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We screened 1,200 living heart, lung, liver, and kidney transplant recipients for hepatitis E virus infection by reverse transcription PCR. In 12 (1%) patients, hepatitis E virus infection was identified; in 11 patients, chronic infection developed. This immunocompromised population is at risk for hepatitis E virus infection. [ABSTRACT FROM AUTHOR]

A retrospective nationwide study on the use of intravenous (IV) zanamivir in patients receiving intensive care who were pretreated with oseltamivir in the Netherlands was performed. In 6 of 13 patients with a sustained reduction of the viral load, the median time to start IV zanamivir was 9 days (range, 4-11 days) compared with 14 days (range, 6-21 days) in 7 patients without viral load reduction (P = .052). Viral load response did not influence mortality. We conclude that IV zanamivir as late add-on therapy has limited effectiveness. The effect of an immediate start with IV zanamivir monotherapy or in combination with other drugs need to be evaluated. [ABSTRACT FROM AUTHOR]

On July 10, 2008, Marburg hemorrhagic fever was confirmed in a Dutch patient who had vacationed recently in Uganda. Exposure most likely occurred in the Python Cave (Maramagambo Forest), which harbors bat species that elsewhere in Africa have been found positive for Marburg virus. A multidisciplinary response team was convened to perform a structured risk assessment, perform risk classification of contacts, issue guidelines for follow-up, provide information, and monitor the crisis response. In total, 130 contacts were identified (66 classified as high risk and 64 as low risk) and monitored for 21 days after their last possible exposure. The case raised questions specific to international travel, postexposure prophylaxis for Marburg virus, and laboratory testing of contacts with fever. We present lessons learned and results of the follow-up serosurvey of contacts and focus on factors that prevented overreaction during an event with a high public health impact. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is ubiquitous in pigs worldwide and may be zoonotic. Previous HEV seroprevalence estimates for groups of people working with swine were higher than for control groups. However, discordance among results of anti-HEV assays means that true seroprevalence estimates, i.e. seroprevalence due to previous exposure to HEV, depends on choice of seroassay. We tested blood samples from three subpopulations (49 swine veterinarians, 153 non-swine veterinarians and 644 randomly selected individuals from the general population) with one IgM and two IgG ELISAs, and subsets with IgG and/or IgM Western blots. A Bayesian stochastical model was used to combine results of all assays. The model accounted for imperfection of each assay by estimating sensitivity and specificity, and accounted for dependence between serological assays. As expected, discordance among assay results occurred. Applying the model yielded seroprevalence estimates of approximately 11% for swine veterinarians,approximately 6% for non-swine veterinarians and approximately 2% for the general population. By combining the results of five serological assays in a Bayesian stochastical model we confirmed that exposure to swine or their environment was associated with elevated HEV seroprevalence. [ABSTRACT FROM AUTHOR]

In response to the mpox outbreak in 2022 and 2023, widespread vaccination with modified vaccinia Ankara-Bavarian Nordic (MVA-BN, also known as JYNNEOS or Imvanex) was initiated. Here, we demonstrate that orthopoxvirus-specific binding and MVA-neutralising antibodies waned to undetectable levels 1 year post vaccination in at-risk individuals who received two doses of MVA-BN administered subcutaneously with an interval of 4 weeks, without prior smallpox or mpox vaccination. Continuous surveillance is essential to understand the impact of declining antibody levels.

Competing Interests: AS is a consultant for AstraZeneca, Calyptus Pharmaceuticals, Darwin Health, EmerVax, EUROIMMUN, F Hoffman-La Roche, Fortress Biotech, Gilead Sciences, Granite Bio, Gritstone Oncology, Guggenheim Securities, Moderna, Pfizer, RiverVest Venture Partners, and Turnstone Biologics. AG is a consultant for Pfizer. La Jolla Institute for Immunology has filed for patent protection for various aspects of T-cell epitope and vaccine design work. All other authors declare no competing interests.

