Deletions in the putative cell receptor-binding domain of Sindbis virus strain MRE16 E2 glycoprotein reduce midgut infectivity in Aedes aegypti.
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.
Myles
Kevin M
KM
Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80526, USA.
Pierro
Dennis J
DJ
Olson
Ken E
KE
D000330:Aedes / Q000821:virology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D008969:Molecular Sequence Data; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012845:Sindbis Virus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics; D014779:Virus Replication
D017931:DNA Primers; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C064950:glycoprotein E2, Sindbis virus