenced the MRE16sp structural genes and found a 90-nucleotide deletion in the E2 glycoprotein that spans the 3' end of the coding region for the putative cell-receptor binding domain (CRBD). We examined the role of this deletion in oral infection of mosquitoes by constructing infectious clones pMRE16icDeltaE200-Y229 and pMRE16ic, representing MRE16 virus genomes with and without the deletion, respectively. A third infectious clone, pMRE16icDeltaE200-C220, was also constructed that contained a smaller deletion extending only to the 3' terminus of the CRBD coding region. Virus derived from pMRE16ic replicated with the same efficiency as parental virus in vertebrate (BHK-21) and mosquito (C6/36) cells and orally infected A. aegypti. Viruses derived from pMRE16icDeltaE200-Y229 and pMRE16icDeltaE200-C220 replicated 10- to 100-fold less efficiently in C6/36 and BHK-21 cells than did MRE16ic virus. Each deletion mutant poorly infected A. aegypti and dramatically reduced midgut infectivity and dissemination. However, all viruses generated nearly equal titers (approximately 6.0 log(10) PFU/ml) in mosquitoes 4 days after infection by intrathoracic inoculation. These results suggest that the deleted portion of the E2 CRBD represents an important determinant of MRE16 virus midgut infectivity in A. aegypti.

FIG. 1.

Strategy for the final assembly of the full-length infectious cDNA clone MRE16ic. Six subclones contained the complete 11,693-nt-long genome, SP6 promoter, 5′ cap, and a poly(A) tail as overlapping amplified cDNA regions. The pBRUC vector was used for all cloning steps.

FIG. 2.

Comparative growth characteristics of MRE16, MRE16ic, MRE16sp, MRE16icΔE200-Y229, and MRE16icΔE200-C220 viruses in BHK-21, C6/36, and Vero cells. (A) Relative plaque sizes in Vero cells. Viruses were grown in 25-cm² tissue culture flasks of BHK-21 (B) and C6/36 (C) cells. Multiplicity of infection was approximately 0.01 PFU/cell. Cell culture medium was harvested from BHK-21 or C6/36 cells infected with MRE16, MRE16ic, MRE16sp, MRE16icΔE200-Y229, or MRE16icΔE200-C220 virus during growth curve experiments and were plaqued on Vero cell monolayers.

FIG. 3.

Deletion identified in MRE16sp and deletions engineered into MRE16icΔE200-Y229 and MRE16icΔE200-C220 viruses. This figure shows the location of the deletion present in the E2 glycoprotein of MRE16sp and MRE16icΔE200-Y229 (bold solid line) and MRE16icΔE200-C220 (bold dotted line). Deduced amino acid residues from prototype AR339 virus sequence are also shown. A total of four amino acid differences are present between AR339 virus and MRE16 virus within the E2 CRDB (E2-172 R→G, E2-178 S→T, E2-197 I→V, and E2-213 T→A). AA, amino acid.

FIG. 4.

Comparative disseminated infection rates of MRE16 and MRE16ic viruses in A. aegypti mosquitoes. Shown are the percentages of mosquitoes orally exposed to either MRE16 or MRE16ic virus with disseminated infections (e.g., positive for SIN virus-specific antigen by IFA of head tissues) at timed intervals (n ≥ 30).

FIG. 5.

Intact A. aegypti midguts assayed for the presence of MRE16, MRE16ic, MRE16icΔE200-Y229, MRE16icΔE200-C220, or MRE16sp virus at various times p.i. (A and B) Composite images of midguts infected with either MRE16 virus (A) or MRE16ic virus (B) at 2 to 3 days p.i. and demonstrating widespread distributions of SIN virus E1 antigen. (C to E) Composite images of midguts infected with MRE16ic ΔE200-Y229 virus at 2 to 3 days p.i. (C), MRE16ic ΔE200-C220 virus at 8 to 9 days p.i. (D), and MRE16sp virus at 8 to 9 days p.i (E). A focus of infection can be seen in the blue square. (SIN virus E1 antigen is shown in green). An Olympus BH2 fluorescent microscope was used. Original magnification, 125×.

FIG. 6.

Comparative replication kinetics of MRE16ic, MRE16icΔE200-Y229, and MRE16icΔE200-C220 viruses in A. aegypti. Mosquitoes were inoculated intrathoracically with between 10 to 15 PFU. Ten mosquitoes were taken at each time point for each virus. Individual virus titers obtained from triturated mosquitoes were averaged for each time point. Time zero was included to confirm the initial inoculating dose. The average inoculating dose, determined at the mosquito zero time point, for MRE16ic virus was 7 PFU. Virus was not recovered from mosquitoes inoculated with the deletion mutants at time zero. Virus was recovered from all of the mosquitoes that were triturated at all subsequent time points.