Assessment of twelve echovirus virus-neutralisation assays in Europe: recommendations for harmonisation of non-polio enterovirus sero-surveillance studies.

Non-polio enteroviruses (NPEV) cause significant disease worldwide. Population-based sero-surveillance, by measuring antibodies against specific NPEV types, provides additional information on past circulation and the prediction for future upsurges. Virus neutralisation assays (VNA), the current method of choice for measuring NPEV type specific antibodies, are not entirely standardised. Via the European Non-Polio Enterovirus Network, we organised a VNA quality assessment in which twelve laboratories participated. We provided five echovirus (E) types (E1, E18, E30 G2, E30 G6 and E6) and intravenous immunoglobulins (IVIG) as a sample for the NPEV VNA quality assessment. Differences in VNA protocols and neutralising Ab (nAb) titres were found between the participating laboratories with geometric coefficients of variation ranging from 10.3-62.9 %. Mixed-effects regression analysis indicated a small but significant effect of type of cell line used. Harmonisation of cell line passage number, however, did not improve variation between laboratories. Calibration by making use of a reference sample, reduced variation between laboratories but differences in nAb titres remained higher than two log2 dilution steps. In conclusion, sero-surveillance data from different laboratories should be compared with caution and standardised protocols are needed.

Molecular characterization of emerging Echovirus 11 (E11) shed light on the recombinant origin of a variant associated with severe hepatitis in neonates.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3Cpro and 3Dpol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections.

Inflammatory damage caused by Echovirus 30 in the suckling mouse brain and HMC3 cells.

Echovirus 30 (E30), a member of the species B Enterovirus family, is a primary pathogen responsible for aseptic meningitis and encephalitis. E30 is associated with severe nervous system diseases and is a primary cause of child illness, disability, and even mortality. However, the mechanisms underlying E30-induced brain injury remain poorly understood. In this study, we used a neonatal mouse model of E30 to investigate the possible mechanisms of brain injury. E30 infection triggered the activation of microglia in the mouse brain and efficiently replicated within HMC3 cells. Subsequent transcriptomic analysis revealed inflammatory activation of microglia in response to E30 infection. We also detected a significant upregulation of polo-like kinase 1 (PLK1) and found that its inhibition could limit E30 infection in a sucking mouse model. Collectively, E30 infection led to brain injury in a neonatal mouse model, which may be related to excessive inflammatory responses. Our findings highlight the intricate interplay between E30 infection and neurological damage, providing crucial insights that could guide the development of interventions and strategies to address the severe clinical manifestations associated with this pathogen.

An In Vivo Model of Echovirus-Induced Meningitis Defines the Differential Roles of Type I and Type III Interferon Signaling in Central Nervous System Infection.

Echoviruses are among the most common worldwide causes of aseptic meningitis, which can cause long-term sequelae and death, particularly in neonates. However, the mechanisms by which these viruses induce meningeal inflammation are poorly understood, owing at least in part to the lack of in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we developed an in vivo neonatal mouse model that recapitulates key aspects of echovirus-induced meningitis. We show that expression of the human homologue of the primary echovirus receptor, the neonatal Fc receptor (FcRn), is not sufficient for infection of the brains of neonatal mice. However, ablation of type I, but not III, interferon (IFN) signaling in mice expressing human FcRn permitted high levels of echovirus replication in the brain, with corresponding clinical symptoms, including delayed motor skills and hind-limb weakness. Using this model, we defined the immunological response of the brain to echovirus infection and identified key cytokines, such as granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6), that were induced by this infection. Lastly, we showed that echoviruses specifically replicate in the leptomeninges, where they induce profound inflammation and cell death. Together, this work establishes an in vivo model of aseptic meningitis associated with echovirus infections that delineates the differential roles of type I and type III IFNs in echovirus-associated neuronal disease and defines the specificity of echoviral infections within the meninges. IMPORTANCE Echoviruses are among the most common worldwide causes of aseptic meningitis, which can cause long-term sequelae or even death. The mechanisms by which echoviruses infect the brain are poorly understood, largely owing to the lack of robust in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we establish a neonatal mouse model of echovirus-induced aseptic meningitis and show that expression of the human homologue of the FcRn, the primary receptor for echoviruses, and ablation of type I IFN signaling are required to recapitulate echovirus-induced meningitis and clinical disease. These findings provide key insights into the host factors that control echovirus-induced meningitis and a model that could be used to test anti-echovirus therapeutics.

