Journal of Virological Methods

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.

ObjectiveGeneration and testing of IGF1 reference materials (RM), suitable for the harmonization of immunoassay (IA) and LC-MS/MS methods for the IGF1 determination in blood. In addition, establishment of age related reference intervals for men and women. MethodsIn a split sample study of 42 patients, and 30 healthy volunteers we tested the commutability of four RMs for IGF1, using four commercial IAs and an LC-MS/MS method. A new set of age dependent reference intervals was established using Lifelines biobank samples, based on the IGF1 LC-MS/MS method. ResultsThe four RMs were found to be commutable, except the RM with the lowest concentration measured with the Siemens Immulite method. The value assignment of the RMs was based on the IGF1 LC-MS/MS method, which was calibrated against WHO international standard 02/254. LC-MS/MS results were on average about 0 to 60% lower than those of the immunoassays. Combining the recalculated IGF1 results in patient samples from a former study with the data from healthy volunteers in this study, showed a reduction in the variation of the data points (standard error of estimate) of 42% and 62% respectively. ConclusionCommutable RMs for IGF1 can be made from serum of healthy blood donors. However, it remains necessary to test the commutability of these RMs in IAs that were not included in this study. By harmonizing methods using the four RMs, the same age-related reference intervals can be used.

BackgroundFlow virometry (FV) - the application of flow cytometry to viruses - has historically been hindered by the inability of cytometers to detect particles below [~]300 nm in size. However, advances in optics and fluidics have enabled cytometers primarily designed for cells to detect viruses and extracellular vesicles (EVs) through light scatter alone. In 2024, the CytoFLEX nano was released, marketed for the detection of particles as small as 40 nm; however, its performance has yet to be compared to a conventional instrument for FV. MethodsFV was utilized to evaluate performance of the CytoFLEX nano and a conventional flow cytometer (CytoFLEX S). Instrument scatter sensitivity was assessed using NIST beads (40-400 nm), and virus stocks [human immunodeficiency virus (HIV), human coronaviruses (HCoV)-229E and HCoV-OC43]. For fluorescence analysis, HIV virions were stained with PE- and BV421-conjugated antibodies targeting virion incorporated proteins (CD38, CD44), individually and in combination. Finally, HIV stocks were labeled with antibodies against the envelope (Env) glycoprotein and tetraspanins (CD9, CD81) to assess EVs within virus preparations. ResultsCompared to the CytoFLEX S, the CytoFLEX nano exhibited substantially greater scatter sensitivity, reflected by up to 50-fold higher signal-to-noise ratio across NIST-traceable beads and virus samples. This enabled clearer resolution of smaller populations, including bead populations < 70 nm that were undetectable on the CytoFLEX S, as well as improved resolution across all viruses. While both instruments reliably detected stained proteins on HIV virions, the CytoFLEX nano revealed a distinct population of tetraspanin-positive EVs within HIV stocks that was undetected on the CytoFLEX S. Using GFP-tagged HIV, we identified Env+ particles lacking GFP, indicating the presence of Env on EVs. ConclusionsThe CytoFLEX nano exhibited markedly improved scatter sensitivity compared to the CytoFLEX S, improving detection of viruses and enabling detection of EV populations that were undetectable on the conventional instrument. While both platforms performed similarly for surface protein labeling, additional consideration of spectral overlap was needed with the CytoFLEX nano in multicolor experiments. These findings highlight that the complementary strengths of each platform can be utilized to more comprehensively characterize virus and EV populations, providing new opportunities to investigate nanoparticle heterogeneity.

The accurate measurement of Ebola virus (EBOV)-specific antibody responses is crucial to assessing immunity induced by EBOV infection or vaccination. For this purpose, the Filovirus Animal Nonclinical Group (FANG) anti-EBOV glycoprotein (GP1,2) ELISA is considered the "gold-standard". However, it has limitations such as high repeat-rates and variability, and low throughput. Here, we describe two new alternative assays: a Single-Molecule Assay Planar EBOV GP1,2 ELISA and a multiplexed EBOV GP1,2, EBOV nucleoprotein, and EBOV Viral Protein 40 Luminex assay, and compare these with two versions of the FANG ELISA. Samples were selected from participants receiving vaccine or placebo in a randomized, placebo-controlled, double-blinded study of two EBOV vaccines (PREVAIL 1), and a longitudinal cohort study of Ebola virus disease (EVD) survivors and their close contacts (PREVAIL 3). All four assays were concordant in their measurements of anti-EBOV GP1,2-specific immunoglobulin G responses, allowing for the determination of conversion equations for antibody measurements across assays. In addition, all four showed a similar ability to distinguish vaccine recipients from placebo recipients and EVD survivors from their close contacts. Compared to the FANG assays, the Quanterix and Luminex assays had lower variability, lower repeat rates, and higher throughput, making them good alternatives for future studies.