BackgroundFollowing the 2022-2023 mpox outbreak, crucial knowledge gaps exist regarding orthopoxvirus-specific immunity in risk groups and its impact on future outbreaks.AimWe combined cross-sectional seroprevalence studies in two cities in the Netherlands with mathematical modelling to evaluate scenarios of future mpox outbreaks among men who have sex with men (MSM).MethodsSerum samples were obtained from 1,065 MSM attending Centres for Sexual Health (CSH) in Rotterdam or Amsterdam following the peak of the Dutch mpox outbreak and the introduction of vaccination. For MSM visiting the Rotterdam CSH, sera were linked to epidemiological and vaccination data. An in-house developed ELISA was used to detect vaccinia virus (VACV)-specific IgG. These observations were combined with published data on serial interval and vaccine effectiveness to inform a stochastic transmission model that estimates the risk of future mpox outbreaks.ResultsThe seroprevalence of VACV-specific antibodies was 45.4% and 47.1% in Rotterdam and Amsterdam, respectively. Transmission modelling showed that the impact of risk group vaccination on the original outbreak was likely small. However, assuming different scenarios, the number of mpox cases in a future outbreak would be markedly reduced because of vaccination. Simultaneously, the current level of immunity alone may not prevent future outbreaks. Maintaining a short time-to-diagnosis is a key component of any strategy to prevent new outbreaks.ConclusionOur findings indicate a reduced likelihood of large future mpox outbreaks among MSM in the Netherlands under current conditions, but emphasise the importance of maintaining population immunity, diagnostic capacities and disease awareness.

The emergence of several bat coronavirus-related disease outbreaks in human and domestic animals has fueled surveillance of coronaviruses in bats worldwide. However, little is known about how these viruses interact with their natural hosts. We demonstrate a Betacoronavirus (subgenus Merbecovirus ), PN-βCoV, in the intestine of its natural host, Nathusius's Pipistrelle Bat ( Pipistrellus nathusii) , by combining molecular and microscopy techniques. Eighty-eight P. nathusii bat carcasses were tested for PN-βCoV RNA by RT-qPCR, of which 25 bats (28%) tested positive. PN-βCoV RNA was more often detected in samples of the intestinal tract than in other sample types. In addition, viral RNA loads were higher in intestinal samples compared to other sample types, both on average and in each individual bat. In one bat, we demonstrated Merbecovirus antigen and PN-βCoV RNA expression in intestinal epithelium and the underlying connective tissue using immunohistochemistry and in situ hybridization, respectively. These results indicate that PN-βCoV has a tropism for the intestinal epithelium of its natural host, Nathusius's Pipistrelle Bat, and imply that the fecal-oral route is a possible route of transmission. IMPORTANCE Virtually all mammal species circulate coronaviruses. Most of these viruses will infect one host species; however, coronaviruses are known to include species that can infect multiple hosts, for example the well-known virus that caused a pandemic, SARS-CoV-2. Chiroptera (bats) include over 1,400 different species, which are expected to harbor a great variety of coronaviruses. However, we know very little about how any of these coronaviruses interact with their bat hosts; for example, we do not know their modes of transmissions, or which cells they infect. Thus, we have a limited understanding of coronavirus infections in this important host group. The significance of our study is that we learned that a bat coronavirus that occurs in a common bat species in Europe has a tropism for the intestines. This implies the fecal-oral route is a likely transmission route.

Infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and vaccinations targeting the spike protein (S) offer protective immunity against coronavirus disease 2019 (COVID-19). This immunity may further be shaped by cross-reactivity with common cold coronaviruses. Mutations arising in S that are associated with altered intrinsic virus properties and immune escape result in the continued circulation of SARS-CoV-2 variants. Potentially, vaccine updates will be required to protect against future variants of concern, as for influenza. To offer potent protection against future variants, these second-generation vaccines may need to redirect immunity to epitopes associated with immune escape and not merely boost immunity toward conserved domains in preimmune individuals. For influenza, efficacy of repeated vaccination is hampered by original antigenic sin, an attribute of immune memory that leads to greater induction of antibodies specific to the first-encountered variant of an immunogen compared with subsequent variants. In this Review, recent findings on original antigenic sin are discussed in the context of SARS-CoV-2 evolution. Unanswered questions and future directions are highlighted, with an emphasis on the impact on disease outcome and vaccine design.

