Search

The cellular composition of body fluids (BF) provides insight into disease pathology and is an important diagnostic parameter. The well-established Sysmex XN haematology analyser (XN) offers automated BF analysis and is in routine use in many laboratories. In this study the performance of the new Sysmex XR analyser (XR) in testing BF is compared to the XN.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 10 4 copies/ml (corresponding to C T values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of C T values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings., Competing Interests: Declaration of competing interest The authors declare to have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mpox is a viral zoonosis with endemic circulation in animals and humans in some West and Central African countries. The disease was imported a few times in the past to countries outside the African continent through infected animals or travelers, one of which resulted in an unprecedented global outbreak sustained by human-to-human transmission in 2022. Although timely and reliable diagnosis is a cornerstone of any disease control, availability of accurate diagnostic assays and comparative performance studies of diagnostic assays remains limited despite of the long-known identification of monkeypox virus (MPXV) as a human pathogen since 1970. We laboratory-developed a real-time PCR test (LDT) and evaluated its performance against the commercial TaqMan™ Monkeypox Virus Microbe Detection Assay (Applied Biosystems, Cat A50137). The limit of detection of the LDT was established at 1.2 genome copies/ml. The sensitivity and specificity of both assays were 99.14% and 100%, respectively, and both are capable of detecting both clade I and clade II of MPXV. Our results demonstrate the validity and accuracy of the LDT for confirmation of MPXV infection from lesion swabs samples., (© 2023. The Author(s).)

In the future, zoonotic spillover events are expected to occur more frequently. Consequences of such events have clearly been demonstrated by recent outbreaks of monkeypox, Ebola virus, and the well-known severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Virus discovery has proven to be an important tool in the preparation against viral outbreaks, generating data concerning the diversity, quantity, and ecology of the vertebrate virome. Orthoparamyxoviruses, a subfamily within the Paramyxoviridae, are important biosurveillance targets, since they include several known animal, human, and zoonotic pathogens such as Nipah virus, measles virus, and Hendra virus. During this study, 127 bat samples, thirty-four rodent samples, and seventeen shrew samples originating from Belgium were screened for orthoparamyxovirus presence using nested reverse transcription-polymerase chain reaction assays and nanopore sequencing. We present here the complete genomes of six putative new viral species, belonging to the genera Jeilongvirus and Henipavirus . Characterization of these genomes revealed significant differences in gene composition and organization, both within viruses of the same genus and between viruses of different genera. Remarkably, a previously undetected gene coding for a protein of unknown function was identified in the genome of a putative new Henipavirus . Additionally, phylogenetic analysis of jeilongviruses and henipaviruses reveals a division of both genera into two clades, one consisting of bat-borne viruses and the other consisting of rodent- and shrew-borne viruses, elucidating the need for proper reclassification., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press.)

Coronavirus Disease 2019 (COVID-19) vaccination has resulted in excellent protection against fatal disease, including in older adults. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases among residents) by combining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosol monitoring, whole-genome phylogenetic analysis and immunovirological profiling of nasal mucosa by digital nCounter transcriptomics. Phylogenetic investigations indicated that each outbreak stemmed from a single introduction event, although with different variants (Delta, Gamma and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 d after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 transcriptomic and genomic signatures uncovered a unique IRF3 low/IRF7 high immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy, including environmental sampling, immunomonitoring and early antiviral therapy, should be considered to prevent post-vaccination COVID-19 mortality in nursing homes., (© 2023. The Author(s).)

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC 50 ) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro . The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.