The co-circulation and rapid expansion of the genus Orthoflavivirus, including dengue virus (DENV), Zika (ZIKV), and Japanese encephalitis virus (JEV), pose significant global health challenges. Developing inclusive pan-genus molecular diagnostics is hindered by high nucleotide divergence (>25%-30%) and the computational limitations of traditional multiple sequence alignment in detecting conserved motifs across large datasets. To overcome these limitations, we developed a systematic alignment-free design pipeline that uses rigorous k-mer analysis and compacted De Bruijn graphs. We analyzed 11,846 RefSeq viral genomes to identify phylogenetically conserved, functionally relevant signatures within the Orthoflavivirus genus as a case study. The pipeline identified a conserved 600-bp region within the non-structural protein 5 gene, facilitating the design of a broad-spectrum TaqMan RT-qPCR assay. Analytical validation against standard reference strains demonstrated a limit of detection of 1-10 copies/{micro}L for DENV1-4, ZIKV, and JEV, with no cross-reactivity against non-target pathogens. In a clinical evaluation of archived samples, the assay achieved 97.33% overall accuracy. It demonstrated 100% sensitivity and specificity for DENV serotypes, yielding significantly earlier cycle threshold (Ct) values compared to a standard commercial kit, while ZIKV detection showed 100% specificity with 71.43% sensitivity. This study validates an alignment-free, k-mer guided approach for uncovering conserved diagnostic targets in highly variable viral genera. The resulting assay offers a robust tool for frontline surveillance, and the computational framework provides a scalable solution for future pandemic preparedness.

Introduction: Herpes simplex virus type 1 (HSV-1) is highly prevalent worldwide, making accurate serological testing essential for both clinical diagnosis and epidemiological surveillance. Automated chemiluminescent immunoassays (CLIAs) offer operational advantages over enzyme-linked immunosorbent assays (ELISAs); however, their diagnostic performance relative to Western blot (WB) confirmation in high-prevalence settings remains insufficiently characterized. Hypothesis/Gap Statement: The comparative diagnostic accuracy of CLIA- and ELISA-based assays for HSV-1 IgG detection, when benchmarked against a WB reference standard in endemic populations, remains unclear. Aim: This study aimed to evaluate HSV-1 IgG seroprevalence and diagnostic performance of one CLIA and two ELISA platforms using Western blot as the reference method. Methodology: Four hundred archived serum samples from adult male craft and manual workers in Qatar were tested using the Mindray CL-900i CLIA, HerpeSelect ELISA, NovaLisa ELISA, and Euroimmun Western blot. Seroprevalence, diagnostic accuracy, and interassay agreement were assessed using WB as the reference standard, with equivocal and indeterminate results excluded from analysis. Results: HSV-1 IgG seroprevalence estimates were comparable across assays: HerpeSelect 72.5%, Mindray 70.5%, NovaLisa 66.3%, and Western blot 66.5%, with no statistically significant differences (all p > 0.05). The Mindray CLIA demonstrated the highest diagnostic performance (sensitivity 95.7%, specificity 88.9%, accuracy 93.4%) and strong agreement with Western blot ({kappa} = 0.85). HerpeSelect showed substantial agreement ({kappa} = 0.81), while NovaLisa exhibited lower specificity. Conclusion: CLIA- and ELISA-based assays produced comparable HSV-1 seroprevalence estimates in this high-prevalence population; however, diagnostic accuracy varied across platforms. The CLIA platform demonstrated the strongest agreement with Western blot, supporting its use in high-throughput settings, while confirmatory testing remains important to minimize misclassification.

