SUMMARY Background Enterovirus D68 (EV-D68) is implicated in a widespread 2014 outbreak of severe respiratory illness across the United States, and has also been sporadically reported in patients with acute flaccid myelitis (AFM). The association between EV-D68 infection and AFM remains unclear. Methods Here we report metagenomic and molecular epidemiological analyses of 25 AFM cases in California and Colorado from 2012−2014. Findings EV-D68 was detected in respiratory secretions from 7 of 11 (64%) patients comprising two temporally and geographically linked AFM clusters at the height of the 2014 outbreak, and from 12 of 25 (48%) investigated AFM cases overall. Phylogenetic analysis revealed that all AFM-associated EV-D68 sequences grouped into a single novel clade B1 strain that originally emerged in 2010. Out of six observed coding polymorphisms in the clade B1 EV-D68 polyprotein, 5 of 6 polymorphisms were shared between neuropathogenic poliovirus and/or EV-D70. One child with AFM and a sibling with only upper respiratory illness were both infected by identical EV-D68 strains, suggesting a potential role for host-specific factors in differential responses to EV-D68 infection. Notably, EV-D68 viremia was identified in a child experiencing acute neurologic progression of his paralytic illness. Deep metagenomic sequencing of CSF from 14 AFM cases failed to reveal evidence of an alternative infectious etiology to EV-D68. Interpretation Taken together, these findings strengthen the putative association between EV-D68 and AFM, as well as the contention that AFM is a rare yet severe clinical manifestation of EV-D68 infection in susceptible hosts.

The Virochip can detect both known and novel variants of viral pathogens present in RTIs. Given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation.

In 2014, the United States experienced an epidemic of acute flaccid myelitis (AFM) cases in children coincident with a nationwide outbreak of enterovirus D68 (EV-D68) respiratory disease. Up to half of the 2014 AFM patients had EV-D68 RNA detected by RT-PCR in their respiratory secretions, although EV-D68 was only detected in cerebrospinal fluid (CSF) from one 2014 AFM patient. Given previously described molecular and epidemiologic associations between EV-D68 and AFM, we sought to develop an animal model by screening seven EV-D68 strains for the ability to induce neurological disease in neonatal mice. We found that four EV-D68 strains from the 2014 outbreak (out of five tested) produced a paralytic disease in mice resembling human AFM. The remaining 2014 strain, as well as 1962 prototype EV-D68 strains Fermon and Rhyne, did not produce, or rarely produced, paralysis in mice. In-depth examination of the paralysis caused by a representative 2014 strain, MO/14-18947, revealed infectious virus, virion particles, and viral genome in the spinal cords of paralyzed mice. Paralysis was elicited in mice following intramuscular, intracerebral, intraperitoneal, and intranasal infection, in descending frequency, and was associated with infection and loss of motor neurons in the anterior horns of spinal cord segments corresponding to paralyzed limbs. Virus isolated from spinal cords of infected mice transmitted disease when injected into naïve mice, fulfilling Koch’s postulates in this model. Finally, we found that EV-D68 immune sera, but not normal mouse sera, protected mice from development of paralysis and death when administered prior to viral challenge. These studies establish an experimental model to study EV-D68-induced myelitis and to better understand disease pathogenesis and develop potential therapies.

Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5 noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 ؋ 10 5 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRVculture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEVpositive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

OBJECTIVE: To characterize the atypical cutaneous presentations in the coxsackievirus A6 (CVA6)–associated North American enterovirus outbreak of 2011–2012. METHODS: We performed a retrospective case series of pediatric patients who presented with atypical cases of hand, foot, and mouth disease (HFMD) from July 2011 to June 2012 at 7 academic pediatric dermatology centers. Patients were included if they tested positive for CVA6 or if they met clinical criteria for atypical HFMD (an enanthem or exanthem characteristic of HFMD with unusual morphology or extent of cutaneous findings). We collected demographic, epidemiologic, and clinical data including history of skin conditions, morphology and extent of exanthem, systemic symptoms, and diagnostic test results. RESULTS: Eighty patients were included in this study (median age 1.5 years, range 4 months–16 years). Seventeen patients were CVA6-positive, and 63 met clinical inclusion criteria. Ninety-nine percent of patients exhibited a vesiculobullous and erosive eruption; 61% of patients had rash involving >10% body surface area. The exanthem had a perioral, extremity, and truncal distribution in addition to involving classic HFMD areas such as palms, soles, and buttocks. In 55% of patients, the eruption was accentuated in areas of eczematous dermatitis, termed “eczema coxsackium.” Other morphologies included Gianotti-Crosti–like (37%), petechial/purpuric (17%) eruptions, and delayed onychomadesis and palm and sole desquamation. There were no patients with serious systemic complications. CONCLUSIONS: The CVA6-associated enterovirus outbreak was responsible for an exanthem potentially more widespread, severe, and varied than classic HFMD that could be confused with bullous impetigo, eczema herpeticum, vasculitis, and primary immunobullous disease.