Background: Only two cases of papillomavirus infections in North American porcupines (Erethizon dorsatum) have been described thus far, and molecular investigation linked these cases to two distinct papillomavirus species., Methods: In this report, we present the clinical, histological and molecular investigation of a third case of a porcupine papillomavirus infection. Papillomatous lesions occurred on the upper and lower lip of an otherwise healthy three-year old female that was kept in captivity. Within one month, the lesions progressed into exophytic black nodules, followed by a temporary stabilization and ultimately spontaneous regression within seven months of their initial observation. PCR-based screening using specific primers for Erethizon dorsatum papillomavirus 1 and 2 revealed the presence of both these virus types, after which nanopore sequencing was used to determine the complete sequences of the two virus genomes., Results: One of the genomes shares 99.9% similarity with the only known sequence for Erethizon dorsatum papillomavirus 1, while the second represents a distinct lineage of Erethizon dorsatum papillomavirus 2, sharing only 93.3% similarity with the previously discovered strain., Conclusions: This report marks the first observation of a papillomavirus co-infection in a North American porcupine, although the individual contribution of the two virus types to the clinical presentation was not assessed., (© 2023. The Author(s).)

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Comparable to the related Ebola virus, Marburg virus is an emerging zoonotic pathogen that causes hemorrhagic fever with a high mortality rate. Therefore, handling of Ebola virus and Marburg virus is limited to biosafety level 4 facilities, of which only a limited number exists worldwide. However, researchers have developed several virus alternatives that are safe to handle in lower biosafety settings. One particularly interesting approach is the engineering of biologically contained Ebola virus by removing an essential gene from the virus genome and providing this missing gene in trans in a specific cell line. Because the virus is confined to this specific cell line, this results in a system that is safe to handle. So far, Ebola virus is the only virus for which biological containment has been reported. Here, we describe the first successful rescue of biologically contained Marburg virus and demonstrate that biological containment is also feasible for other filoviruses. Specifically, we describe the development of containment cell lines for Marburg virus through lentiviral transduction and show the growth and safety characteristics of eGFP-expressing, biologically contained Marburg virus in these cell lines. Additionally, we exploited this newly established Marburg virus system to screen over 500 compounds from available libraries. Lastly, we also validated the applicability of our biologically contained Marburg virus system in a 384-well format, to further illustrate the usefulness of this novel system as an alternative for high-throughput MARV screening of compound libraries., Competing Interests: Declaration of competing interest 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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The subfamily Orthoparamyxovirinae is a group of single-stranded, negative-sense RNA viruses that contains many human, animal, and zoonotic pathogens. While there are currently only forty-two recognized species in this subfamily, recent research has revealed that much of its diversity remains to be characterized. Using a newly developed nested PCR-based screening assay, we report here the discovery of fifteen orthoparamyxoviruses in rodents and shrews from Belgium and Guinea, thirteen of which are believed to represent new species. Using a combination of nanopore and sanger sequencing, complete genomes could be determined for almost all these viruses, enabling a detailed evaluation of their genome characteristics. While most viruses are thought to belong to the rapidly expanding genus Jeilongvirus , we also identify novel members of the genera Narmovirus , Henipavirus, and Morbillivirus . Together with other recently discovered orthoparamyxoviruses, both henipaviruses and the morbillivirus discovered here appear to form distinct rodent-/shrew-borne clades within their respective genera, clustering separately from all currently classified viruses. In the case of the henipaviruses, a comparison of the different members of this clade revealed the presence of a secondary conserved open reading frame, encoding for a transmembrane protein, within the F gene, the biological relevance of which remains to be established. While the characteristics of the viruses described here shed further light on the complex evolutionary origin of paramyxoviruses, they also illustrate that the diversity of this group of viruses in terms of genome organization appears to be much larger than previously assumed., (© The Author(s) 2022. Published by Oxford University Press.)

SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Despite recent advancements in the development of vaccines and monoclonal antibody therapies for Ebola virus disease, treatment options remain limited. Moreover, management and containment of Ebola virus outbreaks is often hindered by the remote nature of the locations in which the outbreaks originate. Small-molecule compounds offer the advantage of being relatively cheap and easy to produce, transport and store, making them an interesting modality for the development of novel therapeutics against Ebola virus disease. Furthermore, the repurposing of small-molecule compounds, previously developed for alternative applications, can aid in reducing the time needed to bring potential therapeutics from bench to bedside. For this purpose, the Medicines for Malaria Venture provides collections of previously developed small-molecule compounds for screening against other infectious diseases. In this study, we used biologically contained Ebola virus to screen over 4,200 small-molecule drugs and drug-like compounds provided by the Medicines for Malaria Venture (i.e., the Pandemic Response Box and the COVID Box) and the Centre for Drug Design and Discovery (CD3, KU Leuven, Belgium). In addition to confirming known Ebola virus inhibitors, illustrating the validity of our screening assays, we identified eight novel selective Ebola virus inhibitors. Although the inhibitory potential of these compounds remains to be validated in vivo, they represent interesting compounds for the study of potential interventions against Ebola virus disease and might serve as a basis for the development of new therapeutics., (Copyright © 2022. Published by Elsevier B.V.)

Acute gastroenteritis is one of the main causes of mortality and morbidity worldwide, affecting mainly children, the immunocompromised and elderly people. Enteric viruses, especially rotavirus A, are considered important etiological agents, while long-term care facilities are considered favorable environments for the occurrence of sporadic cases and outbreaks of acute gastroenteritis. Therefore, it is important to monitor the viral agents present in nursing homes, especially because studies involving the elderly population in Brazil are scarce, resulting in a lack of available virological data. As a result, the causative agent remains unidentified in a large number of reported acute gastroenteritis cases. However, the advent of next-generation sequencing provides new opportunities for viral detection and discovery. The aim of this study was to identify the viruses that circulate among elderly people with and without acute gastroenteritis, living in residential care homes in Belém, Pará, Brazil, between 2017 and 2019. Ninety-three samples were collected and screened by immunochromatography and qPCR. After, the samples were analyzed individually or in pools by next generation sequencing to identify the viruses circulating in this population. In 26 sequenced samples, members of 13 eukaryotic virus families were identified. The most abundantly present virus families were Parvoviridae, Genomoviridae and Smacoviridae. Contigs displaying similarity to pegiviruses were also detected. Furthermore, a near-complete rotavirus A genome was obtained and could be classified as G3P[8] genotype with the equine DS-1-like genetic background. Complete sequences of the VP4 and VP7 genes of a rotavirus C were also detected, belonging to G4P[2]. This study demonstrates the first characterization of the gastrointestinal virome in elderly in Northern Brazil. A diversity of viruses was found to be present in patients with and without diarrhea, reinforcing the need to monitor elderly people residing in long-term care facilities, especially in cases of acute gastroenteritis., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics., (© 2022. The Author(s).)

SignificanceMicrobes colonizing the infant gut during the first year(s) of life play an important role in immune system development. We show that after birth the (nearly) sterile gut is rapidly colonized by bacteria and their viruses (phages), which often show a strong cooccurrence. Most viruses infecting the infant do not cause clinical signs and their numbers strongly increase after day-care entrance. The infant diet is clearly reflected by identification of plant-infecting viruses, whereas fungi and parasites are not part of a stable gut microbiota. These temporal high-resolution baseline data about the gut colonization process will be valuable for further investigations of pathogenic viruses, dynamics between phages and their bacterial host, as well as studies investigating infants with a disturbed microbiota.

We report the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (lineage B.1.1.529) from a Belgian patient with a history of recent travel to Egypt. At the time of writing, this genome constituted the first confirmed case of an infection with the Omicron variant in Europe.

