Use of a short fragment of the C-terminal E gene for detection and characterization of two new lineages of dengue virus 1 in India

Abstract

Here we propose the use of a 216-nucleotide fragment located in the carboxyl terminus of the E gene (E-COOH) and a pairwise-based comparison method for genotyping of dengue virus 1 (DENV-1) strains. We have applied this method to the detection and characterization of DENV-1 in serum samples from travelers returning from the tropics. The results obtained with the typing system correlate with the results obtained by comparison of the sequences of the entire E gene of the strains. The approach demonstrates utility in plotting the distribution and circulation of different genotypes of DENV-1 and also suggests the presence of two new clades of Indian strains. The integration of the method with an online database and a typing characterization tool enhances its strength. Additionally, the analysis of the complete E gene of DENV-1 strains suggested the occurrence of a nondescribed recombination event in the China GD23-95 strain. We propose the use of this methodology as a tool for real-time epidemiological surveillance of dengue virus infections and their pathogenesis.

FIG. 1.

The complete set of nucleotide sequences of the dengue virus 1 glycoprotein E gene (1485 nt, 116 sequences) was aligned by use of the CLUSTAL W program. Phylogenetic analysis was performed by using the best model of nucleotide substitution (according to Modeltest [27] and Tamura and Nei [33], with correction for a gamma distribution of 1.4212) and the neighbor-joining method to reconstruct the phylogenetic tree (MEGA, version 3.0, software package). The statistical significance of the phylogenies constructed was estimated by bootstrap analysis with 1,000 pseudoreplicate data sets. Strains are denoted by number, country of isolation, and year of isolation; genotypes are indicated, and names are given to the right.

FIG. 2.

Phylogenetic tree of the genotype AMAF. Lineages are indicated. Significant bootstrap values (≥60) are indicated. The analysis method was the same as that reported in the legend to Fig. 1.

FIG. 3.

Phylogenetic tree of the genotype ASIA complete E gene. Lineages are indicated. Significant bootstrap values (≥60) are indicated. The analysis method was the same as that reported in the legend to Fig. 1.

FIG. 4.

Phylogenetic tree of the genotype SP complete E gene. Lineages are indicated. Significant bootstrap values (≥60) are indicated. The analysis method was the same as that reported in the legend to Fig. 1.

FIG. 5.

Phylogenetic support for recombination in DENV-1. The neighbor-joining tree supporting the contrasting phylogenetic position of strain GD23-95-China 1995 in the E protein is shown. The 5′ end of the sequence (until position ∼371 of the E gene) groups this strain with genotype ASIA (upper left panel); the 3′ end sequence clustered instead with genotype SOUTH PACIFIC (upper right panel). Diversity plots for putative recombinants and their parents. (Middle left panel) Likelihood surface plot generated by LARD (Likelihood Assisted Recombination Detection). The partition that separates trees with the greatest likelihood score is the most probable recombination breakpoint. (Middle right panel) Pairwise identity plot obtained by the RDP method with parent strains A88-1988-Indonesia (SOUTH PACIFIC) and GD05_99-1995-CHI (ASIA). (Lower left panel) Chimaera results. A chi-square P value plot used to confirm the RDP result. When plotted along the length of the alignment, peaks in these χ² values indicate potential recombination breakpoints. (Lower right panel) DNA distance plot method. Pairwise distances were calculated for each window by using DNADIST (a component of the PHYLIP package), phylogenetic trees were obtained, and bootstrap support for each association is plotted against the position in the alignment of the window's center. The horizontal axis in all graphs is the nucleotide number counting from the 5′ end of the E sequence. The window size and increment were 150 and 3 bp, respectively, in each case.

FIG. 6.

Pairwise analysis of the sequences of four dengue virus strains randomly chosen from the sequences obtained in this study. Samples are SC418VI03, SP genotype, lineages SP-1 and ASIA-4; CNM60VI05, ASIA genotype, lineage ASIA-2; WI22VI05, AMAF genotype, lineage INDIA-1; and SC416VI03, genotype AMAF, lineage INDIA-2. A pairwise Needleman-Wunsch score for the test sequence against all other members of the database was determined. The average score (with the corresponding standard deviation, maximum, and minimum values) against the members of each genotype is plotted.