Search

Bone-related complications are commonly reported following arbovirus infection. These arboviruses are known to disturb bone-remodeling and induce inflammatory bone loss via increased activity of bone resorbing osteoclasts (OCs). We previously showed that Zika virus (ZIKV) could disturb the function of bone forming osteoblasts, but the susceptibility of OCs to ZIKV infection is not known. Here, we investigated the effect of ZIKV infection on osteoclastogenesis and report that infection of pre- and early OCs with ZIKV significantly reduced the osteoclast formation and bone resorption. Interestingly, infection of pre-OCs with a low dose ZIKV infection in the presence of flavivirus cross-reacting antibodies recapitulated the phenotype observed with a high viral dose, suggesting a role for antibody-dependent enhancement in ZIKV-associated bone pathology. In conclusion, we have characterized a primary in vitro model to study the role of osteoclastogenesis in ZIKV pathogenesis, which will help to identify possible new targets for developing therapeutic and preventive measures.

The Omicron BA.1 (B.1.1.529) SARS-CoV-2 variant is characterized by a high number of mutations in the viral genome, associated with immune escape and increased viral spread. It remains unclear whether milder COVID-19 disease progression observed after infection with Omicron BA.1 in humans is due to reduced pathogenicity of the virus or due to pre-existing immunity from vaccination or previous infection. Here, we inoculated hamsters with Omicron BA.1 to evaluate pathogenicity and kinetics of viral shedding, compared to Delta (B.1.617.2) and to animals re-challenged with Omicron BA.1 after previous SARS-CoV-2 614G infection. Omicron BA.1 infected animals showed reduced clinical signs, pathological changes, and viral shedding, compared to Delta-infected animals, but still showed gross- and histopathological evidence of pneumonia. Pre-existing immunity reduced viral shedding and protected against pneumonia. Our data indicate that the observed decrease of disease severity is in part due to intrinsic properties of the Omicron BA.1 variant.

ABSTRACT Continued circulation of A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage in poultry has resulted in the diversification in multiple genetic and antigenic clades. Since 2009, clade 2.3.4.4 hemagglutinin (HA) containing viruses harboring the internal and neuraminidase (NA) genes of other avian influenza A viruses have been detected. As a result, various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8 have been identified. As of January 2023, 83 humans have been infected with A/H5N6 viruses, thereby posing an apparent risk for public health. Here, as part of a risk assessment, the in vitro and in vivo characterization of A/H5N6 A/black-headed gull/Netherlands/29/2017 is described. This A/H5N6 virus was not transmitted between ferrets via the air but was of unexpectedly high pathogenicity compared to other described A/H5N6 viruses. The virus replicated and caused severe lesions not only in respiratory tissues but also in multiple extra-respiratory tissues, including brain, liver, pancreas, spleen, lymph nodes, and adrenal gland. Sequence analyses demonstrated that the well-known mammalian adaptation substitution D701N was positively selected in almost all ferrets. In the in vitro experiments, no other known viral phenotypic properties associated with mammalian adaptation or increased pathogenicity were identified. The lack of transmission via the air and the absence of mammalian adaptation markers suggest that the public health risk of this virus is low. The high pathogenicity of this virus in ferrets could not be explained by the known mammalian pathogenicity factors and should be further studied. IMPORTANCE Avian influenza A/H5 viruses can cross the species barrier and infect humans. These infections can have a fatal outcome, but fortunately these influenza A/H5 viruses do not spread between humans. However, the extensive circulation and reassortment of A/H5N6 viruses in poultry and wild birds warrant risk assessments of circulating strains. Here an in-depth characterization of the properties of an avian A/H5N6 influenza virus isolated from a black-headed gull in the Netherlands was performed in vitro and in vivo, in ferrets. The virus was not transmissible via the air but caused severe disease and spread to extra-respiratory organs. Apart from the detection in ferrets of a mutation that increased virus replication, no other mammalian adaptation phenotypes were identified. Our results suggest that the risk of this avian A/H5N6 virus for public health is low. The underlying reasons for the high pathogenicity of this virus are unexplained and should be further studied.

