Used paper tissues for pathogen identification in acute respiratory infection. (preprint)

We investigated the potential of used paper tissues as a non-invasive sampling method for the diagnosis of acute respiratory infections. The method allowed the identification and typing of respiratory pathogens in symptomatic individuals, as well as in collective samples taken at a community level. The collection of used paper tissues could therefore be useful in epidemiological surveillance for SARS-CoV-2 and other respiratory pathogens such as influenzavirus, respiratory syncytial virus, entero/rhinoviruses and Streptococcus pneumoniae.

Epidemiological surveillance of respiratory pathogens in wastewater in Belgium (preprint)

ABSTRACT Background Detailed information on the circulation of respiratory viruses in the community is crucial to gain better understanding of the burden of respiratory infections on society. Methods By using an in-house respiratory panel for simultaneous detection of 29 respiratory pathogens (22 viruses and 7 bacteria/fungi), we explored the possibility to use wastewater sampling to monitor the circulation of respiratory pathogens at population level. Results We were able to detect all respiratory viruses included in the panel (influenza A, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza viruses (PIV) 1-4, adenovirus (Adv), human bocavirus (HBoV), enterovirus/rhinovirus (EV/RV), enterovirus D68 (EV-D68), parechovirus (HPeV), human coronaviruses (HCoV)-NL63, -229E, -OC43, -HKU-1 and -SARS, cytomegalovirus (CMV) and herpes simplex virus (HSV)-1 and -2), except for influenza B and HCoV MERS which were not circulating in Belgium during the two year study period. An upsurge of EV-D68 infections in Europe in September 2021 was clearly reflected in the wastewater samples. For the viruses where epidemiological data on virus circulation in Belgium were available, these matched the wastewater data. The wastewater pretreatment that was used, optimized for viral enrichment, was as such not suited for the surveillance of bacteria and fungi. Conclusions Community circulation levels of respiratory viruses were well reflected in wastewater samples, indicating that wastewater-based epidemiology can be a valuable tool in the epidemiology and management of respiratory infections.

Sequencing from leftover material of stored rapid Ag Tests (Ag-RDTs): a gamechanger in genomic surveillance? (preprint)

ABSTRACT Archived lateral flow antigen-detection rapid diagnostic tests (Ag-RDTs), used in the diagnosis of COVID-19, were successfully used to extract viral nucleic acids for subsequent RT-qPCR and sequencing by Sanger or Nanopore whole genome sequencing (WGS). The method was successfully applied with different brands of SARS-CoV-2 Ag-RDTs, but also with Ag-RDTs for detection of influenza, rotavirus and adenovirus 40/41. The buffer used in the Ag-RDT had an important influence on the RNA yield from the test stripand the efficiency of subsequent sequencing. Our finding that the test strip in rapid Ag tests is suited to preserve viral genomic material, even for months at room temperature, and therefore can serve as source material for genetic characterization, could improve global coverage of genomic surveillance for SARS-CoV-2 as well as for other viruses.

Environmental circulation of adenovirus 40/41 and SARS-CoV-2 in the context of the emergence of acute hepatitis of unknown origin (preprint)

The recent surge of hepatitis of unknown origin in children is hypothesized to be caused by adenovirus 41 and/or SARS-CoV-2 infections. A relatively high proportion of patients testing positive for these viruses concomitantly with the development of acute hepatitis supports this hypothesis. To formally incriminate these viral infections as causative agents of hepatitis, both a plausible physiopathological pathway and supporting epidemiological dynamics in the community need demonstration. In this study, we measured the level of circulation of adenovirus 40/41 and SARS-CoV-2 in the general population of the city of Leuven in Belgium using wastewater monitoring between December 2020 and May 2022 and indoor air sampling in day care centers between November 2021 and May 2022. We also retrospectively analyzed medical records of 12.672 children attending a tertiary hospital draining the same region between January 2019 and April 2022. Our results demonstrate a recent but modest increase in hepatitis of unknown origin concomitant with a surge of circulating adenovirus 41 and SARS-CoV-2 in the general population, including in children under 5.

Detection Of SARS-CoV-2 Variants Of Concern In Wastewater Of Leuven, Belgium (preprint)

To investigate whether wastewater surveillance can be used as an early warning system to detect a rise in SARS-CoV-2 positive cases, and to follow the circulation of specific variants of concern (VOC) in particular geographical areas, wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. In two residential sampling sites, a rise in viral SARS-CoV-2 copies in wastewater preceded the peaks in positive cases. In the WWTP, peaks in the wastewater viral load were seen simultaneous with the waves op positive cases caused by the original Wuhan SARS-CoV-2 strain, the Alpha variant and the Delta variant. For the Omicron BA.1 variant associated wave, the viral load in wastewater increased to a lesser degree, and much later than the increase in positive cases, which could be attributed to a lower level of fecal excretion, as measured in hospitalized patients. Circulation of SARS-CoV-2 VOCs (Alpha, Delta and Omicron) could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. We found that wastewater-based surveillance is a sensitive tool to monitor SARS-CoV-2 circulation levels and VOCs in larger regions. This can prove to be highly valuable in times of reducing testing capacity. Differences in excretion levels of various SARS-CoV-2 variants should however be taken into account when using wastewater surveillance to monitor SARS-CoV-2 circulation levels in the population.

Comprehensive immunovirological and environmental screening reveals risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks (preprint)

COVID-19 vaccination has resulted in excellent protection against fatal disease, including in the elderly. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases) by combining SARS-CoV-2 aerosol monitoring, whole-genome phylogenetic analysis, and immunovirological profiling by digital nCounter transcriptomics. Phylogenetic investigations indicated each outbreak stemmed from a single introduction event, though with different variants (Delta, Gamma, and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 days 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 signatures and reanalysis of single-cell RNAseq data highlights the unique 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.

