Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages.

Emergence of Novel Norovirus GII.4 Variant.

We detected a novel GII.4 variant with an amino acid insertion at the start of epitope A in viral protein 1 of noroviruses from the United States, Gabon, South Africa, and the United Kingdom collected during 2017-2022. Early identification of GII.4 variants is crucial for assessing pandemic potential and informing vaccine development.

Clinical and molecular epidemiology of enterovirus infections in cerebrospinal fluid samples, 2019-2023.

We performed a comparative, retrospective analysis (March 2019-April 2023) of children diagnosed with non-polio enterovirus (NPEV) central nervous system (CNS) infections (n = 47 vs. 129 contemporaneous controls without NPEV, all <18 years old), requiring cerebrospinal fluid (CSF) testing upon presentation to hospital. We found that showed that admissions decreased during pandemic restrictions (13% vs. controls 33%, p = 0.003). The median age of children with NPEV was 41 days (IQR: 18-72), most were male (n = 76, 59%) and were less likely to present with symptoms of irritability (11% vs. controls 26%, p = 0.04), but more likely to be febrile (93% vs. controls 73%, p = 0.007), have higher respiratory rates (mean 44 bpm, SD 11, vs. controls 36 bpm, SD 14, p = 0.001), higher heart rates (mean 171 bpm, SD 27 vs. controls 141 bpm, SD 36, p < 0.001), higher CSF protein (median 0.66 g/L, interquartile range [IQR] 0.46-1.01, vs. controls 0.53 mg/mL, IQR 0.28-0.89, p = 0.04), higher CSF white cell count (WCC) (median WCC 9.5×106/L, IQR 1-16 vs. controls 3.15×106/L, IQR 2.7-3.6, p < 0.001), but lower CSF glucose (median 2.8 mmol/L, IQR 2.4-3.1 vs. controls 3.1 mmol/L, IQR 2.7-3.6, p < 0.001). Phylogenetic analysis showed that these NPEVs originated from Europe (EV A71, CV B4, E21, E6, CV B3, CV B5, E7, E11, E18), North America (CV B4, E18), South America (E6), Middle East (CV B5), Africa (CV B5, E18), South Asia (E15), East/Southeast Asia (E25, CV A9, E7, E11, E18), and Australia (CV B5).

Increased circulation of GII.17 noroviruses, six European countries and the United States, 2023 to 2024.

We report an increase in GII.17 norovirus outbreaks and sporadic infections of acute gastroenteritis in Austria, Germany, France, Ireland, the Netherlands, England and the United States during the 2023/24 season. A decrease in GII.4 coincided with GII.17 prevalence increasing to between 17% and 64% of all GII detections. Overall, 84% of the GII.17 strains clustered closely with strains first reported in Romania in 2021 and two new sub-lineages were identified. Norovirus surveillance and molecular characterisation should be prioritised this winter.

Increased reports of severe myocarditis associated with enterovirus infection in neonates, United Kingdom, 27 June 2022 to 26 April 2023.

Enteroviruses are a common cause of seasonal childhood infections. The vast majority of enterovirus infections are mild and self-limiting, although neonates can sometimes develop severe disease. Myocarditis is a rare complication of enterovirus infection. Between June 2022 and April 2023, twenty cases of severe neonatal enteroviral myocarditis caused by coxsackie B viruses were reported in the United Kingdom. Sixteen required critical care support and two died. Enterovirus PCR on whole blood was the most sensitive diagnostic test. We describe the initial public health investigation into this cluster and aim to raise awareness among paediatricians, laboratories and public health specialists.

Regeneration Defects in Yap and Taz Mutant Mouse Livers Are Caused by Bile Duct Disruption and Cholestasis.

BACKGROUND AND AIMS: The Hippo pathway and its downstream effectors YAP and TAZ (YAP/TAZ) are heralded as important regulators of organ growth and regeneration. However, different studies provided contradictory conclusions about their role during regeneration of different organs, ranging from promoting proliferation to inhibiting it. Here we resolve the function of YAP/TAZ during regeneration of the liver, where Hippo's role in growth control has been studied most intensely. METHODS: We evaluated liver regeneration after carbon tetrachloride toxic liver injury in mice with conditional deletion of Yap/Taz in hepatocytes and/or biliary epithelial cells, and measured the behavior of different cell types during regeneration by histology, RNA sequencing, and flow cytometry. RESULTS: We found that YAP/TAZ were activated in hepatocytes in response to carbon tetrachloride toxic injury. However, their targeted deletion in adult hepatocytes did not noticeably impair liver regeneration. In contrast, Yap/Taz deletion in adult bile ducts caused severe defects and delay in liver regeneration. Mechanistically, we showed that Yap/Taz mutant bile ducts degenerated, causing cholestasis, which stalled the recruitment of phagocytic macrophages and the removal of cellular corpses from injury sites. Elevated bile acids activated pregnane X receptor, which was sufficient to recapitulate the phenotype observed in mutant mice. CONCLUSIONS: Our data show that YAP/TAZ are practically dispensable in hepatocytes for liver development and regeneration. Rather, YAP/TAZ play an indirect role in liver regeneration by preserving bile duct integrity and securing immune cell recruitment and function.

