Use of base excision sequence scanning for detection of genetic variations in St. Louis encephalitis virus isolates

Abstract

Twenty-two isolates of St. Louis encephalitis (SLE) virus of various geographical origins (Brazil, Argentina, Panama, Texas, Missouri, Maryland, California, and Florida) were examined for genetic variation by the base excision sequence scanning (BESS T-scan) method. A fragment was amplified in the envelope gene with the forward primer labeled in the PCR. The BESS T-scan method determined different clusters according to the profiles generated for the isolates and successfully grouped the isolates according to their geographical origins. Two major clusters, the North American cluster (cluster A) and the South and Central American cluster (cluster B), were defined. Two subgroups, the Texas-California subgroup (subgroup A1) and the Missouri-Maryland-Florida subgroup (subgroup A2), were distinguished within group A. Similarly, group B strains were subclustered to a South American subgroup (subgroup B1) and a Central American subgroup (subgroup B2). These results were consistent with those obtained by DNA sequencing analysis. The ability of the BESS T-scan method to discriminate between strains that present with high degrees of nucleotide sequence similarity indicated that this method provides reliable results and multiple applications for other virus families. The method has proven to be suitable for phylogenetic comparison and molecular epidemiology studies and may be an alternative to DNA sequencing.

FIG. 1

RT-PCR amplification of SLE virus SL-33. Lane M, Marker VIII (Boehringer-Mannheim, Mannheim, Germany); lane 1, PCR product obtained by the standard protocol (with each deoxynucleoside triphosphate at a concentration of 200 μM); lane 2, PCR product obtained by the BESS T-scan protocol (each deoxynucleoside triphosphate at a concentration of 2.5 mM and 200 μM dUTP).

FIG. 2

BESS T-scan profile analysis of fragments detected with Genotyper, version 2.0, software (rows A and B) and the binary sequence (row C) used for phylogenetic analysis. A, size of the marker that comigrated with the sample; B, each value corresponds to the size of a BESS T-scan fragment; C, the binary sequence was deduced from the BESS T-scan fragments (each detected fragment corresponds to a T base); D, DNA sequence of isolate SL-33; E, nucleotide positions are numbered relative to the MSI-7 strain (GenBank accession no. M16614); −, absence of the detected fragment; V, nucleotide A, C, or G.

FIG. 3

Phylogenetic analysis of SLE virus isolates based on a 182-nucleotide fragment between nucleotides 924 and 1105 (numbered after strain MSI-7 [GenBank accession no. M16614]) located in the envelope gene by BESS T-scan technique. Distances and groupings between the 22 isolates were determined by the Jukes-Cantor algorithm and neighbor-joining method with the MEGA software program (10). Bootstrap values are indicated and correspond to 500 replications.

FIG. 4

Phylogenetic analysis of SLE virus isolates based on a 182-nucleotide DNA sequence between nucleotides 924 and 1105 (numbered after strain MSI-7 [GenBank accession no. M16614]) located in the envelope gene. Distances and groupings between the 22 isolates were determined by the Jukes-Cantor algorithm and neighbor-joining method with the MEGA software program (10). Bootstrap values are indicated and correspond to 500 replications.

FIG. 5

Phylogenetic analysis of SLE virus isolates based on a 681-nucleotide DNA sequence between positions 924 to 1604 (numbered after strain MSI-7 [GenBank accession no. M16614]) located in the envelope gene. Distances and groupings between the 22 isolates were determined by the Jukes-Cantor algorithm and neighbor-joining method with the MEGA software program (10). Bootstrap values are indicated and correspond to 500 replications.