We evaluated the BOSCH Vivalytic MPXV assay using serial dilutions of monkeypox virus clade Ib, a new offshoot of clade I identified in 2023. The assay detected viral DNA down to ~100 copies/mL demonstrating comparable analytical sensitivity to our in-house reference PCR and to other commercial platform-based mpox molecular assays. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.

Background The 2026 Andes virus (ANDV) outbreak linked to the cruise ship m/v Hondius triggered prolonged diagnostic follow-up of contacts in several countries. ANDV severity, long incubation, and person-to-person spread complicate case ascertainment. The cross-continent introduction of a rare transmissible virus with limited laboratory readiness is a paramount scenario in pandemic preparedness. To build response capacity under the increased regulatory demands of the European in-vitro diagnostic medical devices regulation (IVDR), we conducted an ad-hoc validation of an investigational RT-PCR product for ANDV detection. Methods Members of the EU Reference Laboratory for Vectorborne Viral Pathogens, the HERA network DURABLE, and the German network PAKOP did a rapid survey of hantavirus diagnostic capacity, a sequence-informed review of molecular in-house assays, and a decentralised validation of a strain-specific research-use-only (RUO) RT-PCR product. We tested biobanked patient samples, differential diagnostic materials, and virus cultures; analytical sensitivity was assessed by probit analysis of quantified in-vitro-transcribed RNA. Findings Networks enabled rapid exchange of methods and reference materials, revealing heterogeneous capacity. The RUO assay was evaluated on 302 clinical specimens and 120 cultured materials covering seven Orthohantavirus species and 67 non-hantavirus pathogens. No specificity issues arose; 37 of 38 ANDV samples were positive. Among non-Andes hantaviruses, only a Sin Nombre virus isolate was positive. The 95% limit of detection was 10·65 copies per reaction. Patient-derived material showed concordance with an established in-house RT-PCR. Interpretation Coordinated expert networks can deliver a swift laboratory response and generate ad-hoc validation data for diagnostic products entering regulatory channels. Network-based validation will probably become essential in future European outbreaks under the IVDR.

Crimean–Congo hemorrhagic fever (CCHF) is a high-fatality zoonotic infection. Iraq has experienced a substantial increase in reported CCHF cases since 2021, yet predictors of mortality and the feasibility of existing severity scores in routine care have not been systematically assessed. We aimed to identify demographic, clinical and laboratory predictors of death among confirmed CCHF patients in Iraq and to develop a simplified severity score suitable for bedside use in resource-constrained settings. We analyzed 273 laboratory-confirmed CCHF cases with known outcomes reported to the Iraqi national surveillance system from 1 January 2021 to 31 December 2024 (273/1,193 confirmed cases with complete standardized forms). Demographic, exposure, clinical, and laboratory variables (including platelet count and cycle threshold [Ct] values) were extracted from harmonized Ministry of Health and Central Public Health Laboratory databases. Univariable and multivariable logistic regression were used to identify mortality predictors. Receiver Operating Characteristic (ROC) curve analysis compared the discriminative performance (area under the curve, AUC) of newly proposed scores with existing CCHF Severity Scoring Index (SSI) and Swanepoel’s Grading Score (SGS). The overall case fatality rate was 12.1

The outbreak of hantavirus disease caused by Andes virus aboard a cruise ship is a reminder of the challenges posed by emerging diseases in the modern era. While Andes virus-associated disease can be particularly severe, it is unlikely to spread extensively beyond the current number of cases or emerge as a large epidemic, especially if public health measures are followed. Nonetheless, the outbreak exemplifies the complexity of international outbreak response with differences in national preparedness frameworks and the rapid spread of mis-/disinformation. We discuss this outbreak in the context of global epidemic and pandemic preparedness and emphasize the importance of sustained, inclusive global collaborative One Health approaches to preparedness and response. We stress the urgent need for global coordination, discuss specific challenges, and provide recommendations for further strengthening of global preparedness.