Background: High incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and low testing uptake were reported in low-income neighbourhoods in Rotterdam. We aimed to improve willingness and access to testing by introducing community-based test facilities, and to evaluate the effectiveness of a rapid antigen detection test (RDT)., Methods: Two to eleven test facilities operated consecutively in three low-income neighbourhoods in Rotterdam, offering the options of walk-in or appointments. Background characteristics were collected at intake and one nasopharyngeal swab was taken and processed using both RDT and reverse transcription polymerase chain reaction (RT-PCR). Visitors were asked to join a survey for evaluation purposes., Results: In total, 19 773 visitors were tested - 9662 (48.9%) without an appointment. Walk-in visitors were older, lived more often in the proximity of the test facilities, and reported coronavirus disease (COVID-19)-related symptoms less often than by-appointment visitors. For 67.7% of the visitors, this was the first time they got tested. A total of 1211 (6.1%) tested SARS-CoV-2-positive with RT-PCR, of whom 309 (25.5%) were asymptomatic. Test uptake increased among residents of the pilot neighbourhoods, especially in the older age groups, compared to people living in comparable neighbourhoods without community-based testing facilities. RDT detected asymptomatic individuals with 71.8% sensitivity, which was acceptable in this high prevalence setting. Visitors reported positive attitudes towards the test facilities and welcomed the easy access., Conclusions: Offering community-based SARS-CoV-2 testing seems a promising approach for increasing testing uptake among specific populations in low-income neighbourhoods., Competing Interests: Disclosure of interest: The authors completed the ICMJE Disclosure of Interest Form (available upon request from the corresponding author) and declare no relevant interests., (Copyright © 2022 by the Journal of Global Health. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Little is known about the interplay between preexisting immunity to endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed antibody reactivity to nucleocapsid and spike antigens and correlated this IgG response to SARS-CoV-2 neutralization. Patients with COVID-19 mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against a seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings indicate a boost of poorly protective CoV-specific antibodies in patients with COVID-19 that correlated with disease severity, revealing "original antigenic sin."

SARS-CoV-2 emerged in China as a zoonotic virus in December 2019. The virus proved to be human-to-human transmissible and its global spread resulted in the ongoing COVID-19 pandemic, associated with high morbidity and mortality. Vaccines were developed at an unprecedented speed and proved to be efficacious in preventing disease, but it remains to be determined if vaccines are able to interrupt transmission. Moreover, virus variants of concern continue to emerge that appear more transmissible and/or less sensitive to virus-specific immune responses. Here, we briefly review the role of animal models in assessing prophylactic and therapeutic options to interrupt SARS-CoV-2 transmission., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

The transmission of direct-acting antiviral resistance-associated substitutions (RAS) could hamper hepatitis C virus (HCV) cure rates and elimination efforts. A phylogenetic analysis of 87 men who have sex with men recently infected with HCV genotype 1a placed one-third (28/87) in a large cluster, in which 96% harbored NS5A M28V RAS., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

On 22 August, a common whitethroat in the Netherlands tested positive for West Nile virus lineage 2. The same bird had tested negative in spring. Subsequent testing of Culex mosquitoes collected in August and early September in the same location generated two of 44 positive mosquito pools, providing first evidence for enzootic transmission in the Netherlands. Sequences generated from the positive mosquito pools clustered with sequences that originate from Germany, Austria and the Czech Republic.

Background: The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur., Aim: We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available., Methods: Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology., Results: The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project., Conclusion: The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

In November 2018, yellow fever was diagnosed in a Dutch traveller returning from a bicycle tour in the Gambia-Senegal region. A complete genome sequence of yellow fever virus (YFV) from the case was generated and clustered phylogenetically with YFV from the Gambia and Senegal, ruling out importation into the Netherlands from recent outbreaks in Brazil or Angola. We emphasise the need for increased public awareness of YFV vaccination before travelling to endemic countries.

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.

