Enterovirus Serotypes Research Articles

Computational Approach for Predicting Common Epitopes in the VP1 Structural Protein of Enterovirus Serotypes EV-D68, EV-D70, and EV-A71

The three human Enterovirus serotypes D-68, D-70, and A-71, are common pathogens that are transmitted by fecal-oral and aerosol routes. These positive RNA viruses were known to exhibit high levels of genetic diversity and variability. Currently, no vaccines are available to protect humans from these three serotypes. Therefore, efforts are needed for the development of a vaccine directed against heterologous viruses. In our study, an immunoinformatics approach is used to identify T- and B-cell epitopes that may help for the generation of a universal vaccine against EV-D70, EV-A71, and EV-D68. B and T cell epitopes were selected based on their length. As a result, 5 B cell epitopes and 18 T cell epitopes were predicted. Our B cell epitope prediction results showed that there are a number of linear regions. Position 150-170 was found to be the most immunogenic for the different strains. Regarding the epitopes of the T lymphocytes, the result of the interactions shows that 95% of the predicted epitopes are common between the 3 sequences and the 5 methods used. These results demonstrate the great immunogenic potential of these sequences and their capacities to trigger immune reactions in people with different HLA alleles. The “VFYDGFAGF” epitope is the most important and most immunogenic for triggering an immune response. Our study results allowed us to identify epitopes to be used in the development of cross-protection vaccines against the three Enterovirus serotypes. However, in vivo and in vitro studies are needed to assess the potential of the epitopes predicted by our study.

Enterovirus Surveillance (EVSurv) in Germany.

The major aim of the enterovirus surveillance (EVSurv) in Germany is to prove the absence of poliovirus circulation in the framework of the Global Polio Eradication Program (GPEI). Therefore, a free-of-charge enterovirus diagnostic is offered to all hospitals for patients with symptoms compatible with a polio infection. Within the quality proven laboratory network for enterovirus diagnostic (LaNED), stool and cerebrospinal fluid (CSF) samples from patients with suspected aseptic meningitis/encephalitis or acute flaccid paralysis (AFP) are screened for enterovirus (EV), typing is performed in all EV positive sample to exclude poliovirus infections. Since 2006, ≈200 hospitals from all 16 German federal states have participated annually. On average, 2500 samples (70% stool, 28% CSF) were tested every year. Overall, the majority of the patients studied are children <15 years. During the 15-year period, 53 different EV serotypes were detected. While EV-A71 was most frequently detected in infants, E30 dominated in older children and adults. Polioviruses were not detected. The German enterovirus surveillance allows monitoring of the circulation of clinically relevant serotypes resulting in continuous data about non-polio enterovirus epidemiology.

Coxsackievirus B4 sewage-isolate induces pancreatitis after oral infection of mice.

ABSTRACTNumerous serotypes which belong to the genus Enterovirus (EV) show variability in their virulence and clinical manifestations. They are also known to undergo changes caused by mutations and recombination during their circulation in the environment and the population. Various EV serotypes are prevalent in groundwater, wastewater and surface waters. Our previous studies showed that oral infection induces pancreatitis depending on specific conditions, such as gravidity, in an outbred murine model. Our aim in the present study was to further explore the pancreatic histopathology in an outbred mouse model following oral infection with clinical isolates from a patient who had aseptic meningitis and an isolate from a treated-sewage sample recovered from the residential area of the patient. The isolates were identified as coxsackievirus B4 (CVB4) in tissue culture. The CVB4 sewage-isolate induced pancreatitis after oral infection. In contrast, pancreatitis was absent following infection with the clinical isolates. Comparison of polyprotein sequences showed that the treated-sewage strains differed from the patient's isolates by 9 and 11 amino acids. We conclude that the isolates of clinical and environmental origin differed in their pathogenic properties and showed genetic variation.

Increased Incidence of Pediatric Type 1 Diabetes With Novel Association With Coxsackievirus A Species in Young Children but Declined Incidence in Adolescents in Taiwan.

