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West Nile virus (WNV) was first detected in the Netherlands in 2020, with circulation observed in birds, mosquitoes, and humans in two geographical areas. Usutu virus (USUV) has been circulating in the Netherlands since 2016. Following the detection of WNV in the Netherlands, we investigated the possible use of petting zoos as urban sentinel sites to examine the extent of WNV and USUV circulation around the two WNV outbreak locations. Chickens at petting zoos and in backyards were sampled within a 15-kilometer radius of the confirmed WNV circulation areas at three timepoints over one year (2021-2022). Sera were analysed using a protein microarray for binding antibodies to orthoflavivirus NS1 antigens and reactive samples were confirmed through micro-focus reduction neutralization tests (mFRNT). Furthermore, mosquitoes at sampling locations were collected to assess their blood feeding behaviour. This serosurvey detected the circulation of USUV and WNV in petting zoo and backyard chickens in 2021, both within and outside the 2020 outbreak areas. The WNV circulation was not detected by other existing surveillance schemes in mosquitoes, wild birds, horses and humans. In addition, the results show rapid decay of USUV antibodies in approximately 20 weeks. Our findings support the utility and the added value of petting zoo chickens as sentinels for monitoring USUV and WNV circulation compared to other available methods. Seroconversions observed in petting zoos and backyard chickens living in or near densely populated urban areas further highlighted potential public health risks that went undetected.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bakker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic B-cell receptor signals incited by cognate antigens are believed to play a crucial role in the pathogenesis of mucosa-associated lymphoid tissue lymphomas. We have explored the immunoglobulin variable regions (IGHV) expressed by 124 ocular adnexal MALT lymphomas (OAML) and tested the in vitro reactivity of recombinant IgM derived from 23 OAMLs. Six of 124 OAMLs (5%) were found to express a high-affinity stereotyped rheumatoid factor. OAMLs have a biased IGHV4-34 usage, which confers intrinsic super auto-antigen reactivity with poly-N-acetyllactosamine (NAL) epitopes, present on cell surface glycoproteins of erythrocytes and B cells. Twenty-one OAMLs (17%) expressed IGHV4-34-encoded B-cell receptors. Five of the 23 recombinant OAML IgMs expressed IGHV4-34, four of which bound to the linear NAL i epitope expressed on B cells but not to the branched NAL I epitope on erythrocytes. One non-IGHV4-34-encoded OAML IgM was also reactive with B cells. Interestingly, three of the 23 OAML IgMs (13%) specifically reacted with proteins of U1-/U-snRNP complexes, which have been implicated as cognate-antigens in various autoimmune diseases such as systemic lupus erythematosus and mixed connective tissue disease. The findings indicate that local autoimmune reactions are instrumental in the pathogenesis of a substantial fraction of OAMLs., (© 2024 Bende et al.)

Background: Recently, Europe has seen an emergence of mosquito-borne viruses (MBVs). Understanding citizens' perceptions of and behaviours towards mosquitoes and MBVs is crucial to reduce disease risk. We investigated and compared perceptions, knowledge, and determinants of citizens' behavioural intentions related to mosquitoes and MBVs in the Netherlands and Spain, to help improve public health interventions., Methods: Using the validated MosquitoWise survey, data was collected through participant panels in Spain (N = 475) and the Netherlands (N = 438). Health Belief Model scores measuring behavioural intent, knowledge, and information scores were calculated. Confidence Interval-Based Estimation of Relevance was used, together with potential for change indexes, to identify promising determinants for improving prevention measure use., Results: Spanish participants' responses showed slightly higher intent to use prevention measures compared to those of Dutch participants (29.1 and 28.2, respectively, p 0.03). Most participants in Spain (92.2%) and the Netherlands (91.8%) indicated they used at least one prevention measure, but differences were observed in which types they used. More Spanish participants indicated to have received information on mosquitoes and MBVs compared to Dutch participants. Spanish participants preferred health professional information sources, while Dutch participants favoured government websites. Determinants for intent to use prevention measures included "Knowledge", "Reminders to Use Prevention Measures", and "Information" in the Netherlands and Spain. Determinants for repellent use included "Perceived Benefits" and "Cues to Action", with "Perceived Benefits" having a high potential for behavioural change in both countries. "Self-Efficacy" and "Knowledge" were determinants in both countries for breeding site removal., Conclusion: This study found differences in knowledge between the Netherlands and Spain but similarities in determinants for intent to use prevention measures, intent to use repellents and intent to remove mosquito breeding sites. Identified determinants can be the focus for future public health interventions to reduce MBV risks., (© 2024. The Author(s).)

SARS-CoV-2 transmission in indoor spaces, where most infection events occur, depends on the types and duration of human interactions, among others. Understanding how these human behaviours interface with virus characteristics to drive pathogen transmission and dictate the outcomes of non-pharmaceutical interventions is important for the informed and safe use of indoor spaces. To better understand these complex interactions, we developed the Pedestrian Dynamics-Virus Spread model (PeDViS), an individual-based model that combines pedestrian behaviour models with virus spread models incorporating direct and indirect transmission routes. We explored the relationships between virus exposure and the duration, distance, respiratory behaviour, and environment in which interactions between infected and uninfected individuals took place and compared this to benchmark 'at risk' interactions (1.5 metres for 15 minutes). When considering aerosol transmission, individuals adhering to distancing measures may be at risk due to the buildup of airborne virus in the environment when infected individuals spend prolonged time indoors. In our restaurant case, guests seated at tables near infected individuals were at limited risk of infection but could, particularly in poorly ventilated places, experience risks that surpass that of benchmark interactions. Combining interventions that target different transmission routes can aid in accumulating impact, for instance by combining ventilation with face masks. The impact of such combined interventions depends on the relative importance of transmission routes, which is hard to disentangle and highly context dependent. This uncertainty should be considered when assessing transmission risks upon different types of human interactions in indoor spaces. We illustrated the multi-dimensionality of indoor SARS-CoV-2 transmission that emerges from the interplay of human behaviour and the spread of respiratory viruses. A modelling strategy that incorporates this in risk assessments can help inform policy makers and citizens on the safe use of indoor spaces with varying inter-human interactions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Atamer Balkan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Mosquito-borne diseases are on the rise. While climatic factors have been linked to disease occurrences, they do not explain the non-random spatial distribution in disease outbreaks. Landscape-related factors, such as vegetation structure, likely play a crucial but hitherto unquantified role., Methods: We explored how three critically important factors that are associated with mosquito-borne disease outbreaks: microclimate, mosquito abundance and bird communities, vary at the landscape scale. We compared the co-occurrence of these three factors in two contrasting habitat types (forest versus grassland) across five rural locations in the central part of the Netherlands between June and September 2021., Results: Our results show that forest patches provide a more sheltered microclimate, and a higher overall abundance of birds. When accounting for differences in landscape characteristics, we also observed that the number of mosquitoes was higher in isolated forest patches., Conclusions: Our findings indicate that, at the landscape scale, variation in tree cover coincides with suitable microclimate and high Culex pipiens and bird abundance. Overall, these factors can help understand the non-random spatial distribution of mosquito-borne disease outbreaks., (© 2024. The Author(s).)