Background: Genetic information of pathogens is an essential input for infectious disease control, public health and for research. Efficiency in preventing and responding to global outbreaks relies on timely access to such information. Still, ownership barriers stand in the way of timely sharing of genetic data from pathogens, frustrating efficient public health responses and ultimately the potential use of such resources in innovations. Under a One Health approach, stakeholders, their interests and ownership issues are manifold and need to be investigated. We interviewed key actors from governmental and non-governmental bodies to identify overlapping and conflicting interests, and the overall challenges for sharing pathogen data, to provide essential inputs to the further development of political and practical strategies for improved data sharing practices., Methods & Findings: To identify and prioritize barriers, 52 Key Opinion Leaders were interviewed. A root-cause analysis was performed to identify causal relations between barriers. Finally, barriers were mapped to the innovation cycle reflecting how they affect the range of surveillance, innovation, and sharing activities. Four main barrier categories were found: compliance to regulations, negative consequences, self-interest, and insufficient incentives for compliance. When grouped in sectors (research institutes, public health organizations, supra-national organizations and industry) stakeholders appear to have similar interests, more than when grouped in domains (human, veterinary and food). Considering the innovation process, most of barriers could be mapped to the initial stages of the innovation cycle as sampling and sequencing phases. These are stages of primary importance to outbreak control and public health response. A minority of barriers applied to later stages in the innovation cycle, which are of more importance to product development., Conclusion: Overall, barriers are complex and entangled, due to the diversity of causal factors and their crosscutting features. Therefore, barriers must be addressed in a comprehensive and integrated manner. Stakeholders have different interests highlighting the diversity in motivations for sharing pathogen data: prioritization of public health, basic research, economic welfare and/or innovative capacity. Broad inter-sectorial discussions should start with the alignment of these interests within sectors. The improved sharing of pathogen data, especially in upstream phases of the innovation process, will generate substantial public health benefits through increased availability of data to inform surveillance systems, as well as to allow the (re-)use of data for the development of medical countermeasures to control infectious diseases.

A seasonal reassortant A(H1N2) influenza virus harbouring genome segments from seasonal influenza viruses A(H1N1)pdm09 (HA and NS) and A(H3N2) (PB2, PB1, PA, NP, NA and M) was identified in March 2018 in a 19-months-old patient with influenza-like illness (ILI) who presented to a general practitioner participating in the routine sentinel surveillance of ILI in the Netherlands. The patient recovered fully. Further epidemiological and virological investigation did not reveal additional cases.

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) has heterologous beneficial effects against non-related infections. The basis of these effects has been poorly explored in humans. In a randomized placebo-controlled human challenge study, we found that BCG vaccination induced genome-wide epigenetic reprograming of monocytes and protected against experimental infection with an attenuated yellow fever virus vaccine strain. Epigenetic reprogramming was accompanied by functional changes indicative of trained immunity. Reduction of viremia was highly correlated with the upregulation of IL-1β, a heterologous cytokine associated with the induction of trained immunity, but not with the specific IFNγ response. The importance of IL-1β for the induction of trained immunity was validated through genetic, epigenetic, and immunological studies. In conclusion, BCG induces epigenetic reprogramming in human monocytes in vivo, followed by functional reprogramming and protection against non-related viral infections, with a key role for IL-1β as a mediator of trained immunity responses., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Together with influenza, the non-influenza RNA respiratory viruses (NIRVs), which include respiratory syncytial virus, parainfluenza viruses, coronavirus, rhinovirus, and human metapneumovirus, represent a considerable global health burden, as recognised by WHO's Battle against Respiratory Viruses initiative. By contrast with influenza viruses, little is known about the contemporaneous global diversity of these viruses, and the relevance of such for development of pharmaceutical interventions. Although far less advanced than for influenza, antiviral drugs and vaccines are in different stages of development for several of these viruses, but no interventions have been licensed. This scarcity of global genetic data represents a substantial knowledge gap and impediment to the eventual licensing of new antiviral drugs and vaccines for NIRVs. Enhanced genetic surveillance will assist and boost research and development into new antiviral drugs and vaccines for these viruses. Additionally, understanding the global diversity of respiratory viruses is also part of emerging disease preparedness, because non-human coronaviruses and paramyxoviruses have been listed as priority concerns in a recent WHO research and development blueprint initiative for emerging infectious diseases. In this Personal View, we explain further the rationale for expanding the genetic database of NIRVs and emphasise the need for greater investment in this area of research., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: Immunocompromised patients can suffer prolonged norovirus symptoms and virus shedding for many years. Little is known about the prevalence of chronic norovirus infection among solid organ transplant (SOT) recipients. In this study, 2182 SOT recipients were retrospectively tested for chronic norovirus infection., Methods: The first and last norovirus positive faecal samples of SOT recipients were sequenced to distinguish between persisting infection and re-infection. Patient charts were reviewed to obtain data on health status and treatments., Results: In all, 101 of 2182 (4.6%) recipients were norovirus infected and 23 (22.8%) of these developed chronic norovirus infection. Chronic norovirus infection was found among allogeneic heart, kidney and lung transplant recipients. The median shedding period at the end of the study period was 218 days (range 32-1164 days)., Conclusions: This study shows that chronic norovirus infection is not a rare phenomenon among SOT recipients in a tertiary-care hospital. Further research is needed to study the risk of norovirus transmission to other immunocompromised patients in the hospital and to the general population., (Copyright © 2016. Published by Elsevier Ltd.)

