Whole Genome Sequencing of Enterovirus species C Isolates by High-Throughput Sequencing: Development of Generic Primers.
Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C) consists of more than 20 types, among which the three serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions. A simple method was developed to quickly sequence the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to being sequenced by a high-throughput technique. The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures. By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.
Unité de Biologie des Virus Entériques, Institut PasteurParis, France; Institut National de la Santé et de la Recherche Médicale, U994Paris, France; WHO Collaborating Center for Research on Enteroviruses and Viral Vaccines, Institut PasteurParis, France.
Maël
M
Bessaud
Centre Pasteur du Cameroun, Service de Virologie Yaoundé, Cameroon.
Serge A
SA
Sadeuh-Mba
Unité de Biologie des Virus Entériques, Institut PasteurParis, France; Institut National de la Santé et de la Recherche Médicale, U994Paris, France; WHO Collaborating Center for Research on Enteroviruses and Viral Vaccines, Institut PasteurParis, France.
Marie-Line
ML
Joffret
Unité de virologie, Institut Pasteur de Madagascar Antananarivo, Madagascar.
Richter
R
Razafindratsimandresy
Unité de Biologie des Virus Entériques, Institut PasteurParis, France; Institut National de la Santé et de la Recherche Médicale, U994Paris, France.
Patsy
P
Polston
Unité de Biologie des Virus Entériques, Institut PasteurParis, France; Institut National de la Santé et de la Recherche Médicale, U994Paris, France.
Romain
R
Volle
Centre d'infectiologie Charles-Mérieux, Université Ankatso Antananarivo, Madagascar.
Mala
M
Rakoto-Andrianarivelo
Unité de Biologie des Virus Entériques, Institut PasteurParis, France; Institut National de la Santé et de la Recherche Médicale, U994Paris, France.
Bruno
B
Blondel
Centre Pasteur du Cameroun, Service de Virologie Yaoundé, Cameroon.
Richard
R
Njouom
Unité de Biologie des Virus Entériques, Institut PasteurParis, France; Institut National de la Santé et de la Recherche Médicale, U994Paris, France; WHO Collaborating Center for Research on Enteroviruses and Viral Vaccines, Institut PasteurParis, France.
Francis
F
Delpeyroux
Enterovirus species C; de novo assembly; high-throughput sequencing; poliovirus; recombination