ABSTRACT Norovirus is the primary cause of viral gastroenteritis (GE). To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of complete genomes. To investigate the potential of shotgun metagenomic sequencing on the Illumina platform for whole-genome sequencing, 71 reverse transcriptase quantitative PCR (RT-qPCR) norovirus positive-feces (threshold cycle [CT],

Zoonotic viral infections that cause severe disease or even death in some people may be asymptomatic or mild in reservoir hosts. Comparison of the pathogenesis of these two host categories may potentially explain the difference in disease. However, infections in reservoir hosts are often neglected. Therefore, we compared the pathogenesis of rabies virus, macacine alphaherpesvirus, West Nile virus, Puumala orthohantavirus, monkeypox virus, Lassa mammarenavirus, H5N1 highly pathogenic avian influenza, Marburg virus, Nipah virus, Middle East respiratory syndrome, and simian/human immunodeficiency viruses in both humans and reservoir hosts. We showed that most aspects of the pathogeneses were remarkably similar. The remaining differences lead to the identification of tipping points in the pathogeneses that are important for explaining the disease outcome in severe human cases. Further elucidating these tipping points by studying zoonotic viral infections in their reservoir hosts may teach us how to reduce the severity of zoonotic viral diseases in humans.

Clinical decision-making regarding isolation of SARS-CoV-2 patients is usually based on semiquantitative cycle-threshold (Ct) values without standardization. However, not all molecular assays produce Ct values, and there is ongoing discussion about whether Ct values can be safely used for decision-making. In this study, we standardized two molecular assays which use different nucleic acid amplification techniques (NAAT): the Hologic Aptima SARS-CoV-2/Flu (TMA) and Roche Cobas 6800 SARS-CoV-2 assays. We calibrated these assays against the first WHO international standard for SARS-CoV-2 RNA by using linear regression of log10 dilution series. These calibration curves were used to calculate viral loads for clinical samples. Clinical performance was assessed retrospectively using samples collected between January 2020 and November 2021, including known positives of the wild-type SARS-CoV-2 virus, the VOCs (alpha, beta, gamma, delta, and omicron) and quality control panels. Linear regression and Bland–Altman analysis showed good correlations for SARS-CoV-2 between Panther TMA and Cobas 6800 when standardized viral loads were used. These standardized quantitative results can benefit clinical decision-making and standardization of infection control guidelines.

Transmission of severe acute respiratory coronavirus-2 (SARS-CoV-2) between livestock and humans is a potential public health concern. We demonstrate the susceptibility of rabbits to SARS-CoV-2, which excrete infectious virus from the nose and throat upon experimental inoculation. Therefore, investigations on the presence of SARS-CoV-2 in farmed rabbits should be considered.

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.

Infections involving antibiotic resistant Staphylococcus aureus (S. aureus) represent a major challenge to successful treatment. Further, although bacteriophages (phages) could be an alternative to antibiotics, there exists a lack of correlation in phage susceptibility results between conventional in vitro and in vivo assays. This discrepancy may hinder the potential implementation of bacteriophage therapy. In this study, the susceptibility of twelve S. aureus strains to three commercial phage cocktails and two single phages was assessed. These S. aureus strains (including ten clinical isolates, five of which were methicillin-resistant) were compared using four assays: the spot test, efficiency of plating (EOP), the optical density assay (all in culture media) and microcalorimetry in human serum. In the spot test, EOP and optical density assay, all cocktails and single phages lysed both methicillin susceptible and methicillin resistant S. aureus strains. However, there was an absence of phage-mediated lysis in high concentrations of human serum as measured using microcalorimetry. As this microcalorimetry-based assay more closely resembles in vivo conditions, we propose that microcalorimetry could be included as a useful addition to conventional assays, thereby facilitating more accurate predictions of the in vivo susceptibility of S. aureus to phages during phage selection for therapeutic purposes.

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.