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa 1-3 . It has since spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of around 32 mutations in spike-located mostly in the N-terminal domain and the receptor-binding domain-that may enhance viral fitness and enable antibody evasion. Here we isolated an infectious Omicron virus in Belgium from a traveller returning from Egypt. We examined its sensitivity to nine monoclonal antibodies that have been clinically approved or are in development 4 , and to antibodies present in 115 serum samples from COVID-19 vaccine recipients or individuals who have recovered from COVID-19. Omicron was completely or partially resistant to neutralization by all monoclonal antibodies tested. Sera from recipients of the Pfizer or AstraZeneca vaccine, sampled five months after complete vaccination, barely inhibited Omicron. Sera from COVID-19-convalescent patients collected 6 or 12 months after symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titres 6-fold to 23-fold lower against Omicron compared with those against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and, to a large extent, vaccine-elicited antibodies. However, Omicron is neutralized by antibodies generated by a booster vaccine dose., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in clinical presentation on the one hand and the host dependent clinical outcome of BPV-1 infection on the other hand indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation., Methods: In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees., Results: We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of them cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis., Conclusions: By generating a significant amount of complete-length BPV genomes, we succeeded to introduce next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample., (© 2022. The Author(s).)

At the end of 2020, several new variants of SARS-CoV-2-designated variants of concern-were detected and quickly suspected to be associated with a higher transmissibility and possible escape of vaccine-induced immunity. In Belgium, this discovery has motivated the initiation of a more ambitious genomic surveillance program, which is drastically increasing the number of SARS-CoV-2 genomes to analyse for monitoring the circulation of viral lineages and variants of concern. In order to efficiently analyse the massive collection of genomic data that are the result of such increased sequencing efforts, streamlined analytical strategies are crucial. In this study, we illustrate how to efficiently map the spatio-temporal dispersal of target mutations at a regional level. As a proof of concept, we focus on the Belgian province of Liège that has been consistently sampled throughout 2020, but was also one of the main epicenters of the second European epidemic wave. Specifically, we employ a recently developed phylogeographic workflow to infer the regional dispersal history of viral lineages associated with three specific mutations on the spike protein (S98F, A222V and S477N) and to quantify their relative importance through time. Our analytical pipeline enables analysing large data sets and has the potential to be quickly applied and updated to track target mutations in space and time throughout the course of an epidemic., (© 2021. The Author(s).)

Recent years have witnessed the discovery of several new viruses belonging to the family Arteriviridae , expanding the known diversity and host range of this group of complex RNA viruses. Although the pathological relevance of these new viruses is not always clear, several well-studied members of the family Arteriviridae are known to be important animal pathogens. Here, we report the complete genome sequences of four new arterivirus variants, belonging to two putative novel species. These new arteriviruses were discovered in African rodents and were given the names Lopma virus and Praja virus. Their genomes follow the characteristic genome organization of all known arteriviruses, even though they are only distantly related to currently known rodent-borne arteriviruses. Phylogenetic analysis shows that Lopma virus clusters in the subfamily Variarterivirinae , while Praja virus clusters near members of the subfamily Heroarterivirinae : the yet undescribed forest pouched giant rat arterivirus and hedgehog arterivirus 1. A co-divergence analysis of rodent-borne arteriviruses confirms that they share similar phylogenetic patterns with their hosts, with only very few cases of host shifting events throughout their evolutionary history. Overall, the genomes described here and their unique clustering with other arteriviruses further illustrate the existence of multiple rodent-borne arterivirus lineages, expanding our knowledge of the evolutionary origin of these viruses.

Recent metagenomics studies have revealed several tick species to host a variety of previously undiscovered RNA viruses. Ixodes ricinus , which is known to be a vector for many viral, bacterial, and protozoan pathogens, is the most prevalent tick species in Europe. For this study, we decided to investigate the virosphere of Belgian I. ricinus ticks. High-throughput sequencing of tick pools collected from six different sampling sites revealed the presence of viruses belonging to many different viral orders and families, including Mononegavirales, Bunyavirales , Partitiviridae , and Reoviridae . Of particular interest was the detection of several new reoviruses, two of which cluster together with members of the genus Coltivirus . This includes a new strain of Eyach virus, a known causative agent of tick-borne encephalitis. All genome segments of this new strain are highly similar to those of previously published Eyach virus genomes, except for the fourth segment, encoding VP4, which is markedly more dissimilar, potentially indicating the occurrence of a genetic reassortment. Further polymerase chain reaction-based screening of over 230 tick pools for 14 selected viruses showed that most viruses could be found in all six sampling sites, indicating the wide spread of these viruses throughout the Belgian tick population. Taken together, these results illustrate the role of ticks as important virus reservoirs, highlighting the need for adequate tick control measures., (© The Author(s) 2021. Published by Oxford University Press.)