Background: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities., Methods: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time., Findings: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways., Conclusion: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Due to climate change and the expanding geographical ranges of key mosquito species, several mosquito-borne viruses (MBVs) have recently emerged in Europe. Understanding people's perceptions and behaviours towards these viruses and the mosquitoes capable of transmitting them is crucial for implementing effective prevention measures and targeted communication campaigns. However, there is currently no appropriate validated survey for European populations to assess this. This study developed and validated a standardized survey, based on the Health Belief Model (HBM), to assess perceptions of mosquitoes and MBVs among Europe's residents. The survey was distributed online to United Kingdom (UK), Dutch and Spanish participants through panel providers. Survey validity and reliability were tested using confirmatory factor analysis (CFA) and Cronbach's alpha. The optimised survey was completed by 336 UK, 438 Dutch and 475 Spanish residents, respectively, and the HBM items passed our validity and reliability testing in all three countries. The final survey has 57 questions, including 19 validated HBM items, and questions to assess demographic characteristics, knowledge, prevention measures and behavioural determinants. Our MosquitoWise survey bridges researchers' understandings of European residents' perceptions and knowledge as a first step to improve preventive behaviour towards mosquitoes and MBVs and guide prevention and communication initiatives., (© 2024. The Author(s).)

Arboviruses such as West Nile Virus (WNV) and Usutu Virus (USUV) are emerging pathogens that circulate between mosquitoes and birds, occasionally spilling over into humans and horses. Current serological screening methods require access to a well-equipped laboratory and are not currently available for on-site analysis. As a proof of concept, we propose here a species-independent lateral flow microarray immunoassay (LMIA) able to quickly detect and distinguish between WNV Non-Structural 1 (NS1) and USUV NS1-specific antibodies. A double antigen approach was used to test sera collected from humans, horses, European jackdaws ( Corvus monedula) , and common blackbirds ( Turdus merula) . Optimization of the concentration of capture antigen spotted on the LMIA membrane and the amount of detection antigen conjugated to detector particles indicated that maximizing both parameters increased assay sensitivity. Upon screening of a larger serum panel, the optimized LMIA showed significantly higher spot intensity for a homologous binding event. Using a Receiver Operating Characteristics (ROC) curve, WNV NS1 LMIA results in humans, horses, and C. monedula showed good correlation when compared to "gold standard" WNV FRNT90. The most optimal derived sensitivity and specificity of the WNV NS1 LMIA relative to corresponding WNV FRNT90-confirmed sera were determined to be 96% and 86%, respectively. While further optimization is required, this study demonstrates the feasibility of developing a species-independent LMIA for on-site analysis of WNV, USUV, and other arboviruses. Such a tool would be useful for the on-site screening and monitoring of relevant species in more remote or low-income regions., Competing Interests: None to declare., (© 2024 The Authors. Published by Elsevier B.V.)

In October 2020, a new lineage of a clade 2.3.4.4b HPAI virus of the H5 subtype emerged in Europe, resulting in the largest global outbreak of HPAI to date, with unprecedented mortality in wild birds and poultry. The virus appears to have become enzootic in birds, continuously yielding novel HPAI virus variants. The recently increased abundance of infected birds worldwide increases the probability of bird-mammal contact, particularly in wild carnivores. Here, we performed molecular and serological screening of over 500 dead wild carnivores and sequencing of RNA positive materials. We show virological evidence for HPAI H5 virus infection in 0.8%, 1.4%, and 9.9% of animals tested in 2020, 2021, and 2022 respectively, with the highest proportion of positives in foxes, polecats and stone martens. We obtained near full genomes of 7 viruses and detected PB2 amino acid substitutions known to play a role in mammalian adaptation in three sequences. Infections were also found in without neurological signs or mortality. Serological evidence for infection was detected in 20% of the study population. These findings suggests that a high proportion of wild carnivores is infected but undetected in current surveillance programmes. We recommend increased surveillance in susceptible mammals, irrespective of neurological signs or encephalitis.

Diagnosis of arbovirus infection or exposure by antibody testing is becoming increasingly difficult due to global expansion of arboviruses, which induce antibodies that may (cross-)react in serological assays. We provide a systematic review of the current knowledge and knowledge gaps in differential arbovirus serology. The search included Medline, Embase and Web of Science databases and identified 911 publications which were reduced to 102 after exclusion of studies not providing data on possible cross-reactivity or studies that did not meet the inclusion criteria regarding confirmation of virus exposure of reference population sets. Using a scoring system to further assess quality of studies, we show that the majority of the selected papers (N = 102) provides insufficient detail to support conclusions on specificity of serological outcomes with regards to elucidating antibody cross-reactivity. Along with the lack of standardization of assays, metadata such as time of illness onset, vaccination, infection and travel history, age and specificity of serological methods were most frequently missing. Given the critical role of serology for diagnosis and surveillance of arbovirus infections, better standards for reporting, as well as the development of more (standardized) specific serological assays that allow discrimination between exposures to multiple different arboviruses, are a large global unmet need., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kasbergen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Climate change is one of several drivers of recurrent outbreaks and geographical range expansion of infectious diseases in Europe. We propose a framework for the co-production of policy-relevant indicators and decision-support tools that track past, present, and future climate-induced disease risks across hazard, exposure, and vulnerability domains at the animal, human, and environmental interface. This entails the co-development of early warning and response systems and tools to assess the costs and benefits of climate change adaptation and mitigation measures across sectors, to increase health system resilience at regional and local levels and reveal novel policy entry points and opportunities. Our approach involves multi-level engagement, innovative methodologies, and novel data streams. We take advantage of intelligence generated locally and empirically to quantify effects in areas experiencing rapid urban transformation and heterogeneous climate-induced disease threats. Our goal is to reduce the knowledge-to-action gap by developing an integrated One Health-Climate Risk framework., Competing Interests: All authors declare no competing interests., (© 2023 The Author(s).)

