Title
Targeted full-genome amplification and sequencing of dengue virus types 1–4 from South America
Date Issued
01 September 2016
Access level
open access
Resource Type
journal article
Author(s)
Naval Medical Research Unit No. 6
Naval Medical Research Unit No. 6
Naval Medical Research Unit No. 6
Publisher(s)
Elsevier B.V.
Abstract
We report optimized workflows for full-genome sequencing of dengue viruses (DENVs) 1–4. Based on alignments of publicly available complete genomes we modified and expanded existing primers sets to amplify DENV genotypes that were previously difficult or impossible to sequence. We also report improvements to streamline laboratory handling, including a dual amplification strategy for easy and difficult to sequence “high-copy” and “low-copy” templates, respectively, and normalization of PCR cycling conditions across serotypes. High-copy templates can be sequenced following amplification of as few as 5 overlapping segments covering the complete viral genome, whereas low-copy templates can be sequenced following amplification of no more than 10 overlapping segments of smaller size. These changes have been validated using a balanced set of wild-type DENV genomes (11 of DENV1, 14 of DENV2, 13 of DENV3 and 7 of DENV4) derived from human serum samples collected throughout South America over the past 15 years. The changes described enable generation of complete DENV genomes from wild-type samples without the need for viral enrichment via passaging through laboratory cell lines. This should facilitate quick and cost-effective generation of DENV full-genome sequences of the type needed for accurate epidemiological surveillance and thorough evolutionary studies of wild-type DENVs.
Start page
158
End page
167
Volume
235
Language
English
OCDE Knowledge area
Enfermedades infecciosas Virología
Scopus EID
2-s2.0-84975865158
PubMed ID
Source
Journal of Virological Methods
ISSN of the container
01660934
Sponsor(s)
The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. We are employees of the U.S. Government. The work was prepared as part of our official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines U.S. Government work as a work prepared by military service members or employees of the U.S. Government as part of that person’s official duties. This work was supported by an award to ML (work unit number 847705.82000.25GB.B0016, Promis ID# MLeguia-PO166-14, for FY2014) from the Global Emerging Infections Surveillance and Response System (GEIS) of the Armed Forces Health Surveillance Center (AFHSC). LL received support from the French Government’s Investissement d’Avenir program Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases grant ANR-10-LABX-62-IBEID, the City of Paris Emergence(s) program in Biomedical Research, and National Institutes of Health grant 1P01AI098670-01A1. The study protocol was approved by NAMRU-6’s IRB in compliance with all applicable Federal regulations governing the protection of human subjects.
Sources of information: Directorio de Producción Científica Scopus