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Aims: Next generation sequencing (NGS) technology has been widely used in monitoring and identifying microbiomes in sewage. We aimed to evaluate the ability of NGS analysis in directly detecting enterovirus (EV) in sewage and to understand the diversity of EVs circulated in the residents in Weishan Lake region., Methods and Results: Fourteen sewage samples were collected in Jining, Shandong Province, China from 2018 to 2019 and were parallelly investigated by the P1 amplicon-based NGS method and cell culture method. The results showed that 20 different serotypes belonging to species Enterovirus A (EV-A) (n = 5), EV-B (n = 13), and EV-C (n = 2) were identified by NGS in the sewage concentrates, which exceeded the number of types detected by cell culture method (n = 9). Echovirus 11 (E11), Coxsackievirus (CV) B5 and CVA9 were the most detected types in those sewage concentrates. Phylogenetic analysis revealed that E11 sequences obtained in this study belonged to genogroup D5 and had close genetic relationship with clinical sequences., Conclusions: Various EV serotypes circulated in populations near Weishan Lake. The combination of NGS technology into environmental surveillance will greatly contribute to improving our knowledge about EV circulation patterns in the population., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

We present the results of environmental surveillance for poliovirus (PV) and non-poliovirus (NPEV) around the switch from trivalent to bivalent oral polio-vaccine (OPV) which occurred in China in May 2016. Sewage samples were collected in Jinan and Linyi city from 2015 to 2017. Enterovirus (EV) isolation, VP1 amplification, Sanger sequencing, and phylogenetic analyses were performed. Among105 sewage samples (36 in Jinan and 69 in Linyi), 101 were positive for EV, with 74.3% (78/105) PV-positive samples and 90.5% (95/105) NPEV-positive samples. A total of 893 EV isolates were obtained, including 326 (36.5%) PVs and 567 (63.5%) NPEVs. Echovirus (E) -11 was the most common serotype out of 18 detected NPEV types (120/567), followed by E-3 (75/567) and E-6 (74/567). PV2 vanished and PV3 came to be the ascendant PV type in sewage after May 2016. Eight PV isolates were judged as pre-vaccine-derived poliovirus (pre-VDPV) and no VDPV or wild PV isolates were monitored. Bayesian phylogenetics demonstrated global E-11 originated in 1876 and evolved with the estimated rate of 4.63 × 10 -3 nucleotide substitutions per site per year (s/s/y). Multiple circulating clusters that originated at different times were coexisting in Shandong province. The most recently common ancestor of global coxsackievirus B5 could date back to 1867, at the evolutionary rate of 3.95 × 10 -3  s/s/y. In conclusion, our study described the changes of PVs and NPEVs around the polio vaccine switch period and provided meaningful global molecular epidemiological data for further studies of EV-related diseases among the population.

Environmental surveillance has been used successfully in monitoring enterovirus (EV) circulation; however cell culture method may introduce a selective bias in those EV strains that are recovered from the environment. In this study, urban sewage samples were collected monthly in Jinan, China in 2018 and concentrated via membrane adsorption/elution method. A P1 seminested RT-PCR (RT-snPCR) and NGS method was developed, by which amplicons of 4000 nucleotide in length covering the entire P1 region of EVs were obtained from sewage concentrates and were further analyzed by next generation sequencing (NGS). In addition, for each sewage concentrate, two other assays - cell culture and NGS based partial VP1 amplicon sequencing - were conducted in parallel and compared. The results showed that the P1 RT-snPCR and NGS method generated the most data, with 32 serotypes identified belonging to species EV-A (n = 11), EV-B (n = 14), and EV-C (n = 7). These serotypes covered all those detected from the methods of cell culture (n = 10) and partial VP1 amplicon sequencing (n = 16). EV serotypes from acute flaccid paralysis surveillance correlated with those from sewage. Phylogenetic analysis on coxsackievirus B5, a common pathogen of meningitis, revealed close genetic relationship between environmental and clinical sequences. These results demonstrate sewage contains different EVs related to a variety of diseases. Traditional cell culture method underestimates the existence of some serotypes. NGS based environmental surveillance provides data which are consistent with those from clinical diseases, greatly improves our understanding on the actual circulation in the population, and should be encouraged for public health surveillance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