Quantitative polymerase chain reaction (qPCR) is limited in measuring absolute nucleic acid copy numbers due to the inherent variability of calibrators. Here, we introduce the Quantal PCR (quPCR), a novel method that eliminates the need for calibrators by defining an intrinsic quantal unit derived from the thermodynamic and kinetic properties of the replication system. This approach first determines amplification efficiency at high template concentrations, which is then used as the replication probability to construct quantification cycle (Cq) distribution profiles. These profiles are compared with those from limiting dilution PCR to derive the Cq value for the minimal quantal-replication unit ("quCq"), enabling calculation of the sample copy number. Validation using a dual-target DNA template showed near-identical copy numbers using two distinct target-specific replication systems. Thus, quPCR represents a new method for absolute nucleic acid quantification at the single-molecule level, offering a calibrator-free alternative for absolute quantification.

The protein sample preparation methods for shotgun proteomics are nowadays well-established unlike the ones for whole protein analysis. The goal of my work has been to create a simple methodology which provides a single uncomplicated sample preparation tool for these two fields. Nowadays the bulk of proteomics work is done using detergents for protein solubilization. The presented concept, which is based on unspecific adsorption of protein molecules on wide pore materials, allows for protein capture and clean-up from solutions of the most commonly used sodium dodecyl sulfate detergent. It could also be applied to proteins in detergent-free solutions. After the capture and clean-up, proteins could be either cleaved for the downstream peptide analysis or eluted for the whole protein analysis. If required, the eluted whole proteins could be recaptured and cleaved into peptides. Depending on the experimental goals, the sample preparation device could be fitted with embedded proteolytic enzymes to simplify routine sample processing and/or reversed phase media for the downstream peptide or protein separation.

Accurate quantification of fungi is important for a myriad of applications but remains challenging. Previously, we demonstrated that an approach called the adenine-HPLC method can quantify bacteria, including those with aggregating properties that are difficult to quantify using conventional methods, by measuring cellular adenine derived from DNA and converting the adenine amount to genome copy number, without being influenced by cell morphology. However, in this study, when this adenine-HPLC method was applied to the quantification of budding yeast as a model fungus, accurate measurement proved impossible. This limitation was attributed to adenine release from other adenine-containing biomolecules, such as RNA and ATP, and we therefore developed a method that suppresses adenine release from these molecules. This method involves reducing the temperature of the acid treatment and prewashing the cells before acid treatment. In addition, we incorporated a process that corrects for the naturally occurring free adenine level as background during total adenine measurement. The improved adenine-HPLC method based on these modifications enables accurate quantification of budding yeast using genomic DNA content in whole cells as the quantification unit.

Enumeration of the heterotrophic plate counts of culturable microorganisms (HPC) is used for the assessment of water quality before, during and after the drinking water treatment processes and monitoring of integrity of the drinking water distribution systems. Any substantial change in HPC is a warning of potential contamination, treatment failure, or intrusion. This study produced data for international standardization of Reasoners 2 Agar (R2A) method. Interlaboratory studies were applied to find out which inoculation technique, spread plate or pour plate, is more applicable, and what is the acceptable transport and storage time between the sampling and initiation of the analysis. Based on the produced interlaboratory data, spread plate inoculation followed by incubation for 7 days at 22 {degrees}C was selected as the standard method, and it is recommended to analyse samples as soon as possible, but the samples may be kept at (5 {+/-} 3) {degrees}C for up to 24 hours after the sampling prior to examination. Performance characteristics of the method were determined in a single laboratory according to ISO 13843:2017 by using process water from waterworks, bottled water, chlorinated and non-chlorinated tap water, and well water intended for human consumption. For quality assurance of the R2A medium, the use of control strains of Bacillus subtilis subsp. spizizenii, Pseudomonas fluorescens and an additional strain of Sphingomonas paucimobilis was verified during the work. The R2A method is especially suitable for determination of micro-organisms forming colonies after a prolonged incubation time, enabling HPC enumeration from biostable waters with low nutrient levels and low temperature. Such waters usually produce zero counts when nutrient-rich formulations of culture media with short incubation times are employed. Further, the nutrient limited conditions of R2A minimize the colony size and overgrowth in all kinds of water providing improvement to the HPC determinations.

