Metagenomic approaches provide an effective high-throughput method to detect uncharacterized virus diversity in a tropical setting from many samples simultaneously. The findings presented in this study further our knowledge of well-characterized and previously unknown viruses present in serum collected from pediatric dengue-like illness patients and advance our understanding of the application of metagenomic approaches to human pathogen detection. Deep sequencing analysis of clinical samples holds tremendous promise as a diagnostic tool by permitting the detection of many different viruses simultaneously, including those present at low-copy numbers and of divergent origin. Major remaining barriers to high-throughput sequencing strategies becoming standard diagnostic practice include prohibitive cost, lengthy sample preparation time, and computationally intensive data analysis requirements. These challenges are magnified in resource-limited settings, such as Nicaragua, but are gradually being addressed. Industry hardware and technical advancements have steadily decreased the per-base cost of deep sequencing, and the results presented here strengthen our expectations of multiplexed sample preparation and bioinformatic data filtering within the framework of current secondgeneration sequencing platforms. Long-term bi-directional partnerships with developing country collaborators facilitate easier access to techniques not currently available on-site, such as deep sequencing, and are also important in providing training opportunities for local scientists and developing relevant pathogen tests and diagnostic policies. This study expands our understanding of the virus diversity in pediatric dengue-like illness in Nicaragua and the application of genomic detection techniques in a tropical setting, findings that are particularly valuable given the pressing need for improved global emerging pathogen surveillance. Figure S1 Virochip sensitivity using poliovirus control RNA. The Virochip can detect one poliovirus gRNA in a background of 10 5 HeLa RNA molecules. Poliovirus RNA was mixed with HeLa total RNA and analyzed on the Virochip. Eighty enterovirus Virochip oligos were found to be responsive to the poliovirus RNA and the mean fold above background of the normalized intensity of these oligos is plotted. Background is defined as the normalized intensity for each oligo in the HeLa-only control sample. The top E-predict hit in the 10 25 to 10 22 samples was human enterovirus C. (PDF) Figure 3 . (PDF)  Effects of a Non-Conservative Sequence on the Properties of β-glucuronidase from Aspergillus terreus Li-20 We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(−1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme. Glycyrrhizin (GL), the main constituent of licorice extract (Glycyrrhiza glabra), is a natural edulcorant as well as an important ingredient of traditional Chinese medicine [1, 2, 3] . By hydrolyzing one or two distal glucuronides, GL can be transformed into glycyrrhetinic acid monoglucuronide (GAMG) or glycyrrhetinic acid (GA) ( Figure 1 ). As an important derivative of GL, GAMG displayed stronger physiological functions than GL such as antiviral, anti-inflammatory, anti-tumor functions, and so on; and it is also 1000-fold sweeter than saccharose [4] . On the other hand, GA is the bioactive substance of GL well known for its pharmacological features [4, 5, 6] . The research on GL biotransformation catalyzed by b-glucuronidase (GUS, EC 3.2.1.31) was reported mainly in animal tissues such as duck [7] and human [8] , whereas studies on GL biotransformation in fungal species are few [9] . In our previous work, a fungal strain, Aspergillus terreus Li-20 was screened, which can use GL as a carbon source and produce GAMG and GA after catalysis by b-glucuronidase (AtGUS). The main disadvantages of AtGUS were low enzyme productivity, low catalytic efficiency, and pathogenicity, which rendered it unsafe for use in the food and medical industries.