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.

Citrus is one of the most important crops cultivated worldwide, representing a strategic source of agricultural income for many countries, including Spain, the main producer within the European Union. The protection of this highly valuable industry against an increasing global movement of pests and pathogens requires effective regulatory measures, including control of plant propagation material, phytosanitary surveillance and risk assessment, which are based not only on knowledge of the established threats but also on potential emerging threats affecting citrus that may circulate unnoticed in the production system. In this work, with the aim of generating knowledge on potential emerging viruses in Spanish citrus orchards, high-throughput sequencing (HTS) analysis has been applied to monitor the sanitary status of several growing areas of one of the main citrus producer regions in Spain, the Valencian Community. The results of this study have revealed a much more complex citrus virome than previously reported, including citrus yellow vein clearing virus (CYVCV), a non-regulated but harmful citrus virus, as well as the T3 genotype of citrus tristeza virus (CTV) and citrus virus A (CiVA), not detected to date in Spain. Moreover, our results indicate the existence of other unknown components of the citrus virome. HTS detection of CYVCV, CTV T3 and CiVA and their presence in Spanish orchards has been confirmed by RT-PCR and Sanger sequencing. These findings have relevant implications in the development of control and regulatory measures against three important viral diseases, tristeza, impietratura and yellow vein clearing diseases, and demonstrate the added value of HTS-based surveillance to discover emerging components of the citrus virome.

Reptarenaviruses are viruses belonging to the genus Reptarenavirus within the family Arenaviridae, which infect snakes and cause inclusion body disease (IBD), a fatal condition characterized by behavioral abnormalities and wasting. Although many reptarenaviruses have been identified thus far, the phylogenetic gaps between reptarenaviruses and the other arenaviruses suggest the existence of yet-to-be-identified reptarenaviruses filling the gaps. In this study, we identified a novel reptarenavirus from publicly available RNA-seq data derived from Amazon coral snake (Micrurus spixii) and tentatively named it Amazon coral snake virus 1 (ACSV-1). We identified four ACSV-1 contigs containing the putative full-length open reading frames of the NP, GP, and L genes, as well as the partial Z gene. Phylogenetic analyses showed that ACSV-1 is highly divergent from known reptarenaviruses. The NP, GP, and L genes showed 48.3%, 42.3%, and 45.7% nucleotide sequence identities, respectively, with those of the closest relatives. Based on the International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria, ACSV-1 can be assigned to a novel species of virus within the genus Reptarenavirus. This study expands our understanding of the diversity and evolution of reptarenaviruses.

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.

BackgroundBoth hemorrhagic fever with renal syndrome (HFRS) and scrub typhus (ST) are acute zoonotic infectious diseases. There is an overlap in their epidemiological characteristics and clinical manifestations, posing challenges for early differential diagnosis. This study aims to identify predictive factors for these two diseases to provide a basis for early diagnosis. Method/FindingsA retrospective analysis was conducted on the clinical data of patients diagnosed with HFRS and ST at the First Affiliated Hospital of Dali University. Logistic regression analysis was employed to explore independent risk factors for the early differential diagnosis of these two diseases, and a nomogram model was constructed based on these risk factors. The performance of the model was evaluated using the area under the receiver operating characteristic curve (AUC). The nomogram was utilized to visually present the predictive variables. Decision curve analysis (DCA) was performed to assess the clinical utility of the model. ResultsA total of 235 patients each with HFRS and ST were included in this study. After adjusting for confounding factors, the results of multivariate logistic regression analysis revealed that sex (male) (adjusted odds ratio [ajOR]: 2.093, 95% confidence interval [CI]: 1.107 - 3.957, P = 0.018), positive proteinuria (ajOR: 4.937, 95% CI: 2.427 - 10.042, P < 0.001), creatinine (CREA) (ajOR: 1.009, 95% CI: 1.003 - 1.015, P = 0.005), heart rate (ajOR: 0.981, 95% CI: 0.966 - 0.997, P = 0.018), and conjunctival congestion (ajOR: 16.167, 95% CI: 5.326 - 49.072, P < 0.001) were independent risk factors for differentiating HFRS from ST. The AUC of the model constructed based on these five independent risk factors was 0.856. ConclusionSex (male), positive proteinuria, elevated CREA, decreased heart rate, and conjunctival congestion are effective predictive factors.

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.