Latent tuberculosis infection (LTBI) remains a significant barrier to global TB control and elimination efforts. The QuantiFERON-TB Gold (QFT) assay is commonly used for the diagnosis of LTBI. However, blood collected in QFT tubes is seldom utilized for molecular and genetic analysis due to the presence of heparin and a dense gel barrier that hinders efficient DNA extraction. To address this limitation, we aimed to develop a method for directly isolating high-quality DNA from blood in QFT tubes, eliminating the need for additional blood sampling and enabling their use in both diagnostic and molecular workflows. In this study, DNA was extracted from blood in EDTA and QFT tubes using a hybrid approach that combined manual lysis with three commercial kits: Thermo Scientific GeneJET, QIAamp DNA Blood Kit, and FavorPrep Blood Genomic DNA Extraction Kit. DNA concentration and purity were measured with a Multiskan SkyHigh Microplate Spectrophotometer, while integrity was assessed through agarose gel electrophoresis. Two nucleic acid amplification techniques (NAATs), ARMS-PCR and whole exome sequencing (WES) were performed to validate applicability of extracted DNA for molecular biology applications. We did not find any differences in the quantity, quality, or application of PCR or sequencing for DNA extracted from EDTA or QFT tubes. The extracted DNA from both EDTA and QFT tubes exhibited A260/280 ratios of 1.7-1.9 and concentrations ranging from 4.9 to 118.5 {micro}g/mL, indicating an adequate yield and purity. Intact genomic DNA and PCR product bands on agarose gel indicated suitability for downstream applications. Additionally, WES produced 6.47-8.71 GB of data per sample, with 42.8-57.7 M reads and GC content between 49.29% and 52.54%. Sequencing metrics were consistently strong, with Q20 values exceeding 98.6% and Q30 values above 95%. Our study presents an optimized and reproducible protocol for extracting high-quality DNA from QFT tubes, producing DNA suitable for both PCR and sequencing technologies. This protocol provides a cost-effective and practical strategy to integrate LTBI diagnosis with genomic research, particularly beneficial in resource-limited settings. This study introduces a novel analytical workflow applicable to diagnostic laboratory settings, enabling the integration of routine LTBI immunodiagnostic testing with downstream genomic analysis. The approach supports improved utilization of clinical specimens in laboratory medicine and may facilitate future biomarker and precision diagnostics research.

Here we evaluated the performance of a previously published tiling PCR primer scheme by Ringlander et al. (2022) for whole-genome amplification of Hepatitis B virus (HBV) in combination with Oxford Nanopore sequencing. The primer set originally developed for Ion Torrent sequencing was adapted by removing platform-specific adapters and tested using clinical serum or plasma samples submitted for routine HBV genotyping and resistance testing. Two multiplexing strategies were compared: a single PCR pool containing all primers and a two-pool strategy with non-overlapping amplicons. Sequencing reads were processed using a Nanopore analysis pipeline, and genome coverage and amplicon performance were compared across samples spanning a wide Ct range and representing HBV genotypes A-E. Across all samples, the median genome coverage was approximately 50%, although recovery varied widely, ranging from complete failure to nearly full genomes. Combining all primers into a single PCR reaction, or separating overlapping amplicons into different reactions, had little overall impact on genome recovery, and no consistent differences between the two pooling strategies were observed. In contrast, amplification efficiency differed markedly between individual amplicons. Amplicons 1-5 generally produced higher sequencing depth, whereas amplicons 6-10 frequently showed low coverage and contributed to incomplete genome recovery. Genome coverage was strongly associated with Ct values, with higher coverage observed in samples with lower Ct values, while coverage was broadly similar across genotypes. These results demonstrate that the Ringlander et al. primer scheme can be adapted for multiplex PCR and Nanopore sequencing of HBV, but uneven amplicon performance limits consistent full-genome recovery and highlights the need for further optimization of HBV tiling PCR designs.

Background: External Quality Assurance (EQA) is an essential component of modern laboratory medicine. Current scientific evidence on EQA focuses primarily on the analyses carried out by EQA providers while relatively little research has been conducted in individual clinical laboratories. Methods: In this retrospective single-center observational study in a clinical laboratory, EQA results were analyzed over a period of four years (2021-2024). The evaluation was based on EQA action reports documented in the institutes internal quality management system. Deviations were classified according to department, type of discrepancy, root cause category (analytical, preanalytical, systemic, unidentifiable), and measures taken. Results: A total of 7226 EQA participations were evaluated during the observation period. The overall error rate remained consistently low, ranging between 0.8% and 1.6%, with no significant change over time (p = 0.87). Most deviations occurred in the departments of clinical chemistry and immuno/autoimmune diagnostics (p < 0.001). These were predominantly quantitative discrepancies (false low/false negative or false high/false positive). Root cause analysis showed a clear dominance of analytical causes (p < 0.001), while preanalytical and systemic causes were identified less frequently. In most cases, corrective measures, such as re-analyses, recalibrations, process adjustments, or staff training, were implemented promptly. Hard structural measures, such as changing methods or discontinuing tests, were rarely necessary. Conclusion: In a clinical laboratory, EQA is an important tool for structured error analysis and continuous quality improvement. Consistent processing of deviating EQA results goes hand in hand with stable analytical performance and a low error rate.