The neonatal Fc receptor is a pan-echovirus receptor.

Echoviruses are amongst the most common causative agents of aseptic meningitis worldwide and are particularly devastating in the neonatal population, where they are associated with severe hepatitis, neurological disease, including meningitis and encephalitis, and even death. Here, we identify the neonatal Fc receptor (FcRn) as a pan-echovirus receptor. We show that loss of expression of FcRn or its binding partner beta 2 microglobulin (ß2M) renders cells resistant to infection by a panel of echoviruses at the stage of virus attachment, and that a blocking antibody to ß2M inhibits echovirus infection in cell lines and in primary human intestinal epithelial cells. We also show that expression of human, but not mouse, FcRn renders nonpermissive human and mouse cells sensitive to echovirus infection and that the extracellular domain of human FcRn directly binds echovirus particles and neutralizes infection. Lastly, we show that neonatal mice expressing human FcRn are more susceptible to echovirus infection by the enteral route. Our findings thus identify FcRn as a pan-echovirus receptor, which may explain the enhanced susceptibility of neonates to echovirus infections.

Early Entry Events in Echovirus 30 Infection.

Echovirus 30 (E30), a member of the enterovirus B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated enterovirus and the cause of several outbreaks all over the world, surprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 strain Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for successful infection, but not the coxsackievirus and adenovirus receptor (CAR) or decay-accelerating factor (DAF), although an interaction with DAF was observed. Double-stranded RNA replication intermediate was generated between 2 and 3 h postinfection (p.i.), and viral capsid production was initiated between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (filipin III) compromised infection, whereas bafilomycin A1, dyngo, U-73122, wortmannin, and nocodazole did not, suggesting the virus follows an enterovirus-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active Rab5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an enterovirus entry pathway, leading to uncoating in early endosomes.IMPORTANCE Echovirus 30 (E30) is a prevalent enterovirus causing regular outbreaks in both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient purified the virus in order to study the early entry events in human cells. We have recently studied other enterovirus B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities between those viruses, allowing us to define a so-called "enterovirus entry pathway." Here, E30 is reminiscent of these viruses, for example, by not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes.

Molecular epidemiological characteristics of echovirus 6 in mainland China: extensive circulation of genotype F from 2007 to 2018.

Echovirus 6 (E6) is associated with various clinical diseases and is frequently detected in environmental sewage. Despite its high prevalence in humans and the environment, little is known about its molecular phylogeography in mainland China. In this study, 114 of 21,539 (0.53%) clinical specimens from hand, foot, and mouth disease (HFMD) cases collected between 2007 and 2018 were positive for E6. The complete VP1 sequences of 87 representative E6 strains, including 24 strains from this study, were used to investigate the evolutionary genetic characteristics and geographical spread of E6 strains. Phylogenetic analysis based on VP1 nucleotide sequence divergence showed that, globally, E6 strains can be grouped into six genotypes, designated A to F. Chinese E6 strains collected between 1988 and 2018 were found to belong to genotypes C, E, and F, with genotype F being predominant from 2007 to 2018. There was no significant difference in the geographical distribution of each genotype. The evolutionary rate of E6 was estimated to be 3.631 × 10-3 substitutions site-1 year-1 (95% highest posterior density [HPD]: 3.2406 × 10-3-4.031 × 10-3 substitutions site-1 year-1) by Bayesian MCMC analysis. The most recent common ancestor of the E6 genotypes was traced back to 1863, whereas their common ancestor in China was traced back to around 1962. A small genetic shift was detected in the Chinese E6 population size in 2009 according to Bayesian skyline analysis, which indicated that there might have been an epidemic around that year.

Genetic characterization of a novel recombinant echovirus 30 strain causing a regional epidemic of aseptic meningitis in Hokkaido, Japan, 2017.