In November 2021, seven western lowland gorillas and four Asiatic lions were diagnosed with COVID-19 at Rotterdam Zoo. An outbreak investigation was undertaken to determine the source and extent of the outbreak and to identify possible transmission routes. Interviews were conducted with staff to identify human and animal contacts and cases, compliance with personal protective equipment (PPE) and potential transmission routes. Human and animal contacts and other animal species suspected to be susceptible to SARS-CoV-2 were tested for SARS-CoV-2 RNA. Positive samples were subjected to sequencing. All the gorillas and lions that could be tested (3/7 and 2/4, respectively) were RT-PCR positive between 12 November and 10 December 2021. No other animal species were SARS-CoV-2 RNA positive. Forty direct and indirect human contacts were identified. Two direct contacts tested RT-PCR positive 10 days after the first COVID-19 symptoms in animals. The zookeepers' viral genome sequences clustered with those of gorillas and lions. Personal protective equipment compliance was suboptimal at instances. Findings confirm transmission of SARS-CoV-2 among animals and between humans and animals but source and directionality could not be established. Zookeepers were the most likely source and should have periodic PPE training. Sick animals should promptly be tested and isolated/quarantined.

Monitoring of SARS-CoV-2 in wastewater (WW) is a promising tool for epidemiological surveillance, correlating not only viral RNA levels with the infection dynamics within the population, but also to viral diversity. However, the complex mixture of viral lineages in WW samples makes tracking of specific variants or lineages circulating in the population a challenging task. We sequenced sewage samples of 9 WW-catchment areas within the city of Rotterdam, used specific signature mutations from individual SARS-CoV-2 lineages to estimate their relative abundances in WW and compared them against those observed in clinical genomic surveillance of infected individuals between September 2020 and December 2021. We showed that especially for dominant lineages, the median of the frequencies of signature mutations coincides with the occurrence of those lineages in Rotterdam's clinical genomic surveillance. This, along with digital droplet RT-PCR targeting signature mutations of specific variants of concern (VOCs), showed that several VOCs emerged, became dominant and were replaced by the next VOC in Rotterdam at different time points during the study. In addition, single nucleotide variant (SNV) analysis provided evidence that spatio-temporal clusters can also be discerned from WW samples. We were able to detect specific SNVs in sewage, including one resulting in the Q183H amino acid change in the Spike gene, that was not captured by clinical genomic surveillance. Our results highlight the potential use of WW samples for genomic surveillance, increasing the set of epidemiological tools to monitor SARS-CoV-2 diversity., Competing Interests: Declaration of competing interest All the authors declare no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2023. Published by Elsevier B.V.)

Background: Healthcare facilities have been challenged by the risk of SARS-CoV-2 transmission between healthcare workers (HCW) and patients. During the first wave of the COVID-19 pandemic, infections among HCW were observed, questioning infection prevention and control (IPC) measures implemented at that time., Aim: This study aimed to identify nosocomial transmission routes of SARS-CoV-2 between HCW and patients in a tertiary care hospital., Methods: All SARS-CoV-2 PCR positive HCW and patients identified between 1 March and 19 May 2020, were included in the analysis. Epidemiological data were collected from patient files and HCW contact tracing interviews. Whole genome sequences of SARS-CoV-2 were generated using Nanopore sequencing (WGS). Epidemiological clusters were identified, whereafter WGS and epidemiological data were combined for re-evaluation of epidemiological clusters and identification of potential transmission clusters. HCW infections were further classified into categories based on the likelihood that the infection was acquired via nosocomial transmission. Secondary cases were defined as COVID-19 cases in our hospital, part of a transmission cluster, of which the index case was either a patient or HCW from our hospital., Findings: The study population consisted of 293 HCW and 245 patients. Epidemiological data revealed 36 potential epidemiological clusters, with an estimated 222 (75.7%) HCW as secondary cases. WGS results were available for 195 HCW (88.2%) and 20 patients (12.8%) who belonged to an epidemiological cluster. Re-evaluation of the epidemiological clusters, with the available WGS data identified 31 transmission clusters with 65 (29.4%) HCW as secondary cases. Transmission clusters were all part of 18 (50.0%) previously determined epidemiological clusters, demonstrating that several larger outbreaks actually consisted, of several smaller transmission clusters. A total of 21 (7.2%) HCW infections were classified as from confirmed nosocomial, of which 18 were acquired from another HCW and 3 from a patient., Conclusion: The majority of SARS-CoV-2 infections among HCW could be attributed to community-acquired infection. Infections among HCW that could be classified as due to nosocomial transmission, were mainly caused by HCW-to-HCW transmission rather than patient-to-HCW transmission. It is important to recognize the uncertainties of cluster analyses based solely on epidemiological data., (© 2023. The Author(s).)

Drivers are factors that have the potential to directly or indirectly influence the likelihood of infectious diseases emerging or re-emerging. It is likely that an emerging infectious disease (EID) rarely occurs as the result of only one driver; rather, a network of sub-drivers (factors that can influence a driver) are likely to provide conditions that allow a pathogen to (re-)emerge and become established. Data on sub-drivers have therefore been used by modellers to identify hotspots where EIDs may next occur, or to estimate which sub-drivers have the greatest influence on the likelihood of their occurrence. To minimise error and bias when modelling how sub-drivers interact, and thus aid in predicting the likelihood of infectious disease emergence, researchers need good-quality data to describe these sub-drivers. This study assesses the quality of the available data on sub-drivers of West Nile virus against various criteria as a case study. The data were found to be of varying quality with regard to fulfilling the criteria. The characteristic with the lowest score was completeness, i.e. where sufficient data are available to fulfil all the requirements for the model. This is an important characteristic as an incomplete data set could lead to erroneous conclusions being drawn from modelling studies. Thus, the availability of good-quality data is essential to reduce uncertainty when estimating the likelihood of where EID outbreaks may occur and identifying the points on the risk pathway where preventive measures may be taken.

