title: Molecular Evolution and Intraclade Recombination of Enterovirus D68 during the 2014 Outbreak in the United States.
abstract: In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies.
  
  
  OBJECTIVE
  Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tan
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Mercy Hospitals and Clinics, Kansas City, Missouri, USA.
authors.forename: Ferdaus
authors.initials: F
authors.lastname: Hassan
authors.affiliation: Department of Pediatrics, Children's Mercy Hospitals and Clinics, Kansas City, Missouri, USA.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Schuster
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Ari
authors.initials: A
authors.lastname: Simenauer
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Mercy Hospitals and Clinics, Kansas City, Missouri, USA.
authors.forename: Rangaraj
authors.initials: R
authors.lastname: Selvarangan
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Halpin
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Xudong
authors.initials: X
authors.lastname: Lin
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Nadia
authors.initials: N
authors.lastname: Fedorova
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA.
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Stockwell
authors.affiliation: Centre of Influenza Research, School of Public Health, The University of Hong Kong, Hong Kong, China.
authors.forename: Tommy Tsan-Yuk
authors.initials: TT
authors.lastname: Lam
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: James D
authors.initials: JD
authors.lastname: Chappell
authors.affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Tina V
authors.initials: TV
authors.lastname: Hartert
authors.affiliation: Marie Bashir Institute for Infectious Diseases & Biosecurity, Charles Perkins Centre, School of Biological Sciences and Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Holmes
authors.affiliation: Infectious Diseases Group, J. Craig Venter Institute, Rockville, Maryland, USA sdas@jcvi.org.
authors.forename: Suman R
authors.identifier: http://orcid.org/0000-0003-2496-9724
authors.initials: SR
authors.lastname: Das
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D016000:Cluster Analysis; D004196:Disease Outbreaks; D030016:Enterovirus D, Human / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004769:Enterovirus Infections / Q000453:epidemiology* / Q000821:virology*; D019143:Evolution, Molecular; D005260:Female; D014644:Genetic Variation; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011446:Prospective Studies; D011995:Recombination, Genetic; D012141:Respiratory Tract Infections / Q000453:epidemiology / Q000821:virology; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D014481:United States / Q000453:epidemiology