A regional epidemic of aseptic meningitis caused by echovirus 30 (E30) occurred in Hokkaido, Japan, during the period of August-December 2017. To investigate their phylogenetic relationship to other human enteroviruses, we determined the complete genomic nucleotide sequences of isolates from this outbreak. Phylogenetic analysis of the viral capsid protein 1 gene showed that the strains were most closely related to E30 strains detected in Germany, France, and Russia in 2013. In contrast, the region encoding the viral protease and the RNA-dependent RNA polymerase had a close phylogenetic relationship to non-E30 enteroviruses detected in the United Kingdom and Switzerland in 2015-2017, suggesting that a recombination event had occurred.

Intra- and inter-serotypic recombinations in the 5΄ UTR-VP4 region of Echovirus 30 strains.

Recombination has been recognized as a major mechanism of evolution in enteroviruses. The Echovirus 30 (E-30) strain Gior was sequenced and phylogenetically compared to all available E-30 sequences to detect recombination events between the 5΄UTR and VP1 genomic regions. The comparison of phylogenetic trees of the 5΄UTR and VP1 revealed incongruences concerning strains, lineages and sub-lineages. Comparative analysis of 62 E-30 sub-genomic sequences revealed six different recombination events that almost all occurred in the same region, having a start point in the 3΄end of the 5΄ UTR and end point in VP4. The only exception was the sub-lineage of Gior for which both borders of recombination were located in the 5΄UTR. These results describe for the first time recombination events in this region in circulating EV-B strains, revealing the exact points of these recombination events, highlighting the impact of such events on the evolution and epidemiology of enteroviruses.

Upsurge in echovirus 30 detections in five EU/EEA countries, April to September, 2018.

An upsurge in Echovirus 30 (E30) infections, associated with meningitis/meningoencephalitis, has been observed in Denmark, Germany, the Netherlands, Norway and Sweden in the period April to September 2018, compared with 2015-2017. In total, 658 E30 infections among 4,537 enterovirus infections were detected in 15 countries between January and September 2018 and affected mainly newborns and 26-45 year-olds. National public health institutes are reminded to remain vigilant and inform clinicians of the ongoing epidemic.

Human Rhinoviruses A9, A49, B14 and Echovirus 3, 9 among the patients with acute respiratory infection.

  Background: Acute respiratory infection result in high mortality and morbidity worldwide. There are several viral factors that originate respiratory diseases among them Enteroviruses(EVs) and Human Rhinoviruses(HRVs) can be mentioned. HRVs and EVs belong to Picornaviridae family and they have been recently classified under Enteroviruses. The pattern of respiratory infections generating organisms varies according to geographical locations. Therefore, it seems necessary to organize an appropriate plan to manage common viral diseases exclusively about Rhinoviruses and Enteroviruses. PATIENT AND METHODS: A total of 100 samples were collected from patients with acute respiratory infections (ARIs) who were hospitalized in Ahvaz city hospitals during December 2012 to November 2013 (one year longitude). Semi-Nested PCR was done on samples for detection of HRVs and EVs using region gene of VP4/VP2. Phylogenetic and molecular evolutionary analyses performed with MEGA version 5 software find out the sequence homology among the detected HRV and EV serotype. RESULTS: The results of this study revealed that from of 100 cases of ARIs 19 patients (19%) were HRV positive and 3 (3%) patients positive for EVs. Most positive cases of HRVs were observed in the autumn season while 3 positive cases of EVs were equally found in spring, summer and autumn. Phylogenetic analyses showed that the HRV strains were HRV-A9, HRV-A49, HRV-B14 and EV strains were Echo3 and 9. CONCLUSION: The results of this study revealed that high prevalence of 19% HRVs, HRV-A9, HRV-A49, HRV-B14 serotypes and low frequency of 3% Echo Viruses, Echo3 and Echo 9 serotypes have been detected in patients with ARI.

Molecular characterization of environmental and clinical echovirus 6 isolates from Poland, 2006-2014.

The aim of this study was to investigate the genetic variability of echovirus 6 (E6) isolates from environmental samples and clinical cases of aseptic meningitis from 2006 to 2014. The analysis of the VP1 region showed the extensive diversity (up to 18.8%) and revealed that E6 circulating in Poland belong to four groups. Environmental strains clustered in three groups excepting the 2012 outbreak group, which shows the sudden introduction of new epidemic variant with Asiatic origin. Data from the study established relationships of E6 from Poland with previously characterized strains and confirmed the importance of both clinical and environmental surveillance. J. Med. Virol. 89:936-940, 2017. © 2016 Wiley Periodicals, Inc.

