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.

Paediatric acute hepatitis of unknown aetiology: a national investigation and adenoviraemia case-control study in the UK.

BACKGROUND: An increase in acute severe hepatitis of unknown aetiology in previously healthy children in the UK in March, 2022, triggered global case-finding. We aimed to describe UK epidemiological investigations of cases and their possible causes. METHODS: We actively surveilled unexplained paediatric acute hepatitis (transaminase >500 international units per litre) in children younger than 16 years presenting since Jan 1, 2022, through notifications from paediatricians, microbiologists, and paediatric liver units; we collected demographic, clinical, and exposure information. Then, we did a case-control study to investigate the association between adenoviraemia and other viruses and case-status using multivariable Firth penalised logistic regression. Cases aged 1-10 years and tested for adenovirus were included and compared with controls (ie, children admitted to hospital with an acute non-hepatitis illness who had residual blood samples collected between Jan 1 and May 28, 2022, and without known laboratory-confirmed diagnosis or previous adenovirus testing). Controls were frequency-matched on sex, age band, sample months, and nation or supra-region with randomised selection. We explored temporal associations between frequency of circulating viruses identified through routine laboratory pathogen surveillance and occurrence of cases by linear regression. SARS-CoV-2 seropositivity of cases was examined against residual serum from age-matched clinical comparison groups. FINDINGS: Between Jan 1 and July 4, 2022, 274 cases were identified (median age 3 years [IQR 2-5]). 131 (48%) participants were male, 142 (52%) were female, and one (<1%) participant had sex data unknown. Jaundice (195 [83%] of 235) and gastrointestinal symptoms (202 [91%] of 222) were common. 15 (5%) children required liver transplantation and none died. Adenovirus was detected in 172 (68%) of 252 participants tested, regardless of sample type; 137 (63%) of 218 samples were positive for adenovirus in the blood. For cases that were successfully genotyped, 58 (81%) of 72 had Ad41F, and 57 were identified as positive via blood samples (six of these were among participants who had undergone a transplant). In the case-control analysis, adenoviraemia was associated with hepatitis case-status (adjusted OR 37·4 [95% CI 15·5-90·3]). Increases in the detection of adenovirus from faecal samples, but not other infectious agents, in routine laboratory pathogen surveillance correlated with hepatitis cases 4 weeks later, which independently suggested an association (ß 0·06 [95% CI 0·02-0·11]). No association was identified for SARS-CoV-2 antibody seropositivity. INTERPRETATION: We observed an association between adenovirus 41F viraemia and paediatric acute hepatitis. These results can inform diagnostic testing recommendations, clinical management, and exploratory in vitro or clinical studies of paediatric acute hepatitis of unknown aetiology. The role of potential co-factors, including other viruses and host susceptibility, requires further investigation. FUNDING: None.

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.

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.

Immune Imprinting Drives Human Norovirus Potential for Global Spread.

Understanding the complex interactions between virus and host that drive new strain evolution is key to predicting the emergence potential of variants and informing vaccine development. Under our hypothesis, future dominant human norovirus GII.4 variants with critical antigenic properties that allow them to spread are currently circulating undetected, having diverged years earlier. Through large-scale sequencing of GII.4 surveillance samples, we identified two variants with extensive divergence within domains that mediate neutralizing antibody binding. Subsequent serological characterization of these strains using temporally resolved adult and child sera suggests that neither candidate could spread globally in adults with multiple GII.4 exposures, yet young children with minimal GII.4 exposure appear susceptible. Antigenic cartography of surveillance and outbreak sera indicates that continued population exposure to GII.4 Sydney 2012 and antigenically related variants over a 6-year period resulted in a broadening of immunity to heterogeneous GII.4 variants, including those identified here. We show that the strongest antibody responses in adults exposed to GII.4 Sydney 2012 are directed to previously circulating GII.4 viruses. Our data suggest that the broadening of antibody responses compromises establishment of strong GII.4 Sydney 2012 immunity, thereby allowing the continued persistence of GII.4 Sydney 2012 and modulating the cycle of norovirus GII.4 variant replacement. Our results indicate a cycle of norovirus GII.4 variant replacement dependent upon population immunity. Young children are susceptible to divergent variants; therefore, emergence of these strains worldwide is driven proximally by changes in adult serological immunity and distally by viral evolution that confers fitness in the context of immunity. IMPORTANCE In our model, preepidemic human norovirus variants harbor genetic diversification that translates into novel antigenic features without compromising viral fitness. Through surveillance, we identified two viruses fitting this profile, forming long branches on a phylogenetic tree. Neither evades current adult immunity, yet young children are likely susceptible. By comparing serological responses, we demonstrate that population immunity varies by age/exposure, impacting predicted susceptibility to variants. Repeat exposure to antigenically similar variants broadens antibody responses, providing immunological coverage of diverse variants but compromising response to the infecting variant, allowing continued circulation. These data indicate norovirus GII.4 variant replacement is driven distally by virus evolution and proximally by immunity in adults.

