Targeted full-genome amplification and sequencing of dengue virus types 1–4 from South America

International audienceWe 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

Full-genome amplification and sequencing of Zika viruses using a targeted amplification approach

© 2017 We have developed methods for full-genome sequencing of Zika viruses (ZIKVs) based on a targeted amplification approach. We used alignments of publicly available complete genome data to design a primer set that selectively amplifies ZIKVs. The approach includes amplification strategies for templates present at both high- and low-copy number, and PCR cycling conditions that have been normalized across genome fragments in order to streamline laboratory handling. Abundant templates can be amplified using a strategy that uses 6 overlapping amplicons to cover the complete viral genome, whereas scarce templates can be amplified using a strategy that uses 11 overlapping amplicons of smaller size. The workflow is sequencing platform agnostic, and thus, can be used in low resource settings where access to traditional Sanger sequencing is the only option available. Given the scarcity of tools for ZIKV, this approach should facilitate epidemiological surveillance and other studies that require the generation of complete viral genomic information quickly and cost-effectively

Distribución y hospederos de pulgas (siphonaptera) en la provincia de Ayabaca, Piura - 1999

Estudio descriptivo transversal realizado en la provincia de Ayabaca (zona endémica de peste bubónica), departamento de Piura, Perú; con el objetivo de ampliar los conocimientos sobre las especies de pulgas presentes. Fueron colectados 10 152 especímenes de pulgas en 46 localidades pertenecientes a seis distritos de la provincia de Ayabaca (Suyo, Sapillica, Montero, Paimas, Lagunas y Ayabaca), entre los meses de enero a julio de 1999. El muestreo se realizó seleccionando para cada vivienda un mínimo de cinco ropas de cama y cinco cuyes (Cavia porcellus) además de la totalidad de los roedores capturados en las viviendas y en el área silvestre. Ocho especies de pulgas fueron identificadas (Pulex irritans, Tiamastus cavicola, Polygenis litargus, Xenopsylla cheopis, Ctenocephalides felis felis, Craneopsylla minerva minerva, Leptopsylla segnis y Cediopsylla spillmanni) de éstas, P. irritans se reportó en todos los distritos muestreados, seguida de X. cheopis y P. litargus, encontradas en cinco de los seis distritos

Novel Echarate Virus Variant Isolated from Patient with Febrile Illness, Chanchamayo, Peru

A new phlebovirus variant was isolated from an acute febrile patient in Chanchamayo, Peru. Genome characterization and p-distance analyses based on complete open reading frames revealed that the virus is probably a natural reassortant of the Echarate virus (large and small segments) with a yet-unidentified phlebovirus (M segment)