We report here the complete genome sequence of ruloma virus, a novel paramyxovirus detected in a Machangu's brush-furred rat from Tanzania. Ruloma virus has the longest orthoparamyxovirus genome reported to date and forms a sister clade to all currently known members of the genus Jeilongvirus ., (Copyright © 2021 Vanmechelen et al.)

Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of SARS-CoV-2 have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analyzed with gold-standard phylogeographic approaches. To tackle this practical limitation, we here describe and apply a rapid analytical pipeline to analyze the spatiotemporal dispersal history and dynamics of SARS-CoV-2 lineages. As a proof of concept, we focus on the Belgian epidemic, which has had one of the highest spatial densities of available SARS-CoV-2 genomes. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

We investigated the genetic profiles of killer cell immunoglobulin-like receptors (KIRs) in Ebola virus-infected patients. We studied the relationship between KIR-human leukocyte antigen (HLA) combinations and the clinical outcomes of patients with Ebola virus disease (EVD). We genotyped KIRs and HLA class I alleles using DNA from uninfected controls, EVD survivors, and persons who died of EVD. The activating 2DS4-003 and inhibitory 2DL5 genes were significantly more common among persons who died of EVD; 2DL2 was more common among survivors. We used logistic regression analysis and Bayesian modeling to identify 2DL2, 2DL5, 2DS4-003, HLA-B-Bw4-Thr, and HLA-B-Bw4-Ile as probably having a significant relationship with disease outcome. Our findings highlight the importance of innate immune response against Ebola virus and show the association between KIRs and the clinical outcome of EVD.

Marburg virus (MARV) is the only known pathogenic filovirus not belonging to the genus Ebolavirus. Minigenomes have proven a useful tool to study MARV, but all existing MARV minigenomes are dependent on the addition of an exogenous T7 RNA polymerase to drive minigenome expression. However, exogenous expression of a T7 polymerase is not always feasible and can act as a confounding factor in compound screening assays. We have developed an alternative minigenome that is controlled by the natively expressed RNA polymerase II. We demonstrate here the characteristics of this new system and its applicability in a wide range of cell types. Our system shows a clear concentration-dependent activity and shows comparable activity to the existing T7 polymerase-based system at higher concentrations, also in difficult-to-transfect cell lines. In addition, we show that our system can be used for compound screening in a 96-well format, thereby providing an attractive alternative to previously developed MARV minigenomes., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus-host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE.

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

Common or European hedgehogs can be found throughout Western Europe. They are known carriers of a variety of parasitic and bacterial pathogens, and have also been shown to carry several viruses, including morbilli-like paramyxoviruses, although the pathogenic and zoonotic potential of some of these viruses has yet to be determined. We report here the discovery of a novel paramyxovirus in Belgian hedgehogs, named Belerina virus. The virus was detected by nanopore sequencing of RNA isolated from hedgehog tissue. Out of 147 animals screened in this study, 57 tested positive for Belerina virus (39%), indicating a high prevalence of this virus in the Belgian hedgehog population. Based on its divergence from other known paramyxovirus species, Belerina virus is thought to represent a new species in the family Paramyxoviridae. Phylogenetic analysis groups Belerina virus together with the bat-borne Shaan virus within the genus Jeilongvirus, although expanding the tree with partial genomes shows Belerina virus forming a separate subclade within this genus, alongside a yet-unnamed paramyxovirus isolated from a greater tube-nosed bat. In summary, we discuss the complete genome sequence of Belerina virus, a putative new paramyxovirus species commonly found in Belgian hedgehogs.