Many arboviruses, including viruses of the Flavivirus genus, are known to cause severe neurological disease in humans, often with long-lasting, debilitating sequalae in surviving patients. These emerging pathogens impact millions of people worldwide, yet still relatively little is known about the exact mechanisms by which they gain access to the human central nervous system. This review focusses on potential haematogenous and transneural routes of neuroinvasion employed by flaviviruses and identifies numerous gaps in knowledge, especially regarding lesser-studied interfaces of possible invasion such as the blood–cerebrospinal fluid barrier, and novel routes such as the gut–brain axis. The complex balance of pro-inflammatory and antiviral immune responses to viral neuroinvasion and pathology is also discussed, especially in the context of the hypothesised Trojan horse mechanism of neuroinvasion. A greater understanding of the routes and mechanisms of arboviral neuroinvasion, and how they differ between viruses, will aid in predictive assessments of the neuroinvasive potential of new and emerging arboviruses, and may provide opportunity for attenuation, development of novel intervention strategies and rational vaccine design for highly neurovirulent arboviruses.

Background: In the Netherlands, unrestricted access to direct-acting antivirals (DAAs) halved the incidence of acute hepatitis C virus (HCV) infections among HIV-infected men who have sex with men (MSM). To develop strategies that can further reduce the spread of HCV, it is important to understand the transmission dynamics of HCV. We used phylogenetic analysis of a dense sample of MSM to provide insight into the impact of unrestricted access to DAAs on HCV transmission in the Netherlands and in Belgium. Methods: We included 89 MSM that were recently infected with HCV genotype 1a in ten Dutch and one Belgian HIV treatment centers. Sequences were generated using next gene sequencing and Sanger sequencing. Maximum likelihood phylogenetic analysis (general time reversible model) was performed on concatenated NS5A and NS5B sequences and a reference set of 389 highly similar control sequences selected from GenBank. A cluster was based on a minimum bootstrap support of 90% and a 3% genetic distance threshold. Results: We found that 78 (88%) of individuals were part of seven major clusters. All clusters included individuals from across the study region, however, different cities were part of different clusters. In three clusters, HIV-negative MSM clustered with sequences from HIV-positive MSM. All clusters that were observed before the introduction of DAAs persisted after unrestricted access to DAAs became available. Conclusion: Recently acquired HCV infections among MSM in the Netherlands and Belgium are strongly clustered and therefore highly suitable for targeted prevention strategies, such as contact tracing and partner notification. Importantly, despite an HCV incidence reduction after high DAA uptake and continuously monitoring, HCV transmission persisted in the same clusters.

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.

Puumala orthohantavirus (PUUV) is endemic in Europe and can cause hemorrhagic fever with renal syndrome (nephropathia epidemica). Disease features include fever, thrombocytopenia, and acute kidney injury (AKI). This retrospective cohort study of forty PUUV patients aims to characterize associations of serum immunological, hemostatic or kidney injury markers to disease severity. While interleukin-18 (IL-18) was significantly increased in severely thrombocytopenic patients (9 platelets/L) compared to patients with higher platelet counts, RANTES was significantly decreased in these patients. These data suggest that patients with significant thrombocytopenia might have experienced pronounced Th1 immune responses. When kidney dysfunction was used as the primary disease outcome, recently identified AKI biomarkers (Cystatin C, insulin-like growth factor-binding protein 7, Nephrin, and trefoil factor 3) were significantly upregulated in patients with severe PUUV infection, defined as the estimated glomerular filtration rate (eGFR) below 30 m/min/1.73 m2. The increased expression of these markers specifically indicates pathology in glomeruli and proximal tubuli. Furthermore, E-selectin was significantly higher while interferon gamma-induced protein 10 (IP-10) was significantly lower in PUUV patients with more severe kidney dysfunction compared to patients with higher eGFR-values. Increased E-selectin illustrates the central role of endothelial cell activation, whereas decreased IP-10 could indicate a less important role of this cytokine in the pathogenesis of kidney dysfunction.

ABSTRACT Humans are infected with paramyxoviruses of different genera early in life, which induce cytotoxic T cells that may recognize conserved epitopes. This raises the question of whether cross-reactive T cells induced by antecedent paramyxovirus infections provide partial protection against highly lethal zoonotic Nipah virus infections. By characterizing a measles virus-specific but paramyxovirus cross-reactive human T cell clone, we discovered a highly conserved HLA-B*1501-restricted T cell epitope in the fusion protein. Using peptides, tetramers, and single cell sorting, we isolated a parainfluenza virus-specific T cell clone from a healthy adult and showed that both clones cleared Nipah virus-infected cells. We identified multiple conserved hot spots in paramyxovirus proteomes that contain other potentially cross-reactive epitopes. Our data suggest that, depending on HLA haplotype and history of paramyxovirus exposures, humans may have cross-reactive T cells that provide protection against Nipah virus. The effect of preferential boosting of these cross-reactive epitopes needs to be further studied in light of paramyxovirus vaccination studies. IMPORTANCE Humans encounter multiple paramyxoviruses early in life. This study shows that infection with common paramyxoviruses can induce T cells cross-reactive with the highly pathogenic Nipah virus. This demonstrates that the combination of paramyxovirus infection history and HLA haplotype affects immunity to phylogenetically related zoonotic paramyxoviruses.

