Phylogeny of the genus Flavivirus

Abstract

We undertook a comprehensive phylogenetic study to establish the genetic relationship among the viruses of the genus Flavivirus and to compare the classification based on molecular phylogeny with the existing serologic method. By using a combination of quantitative definitions (bootstrap support level and the pairwise nucleotide sequence identity), the viruses could be classified into clusters, clades, and species. Our phylogenetic study revealed for the first time that from the putative ancestor two branches, non-vector and vector-borne virus clusters, evolved and from the latter cluster emerged tick-borne and mosquito-borne virus clusters. Provided that the theory of arthropod association being an acquired trait was correct, pairwise nucleotide sequence identity among these three clusters provided supporting data for a possibility that the non-vector cluster evolved first, followed by the separation of tick-borne and mosquito-borne virus clusters in that order. Clades established in our study correlated significantly with existing antigenic complexes. We also resolved many of the past taxonomic problems by establishing phylogenetic relationships of the antigenically unclassified viruses with the well-established viruses and by identifying synonymous viruses.

FIG. 1

Relative genomic positions of primers used for amplification of DNA templates from the NS5 gene of flaviviruses and for sequencing. ∗, From reference . ∗∗, The name in parentheses indicates a degenerate primer at the same location corresponding to the primer immediately above. ∗∗∗, From reference .

FIG. 2

Electron micrograph of Vero cells infected with Tamana bat virus (magnification, × 121,125).

FIG. 3

Phylogenetic tree of the genus Flavivirus, using nucleotide sequence. The tree was constructed by the neighbor-joining method of MEGA. Each number at nodes is the percentage of 500 bootstrap replicate support; ∗ indicates confidence probability higher than 90%. Vertical length is arbitrary. Scale is percentage of genetic distance.

FIG. 4

Phylogenetic tree of the genus Flavivirus, using amino acid sequence. The tree was constructed by the neighbor-joining method using MEGA. Each number at nodes is the percentage of 500 bootstrap replicates. Vertical length is arbitrary. Scale is percentage of amino acid distance.

FIG. 5

Multiple amino acid sequence alignment of the CFA, Apoi, and Kedougou viruses and one member representing each clade of the genus Flavivirus. Amino acid 1 corresponds to nucleotides 9018 to 9020 of YF virus. A dash indicates missing one amino acid. A dot indicates that the amino acid (or absence of it) in a given amino acid sequence is the same as in the corresponding sequence of CFA virus above of the aligned sequences.

FIG. 6

Pairwise nucleotide sequence identity relationship among three clusters of the genus Flavivirus. ∗, Number of pairs with 63 to 65% nucleotide sequence identity/total number of pair sequence compared.