Introduction: In 2020, the first Dutch West Nile virus (WNV) infected birds were detected through risk-targeted surveillance of songbirds. Retrospective testing of patients with unexplained neurological disease revealed human WNV infections in July and August 2020. Bird ringers are highly exposed to mosquito bites and possibly avian excrements during ringing activities. This study therefore investigates whether bird ringers are at higher risk of exposure to WNV and Usutu virus (USUV)., Methods: Dutch bird ringers were asked to provide a single serum sample (May - September 2021) and to fill out a survey. Sera were screened by protein microarray for presence of specific IgG against WNV and USUV non-structural protein 1 (NS1), followed by focus reduction virus neutralization tests (FRNT). Healthcare workers (2009-2010), the national immunity cohort (2016-2017) and blood donors (2021) were used as control groups without this occupational exposure., Results: The majority of the 157 participating bird ringers was male (132/157, 84%) and the median age was 62 years. Thirty-seven participants (37/157, 23.6%) showed WNV and USUV IgG microarray signals above background, compared to 6.4% (6/94) in the community cohort and 2.1% (2/96) in blood donors ( p  < 0.01). Two seroreactive bird ringers were confirmed WNV or USUV positive by FRNT. The majority of seroreactive bird ringers travelled to EU countries with reported WNV human cases (30/37, 81%) ( p  = 0.07). No difference was observed between bird ringers with and without previous yellow fever vaccination., Discussion: The higher frequency of WNV and/or USUV IgG reactive bird ringers indicates increased flavivirus exposure compared to the general population, suggesting that individuals with high-exposure professions may be considered to complement existing surveillance systems. However, the complexity of serological interpretation in relation to location-specific exposure (including travel), and antibody cross-reactivity, remain a challenge when performing surveillance of emerging flaviviruses in low-prevalence settings., Competing Interests: The authors declare that they have no financial and/or competing interests., (© 2023 The Authors.)

Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus causing mortality in Eurasian blackbirds ( Turdus merula ) in Europe. In dead blackbirds, avian malaria co-infection due to mosquito-borne hemosporidians (e.g., Plasmodium spp.) has been reported. In humans, a similar co-infection of a flavivirus, Dengue virus, and Plasmodium spp. is causing increased severity of clinical disease. Currently, the effects of co-infection of arboviruses and hemosporidians in blackbirds remain unclear. This study investigates the rate of USUV and Plasmodium spp. co-infection in found-dead blackbirds ( n  = 203) from 2016 to 2020 in the Netherlands. Presence of Plasmodium spp. was evaluated by cytology (43/203; 21,2%), histopathology (94/186; 50,5%) and qPCR (179/203; 88,1%). The severity of histological lesions in USUV and Plasmodium spp. co-infected dead blackbirds (121/203; 59,6%) were compared with those in Plasmodium spp. single-infected cases. Additionally, since no knowledge is present on the infection rate on live birds and mosquitoes in the Netherlands, a small group of live blackbirds ( n  = 12) and selected in the field-collected mosquito pools ( n  = 96) in 2020 were tested for the presence of Plasmodium spp. The latter was detected in the tested live blackbirds by qPCR (8/10; 80%), and cytology (3/11; 27,3%) and in the mosquito pools by qPCR (18/96; 18,7%). For this study, co-infection between USUV and Plasmodium spp. was observed only in the dead blackbirds. The high Plasmodium spp. presence, associated with lower lesions score, in single infected found dead birds suggest a predominantly smaller pathogenic role as single agent. On the other hand, the higher histological lesion scores observed in USUV and Plasmodium spp. co-infected birds suggests a major pathogenic role for the virus or an increased severity of the lesions due to a possible interplay of the two agents., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Objective: We aimed to assess SARS-CoV-2 contamination of air and surfaces to gain insight into potential occupational exposure in a large meat processing plant experiencing COVID-19 clusters. Methods: Oro-nasopharyngeal SARS-CoV-2 screening was performed in 76 workers. Environmental samples ( n = 275) including air, ventilation systems, sewage, and swabs of high-touch surfaces and workers' hands were tested for SARS-CoV-2 RNA by real-time quantitative polymerase chain reaction. Results: Twenty-seven (35.5%) of the (predominantly asymptomatic) workers tested positive with modest to low viral loads (cycle threshold ≥ 29.7). Six of 203 surface swabs, 1 of 12 personal air samples, and one of four sewage samples tested positive; other samples tested negative. Conclusions: Although one third of workers tested positive, environmental contamination was limited. Widespread SARS-CoV-2 transmission via air and surfaces was considered unlikely within this plant at the time of investigation while strict COVID-19 control measures were already implemented., Competing Interests: Conflict of interest: The authors are employed by the respective affiliations mentioned. The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Occupational and Environmental Medicine.)

Background: Outbreaks of zoonotic emerging infectious diseases (EIDs) require rapid identification of potential reservoir hosts and mapping disease spread in these hosts to inform risk assessment and adequate control measures. Animals are often understudied when a novel EID is detected in humans and acquisition of animal samples is hampered by practical, ethical, and legal barriers, of which there is currently no clear overview. Therefore, the three aims of this study are (1) to map potentially available collections of animal samples, (2) to assess possibilities and barriers for reuse of these samples and (3) to assess possibilities and barriers for active animal and environmental sampling in the Netherlands., Methods: A literature search was performed to identify ongoing sampling activities and opportunities for reuse or active sampling. Semi-structured interviews with stakeholder organizations were conducted to gain further insight into the three research questions., Results: Various sample collections of surveillance, diagnostic and research activities exist in the Netherlands. Sample size, coverage, storage methods and type of samples collected differs per animal species which influences reuse suitability. Organizations are more likely to share samples, for reuse in outbreak investigations, when they have a pre-existing relationship with the requesting institute. Identified barriers for sharing were, among others, unfamiliarity with legislation and unsuitable data management systems. Active sampling of animals or the environment is possible through several routes. Related barriers are acquiring approval from animal- or property owners, conflicts with anonymization, and time needed to acquire ethical approval., Conclusion: The animal sample collections identified would be very valuable for use in outbreak investigations. Barriers for sharing may be overcome by increasing familiarity with legislation, building (international) sharing networks and agreements before crises occur and developing systems for sample registration and biobanking. Proactive setting up of ethical approvals will allow for rapid animal sample collection to identify EID hosts and potential spillovers., Competing Interests: The authors do not declare any conflicts of interest., (© 2023 Published by Elsevier B.V.)