The global expansion of highly pathogenic avian influenza (HPAI) virus A(H5N1) underscores the need for rapid surveillance at high-risk wildlife interfaces. Taiaroa Head (45.7828{degrees} S, 170.7333{degrees} E) in the South Island of Aotearoa New Zealand hosts a plethora of aquatic wildlife including a large red-billed gull (Chroicocephalus novaehollandiae scopulinus) colony as well as the only mainland breeding colony of northern royal albatross (Diomedea sanfordi). The Royal Albatross Centre is also a major nature tourism destination, attracting tens of thousands of visitors annually, thereby creating a dense ecological and human-wildlife interface vulnerable to viral incursion. We evaluated the GeneXpert II platform using the Xpert(R) Xpress Flu/RSV cartridge as a field-deployable tool for avian influenza virus detection in environmental and wildlife-associated samples. The assay detected synthetic influenza A viral RNA and multiple endemic low pathogenic avian influenza virus subtypes (A(H3N8), A(H1N9), A(H5N2) and A(H7N7)) circulating in New Zealand birds. Influenza A virus was reliably identified in spiked environmental water samples with no consistent PCR inhibition as well as naturally occurring avian influenza virus in duck pond water. Field deployment demonstrated that the system could be operated by non-laboratory personnel with minimal training in a non-clinical setting. This study establishes the feasibility of near-real-time environmental monitoring. Repurposing clinical cartridge-based point-of-care diagnostics offers a practical early warning approach for avian influenza virus surveillance at ecologically and economically significant locations.

BackgroundGenetic drift and host-associated adaptation in influenza A viruses threaten the long-term reliability of RT-qPCR-based diagnostics, particularly when nucleotide mismatches arise within primer and probe binding regions. Conventional assay evaluations often emphasize sequence conservation but rarely assess functional mismatch tolerance across divergent subtypes and hosts. MethodsWe performed an in silico evaluation of a matrix (M) gene-targeted RT-qPCR assay by aligning primer and probe binding regions against 22 H1N1 isolates and representative H3N2 and H5N1 reference strains, including recent zoonotic isolates from avian and bovine hosts. Nucleotide mismatches were identified, quantified, and mapped relative to assay components and oligonucleotide termini. Mismatch burden was summarized by subtype and assay region. ResultsH1N1 isolates exhibited complete conservation across primer and probe regions. In contrast, H3N2 and H5N1 strains demonstrated subtype-specific sequence variability, with a total of eleven mismatches identified across seven non-H1N1 isolates (mean mismatch per isolate = 2.43). Probe mismatches predominated (63.6%), occurring primarily at internal positions, while primer mismatches were infrequent and largely avoided 3' terminal nucleotides. Recent H5N1 isolates (2023-2024) shared conserved internal mismatches in the probe and forward primer, whereas a historical H5N1 isolate (2016) exhibited a distinct profile including a terminal probe mismatch. Despite this variability, mismatch patterns were consistent with preserved amplification potential. ConclusionThis study demonstrates that the evaluated influenza A M gene RT-qPCR assay exhibits inherent mismatch tolerance across human and zoonotic subtypes. By shifting diagnostic evaluation from strict sequence identity to functional resilience, our findings provide a framework for designing and maintaining robust molecular assays suitable for surveillance and pandemic preparedness amid ongoing viral evolution. Graphical AbstractIn silico evaluation of an influenza A matrix gene RT-qPCR assay demonstrates subtype-specific primer and probe mismatches across H3N2 and H5N1 strains, including recent zoonotic isolates. Despite observed variability, mismatches predominantly occur at internal positions and spare primer 3' termini, supporting inherent assay mismatch tolerance and suitability for surveillance applications. O_FIG O_LINKSMALLFIG WIDTH=150 HEIGHT=200 SRC="FIGDIR/small/707407v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@e48677org.highwire.dtl.DTLVardef@1380ddcorg.highwire.dtl.DTLVardef@11606f0org.highwire.dtl.DTLVardef@121b4ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