We reviewed the epidemiological and clinical characteristics of 927 aseptic meningitis patients in Shandong in 2014, and the phylogeny of predominant enterovirus (EV) types causing this disease was analyzed. A total of 209 patients that were positive for EV were identified by both cell culture and a reverse transcription-seminested PCR in cerebrospinal fluid samples. The positive patients were most likely to be children within 15 years of age, had symptoms such as fever, vomiting and nausea (P＜ .05). The 209 EV sequences belonged to 11 types, and coxsackievirus B5, echovirus types 6 and 30 were predominant types. VP1 analysis exhibited multiple lineages were co-circulating. The significance of the study could come from the fact that surveillance is important to monitor the prevalence of EV types in population, which shows enterovirus meningitis maintains an important public health problem in China., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Coxsackievirus A4 (CV-A4) has been reported frequently in association with many infectious diseases and cases of hand, foot, and mouth disease potentially associated with CV-A4 infection are also identified. This study summarized the Shandong CV-A4 strains isolated from 25years acute flaccid paralysis surveillance, with an emphasis on exploring the phylogenetic analyses and spatiotemporal dynamics of CV-A4 at the global scale. We sampled 43 CV-A4 isolates and utilized VP1 gene to construct phylogenetic trees. Further extensive Bayesian phylogeographic analysis was carried out to investigate the evolution of CV-A4 and understand the spatiotemporal diffusion around the world using BEAST and SPREAD software. Phylogenetic trees showed that CV-A4 emerged to be more active in recent decades and multiple transmission chains were co-circulating. The molecular clock analysis estimated a mean evolutionary rate of 6.4×10 -3 substitutions/site/year, and the estimated origin of CV-A4 around 1944. The phylogeographic analyses suggested the origin of CV-A4 could be in the USA, however regional dissemination was mainly located around the Asia-Europe region. The spatiotemporal dynamics of CV-A4 exhibited frequent viral traffic among localities, and virus from Shandong province seemed to have played a central role in spreading around China and neighboring countries. Our phylogenetic description and phylogeographic analyses indicate the importance of large spatial- and temporal-scale studies in understanding epidemiological dynamics of CV-A4, particularly the diffusion routes will be of great importance to global control efforts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study described the results of environmental enterovirus surveillance conducted in Shandong Province of China in 2013. Altogether 39 sewage samples were collected and 873 enterovirus isolates (including 334 polioviruses) belonging to 22 serotypes were obtained. Echovirus (E) -7, coxsackievirus (CV) -B5, E-11, E-6, and E-3 were the most commonly detected non-polio enterovirus serotypes, and phylogeny of E-7 and CV-B5 was described. The numbers of isolates of different serotypes from sewage supernatant were compared with those from the solids. Interestingly, dramatic divergence was observed between the supernatant and solids origin for the serotypes of E-3 and E-6, which were prone to the solids and supernatant, respectively. A following adsorption test with E-3 and E-6 added sewage specimens confirmed the different preference. Furthermore, the adsorption of Sabin poliovirus type 1 to the solids under different conditions was investigated, and the results showed that acid medium, cold temperature, and high solids concentration facilitated the viral adsorption to the solids, whereas change of virus titer did not influence the proportion of adsorption. These results highlighted the importance of combining the enterovirus isolates from the supernatant and solids together in environmental surveillance so as to better understand the local circulation of different serotypes.

Coxsackievirus A21 (CV-A21) is a rarely detected serotype belonging to the species Enterovirus C (EV-C). In this study, we report the isolation and genetic characterization of CV-A21 in Shandong Province, China, during 1997 to 2013. A total of 13 strains were obtained from surveillance of cases of acute flaccid paralysis (AFP) (n = 9) and from environmental sewage (n = 4). Sequence comparison of the VP1 genes revealed high nucleotide sequence similarity (94.1 % to 99.8 % identity) among these Shandong strains during the period of 17 years and 75.8 % to 98.5 % sequence identity to foreign strains. Bayesian phylodynamic evolutionary analysis of Shandong and global CV-A21 VP1 sequences revealed that the inferred CV-A21 ancestral sequence dated back to 1750 (1643-1841) and evolved with 2.943 × 10(-3) substitutions per site per year. Alignment of the deduced VP1 amino acid sequences revealed changes that might alter the hydropathicity of the encoded protein. The complete genome of one strain from 2013 was sequenced and evidence of recombination was detected by similarity plot and bootscanning analyses. This study describes the complete genome characterization and molecular epidemiology of CV-A21 in China and gives further insight into CV-A21 evolution.

Enteroviruses (EVs) are important human pathogens associated with various clinical syndromes. This study represents an overview of non-polio enteroviruses (NPEVs) isolated from acute flaccid paralysis (AFP) surveillance in Shandong Province, China from 1988 to 2013. Altogether 792 and 170 NPEV isolates were isolated from stool specimens of 9263 AFP cases and 1059 contacts, respectively. Complete VP1 sequencing and typing on all 962 isolates revealed 53 NPEV types in which echovirus (E) 6 (7.6%), E14 (7.6%), E11 (7.4%), coxsackievirus (CV) B3 (7.4%), E25 (5.6%), CVB5 (4.9%), E7 (4.5%) and EV-A71 (4.4%) were the eight most commonly reported serotypes. Distinct summer-fall seasonality was observed, with June-October accounting for 79.3% of isolation from AFP cases with known month of specimen collection. Increase of isolation of EV-A71 and CVA--the predominant pathogens for the hand, foot, and mouth disease--was observed in recent years. Sequence analysis on VP1 coding region of EV-A71 and E6 suggested Shandong strains had great genetic divergence with isolates from other countries. The results described in this study provide valuable information on the circulation and emergence of different EV types in the context of limited EV surveillance in China.