A Dutch traveller returning from Suriname in early March 2017, presented with fever and severe acute liver injury. Yellow fever was diagnosed by (q)RT-PCR and sequencing. During hospital stay, the patient's condition deteriorated and she developed hepatic encephalopathy requiring transfer to the intensive care. Although yellow fever has not been reported in the last four decades in Suriname, vaccination is recommended by the World Health Organization for visitors to this country., (This article is copyright of The Authors, 2017.)

Background: Patients treated with chemotherapy have an impaired response to influenza virus vaccination compared to healthy controls. Little is known about the broadness of the antibody response in these patients., Methods: Breast cancer patients on FEC (5-fluorouracil, epirubicin and cyclophosphamide) chemotherapy regimens were vaccinated with influenza virus vaccine. Sera were obtained before and three weeks after vaccination. In addition to the determination of virus-specific antibody titres by hemagglutination inhibition assay, the broadness of the response was assessed by the use of a protein microarray and baseline titres were compared with an age-matched reference group., Results: We included 38 breast cancer patients and found a wide variety in serum antibody response after vaccination. Patients with a history of influenza vaccination had higher pre-vaccination titres, which were comparable to the reference group. Increasing number of cycles of chemotherapy did not have a negative effect on influenza array antibody levels, nor on the HI antibody response., Conclusions: Overall there was a broad serum antibody response to the influenza virus vaccine in patients treated with chemotherapy for breast cancer., (Copyright © 2017. Published by Elsevier Ltd.)

A plethora of viruses can be transmitted by the food- and waterborne route. However, their recognition is challenging because of the variety of viruses, heterogeneity of symptoms, the lack of awareness of clinicians, and limited surveillance efforts. Classical food- and waterborne viral disease outbreaks are mainly caused by caliciviruses, but the source of the virus is often not known and the foodborne mode of transmission is difficult to discriminate from human-to-human transmission. Atypical food- and waterborne viral disease can be caused by viruses such as hepatitis A and hepatitis E. In addition, a source of novel emerging viruses with a potential to spread via the food- and waterborne route is the repeated interaction of humans with wildlife. Wildlife-to-human adaptation may give rise to self- limiting outbreaks in some cases, but when fully adjusted to the human host can be devastating. Metagenomic sequencing has been investigated as a promising solution for surveillance purposes as it detects all viruses in a single protocol, delivers additional genomic information for outbreak tracing, and detects novel unknown viruses. Nevertheless, several issues must be addressed to apply metagenomic sequencing in surveillance. First, sample preparation is difficult since the genomic material of viruses is generally overshadowed by host- and bacterial genomes. Second, several data analysis issues hamper the efficient, robust, and automated processing of metagenomic data. Third, interpretation of metagenomic data is hard, because of the lack of general knowledge of the virome in the food chain and the environment. Further developments in virus-specific nucleic acid extraction methods, bioinformatic data processing applications, and unifying data visualization tools are needed to gain insightful surveillance knowledge from suspect food samples.