OBJECTIVEType 1 diabetes (T1D) has been linked to enterovirus infection in small population-based epidemiological studies. We investigated the secular relationship of T1D incidence with enterovirus infection and enterovirus species using nationwide population-based analysis.RESEARCH DESIGN AND METHODSWe accessed the National Health Insurance Research Database of Taiwan to identify T1D and enterovirus infection cases from 2001 to 2015. Enterovirus serotype isolation rates were obtained from the nationwide laboratory surveillance systems. Negative binomial regression models assessed the incidence trend, and extended Cox proportional hazards models analyzed the association of enterovirus infection with T1D incidence. Spearman correlation coefficients evaluated the correlation between T1D incidence and circulating enterovirus species.RESULTST1D incidence rates in youth younger than 20 years were 6.30 and 5.02 per 100,000 person-years in 2001 and 2015 (P = 0.287), respectively. T1D incidence increased significantly in children aged 0–6 years (P < 0.001) but decreased in adolescents aged 13–19 years (P = 0.011). The T1D risk in children aged 0–6 years with enterovirus infection was significantly higher than that in noninfected subjects (hazard ratio 1.46; 95% CI 1.35–1.58; P < 0.001). Additionally, TID incidence in children aged 0–6 years was significantly correlated with the isolation rates of coxsackievirus A species (r = 0.60; P = 0.017), but no association was found beyond the age of 7.CONCLUSIONSWe demonstrated that T1D incidence increased in children aged 0–6 years but decreased in adolescents aged 13–19 years in Taiwan. Enterovirus-infected subjects younger than 7 years had a higher risk of T1D than noninfected subjects.

An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes

Enterovirus (EV) infectivity is typically measured as a bulk parameter, yet EV serotypes vary in their susceptibility to natural and engineered stressors. Here we developed an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously and specifically quantify the infectious concentrations of eight EV serotypes commonly encountered in sewage (coxsackieviruses A9, B1, B2, B3, B4 and B5, and echoviruses 25 and 30). The method uses two cell lines for virus replication and serotype-specific qPCR primers for quantification. Primers were designed to target multiple environmental strains of a given serotype and displayed high specificity. The ICC-RTqPCR method exhibited a linear calibration range between 50 and 1000 (echoviruses) or 5000 (coxsackieviruses) infectious units per mL. Over this range, measurements were not influenced by the presence of non-target serotypes, and calibration slopes were reproducible for different virus batches and cell ages. The ICC-RTqPCR method was able to accurately quantify the infectious concentration of a virus after inactivation by heat, and the concentration of a virus within a wastewater matrix. This method will be valuable to assess the differing fates of EV serotypes in natural or engineered systems, and to portray the associated changes in EV population composition.

Epidemiological Characteristics of Hand, Foot, and Mouth Disease Outbreaks in Qingdao, 2009-2018.

Background:In China, hand, foot, and mouth disease (HFMD) outbreaks have become an important issue recent years. We analyzed the epidemiological characteristics of HFMD outbreaks in Qingdao during 2009–2018, and provided evidences for prevention and control of the disease.Methods:Data were analyzed by descriptive analysis and correlation analysis, and throat swabs were detected for enterovirus RNA using RT-PCR.Results:Overall, 116 HFMD outbreaks were reported in Qingdao during 2009–2018, with the epidemic of the outbreaks exhibiting a decreasing tendency. The characteristics of outbreaks presented two patterns, including two-peak pattern and rural area to urban-rural fringe area to urban areas pattern. Male patients were predominant in these outbreaks. The location of the outbreaks changed from nursery to community. Non-EV71/CA16 enteroviruses were gradually becoming predominant enteroviruses serotypes. The durations of outbreaks were positively correlated with response times and the number of cases.Conclusion:The epidemiological characteristics analysis of HFMD outbreaks could provide a scientific basis for the prevention and control the disease. Reporting and handling promptly are the keys to control epidemic outbreaks of HFMD.

Molecular epidemiology of enteroviruses and predominance of echovirus 30 in an Iranian population with aseptic meningitis.

Human enteroviruses are the most prevalent causes of aseptic meningitis worldwide. However, despite such predominancy, defining the enteroviral etiology of aseptic meningitis remains a diagnostic dilemma for the clinician in Iran. Therefore, this study was conducted to characterize the prevalence and clinical significance of enteroviral aseptic meningitis as well as the predominant enterovirus serotypes among patients with aseptic meningitis in the South of Iran.Cerebrospinal fluid (CSF) specimens were obtained from 73 patients with aseptic meningitis (52.1% males and 47.9% females), ages ranging from 1month to 88years. Following the extraction of nucleic acid, the detection of enteroviruses was performed by RT-PCR, targeting the 5' untranslated region of the genome, and sequencing. Enteroviruses were found in 46.6% of samples (34/73). The most predominant serotype was echovirus 30, followed by coxsackievirus B5 and poliovirus type 1 Sabin strain. The enterovirus infections were more prevalent among female patients (58.8%) and those below 5years of age (52.9%). Although enterovirus infections were observed throughout the year, the infections were more prevalent during autumn with fever as the predominant clinical symptom. The outcomes revealed that enteroviruses are significant causes of aseptic meningitis in the South of Iran, while suspected cases of aseptic meningitis are usually monitored by bacterial culture and biochemical testing of CSF samples. Therefore, the etiology remains unknown in most cases. Molecular detection of viral pathogens should be included as a common approach in the screening of patients with aseptic meningitis to prevent unnecessary treatment and to improve clinical management.

