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Background: Ticks carry a variety of microorganisms, some of which are pathogenic to humans. The human risk of tick-borne diseases depends on, among others, the prevalence of pathogens in ticks biting humans. To follow-up on this prevalence over time, a Belgian study from 2017 was repeated in 2021., Methods: During the tick season 2021, citizens were invited to have ticks removed from their skin, send them and fill in a short questionnaire on an existing citizen science platform for the notification of tick bites (TekenNet). Ticks were morphologically identified to species and life stage level and screened using multiplex qPCR targeting, among others, Borrelia burgdorferi (sensu lato), Anaplasma phagocytophilum, Borrelia miyamotoi, Neoehrlichia mikurensis, Babesia spp., Rickettsia helvetica and tick-borne encephalitis virus (TBEV). The same methodology as in 2017 was used., Results: In 2021, the same tick species as in 2017 were identified in similar proportions; of 1094 ticks, 98.7% were Ixodes ricinus, 0.8% Ixodes hexagonus and 0.5% Dermacentor reticulatus. A total of 928 nymphs and adults could be screened for the presence of pathogens. Borrelia burgdorferi (s.l.) was detected in 9.9% (95% CI 8.2-12.0%), which is significantly lower than the prevalence of 13.9% (95% CI 12.2-15.7%) in 2017 (P = 0.004). The prevalences of A. phagocytophilum (4.7%; 95% CI 3.5-6.3%) and R. helvetica (13.3%; 95% CI 11.2-15.6%) in 2021 were significantly higher compared to 2017 (1.8%; 95% CI 1.3-2.7% and 6.8%; 95% CI 5.6-8.2% respectively) (P < 0.001 for both). For the other pathogens tested, no statistical differences compared to 2017 were found, with prevalences ranging between 1.5 and 2.9% in 2021. Rickettsia raoultii was again found in D. reticulatus ticks (n = 3/5 in 2021). Similar to 2017, no TBEV was detected in the ticks. Co-infections were found in 5.1% of ticks. When combining co-infection occurrence in 2017 and 2021, a positive correlation was observed between B. burgdorferi (s.l.) and N. mikurensis and B. burgdorferi (s.l.) and B. miyamotoi (P < 0.001 for both)., Conclusions: Although the 2021 prevalences fell within expectations, differences were found compared to 2017. Further research to understand the explanations behind these differences is needed., (© 2024. The Author(s).)

We conducted a multicentre hospital-based test-negative case-control study to measure the effectiveness of adapted bivalent COVID-19 mRNA vaccines against PCR-confirmed SARS-CoV-2 infection during the Omicron XBB lineage-predominant period in patients aged ≥ 60 years with severe acute respiratory infection from five countries in Europe. Bivalent vaccines provided short-term additional protection compared with those vaccinated > 6 months before the campaign: from 80% (95% CI: 50 to 94) for 14-89 days post-vaccination, 15% (95% CI: -12 to 35) at 90-179 days, and lower to no effect thereafter.

Background: To support the COVID-19 pandemic response, many countries, including Belgium, implemented baseline genomic surveillance (BGS) programs aiming to early detect and characterize new SARS-CoV-2 variants. In parallel, Belgium maintained a sentinel network of six hospitals that samples patients with severe acute respiratory infections (SARI) and integrated SARS-CoV-2 detection within a broader range of respiratory pathogens. We evaluate the ability of the SARI surveillance to monitor general trends and early signals of viral genetic evolution of SARS-CoV-2 and compare it with the BGS as a reference model., Methods: Nine-hundred twenty-five SARS-CoV-2 positive samples from patients fulfilling the Belgian SARI definition between January 2020 and December 2022 were sequenced using the ARTIC Network amplicon tiling approach on a MinION platform. Weekly variant of concern (VOC) proportions and types were compared to those that were circulating between 2021 and 2022, using 96,251 sequences of the BGS., Results: SARI surveillance allowed timely detection of the Omicron (BA.1, BA.2, BA.4, and BA.5) and Delta (B.1.617.2) VOCs, with no to 2 weeks delay according to the start of their epidemic growth in the Belgian population. First detection of VOCs B.1.351 and P.1 took longer, but these remained minor in Belgium. Omicron BA.3 was never detected in SARI surveillance. Timeliness could not be evaluated for B.1.1.7, being already major at the start of the study period., Conclusions: Genomic surveillance of SARS-CoV-2 using SARI sentinel surveillance has proven to accurately reflect VOCs detected in the population and provides a cost-effective solution for long-term genomic monitoring of circulating respiratory viruses., Competing Interests: The authors declare no conflict of interest., (© 2023 Sciensano and The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (10 7 , 10 5 and 10 3 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Purple non-sulfur bacteria (PNSB) are recognized as a highly versatile group of bacteria that assimilate a broad range of carbon sources. Growing heterotrophically, PNSB such as Rhodospirillum rubrum ( Rs. rubrum ) generate reduced equivalents that are used for biomass production. However, under photoheterotrophic conditions, more reduced electron carriers than required to produce biomass are generated. The excess of reduced equivalents still needs to be oxidized for the metabolism to optimally operate. These metabolic reactions are known as electron sinks. Most PNSB rely on the CO 2 -fixing Calvin cycle and H 2 production to oxidize these reduced equivalents. In addition to these well-described electron sinks, the involvement of some pathways, such as polyhydroxyalkanoate (PHA) biosynthesis, in redox poise is still controversial and requires further studies. Among them, isoleucine biosynthesis has been recently highlighted as one of these potential pathways. Here, we explore the role of isoleucine biosynthesis in Rs. rubrum . Our results demonstrate that the isoleucine content is higher under illuminated conditions and that submitting Rs. rubrum to light stress further increases this phenomenon. Moreover, we explore the production of (p)ppGpp in Rs. rubrum and its potential link with light stress. We further demonstrate that a fully functional isoleucine biosynthesis pathway could be an important feature for the onset of Rs. rubrum growth under photoheterotrophic conditions even in the presence of an exogenous isoleucine source. Altogether, our data suggest that isoleucine biosynthesis could play a key role in redox homeostasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bayon-Vicente, Marchand, Ducrotois, Dufrasne, Hallez, Wattiez and Leroy.)

