A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012-14): a retrospective cohort study

Abstract

Background: Enterovirus D68 was implicated in a widespread outbreak of severe respiratory illness across the USA in 2014 and has also been reported sporadically in patients with acute flaccid myelitis. We aimed to investigate the association between enterovirus D68 infection and acute flaccid myelitis during the 2014 enterovirus D68 respiratory outbreak in the USA.

Methods: Patients with acute flaccid myelitis who presented to two hospitals in Colorado and California, USA, between Nov 24, 2013, and Oct 11, 2014, were included in the study. Additional cases identified from Jan 1, 2012, to Oct 4, 2014, via statewide surveillance were provided by the California Department of Public Health. We investigated the cause of these cases by metagenomic next-generation sequencing, viral genome recovery, and enterovirus D68 phylogenetic analysis. We compared patients with acute flaccid myelitis who were positive for enterovirus D68 with those with acute flaccid myelitis but negative for enterovirus D68 using the two-tailed Fisher's exact test, two-sample unpaired t test, and Mann-Whitney U test.

Findings: 48 patients were included: 25 with acute flaccid myelitis, two with enterovirus-associated encephalitis, five with enterovirus-D68-associated upper respiratory illness, and 16 with aseptic meningitis or encephalitis who tested positive for enterovirus. Enterovirus D68 was detected in respiratory secretions from seven (64%) of 11 patients comprising two temporally and geographically linked acute flaccid myelitis clusters at the height of the 2014 outbreak, and from 12 (48%) of 25 patients with acute flaccid myelitis overall. Phylogenetic analysis revealed that all enterovirus D68 sequences associated with acute flaccid myelitis grouped into a clade B1 strain that emerged in 2010. Of six coding polymorphisms in the clade B1 enterovirus D68 polyprotein, five were present in neuropathogenic poliovirus or enterovirus D70, or both. One child with acute flaccid myelitis and a sibling with only upper respiratory illness were both infected by identical enterovirus D68 strains. Enterovirus D68 viraemia was identified in a child experiencing acute neurological progression of his paralytic illness. Deep metagenomic sequencing of cerebrospinal fluid from 14 patients with acute flaccid myelitis did not reveal evidence of an alternative infectious cause to enterovirus D68.

Interpretation: These findings strengthen the putative association between enterovirus D68 and acute flaccid myelitis and the contention that acute flaccid myelitis is a rare yet severe clinical manifestation of enterovirus D68 infection in susceptible hosts.

Funding: National Institutes of Health, University of California, Abbott Laboratories, and the Centers for Disease Control and Prevention.

Figure 1. Study patients in California and Colorado from 2012–2014

(A) Flow chart of patients whose samples were analyzed in this study, including the sample provider, number of cases, and methods used for analysis. (B) Timeline of patients investigated by the CDPH and CHLA (California) and CHCO (Colorado) meeting the case definition of AFM. In California, cases of AFM were reported to CDPH beginning in Jun 2012 when surveillance and testing were initiated. As of Oct 2014, 37 AFM patients had been tested at the CDPH, 1 before (in Dec 2012) and 36 during the surveillance period (upper panel). At the CHCO in Aurora, Colorado, there were 16 hospitalized pediatric patients meeting the case definition of AFM from Jan 2012 to Oct 2014 (lower panel). Circles denote EV-D68-positive AFM patients (red), EV-D68-negative AFM patients (pink), EV-D68-positive URI patients (blue), non-AFM patients with encephalitis and/or meningitis (orange), or AFM patients not included in the study due to lack of sample availability or consent (translucent; either grey, marked with "x", or clear). The dashed boxes outline temporally linked clusters of AFM cases seen at CHLA and CHCO in Aug to Oct 2014, or additional sporadic AFM cases. Abbreviations: AFM, acute flaccid myelitis; URI, upper respiratory infection; CDPH, California Department of Public Health; CHLA, Children's Hospital Los Angeles; CHCO, Children's Hospital Colorado; NGS, next-generation sequencing.

Figure 2. Phylogeny of EV-D68 by VP1 gene sequence

All 180 complete EV-D68 VP1 sequences available in GenBank as of Dec 2014, including the 17 new EV-D68 VP1 gene sequences in this study (boldface), were aligned using MUSCLE, and phylogenetic trees were constructed using the MrBayes algorithm. EV-D68 strains from AFM patients are grouped together in a novel clade (clade B1) and include sequences from patients with severe respiratory illness from the 2014 outbreak. AFM cases are marked in red text, encephalitis in orange text, and respiratory illness only in blue text. Abbreviations: nt, nucleotide.

Figure 3. Coding polymorphisms associated with EV-D68 clade B1

(A) Eight EV-D68 genomes were recovered from clinical NP/OP samples harboring EV-D68 at levels sufficient for genome recovery (boldface). Translated polyproteins were aligned using MUSCLE and phylogenetically clustered using MrBayes with the 9 other complete EV-D68 genomes in GenBank as of Dec 2014 and the genomes of other representative enteroviruses. AFM cases are marked in red text, encephalitis in orange text, and respiratory illness only in blue text. The scale bar shows the number of amino acid substitutions per site. (B) Pairwise identity plots of a clade B1 EV-D68 genome (US/CA/14-6100) versus other enteroviruses. (C) Coding polymorphisms associated with the EV-D68 clade B1 polyprotein compared to non-clade B1 EV-D68 and other representative enteroviruses. Abbreviations: PV, poliovirus; HRV, human rhinovirus; EV, enterovirus.