Towards reliable whole genome sequencing for outbreak preparedness and response.

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.

Detection of West Nile virus in a common whitethroat (Curruca communis) and Culex mosquitoes in the Netherlands, 2020.

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

Whole-Blood Testing for Diagnosis of Acute Zika Virus Infections in Routine Diagnostic Setting.

We evaluated the benefit of whole blood versus plasma to detect acute Zika virus infections. Comparison of Zika virus quantitative reverse transcription PCR results in single timepoint whole blood-plasma pairs from 227 patients with suspected Zika virus infection resulted in confirmation of 8 additional patients with Zika virus infection.

MERS-CoV in Camels but Not Camel Handlers, Sudan, 2015 and 2017.

We tested samples collected from camels, camel workers, and other animals in Sudan and Qatar in 2015 and 2017 for evidence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. MERS-CoV antibodies were abundant in Sudan camels, but we found no evidence of MERS-CoV infection in camel workers, other livestock, or bats.

A daily-life-oriented intervention to improve prospective memory and goal-directed behaviour in ageing: a pilot study.

Difficulties in the execution of goal-directed behaviours, and particularly their prospective memory component, can arise in ageing and have important consequences for autonomy. The first objective of this article is to present an intervention that trained older individuals who reported prospective memory or goal-directed behaviour problems to use "implementation intentions". This technique, which has been shown to improve different aspects of goal-directed behaviour enactment, consists of establishing a mental (verbal and/or visual) link between the action that must be performed and the situation in which it must be performed. Our programme proposes exercises of progressively increasing difficulty that are targeted at daily life situations. Our second objective was to test the programme in small groups of older adults. Preliminary data regarding the programme's feasibility and its initial efficacy show a significant improvement in the main outcome measure, a questionnaire assessing goal-directed behaviours in everyday life. The participants also reported being significantly less bothered by their difficulties, although there were no significant changes in quality of life, self-esteem, anxiety or depression. Two participants with different psychological profiles, who benefited differently from the intervention, are then presented in more detail.

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.

Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets.

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

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.

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.

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.

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.

Secondary substitutions in the hemagglutinin and neuraminidase genes associated with neuraminidase inhibitor resistance are rare in the Influenza Resistance Information Study (IRIS).

Amino acid substitutions in influenza virus neuraminidase (NA) that cause resistance to neuraminidase inhibitors (NAI) generally result in virus attenuation. However, influenza viruses may acquire secondary substitutions in the NA and hemagglutinin (HA) proteins that can restore viral fitness. To assess to which extent this happens, the emergence of NAI resistance substitutions and secondary - potentially compensatory - substitutions was quantified in influenza viruses of immunocompetent individuals included in the Influenza Resistance Information Study (IRIS; NCT00884117). Known resistance substitutions were detected by mutation specific RT-PCR in viruses of 57 of 1803 (3.2%) oseltamivir-treated individuals, including 39 individuals infected with A/H1N1pdm09 [H275Y] virus and 18 with A/H3N2 [R292K] virus. A total of fifteen and ten other amino acid substitutions were acquired in HA and NA respectively, of A/H1N1pdm09, A/H3N2 and influenza B viruses upon treatment with oseltamivir but none of these was associated with resistance to oseltamivir. All cultured viruses with the known resistance substitutions H275Y or R292K showed reduced susceptibility to oseltamivir in the NA-star assay. Upon next-generation sequencing, the vast majority of NAI resistant A/H1N1pdm09 and A/H3N2 viruses had no resistance-associated secondary substitutions at high frequency. Only in two A/H1N1pdm09 [H275Y] viruses, the potentially compensatory secondary substitutions HA-D52N and NA-R152K were detected. We conclude that the emergence of secondary substitutions that may restore viral fitness upon the emergence of known influenza virus NAI resistance substitutions was a rare event in this immunocompetent population.

Apathy and executive dysfunction in Alzheimer disease.

Apathy, defined as a reduction in voluntary goal-directed behaviors, is one of the most common behavioral symptoms encountered in Alzheimer disease (AD). However, the processes underlying the different components of apathy are still unclear. The aim of this study was to explore a particularly important aspect of executive function in daily life: multitasking [assessed with the Modified Six Elements Task (MSET)], and its relationship with apathy in AD. Sixty-seven participants (37 AD patients matched with 30 control participants) were screened using the MSET. Simultaneously, a close relative of each patient was given the Apathy Inventory, which assesses 3 distinct dimensions of apathy (lack of initiative, lack of interest, and emotional blunting). AD patients presented significantly more multitasking deficits than control participants. In addition, regression analyses revealed that the number of rule breaks on the MSET (inability to perform several tasks in a predefined time observing a number of rules) was the best predictor of apathy, and especially of lack of initiative. These results suggest that the relation between lack of initiative and multitasking has a specific character and that mechanisms underlying multitasking constitute a key component of goal-directed behaviors.

Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands.

In late December 2019, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands.

Author Correction: Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

First molecular analysis of rabies virus in Qatar and clinical cases imported into Qatar, a case report.