SARS-CoV-2 prevention and control measures did not only impact SARS-CoV-2 circulation, but also the timing and prevalence of other seasonal respiratory viruses. Especially in children, information on exposure and infections to seasonal coronaviruses as well as SARS-CoV-2 in the first year of the pandemic is largely lacking. Therefore, we set up a one-year serological survey in a large tertiary hospital in the Netherlands. We show that seasonal coronavirus seroprevalence significantly decreased in 2021 in children less than one year, most likely due to COVID-19 control measures. The SARS-CoV-2 seroprevalence in children and adolescents increased from 0.4% to 11.3%, the highest in adolescents. This implies higher exposure rates in adolescents as compared to the general population (>18 years old). It is clear that there have been significant changes in the circulation and subsequent immunity against most respiratory pathogens as a result of the mitigation measures. The implications on shorter as well as longer term are still largely unknown, but the impact of the SARS-CoV-2 pandemic and subsequent control measures will continue to affect the dynamics of other pathogens.

In some areas in temperate Europe, genomic analyses of mosquito-borne virus outbreaks have revealed the presence of similar virus strains over several years, indicating local overwintering of these viruses. However, it remains unclear how mosquito-borne viruses can persist in winter, when conditions are generally unfavourable for virus circulation. One of the presumed routes of virus persistence is via diapausing mosquitoes. Here, we set out to study whether arbovirus persistence of West Nile virus (WNV), Usutu virus (USUV) and Sindbis virus (SINV) occurs in diapausing mosquitoes in the Netherlands. To this end, mosquito collections were carried out in the winter of 2020 and 2021, in hibernacula located in two areas with previously observed WNV and/or USUV activity. In total, we collected 4200 mosquitoes belonging to four species ( Culex pipiens , Culiseta annulata , Anopheles maculipennis s.l., and Culex territans ), which were pooled in 490 monospecific pools. These pools were subjected to WNV-, USUV- and SINV-screening using a multiplex real-time RT-PCR assay. All mosquito pools tested negative for the presence of WNV, USUV and SINV RNA. Consequently, we did not find evidence of arbovirus persistence in diapausing mosquitoes in the Netherlands, even though USUV and WNV have re-appeared in birds and/or mosquitoes during the summer seasons of 2020-2022. Concluding, given the persistence of USUV and WNV in the Netherlands and SINV in other temperate regions, this study highlights the importance of further research on (alternative) arbovirus overwintering routes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Rabies virus (RABV) has a broad host range and infects multiple cell types throughout the infection cycle. Next-generation sequencing (NGS) and minor variant analysis are powerful tools for studying virus populations within specific hosts and tissues, leading to novel insights into the mechanisms of host-switching and key factors for infecting specific cell types. In this study we investigated RABV populations and minor variants in both original (non-passaged) samples and in vitro-passaged isolates of various CNS regions (hippocampus, medulla oblongata and spinal cord) of a fatal human rabies case, and of multiple CNS and non-CNS tissues of experimentally infected mice. No differences in virus populations were detected between the human CNS regions, and only one non-synonymous single nucleotide polymorphism (SNP) was detected in the fifth in vitro passage of virus isolated from the spinal cord. However, the appearance of this SNP shows the importance of sequencing newly passaged virus stocks before further use. Similarly, we did not detect apparent differences in virus populations isolated from different CNS and non-CNS tissues of experimentally infected mice. Sequencing of viruses obtained from pharyngeal swab and salivary gland proved difficult, and we propose methods for improving sampling.

Wild birds are reservoirs of several zoonotic arboviruses including West Nile virus (WNV) and Usutu virus (USUV), and are often monitored as indicators for virus introduction and spread. To optimize the bird surveillance for arboviruses in the Netherlands and to explore the possibilities for citizen science in surveillance, we investigated the suitability of using alternative sample types from live and dead birds. The sensitivity of molecular detection via RT-PCR of viral RNA in feather, heart, lung, throat and cloaca swabs from dead birds, and serum, dried blood spots (DBS) and throat and cloaca swabs from live birds were compared. IgY antibody detection was also assessed from DBS relative to serum on protein-microarray and virus neutralization test. Feathers showed a high detection sensitivity for USUV RNA in both live and dead birds, and no significant decrease was observed in the RNA loads in the feathers after being stored dry at room temperature for 43 days. Additionally, viral RNAs extracted from feathers of day 0 and 43 were successfully sequenced. The results indicated no statistical significant difference in sensitivity and viral loads detection in heart, spleen, and lung relative to corresponding brain samples in dead birds. In live birds, viral RNA loads did not differ between throat and cloaca swabs. This study identified less-invasive sample types that allows involvement of citizens in collecting samples from wild birds for arbovirus surveillance. Sensitivity and specificity of DBS-based antibody detections were significantly lower and therefore need optimization., Competing Interests: None declared., (© 2022 The Authors.)

