Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum.

Mary Dias,Barbara Muehlemann,Terry Jones,Anita Shet,Derek Smith,Tom Solomon,Sudhir Krishna,Shilpa Siddappa,Chitra Pattabiraman,Krishnapriya Tamma,Malali Gowda

doi:10.12688/wellcomeopenres.14438.2

Abstract

Background: Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics. Methods: Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014. Results: We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine. Conclusions: We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause.

Highlights

Acute undifferentiated febrile illness refers to a sudden onset of high fever without localized organ-specific clinical features[1]
We sequenced RNA extracted from the serum of four patients hospitalized with severe febrile illness and from one plasma sample from a patient hospitalized with prolonged febrile illness (Table 1)
A BLAST32 similarity search, mapping all sequenced reads to a database of NCBI reference viral sequences (Table 1), identified 19,120 DENV3 sequence reads and 14 Japanese encephalitis virus (JEV) sequence reads in sample F2, and 12 JEV sequence reads in sample F5

Summary

Introduction

Acute undifferentiated febrile illness refers to a sudden onset of high fever without localized organ-specific clinical features[1]. The unbiased metagenomic sequencing of clinical material from patients with acute fever can overcome these limitations[3,11]. We identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. Conclusions: We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile

Methods

Results

Conclusion