Molecular evolution of two asymptomatic echovirus 6 strains that constitute a novel branch of recently epidemic echovirus 6 in China.

BACKGROUND: Echovirus 6 (E6) infections are associated with aseptic meningitis and acute flaccid paralysis (AFP). But some infections, sometimes most of them, are asymptomatic. The mechanism of E6 virulence is unknown. Analyses of the molecular evolution of asymptomatic E6 may help understand why the infections show different manifestations. METHODS: Ninety-six stool samples of healthy children in Yunnan, China were collected and two E6 strains were isolated from them. The whole genomes of these two E6 strains were sequenced, and their molecular evolution was analyzed. RESULTS: The results showed that the two E6 strains may be derived from KJ7724XX strains, which were predominant in AFP patients in Shangdong in 2011. The evolution was accelerated when the two E6 strains formed, although no positive selection site was found. The 11 exclusive mutations on which selection force significantly changed were found in the 2C, 3AB and 3C genes. CONCLUSION: There are some E6 strains which did not cause the disease in the children of Yunnan. These E6 strains maybe come from a recombinant E6 strain which was associated with the outbreak of AFP in Shangdong in 2011. However, some new mutations were found in the 2C, 3AB and 3C genes of these asymptomatic strains, and these mutations may be constraint by the natural selection and could be potentially responsible for clinical presentations.

Increase in ECHOvirus 6 infections associated with neurological symptoms in the Netherlands, June to August 2016.

The Dutch virus-typing network VIRO-TypeNed reported an increase in ECHOvirus 6 (E-6) infections with neurological symptoms in the Netherlands between June and August 2016. Of the 31 cases detected from January through August 2016, 15 presented with neurological symptoms. Ten of 15 neurological cases were detected in the same province and the identified viruses were genetically related. This report is to alert medical and public health professionals of the circulation of E-6 associated with neurological symptoms.

Genetic and antigenic structural characterization for resistance of echovirus 11 to pleconaril in an immunocompromised patient.

Pleconaril is a capsid inhibitor used previously to treat enterovirus infections. A pleconaril-resistant echovirus 11 (E11) strain was identified before pleconaril treatment was given in an immunocompromised patient. The patient was also treated with intravenous Ig (IVIg) for a long period but remained unresponsive. The pleconaril-resistant strains could not be neutralized in vitro, confirming IVIg treatment failure. To identify the basis of pleconaril resistance, genetic and structural analyses were conducted. Analysis of a modelled viral capsid indicated conformational changes in the hydrophobic pocket that could prevent pleconaril docking. Substitutions (V117I, V119M and I188L) in the pleconaril-resistant viruses were found in the pocket region of VP1. Modelling suggested that V119M could confer resistance, most probably due to the protruding sulfate side chain of methionine. Although pleconaril resistance induced in vitro in a susceptible E11 clinical isolate was characterized by a different substitution (I183M), resistance was suggested to also result from a similar mechanism, i.e. due to a protruding sulfate side chain of methionine. Our results showed that resistant strains that arise in vivo display different markers from those identified in vitro and suggest that multiple factors may play a role in pleconaril resistance in patient strains. Based on IVIg treatment failure, we predict that one of these factors could be immune related. Thus, both IVIg and capsid inhibitors target the viral capsid and can induce mutations that can be cross-reactive, enabling escape from both IVIg and the drug. This could limit treatment options and should be investigated further.

Serological misdiagnosis of acute liver failure associated with echovirus 25 due to immunological similarities to hepatitis A virus and prozone effect.

We describe a case of acute liver failure caused by echovirus 25 (E25) in a previously healthy 2-year-old boy. Initial serological studies were consistent with hepatitis A virus (HAV), with prozone phenomenon. The similarity of E25 to HAV may obscure accurate diagnosis in some cases of hepatitis.

Elucidation of echovirus 30's origin and transmission during the 2012 aseptic meningitis outbreak in Guangdong, China, through continuing environmental surveillance.

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.

Acute meningoencephalitis associated with echovirus 9 infection in Sri Lanka, 2009.