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.

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.

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.

Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing.

There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.

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.

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.

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.

In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release.

Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal "hand-shaped" polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two "helicase" domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.

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.

Rotavirus Genomic RNA Complex Forms via Specific RNA-RNA Interactions: Disruption of RNA Complex Inhibits Virus Infectivity.

Rotavirus (RV), a member of the Reoviridae family, causes infection in children and infants, with high morbidity and mortality. To be viable, the virus particle must package a set of eleven RNA segments. In order to understand the packaging mechanism, here, we co-synthesized sets of RNA segments in vitro in different combinations and detected by two alternate methods: the electrophoretic mobility shift assay (EMSA) and the RNA-bead pull-down assay. We showed that viral positive-sense RNA segments interact with each other in a specific manner, forming RNA complexes, and that the RNA-RNA interactions followed a sequential order initiated by small RV segments. Further, we demonstrated that RNA complexes were perturbed by targeted specific antisense oligoribonucleotides (ORNs) complementary to short RNA sequences, indicating that the RNA-RNA interactions between different segments were sequence-specific. The same inhibitory ORNs also had the capability to inhibit virus replication. The combined in vitro and in vivo data inferred that RNA-RNA interactions and specific complex formation are essential for sorting different segments, possibly prior to, or during, genome packaging. As genome assembly is a universal requirement in the Reoviridae family, this work offers an approach towards a further understanding of the sorting and packaging mechanisms of RV and related dsRNA (double-stranded RNA) viruses.

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.

Atomic model of a nonenveloped virus reveals pH sensors for a coordinated process of cell entry.

Viruses sense environmental cues such as pH to engage in membrane interactions for cell entry during infection, but how nonenveloped viruses sense pH is largely undefined. Here, we report both high- and low-pH structures of bluetongue virus (BTV), which enters cells via a two-stage endosomal process. The receptor-binding protein VP2 possesses a zinc finger that may function to maintain VP2 in a metastable state and a conserved His866, which senses early-endosomal pH. The membrane-penetration protein VP5 has three domains: dagger, unfurling and anchoring. Notably, the ß-meander motif of the anchoring domain contains a histidine cluster that can sense late-endosomal pH and also possesses four putative membrane-interaction elements. Exposing BTV to low pH detaches VP2 and dramatically refolds the dagger and unfurling domains of VP5. Our biochemical and structure-guided-mutagenesis studies support these coordinated pH-sensing mechanisms.

Influence of cellular trafficking pathway on bluetongue virus infection in ovine cells.

Bluetongue virus (BTV), a non-enveloped arbovirus, causes hemorrhagic disease in ruminants. However, the influence of natural host cell proteins on BTV replication process is not defined. In addition to cell lysis, BTV also exits non-ovine cultured cells by non-lytic pathways mediated by nonstructural protein NS3 that interacts with virus capsid and cellular proteins belonging to calpactin and ESCRT family. The PPXY late domain motif known to recruit NEDD4 family of HECT ubiquitin E3 ligases is also highly conserved in NS3. In this study using a mixture of molecular, biochemical and microscopic techniques we have analyzed the importance of ovine cellular proteins and vesicles in BTV infection. Electron microscopic analysis of BTV infected ovine cells demonstrated close association of mature particles with intracellular vesicles. Inhibition of Multi Vesicular Body (MVB) resident lipid phosphatidylinositol-3-phosphate resulted in decreased total virus titre suggesting that the vesicles might be MVBs. Proteasome mediated inhibition of ubiquitin or modification of virus lacking the PPXY in NS3 reduced virus growth. Thus, our study demonstrated that cellular components comprising of MVB and exocytic pathways proteins are involved in BTV replication in ovine cells.