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The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8·2%) sequences belonged to genotype GI, 15 256 (91·7%) to GII, and seven (<0·1%) to GIV.1. During this period, 26 different norovirus capsid genotypes circulated and 22 different recombinant genomes were found. GII.4 drift variants emerged with 2–3-year periodicity up to 2012, but not afterwards. Instead, the GII.4 Sydney capsid seems to persist through recombination, with a novel recombinant of GII.P16–GII.4 Sydney 2012 variant detected in 2014 in Germany (n=1) and the Netherlands (n=1), and again in 2016 in Japan (n=2), China (n=8), and the Netherlands (n=3). The novel GII.P17–GII.17, first reported in Asia in 2014, has circulated widely in Europe in 2015–16 (GII.P17 made up a highly variable proportion of all sequences in each country [median 11·3%, range 4·2–53·9], as did GII.17 [median 6·3%, range 0–44·5]). GII.4 viruses were more common in outbreaks in health-care settings (2239 [37·2%] of 6022 entries) compared with other genotypes (101 [12·5%] of 809 entries for GI and 263 [13·5%] of 1941 entries for GII non-GII.Pe–GII.4 or GII.P4–GII.4). Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions. European Union's Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund.

Key Points Norovirus infections pose a substantial risk to human health worldwide. Modes of viral transmission, the severity of illness and evolutionary pressures all contribute to this risk and can vary between viral genotypes. Many details about the transmission of noroviruses remain unknown, especially regarding the origin of newly emerging strains. The recent emergence of genotype GII.P17-GII.17 noroviruses in Asia should serve as a warning that future risks from norovirus outbreaks might arise from genotypes other than those currently targeted by vaccine development. Bacteria in the host microbiota might influence human norovirus infections by providing HBGA-like sugars for norovirus attachment and by modulating host immunity. B cells support norovirus replication in the presence of bacteria that express histo-blood group antigen (HBGA)-like sugars. A recently described cell culture system for the study of noroviruses in B cells will hopefully advance our understanding of many aspects of human noroviruses, ranging from the molecular characterization of their life cycle to the development of improved vaccines. In the modern world, several factors have increased the global health challenge posed by noroviruses. In this Review, Koopmans and colleagues describe advances in the study of norovirus transmission, pathogenesis and evolution, and consider future prospects for therapeutics. Norovirus infections are a major cause of gastroenteritis, and outbreaks occur frequently. Several factors are currently increasing the challenge posed by norovirus infections to global health, notably the increasing number of infections in immunocompromised individuals, who are more susceptible to disease, and the globalization of the food industry, which enables large norovirus outbreaks to occur on an international scale. Furthermore, the rapid rate of the genetic and antigenic evolution of circulating noroviruses complicates the development of vaccines and therapies that are required to counter these challenges. In this Review, we describe recent advances in the study of the transmission, pathogenesis and evolution of human noroviruses, and consider the ongoing risk of norovirus outbreaks, together with the future prospects for therapeutics, in a rapidly changing world.

During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5-6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.

Norovirus infections are a leading cause of acute gastroenteritis worldwide, affecting people of all ages. There are 10 norovirus genogroups (GI-GX) that infect humans and animals in a host-specific manner. New variants and genotypes frequently emerge, and their origin is not well understood. One hypothesis is that new human infections may be seeded from an animal reservoir, as human noroviruses have occasionally been detected in animal species. The majority of these sequences were identified as older GII.4 variants, but a variety of other GIIs and GIs have been detected as well. While these sequences share at least 94% nt similarity with human strains, most of them are >98% identical to human strains. The fact that these strains were detected in animals after they had been detected through human surveillance to be already circulating in humans suggests human-to-animal transmission.

Infections involving antibiotic resistant Staphylococcus aureus (S. aureus) represent a major challenge to successful treatment. Further, although bacteriophages (phages) could be an alternative to antibiotics, there exists a lack of correlation in phage susceptibility results between conventional in vitro and in vivo assays. This discrepancy may hinder the potential implementation of bacteriophage therapy. In this study, the susceptibility of twelve S. aureus strains to three commercial phage cocktails and two single phages was assessed. These S. aureus strains (including ten clinical isolates, five of which were methicillin-resistant) were compared using four assays: the spot test, efficiency of plating (EOP), the optical density assay (all in culture media) and microcalorimetry in human serum. In the spot test, EOP and optical density assay, all cocktails and single phages lysed both methicillin susceptible and methicillin resistant S. aureus strains. However, there was an absence of phage-mediated lysis in high concentrations of human serum as measured using microcalorimetry. As this microcalorimetry-based assay more closely resembles in vivo conditions, we propose that microcalorimetry could be included as a useful addition to conventional assays, thereby facilitating more accurate predictions of the in vivo susceptibility of S. aureus to phages during phage selection for therapeutic purposes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a major global health problem, and public health surveillance is crucial to monitor and prevent virus spread. Wastewater-based epidemiology has been proposed as an addition to disease-based surveillance because virus is shed in the feces of ≈40% of infected persons. We used next-generation sequencing of sewage samples to evaluate the diversity of SARS-CoV-2 at the community level in the Netherlands and Belgium. Phylogenetic analysis revealed the presence of the most prevalent clades (19A, 20A, and 20B) and clustering of sewage samples with clinical samples from the same region. We distinguished multiple clades within a single sewage sample by using low-frequency variant analysis. In addition, several novel mutations in the SARS-CoV-2 genome were detected. Our results illustrate how wastewater can be used to investigate the diversity of SARS-CoV-2 viruses circulating in a community and identify new outbreaks.