Background Following an increase in mpox clade Ib cases in several African countries, the World Health Organization recognized mpox as a public health emergency of international concern, highlighting the need for reliable and accessible diagnostic tools. As several mpox clades are co-circulating in endemic countries, the analytical sensitivity of diagnostic assays should be evaluated for all of them in a comparative manner. Methods This study evaluated the analytical sensitivity of three rapid tests (Flowflex Monkeypox virus rapid test from ACON Biotech, Monkeypox antigen rapid test from Assure Tech, and Standard Q Monkeypox Ag Test from SD Biosensor) and two point-of care molecular tests (SD Biosensor's M10 and Cepheid's Xpert MPOX assays) using serial dilutions of viral stocks from the four clades of Monkeypox virus (MPXV:) Ia, Ib, IIa, and IIb. Findings Upon our analytical comparative benchmarking, the three rapid tests demonstrated comparable analytical sensitivity for all four clades, with a limit of detection of approximately 1,000,000 DNA copies/mL or 1,000 FFU/mL. The two molecular tests demonstrated also comparable analytical sensitivity to an in-house PCR assay for all four clades, detecting concentrations down to 10-100 DNA copies per mL, corresponding to a non-detectable titer of infectious particles. The Xpert assay detected the Clade Ib strain only through its orthopoxvirus target and not its MPXV target, and none of the assays could distinguish between MPXV clades. Interpretation No differences in sensitivity was observed across MPXV clades neither for Ag-RDTs nor for molecular POCTs. However, the potential of simple, affordable tests, such as Ag-RDTs, is limited by poor sensitivity while the use of highly sensitive POC molecular platforms remains limited by their cost. To date, the lack of accurate, affordable POC MPXV-specific assays with potential to differentiate clades, limits diagnostic capacities as well as our understanding of the virus and its epidemiology. Funding This work was supported by FIND and internal funds of the Centre for Emerging Viral Diseases. Mpox diagnostic tests were provided by FIND and the World Health Organization (WHO). MOD was supported by Schmidt Science Fellows, in partnership with the Rhodes Trust. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by FIND and internal funds of the Centre for Emerging Viral Diseases. Mpox diagnostic tests were provided by FIND and the World Health Organization (WHO). MOD was supported by Schmidt Science Fellows, in partnership with the Rhodes Trust. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript

Hybrid immunity offers stronger and more durable antibody-mediated protection against symptomatic SARS-CoV-2 infection. In the present study, we report the development of a durable antibody response in an individual with hybrid immunity who also received three doses of the prototype COVID-19 vaccine. Polyclonal plasma antibody obtained from this donor also showed extraordinary neutralization breadth against contemporary Omicron variants. One of the functional monoclonal antibodies (ATHS-C30) isolated from this individual, representing the IGHV4-30 lineage-specific B cell with strong binding affinity to JN.1 spike protein, showed extraordinary neutralization breadth, including variants of Omicron lineages that emerged beyond JN.1, such as KP.2, KP.3.1.1, KP.3.2, KP.3.3, LB.1 and XEC. ATHS-C30 was found to bind RBD with high affinity and showed distinct epitope specificity to the other neutralizing mAbs isolated from the same donor through the epitope binning assay. Molecular modelling of the CDHR3 sequence using existing structures indicated that ATHSC-30 belongs to the class 4 antibody, a feature that contributes to breadth, while epitope conservation analysis indicated that the majority of RBD-interacting residues of ATHSC-30 are evolutionarily conserved. Taken together, our study indicate that ATHS-C30 forms the basis of development of a robust and broadly neutralizing antibody response in this individual with hybrid immunity, which overcomes the antigenic variation by targeting highly conserved and cryptic epitopes in destabilizing the spike structure.

