Detection by Direct Next Generation Sequencing Analysis of Emerging Enterovirus D68 and C109 Strains in an Environmental Sample From Scotland

Abstract

Background: Human enteroviruses (EVs) have been linked with severe disease and syndromes as varied as acute respiratory illness, myocarditis, and flaccid paralysis. With global polio eradication on sight the focus of clinical investigations has expanded to the identification of other EV serotypes associated with severe neurological conditions such as EV-D68, responsible for large outbreaks in 2014 and 2016 that spread worldwide and were related with severe respiratory disease leading to acute myelitis in some cases. New EV serotypes with epidemic potential continue to emerge such as EV-C104, EV-C105, EV-C109, and EV-C117 identified in respiratory samples in recent years.Methods: We used a next generation sequencing (NGS) approach to detect multiple EV serotypes directly in a sewage concentrate from Glasgow (Scotland, United Kingdom) generating whole-capsid nucleotide sequences that were compared to sequences of cell culture isolates from this sewage sample and clinical EV isolates from GenBank.Results: Thirteen different serotypes belonging to all four A, B, C, and D EV species were identified in the sewage concentrate. EV strains closely related to EV-D68 epidemic isolates of B3 lineage reported in the United States and Europe in 2016 and to EV-C109 respiratory isolates found in Denmark and Netherlands in 2015 were identified.Conclusion: Environmental surveillance (ES) can effectively detect EV circulation in human populations. The use of NGS for ES can help overcoming the limitations of traditional cell culture and sequencing methods, which are selective and biased, and can contribute to the early detection and assessment of spread of emerging EV pathogens.

Tracking SARS-CoV-2 in Sewage: Evidence of Changes in Virus Variant Predominance during COVID-19 Pandemic

“…The samples were transported to the laboratory in chilled packages on the day of sampling and were stored at −80 °C until use. Each sample was processed using a filtration–centrifugation method described before and previously validated for the detection of polio and non-polio enteroviruses during routine ES for poliovirus as part of our role as a WHO Global Specialized polio network laboratory [ 43 , 44 ]. Briefly, following the removal of solids by centrifugation at 3000× g , wastewater was filtered through a 500 mL Nalgene Rapid-flow™ 0.45 µM filter (Thermo Fisher Scientific, Waltham, MA, USA) and concentrated using Centriprep centrifugal filter units with a 10 kDa molecular weight cutoff (Merck Life Science UK Limited, Gillingham, UK) following manufacturer’s instructions.…”

Commonly setting biological standards in rare diseases

“…NIBSC as part of the WHO Small Working Group for polio diagnosis and in collaboration with Imperial College in London (UK), supported by the Bill and Melinda Gates Foundation (BMGF), is actively pursuing these aims. We have recently developed methods that can detect and identify not only poliovirus but also human enteroviruses directly from cell cultures, stool extracts and sewage concentrates using novel Next Generation Sequencing (NGS) approaches [11,[14][15][16][17][18][19]. This allows us to identify multiple enterovirus serotypes in sewage samples and hence in the human population, including some of public health relevance such as poliovirus, EV-D68, and EV-A71, as well as novel serotypes with potential to cause outbreaks such as EV-C104, EV-C105 and EV-C109.…”

Enterovirus D68: molecular biological characteristics, features of infection

“…В названиях последовательностей указываются номер присоединения, место и год изоляции. Представлены 4 основных клады от A до D. Клада B дополнительно разделена на B1-B3 [25]. Сезонное распределение обнаружения EV-D68 (абс.)…”

“…В настоящее время штаммы EV-D68 разделены на четыре субтипа и множество линий, включающих первоначальные классические подтипы и недавние эпидемиологически значимые линии: субтип A, субтип B, субтип C и субтип D (рис. 3) [25].…”