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Enterovirus D68 (EV-D68) infection has been associated with outbreaks of severe respiratory illness and increased cases of nonpolio acute flaccid myelitis. The patterns of EV-D68 circulation and molecular epidemiology are not fully understood. In this study, nasopharyngeal (NP) specimens collected from patients in the Lower Hudson Valley, New York, from 2014 to 2018 were examined for rhinovirus/enterovirus (RhV/EV) by the FilmArray respiratory panel. Selected RhV/EV-positive NP specimens were analyzed using two EV-D68-specific real-time RT-PCR assays, Sanger sequencing and metatranscriptomic next-generation sequencing. A total of 2,398 NP specimens were examined. EV-D68 was detected in 348 patients with NP specimens collected in 2014 (n = 94), 2015 (n = 0), 2016 (n = 160), 2017 (n = 5), and 2018 (n = 89), demonstrating a biennial upsurge of EV-D68 infection in the study area. Ninety-one complete or nearly complete EV-D68 genome sequences were obtained. Genomic analysis of these EV-D68 strains revealed dynamics and evolution of circulating EV-D68 strains since 2014. The dominant EV-D68 strains causing the 2014 outbreak belonged to subclade B1, with a few belonging to subclade B2. New EV-D68 subclade B3 strains emerged in 2016 and continued in circulation in 2018. Clade D strains that are rarely detected in the United States also arose and spread in 2018. The establishment of distinct viral strains and their variable circulation patterns provide essential information for future surveillance, diagnosis, vaccine development, and prediction of EV-D68-associated disease prevalence and potential outbreaks.

Disease caused by Neisseria meningitidis is associated with serious complications and a high fatality rate. Asymptomatic individuals can harbor the bacterium in the throat, a state known as “carriage,” which can lead to person-to-person spread of the pathogen. This study examined N. meningitidis carriage from 2010 to 2013 among 2 groups in the Quebec City region: ninth-grade students (aged 13 to 15 years), who were also followed in their last year of high school (eleventh grade/college entry; 16 to 18 years), and university students (18 to 25 years); both groups have been shown in some other geographic regions to have high rates of carriage. This study demonstrated that N. meningitidis carriage rates were higher among university students in dormitories than ninth-grade and eleventh-grade/college entry students. Understanding carriage rates in these age groups leads to better strategies to control N. meningitidis by targeting vaccination to those responsible for transmission within the population., Neisseria meningitidis carriage data are necessary to inform serogroup B (NmB) immunization program implementation. This longitudinal study compared detection methods to measure N. meningitidis throat carriage prevalence in Quebec from November 2010 to December 2013 using cultured swab isolates and direct swab PCR from students in ninth grade (aged 13 to 15 years; n = 534) and eleventh grade/college entry (16 to 18 years; n = 363) and in university students in dormitories (18 to 25 years; n = 360) at 3 time points per group. Meningococcal and NmB carriage rates were lower in ninth- and eleventh-grade/college entry students than university students, regardless of methodology. Genotyping cultured isolates by PCR detected NmB and non-NmB in 2.1% and 7.3% of ninth-grade students, in 1.7% and 7.2% of eleventh-grade/college entry students, and in 7.5% and 21.9% of university students, respectively. NmB acquisition rates were 1.9, 0.7, and 3.3 per 1,000 person-months across respective age groups. Most NmB isolates (94.7%, 76.9%, and 86.8%, respectively) expressed subfamily A factor H binding-protein (fHBP) variants. The most common non-NmB serogroups were NmY (1.7%/1.1%) from ninth grade and eleventh grade/college entry and NmW (2.8%) from university students. Genomic analyses detected disease-associated sequence types in carriage isolates, and carriage could persist for months. This is the largest longitudinal carriage study in Canada and the first to report fHBP variants in NmB carriage isolates in healthy Canadians. These data contribute to identification of the optimal window for NmB vaccination in precollege adolescents and provide a baseline for investigating NmB vaccination effects on carriage in this population. IMPORTANCE Disease caused by Neisseria meningitidis is associated with serious complications and a high fatality rate. Asymptomatic individuals can harbor the bacterium in the throat, a state known as “carriage,” which can lead to person-to-person spread of the pathogen. This study examined N. meningitidis carriage from 2010 to 2013 among 2 groups in the Quebec City region: ninth-grade students (aged 13 to 15 years), who were also followed in their last year of high school (eleventh grade/college entry; 16 to 18 years), and university students (18 to 25 years); both groups have been shown in some other geographic regions to have high rates of carriage. This study demonstrated that N. meningitidis carriage rates were higher among university students in dormitories than ninth-grade and eleventh-grade/college entry students. Understanding carriage rates in these age groups leads to better strategies to control N. meningitidis by targeting vaccination to those responsible for transmission within the population.

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.

Complete genome sequences of nine enterovirus D68 (EV-D68) strains from patients in New York were obtained in 2016 by metagenomic next-generation sequencing. Comparative genomic analysis suggests that a new subclade B3, with ~4.5% nucleotide divergence from subclade B1 strains causing the 2014 outbreak, is circulating in the United States in 2016.

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro . Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.