Orthohantaviruses are zoonotic viruses that are naturally maintained by persistent infection in specific reservoir species. Although these viruses mainly circulate among rodents worldwide, spill-over infection to humans occurs. Orthohantavirus infection in humans can result in two distinct clinical outcomes: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). While both syndromes develop following respiratory transmission and are associated with multi-organ failure and high mortality rates, little is known about the mechanisms that result in these distinct clinical outcomes. Therefore, it is important to identify which cell types and tissues play a role in the differential development of pathogenesis in humans. Here, we review current knowledge on cell tropism and its role in pathogenesis during orthohantavirus infection in humans and reservoir rodents. Orthohantaviruses predominantly infect microvascular endothelial cells (ECs) of a variety of organs (lungs, heart, kidney, liver, and spleen) in humans. However, in this review we demonstrate that other cell types (e.g., macrophages, dendritic cells, and tubular epithelium) are infected as well and may play a role in the early steps in pathogenesis. A key driver for pathogenesis is increased vascular permeability, which can be direct effect of viral infection in ECs or result of an imbalanced immune response in an attempt to clear the virus. Future studies should focus on the role of identifying how infection of organ-specific endothelial cells as well as other cell types contribute to pathogenesis.

Background: Although the risk for transplacental transmission of SARS-CoV-2 is rare, placental infections with adverse functional consequences have been reported. This study aims to analyse histological placental findings in pregnancies complicated by SARS-CoV-2 infection and investigate its correlation with clinical symptoms and perinatal outcomes. We want to determine which pregnancies are at-risk to prevent adverse pregnancy outcomes related to COVID-19 in the future. Methods: A prospective, longitudinal, multicentre, cohort study. All pregnant women presenting between April 2020 and March 2021 with a nasopharyngeal RT-PCR-confirmed SARS-CoV-2 infection were included. Around delivery, maternal, foetal and placental PCR samples were collected. Placental pathology was correlated with clinical maternal characteristics of COVID-19. Results: Thirty-six patients were included, 33 singleton pregnancies (n = 33, 92%) and three twin pregnancies (n = 3, 8%). Twenty-four (62%) placentas showed at least one abnormality. Four placentas (4/39, 10%) showed placental staining positive for the presence of SARS-CoV-2 accompanied by a unique combination of diffuse, severe inflammatory placental changes with massive perivillous fibrin depositions, necrosis of syncytiotrophoblast, diffuse chronic intervillositis, and a specific, unprecedented CD20+ B-cell infiltration. This SARS-CoV-2 placental signature seems to correlate with foetal distress (75% vs. 15.6%, p = 0.007) but not with the severity of maternal COVID-19 disease. Conclusion: We describe a unique placental signature in pregnant patients with COVID-19, which has not been reported in a historical cohort. We show that the foetal environment can be seriously compromised by disruption of placental function due to local, devastating SARS-CoV-2 infection. Maternal clinical symptoms did not predict the severity of the SARS-CoV-2-related placental signature, resulting in a lack of adequate identification of maternal criteria for pregnancies at risk. Close foetal monitoring and pregnancy termination in case of foetal distress can prevent adverse pregnancy outcomes due to COVID-19 related placental disease.

Background: In 2015–2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. Methods: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. Results: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. Conclusions: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.