Seroprevalence and Virologic Surveillance of Enterovirus 71 and Coxsackievirus A6, United Kingdom, 2006-2017.

Enterovirus A71 (EV-A71) and coxsackievirus A6 (CVA6) cause hand, foot and mouth disease (HFMD) and are occasionally linked to severe neurologic complications and large outbreaks worldwide. We estimated EV-A71 and CVA6 seroprevalence using cross-sectional age-stratified samples collected in 2006, 2011, and 2017. Seroprevalences of EV-A71 and CVA6 increased from 32% and 54% at 6-11 months to >75% by 10 years of age. Antibody titers declined after 20 years, which could indicate infrequent re-exposure in older populations. Age profiles for acquiring infections and mean titers were comparable in the 3 testing years, despite the marked increase in incidence of CVA6-related HFMD from 2010. The uncoupling of changes in disease severity from the infection kinetics of CVA6 as we inferred from the seroprevalence data, rather than incidence of infection over the 11-year study period, provides further evidence for a change in its pathogenicity.

Detection of Norovirus Variant GII.4 Hong Kong in Asia and Europe, 2017-2019.

We report a new norovirus GII.4 variant, GII.4 Hong Kong, with low-level circulation in 4 Eurasia countries since mid-2017. Amino acid substitutions in key residues on the virus capsid associated with the emergence of pandemic noroviruses suggest that GII.4 Hong Kong has the potential to become the next pandemic variant.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Mapping the pH Sensors Critical for Host Cell Entry by a Complex Nonenveloped Virus.

Bluetongue virus (BTV), in the family Reoviridae, is an insect-borne, double-capsid virus causing hemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.IMPORTANCE Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses.

Retrospective analysis on confirmation rates for referred positive rotavirus samples in England, 2016 to 2017: implications for diagnosis and surveillance.

BackgroundRapid diagnostic tests are commonly used by hospital laboratories in England to detect rotavirus (RV), and results are used to inform clinical management and support national surveillance of the infant rotavirus immunisation programme since 2013. In 2017, the Public Health England (PHE) national reference laboratory for enteric viruses observed that the presence of RV could not be confirmed by PCR in a proportion of RV-positive samples referred for confirmatory detection.AimWe aimed to compare the positivity rate of detection methods used by hospital laboratories with the PHE confirmatory test rate.MethodsRotavirus specimens testing positive at local hospital laboratories were re-tested at the PHE national reference laboratory using a PCR test. Confirmatory results were compared to original results from the PHE laboratory information management system.ResultsHospital laboratories screened 70.1% (2,608/3,721) of RV samples using immunochromatographic assay (IC) or rapid tests, 15.5% (578/3,721) using enzyme immunoassays (EIA) and 14.4% (535/3,721) using PCR. Overall, 1,011/3,721 (27.2%) locally RV-positive samples referred to PHE in 2016 and 2017 failed RV detection using the PHE reference laboratory PCR test. Confirmation rates were 66.9% (1,746/2,608) for the IC tests, 87.4% (505/578) for the EIA and 86.4% (465/535) for the PCR assays. Seasonal confirmation rate discrepancies were also evident for IC tests.ConclusionsThis report highlights high false positive rates with the most commonly used RV screening tests and emphasises the importance of implementing verified confirmatory tests for RV detections. This has implications for clinical diagnosis and national surveillance.

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.

Rotavirus mRNAS are released by transcript-specific channels in the double-layered viral capsid.

Rotaviruses are the single most common cause of fatal and severe childhood diarrheal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialize in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organization. To this end, we developed a single molecule assay for capturing and identifying transcripts extruded from transcriptionally active viral particles. Our findings support a model in which each channel specializes in extruding transcripts of a specific segment that in turn is linked to a single transcription complex. Our approach can be extended to study other viruses and transcription systems.

Structural constraints in the packaging of bluetongue virus genomic segments.

The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5' and 3' ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was packaged successfully, confirming that the motif identified directs the correct packaging of the segment.

Rapid generation of replication-deficient monovalent and multivalent vaccines for bluetongue virus: protection against virulent virus challenge in cattle and sheep.

Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.

Epidemiological trends in viral meningitis in England: Prospective national surveillance, 2013-2023.

