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

Abstract

BackgroundMosquito-borne flaviviruses causing diseases such as dengue and Japanese encephalitis are devastating, particularly in the tropics. Although, multiple flaviviruses are known to co-circulate in India, when a patient presents with febrile illness, testing is usually limited to specific pathogens. Unbiased metagenomic sequencing of febrile cases can reveal the presence of multiple pathogens and provide complete genome information. Sequence information, a cornerstone for tracing virus evolution, is relevant for the design of vaccines and therapeutics. In order to assess the usefulness of unbiased metagenomic sequencing for the identification of viruses associated with febrile illness, we sequenced serum from four individuals and plasma from one individual, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illnesses, together with one healthy control in 2014.\n\nResultsWe identified and assembled a complete dengue virus type 3 (DENV3) sequence from the serum of a case classified as severe dengue. We also found a small number of Japanese encephalitis virus (JEV) sequences in the serum of two adults with febrile illness, including the one who had dengue. Phylogenetic analysis of the dengue sequence indicates that it belongs to a predominantly Asian, DENV3, genotype III clade. It had an estimated divergence time of 13.86 years (95% Highest Posterior Densities 12.94 - 14.83 years) with the closest Indian strain. Amino acid substitutions were present throughout the sequenced genome, including 11 substitutions in the antigenic envelope protein compared to the strain used for the development of the first commercial dengue vaccine. Of these one substitution (E361D) was unique and six were in critical antigenic sites.\n\nConclusionsWe demonstrate that both genome assembly and detection of a low number of viral sequences are possible by unbiased sequencing of clinical material. Complete dengue virus sequence analysis places the sequenced genome in a recent, predominantly Asian clade within genotype III of DENV3. The detection of JEV, an agent not routinely tested in febrile illness in India, warrants further analysis and highlights the need to study co-circulating flaviviruses in parallel.