Virology Journal

Autophagy is a catabolic process used for the degradation of organelles and proteins. Macroautophagy involves the formation of autophagosomes and subsequent fusion with lysosomes to mediate cargo degradation. It also functions as a cellular defence mechanism, known as xenophagy, during infection. Previous studies show that different viruses manipulate the autophagy pathway of the host cell to assure successful replication and/or virion assembly. Vaccinia virus (VACV), the prototypic poxvirus, replicates exclusively in the cytoplasm of host cells. It is known that VACV infection causes LC3 lipidation and prevents autophagosome formation, yet the double membrane vesicles formed during autophagy do not serve as the source of the mature VACV membrane. To date the viral protein(s) causing increased LC3 lipidation have not been identified. Here we developed an image-based screening approach based on LC3 granularity to identify candidate VACV genes affecting its lipidation. We identify several candidate viral membrane proteins as effectors of LC3 lipidation, suggesting that the interplay between VACV and autophagy is more directed than previously thought.

COVID-19 has spread rapidly and caused a global pandemic making it one of the deadliest in history. Early identification of patients with coronavirus disease 2019 who may develop critical illness is of immense importance. Therefore, novel biomarkers were needed to identify patients who will suffer rapid disease progression to severe complications and death. Many treatments were adopted including the antiviral Remdesivir, the antiretroviral Lopinavir /Ritonavir and Tocilizumab. Our study aimed not only to specify high-risk factors and biomarkers of fatal outcome in hospitalized subjects with coronavirus but also to compare the efficacy of the three considered treatments to help clinicians better choose a therapeutic strategy and reduce mortality. We divided the population (n=711) into four main groups based according to the WHO ordinal severity scale. The percentage of mortality, in and out the hospital, the length of stay in the hospital, the pulmonary inflammatory lesion and its distribution, the SARS-CoV-2 IgM and IgG variations at admission, the inflammatory markers, the complete blood count, the coagulation factors and enzymes, proteins and electrolytes profile, glucose and lipid profile, and other relevant markers were measured. The significance of the observed variation was assessed by multivariate and ANOVA analyses. We succeeded to establish a novel predictive scoring model of disease progression based on a cohort of Lebanese hospitalized patients relying on the pulmonary inflammatory lesions, inflammation biomarkers such as LDH, D-Dimer, CRP, IL-6 and the lymphocyte count, the number of comorbidities and the age of the patient which all were significantly correlated with the illness severity showing best outcomes with immunomodulatory and anticoagulant treatments by the results. As top tier, Tocilizumab was more efficient than the two other treatments in non-severe cases but none of the used treatments was insanely effective alone to reduce mortality in severe cases.

Background: Mobile laboratory diagnostic technologies for Nipah virus outbreak prevention, mitigation and response remain limited, despite the critical need for such capacities in remote, low-resource regions where most cases occur. We aim to address this gap by implementing a workflow that includes method development, laboratory validation, and field demonstration of a mobile Nipah virus complex diagnostic solution. Methods: We developed a flexible mobile laboratory workflow incorporating PCR capacity, a novel amplicon-based sequencing protocol, and a validated Nipah virus inactivation procedure. Following development and validation, we demonstrated the feasibility of this workflow through repeated field sampling of bat colonies in Nipah virus endemic regions of Bangladesh across multiple field campaigns. Findings: We demonstrated the feasibility of this system for early outbreak response and as a potential early warning tool prior to the emergence of human cases. We detected two urine samples from flying foxes that tested positive and performed full-scale on-site analysis, including qPCR diagnostics and NGS sequencing, within 24 hours. Interpretation: As highlighted in the present study, active surveillance enables outbreak prevention by identifying bat colonies that are actively shedding viruses in real time, even in rural settings. Also, this method can provide rapid, on-site sequence data to track and better understand the genomic diversity of Nipah virus in natural reservoirs during both outbreak and non-outbreak periods. In this study we aimed to establish the foundations of a standard procedure for safe and rapid field testing of Nipah virus in remote areas.

