Development And Preliminary Evaluation Of A Multiplexed Amplification And Next Generation Sequencing Method For Viral Hemorrhagic Fever Diagnostics

Abstract

Viral hemorrhagic fever is a severe and potentially lethal disease, characterized by fever, malaise, vomiting, mucosal and gastrointestinal bleeding, and hypotension, in which multiple organ systems are affected. Due to modern transportation and global trade, outbreaks of viral hemorrhagic fevers have the potential to spread rapidly and affect a significant number of susceptible individuals. Thus, urgent and robust diagnostics with an identification of the causative virus is crucial. However, this is challenged by the number and diversity of the viruses associated with hemorrhagic fever. Several viruses classified in Arenaviridae, Filoviridae, and Flaviviridae families and Bunyavirales order may cause symptoms of febrile disease with hemorrhagic symptoms. We have developed and evaluated a novel method that can potentially identify all viruses and their genomic variants known to cause hemorrhagic fever in humans. The method relies on selected amplification of the target viral nucleic acids and subsequent high throughput sequencing technology for strain identification. Computer-based evaluations have revealed very high sensitivity and specificity, provided that the primer design is kept updated. Laboratory tests using several standard hemorrhagic virus strains and patient specimens have demonstrated excellent suitability of the assay in various sequencing platforms, which can achieve a definitive diagnosis in less than 3.5 hours.