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High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections.

ABSTRACT: Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.

SUBMITTER: Thai KT

PROVIDER: S-EPMC3255838 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections.

Thai Khoa T D KT Henn Matthew R MR Zody Michael C MC Tricou Vianney V Nguyet Nguyen Minh NM Charlebois Patrick P Lennon Niall J NJ Green Lisa L de Vries Peter J PJ Hien Tran Tinh TT Farrar Jeremy J van Doorn H Rogier HR de Jong Menno D MD Birren Bruce W BW Holmes Edward C EC Simmons Cameron P CP

Journal of virology 20111116 2

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in s ...[more]

PMID: 22090119

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Project description:Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. Intending to shed light on the viral determinants for severe dengue pathogenesis, we sought to analyze the DENV-2 intrahost genetic diversity in 68 patient cases clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18). Unlike previous DENV intrahost diversity studies whose approaches employed PCR, here we performed viral whole-genome deep sequencing from clinical samples with an amplicon-free approach, representing the real intrahost diversity scenario. Striking differences were detected in the viral population structure between the three clinical categories, which appear to be driven mainly by different infection times and selection pressures, rather than being linked with the clinical outcome itself. Diversity in the NS2B gene, however, showed to be constrained, irrespective of clinical outcome and infection time. Finally, 385 non-synonymous intrahost single-nucleotide variants located along the viral polyprotein, plus variants located in the untranslated regions, were consistently identified among the samples. Of them, 124 were exclusively or highly detected among cases with warning signs and among severe cases. However, there was no variant that by itself appeared to characterize the cases of greater severity, either due to its low intrahost frequency or the conservative effect on amino acid substitution. Although further studies are necessary to determine their real effect on viral proteins, this heightens the possibility of epistatic interactions. The present analysis represents an initial effort to correlate DENV-2 genetic diversity to its pathogenic potential and thus contribute to understanding the virus's dynamics within its human host.

Project description: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.

Dataset Information

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections.

Publications

High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections.

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