Defective interfering particles (DIPs) derived from the influenza A virus (IAV) are a promising antiviral agent due to their strong antiviral efficacy demonstrated in various animal models. OP7 is an unconventional IAV DIP with multiple point mutations in the viral RNA (vRNA) of genome segment 7, as opposed to the large internal genomic deletions typically found in conventional IAV DIPs. Further, OP7 showed an even higher interfering efficacy than conventional DIPs. However, the inhibitory effect of OP7 on standard virus (STV) replication has primarily been investigated in Madin-Darby Canine Kidney (MDCK) cells, which lack a functional myxovirus resistance (Mx)-mediated antiviral activity against IAV. In this study, we examined the antiviral activity and mechanism of antiviral action of OP7 in an interferon (IFN)-competent human lung carcinoma cell line (Calu-3) in vitro. We performed STV and OP7 co-infection experiments using a variety of infection conditions and measured the time-resolved dynamics in viral titer, vRNA, protein level, and host cell gene expression. We observed that OP7 co-infection results in enhanced type I IFN responses and markedly reduced infectious virus release, even at low doses. Additionally, we found that at a high STV multiplicity of infection (MOI), the replication interference of OP7, suppressing the replication of STV vRNA, appears to be the dominant mechanism of its antiviral action. At a low MOI, however, IFN induction seems to be more important. Furthermore, we examined the efficacious co-infection time window for potential prophylactic and therapeutic antiviral treatment. We observed an antiviral effect exerted by OP7 infection for up to seven days before STV infection and up to 24 hours after STV infection. Together, these findings demonstrate that OP7 is a potent antiviral DIP. Therefore, this work supports the further development of OP7 as a therapeutic and prophylactic antiviral agent.

Background: To investigate the safety and efficacy of CTguided lung nodule localization needles for the preoperative localization of small pulmonary nodules. Methods: A retrospective study was conducted on 102 patients with a total of 113 small pulmonary nodules who underwent preoperative localization at Jinan Fourth People's Hospital from January 2024 to December 2025. Nodule diameter and depth, localization time, the number of pleural punctures, the localization success rate, and postoperative complications (hook dislodgement, hemorrhage, and pneumothorax) were recorded. All patients underwent video assisted thoracoscopic surgery (VATS) after localization. Results: The mean nodule diameter was 0.97{+/-}0.36 cm, the mean depth was 1.26{+/-}0.48 cm, and the mean localization time was 9.8{+/-}3.65 minutes. The hook dislodgement rate was 0.98% (1/102), the intrapulmonary hemorrhage rate was 14.71% (15/102), and the pneumothorax rate was 16.67% (17/102). All pulmonary nodules were successfully resected by VATS at 73.82{+/-}13.83 minutes after localization, and no severe complications occurred. Conclusions: The use of a CTguided lung nodule localization needle for the preoperative localization of small pulmonary nodules decreases the time needed for intraoperative nodule detection and operation time. This strategy is a simple, safe, and accurate preoperative localization method that is worthy of increased clinical use.

Hantavirids, specifically the members within the genus Orthohantavirus, represent a significant global public health threat, with bat-associated lineages challenging traditional reservoir paradigms. To investigate the genetic diversity of hantavirids in Southeast Asia, we conducted an expanded surveillance program in Lao PDR from May 2023 to October 2025 in bat populations and wild animals from local wet markets. Using molecular screening and deep sequencing to characterize hantavirids from bat populations and wild animals from local wet markets, we identified 20 positive samples across four bat species, recovering coding-complete genomes for multiple novel variants. Phylogenetic analysis confirmed that these viruses form a monophyletic group within Mobatvirus, resolving into two major subclades. The first subclade clustered with Quezon and Robina viruses found in fruit-eating bats. The second subclade further split into two lineages corresponding to Thakrong and Xuan Son viruses, which are associated with trident and leaf-nosed bats, respectively. Despite the strong host specificity observed, the detection of these viruses in a wet market, a critical interface for human-wildlife contact, indicates a potential zoonotic risk. These findings significantly expand the known diversity of mobatviruses in Laos and highlight the urgent need for serological surveillance in at-risk human populations to assess the potential for spillover.

