Biennial Upsurge and Molecular Epidemiology of Enterovirus D68 Infection in New York, USA, 2014 to 2018.

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.

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014.

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.

External surface area determination of lipid vesicles using trinitrobenzene sulfonate and ultraviolet/visible spectrophotometry.

The lamellarity of liposomes is an important parameter to be controlled in liposomal delivery-release applications. A practical estimate of the degree of liposome lamellarity can be obtained by measuring the relative external surface area of the liposomes using a chemical assay. All such assays are based on a signal change caused by exposed marker lipids on reaction with a specific externally added reagent. However, a quantitative determination is often distorted by background reactions and contributions of internal lipid labeling. In the so-called TNBS assay, the marker lipid is phosphatidylethanolamine (PE) and the externally added reagent is TNBS (2,4,6-trinotrobenzene sulfonate). Mechanistic aspects of the TNBS assay were considered for improving the assay. Internal lipid labeling via PE flip-flop and/or TNBS permeation was minimal not only in cholesterol-containing liposomes but also in cholesterol-free liposomes if in the latter case membrane fluidity was decreased by slightly increasing the PE content. Compared with earlier versions of the TNBS assay, the amount of marker lipid and the time for analysis could be reduced considerably. The elaborated protocol was also applied to liposomes prepared from lipidic egg yolk isolates, offering a simple and inexpensive method for the development and in-process control of new liposome formation technologies.

Giant vesicles: preparations and applications.

There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.