In the Netherlands, 69 of the 126 (55%) mink farms in total became infected with SARS-CoV-2 in 2020. Despite strict biosecurity measures and extensive epidemiological investigations, the main transmission route remained unclear. A better understanding of SARS-CoV-2 transmission between mink farms is of relevance for countries where mink farming is still common practice and can be used as a case study to improve future emerging disease preparedness. We assessed whether SARS-CoV-2 spilled over from mink to free-ranging animals, and whether free-ranging animals may have played a role in farm-to-farm transmission in the Netherlands. The study encompassed farm visits, farm questionnaires, expert workshops and SARS-CoV-2 RNA and antibody testing of samples from target animal species (bats, birds and free-ranging carnivores). In this study, we show that the open housing system of mink allowed access to birds, bats and most free-ranging carnivores, and that direct and indirect contact with mink was likely after entry, especially for free-ranging carnivores and birds. This allowed SARS-CoV-2 exposure to animals entering the mink farm, and subsequent infection or mechanical carriage by the target animal species. Moreover, mink can escape farms in some cases, and two SARS-CoV-2-positive mink were found outside farm premises. No other SARS-CoV-2-RNA-positive free-ranging animals were detected, suggesting there was no abundant circulation in the species tested during the study period. To investigate previous SARS-CoV-2 infections, SARS-CoV-2 antibody detection using lung extracts of carcasses was set up and validated. One tested beech marten did have SARS-CoV-2 antibodies, but the closest SARS-CoV-2-infected mink farm was outside of its home range, making infection at a mink farm unlikely. Knowing that virus exchange between different species and the formation of animal reservoirs affects SARS-CoV-2 evolution, continued vigilance and monitoring of mink farms and surrounding wildlife remains vital., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2-positive cats and dogs in 10 infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of 10 farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all 10 farms. This study shows evidence of SARS-CoV-2 infection in 12 feral cats and 2 dogs. Eleven cats (18%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10%-18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

Background: To understand the dynamics of infectious diseases, genomic epidemiology is increasingly advocated, with a need for rapid generation of genetic sequences during outbreaks for public health decision making. Here, we explore the use of metagenomic sequencing compared to specific amplicon- and capture-based sequencing, both on the Nanopore and the Illumina platform for generation of whole genomes of Usutu virus, Zika virus, West Nile virus, and Yellow Fever virus., Results: We show that amplicon-based Nanopore sequencing can be used to rapidly obtain whole genome sequences in samples with a viral load up to Ct 33 and capture-based Illumina is the most sensitive method for initial virus determination., Conclusions: The choice of sequencing approach and platform is important for laboratories wishing to start whole genome sequencing. Depending on the purpose of genome sequencing the best choice can differ. The insights presented in this work and the shown differences in data characteristics can guide labs to make a well informed choice., (© 2022. The Author(s).)

Novel safe, immunogenic, and effective vaccines are needed to control the COVID-19 pandemic, caused by SARS-CoV-2. Here, we describe the safety, robust immunogenicity, and potent efficacy elicited in rhesus macaques by a modified vaccinia virus Ankara (MVA) vector expressing a full-length SARS-CoV-2 spike (S) protein (MVA-S). MVA-S vaccination was well tolerated and induced S and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against SARS-CoV-2 and several variants of concern. S-specific IFNγ, but not IL-4, -producing cells were also elicited. After SARS-CoV-2 challenge, vaccinated animals showed a significant strong reduction of virus loads in bronchoalveolar lavages (BAL) and decreased levels in throat and nasal mucosa. Remarkably, MVA-S also protected macaques from fever and infection-induced cytokine storm. Computed tomography and histological examination of the lungs showed reduced lung pathology in MVA-S-vaccinated animals. These findings favor the use of MVA-S as a potential vaccine for SARS-CoV-2 in clinical trials., Competing Interests: Author EP was employed by Biofabri. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mooij, García-Arriaza, Pérez, Lázaro-Frías, Verstrepen, Böszörményi, Mortier, Fagrouch, Kiemenyi-Kayere, Niphuis, Acar, Meijer, Stammes, Kondova, Verschoor, GeurtsvanKessel, de Bruin, Sikkema, Luczkowiak, Delgado, Montenegro, Puentes, Rodríguez, Bogers, Koopman and Esteban.)

Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches., (Copyright © 2021. Published by Elsevier B.V.)

Assays to measure SARS-CoV-2-specific neutralizing antibodies are important to monitor seroprevalence, to study asymptomatic infections and to reveal (intermediate) hosts. A recently developed assay, the surrogate virus-neutralization test (sVNT) is a quick and commercially available alternative to the "gold standard" virus neutralization assay using authentic virus, and does not require processing at BSL-3 level. The assay relies on the inhibition of binding of the receptor binding domain (RBD) on the spike (S) protein to human angiotensin-converting enzyme 2 (hACE2) by antibodies present in sera. As the sVNT does not require species- or isotype-specific conjugates, it can be similarly used for antibody detection in human and animal sera. In this study, we used 298 sera from PCR-confirmed COVID-19 patients and 151 sera from patients confirmed with other coronavirus or other (respiratory) infections, to evaluate the performance of the sVNT. To analyze the use of the assay in a One Health setting, we studied the presence of RBD-binding antibodies in 154 sera from nine animal species (cynomolgus and rhesus macaques, ferrets, rabbits, hamsters, cats, cattle, mink and dromedary camels). The sVNT showed a moderate to high sensitivity and a high specificity using sera from confirmed COVID-19 patients (91.3% and 100%, respectively) and animal sera (93.9% and 100%), however it lacked sensitivity to detect low titers. Significant correlations were found between the sVNT outcomes and PRNT 50 and the Wantai total Ig and IgM ELISAs. While species-specific validation will be essential, our results show that the sVNT holds promise in detecting RBD-binding antibodies in multiple species., Competing Interests: The authors declare that there is no conflict of interest., (© 2021 The Author(s).)

Objective: Unprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure., Methods: Air sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks' housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR., Results: Inside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000-10 000 copies/m 3 ). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks' housing units and surfaces contained SARS-CoV-2 RNA., Conclusions: Infected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and humans on farms. High number of farm infections (68/126) in minks and farm workers (>50% of farms) were detected, with limited community spread. Three of five initial introductions of SARS-CoV-2 led to subsequent spread between mink farms until November 2020. Viruses belonging to the largest cluster acquired an amino acid substitution in the receptor binding domain of the Spike protein (position 486), evolved faster and spread longer and more widely. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combining genetic information with epidemiological information when investigating outbreaks at the animal-human interface., (© 2021. The Author(s).)

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global&#95;report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 O'Toole Á et al.)

