Abstract
Dengue virus, a positive-sense single-stranded RNA virus, continuously threatens human health. Although several criteria for evaluation of severe dengue have been recently established, the ability to prognose the risk of severe outcomes for dengue patients remains limited. Mutant spectra of RNA viruses, including single nucleotide variants (SNVs) and defective virus genomes (DVGs), contribute to viral virulence and growth. Here, we determine the potency of intrahost viral population in dengue patients with primary infection that progresses into severe dengue. A total of 65 dengue virus serotype 2 infected patients in primary infection including 17 severe cases were enrolled. We utilized deep sequencing to directly define the frequency of SNVs and detection times of DVGs in sera of dengue patients and analyzed their associations with severe dengue. Among the detected SNVs and DVGs, the frequencies of 9 SNVs and the detection time of 1 DVG exhibited statistically significant differences between patients with dengue fever and those with severe dengue. By utilizing the detected frequencies/times of the selected SNVs/DVG as features, the machine learning model showed high average with a value of area under the receiver operating characteristic curve (AUROC, 0.966 ± 0.064). The elevation of the frequency of SNVs at E (nucleotide position 995 and 2216), NS2A (nucleotide position 4105), NS3 (nucleotide position 4536, 4606), and NS5 protein (nucleotide position 7643 and 10067) and the detection times of the selected DVG that had a deletion junction in the E protein region (nucleotide positions of the junction: between 969 and 1022) increased the possibility of dengue patients for severe dengue. In summary, we demonstrated the detected frequencies/times of SNVs/DVG in dengue patients associated with severe disease and successfully utilized them to discriminate severe patients using machine learning algorithm. The identified SNVs and DVGs that are associated with severe dengue will expand our understanding of intrahost viral population in dengue pathogenesis.
Highlights
Dengue virus (DENV), is a mosquito-borne pathogen which causes more than 90 million acute infection cases and 0.5 million fatalities worldwide each year (Bhatt et al, 2013)
All sera in this study were collected on the first day of arrival at the hospital from 65 individuals, who were primarily infected by DENV2 according to the undetectable anti-DENV antibody by rapid test, during the 2015 dengue outbreak in Tainan city, Taiwan (Tsai et al, 2016)
Regarding the abundance of viral RNA copies, viral RNA amount in the collected sera ranged from 9.7 × 104 to 8.8 × 107 copies (Supplementary Table 1), and no significant difference was found between mild and severe dengue groups (Figure 1A) by using the Mann–Whitney U test, which suggested that DENV genomes were present in similar amounts in the sera of mild and severe dengue patients
Summary
Dengue virus (DENV), is a mosquito-borne pathogen which causes more than 90 million acute infection cases and 0.5 million fatalities worldwide each year (Bhatt et al, 2013). Dengue virus is transmitted by female mosquitoes mainly of the species Aedes aegypti and, to a lesser extent, Ae. albopictus. The structural proteins play important roles in viral entry into cells, such as E protein in viral attachment, prM/M and E protein in viral fusion, C protein in virion assembly, and prM and E in virus release. According to the antigenic properties mainly contributed by the E protein, DENV has been classified into four serotypes, i.e., DENV1 to DENV-4, which have recently been found across tropical and subtropical regions worldwide (Roehrig, 2003). The nonstructural proteins have multiple roles in virus replication in hosts, including the assembly of replication complex, immune response modulation, and protease activities (Lindenbarch, 2007)
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