Genomic Approaches to Virus Detection and Discovery in Acute Pediatric Illness in Nicaragua

Monitoring the global emergence and spread of novel human pathogens is of vital interest to public health efforts aimed at preventing and treating infectious disease. Viral surveillance studies yield important information about the prevalence and seasonality of circulating virus species and the possible existence of novel species, yet many large screening studies that have been conducted in temperate countries have not been repeated in tropical settings. This thesis describes experiments aimed at expanding our understanding of the virus diversity associated with acute pediatric illness in a tropical environment using comprehensive genomic detection strategies./nEnteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In Chapter 2, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acid from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that HEV109 is most closely related to serotypes of enterovirus species C (HEV-C) in all genomic regions except the 5 ́-untranslated region (5 ́UTR). Bootstrap analysis indicates that the 5' UTR of HEV109 is likely the product of an inter-species recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the HEV109 coding region shares 67-72% nucleotide sequence identity with its nearest relatives. HEV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of HEV109./nDengue virus is an archetypal emerging viral disease that infects an estimated 50-100 million people annually worldwide, yet current diagnostic practices cannot detect an etiologic agent in ~40% of dengue-like illness cases. Metagenomic approaches to pathogen detection, such as viral microarrays and deep sequencing, are promising tools to address emerging and non- diagnosable disease challenges, but have not been fully utilized in cases of tropical illness. In Chapter 3, we used the Virochip microarray and deep sequencing to characterize the viral spectrum in human sera from Nicaraguan patients presenting with dengue-like symptoms but testing negative for dengue virus. We utilized a barcoding strategy to simultaneously sequence multiple serum specimens, generating an average of 5.1 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 GenBank searches. Virus sequence was detected in 35% (12/34) of previously negative cases using deep sequencing, including six samples with Human Herpesvirus 6 sequence and five samples containing sequence from a putative novel virus related to the Circoviridae family. These results demonstrate the utility of two metagenomic strategies, the Virochip and deep sequencing, as comprehensive platforms to detect known and divergent viruses in the study of tropical febrile illness.

Advisor: Harris, Eva