Detection and Serotyping of Dengue virus Infection using third generation sequencing technology

Authors

• LC Perinet
• AN Henning
• PC Baré
• MJ Miller
• V De Giorgi

Abstract

Dengue virus (DENV) is a significant public health concern globally. It is considered an emerging or reemerging infectious disease due to several factors: geographic spread, climate change, urbanization, vector control challenges and epidemic dynamics. By implementing rapid, accurate and scalable screening strategies, countries can enhance their ability to detect Dengue virus early, improve case management, and effectively mitigate the impact of Dengue outbreaks on public health. We utilized Oxford Nanopore technology (ONT) to investigate its performance detecting and serotyping DENV in specimens collected in a real epidemiological context. Twenty-four plasma samples collected in Argentina during a 2016 DENV outbreak with a range of Ct values were tested. Complementary DNA (cDNA) was prepared and amplified to increase assay sensitivity. Sequencing libraries using amplified total cDNA were prepared and run on the ONT Mk1C, individually and in groups of 6 (multiplexed) and the results were analyzed. Whether single or multiplexed, all samples mapped to Dengue serotype 1 (DENV-1). Lower Ct values generally resulted in more viral reads and higher genome coverage. Single runs proved most successful, 14/24 samples had over 91% genome coverage and except for one outlier, all samples had over 53% coverage. Multiplexed samples resulted in fewer reads and lower genome coverage, however these were sufficient to detect and determine DENV-1 in all samples. The assay showed high specificity, detecting DENV-1 in all samples and the portable ONT Mk1C device provided simplified real time sequencing, showing its potential for use as a point-of-care assay.

Scientific Focus Area: Microbiology and Infectious Diseases

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