BackgroundThere is a need for synthetic peptide-based serologic assays that exploit avidity to replace whole antigens while enabling low-cost diagnostics in resource-limited settings. ObjectiveTo evaluate the diagnostic accuracy of a polymeric peptide-based ELISA leveraging avidity to enhance signal. MethodA 15-member SARS-CoV-2 peptide library corresponding to multiple epitope clusters and proteins was screened by indirect ELISA using pooled sera from RT-PCR-confirmed COVID-19 patients to identify peptides with possible diagnostic utility. The identified lead candidate, S559, possessed terminal cysteine-substitution to allow disulfide polymerization, and the resulting avidity gain was evaluated by comparing the apparent dissociation constant (KDapp) before and after depolymerization with N-acetylcysteine. The performance of an optimized ELISA using S559 was evaluated on 1,222 prospectively collected COVID-19 serum samples and 218 biobanked pre-COVID control serum samples. ResultsPolymeric S559 with a KDapp of 29.26 nM-1was demonstrated to have a 218% avidity gain relative to the completely depolymerized form. At pre-defined thresholds, the optimized S559 ELISA has a sensitivity and specificity of 83.39% (95%CI: 81.18% and 85.43%) and 96.79% (95%CI: 93.50% and 98.70%), respectively. At post hoc thresholds determined by Youden index, sensitivity and specificity reached 95.01 (95% CI: 93.63% - 96.16%) and 100.00% (95% CI: 98.32% - 100.00%), respectively. ConclusionHomomultivalent epitope presentation using polymeric S559 allows a highly specific immunoassay using human sera that may have important value in detecting antibodies, whether for diagnosing infection, confirming vaccination status or conducting surveillance.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

The ubiquitin-conjugating enzyme UBE2J1 functions in the proteasomal degradation of proteins at the ER. Existing evidence suggests that it plays a role during viral infection, with elevated UBE2J1 levels generally associated with increased infection. This is particularly relevant for some RNA viruses; however, the regulation of UBE2J1 during infection has not been well studied. Here, we used a BHK21 cell model to demonstrate that UBE2J1 overexpression promotes the replication of Vesicular Stomatitis Virus (VSV), as indicated by a significant increase in viral titres. To better understand the underlying molecular processes, cells were co-transfected to express the VSV-G protein and wild-type UBE2J1 protein, and we observed a significant increase in the syncytial fusion area. This effect was not observed when catalytically inactive (C91S) or phospho-deficient (S184A) forms of the protein were used. Interestingly, overexpression of a truncated, non-ER localized form of UBE2J1 ({Delta}TM) led to the largest increase in the syncytial fusion area. This arose as a result of increased syncytia size, and may indicate an enhanced cellular role if soluble forms of UBE2J1 are not anchored to the ER. Additional studies using truncated, mutated and wild-type proteins confirmed that UBE2J1 increases VSV viral replication and is associated with an increase in the number of infection plaques. Considering the emerging evidence for UBE2J1 involvement in viral infection, our finding should help in understanding the role of this protein in viral pathogenesis and cellular processes linked to syncytialization.

Bacterial and Viral infections have identical symptoms, making it difficult to diagnose based on visible symptoms. This results in the overprescription of antibiotics, even in cases of viral infections, to avoid missing bacterial infections that could progress into a more severe condition and/or sepsis. This diagnostic gap contributes to the overuse of antimicrobials. To address the issue, a deployable diagnostic test has been developed and evaluated that can help differentiate between bacterial and viral infections. The approach is centred on three crucial biological markers: CD64 expression on leukocytes to detect bacterial infections, CD169 expression on leukocytes to detect viral infections and total leukocyte count as a general health indicator of the patient. ABxSure comprises a cartridge for sample processing, a device with in-built pneumatics, and an optical reader for quantifying biomarkers from blood. The ABxSure performance was tested and compared with a commercial plate reader and flow cytometer, yielding a promising correlation of 0.89. This comprehensive test will potentially provide clinicians with valuable information for effective and timely treatment.