Virus Identification in Unknown Tropical Febrile Illness Cases Using Deep SequencingNathan L. Yozwiak., Peter Skewes-Cox, Mark D. Stenglein., Angel Balmaseda, Eva Harris, Joseph L. DeRisi
PLoS Neglected Tropical Diseases, 2012Abstract: 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.