A novel human coronavirus (HCoV-EMC/2012) was isolated from a man with acute pneumonia and renal failure in June 2012. This report describes the complete genome sequence, genome organization, and expression strategy of HCoV-EMC/2012 and its relation with known coronaviruses. The genome contains 30,119 nucleotides and contains at least 10 predicted open reading frames, 9 of which are predicted to be expressed from a nested set of seven subgenomic mRNAs. Phylogenetic analysis of the replicase gene of coronaviruses with completely sequenced genomes showed that HCoV-EMC/2012 is most closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4) and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), which prototype two species in lineage C of the genus Betacoronavirus. In accordance with the guidelines of the International Committee on Taxonomy of Viruses, and in view of the 75% and 77% amino acid sequence identity in 7 conserved replicase domains with BtCoV-HKU4 and BtCoV-HKU5, respectively, we propose that HCoV-EMC/2012 prototypes a novel species in the genus Betacoronavirus. HCoV-EMC/2012 may be most closely related to a coronavirus detected in Pipistrellus pipistrellus in The Netherlands, but because only a short sequence from the most conserved part of the RNA-dependent RNA polymerase-encoding region of the genome was reported for this bat virus, its genetic distance from HCoV-EMC remains uncertain. HCoV-EMC/2012 is the sixth coronavirus known to infect humans and the first human virus within betacoronavirus lineage C. Coronaviruses are capable of infecting humans and many animal species. Most infections caused by human coronaviruses are relatively mild. However, the outbreak of severe acute respiratory syndrome (SARS) caused by SARS-CoV in 2002 to 2003 and the fatal infection of a human by HCoV-EMC/2012 in 2012 show that coronaviruses are able to cause severe, sometimes fatal disease in humans. We have determined the complete genome of HCoV-EMC/2012 using an unbiased virus discovery approach involving next-generation sequencing techniques, which enabled subsequent state-of-the-art bioinformatics, phylogenetics, and taxonomic analyses. By establishing its complete genome sequence, HCoV-EMC/2012 was characterized as a new genotype which is closely related to bat coronaviruses that are distant from SARS-CoV. We expect that this information will be vital to rapid advancement of both clinical and vital research on this emerging pathogen.

Norovirus is the most common cause of non-bacterial gastroenteritis and is a burden worldwide. The increasing norovirus diversity is currently categorized into at least 10 genogroups which are further classified into more than 40 genotypes. In addition to humans, norovirus can infect a broad range of hosts including livestock, pets, and wild animals, e.g., marine mammals and bats. Little is known about norovirus infections in most non-human hosts, but the close genetic relatedness between some animal and human noroviruses coupled with lack of understanding where newly appearing human norovirus genotypes and variants are emerging from has led to the hypothesis that norovirus may not be host restricted and might be able to jump the species barrier. We have systematically reviewed the literature to describe the diversity, prevalence, and geographic distribution of noroviruses found in animals, and the pathology associated with infection. We further discuss the evidence that exists for or against interspecies transmission including surveillance data and data from in vitro and in vivo experiments.

Zoonotic influenza A viruses originating from the animal reservoir pose a threat for humans, as they have the ability to trigger pandemics upon adaptation to and invasion of an immunologically naive population. Of particular concern are the H5N1 viruses that continue to circulate in poultry in numerous countries in Europe, Asia and Africa, and the recently emerged H7N9 viruses in China, due to their relatively high number of human fatalities and pandemic potential. To start a pandemic, zoonotic influenza A viruses should not only acquire the ability to attach to, enter and replicate in the critical target cells in the respiratory tract of the new host, but also efficiently spread between humans by aerosol or respiratory droplet transmission. Here, we discuss the latest advances on the genetic and phenotypic determinants required for avian influenza A viruses to adapt to and transmit between mammals.

Sewage metagenomics has risen to prominence in urban population surveillance of pathogens and antimicrobial resistance (AMR). Unknown species with similarity to known genomes cause database bias in reference-based metagenomics. To improve surveillance, we seek to recover sewage genomes and develop a quantification and correlation workflow for these genomes and AMR over time. We use longitudinal sewage sampling in seven treatment plants from five major European cities to explore the utility of catch-all sequencing of these population-level samples. Using metagenomic assembly methods, we recover 2332 metagenome-assembled genomes (MAGs) from prokaryotic species, 1334 of which were previously undescribed. These genomes account for ~69% of sequenced DNA and provide insight into sewage microbial dynamics. Rotterdam (Netherlands) and Copenhagen (Denmark) show strong seasonal microbial community shifts, while Bologna, Rome, (Italy) and Budapest (Hungary) have occasional blooms of Pseudomonas -dominated communities, accounting for up to ~95% of sample DNA. Seasonal shifts and blooms present challenges for effective sewage surveillance. We find that bacteria of known shared origin, like human gut microbiota, form communities, suggesting the potential for source-attributing novel species and their ARGs through network community analysis. This could significantly improve AMR tracking in urban environments. In this study, the authors use metagenomics to analyze sewage samples from five European cities over time and report enhanced detection of rare taxa through combined assembly methods, significant seasonal and geographical microbiome variations, and the potential to attribute microbial sequences to specific sources.