Recent advances in sequencing technology and transcriptome mining have revealed highly divergent hepaciviruses in birds. However, only a limited number of avian hepaciviruses have been identified to date, leaving their diversity and evolutionary history poorly understood. Moreover, deep phylogenetic gaps among known avian hepaciviruses suggest that additional lineages remain undiscovered. Here, we screened publicly available RNA-seq data and identified three previously undescribed hepaciviruses from rock pigeon (Columba livia), rusty-margined flycatcher (Myiozetetes cayanensis), and Hispaniolan amazon (Amazona ventralis), named rock pigeon hepacivirus (RpHV), rusty-margined flycatcher hepacivirus (RfHV), and Hispaniolan amazon hepacivirus (HaHV). Although these three viruses meet the ICTV species demarcation criteria relative to their closest known relatives, the NS5B-based criterion was not satisfied between RfHV and HaHV. Notably, however, their genome sequence identity is low at 43.2%, and their hosts differ at the order level, suggesting that their classification warrants further consideration. Our phylogenetic analysis showed that avian hepaciviruses, including those found in this study, are monophyletic, but phylogenetic incongruence was observed between avian hepaciviruses and their hosts, suggesting past cross-species transmission among avian hepaciviruses. Overall, this study provides novel insights into the diversity and evolution of hepaciviruses.

Small ruminants (sheep and goats) are one of the few mammals in which an exogenous retrovirus (XRV) and closely related endogenous retroviral elements (ERV) coexist within the same host genome. The betaretroviruses Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause pulmonary and nasal adenocarcinomas, respectively, and share extensive sequence similarity with their endogenous counterparts. Consequently, molecular surveillance must rely on assays that can unequivocally distinguish true exogenous infection from ERV-derived templates; failure to do so compromises diagnosis, phylogenetic inference, and epidemiological conclusions. We retrieved all complete JSRV, ENTV-1/2, and related ERV genomes deposited in public repositories and performed a comprehensive alignment. Only a limited number of genomic segments were capable of distinguishing exogenous from endogenous sequences. We refer to these as discriminating regions (DRs). Phylogenies built using DRs revealed that several entries annotated as XRV are, in fact, ERV-derived or chimeric artefacts generated by short-amplicon reconstruction. A systematic literature review of over 100 articles identified 286 distinct primers and probes used for the XRV amplification. In-silico mapping of each oligonucleotide onto the full alignment showed that only 28 % reliably differentiate XRV from ERV. We experimentally validated the predictive power of this approach for 17 primer/probe sets, confirming that non-discriminating assays produce false-positive signals from endogenous templates. The misannotation of ERV sequences as exogenous viruses has resulting in the population of databases with dubious entries, fostering erroneous hypotheses such as vector-borne transmission of JSRV and ENTV. To address this issue, we propose a concise set of criteria for assay design, validation, and database annotation emphasizing DR targeting, specificity testing against endogenous templates, and transparent reporting. Although this framework was developed for small ruminants, it is readily applicable to any host-virus system in which exogenous viruses coexist with endogenous viral elements. This will strengthen viral surveillance, phylogenetics, and the One Health initiatives.

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.

In the past decade, dengue fever has emerged as a major public health on Reunion Island in the Southwest Indian Ocean. During the 2018-2022 outbreak, an unusual increase in ocular complications was reported in some patients. To investigate a potential viral cause, we analysed 447 blood samples from hospitalized patients with and without ophthalmic symptoms. Genetic sequencing revealed the co-circulation of two strains of dengue virus serotype 1, both genetically linked to strains previously identified in Asia. Notably, all patients with ophthalmic symptoms were infected with viruses from a single cluster within genotype I, which harbored several unique mutations. These findings suggest that the rare ocular complications observed during this outbreak may be associated with specific viral cluster. Further laboratory studies are required to confirm this potential link.

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.

Pneumonia virus of mice (PVM), the mouse homolog to respiratory syncytial virus (RSV), is increasingly used as surrogate model to study pneumovirus pathogenesis in a more natural pathogen-host relation. Two major strains of PVM, strain 15 and J3666 are currently used in laboratories, with preferences for either one or the other based on the well-documented isolation history of strain 15 or the suggested higher virulence of strain J3666. Using conventional and long read sequencing, we found that the PVM strain J3666 represents two distinct virus populations, which are defined by sequence and structure of the G and SH genes encoding the putative attachment and small hydrophobic proteins, in addition to further nucleotide polymorphisms. Specifically, a nucleotide polymorphism at position 65 in the G gene results in either an upstream open reading frame (uORF) preceding the main ORF in frame, or an extension of the major G ORF by 18 codons. The impact of the different forms of the J3666-G genes on PVM was examined by generating recombinant PVMs differing exclusively in the distinctive 5 portion of the respective G gene. This revealed that the population expressing a G protein with an extended main G ORF was more virulent, whereas the presence of a uORF attenuated virulence. The virulence of PVM correlated with increased expression levels of G, whereas attenuation was rather associated with downregulated expression of G due to the presence of a uORF. Thus, modulation of G protein levels may be an important mechanism by which pneumoviruses modulate virulence. ImportanceThe pneumonia virus of mice strain J3666 is considered a more virulent and more suitable model for severe lower respiratory tract infections. The organization of the gene for the attachment protein G is reported to contain a small upstream open reading frame (uORF) preceding the main G ORF in frame. The translated G protein is predicted to comprise 396 amino acids. We report that this virus strain may be a mixture of two different populations, each with differing virulence. The more virulent population encodes a G protein of potentially 414 amino acids instead of a small uORF. This G gene organization is associated with an increased G protein expression. Importantly, this organization of the G gene is in line with that of several newly identified pneumoviruses, i.e., canine and swine pneumoviruses. These viruses may comprise a distinct group within the Pneumoviridae family.