Mpox virus (MPXV) gained increased attention following the declaration of two Public Health Emergencies of International Concern (PHEICs) in 2022 and 2024. The rapid spread of MPXV and the increase in human-to-human transmission highlighted the need for improved diagnostic tools for characterizing infection patterns and transmission dynamics. While PCR is effective for detecting active infections, serological approaches can help identify previous or asymptomatic infections and support retrospective surveillance. However, many serological assays developed during recent outbreaks have not been evaluated in endemic settings such as the Democratic Republic of the Congo (DRC). This study aims to define antigen-specific serological cutoff values to differentiate MPXV-seroreactive individuals from those with other orthopoxvirus (OPXV) exposure or different vaccination histories, specifically for use in the DRC. Here, we analyzed 134 individuals, divided into six distinct cohorts with different exposures. Serum samples were tested using Mesoscale Discovery (MSD) to screen for five MPXV and vaccinia virus (VACV) orthologous antigens: A29L/A27L, A35R/A33R, B6R/B5R, E8L/D8L, and M1R/L1R. Receiver operating characteristic (ROC) analysis identified the best-performing antigens and established seroreactivity cutoff values. A binary composite rule was also evaluated to improve the classification of these results. We identified three MPXV antigens, E8L (cut-off=12.33 AU/mL), A35R (cut-off=5.22 AU/mL), and B6R (cut-off=9.77 AU/mL), that showed the strongest discriminatory performance in the dataset. Collectively, these three antigens form a significant panel that demonstrated clear separation between our mpox survivor cohort and other OPXV-exposed individuals.

This study aimed to evaluate DARO Systems detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) against serum and oral fluid surveillance methods within a controlled study consisting of one PRRSV infected seeder pig and 46 naive nursery pigs. Findings showed DARO Systems comprehensive herd-level surveillance approach detected PRRSV earlier than traditional testing methods.

Background & AimsAntiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure. Understanding HBV-host cell interaction is crucial for developing novel therapeutic approaches. Here, we report host cell proteins associated with HBV virions and filamentous subviral particles (fSVPs) and characterize one of them, apolipoprotein C1 (ApoC1), mechanistically. MethodsHighly purified HBV virions and fSVPs were obtained by sequential use of several biophysical methods. Particles were analyzed by mass spectrometry and associated proteins were evaluated phenotypically using an HBV infection model. The top hit, ApoC1 was characterized in detail. ResultsAssociated with virions and fSVPs, we identified in addition to known chaperones such as HSP90AB1 and HSC70, several apolipoprotein-related factors. RNAi-based phenotypic validation identified strongest effects for ApoC1, likely due to two complementary effects. First, ApoC1 depletion reduced intracellular cholesterol level impairing HBV infection and SVP production, which was compensated by exogenous cholesterol substitution. Second, ApoC1 that is mainly enriched in high-density lipoprotein (HDL), associates with HBV virions and fSVPs and increases HBV infectivity. The same was found for hepatitis D virus (HDV), a satellite virus utilizing HBV envelopes. Supplementation of exogenous HDL enhanced infection most likely via scavenger receptor class B type 1 (SR-B1), the natural HDL receptor. Consistently, inhibition of SR-B1 suppressed HBV and HDV infection. ConclusionsWe established a method for obtaining highly purified HBV virions and fSVPs and identified the HDL component ApoC1 to associate with both particle types. ApoC1 promotes HBV and HDV infection most likely via SR-B1 facilitating viral entry.

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.