BACKGROUND: In the conjugate vaccine era, viruses are the most common cause of meningitis. Here, we evaluated epidemiological trends in laboratory-confirmed viral meningitis across all age-groups over an 11-year period in England. METHODS: In England, hospital laboratories routinely report laboratory-confirmed infections electronically to the UK Health Security Agency. Records of positive viral detections in cerebrospinal fluid during 2013-2023 were extracted. Incidence rates with confidence intervals were calculated using mid-year resident population estimates. RESULTS: There were 22,114 laboratory-confirmed viral meningitis cases, including 15,299 cases during 2013-19 (pre COVID-19), with a gradual increase in incidence from 3.5/100,00 (95%CI: 3.3-3.6) to 3.9/100,000 (95%CI: 3.6-4.1). During 2020-21 when pandemic restrictions were in place, there were 2061 cases (1.8/100,000; 1.7-1.9), which increased to 4754 (4.2/100,000; 4.0-4.3) during 2022-23 (post pandemic restrictions). Infants aged <3 months accounted for 39.4% (8702/22,048) of all cases, with a stable incidence 2013-19 (504/100,000, 95%CI: 491-517), followed by a significant decline during 2020-21 (204/100,000; 188-221) and then an increase during 2022-23 (780/100,000; 749-812), with enteroviruses being the commonest cause (84.9%, 7387/8702; 424.74/100,000; 95%CI: 415.12-434.51), followed by parechoviruses (9.1%, 792/8702; 45.54/100,000; 95%CI: 42.42-48.82) and herpes simplex virus (4.4%, 380/8702; 21.85/100,000; 95%CI: 19.71-24.16). Pandemic restrictions were associated with significant declines in the incidence of enterovirus (77.7%) and parechoviruses (64% lower), with rebounds after societal restrictions were lifted. CONCLUSIONS: Rates of viral meningitis have returned to pre-pandemic levels since societal restrictions were lifted. The highest incidence of viral meningitis remains in infants aged <3 months and most commonly due to enteroviral infection.

Suicidal ideation among prisoners: analysis with a gender perspective in a five-year follow-up (2017-2022).

INTRODUCTION: There is a high incidence of self-harming behavior in the prison setting, with a suicide rate that is higher than that of the general population. Previous studies describe the association of sociodemographic, clinical, and criminological factors with the risk of suicide in the male prison population, but there is little research that specifically analyses suicidal behavior among women. The objective of this study is to analyze the characteristics of inmates who are admitted to a psychiatric unit for suicidal thoughts or attempted suicide. MATERIAL AND METHOD: Descriptive and comparative analysis of 97 inmates (68 men, 29 women) admitted to the Unidad de Hospitalización Psiquiátrica Penitenciaria de Cataluña (UHPP-C), for suicidal ideation, between January 1, 2017, and December 31, 2022. RESULTS: There are differences in terms of place of birth, with a more significant presence of African nationalities in non-national males, while foreign inmates tend to come from Latin American countries. Men have a lower mean age, longer admissions, and a higher readmission rate. They also suffer from more psychotic and addictive disorders. Women have a higher prevalence of personality disorders and affective symptoms. CONCLUSIONS: There are sociodemographic and clinical differences between male and female prison inmates who require admission for suicidal ideation. Including a gender perspective in studies on suicide risk in the prison population can provide a solid foundation for future studies, thus allowing a more complete understanding of suicidal ideation and intervention needs in the prison population.

Recovery of African horse sickness virus from synthetic RNA.

African horse sickness virus (AHSV) is an insect-vectored emerging pathogen of equine species. AHSV (nine serotypes) is a member of the genus Orbivirus, with a morphology and coding strategy similar to that of the type member, bluetongue virus. However, these viruses are distinct at the genetic level, in the proteins they encode and in their pathobiology. AHSV infection of horses is highly virulent with a mortality rate of up to 90 %. AHSV is transmitted by Culicoides, a common European insect, and has the potential to emerge in Europe from endemic countries of Africa. As a result, a safe and effective vaccine is sought urgently. As part of a programme to generate a designed highly attenuated vaccine, we report here the recovery of AHSV from a complete set of RNA transcripts synthesized in vitro from cDNA clones. We have demonstrated the generation of mutant and reassortant AHSV genomes, their recovery, stable passage, and characterization. Our findings provide a new approach to investigate AHSV replication, to design AHSV vaccines and to aid diagnosis.

Asymptomatic immunodeficiency-associated vaccine-derived poliovirus infections in two UK children.

Increasing detections of vaccine-derived poliovirus (VDPV) globally, including in countries previously declared polio free, is a public health emergency of international concern. Individuals with primary immunodeficiency (PID) can excrete polioviruses for prolonged periods, which could act as a source of cryptic transmission of viruses with potential to cause neurological disease. Here, we report on the detection of immunodeficiency-associated VDPVs (iVDPV) from two asymptomatic male PID children in the UK in 2019. The first child cleared poliovirus with increased doses of intravenous immunoglobulin, the second child following haematopoetic stem cell transplantation. We perform genetic and phenotypic characterisation of the infecting strains, demonstrating intra-host evolution and a neurovirulent phenotype in transgenic mice. Our findings highlight a pressing need to strengthen polio surveillance. Systematic collection of stool from asymptomatic PID patients who are at high risk for poliovirus excretion could improve the ability to detect and contain iVDPVs.