Porcine reproductive and respiratory syndrome (PRRS) is a globally distributed disease caused by Betaarterivirus europensis (PRRSV-1) and Betaarterivirus americense (PRRSV-2). Its clinical presentation ranges from subclinical infection to severe disease, depending on viral evolution and the emergence of novel variants. The aim of this study was to characterize the genetic diversity and identify recombination events in the ORF7 (nucleocapsid, N) gene of ten PRRSV-2 strains circulating in pig farms in Lima, Peru. Bioinformatic analyses were performed using DNAMAN v10.0, MEGA 6, BepiPred-2.0, DnaSP v6, and RDP v4.101. Phylogenetic analysis revealed two well-defined lineages: eight strains clustered within lineage 1A (NADC34-like), and two within lineage 5A (VR2332-like), demonstrating the co-circulation of genetically distinct variants in the region. Comparative sequence analysis identified significant amino acid substitutions in eight strains (15, 16, 17, 20, 21, 22, 23, and 24), with strain 24 being the most divergent, accumulating multiple substitutions, including T81I, R109S, I115F, R116S, and A119K within the C-terminal region encompassing antigenic domains I-V. B-cell epitope prediction using BepiPred-2.0 identified six epitope patterns (A-F) comprising nine potential B-cell epitope regions (positions 5-19, 33-72, 33-73, 84-85, 87-98, 84-98, 87-97, 84, and 119). Patterns B, E, and F exhibited four to five predicted epitope sites and corresponded to strains 21, 22, 23, and 24. Recombination analysis using RDP v4.101 detected a statistically robust recombination event in strain 18_montana2020-R (lineage 5A), with strain 24_montana2020-WT (lineage 1A) identified as the putative major parent (100% similarity) and the vaccine-like VR2332 strain (lineage 5A) as the minor parent (99.3% similarity). Secondary evidence of the same recombination event was observed in strain 19_montana2020-R. Genetic diversity analysis of the ORF7 gene identified 50 polymorphic nucleotide sites and 52 mutations. Overall, these findings demonstrate substantial genetic variability in the ORF7 gene of PRRSV-2 circulating in Lima, Peru, characterized by lineage co-circulation and inter-lineage recombination. Continuous molecular surveillance is warranted to monitor viral evolution, assess potential antigenic implications, and support effective PRRS control strategies in the Peruvian swine industry.

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.

BackgroundA critical radiologist shortage exists in India, leading to delayed chest radiograph (CXR) interpretation. This leads to disease progression, higher morbidity, and mortality. Artificial intelligence-based CXR interpretation by Lenek Intelligent Radiology Assistant (LIRA) is a promising solution. This study aims to establish the screening and triaging capabilities of LIRA by assessing its accuracy in detecting abnormalities and pathologies in CXRs from geographically diverse institutions. MethodsWe conducted a retrospective multi-source validation of the diagnostic accuracy of LIRA for the detection of general abnormalities, tuberculosis, consolidation, pleural effusion, pneumothorax, and cardiomegaly. De-identified chest radiographs were input into LIRA models. The obtained interpretations were compared to the established ground truth reporting for the calculation of sensitivity, specificity, and AUROC with 95% CI for individual pathologies across varying probability thresholds. ResultsLIRA demonstrated high sensitivity for general abnormality detection (AUROC 0.93-0.986, 84.4-97.1% sensitivity, 88.9-92.4% specificity) and tuberculosis triaging (Shenzhen & Montgomery: 88.5-89.7% sensitivity, 89.9-90.5% specificity; Jaypee: 98.7% sensitivity, 63.6% specificity). For consolidation (AUROC 0.884-0.895, 96.4-96.9% sensitivity, 70.8-77.1% specificity), pleural effusion (AUROC 0.942-0.967, 79.7-99.1% sensitivity, 81.2-87.7% specificity), pneumothorax (AUROC 0.87, 90.6-94.8% sensitivity, 79.5-82.7% specificity) and cardiomegaly (AUROC 0.883, 95.1% sensitivity, 81.6% specificity), the model exhibited commendable accuracy as well. ConclusionsThe diagnostic performance of LIRA was consistent across various pathologies and chest radiographs from diverse geographic locations, with particular strengths in abnormality detection and tuberculosis screening. The risk-stratified triaging and high sensitivity of LIRA make it a reliable adjunct solution to address radiologist shortages, reduce turnaround times, and support Indias tuberculosis elimination goals.

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