Human norovirus is a leading cause of gastroenteritis and is efficiently transmitted between humans and around the globe. The burden of norovirus infections in the global community and in health-care settings warrant the availability of outbreak prevention strategies and control measures that are tailored to the pathogen, outbreak setting and population at risk. A better understanding of viral and host determinants of transmission would aid in developing and fine-tuning such efforts. Here, we describe mechanisms of transmission, available model systems for studying norovirus transmission and their strengths and weaknesses as well as future research strategies., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) is genetically highly divergent and classified in ten genotypes and forty subgenotypes in distinct ethno-geographic populations worldwide. Ethiopia is a country with high HBV prevalence; however, little is known about the genetic variability of HBV strains that circulate. Here, we characterize the complete genome of 29 HBV strains originating from five Ethiopian regions, by 454 deep sequencing and Sanger sequencing. Phylogenetically, ten strains were classified as genotype A1 and nineteen as genotype D. Fifteen genotype D strains, provisionally named subgenotype D10, showed a novel distinct cluster supported by high bootstrap value and >4% nucleotide divergence from other known subgenotypes. In addition, the novel D10 strains harboured nine unique amino acid signatures in the surface, polymerase and X genes. Seventy-two per cent of the genotype D strains had the precore premature stop codon G1896A. In addition, 63% genotype A and 33% genotype D strains had the basal core promoter mutations, A1762T/G1764A. Furthermore, four pre-S deletion variants and two recombinants were identified in this study. In conclusion, we identified a novel HBV subgenotype D10 circulating in Ethiopia, underlining the high genetic variability of HBV strains in Africa., (© 2016 John Wiley & Sons Ltd.)

In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R 0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak., (This article is copyright of The Authors, 2017.)

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A norovirus was detected in harbor porpoises, a previously unknown host for norovirus. This norovirus had low similarity to any known norovirus. Viral RNA was detected primarily in intestinal tissue, and specific serum antibodies were detected in 8 (24%) of 34 harbor porpoises from the North Sea.

Background: Dengue virus (DENV) infection is widespread and its disease burden has increased in past decades. However, little is known about the epidemiology of dengue in the Middle East and North Africa (MENA)., Methodology / Principal Findings: Following Cochrane Collaboration guidelines and reporting our findings following PRISMA guidelines, we systematically reviewed available records across MENA describing dengue occurrence in humans (prevalence studies, incidence studies, and outbreak reports), occurrence of suitable vectors (Aedes aegypti and Aedes albopictus), and DENV vector infection rates. We identified 105 human prevalence measures in 13 of 24 MENA countries; 81 outbreaks reported from 9 countries from 1941-2015; and reports of Ae. aegypti and/or Ae. albopictus occurrence in 15 countries. The majority of seroprevalence studies were reported from the Red Sea region and Pakistan, with multiple studies indicating >20% DENV seroprevalence in general populations (median 25%, range 0-62%) in these subregions. Fifty percent of these studies were conducted prior to 1990. Multiple studies utilized assays susceptible to serologic cross-reactions and 5% of seroprevalence studies utilized viral neutralization testing. There was considerable heterogeneity in study design and outbreak reporting, as well as variability in subregional study coverage, study populations, and laboratory methods used for diagnosis., Conclusions / Significance: DENV seroprevalence in the MENA is high among some populations in the Red Sea region and Pakistan, while recent outbreaks in these subregions suggest increasing incidence of DENV which may be driven by a variety of ecologic and social factors. However, there is insufficient study coverage to draw conclusions about Aedes or DENV presence in multiple MENA countries. These findings illustrate the epidemiology of DENV in the MENA while revealing priorities for DENV surveillance and Aedes control., Competing Interests: The authors have declared that no competing interests exist.

The recent mumps outbreaks among MMR vaccinated persons have raised questions about the biological mechanisms related to mumps symptoms and complications in the background of waning immunity. Contrary to other paramyxoviruses, the understanding of mumps virus pathogenesis is limited, and further in-depth clinical studies are required to provide answers to important research questions.

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus, but the exact modes of transmission of MERS-CoV to humans remain unclear. In February 2014, an assessment was made of the suitability and sensitivity of different types of sample for the detection of MERSCoV by real-time reverse-transcription polymerase chain reaction (RT-PCR) for three gene targets: UpE (upstream of the E gene), the N (nucleocapsid) gene and open reading frame (ORF) 1a. Fifty-three animals presented for slaughter were sampled. A high percentage of the sampled camels (79% [95% confidence interval 66.9-91.5%, standard error 0.0625]; 42 out of 53) were shown to be shedding MERS-CoV at the time of slaughter, yet all the animals were apparently healthy. Among the virus-positive animals, nasal swabs were most often positive (97.6%). Oral swabs were the second most frequently positive (35.7%), followed by rectal swabs (28.5%). In addition, the highest viral load, expressed as a cycle threshold (Ct) value of 11.27, was obtained from a nasal swab. These findings lead to the conclusion that nasal swabs are the candidate sample of choice for detecting MERS-CoV using RT-PCR technology in apparently healthy camels., (© OIE (World Organisation for Animal Health), 2016.)