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

Metagenomic sequencing is a promising method to determine the virus diversity in environmental samples such as sewage or shellfish. However, to identify the short RNA genomes of human enteric viruses among the large diversity of nucleic acids present in such complex matrices, method optimization is still needed. This work presents methodological developments focused on norovirus, a small ssRNA non-enveloped virus known as the major cause of human gastroenteritis worldwide and frequently present in human excreta and sewage. Different elution protocols were applied and Illumina MiSeq technology were used to study norovirus diversity. A double approach, agnostic deep sequencing and a capture-based approach (VirCapSeq-VERT) was used to identify norovirus in environmental samples. Family-specific viral contigs were classified and sorted by SLIM and final norovirus contigs were genotyped using the online Norovirus genotyping tool v2.0. From sewage samples, 14 norovirus genogroup I sequences were identified of which six were complete genomes. For norovirus genogroup II, nine sequences were identified and three of them comprised more than half of the genome. In oyster samples bioaccumulated with these sewage samples, only the use of an enrichment step during library preparation allowed successful identification of nine different sequences of norovirus genogroup I and four for genogroup II (>500 bp). This study demonstrates the importance of method development to increase virus recovery, and the interest of a capture-based approach to be able to identify viruses present at low concentrations.

Zika virus (ZIKV) infection is typically characterized by a mild disease presenting with fever, maculopapular rash, headache, fatigue, myalgia, and arthralgia. A recent animal study found that ZIKV-infected pregnant Ifnar−/−mice developed vascular damage in the placenta and reduced amount of fetal capillaries. Moreover, ZIKV infection causes segmental thrombosis in the umbilical cord of pregnant rhesus macaques. Furthermore, several case reports suggest that ZIKV infection cause coagulation disorders. These results suggest that ZIKV could cause an alteration in the host hemostatic response, however, the mechanism has not been investigated thus far. This paper aims to determine whether ZIKV infection on HUVECs induces apoptosis and elevation of tissue factor (TF) that leads to activation of secondary hemostasis. We infected HUVECs with two ZIKV strains and performed virus titration, immunostaining, and flow cytometry to confirm and quantify infection. We measured TF concentrations with flow cytometry and performed thrombin generation test (TGT) as a functional assay to assess secondary hemostasis. Furthermore, we determined the amount of cell death using flow cytometry. We also performed enzyme-linked immunosorbent assay (ELISA) to determine interleukin (IL)-6 and IL-8 production and conducted blocking experiments to associate these cytokines with TF expression. Both ZIKV strains infected and replicated to high titers in HUVECs. We found that infection induced elevation of TF expressions. We also showed that increased TF expression led to shortened TGT time. Moreover, the data revealed that infection induced apoptosis. In addition, there was a significant increase of IL-6 and IL-8 production in infected cells. Here we provide in vitro evidence that infection of HUVECs with ZIKV induces apoptosis and elevation of TF expression that leads to activation of secondary hemostasis.

Experiments in which complex virome sequencing data is generated remain difficult to explore and unpack for scientists without a background in data science. The processing of raw sequencing data by high throughput sequencing workflows usually results in contigs in FASTA format coupled to an annotation file linking the contigs to a reference sequence or taxonomic identifier. The next step is to compare the virome of different samples based on the metadata of the experimental setup and extract sequences of interest that can be used in subsequent analyses. The viromeBrowser is an application written in the opensource R shiny framework that was developed in collaboration with end-users and is focused on three common data analysis steps. First, the application allows interactive filtering of annotations by default or custom quality thresholds. Next, multiple samples can be visualized to facilitate comparison of contig annotations based on sample specific metadata values. Last, the application makes it easy for users to extract sequences of interest in FASTA format. With the interactive features in the viromeBrowser we aim to enable scientists without a data science background to compare and extract annotation data and sequences from virome sequencing analysis results.

Norovirus infections are a leading cause of acute gastroenteritis worldwide, affecting people of all ages. There are 10 norovirus genogroups (GI-GX) that infect humans and animals in a host-specific manner. New variants and genotypes frequently emerge, and their origin is not well understood. One hypothesis is that new human infections may be seeded from an animal reservoir, as human noroviruses have occasionally been detected in animal species. The majority of these sequences were identified as older GII.4 variants, but a variety of other GIIs and GIs have been detected as well. While these sequences share at least 94% nt similarity with human strains, most of them are >98% identical to human strains. The fact that these strains were detected in animals after they had been detected through human surveillance to be already circulating in humans suggests human-to-animal transmission.