Detection of non-polio and polio enteroviruses in Acute Flaccid Paralysis surveillance in Turkey.

Poliomyelitis was a disease feared worldwide, striking suddenly and paralysing mainly children for life. Monitoring of suspected cases of poliomyelitis is carried out with Acute Flaccid Paralysis (AFP) surveillance in Turkey. This study examines national data of AFP surveillance and the epidemiology of enteroviruses (EV) in Turkey from 2000 to 2019 and gives an overview of the detected serotypes of EVs. A total of 13,640 samples collected from patients with 5216 AFP pre-diagnosed cases (2 samples from each patient) and 3,208 contacts, during a 20-year period (2000-2019) were investigated. All isolated polioviruses were tested for their wild or vaccine origin according to the WHO recommended protocol by PCR and sequencing analysis were performed. Enterovirus positivity was detected in a total of 915 cases, which were identified as 204 Sabin-like polio virus (SLPV) and 711 non-polio enterovirus (NPEV). Of the 204 SLPV, 141 (69.1%) AFP were detected in patients and 63 (30.9%) were detected in samples taken from their contacts. Of the 711 NPEVs, 516 (72.5%) were from AFP cases and 195 (27.5%) were detected in samples taken from their contacts. It is concluded that the reason for the higher detection rate of NPEV in samples from AFP pre-diagnosed cases is attributed to the polio vaccination rates reaching 97% between 2008 and 2019 in Turkey. The most frequently detected NPEV serotypes were Coxackie A24, B3, and Echo 30. This retrospective study is the first comprehensive study in Turkey to evaluate the results of the AFP surveillance in the last 20 years.

High Diversity of Human Non-Polio Enterovirus Serotypes Identified in Contaminated Water in Nigeria

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017–2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.

Enterovirus and gut virome profile in Indian infants and immune response to oral rotavirus vaccine

Background: Oral vaccines such as oral poliovirus vaccine and oral rotavirus vaccine perform suboptimally in low-and-middle income settings such as India. Asymptomatic infection with enteric viruses is associated with impaired immune response to OPV. Whether there is similar interference of asymptomatic presence of enteric viruses with response to other live viral vaccines such as oral rotavirus vaccine (ORV) is unclear. Methods and materials: We included stool samples collected at/before dose 1 and 2 of Rotarix™ from south Indian infants participating in a study evaluating the role of the bacterial gut microbiome in ORV (Rotarix) response. Serum anti-rotavirus IgA levels were measured at 6 weeks age before administration of first ORV dose and at 14 weeks age, 4 weeks after second ORV dose. Shedding of Rotarix vaccine virus was assessed at 7 weeks and 10 weeks of age (1 week after dose 1 ORV and dose 2 ORV, respectively). Enterovirus (EV) infection and carriage was detected from stool samples collected on the day of ORV administration (dose 1 at 6 weeks age and dose 2 at 10 weeks age) from N = 101 infants at 6 and 10 weeks by pan-Enterovirus qPCR targeting the 5′ UTR region followed by shotgun metagenomic NGS for profiling EV serotypes and the gut virome. Bioinformatic analysis was performed using the ViromeScan pipeline with a specifically created database including most enterovirus serotypes. Results: At 6 weeks age, 27.72% (28/101) of all infants were positive for enterovirus carriage while at 10 weeks age, 16.83% (17/101) of all infants were positive for the pan-EV target (5′ UTR). Infants harboring non-polio enteroviruses (NPEV) had significantly higher odds of remaining seronegative to ORV (Odds ratio = 2.53; 95% CI 1.02–6.51), p = 0.03 when compared to infants free of NPEV at both time points. 39 different non-polio enterovirus serotypes were detected in 6–10 week infants with Echovirus-14 and Coxsackievirus-A4 being the commonest serotypes. Conclusion: Infection with NPEV serotypes at the time of any of the two ORV doses has an inhibitory effect on the seroresponse to ORV but is not confined to any particular NPEV serotype.