Background: Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of global mortality, with persistent lung inflammation contributing to disease progression. This inflammation is partly associated with reduced levels of histone deacetylase 2 (HDAC2). Previous studies suggest that Vitamin D may modulate HDAC2 levels. This study aimed to evaluate the effect of Vitamin D supplementation on HDAC2 expression in stable COPD patients. This experimental study aimed to evaluate the effect of vitamin D supplementation on HDAC2 expression in stable COPD patients at Jemursari Islamic Hospital. Methods: Five COPD patients received a daily dose of 5000 IU of Vitamin D for three months. Serum levels of 25(OH)D3 and HDAC2 were measured before and after the intervention. Results: Vitamin D supplementation resulted in a significant increase in both 25(OH)D and HDAC2 levels. Pulmonary function parameters showed an increasing trend, however, no statistically significant differences were observed. Conclusion: Vitamin D supplementation was associated with increased HDAC2 levels, suggesting a potential anti-inflammatory effect. However, no significant improvement in pulmonary function was observed. Further studies are needed to determine its clinical impact.

BackgroundOropouche virus is an emerging arbovirus increasingly associated with neurological complications, but its human cellular tropism and potential routes to the central nervous system remain poorly defined. This study aimed to characterize infection across clinically relevant human cell types and to investigate interactions with a human blood-brain barrier model and human neuronal/glial cells. MethodsA panel of human cell lines and primary human cells relevant to systemic and neurological disease was infected with Oropouche virus. Viral replication and production of infectious particles were quantified using molecular assays and infectivity titrations, and viral protein expression was assessed by immunoblotting and immunofluorescence. Barrier crossing was evaluated using a Transwell brain endothelial model with permeability monitoring, and infection dynamics in neuronal/glial cultures derived from human neural progenitors were quantified by imaging-based analyses. Group comparisons used non-parametric tests with Dunn-Bonferroni correction and Mann-Whitney tests; neuronal/glial cell counts were analysed using linear models with Fisher tests for interaction terms and multiplicity-adjusted post hoc comparisons. ResultsOropouche virus productively infected hepatocyte-like and intestinal epithelial cells, with high viral RNA output and release of infectious progeny. Primary synoviocytes, chondrocytes and skeletal muscle cells were permissive but produced lower infectious titers. Brain endothelial cells were inoculated and virus was progressively detected in the basolateral compartment, while endothelial permeability remained unchanged, indicating barrier crossing without disruption. In neuronal/glial cultures, both neurons and astrocytes were susceptible; infection was associated with marked cytopathic changes and a preferential, accelerated decline in neuron abundance over time. ConclusionsThese findings demonstrate broad human cell tropism and support blood-brain barrier crossing without major loss of barrier integrity, alongside pronounced neuronal vulnerability. The described models provide a platform to dissect mechanisms of neuroinvasion and to evaluate targeted antiviral strategies.

The global ambition to end the human immunodeficiency virus (HIV) epidemic requires understanding which system-level policy levers, enacted under the framework of Universal Health Coverage (UHC), are most effective in achieving both transmission reduction and diagnostic coverage. This study addresses an important evidence gap by quantifying the within-country association between measurable UHC policy indicators and the estimated rate of new HIV infections across nine Southeast Asian countries between 2013 and 2022. Employing a Fixed-Effects panel data methodology, the analysis controls for time-invariant national heterogeneity, ensuring reliable estimates of policy impact. We found that marginal changes in total current health expenditure (CHE) as a percentage of gross domestic product (GDP) were not statistically significantly associated with changes in HIV incidence. However, increases in the UHC Infectious Disease Service Coverage Index were statistically significantly associated with concurrent reductions in HIV incidence (p < 0.001), suggesting the efficacy of targeted service implementation as the principal driver of curbing new HIV infections. In addition, the UHC Reproductive, Maternal, Newborn, and Child Health Service Coverage Index exhibited a statistically significant positive association with changes in HIV incidence (p < 0.01), which is interpreted as a vital surveillance artefact resulting from expanded detection and reporting of previously undiagnosed HIV cases. Furthermore, out-of-pocket (OOP) health expenditure as a percentage of CHE showed a counter-intuitive negative association with changes in HIV incidence (p < 0.01), suggesting this metric primarily shows ongoing indirect cost burdens on the established patient cohort, or, alternatively, presents a diagnostic access barrier that results in lower case finding. These findings suggest that policymakers should prioritise investment in targeted infectious disease service efficacy over aggregate fiscal commitment and utilise integrated sexual health platforms for strengthened HIV surveillance and case identification.