Because of the enormous variation in their genome sequence, genotyping enteroviruses by standard methods can prove to be quite challenging. Nanopore sequencing offers the potential to overcome the limitations of older techniques, but thus far, only amplicon-based strategies have been used to sequence complete enterovirus genomes. By combining a sequence-independent, single primer amplification (SISPA) for cDNA generation with next-generation sequencing using the Oxford Nanopore MinION, complete enterovirus genomes can be obtained in an easy-to-use, sequence-independent manner. To demonstrate its usability, we applied this technique to determine the complete genome sequence of an enterovirus C104 strain, representing the first documented occurrence of this uncommon enterovirus strain in Belgium., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ebolaviruses pose a substantial threat to wildlife populations and to public health in Africa. Evolutionary analyses of virus genome sequences can contribute significantly to elucidate the origin of new outbreaks, which can help guide surveillance efforts. The reconstructed between-outbreak evolutionary history of Zaire ebolavirus so far has been highly consistent. By removing the confounding impact of population growth bursts during local outbreaks on the free mixing assumption that underlies coalescent-based demographic reconstructions, we find-contrary to what previous results indicated-that the circulation dynamics of Ebola virus in its animal reservoir are highly uncertain. Our findings also accentuate the need for a more fine-grained picture of the Ebola virus diversity in its reservoir to reliably infer the reservoir origin of outbreak lineages. In addition, the recent appearance of slower-evolving variants is in line with latency as a survival mechanism and with bats as the natural reservoir host., Competing Interests: The authors have declared that no competing interests exist.

A wealth of viral data sits untapped in publicly available metagenomic data sets when it might be extracted to create a usable index for the virological research community. We hypothesized that work of this complexity and scale could be done in a hackathon setting. Ten teams comprised of over 40 participants from six countries, assembled to create a crowd-sourced set of analysis and processing pipelines for a complex biological data set in a three-day event on the San Diego State University campus starting 9 January 2019. Prior to the hackathon, 141,676 metagenomic data sets from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) were pre-assembled into contiguous assemblies (contigs) by NCBI staff. During the hackathon, a subset consisting of 2953 SRA data sets (approximately 55 million contigs) was selected, which were further filtered for a minimal length of 1 kb. This resulted in 4.2 million (Mio) contigs, which were aligned using BLAST against all known virus genomes, phylogenetically clustered and assigned metadata. Out of the 4.2 Mio contigs, 360,000 contigs were labeled with domains and an additional subset containing 4400 contigs was screened for virus or virus-like genes. The work yielded valuable insights into both SRA data and the cloud infrastructure required to support such efforts, revealing analysis bottlenecks and possible workarounds thereof. Mainly: (i) Conservative assemblies of SRA data improves initial analysis steps; (ii) existing bioinformatic software with weak multithreading/multicore support can be elevated by wrapper scripts to use all cores within a computing node; (iii) redesigning existing bioinformatic algorithms for a cloud infrastructure to facilitate its use for a wider audience; and (iv) a cloud infrastructure allows a diverse group of researchers to collaborate effectively. The scientific findings will be extended during a follow-up event. Here, we present the applied workflows, initial results, and lessons learned from the hackathon.

Genomic sequencing for early identification of Ebola virus remains a big challenge in low-income countries. Here, we report the complete genome sequence of an Ebola virus strain obtained during the 2017 Likati outbreak in the Democratic Republic of the Congo (DRC) by using the Oxford Nanopore Technologies (ONT) MinION sequencer., (Copyright © 2019 Wawina-Bokalanga et al.)

Hantaviruses are zoonotic hemorrhagic fever viruses for which prevention of human spillover remains the first priority in disease management. Tailored intervention measures require an understanding of the drivers of enzootic dynamics, commonly inferred from distorted human incidence data. Here, we use longitudinal sampling of approximately three decades of Puumala orthohantavirus (PUUV) evolution in isolated reservoir populations to estimate PUUV evolutionary rates, and apply these to study the impact of environmental factors on viral spread. We find that PUUV accumulates genetic changes at a rate of ∼10 -4 substitutions per site per year and that land cover type defines the dispersal dynamics of PUUV, with forests facilitating and croplands impeding virus spread. By providing reliable short-term PUUV evolutionary rate estimates, this work facilitates the evaluation of spatial risk heterogeneity starting from timed phylogeographic reconstructions based on virus sampling in its animal reservoir, thereby side-stepping the need for difficult-to-collect human disease incidence data.