BACKGROUND:Mpox has spread to 36 countries in Africa, yet many face challenges achieving nationwide PCR-based testing due to cost and limited access in remote and rural areas. Point-of-care antigen-based rapid diagnostic tests (RDTs) might help improve diagnostic access. The aim of this study was to evaluate the diagnostic accuracy of five mpox antigen-based RDTs. METHODS:We performed a retrospective diagnostic accuracy study using lesion swabs from patients from a WHO transmission study with suspected mpox in nine health zones of Kinshasa province in DR Congo from Feb 12 to June 28, 2025, supplemented by PCR-negative swabs from consenting patients from other research studies. Five antigen-based RDTs were assessed, with results compared against the RADI FAST Mpox PCR assay. The parent study took swabs from people of all ages with suspected and probable case infection who presented to health-care facilities, as well as those within the community or in households. For each participant, six lesion swabs were collected: one was used for reference testing and each remaining swab was used for a different RDT. The primary outcome of diagnostic accuracy, as measured by sensitivity and specificity in tests from all patients, was assessed in DR Congo by operators masked to the results of other assays. Analytical sensitivity was evaluated using MPXV clade Ib isolates at the Geneva University Hospitals (Geneva, Switzerland). FINDINGS:We tested lesion swabs from 190 patients (153 from the WHO transmission study and 37 additional patients). The best performing assay, from Guangdong Wesail Biotech, had a sensitivity of 77·3% (95% CI 68·0-84·5; 75 of 97) and specificity of 93·5% (86·6-97·0; 87 of 93). The Hangzhou Testsea Biotechnology assay had a similar performance (sensitivity 72·2% [62·5-80·1]; 70 of 97); specificity 93·5% [86·6-97·0]; 87 of 93). Tests with moderate sensitivity and high specificity were by Beijing Hotgen Biotech (sensitivity 59·8% [49·8-69·0]; 58 of 97; specificity 96·8% [90·9-98·9]; 90 of 93) and Contipharma (sensitivity 50·5% [40·7-60·3]; 49 of 97; specificity 95·7% [89·5-98·3]; 89 of 93). The NG Biotech assay had the lowest sensitivity (39·2% [30·1-49·1]; 38 of 97) but a similarly high specificity (96·8% [90·9-98·9]; 90 of 93). INTERPRETATION:Several antigen-based RDTs show high positive predictive values for mpox screening in high-prevalence settings, in which positive test results could support confirmation of infection in the absence of PCR but negative results cannot rule out infection. FUNDING:Ministry of Health, Labour, and Welfare of the Government of Japan.

ABSTRACT The recombinant vesicular stomatitis virus–vectored Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine, while effective and well-tolerated, exhibits notable reactogenicity, manifesting in expected adverse events (AEs), such as fever, headache, and pain, along with rare, unexpected AEs, including skin lesions, cutaneous vasculitis, and transient arthritis. The presence or absence of AEs following rVSVΔG-ZEBOV-GP vaccination is associated with a specific innate plasma signature. This study aims to elucidate in vitro the tropism of the vaccine for different cell types derived from tissues previously reported to be involved in the unexpected AEs. Upon in vitro infection with rVSVΔG-ZEBOV-GP, various cell types, such as synoviocytes, fibroblasts, keratinocytes, and endothelial cells (except chondrocytes), demonstrate productive infection, which in dermal fibroblasts triggered the release of many innate plasma signature markers, including keratinocytes’ pro-inflammatory and proapoptotic cytokines such as OSM and TRAIL. Infected monocytes from buffy coats, activated by infection, produce most innate plasma signature markers. In co-culture, rVSVΔG-ZEBOV-GP-infected monocytes serve as a source to synoviocyte infection, resulting in distinct kinetics modulation in innate biomarkers (transcription and secretion) and upregulation of specific genes, such as NEDD8 and SIGLEC-1, which have been associated with inflammatory arthritis in animal models. Altogether, our work, based on in vitro studies, provides insights into the possible mechanisms of rVSVΔG-ZEBOV-GP underlying the observed reactogenicity by showing tropism of the vaccine for off-target cells derived from AE-affected compartments (skin, joints, vessels). Furthermore, in vitro interaction with infected monocytes modulates the innate response of synoviocytes.IMPORTANCEOur study expanded knowledge about the cellular tropism of rVSVΔG-ZEBOV-GP vaccine toward peripheral blood mononuclear cells and cell lines derived from tissues (skin, vessels, and joints) associated with unexpected AEs, as well as their possible contribution to the vaccine-induced innate response. Using in vitro infection and co-culture techniques, we showed that rVSVΔG-ZEBOV-GP-infected monocytes can transmit the virus to synoviocytes, and how infection affects human monocytes and synoviocytes at the protein and transcriptomic levels. Our findings provide insights into the in vitro off-target infection dynamics and innate immune response triggered by rVSVΔG-ZEBOV-GP vaccine. While these results may enhance understanding of rVSV-based vectors, in vivo relevance remains unclear, as does whether the effects come from the VSV backbone or Ebola GP. These findings support the evaluation of off-target effects of rVSV-based vaccine candidates, including those under development for hemorrhagic fever viruses, such as Marburg virus, Sudan virus, and Lassa virus, especially if similar AEs are observed.