Epidemical and etiological study on hand, foot and mouth disease following EV-A71 vaccination in Xiangyang, China

Coxsackievirus A6 (CV-A6) and Coxsackievirus A10 (CV-A10) have been emerging as the prevailing serotypes and overtaking Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) in most areas as main pathogens of hand, foot and mouth disease (HFMD) in China since 2013. To investigate whole etiological spectrum following EV-A71 vaccination of approximate 40,000 infants and young children in Xiangyang, enteroviruses were serotyped in 4415 HFMD cases from October 2016 to December 2017 using Real Time and conventional PCR and cell cultures. Of the typeable 3201 specimen, CV-A6 was the predominant serotype followed by CV-A16, CV-A10, CV-A5, CV-A2 and EV-A71 with proportions of 59.54%, 15.31%, 11.56%, 4.56%, 3.78% and 3.03%, respectively. Other 12 minor serotypes were also detected. The results demonstrated that six major serotypes of enteroviruses were co-circulating, including newly emerged CV-A2 and CV-A5. A dramatic decrease of EV-A71 cases was observed, whereas the total cases remained high. Multivalent vaccines against major serotypes are urgently needed for control of HFMD.

Hand, foot and mouth disease in southern Vietnam during 2015–2018

Background: Hand, foot and mouth disease (HFMD) continues to challenge Asia with pandemic potential. In Vietnam, there have been two major outbreaks occurring during 2011–2012 (>200,000 hospitalizations and >200 deaths) and more recently in 2018 (>130,000 hospitalizations and 17 deaths). Given the high burden and the complex epidemic dynamics of HFMD, synthesizing its clinical and epidemiological data remains essential to inform the development of appropriate interventions and design public health measures. Methods and materials: Methodology/principal findings: We report the results of a hospital-based study conducted during 2015–2018, covering the severe HFMD outbreak recently documented in Vietnam in 2018. The study was conducted at three major hospitals responsible for receiving HFMD patients from southern Vietnam with a population of over 40 million. A total of 19 enterovirus serotypes were detected in 1196 HFMD patients enrolled in the clinical study during 2015–2018, with enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), CV-A10 and CV-A16 being the major causes. Despite the emergence of coxsackieviruses, EV-A71 remains the leading cause of severe HFMD in Vietnam. EV-A71 was consistently detected at a higher frequency during the second half of the years. Results: The emergence of EV-A71 subgenogroup C4 in late 2018 was preceded by its low activity during 2017–early 2018. Compared with EV-A71 subgenogroup B5, C4 was more likely to be associated with severe HFMD, representing the first report demonstrating the difference in clinical severity between subgenogroup C4 and B5, the two predominant EV-A71 subgenogroups causing HFMD worldwide. Conclusion: Conclusion/significance: Our data have provided significant insights into important aspects of HFMD over six years (2013–2018) in Vietnam, and emphasize active surveillance for pathogen circulation remains essential to inform the local public health authorities in the development of appropriate intervention strategies to reduce the burden of this emerging infections. Multivalent vaccines are urgently needed to control HFMD.

Molecular strategy for the direct detection and identification of human enteroviruses in clinical specimens associated with hand, foot and mouth disease.

BackgroundDiseases caused by human enteroviruses (EVs) are a major global public health problem. Thus, the effective diagnosis of all human EVs infections and the monitoring of epidemiological and ecological dynamic changes are urgently needed.MethodsBased on two comprehensive virological surveillance systems of hand, foot and mouth disease (HFMD), real-time PCR and nested RT-PCR (RT-snPCR) methods based on the enteroviral VP1, VP4-VP2 and VP4 regions were designed to directly detect all human EVs serotypes in clinical specimens.ResultsThe results showed that the proposed serotyping strategy exhibit very high diagnostic efficiency (Study 1: 99.9%; Study 2: 89.5%), and the variance between the study was due to inclusion of the specific Coxsackie virus A6 (CVA6) real-time RT-PCR and VP4 RT-snPCR in Study 1 but not Study 2. Furthermore, only throat swabs were collected and analyzed in Study 2, whereas in Study 1, if a specific EV serotype was not identified in the primary stool sample, other sample types (rectal swab and throat swab) were further tested where available. During the study period from 2013 to 2018, CVA6 became one of the main HFMD causative agents, whereas the level of enterovirus A71 (EV-A71) declined in 2017.ConclusionThe findings of this study demonstrate the appropriate application of PCR methods and the combination of biological sample types that are useful for etiological studies and propose a molecular strategy for the direct detection of human EVs in clinical specimens associated with HFMD.