Background: Clinical signs and symptoms of different airway pathogens are generally indistinguishable, making laboratory tests essential for clinical decisions regarding isolation and antiviral therapy. Immunochromatographic tests (ICT) and direct immunofluorescence assays (DFA) have lower sensitivities and specificities than molecular assays, but have the advantage of quick turnaround times and ease-of-use., Objective: To evaluate the performance of a rapid molecular assay, ARIES FluA/B & RSV, using laboratory developed RT-PCR assays (LDA), ICT (BinaxNOW) and DFA., Methods: Analytical and clinical performance were evaluated in a retrospective study arm (stored respiratory samples obtained between 2006-2015) and a prospective study arm (unselected fresh clinical samples obtained between December 2015 and March 2016 tested in parallel with LDAs)., Results: Genotype inclusivity and analytical specificity was 100%. However, ARIES was 0.5 log, 1-2logs and 2.5logs less sensitive for fluA, RSV and fluB respectively, compared to LDA. In total, 447 clinical samples were included, of which 15.4% tested positive for fluA, 9.2% for fluB and 26.0% for RSV, in both LDA and ARIES. ARIES clinical sensitivity compared to LDA was 98.6% (fluA), 93.3% (fluB) and 95.1% (RSV). Clinical specificity was 100% for all targets. ARIES detected 10.6% (4 fluA, 8 fluB, 11 RSV) and 26.9% (7 fluA, 3 fluB, 22 RSV) more samples compared to DFA and ICT, all confirmed by LDA., Conclusion: Although analytically ARIES is less sensitive than LDA, the clinical performance of the assay in our tertiary care setting was comparable, and significantly better than that of the established rapid assays., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In October 2016, a severe infection with swine influenza A(H1N1) virus of the Eurasian avian lineage occurred in a child with a previous history of eczema in the Netherlands, following contact to pigs. The patient's condition deteriorated rapidly and required life support through extracorporeal membrane oxygenation. After start of oseltamivir treatment and removal of mucus plugs, the patient fully recovered. Monitoring of more than 80 close unprotected contacts revealed no secondary cases., (This article is copyright of The Authors, 2016.)

Purpose: We report 18 cases of confirmed Zika virus (ZIKV) infection in travellers returning to the Netherlands from Surinam (South America, bordering northern Brazil) and the Dominican Republic., Methods: In a multi-centre study, we collected epidemiological, virological and clinical characteristics, as well as data on travel history, underlying illness and laboratory results of the 18 imported ZIKV infection cases using a standardised form., Results: Most cases had a self-limiting course of disease, two patients developed complications, one had Guillain-Barré and another had severe thrombocytopenia. Four patients had underlying illness. One of the reported cases was pregnant. Three of 13 patients tested had a weak-positive result for dengue IgM. The majority of patients were born in Suriname and/or visiting friends and relatives (VFR)., Conclusions: Providing pre-travel advice among travellers, especially VFR travellers, is needed to enhance the use of preventive measures against ZIKV infection. Further evidence on health risks associated with ZIKV infection is urgently needed., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interest. The authors declare that the final manuscript has not been published before and the work is not under consideration for publication elsewhere.

Objective: To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection., Methods: We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays' target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays., Findings: Oligonucleotides of the published real-time RT-PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 10 4 RNA copies/mL of blood and 2 × 10 4 RNA copies/mL of urine., Conclusion: We provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.

VIRO-TypeNed is a collaborative molecular surveillance platform facilitated through a web-based database. Genetic data in combination with epidemiological, clinical and patient data are shared between clinical and public health laboratories, as part of the surveillance underpinning poliovirus eradication. We analysed the combination of data submitted from 2010 to 2014 to understand circulation patterns of non-polio enteroviruses (NPEV) of public health relevance. Two epidemiological patterns were observed based on VIRO-TypeNed data and classical surveillance data dating back to 1996: (i) endemic cyclic, characterised by predictable upsurges/outbreaks every two to four years, and (ii) epidemic, where rare virus types caused upsurges/outbreaks. Genetic analysis suggests continuous temporal displacement of virus lineages due to the accumulation of (silent) genetic changes. Non-synonymous changes in the antigenic B/C loop suggest antigenic diversification, which may affect population susceptibility. Infections were frequently detected at an age under three months and at an older, parenting age (25-49 years) pointing to a distinct role of immunity in the circulation patterns. Upsurges were detected in the summer and winter which can promote increased transmissibility underlying new (cyclic) upsurges and requires close monitoring. The combination of data provide a better understanding of NPEV circulation required to control and curtail upsurges and outbreaks., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of The Authors, 2016.)