Abstract Background In settings where hepatitis E virus (HEV) is highly endemic, anti-HEV IgG seroprevalence approaches saturation, yet how often disease occurs in previously infected individuals, and how protective natural genotype 1 immunity is, remains unmeasured. IgG concentration cannot answer this as levels rise within days of symptom onset masking any pre-existing levels; IgG avidity, maturing over months, may serve as a useful tool to understand reinfections. Methods We enrolled 1007 suspected HEV patients and measured anti-HEV IgG avidity in IgG-positive confirmed cases and an age-stratified sample of IgG-positive test-negatives (n = 366) in enhanced surveillance around a 2022 vaccination campaign in Bentiu, South Sudan. Using high avidity (avidity index ≥50%) as a marker of past infection, we estimated (i) the reinfection fraction, (ii) protection from prior infection using a test negative design and (iii) associations with viral load and infection severity. Results Median avidity index was 11% in cases versus 77% in test-negatives. Among unvaccinated cases, 11.6% (28/242) carried high-avidity IgG (indicative of mature immunity and therefore reinfection), rising with age (odds ratio 1.6 per decade, p=0.006). Mature immunity was associated with 95% lower odds of disease (adjusted OR 0.05, 95% CI 0.03–0.08), robust to threshold and case-definition. High-avidity cases were less often viremic (29% vs 78%) at matched time since onset, though we found no differences in liver function biomarkers between patients with high-avidity and low-avidity. Conclusions Most HEV disease occurred in previously uninfected individuals, and prior immunity was strongly protective against detected disease. Avidity provides a population-level tool to read the immune landscape where seroprevalence may be uninformative. Short Summary Using IgG avidity to distinguish between past from recent hepatitis E infection in a highly endemic setting, we found most disease occurred in previously uninfected individuals. Prior infection was associated with 95% lower odds of disease, demonstrating strong natural immunity.

Background SARS-CoV-2 Omicron lineage variants continue to evolve antigenically, potentially compromising antibody-mediated immunity despite widespread vaccination and infection. We investigated whether SARS-CoV-2-specific antibody persistence translates into sustained neutralizing breadth against contemporary Omicron lineage variants. Methods We conducted a longitudinal study of 83 adults sampled at days 0, 90, 180, and 360. Binding and live-virus neutralizing antibody responses were measured against ancestral SARS-CoV-2 and successive Omicron lineages, including JN.1 and its descendants. Anti-nucleoprotein antibodies were assessed as evidence of previous natural exposure. Findings Anti-nucleoprotein antibodies were detected in vaccinated participants, consistent with previous natural exposure. Neutralizing antibodies against ancestral Wuhan virus had an estimated half-life of 334 days, compared with 171 days for XBB.1.16 and 151 days for JN.1. Binding antibodies persisted longer than neutralizing antibodies, although JN.1 RBD-binding antibody durability was also reduced. Neutralization of JN.1 descendants declined during follow-up; at day 180, 54.2% of participants had titres below the detection threshold for XEC. Neutralization partially recovered by day 360 for several descendants. Interpretation Hybrid immunity generated persistent antibody responses, but functional neutralization became less durable as antigenic divergence increased. Persistent binding antibodies should therefore not be assumed to represent equivalent functional immunity against contemporary SARS-CoV-2 variants. Variant-specific neutralization surveillance, alongside genomic surveillance, could inform assessment of population immunity and updated vaccine strategies.

BACKGROUND:Diagnostics are essential for understanding hepatitis E epidemiology, but the field performance of available tests remains unclear. We evaluated the performance of polymerase chain reactions (PCR), IgM ELISA, and the Assure hepatitis E virus (HEV) IgM rapid diagnostic test (RDT) during a HEV genotype 1 outbreak and assessed the duration of viremia and antibodies responses. METHODS:We used data from enhanced surveillance at a health facility in Bentiu internally displaced persons camp, South Sudan (March-December 2022). As part of a vaccine effectiveness study, suspected hepatitis E cases underwent testing with all 3 diagnostics at enrollment with a follow-up sample. We used a latent class model to estimate test performance and accelerated failure time models to estimate time from jaundice onset to a negative test for PCR and ELISA. RESULTS AND CONCLUSIONS:Among 893 suspected cases, test sensitivity declined with time from jaundice onset. Within 30 days of jaundice onset, PCR sensitivity was 73% (95% Credible Interval [CrI] 27, 90), compared to 86% for RDT (95% CrI: 74, 93), and 95% for ELISA (95% CrI: 91, 98). Specificity was high across tests: PCR at 98% (95% CrI: 98, 99), RDT at 95% (95% CrI: 93, 96), and ELISA at 95% (95% CrI: 93, 96). Median time from jaundice onset to negative test was 19 days (95% CI: 17, 21) for PCR and 113 days (95% CI: 87, 163) for ELISA. The Assure IgM RDT showed higher sensitivity for identifying hepatitis E than PCR and similar specificity to IgM ELISA, supporting its use in surveillance. Care seeking delays can greatly influence the interpretation of diagnostic tests.

