Lien vers Pubmed [PMID] – 27334982
J. Virol. Methods 2016 Jun;235:158-167
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.