The aetiology of acute meningoencephalitis in Sri Lankan children and adults is poorly understood. This study was carried out to determine pathogens responsible for meningoencephalitis in Sri Lanka. A hospital-based cross-sectional study was performed using cerebrospinal fluid samples (22 adult and 17 pediatric) collected from August to December 2009 from patients clinically diagnosed with acute meningoencephalitis at two tertiary care hospitals in Sri Lanka. Routine microbiology for bacterial pathogens together with in-house RT-PCR and PCR assays for the detection of dengue viruses, Japanese encephalitis virus, West Nile virus, chikungunya virus, enteroviruses, mumps virus, measles virus, herpes simplex viruses types 1 and 2, and varicella zoster virus were performed. Bacterial pathogens were not isolated from any patient specimens. However, from nine of the paediatric patients aged 1 month to 10 years (mean age 5.2 years) echovirus 9 (E-9; family Picornaviridae, genus Enterovirus,species Enterovirus B ) was detected by RT-PCR. All nine patients presented with fever, six had headache, and seven had vomiting. Neck stiffness indicating meningitis was present in six of the patients. Phylogenetic analysis of partial VP1 and VP4-VP2 genes showed these E-9 strains to be most closely related to E-9 strains detected in CSF from Korea and France in 2005 and 2006. The remaining patients were negative for all other viruses tested. E-9 was the most common cause of acute meningoencephalitis in the tested paediatric population from Sri Lanka in 2009, which likely reflects circulation of this E-9 strain between Europe and Asia over several years.

Aseptic meningitis outbreak associated with echovirus 30 among high school football players--Los Angeles County, California, 2014.

On August 4, 2014, the Acute Communicable Disease Control Program of the Los Angeles County Department of Public Health received a report of three aseptic meningitis cases among football players at a county high school. An investigation was conducted to determine the extent of the outbreak, identify potential exposures, and recommend control measures. An outbreak-associated aseptic meningitis case was defined as an illness of any team or family member with onset during July 28-August 11 with 1) cerebrospinal fluid pleocytosis and negative bacterial culture or 2) an emergency department visit with headache, fever, and stiff neck. Ten cases were identified; nine in males, and one in a female; patient ages ranged from 13 to 17 years. All the patients sought care at an emergency department, and five were hospitalized, resulting in 12 total hospital days. All 10 patients have recovered. Eight patients were football players, and two were siblings of football players. The most affected subgroup was the junior varsity football team, with seven cases out of 57 players (attack rate = 12.3%); the relative risk for aseptic meningitis was higher among players who were linemen than among those who were not linemen (relative risk = 5.4 [p = 0.03]). Of the 10 patients, eight tested positive by polymerase chain reaction for enterovirus, and two were not tested. Echovirus testing was performed at the California Viral and Rickettsial Disease Laboratory. Of the eight specimens testing positive for enterovirus, seven tested positive for echovirus 30, and one specimen could not be typed because of insufficient quantity.

Recombination and evolutionary dynamics of human echovirus 6.

Enterovirus (EV) infections are associated with a wide array of often severe disease presentations including aseptic meningitis, encephalitis, and acute flaccid paralysis. Surveillance for polioviruses and other EVs is therefore important as a public health measure both for patient management and epidemiological studies. From 1988 to 2008, echovirus (E) 30 was the predominant genotype in Spain (33.7% of the total typed EVs). E6 was also endemic throughout this period although isolated less frequently (12.5%). In 2009, however, a substantial increase in the incidence of E6 was detected (60%), displacing E30 type (2%). To investigate the evolution and recombination in the epidemiology and transmission of E6 in Spain, a genetic analysis in VP1 and 3Dpol regions of 67 Spanish strains collected during the period 2004-2010 was performed. All VP1 sequences clustered monophyletically in the assigned genogroup C, subgroup 9, currently the predominant circulating strains identified in Europe and elsewhere in the last 10 years. 3Dpol sequences were interspersed with other species B EVs resulting from several recombination events that generated at least 12 different recombinant forms (RFs) among study samples. These showed typically minimal divergence in VP1. The co-circulation of different lineages of E6 in the same geographical area associated with its mainly endemic pattern of transmission may have contributed to the extremely short estimated half-life of E6 RFs (0.87 years). This pattern contrasts markedly with other species B EVs and EV71 where VP1 lineage expansion and extinction occurred in step with defined recombination events and periodic changes in incidence.