We report here the complete sequence and genome organization of a new papillomavirus, Erethizon dorsatum papillomavirus 2 (EdPV2), which was isolated from cutaneous lesions observed on the muzzle of a North American porcupine. The complete genome is 8809 nucleotides long and encodes five early (E6-E7-E1-E2-E4) and two late proteins (L2-L1). In addition to the upstream regulatory region, the EdPV2 genome contains an exceptionally large secondary non-coding region with no apparent functional relevance. EdPV2 is strongly divergent from the previously described porcupine papillomavirus EdPV1 and phylogenetic analysis shows EdPV2 clustering near members of the genus Pipapillomavirus, a group of rodent papillomaviruses. Pairwise sequence comparison based on the L1 open reading frame identifies Rattus norvegicus papillomavirus 1 as the closest related virus (59.97 % similarity). Based on its low sequence similarity to other known papillomaviruses, EdPV2 is thought to represent a new genus in the family Papillomaviridae.

Background: In the past decade, many new paramyxoviruses that do not belong to any of the seven established genera in the family Paramyxoviridae have been discovered. Amongst them are J-virus (JPV), Beilong virus (BeiPV) and Tailam virus (TlmPV), three paramyxovirus species found in rodents. Based on their similarities, it has been suggested that these viruses should compose a new genus, tentatively called 'Jeilongvirus'., Results: Here we present the complete genomes of three newly discovered paramyxoviruses, one found in a bank vole (Myodes glareolus) from Slovenia and two in a single, co-infected Rungwe brush-furred rat (Lophuromys machangui) from Mozambique, that represent three new, separate species within the putative genus 'Jeilongvirus'. The genome organization of these viruses is similar to other paramyxoviruses, but like JPV, BeiPV and TlmPV, they possess an additional open reading frame, encoding a transmembrane protein, that is located between the F and G genes. As is the case for all Jeilongviruses, the G genes of the viruses described here are unusually large, and their encoded proteins are characterized by a remarkable amino acid composition pattern that is not seen in other paramyxoviruses, but resembles certain motifs found in Orthopneumovirus G proteins., Conclusions: The phylogenetic clustering of JPV, BeiPV and TlmPV with the viruses described here, as well as their shared features that set them apart from other paramyxoviruses, provide additional support for the recognition of the genus 'Jeilongvirus'.

The family Arteriviridae harbors a rapidly expanding group of viruses known to infect a divergent group of mammals, including horses, pigs, possums, primates, and rodents. Hosts infected with arteriviruses present with a wide variety of (sub) clinical symptoms, depending on the virus causing the infection and the host being infected. In this study, we determined the complete genome sequences of three variants of a previously unknown virus found in Olivier's shrews (Crocidura olivieri guineensis) sampled in Guinea. On the nucleotide level, the three genomes of this new virus, named Olivier's shrew virus 1 (OSV-1), are 88-89% similar. The genome organization of OSV-1 is characteristic of all known arteriviruses, yet phylogenetic analysis groups OSV-1 separately from all currently established arterivirus lineages. Therefore, we postulate that OSV-1 represents a member of a novel arterivirus genus. The virus described here represents the first discovery of an arterivirus in members of the order Eulipotyphla, thereby greatly expanding the known host spectrum of arteriviruses.

Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet's microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10 6.42 -10 7.01 copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded.