Genetic diversity and evolution of SARS-CoV-2 in Belgium during the first wave outbreak (preprint)

SARS-CoV-2, the causative agent of COVID-19 was first detected in Belgium on 3rd February 2020, albeit the first epidemiological wave started in March and ended in June 2020. One year after the first epidemiological wave hit the country data analyses reveled the temporal and variant distribution of SARS-CoV-2 and its implication with Belgian epidemiological measures. In this study, 766 complete SARS-CoV-2 genomes of samples originating from the first epidemiological were sequenced to characterize the temporal and geographic distribution of the COVID-19 pandemic in Belgium through phylogenetic and variant analysis. Our analysis reveals the presence of the major circulating SARS-CoV-2 clades (G, GH and GR) and lineages circulating in Belgium at that time. Moreover, it contextualizes the density of SARS-CoV-2 cases over time with non-intervention measures taken to prevent the spread of SARS-CoV-2 in Belgium, specific international case imports and the functional implications of the most representative non-synonymous mutations present in Belgium between February to June 2020.

The clinically validated viral superinfection therapy (SIT) platform technology could cure early cases of COVID-19 disease (preprint)

Currently, SARS-CoV-2 infection which is the causative agent for COVID-19 disease is a worldwide pandemic with more than 100 million global cases and more than 2.0 million deaths (as of January, 2021). While several vaccines for prevention of COVID-19 have already been registered by the regulatory authorities, the problem is that the substitution rate of this virus is estimated to be one change per 2 weeks, thus mutations could arise that threaten the efficacy of vaccines. Unfortunately, there is no current evidence from random clinical trials to recommend any specific post-exposure treatment for patients with suspected or confirmed COVID-19 disease. Here we propose an innovative superinfection therapeutic (SIT) strategy, which could complement the development of prophylactic vaccines. SIT is based on clinical observations that unrelated harmless viruses might interact in patients infected with pathogenic virus. During SIT, the patient benefits from superinfection with an apathogenic double-stranded RNA (dsRNA) virus such as the infectious bursal disease virus (IBDV), which is a powerful activator of the interferon-dependent antiviral gene program. An attenuated vaccine strain of IBDV was already successfully administered to resolve acute and persistent infections induced by two completely different viruses, the hepatitis B (DNA) and C (RNA) viruses (HBV/HCV). The safety of orally administered acid-resistant IBDV strain R903/78 reverse engineered viral drug candidate was demonstrated in 10 stage IV cancer patients who exhausted all conventional therapy. Following repeated oral administration of the virus up to 109 infectious units (IU)/ dose, only mild flu-like side effects were reported in some patients. Proof-of-principle efficacy was demonstrated in an early COVID-19 patient who was successfully treated with 3x106 IU of an attenuated IBDV vaccine. A small scale dose-finding Phase I safety study is proposed.

At least seven distinct rotavirus genotype constellations in bats with evidence of reassortment and zoonotic transmissions (preprint)

ABSTRACT Bats host many viruses pathogenic to humans, and increasing evidence suggests that Rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America and Africa were PCR-positive for RVA and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, including evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite varying levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses. Importance The increased research on bat coronaviruses after SARS-CoV and MERS-CoVallowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents shed light on the vast genetic diversity of rotaviruses and also hinted at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

Early experiences with antibody testing in a Flemish nursing home during an acute COVID-19 outbreak: a retrospective cohort study. (preprint)

objectives: to assess the prevalence of COVID-19 (PCR-test) in residents and staff of a nursing home. To examine the presence of IgM and IgG antibodies in the sample and the relation between PCR and antibody test results. design: cross-sectional and (retrospective) cohort study setting: a nursing home for the elderly Bessemerberg in Lanaken (Belgium) with up to 130 beds. Lanaken is situated in the Belgian province with the highest COVID-19 prevalence. participants: residents (N=108) and staff members (N=93) of the nursing home outcomes: PCR, IgM and IgG results: the prevalence of COVID-19, based on PCR test was 34% (N=40) for residents and 13% (N=11) for staff members, respectively. Of the residents, 13% showed positive IgM results and 15% positive IgG results. In 17% of the residents, at least one of the antibodies was positive. In total 13% of the staff members had positive IgM and 16% had a positive IgG. In 20% of the staff members at least one of these antibody tests was positive. In PCR positive residents, the percentage of IgM positive, IgG positive, and at least one of both was 28%, 34%, and 41%. In PCR positive staff, we found 30%, 60%, and 60%. Additional antibody tests were performed in nine residents between day 11 and 14 after the positive PCR test. Of those, 7 (78%) tested positive on at least one antibody. When retesting three weeks later, all remaining residents also tested positive. conclusions: Recently it was reported that in Belgium antibodies are present in 3-4% of the general population. Although, the prevalence in our residents is higher, the number is largely insufficient for herd immunity. In staff members of the regional hospital the prevalence of antibodies was 6%. The higher prevalence in nursing home staff (21%) may be related to the complete absence of good quality protection in the first weeks of the outbreak.

STAT2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in SARS-CoV-2 infected hamsters (preprint)

Introductory paragraphSince the emergence of SARS-CoV-2 causing COVID-19, the world is being shaken to its core with numerous hospitalizations and 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 productive SARS-CoV-2 infection in the lungs of mice is limited and restricted by early type I interferon responses. In contrast, we show that Syrian hamsters are highly permissive to SARS- CoV-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. Moreover, we identify an exuberant innate immune response as a 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. Finally, we assess SARS-CoV- 2-induced lung pathology in hamsters by micro-CT alike used in clinical practice. 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.