Identifying the origin of the rabies virus (RABV) infection may have significant implications for control measures. Here, we identified the source of a RABV infection of two Nepalese migrants in Qatar by comparing their RABV genomes with RABV genomes isolated from the brains of a RABV infected camel and fox from Qatar.

COVID-19 in health-care workers in three hospitals in the south of the Netherlands: a cross-sectional study.

BACKGROUND: 10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers. METHODS: We did a cross-sectional study at three of the nine hospitals located in the south of the Netherlands. We screened health-care workers at the participating hospitals for SARS-CoV-2 infection, based on clinical symptoms (fever or mild respiratory symptoms) in the 10 days before screening. We obtained epidemiological data through structured interviews with health-care workers and combined this information with data from whole-genome sequencing of SARS-CoV-2 in clinical samples taken from health-care workers and patients. We did an in-depth analysis of sources and modes of transmission of SARS-CoV-2 in health-care workers and patients. FINDINGS: Between March 2 and March 12, 2020, 1796 (15%) of 12 022 health-care workers were screened, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete and near-complete genome sequences from 50 health-care workers and ten patients. Most sequences were grouped in three clusters, with two clusters showing local circulation within the region. The noted patterns were consistent with multiple introductions into the hospitals through community-acquired infections and local amplification in the community. INTERPRETATION: Although direct transmission in the hospitals cannot be ruled out, our data do not support widespread nosocomial transmission as the source of infection in patients or health-care workers. FUNDING: EU Horizon 2020 (RECoVer, VEO, and the European Joint Programme One Health METASTAVA), and the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Assessing impulsivity changes in Alzheimer disease.

Impulsive behaviors are common in brain-damaged patients including those with neurodegenerative diseases such as Alzheimer disease (AD). The objective of this study was to develop and validate a short version of the UPPS Impulsive Behavior Scale assessing changes on 4 different dimensions of impulsivity, namely urgency, (lack of) premeditation, (lack of) perseverance, and sensation seeking, arising in the course of a neurodegenerative disease. To this end, caregivers of 83 probable AD patients completed a short questionnaire adapted from the UPPS Impulsive Behavior Scale. Exploratory and confirmatory factor analyses of the data were performed and revealed that a model with 4 distinct but related latent variables corresponding to 4 different dimensions of impulsivity fit the data best. Furthermore, the results showed that lack of perseverance, followed by lack of premeditation and urgency, increased after the onset of the disease, whereas sensation seeking decreased. Overall, the multifaceted nature of impulsivity was confirmed in a sample of AD patients, whose caregivers reported significant changes regarding each facet of impulsivity. Consequently, the short version of the UPPS Impulsive Behavior Scale opens up interesting prospects for a better comprehension of behavioral symptoms of dementia.

A life-course and multifactorial approach to Alzheimer's disease: Implications for research, clinical assessment and intervention practices.

According to the dominant biomedical view, Alzheimer's disease (AD) has a precise, necessary and unifying neurobiological cause, which distinguishes it from other neurodegenerative diseases and normal ageing. However, different types of evidence specifically lead to questioning the foundations of this essentialist and category-based approach to AD. It seems more and more evident that AD represents a heterogeneous state, determined by multiple factors and mechanisms that interact and intervene throughout life. This other way of conceiving AD not only requires a change of research objectives, but also a profound modification of clinical assessment and intervention practices. It also appeals to follow the path of prevention.

Influenza A/H3N2 virus infection in immunocompromised ferrets and emergence of antiviral resistance.

Influenza viruses can cause severe life threatening infections in high-risk patients, including young children, the elderly and patients with compromised immunity due to underlying medical conditions or immunosuppressive treatment. The impaired immunity of these patients causes prolonged virus infection and combined with antiviral treatment facilitates the emergence of viruses with resistance mutations. The diverse nature of their immune status makes them a challenging group to study the impact of influenza virus infection and the efficacy of antiviral therapy. Immunocompromised ferrets may represent a suitable animal model to assess influenza virus infection and antiviral treatment strategies in immunocompromised hosts. Here, ferrets were given a daily oral solution of mycophenolate mofetil, tacrolimus and prednisolone sodium phosphate to suppress their immune system. Groups of immunocompromised and immunocompetent ferrets were inoculated with an A/H3N2 influenza virus and were subsequently treated with Oseltamivir or left untreated. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on the throat and nose specimens to study virus replication during the course of infection. All immunocompromised ferrets had prolonged presence of viral RNA and a higher total amount of virus shedding compared to the immunocompetent ferrets. Although Oseltamivir reduced the total amount of virus shedding from the nose and throat of treated ferrets, it also resulted in the emergence of the neuraminidase R292K resistance substitution in all these animals, as determined by mutation specific RT-PCR and next-generation sequencing. No additional mutations that could be associated with the emergence of the R292K resistance mutation were detected. The immunocompromised ferret model can be used to study A/H3N2 virus shedding and is a promising model to study new antiviral strategies and the emergence of antiviral resistance in immunocompromised hosts.