Rationale: Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives: To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods: We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results: Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of [&ge;]2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions: Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance.

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

Dengue virus (DENV) is a major burden to global public health, affecting hundreds of millions annually. Children represent the major proportion of global dengue cases, ranging from asymptomatic or subclinical presentation to dengue fever (DF) and severe dengue hemorrhagic fever or shock syndrome (DHF/DSS). The factors that distinguish this range of disease severity are still poorly understood. To identify biomarkers of severity, we analyzed the plasma proteome of acute DENV infected children including both subclinical and hospitalized cases. Proteins associated with the acute-phase response, innate immune and lysosomal activation, and components of the coagulation cascade showed marked differences between hospitalized and subclinical cases during early infection. Longitudinal profiling demonstrated that endothelial dysfunction emerges early, with PTX3 showing the strongest and most rapid upregulation in hospitalized patients, supporting its potential role as a marker of imminent vascular involvement. When comparing severe (DHF/DSS) and classical DF hospitalized cases, CLEC11A displayed the highest fold change at hospital admittance. We used machine-learning analysis to predict disease severity at the acute phase of infection, distinguishing subclinical from hospitalized cases and patients that develop classical dengue fever or severe disease based on the identified complement regulators and inflammatory markers. The panel of identified plasma proteins shed light on the mechanisms of dengue related disease progression and may provide a handle to predict disease severity based on blood markers present during the acute phase of infection.

Dengue virus (DENV) infection is a major global health threat, affecting more than half of the worlds population. Severe dengue is a life-threatening condition characterised by systemic bleeding, vascular leakage, and interstitial fluid accumulation that can progress to hypovolaemic shock. Circulating DENV non-structural protein 1 (NS1) has long been implicated in driving vascular hyperpermeability through its disruptive effects on endothelial cell junctions and the glycocalyx. The lymphatic system, which runs alongside the vascular network, plays a critical role in resorbing and recirculating interstitial fluid and immune cells extravasated from blood vessels. Despite its importance in maintaining tissue fluid homeostasis, the impact of dengue disease on lymphatic vessels has not previously been explored. Here, we present the first evidence that DENV-2 NS1 induces marked hyperpermeability in lymphatic endothelial cells, as measured by transendothelial electrical resistance, and impairs lymphangiogenesis in vitro. These effects were not attributable to changes in cell viability, morphology, or metabolic activity, as assessed by live/dead and metabolic assays and image analysis. Instead, we observed a defect in lymphatic endothelial cell migration, measured by scratch assay, which may underlie the reduced lymphangiogenic potential. Bulk RNA-seq, immunocytochemistry, and advanced image analysis further demonstrated pronounced reorganisation of cell-cell junctions, the cytoskeleton, and focal adhesions. Notably, junctional proteins including VE-cadherin, ZO-1, and Claudin-5 were not downregulated but instead displayed disorganised distribution along the cell junctions or aberrant cytoplasmic localisation. These structural disruptions became even more pronounced under flow conditions produced using a microfluidic system. Together, these findings demonstrate for the first time that DENV-2 NS1 directly disrupts lymphatic endothelial cell function, leading to junctional disorganisation and hyperpermeability. Such impairment of lymphatic drainage may contribute to the pathophysiology of severe dengue. Author SummaryDengue is a rapidly expanding mosquito-borne disease that now affects many tropical and subtropical regions worldwide. Severe cases can lead to extensive fluid leakage from blood vessels, which causes tissue swelling and, in the most dangerous situations, shock. Although much research has focused on how dengue damages the blood vascular system, almost nothing is known about its impact on the lymphatic system, which is responsible for removing fluid from tissues and returning it to the bloodstream. Because both systems work together to maintain fluid balance, understanding how dengue affects lymphatic vessels is important for explaining why fluid accumulation becomes so severe in critical disease. In our study, we examined whether the viral protein NS1, which circulates during infection, directly affects the cells that line lymphatic vessels. We found that NS1 increases the permeability of these cells and reduces their ability to form new vessel structures. These effects were not caused by cell death but by disruptions in how the cells organise their junctions, internal scaffolding, and interactions with neighbouring cells. By showing that NS1 can directly impair lymphatic vessel function, our work identifies a previously overlooked mechanism that may contribute to fluid build-up in severe dengue and suggests new avenues for future therapeutic research.

