ABSTRACT
Wild-type Sindbis virus (SINV) strain MRE16 efficiently infects Aedes aegypti midgut epithelial cells (MEC), but laboratory-derived neurovirulent SINV strain TE/5'2J infects MEC poorly. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of MRE16 and TE/5'2J differ at 60 residue sites. To identify the genetic determinants of MEC infection of MRE16, the TE/5'2J 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/5'2J 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.
Subject(s)
Aedes/virology , Alphavirus Infections/genetics , Conserved Sequence , Insect Vectors , Sindbis Virus/pathogenicity , Viral Envelope Proteins/genetics , Alphavirus Infections/virology , Amino Acid Sequence , Animals , Cell Line , Flaviviridae/genetics , Molecular Sequence Data , Sequence Homology, Amino Acid , Sindbis Virus/genetics , Sindbis Virus/physiology , Viral Envelope Proteins/chemistry , Virus ReplicationABSTRACT
Mosquito midgut epithelial cells (MEC) play a major role in determining whether an arbovirus can successfully infect and be transmitted by mosquitoes. The Sindbis virus (SINV) strain TR339 efficiently infects Aedes aegypti MEC but the SINV strain TE/5'2J poorly infects MEC. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of TR339 and TE/5'2J differ at two sites, E2-55 and E2-70. We have altered the TE/5'2J virus genome by site-directed mutagenesis to contain two TR339 residues, E2-55 H-->Q (histidine to glutamine) and E2-70 K-->E (lysine to glutamic acid). We have characterized the growth patterns of derived viruses in cell culture and determined the midgut infection rate (MIR) in A. aegypti mosquitoes. Our results clearly show that the E2-55 H-->Q and the E2-70 K-->E mutations in the TE/5'2J virus increase MIR both independently and in combination. TE/5'2J virus containing both TR339 E2 residues had MIRs similar to the parental TR339 virus. In addition, SINV propagated in a mammalian cell line had a significantly lower A. aegypti midgut 50 % infectious dose than virus propagated in a mosquito cell line.