Pre-existing immunity played a significant role in protection during the latest influenza A virus H1N1 pandemic, especially in older age groups. Structural similarities were found between A(H1N1)2009 and older H1N1 virus strains to which humans had already been exposed. Broadly cross-reactive antibodies capable of neutralizing the A(H1N1)2009 virus have been implicated in this immune protection in adults. We investigated the serological profile of a group of young children aged 9 years (n=55), from whom paired blood samples were available, just prior to the pandemic wave (March 2009) and shortly thereafter (March 2010). On the basis of A(H1N1)2009 seroconversion, 27 of the 55 children (49 %) were confirmed to be infected between these two time points. Within the non-infected group of 28 children (51 %), high levels of seasonal antibodies to H1 and H3 HA1 antigens were detected prior to pandemic exposure, reflecting past infection with H1N1 and H3N2, both of which had circulated in The Netherlands prior to the pandemic. In some children, this reactivity coincided with specific antibody reactivity against A(H1N1)2009. While these antibodies were not able to neutralize the A(H1N1)2009 virus, they were able to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro upon interaction with the A(H1N1)2009 virus. This finding suggests that cross-reactive antibodies could contribute to immune protection in children via ADCC.

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients. Immunosuppressants used to prevent alloreactivity can also interfere with virus infection, but the direct effects of the specific type of immunosuppressants on rotavirus infection are still unclear. Here we profiled the effects of different immunosuppressants on rotavirus using a 2D culture model of Caco2 human intestinal cell line and a 3D model of human primary intestinal organoids inoculated with laboratory and patient-derived rotavirus strains. We found that the responsiveness of rotavirus to Cyclosporine A treatment was moderate and strictly regulated in an opposite direction by its cellular targets cyclophilin A and B. Treatment with mycophenolic acid (MPA) resulted in a 99% inhibition of viral RNA production at the clinically relevant concentration (10 μg/ml) in Caco2 cells. This effect was further confirmed in organoids. Importantly, continuous treatment with MPA for 30 passages did not attenuate its antiviral potency, indicating a high barrier to drug resistance development. Mechanistically, the antiviral effects of MPA act via inhibiting the IMPDH enzyme and resulting in guanosine nucleotide depletion. Thus for transplantation patients at risk for rotavirus infection, the choice of MPA as an immunosuppressive agent appears rational., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Influenza A viruses can replicate in the olfactory mucosa and subsequently use the olfactory nerve to enter the central nervous system (CNS). It is currently unknown whether intervention strategies are able to reduce or prevent influenza virus replication within the olfactory mucosa and subsequent spread to the CNS. Therefore, we tested the efficacy of homologous vaccination and prophylactic oseltamivir to prevent H5N1 virus CNS invasion via the olfactory nerve in our ferret model., Methods: Ferrets were vaccinated intramuscularly or received oseltamivir (5 mg/kg twice daily) prophylactically before intranasal inoculation of highly pathogenic H5N1 virus (A/Indonesia/05/2005) and were examined using virology and pathology., Results: Homologous vaccination reduced H5N1 virus replication in the olfactory mucosa and prevented subsequent virus spread to the CNS. However, prophylactic oseltamivir did not prevent H5N1 virus replication in the olfactory mucosa sufficiently, resulting in CNS invasion via the olfactory nerve causing a severe meningoencephalitis., Conclusions: Within our ferret model, vaccination is more effective than prophylactic oseltamivir in preventing CNS invasion by H5N1 virus via the olfactory nerve. This study highlights the importance of including the olfactory mucosa, olfactory nerve, and CNS tissues in future vaccine and antiviral studies, especially for viruses with a known neurotropic potential., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

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.