Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing.

Md Abu Choudhury,John Aaskov,Rick Twee-Hee Ong,Shahera Banu,William B Lott,Yik-Ying Teo,Anthony Youzhi Cheng

doi:10.1371/journal.pone.0142473

Abstract

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009–0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.

Highlights

RNA virus populations are characterised by a high frequency of mutations introduced into their genomes; this ranges from 10−3 to 10−5 substitutions per nucleotide per round of replication [1, 2]
RNA virus populations must strike a fine balance between the genetic diversity necessary to survive in changing host environments [9] and the genetic diversity leading to error catastrophe
Descriptions of the nature and magnitude of genetic diversity in Dengue virus (DENV) populations [15, 17, 38,39,40] have suffered from two technological constraints: the limited size of the genome sequence that can be cloned into a single plasmid for subsequent sequencing, and the logistics of sequencing statistically large numbers of individually cloned genome fragments

Summary

Introduction

RNA virus populations are characterised by a high frequency of mutations introduced into their genomes; this ranges from 10−3 to 10−5 substitutions per nucleotide per round of replication [1, 2]. This effect is compounded by large population sizes and short generation times [3]. RNA viruses, live on the edge of the “error catastrophe” [12], striking a delicate balance between the genetic diversity required to ensure survival in mutable and hostile host environments and the accumulation of deleterious mutations that would lead to extinction

Methods

Results

Conclusion