Viral respiratory infections (VRIs) are a persistent global health concern, driven by the year-round circulation of diverse viruses. During the coronavirus disease 2019 (COVID-19) pandemic, the prevalence of most respiratory viruses declined sharply following nonpharmaceutical interventions. Their subsequent resurgence and co-circulation with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) created a novel and unpredictable VRI landscape. We investigated the respiratory virus dynamics and co-occurrence patterns in symptomatic outpatients in Switzerland, using nasopharyngeal specimens collected between August 2022 and July 2024 by the National Reference Center for Influenza. In total, 5,369 specimens were tested by PCR or RT-PCR for 16 respiratory viruses. Co-detections were identified in 9% of positive samples and occurred year-round. The majority involved two viruses (92%), although triple and quadruple detections were also observed. Statistical analysis showed that multiple detections were significantly more common in children. Their incidence was higher not only with the most prevalent ones, rhinoviruses (RVs) and SARS-CoV-2, but also with bocaviruses (hBoV) and adenoviruses (ADVs). A statistically significant decrease in viral load was observed when a second virus was co-detected with SARS-CoV-2 or respiratory syncytial virus (RSV), but not with influenza viruses. This effect was most pronounced in early- and middle-aged adults. Through the systematic investigation of viral co-occurrence, this study emphasizes the importance of community-based surveillance. Beyond monitoring respiratory disease burden, such surveillance provides a foundation for generating hypotheses to better understand viral interactions and the virus-host interplay during infection.IMPORTANCEViral respiratory infections (VRIs) are a major public health challenge due to their constant circulation and significant impact on vulnerable populations. The coronavirus disease 2019 (COVID-19) pandemic profoundly altered the dynamics of respiratory viruses, leading first to a sharp decline in their prevalence, followed by an unpredictable resurgence in co-circulation with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In this context, understanding viral interactions and their co-detection in the community has become essential. This project provides novel insights into the frequency and characteristics of viral co-infections in a real-world outpatient setting. It highlights age-related differences and demonstrates statistically significant effects on viral load, particularly for SARS-CoV-2 and respiratory syncytial virus (RSV). These findings underscore the importance of community-based surveillance, not only to monitor the overall burden of respiratory diseases but also to generate new hypotheses on virus-host interactions. Ultimately, this knowledge can guide the development of more effective prevention and management strategies.

Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne disease with a case fatality rate of up to 40% in humans, posing a significant health threat. This study investigates the 2022-23 CCHF outbreaks in Iraq, the highest recorded to date, and analyzes potential factors at the human-animal-environmental interface. Data from the Iraqi government, the World Health Organization, and the World Bank were used to analyze CCHF trends and affecting factors. This included epidemiological reports, clinical data, tick infestation and seroprevalence studies, and climate data. Descriptive and statistical analyses examined case trends, geographic and demographic characteristics, clinical manifestations, risk factors, seasonal patterns, and influencing factors. A sudden rise in CCHF cases began in southern Iraq in April 2022 and expanded across all governorates, with a shift toward urban areas. Higher incidence was observed among males, aged 25-44, and those involved in slaughtering. The most common clinical manifestation was fever (97%), followed by hemorrhagic symptoms (54%). Bleeding from the gums or mouth and subcutaneous bleeding were more frequent in patients with fatal outcomes. Seasonal patterns showed peaks during spring and fall, correlating with tick activity and potentially exacerbated by climate change. Tick infestation and seroprevalence studies indicated a high prevalence of Hyalomma ticks and CCHF seropositivity among domestic animals in southern Iraq (60%), consistent with the distribution of CCHF human cases. Iraq's ongoing CCHF outbreak demands multidisciplinary One Health strategies. The Iraqi government has adopted such a control strategy, contributing to regional and global efforts to enhance pandemic preparedness.