Background: Sars-CoV-2 outbreaks resulted in a high case fatality rate in nursing homes (NH) worldwide. It is unknown to which extent presymptomatic residents and staff contribute to the spread of the virus., Aims: To assess the contribution of asymptomatic and presymptomatic residents and staff in SARS-CoV-2 transmission during a large outbreak in a Dutch NH., Methods: Observational study in a 185-bed NH with two consecutive testing strategies: testing of symptomatic cases only, followed by weekly facility-wide testing of staff and residents regardless of symptoms. Nasopharyngeal and oropharyngeal testing with RT-PCR for SARs-CoV-2, including sequencing of positive samples, was conducted with a standardised symptom assessment., Results: 185 residents and 244 staff participated. Sequencing identified one cluster. In the symptom-based test strategy period, 3/39 residents were presymptomatic versus 38/74 residents in the period of weekly facility-wide testing (P-value < 0.001). In total, 51/59 (91.1%) of SARS-CoV-2 positive staff was symptomatic, with no difference between both testing strategies (P-value 0.763). Loss of smell and taste, sore throat, headache or myalga was hardly reported in residents compared to staff (P-value <0.001). Median Ct-value of presymptomatic residents was 21.3, which did not differ from symptomatic (20.8) or asymptomatic (20.5) residents (P-value 0.624)., Conclusions: Symptoms in residents and staff are insufficiently recognised, reported or attributed to a possible SARS-CoV-2 infection. However, residents without (recognised) symptoms showed the same potential for viral shedding as residents with symptoms. Weekly testing was an effective strategy for early identification of SARS-Cov-2 cases, resulting in fast mitigation of the outbreak., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The current coronavirus disease 2019 (COVID-19) pandemic is the first to apply whole-genome sequencing near to real time, with over 2 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequences generated and shared through the GISAID platform. This genomic resource informed public health decision-making throughout the pandemic; it also allowed detection of mutations that might affect virulence, pathogenesis, host range or immune escape as well as the effectiveness of SARS-CoV-2 diagnostics and therapeutics. However, genotype-to-phenotype predictions cannot be performed at the rapid pace of genomic sequencing. To prepare for the next phase of the pandemic, a systematic approach is needed to link global genomic surveillance and timely assessment of the phenotypic characteristics of novel variants, which will support the development and updating of diagnostics, vaccines, therapeutics and nonpharmaceutical interventions. This Review summarizes the current knowledge on key viral mutations and variants and looks to the next phase of surveillance of the evolving pandemic., (© 2021. Springer Nature America, Inc.)

Objectives: To obtain insight into SARS-CoV-2 clustering and transmission routes during outbreaks in the predominantly migrant workforce of the fruit and vegetable packaging industry of South Holland, the Netherlands, May to July 2020., Design: This mixed-methods study applied direct observation and interviews, epidemiologic investigation, source and contact data analysis and whole-genome sequencing., Results: We detected 46 SARS-CoV-2 cases and 4 outbreaks with a proportional representation of labour migrant and native workers in 6 unrelated facilities. Complete viral genome sequences revealed at least 3 clusters of native workers and labour migrants, 2 within and 1 between facilities. On-site inspections found adequate implementation of preventative measures to which both native workers and labour migrants showed suboptimal adherence. Being a labour migrant was associated with living in shared housing, but not with more contacts or different sources., Conclusions: The fruit and vegetable packaging industry gave the impression of sufficient preparedness and control. Suboptimal adherence to the facilities' preventative guidelines could have facilitated work floor transmission. Community and household transmission are likely to have contributed to outbreaks. We encourage further research into risk factors for transmission in labour migrants and application of these insights into targeted public health policy., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: An outbreak of coronavirus disease 2019 (COVID-19) in a nursing home in the Netherlands, following an on-site church service held on 8 March 2020, triggered an investigation to unravel sources and chain(s) of transmission., Methods: Epidemiological data were collected from registries and through a questionnaire among church attendees. Symptomatic residents and healthcare workers (HCWs) were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse-transcription polymerase chain reaction and subjected to whole genome sequencing (WGS). Sequences from a selection of people from the same area were included as community reference., Results: After the church service, 30 of 39 attendees (77%) developed symptoms; 14 (11 residents and 3 nonresidents) were tested and were positive for COVID-19. In the following 5 weeks, 62 of 300 residents (21%) and 30 of 640 HCWs (5%) tested positive for COVID-19; 21 of 62 residents (34%) died. The outbreak was controlled through a cascade of measures. WGS of samples from residents and HCWs identified a diversity of sequence types, grouped into 8 clusters. Seven resident church attendees all were infected with distinct viruses, 4 of which belonged to 2 larger clusters in the nursing home., Conclusions: Although initial investigation suggested the church service as the source of the outbreak, detailed analysis showed a more complex picture, most consistent with widespread regional circulation of the virus in the weeks before the outbreak, and multiple introductions into the nursing home before the visitor ban. The findings underscore the importance of careful outbreak investigations to understand SARS-CoV-2 transmission to develop evidence-based mitigation measures., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus. Despite its continuous circulation in Europe, knowledge on the pathology, cellular and tissue tropism and pathogenetic potential of different circulating viral lineages is still fragmentary. Here, macroscopic and microscopic evaluations are performed in association with the study of cell and tissue tropism and comparison of lesion severity of two circulating virus lineages (Europe 3; Africa 3) in 160 Eurasian blackbirds (Turdus merula) in the Netherlands. Results confirm hepatosplenomegaly, coagulative necrosis and lymphoplasmacytic inflammation as major patterns of lesions and, for the first time, vasculitis as a novel virus-associated lesion. A USUV and Plasmodium spp. co-infection was commonly identified. The virus was associated with lesions by immunohistochemistry and was reported most commonly in endothelial cells and blood circulating and tissue mononucleated cells, suggesting them as a major route of entry and spread. A tropism for mononuclear phagocytes cells was further supported by viral labeling in multinucleated giant cells. The involvement of ganglionic neurons and epithelial cells of the gastrointestinal tract suggests a possible role of oral transmission, while the involvement of feather follicle shafts and bulbs suggests their use as a diagnostic sample for live bird testing. Finally, results suggest similar pathogenicity for the two circulating lineages.