Background: HIV/AIDS remains a major public health challenge in Tanzania, where viral load suppression among adults on ART stands at 78% and HVL testing uptake among eligible patients is approximately 22%. Since the introduction of the National HVL Testing Guideline in 2015, little has been done to systematically evaluate its implementation. Objective: To evaluate adherence to the National HVL Testing Guideline across CTC clinics in Dar es Salaam Region, covering ART monitoring, documentation, turnaround time, and factors affecting implementation. Methods: A cross-sectional study was conducted in 2021 across 15 public health facilities with CTC clinics in all five Dar es Salaam districts. A total of 330 PLHIV on ART for more than six months were selected through systematic random sampling with proportional to size allocation, and 45 healthcare providers through convenient sampling. Data were collected via abstraction forms and self-administered questionnaires, and analysed using SPSS Version 23 with descriptive statistics, bivariate analysis, and binary logistic regression. Results: Only 25.1% of patients had their first HVL sample taken at six months as per guideline, with 68.8% delayed beyond six months. Second and third samples were similarly delayed. MoHCDGEC sample tracking forms were absent in 96.7% of facilities and incomplete in 99.1%, and no facility captured specimen acceptance or rejection as site feedback. Turnaround time exceeded the 14-day guideline threshold in 64.5%, 66.7%, and 69.4% of first, second, and third results respectively. Patient negligence (AOR=9.84; 95% CI: 1.83-52.77) and storage (AOR=5.72; 95% CI: 0.94-35.0) were independently associated with guideline adherence. Conclusion: Adherence to the National HVL Testing Guideline in Dar es Salaam is suboptimal across testing timelines, documentation, and turnaround time, with patient negligence and storage capacity as significant determinants. Targeted interventions are needed to strengthen patient education, improve storage infrastructure, enhance documentation systems, and support providers in adhering to guideline-specified timelines.

Measurement of antibody responses to viral infection is essential for surveillance, diagnostics, epidemiological research, and natural history of infection studies. However, current methods to detect virus-specific antibodies are often resource-intensive and impractical for deployment in outbreak settings or in field-based studies. This manuscript presents two economical, high-throughput immunoassays--the cytoblot immunoassay (CBA) and strip immunoblot assay (SIA)--for detecting and quantifying anti-lymphocytic choriomeningitis mammarenavirus (LCMV) antibodies in mouse serum. To validate, we tested serum from acutely or persistently experimentally infected mice. Both assays detected LCMV-specific IgG and IgM antibodies with high sensitivity and specificity across multiple timepoints. By facilitating the study of immune responses in rodent reservoirs, these tools can enhance our understanding of zoonotic viral transmission, provide scalable platforms for outbreak preparedness, and serve as adaptable models for the development of rapid serological assays for other viral pathogens.

Hantaviruses are zoonotic negative-sense RNA viruses that cause two severe diseases; haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) for which no approved antiviral therapies are available. To identify host-directed modulators of hantavirus infection in the available annotated drug space, we performed a drug repurposing screen in A549 cells and HUVECs, using live Puumala virus (PUUV). We identified and validated 70 drugs with antiviral activity across these 2 different cell systems. Functional clustering confirmed the known infection-inhibitory effect of several group of compounds, including inhibitors of heat shock proteins, mTOR pathway or nucleotide synthesis. In addition, we also identified compounds yet unexplored as antivirals against Hantaviruses, such as certain antibiotics. This dataset provides a systematic map of host pathways influencing PUUV infection and highlights candidate compounds and cellular processes that warrant further investigation.