School-based interventions during epidemics are often controversial, as experienced during the COVID-19 pandemic, where reducing transmission had to be weighed against the adverse effects on young children. However, it remains unclear how the broader epidemiologic context influences the effectiveness of these interventions and when they should be implemented. Through integrated modeling of epidemiological and genetic data from a longitudinal school-based surveillance study of SARS-CoV-2 in 2021–2022 (N children = 336, N adults = 51) and scenario simulations, we show how transmission dynamics in schools changed markedly due to strong increases in community-acquired infections in successive periods of viral variants, ultimately undermining the potential impact of school-based interventions in reducing infection rates in the school-aged population. With pandemic preparedness in mind, this study advocates for a dynamic perspective on the role and importance of schools in infectious disease control, one that adapts to the evolving epidemiological landscape shaped by pathogen characteristics and evolution, shifting public health policies, and changes in human behavior.

While Influenza Virus and Respiratory Syncytial Virus (RSV) are considered as a significant health burden in children, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes milder diseases in this age group compared to adults. To investigate the involvement of the upper respiratory tract human airway epithelium (HAE) in this pattern, we established an in-house model of reconstituted HAE cultured in air-liquid interface from nasal swabs of children and adults and characterised it before and after ex vivo respiratory viral infections using focused and unbiased approaches. Fully differentiated paediatric HAE exhibited an increasing induction level of genes related to mucociliary clearance, while higher expression of innate immune pathways was found in the ones from adults. While similar viral replication kinetics in both age groups were shown for SARS-CoV-2, Influenza A Virus (IAV), RSV and Rhinovirus (RV) infection, transcriptomic analysis showed stronger and earlier induction of IFN-related pathways in SARS-CoV-2-infected HAE from children compared to IAV, RSV and RV. IAV and RSV had the weakest innate immune response increase in HAE from children versus adults. RV infection showed an intermediate pattern, resembling SARS-CoV-2 more than RSV or IAV. Our work demonstrates a distinct sensing of SARS-CoV-2 compared to other respiratory viruses ex vivo, which may contribute to the milder course of disease in SARS-CoV-2 infected children and argues for a role of early virus-HAE interaction in shaping viral pathogenesis. Furthermore, we show that innate immune responses towards respiratory viruses are virus-specific and differ between age groups. Hence, findings on SARS-CoV-2 cannot be extrapolated to other respiratory viruses. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Nucleic acid-based assays are the diagnostic gold standard for filoviruses, including Ebola (EBOV) and Sudan (SUDV) viruses. However, outbreaks in areas with limited laboratory infrastructure highlight the need for simpler diagnostic tests that can be rapidly and safely used in the field. METHODS:We evaluated eight antigen rapid diagnostic tests (Ag-RDTs) for their ability to detect EBOV and SUDV. Analytical panels using virus cell slurries were used to assess limit of detection, and clinical samples were tested to determine sensitivity and specificity. RESULTS:Five Ag-RDTs detected EBOV and three detected SUDV, although clinical sensitivity was low (20-40 % for EBOV, 33 % for SUDV), improving only with higher viral loads. All assays demonstrated 100 % clinical specificity with no cross-reactivity. DISCUSSION:Although none of the evaluated Ag-RDTs are suitable for routine diagnosis, some may be useful in high viral load contexts such as cadaver testing. Our findings highlight the need to improve Ag-RDT sensitivity or develop high-sensitivity point-of-care molecular diagnostics.

The true burden of hepatitis E is poorly understood due to a lack of routine diagnostic testing and nonspecific symptoms. We evaluated the sensitivity and specificity of international case definitions for suspected hepatitis E and explored whether alternative case definitions could more accurately identify those with true hepatitis E, thus improving our ability to interpret surveillance data. We used data from acute jaundice surveillance in South Sudan (March-December 2022) and Bangladesh (December 2014-September 2017). Individuals seeking care with acute jaundice syndrome (AJS) were asked about signs/symptoms of hepatitis E and underwent testing with anti-hepatitis E virus (HEV) IgM ELISA. We used an ensemble of classification models to assess how well signs/symptoms could distinguish between AJS with and without HEV. To explore alternative case definitions, we estimated the sensitivity and specificity of all combinations of signs/symptoms. Among patients with AJS, 20% in South Sudan and 38% in Bangladesh had IgM antibodies. International case definitions for suspected hepatitis E had variable sensitivity (53-96%) and specificity (6-60%) across study populations. Alternative case definitions had poor discrimination in both populations (AUC SouthSudan =0.64; 95% Confidence Interval (CI): 0.57, 0.71; AUC Bangladesh = 0.60; 95% CI: 0.57, 0.63). No alternative case definitions had both sensitivity and specificity above 60% in both study populations. Acute jaundice surveillance in two distinct populations revealed that signs/symptoms alone are insufficient to distinguish HEV-related cases from other causes of acute jaundice. Accurately characterizing the burden of hepatitis E and assessing the cost effectiveness of vaccines will require expanded use of diagnostic testing.