Background: The Dutch province of Limburg borders the German district of Heinsberg, which had a large cluster of COVID-19 cases linked to local carnival activities before any cases were reported in the Netherlands. However, Heinsberg was not included as an area reporting local or community transmission per the national case definition at the time. In early March, two residents from a long-term care facility (LTCF) in Sittard, a Dutch town located in close vicinity to the district of Heinsberg, tested positive for COVID-19. In this study we aimed to determine whether cross-border introduction of the virus took place by analysing the LTCF outbreak in Sittard, both epidemiologically and microbiologically., Methods: Surveys and semi-structured oral interviews were conducted with all present LTCF residents by health care workers during regular points of care for information on new or unusual signs and symptoms of disease. Both throat and nasopharyngeal swabs were taken from residents suspect of COVID-19, based on regional criteria, for the detection of SARS-CoV-2 by Real-time Polymerase Chain Reaction. Additionally, whole genome sequencing was performed using a SARS-CoV-2 specific amplicon-based Nanopore sequencing approach. Moreover, twelve random residents were sampled for possible asymptomatic infections., Results: Out of 99 residents, 46 got tested for COVID-19. Out of the 46 tested residents, nineteen (41%) tested positive for COVID-19, including 3 asymptomatic residents. CT-values for asymptomatic residents seemed higher compared to symptomatic residents. Eleven samples were sequenced, along with three random samples from COVID-19 patients hospitalized in the regional hospital at the time of the LTCF outbreak. All samples were linked to COVID-19 cases from the cross-border region of Heinsberg, Germany., Conclusions: Sequencing combined with epidemiological data was able to virtually prove cross-border transmission at the start of the Dutch COVID-19 epidemic. Our results highlight the need for cross-border collaboration and adjustment of national policy to emerging region-specific needs along borders in order to establish coordinated implementation of infection control measures to limit the spread of COVID-19.

Aim: To investigate the sources of infection among healthcare workers (HCWs) and patients in a teaching hospital in the Netherlands during the early stages of the coronavirus disease 2019 (COVID-19) pandemic using epidemiological and whole-genome sequencing data., Methods: From 3 rd April to 11 th May 2020, 88 HCWs and 215 patients were diagnosed with COVID-19. Whole-genome sequences were obtained for 30 HCWs and 20 patients., Results: Seven and 11 sequence types were identified in HCWs and patients, respectively. Cluster A was the most common sequence type, detected in 23 (77%) HCWs; of these, 14 (61%) had direct patient contact and nine (39%) had indirect patient contact. In addition, seven patients who were not hospitalized in the COVID-19 cohort isolation ward who became positive during their admission were infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cluster A. Following universal masking of all HCWs and emphasis on physical distancing during meals and breaks, no further evidence was found for patient-to-HCW or HCW-to-HCW transmission or vice versa., Conclusion: The finding that patients and HCWs were infected with SARS-CoV-2 cluster A suggests both HCW-to-HCW and HCW-to-patient transmission., (Copyright © 2021 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Animal experiments have shown that nonhuman primates, cats, ferrets, hamsters, rabbits, and bats can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, SARS-CoV-2 RNA has been detected in felids, mink, and dogs in the field. Here, we describe an in-depth investigation using whole-genome sequencing of outbreaks on 16 mink farms and the humans living or working on these farms. We conclude that the virus was initially introduced by humans and has since evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period, several weeks before detection. Despite enhanced biosecurity, early warning surveillance, and immediate culling of animals in affected farms, transmission occurred between mink farms in three large transmission clusters with unknown modes of transmission. Of the tested mink farm residents, employees, and/or individuals with whom they had been in contact, 68% had evidence of SARS-CoV-2 infection. Individuals for which whole genomes were available were shown to have been infected with strains with an animal sequence signature, providing evidence of animal-to-human transmission of SARS-CoV-2 within mink farms., (Copyright © 2021, American Association for the Advancement of Science.)

Background: People on ships are at high risk for outbreaks of infectious diseases including coronavirus disease 2019 (COVID-19). A rapid and well-coordinated response is important to curb transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We studied an outbreak on an industrial ship to improve outbreak control for ships and coordination between participating harbour partners., Materials and Methods: Public Health Service (PHS) Rotterdam-Rijnmond performed an epidemiological investigation during the outbreak of COVID-19 among 77 seafarers on a ship in their port. The captain was interviewed about ship details and his experiences during the outbreak. The seafarers were asked to fill in questionnaires about symptoms suspicious of COVID-19 and date of symptom onset. Information about stakeholders involved in outbreak control was registered., Results: The captain first contacted PHS about probable cases on March 31st 2020 via a physician ashore. One crewmember was hospitalised on April 8th and another died unexpectedly aboard on April 10th. Questionnaires distributed mid-April to the 75 remaining seafarers showed that 38 of 60 responders (63%) had had suspicious symptoms between February 15th and April 13th. None of them were tested but a total of 8 other crewmembers tested positive for COVID-19 after leaving the ship, including the hospitalised crewmember and the one who died aboard. On May 5th, the last case left isolation and the quarantine ended. Many different stakeholders were involved in the outbreak response and responsibilities were not always fully clear beforehand, causing coordination issues., Conclusions: Testing crew with COVID-19 symptoms underpins control measures and clarifies communication between stakeholders. Building a network beforehand to develop outbreak guidelines tailored to ships and local circumstances is essential to control future outbreaks on ships.

The first highly pathogenic (HP) influenza A/H7N9 was reported in Guangdong in January 2017. To investigate the emergence and spread of HP A/H7N9 in Guangdong province, we sequenced 297 viruses (58 HP A/H7N9, 19 low pathogenic (LP) A/H7N9, and 220 A/H9N2) during 2016-2017. Our analysis showed that during the fifth wave, three A/H7N9 lineages were co-circulating in Guangdong: the local LP Pearl River Delta (PRD) lineage (13%), the newly imported LP Yangtze River Delta (YRD) lineage (23%), and the HP YRD lineage (64%). Previously circulating YRD-lineage LP during the third wave evolved to the YRD-lineage HP A/H7N9 in Guangdong. All YRD-lineage LP detected during the fifth wave most likely originated from newly imported viruses into Guangdong. Genotype comparison of HP A/H7N9 suggests limited outward spread of HP A/H7N9 to other provinces. The distribution of HP A/H7N9 cleavage site variants on live poultry markets differed from that found in humans, suggesting a V1-type cleavage site may facilitate human infections., (© The Author(s) 2020. Published by Oxford University Press.)