Serotype specific epitopes identified by neutralizing antibodies underpin immunogenic differences in Enterovirus B

Echovirus 30 (E30), a serotype of Enterovirus B (EV-B), recently emerged as a major causative agent of aseptic meningitis worldwide. E30 is particularly devastating in the neonatal population and currently no vaccine or antiviral therapy is available. Here we characterize two highly potent E30-specific monoclonal antibodies, 6C5 and 4B10, which efficiently block binding of the virus to its attachment receptor CD55 and uncoating receptor FcRn. Combinations of 6C5 and 4B10 augment the sum of their individual anti-viral activities. High-resolution structures of E30-6C5-Fab and E30-4B10-Fab define the location and nature of epitopes targeted by the antibodies. 6C5 and 4B10 engage the capsid loci at the north rim of the canyon and in-canyon, respectively. Notably, these regions exhibit antigenic variability across EV-Bs, highlighting challenges in development of broad-spectrum antibodies. Our structures of these neutralizing antibodies of E30 are instructive for development of vaccines and therapeutics against EV-B infections.

Clinical, etiological and epidemiological investigations of hand, foot and mouth disease in southern Vietnam during 2015 - 2018.

Hand, foot and mouth disease (HFMD) continues to challenge Asia with pandemic potential. In Vietnam, there have been two major outbreaks occurring during 2011–2012 (>200,000 hospitalizations and >200 deaths) and more recently in 2018 (>130,000 hospitalizations and 17 deaths). Given the high burden and the complex epidemic dynamics of HFMD, synthesizing its clinical and epidemiological data remains essential to inform the development of appropriate interventions and design public health measures. We report the results of a hospital-based study conducted during 2015–2018, covering the severe HFMD outbreak recently documented in Vietnam in 2018. The study was conducted at three major hospitals responsible for receiving HFMD patients from southern Vietnam with a population of over 40 million. A total of 19 enterovirus serotypes were detected in 1196 HFMD patients enrolled in the clinical study during 2015–2018, with enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), CV-A10 and CV-A16 being the major causes. Despite the emergence of coxsackieviruses, EV-A71 remains the leading cause of severe HFMD in Vietnam. EV-A71 was consistently detected at a higher frequency during the second half of the years. The emergence of EV-A71 subgenogroup C4 in late 2018 was preceded by its low activity during 2017–early 2018. Compared with EV-A71 subgenogroup B5, C4 was more likely to be associated with severe HFMD, representing the first report demonstrating the difference in clinical severity between subgenogroup C4 and B5, the two predominant EV-A71 subgenogroups causing HFMD worldwide. Our data have provided significant insights into important aspects of HFMD over four years (2015–2018) in Vietnam, and emphasize active surveillance for pathogen circulation remains essential to inform the local public health authorities in the development of appropriate intervention strategies to reduce the burden of this emerging infections. Multivalent vaccines are urgently needed to control HFMD.

Genetic Variation of Multiple Serotypes of Enteroviruses Associated with Hand, Foot and Mouth Disease in Southern China

Enteroviruses (EVs) species A are a major public health issue in the Asia–Pacific region and cause frequent epidemics of hand, foot and mouth disease (HFMD) in China. Mild infections are common in children; however, HFMD can also cause severe illness that affects the central nervous system. To molecularly characterize EVs, a prospective HFMD virological surveillance program was performed in China between 2013 and 2016. Throat swabs, rectal swabs and stool samples were collected from suspected HFMD patients at participating hospitals. EVs were detected using generic real-time and nested reverse transcription-polymerase chain reactions (RT-PCRs). Then, the complete VP1 regions of enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16) and CVA6 were sequenced to analyze amino acid changes and construct a viral molecular phylogeny. Of the 2836 enrolled HFMD patients, 2,517 (89%) were EV positive. The most frequently detected EVs were CVA16 (32.5%, 819), CVA6 (31.2%, 785), and EV-A71 (20.4%, 514). The subgenogroups CVA16_B1b, CVA6_D3a and EV-A71_C4a were predominant in China and recombination was not observed in the VP1 region. Sequence analysis revealed amino acid variations at the 30, 29 and 44 positions in the VP1 region of EV-A71, CVA16 and CVA6 (compared to the respective prototype strains BrCr, G10 and Gdula), respectively. Furthermore, in 21 of 24 (87.5%) identified EV-A71 samples, a known amino acid substitution (D31N) that may enhance neurovirulence was detected. Our study provides insights about the genetic characteristics of common HFMD-associated EVs. However, the emergence and virulence of the described mutations require further investigation.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00266-7) contains supplementary material, which is available to authorized users.