The cytoplasmic tail (CT) of the HIV-2 envelope glycoprotein (Env) includes amino acid (aa) sequences that are similar to lentiviral lytic peptides (LLP) described in other lentiviruses. Within the putative LLP-2 region, we previously observed insertions of 3 or 7 aa in sequences deduced from plasma viral RNA of symptomatic HIV-2-infected individuals. Based on these observations, we reproduced the insertions in a molecular clone to assess their impact on replicative fitness and cell death in vitro. Using a molecular clone of the HIV-2 ROD reference strain, site-directed mutagenesis experiments allowed the generation of plasmids with the insertion L 791 TAI or L 791 QRALTAI in the Env protein. The clone with 7 aa insertion enhanced viral release 8 to 11 times in infected T cells and cell viability was impaired by more than 20%, compared with the wild-type HIV-2 ROD virus. The effect of the 3 aa insertion was milder, with a nonsignificant trend to enhance viral replication and cell death compared with the wild-type virus. Interestingly, the insertions in the Env proteins did not induce a significant increase of viral infectivity, as revealed by the infectivity assay using TZM-bl cells. The insertions in the Env CT observed in vivo from disease progressors may, therefore, be involved in the higher viral load observed in these individuals. This study may open the way to the development of a prognostic marker related to the HIV-2 infection progression.

Introduction: The WHO urges action against the threat posed by HIV drug resistance. It is well known that the sensitivity of Next-Generation Sequencing (NGS) is greater than that of Sanger Sequencing (SS). The objective of this study was to evaluate the performance of the novel NGS HIV-1 drug resistance monitoring system., Materials & Methods: NGS analyses were performed on 67 plasma samples from HIV-1 infected patients using the Sentosa SQ HIV Genotyping Assay from Vela-Dx. This kit was used on a semi-automated Ion Torrent-based platform. Sequences were compared to those obtained by SS. Samples were analysed in the same and in separate runs. Quality controls (QC) were added to control sequencing processes of protease (PRO), reverse transcriptase (RT) and integrase (INT) regions., Results: Of the 41 analysed samples, 33 (80.5%) had identical drug resistance interpretation reports. Discrepant results were observed for eight samples. Five of them were only detected by NGS and had drug resistance mutations (DRMs) with an allelic frequency below the limit of detection of the SS method (between 6.3 to 20.5%). Two DRMs were only identified using the SS method. The sequences were similar in 98.2% of cases (counting variants as mismatches) and homologous in 99.9% if missed variants. Duplicated samples in a single run were similar in 95.7% (99.9%) of cases. Duplicated samples in two different runs were 98% (100%) homologous. QC results were manually assessed with a score of 340/340 for detection of DRMs in PRO and RT and 100% for INT sequencing., Conclusions: This is the first preliminary evaluation in Belgium employing the Sentosa SQ HIV Genotyping Assay. The NGS appears to be a promising tool for the detection of DRMs in HIV-1 patients and showed a higher sensitivity compared to SS. Large studies assessing the clinical relevance of low frequency DRMs are needed., Competing Interests: The authors have declared that no competing interests exist.

The HIVs have evolved by selecting means to hijack numerous host cellular factors. HIVs exploit the transcription factor NF-κB to ensure efficient LTR-driven gene transcription. However, NF-κB is primarily known to act as a key regulator of the proinflammatory and antiviral responses. Interestingly, retroviruses activate NF-κB during early stages of infection to initiate proviral genome expression while suppressing it at later stages to restrain expression of antiviral genes. During HIV-1 infection, diverse viral proteins such as Env, Nef and Vpr have been proposed to activate NF-κB activity, whereas Vpu has been shown to inhibit NF-κB activation. It is still unclear how HIV-2 regulates NF-κB signaling pathway during its replication cycle. Here we confirm that human BST-2 and HIV-1 Env proteins can trigger potent activation of NF-κB. Importantly, we demonstrate for the first time that the HIV-2 Env induces NF-κB activation in HEΚ293T cells. Furthermore, the anti-BST-2 activity of the HIV-2 Env is not sufficient to completely inhibit NF-κB activity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail., Competing Interests: The authors declare no conflict of interest.