The multi-dimensional challenges of controlling respiratory virus transmission in indoor spaces: Insights from the linkage of a microscopic pedestrian simulation and SARS-CoV-2 transmission model.

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.

Landscape level associations between birds, mosquitoes and microclimates: possible consequences for disease transmission?

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.

West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

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.

Development and validation of the MosquitoWise survey to assess perceptions towards mosquitoes and mosquito-borne viruses in Europe.

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.

A species-independent lateral flow microarray immunoassay to detect WNV and USUV NS1-specific antibodies in serum.

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.

High number of HPAI H5 virus infections and antibodies in wild carnivores in the Netherlands, 2020-2022.

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.

The increasing complexity of arbovirus serology: An in-depth systematic review on cross-reactivity.

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.

Decision-support tools to build climate resilience against emerging infectious diseases in Europe and beyond.

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.

SARS-CoV-2 in lions, gorillas and zookeepers in the Rotterdam Zoo, the Netherlands, a One Health investigation, November 2021.

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.

The pathology of co-infection with Usutu virus and Plasmodium spp. in naturally infected Eurasian blackbirds (Turdus merula).

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.

Assessing West Nile virus (WNV) and Usutu virus (USUV) exposure in bird ringers in the Netherlands: a high-risk group for WNV and USUV infection?

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.

Combining epidemiological data and whole genome sequencing to understand SARS-CoV-2 transmission dynamics in a large tertiary care hospital during the first COVID-19 wave in The Netherlands focusing on healthcare workers.

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.

Rapid response screening for emerging zoonotic pathogens, barriers and opportunities: A study for enhanced preparedness of the Netherlands.

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.

Rise and fall of SARS-CoV-2 variants in Rotterdam: Comparison of wastewater and clinical surveillance.

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.

Reduced Seasonal Coronavirus Antibody Responses in Children Following COVID-19 Mitigation Measures, The Netherlands.

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.

A Comprehensive Sampling Study on SARS-CoV-2 Contamination of Air and Surfaces in a Large Meat Processing Plant Experiencing COVID-19 Clusters in June 2020.

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.

Arbovirus persistence in North-Western Europe: Are mosquitoes the only overwintering pathway?

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.

Rabies Virus Populations in Humans and Mice Show Minor Inter-Host Variability within Various Central Nervous System Regions and Peripheral Tissues.

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.

Evaluation of the use of alternative sample types for mosquito-borne flavivirus surveillance: Using Usutu virus as a model.

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.

Manifestation of SARS-CoV-2 Infections in Mink Related to Host-, Virus- and Farm-Associated Factors, The Netherlands 2020.

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.