Abstract
Dengue virus is an emerging infectious agent that infects an estimated 50–100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic pathogen in ∼40% of dengue-like illnesses. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non-diagnosable disease challenges. In this study, we used the Virochip microarray and deep sequencing to characterize the spectrum of viruses present in human sera from 123 Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously deep sequence multiple serum specimens, generating on average over 1 million reads per sample. We then implemented a stepwise bioinformatic filtering pipeline to remove the majority of human and low-quality sequences to improve the speed and accuracy of subsequent unbiased database searches. By deep sequencing, we were able to detect virus sequence in 37% (45/123) of previously negative cases. These included 13 cases with Human Herpesvirus 6 sequences. Other samples contained sequences with similarity to sequences from viruses in the Herpesviridae, Flaviviridae, Circoviridae, Anelloviridae, Asfarviridae, and Parvoviridae families. In some cases, the putative viral sequences were virtually identical to known viruses, and in others they diverged, suggesting that they may derive from novel viruses. These results demonstrate the utility of unbiased metagenomic approaches in the detection of known and divergent viruses in the study of tropical febrile illness.
Highlights
Viral infections pose a significant global health burden, especially in the developing world where most infectious disease deaths occur in children and are commonly due to preventable or treatable agents
Traditional diagnostic tests for viruses depend on amplification of conserved portions of the viral genome, detection of the binding of antibodies to viral proteins, or replication of the virus in cell cultures
We use two approaches, microarrays and deep sequencing, to virus identification that are less susceptible to such shortcomings
Summary
Viral infections pose a significant global health burden, especially in the developing world where most infectious disease deaths occur in children and are commonly due to preventable or treatable agents. Effective diagnostic and surveillance tools are crucial for reducing disability-adjusted-life-years (DALYs) due to infectious agents and for bolstering elimination and treatment programs [1]. Unrecognized and novel pathogens continually emerge due to globalization, climate change, and environmental encroachment, and pose important diagnostic challenges [2,3]. Dengue virus (DENV) infection is the most common arthropodborne viral disease of humans, with an estimated 50–100 million clinical infections occurring annually worldwide [4]. Dengue diagnostics are of crucial importance due to its broad spectrum of clinical presentations, global emergence and spread, unique disease epidemiology, and possible clinical relation to other as-yet unknown tropical febrile pathogens
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