Pregnancy is associated with an increased risk of severe COVID-19. In addition, SARS-CoV-2 infection during gestation has been linked to adverse obstetrical outcomes and placental abnormalities. Nevertheless, the susceptibility of early trophoblast cells to SARS-CoV-2 and the potential consequences of infection on trophoblast function remain unclear. In this study, we assessed the permissiveness of first trimester trophoblast cells to SARS-CoV-2 infection and its impact on trophoblast cells fusion. To address this, we isolated primary cytotrophoblast (CTB) cells from first trimester human placentas and allow their differentiation into STB in vitro. These cells were infected with SARS-CoV-2 variants of concern, including Delta and Omicron (BA.1, BA.2, BA.5). Viral replication was assessed by RT-qPCR and immunofluorescence, while host cell responses, including expression of viral entry receptors and innate immunity genes, were measured by RT-qPCR. Trophoblast fusion was evaluated by staining and calculating the fusion index. In parallel, placental tissues from SARS-CoV-2-infected pregnancies were analyzed by immunohistochemistry to quantify syncytial knots (SK) formation in vivo. Our results demonstrate that both first trimester CTB and STB are permissive to SARS-CoV-2 infection in a variant- and donor-dependent manners, with Delta exhibiting higher replication efficiency compared to Omicron variants. In STB, viral replication did not correlate with the induction of entry receptors or type III interferon responses. However, in CTB, viral replication was significantly associated with enhanced cell fusion. In parallel, an increased number of SK was observed in infected placental areas in vivo compared to non-infected regions from the same placenta and to gestational age-matched controls. Altogether, these in vitro and in vivo results suggest that SARS-CoV-2 infection in early pregnancy may alter STB turnover, potentially contributing to placental dysfunction and adverse pregnancy outcomes.

Background Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis, particularly in Asia and Africa, where HEV genotypes 1 and 2 are prevalent. Although a recombinant vaccine, Hecolin, is available, it has not been used to control outbreaks. The licensed three-dose regimen might pose challenges for it to be an impactful outbreak control tool. Our study aimed to estimate the effectiveness of two doses of Hecolin in the context of the first-ever reactive use of the vaccine. Methods We conducted a case-control study during an HEV outbreak in the Bentiu internally displaced persons camp, South Sudan. Patients with acute jaundice syndrome (suspected cases) seeking care at the M & eacute;decins Sans Fronti & egrave;res hospital were screened for study eligibility. Eligible participants were those that had been eligible for vaccination (ie, living in the camp and aged 16-40 years). Confirmed cases were defined as individuals who tested positive for hepatitis E by RT-PCR or anti-HEV IgM ELISA. Each case was matched to six controls by age, sex, pregnancy status, and residence. Self-reported vaccination status was verified through vaccination cards. The primary analysis was two-dose vaccine effectiveness, which we estimated with a matched case-control design using conditional logistic regression models. In secondary analyses we estimated vaccine effectiveness using a test-negative design and the screening method. We used test-negative cases and their matched controls as a bias indicator analysis to help quantify potential health seeking behaviour biases. Findings Between May 10 and Dec 30, 2022, we identified 859 patients with suspected hepatitis E. Of these, 201 met the eligibility criteria and 21 cases had laboratory confirmed hepatitis E. Among the confirmed cases, 10 (48%) were unvaccinated compared with 33 (27%) of 121 matched controls. In the primary analysis we estimated an unadjusted two-dose vaccine effectiveness of 678% (95% CI-286 to 919), and a two-dose vaccine effectiveness of 840% (-2085 to 992) after adjustment for potential confounders. The bias indicator analysis suggested that test-negative cases might have been more likely to have been vaccinated than their matched community controls due to different health-care seeking behaviours, potentially meaning underestimation of effectiveness estimates. The test-negative design, which uses facility-matched controls, led to an adjusted two-dose effectiveness of 894% (564 to 980). Interpretation Despite the small sample size, our estimates provide evidence of effectiveness of a two-dose regimen against HEV genotype 1 during a protracted outbreak, supporting its use in similar contexts. Funding M & eacute;decins Sans Fronti & egrave;res. Copyright (c) 2025 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.