Human enterovirus B73 (EV-B73) is a member of species Enterovirus B. To date, only one complete genome sequence of prototype strain CA55-1988 from California has been available. In this study, the complete genome analysis of an EV-B73 strain 088/SD/CHN/04 isolated from an acute flaccid paralysis case in Shandong Province, China in 2004 is conducted. It had 75.6 and 79.3 % nucleotide similarity with prototype strain CA55-1988 in the VP1 coding region and the complete genome, respectively. It had great VP1 nucleotide divergence (16.7-24.4 %) with EV-B73 strains from other parts of the world. Similarity plot and bootscanning analyses provided evidence of recombination with other EV-B viruses.

Environmental surveillance is an effective approach in investigating the circulation of polioviruses (PVs) and other human enteroviruses (EVs) in the population. The present report describes the results of environmental surveillance conducted in Shandong Province, China, from 2008 to 2012. A total of 129 sewage samples were collected, and 168 PVs and 1,007 nonpolio enteroviruses (NPEVs) were isolated. VP1 sequencing and typing were performed on all isolates. All PV strains were Sabin-like, with the numbers of VP1 substitutions ranging from 0 to 7. The NPEVs belonged to 19 serotypes, and echovirus 6 (E6), E11, coxsackievirus B3 (CVB3), E3, E12, and E7 were the six main serotypes, which accounted for 18.3%, 14.8%, 14.5%, 12.9%, 9.0%, and 5.7% of NPEVs isolated, respectively. Typical summer-fall peaks of NPEV were observed in the monthly distribution of isolation, and an epidemic pattern of annual circulation was revealed for the common serotypes. Phylogenetic analysis was performed on environmental CVB3 and E3 strains with global reference strains and local strains from aseptic meningitis patients. Shandong strains formed distinct clusters, and a close relationship was observed between local environmental and clinical strains. As an EV-specific case surveillance system is absent in China and many other countries, continuous environmental surveillance should be encouraged to investigate the temporal circulation and phylogeny of EVs in the population.

The Flinders Technology Australia (FTA) Elute Card is a commercial product that facilitates the collection, transport, archiving and processing of nucleic acids from a wide variety of biological samples at room temperature. While the cards have been designed so that sterile/deionized water can elute DNA easily, they are not suitable for some less stable RNAs. This study was undertaken to determine the optimal conditions such as the buffer type, buffer pH and incubation temperature for the elution of enteroviral RNA from FTA Elute Cards prior to quantitative analysis using real-time PCR (qPCR) or consensus degenerate hybrid oligonucleotide primer VP1 RT-semi nested PCR (CODEHOP VP1 RT-snPCR). TE-1 (pH 8.0), rather than sterile water, was the best buffer for high efficiency elution of enteroviral RNA at 95°C. However, as the estimated recovery rate of viral RNA eluted from the cards averaged to be only 6.1%, enterovirus assays using FTA elution should be considered qualitative, especially at low virus titers, and therefore the results of the assay should be interpreted carefully., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Enterovirus 76 (EV76) is a new member of species Human Enterovirus A (HEV-A). So far, the only complete genome sequence of the prototype strain from France has been available. In this study, we determined the complete nucleotide sequence of strain 04360, isolated from an acute flaccid paralysis patient in Shandong province, China in 2004. Sequence analysis revealed 80.7-94.7% VP1 nucleotide identity to other EV76 strains and provided evidence of recombination with other types of HEV-A in the P2 and P3 coding regions.

In 2009, a major outbreak of aseptic meningitis was noted in Linyi city, Shandong province, China. From June to September 2009, a total of 2,104 cases were involved in this outbreak, and 98.6% of patients were <16 years of age. To determine the pathogen of the outbreak, 42 cerebrospinal fluid specimens collected from aseptic meningitis cases were tested for cell culture, and 17 (40.5%) enteroviruses were isolated and identified as Coxsackievirus B5 (CVB5). Homologous comparison indicated that these isolates had 0-7.7% nucleotide divergence with each other. Phylogenetic reconstruction showed global CVB5 could be separated into four genogroups, and all Linyi CVB5 isolates belonged to the genogroup C which had been circulating for recent 27 years in Asia and Europe. Interestingly, two distinct lineages were observed for the 17 isolates in the phylogenetic tree, indicating that at least two different transmission chains of CVB5 were responsible for this outbreak. This study showed that CVB5-associated aseptic meningitis is an emerging concern in China. J. Med. Virol. 85:483-489, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]