A Serotype-Specific and Multiplex PCR Method for Whole-Genome Sequencing of Dengue Virus Directly from Clinical Samples

Su, Wenzhe; Jiang, Liyun; Lu, Weizhi; Xie, Hua; Cao, Yiming; Di, Biao; Li, Yan; Nie, Kai; Wang, Huanyu; Zhang, Zhoubin; Xu, Songtao

doi:10.1128/spectrum.01210-22

Cited by 8 publications

(4 citation statements)

References 48 publications

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“…The development of PrimalScheme has enabled researchers to develop custom primer schemes for amplicon-based sequencing. DengueSeq is not the first nor the only primer scheme available for dengue virus, and several other custom dengue virus primer schemes have recently been developed [23][24][25][26][27][28]. What makes DengueSeq different from other custom primer schemes is that we (1) designed our primer schemes to include global genetic diversity within each of the serotypes, (2) performed comprehensive validation with samples representing genetic diversity within each serotype, different sample types, and a range of virus copies, and (3) validated a pan-serotype approach that can be used to perform whole genome sequencing of all four serotypes without prior serotyping.…”

Section: Discussionmentioning

confidence: 99%

DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus

Vogels,

Hill,

Breban

et al. 2023

Preprint

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BackgroundThe increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes.ResultsWe developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 101-102RNA copies/μL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments.ConclusionsDengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

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Section: Discussionmentioning

confidence: 99%

DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus

Vogels,

Hill,

Breban

et al. 2023

Preprint

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“…However, no DENV transmission dynamic inferences were conducted in those studies, which limits the usage of whole genome data of DENV. In 2022, Su et al ( 43 ) developed a multiplex PCR strategy to obtain the whole genome from all four serotypes of DENV and evaluated it in clinical samples (threshold cycle range, 23.91–35.11; coverage, 97.34–99.52%; depth, 20). This makes it possible to sequence the whole genome of DENV in low-equipped laboratories.…”

Section: Discussionmentioning

confidence: 99%

Genomic epidemiology of dengue in Shantou, China, 2019

Tian

Liang

Huang

et al. 2023

Front. Public Health

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ObjectivesDengue has been endemic in Southeast Asian countries for decades. There are few reports tracing the dynamics of dengue in real time. In this study, we generated hundreds of pathogen genomes to understand the genomic epidemiology of an outbreak in a hyper-endemic area of dengue.MethodsWe leveraged whole-genome short-read sequencing (PE150) to generate genomes of the dengue virus and investigated the genomic epidemiology of a dengue virus transmission in a mesoscale outbreak in Shantou, China, in 2019.ResultsThe outbreak was sustained from July to December 2019. The total accumulated number of laboratory-confirmed cases was 944. No gender bias or fatalities were recorded. Cambodia and Singapore were the main sources of imported dengue cases (74.07%, n = 20). A total of 284 dengue virus strains were isolated, including 259 DENV-1, 24 DENV-2, and 1 DENV-3 isolates. We generated the entire genome of 252 DENV isolates (229 DENV-1, 22 DENV-2, and 1 DENV-3), which represented 26.7% of the total cases. Combined epidemiological and phylogenetic analyses indicated multiple independent introductions. The internal transmission evaluations and transmission network reconstruction supported the inference of phylodynamic analysis, with high Bayes factor support in BSSVS analysis. Two expansion founders and transmission chains were detected in CCH and LG of Shantou.ConclusionsWe observed the instant effects of genomic epidemiology in monitoring the dynamics of DENV and highlighted its prospects for real-time tracing of outbreaks of other novel agents in the future.

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“…Traditional epidemiology employs observational or descriptive methods to uncover patterns and risk factors for diseases in populations. This includes gathering information on demographics, symptoms, location, time, and exposure to dengue vectors, as well as utilizing serology tests to minimize misdiagnosis or overdiagnosis [59]. However, there are still lots of limitations.…”

Section: Post-analytical Phasementioning

confidence: 99%

Molecular Detection and Genotyping of Dengue Viruses: Current Techniques and Future Prospect

Lee,

Morshed

2024

Bangla. J. Microbiol.

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The Dengue virus is a prevalent mosquito-borne disease, and its global incidence has been steadily increasing due to the favorable environmental conditions that promote mosquito breeding, primarily influenced by rising temperatures. Bangladesh has been particularly hard-hit by an intense and ongoing outbreak, resulting in a surge of cases and fatalities. Effective management of this disease necessitates the implementation of robust public health measures, including rigorous surveillance and early diagnosis. While serological tests are commonly employed in clinical diagnosis, molecular methods hold a critical role in identifying the specific Dengue virus strain, thereby contributing to a more comprehensive understanding of disease severity. This article serves as an extensive review, delving into various molecular testing techniques employed for both surveillance and clinical diagnosis. It offers valuable insights for research and clinical laboratories engaged in the detection of Dengue virus RNA in mosquitoes, environmental samples, and clinical specimens. The methods covered encompass a spectrum of approaches, including conventional PCR, isothermal amplification, real-time RT-PCR, Sanger sequencing, and whole-genome sequencing, providing a holistic overview of the available techniques. These methods play pivotal roles in clinical diagnosis, outbreak analysis, vector surveillance, and vaccine development. Furthermore, the article underscores the importance of integrating these techniques into existing healthcare systems, emphasizing their significance in ensuring precise dengue diagnostics to enhance the efficiency of disease management. These molecular methods are indispensable tools that contribute to accurate diagnosis, enable effective outbreak investigation, facilitate vector surveillance, and support vaccine preparation, thereby enhancing the overall management and control of dengue, ultimately working toward mitigating its impact on public health. Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 41-49

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A Serotype-Specific and Multiplex PCR Method for Whole-Genome Sequencing of Dengue Virus Directly from Clinical Samples

Abstract: Dengue virus (DENV) is becoming the most globally prevalent arbovirus. The number of people living under the threat of DENV is increasing year by year.

Cited by 8 publications

References 48 publications

DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus

DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus

Genomic epidemiology of dengue in Shantou, China, 2019

Molecular Detection and Genotyping of Dengue Viruses: Current Techniques and Future Prospect

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