Background: Hantavirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaviruses are found on the American continent where the most prevalent viruses like Andes virus and Sin Nombre virus are known to cause hantavirus pulmonary syndrome. New World hantavirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes virus and Maporal virus; the only hantaviruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantavirus pulmonary syndrome in humans., Methodology/principal Findings: Hamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantavirus strains (Bayou virus, Black Creek Canal virus, Caño Delgadito virus, Choclo virus, Laguna Negra virus, and Maporal virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantavirus results in an acute disease that precisely mimics both hantavirus disease in humans and Andes virus infection of hamsters., Conclusions/ Significance: Infected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaviruses reaching a lethal outcome with signs of disease mimicking human disease.

We report here the draft genome of a novel nairovirus, Grotenhout virus, isolated from deer ticks ( Ixodes ricinus ) in Belgium. The genome consists of two segments, L and S, and is most similar to the tick-borne South Bay virus, with amino acid identities ranging from 60 to 64%., (Copyright © 2017 Vanmechelen et al.)

Papillomaviridae form a large family of viruses that are known to infect a variety of vertebrates, including mammals, reptiles, birds and fish. Infections usually give rise to minor skin lesions but can in some cases lead to the development of malignant neoplasia. In this study, we identified a novel species of papillomavirus (PV), isolated from warts of four giraffes (Giraffa camelopardalis). The sequence of the L1 gene was determined and found to be identical for all isolates. Using nanopore sequencing, the full sequence of the PV genome could be determined. The coding region of the genome was found to contain seven open reading frames (ORF), encoding the early proteins E1, E2 and E5-E7 as well as the late proteins L1 and L2. In addition to these ORFs, a region located within the E2 gene is thought, based on sequence similarities to other papillomaviruses, to encode an E4 protein, although no start codon could be identified. Based on the sequence of the L1 gene, this novel PV was found to be most similar to Capreolus capreolus papillomavirus 1 (CcaPV1), with 67.96% nucleotide identity. We therefore suggest that the virus identified here is given the name Giraffa camelopardalis papillomavirus 1 (GcPV1) and is classified as a novel species within the genus Deltapapillomavirus, in line with the current guidelines for the nomenclature and classification of PVs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Many methods for the discovery of novel viruses are based on amplification of the virus using consensus or degenerate PCR primers. A downside of this approach is that it requires prior knowledge of the viral nucleotide sequence to be applicable. Presented in this unit is a method for the sequence-independent amplification of circular viral genomes that is based on the rolling-circle mechanism used by certain viruses in their natural replication cycle. The amplification of the virus of interest is coupled to the isolation of the viral genome by gel extraction following a restriction digestion. Once isolated, the sequence of the viral genome can be determined by nanopore sequencing, a rapid and inexpensive next-generation sequencing technology that generates long reads in real time. The method described in this unit was originally developed for the discovery of papillomaviruses, but can be used for the identification of all types of circular DNA viruses. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Over the last decade, the recognized host range of hantaviruses has expanded considerably with the discovery of distinct hantaviruses in shrews, moles and bats. Unfortunately, in-depth studies of these viruses have been limited. Here we describe a comprehensive analysis of the spatial distribution, genetic diversity and evolution of Nova virus, a hantavirus that has the European mole as its natural host. Our analysis demonstrated that Nova virus has a high prevalence and widespread distribution in Belgium. While Nova virus displayed relatively high nucleotide diversity in Belgium, amino acid changes were limited. The nucleocapsid protein was subjected to strong purifying selection, reflecting the strict evolutionary constraints placed upon Nova virus by its host. Spatio-temporal analysis using Bayesian evolutionary inference techniques demonstrated that Nova virus had efficiently spread in the European mole population in Belgium, forming two distinct clades, representing east and west of Belgium. The influence of landscape barriers, in the form of the main waterways, on the dispersal velocity of Nova virus was assessed using an analytical framework for comparing Bayesian viral phylogenies with environmental landscape data. We demonstrated that waterways did not act as an environmental resistance factor slowing down Nova virus diffusion in the mole population. With this study, we provide information about the spatial diffusion of Nova virus and contribute sequence information that can be applied in further functional studies., (© 2016 John Wiley & Sons Ltd.)