Multiplexed High-Throughput Serological Assay for Human Enteroviruses.

Immunological assays detecting antibodies against enteroviruses typically use a single enterovirus serotype as antigen. This limits the ability of such assays to detect antibodies against different enterovirus types and to detect possible type-specific variation in antibody responses. We set out to develop a multiplexed assay for simultaneous detection of antibodies against multiple enterovirus and rhinovirus types encompassing all human infecting species. Seven recombinant VP1 proteins from enteroviruses EV-A to EV-D and rhinoviruses RV-A to RV-C species were produced. Using Meso Scale Diagnostics U-PLEX platform we were able to study antibody reactions against these proteins as well as non-structural enterovirus proteins in a single well with 140 human serum samples. Adults had on average 33-fold stronger antibody responses to these antigens (p < 10−11) compared to children, but children had less cross-reactivity between different enterovirus types. The results suggest that this new high-throughput assay offers clear benefits in the evaluation of humoral enterovirus immunity in children, giving more exact information than assays that are based on a single enterovirus type as antigen.

Detection of enteroviruses in urban sewage by next generation sequencing and its application in environmental surveillance

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.

Molecular characterization of enteroviruses among hospitalized patients in Greece, 2013–2015

BackgroundThere are only sporadic data for the circulation of Enteroviruses (EVs) in Greece with previous studies reporting mainly the presence of Echoviruses (E) and Coxsackie viruses (CV) B. ObjectivesWe carried out a surveillance study for the molecular characterization of EVs detected in hospitalized patients throughout Greece as well as a phylogenetic analysis of the most frequently encountered serotypes. Study designStools, cerebrospinal fluids, throat swabs and blood samples were collected from hospitalized patients with suspicion of EV infection. All samples were tested for EVs by rRT-PCR targeting the 5′ untranslated region of EV genome. For positive samples, PCR amplification and sequencing targeting a part of VP1 region was performed. ResultsWe examined 831 samples and 209 were positive for EVs with Enterovirus B species being the most frequently amplified. E30, CVB5 and E9 were the most frequent serotypes of Enterovirus B species, whereas CVA6 and EV-A71 the most frequent serotypes of Enterovirus A species. Evs were significantly detected more frequently in stool samples compared to other types of specimens. Phylogenetic analysis revealed that most EV-A71 strains clustered in the subgenogroups C2 whereas all the CVA6 strains belonged to sub-genotype D3. Additionally, two different lineages of E30 and three different clusters of E9 viruses circulated simultaneously in Greece. Our data indicated that most EV strains from Greece were similar to strains circulating throughout Europe during the same period. ConclusionsWe provide a comprehensive picture of EVs circulating in Greece which can be helpful to interpret trends in EV diseases by associating them with circulating serotypes.

Epidemiological and aetiological characteristics of hand, foot, and mouth disease in Sichuan Province, China, 2011\u20132017

Hand, foot, and mouth disease (HFMD) remains a threat to the Asia-Pacific region. The epidemiological characteristics and pathogen spectrum of HFMD vary with space and time. These variations are crucial for HFMD interventions but poorly understood in Sichuan Province, China, particularly after the introduction of the EV-A71 vaccine. Using descriptive methods, regression analyses, spatial autocorrelation analysis, and space-time scan statistics, we analysed the epidemiological and aetiological characteristics of HFMD surveillance data in Sichuan Province between 2011 and 2017 to identify spatio-temporal variations. The dominant serotypes of HFMD have changed from enterovirus 71 and coxsackievirus A16 to other enteroviruses since 2013. The seasonal pattern of HFMD showed two peaks generally occurring from April to July and November to December; however, the seasonal pattern varied by prefecture and enterovirus serotype. From 2011 to 2017, spatio-temporal clusters were increasingly concentrated in Chengdu, with several small clusters in northeast Sichuan. The clusters observed in southern Sichuan from 2011 to 2015 disappeared in 2016–2017. These findings highlight the importance of pathogen surveillance and vaccination strategies for HFMD interventions; future prevention and control of HFMD should focus on Chengdu and its vicinity.