A majority of emerging infectious diseases are of zoonotic origin. Metagenomic Next-Generation Sequencing (mNGS) has been employed to identify uncommon and novel infectious etiologies and characterize virus diversity in human, animal, and environmental samples. Here, we systematically reviewed studies that performed viral mNGS in common livestock (cattle, small ruminants, poultry, and pigs). We identified 2481 records and 120 records were ultimately included after a first and second screening. Pigs were the most frequently studied livestock and the virus diversity found in samples from poultry was the highest. Known animal viruses, zoonotic viruses, and novel viruses were reported in available literature, demonstrating the capacity of mNGS to identify both known and novel viruses. However, the coverage of metagenomic studies was patchy, with few data on the virome of small ruminants and respiratory virome of studied livestock. Essential metadata such as age of livestock and farm types were rarely mentioned in available literature, and only 10.8% of the datasets were publicly available. Developing a deeper understanding of livestock virome is crucial for detection of potential zoonotic and animal pathogens and One Health preparedness. Metagenomic studies can provide this background but only when combined with essential metadata and following the “FAIR” (Findable, Accessible, Interoperable, and Reusable) data principles.

ABSTRACT Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection). IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.

Metagenomics poses opportunities for clinical and public health virology applications by offering a way to assess complete taxonomic composition of a clinical sample in an unbiased way. However, the techniques required are complicated and analysis standards have yet to develop. This, together with the wealth of different tools and workflows that have been proposed, poses a barrier for new users. We evaluated 49 published computational classification workflows for virus metagenomics in a literature review. To this end, we described the methods of existing workflows by breaking them up into five general steps and assessed their ease-of-use and validation experiments. Performance scores of previous benchmarks were summarized and correlations between methods and performance were investigated. We indicate the potential suitability of the different workflows for (1) time-constrained diagnostics, (2) surveillance and outbreak source tracing, (3) detection of remote homologies (discovery), and (4) biodiversity studies. We provide two decision trees for virologists to help select a workflow for medical or biodiversity studies, as well as directions for future developments in clinical viral metagenomics.

ABSTRACT Recent Zika virus (ZIKV) infections have been associated with a range of neurological complications, in particular congenital microcephaly. Human neural progenitor cells (hNPCs) are thought to play an important role in the pathogenesis of microcephaly, and experimental ZIKV infection of hNPCs has been shown to induce cell death. However, the infection efficiency and rate of cell death have varied between studies, which might be related to intrinsic differences between African and Asian lineage ZIKV strains. Therefore, we determined the replication kinetics, including infection efficiency, burst size, and ability to induce cell death, of two Asian and two African ZIKV strains. African ZIKV strains replicated to higher titers in Vero cells, human glioblastoma (U87MG) cells, human neuroblastoma (SK-N-SH) cells, and hNPCs than Asian ZIKV strains. Furthermore, infection with Asian ZIKV strains did not result in significant cell death early after infection, whereas infection with African ZIKV strains resulted in high percentages of cell death in hNPCs. The differences between African and Asian lineage ZIKV strains highlight the importance of including relevant ZIKV strains to study the pathogenesis of congenital microcephaly and caution against extrapolation of experimental data obtained using historical African ZIKV strains to the current outbreak. Finally, the fact that Asian ZIKV strains infect only a minority of cells with a relatively low burst size together with the lack of early cell death induction might contribute to its ability to cause chronic infections within the central nervous system (CNS). IMPORTANCE The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells—which are important target cells in the development of congenital microcephaly—less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly.

Norovirus is the most common cause of non-bacterial gastroenteritis and is a burden worldwide. The increasing norovirus diversity is currently categorized into at least 10 genogroups which are further classified into more than 40 genotypes. In addition to humans, norovirus can infect a broad range of hosts including livestock, pets, and wild animals, e.g., marine mammals and bats. Little is known about norovirus infections in most non-human hosts, but the close genetic relatedness between some animal and human noroviruses coupled with lack of understanding where newly appearing human norovirus genotypes and variants are emerging from has led to the hypothesis that norovirus may not be host restricted and might be able to jump the species barrier. We have systematically reviewed the literature to describe the diversity, prevalence, and geographic distribution of noroviruses found in animals, and the pathology associated with infection. We further discuss the evidence that exists for or against interspecies transmission including surveillance data and data from in vitro and in vivo experiments.