Background: As of 2024, three approved respiratory syncytial virus (RSV) vaccines are licensed for use in adults in the United States: Arexvy™, Abrysvo™, and mRESVIA™. These vaccines are specifically designed to prevent lower respiratory tract disease caused by RSV in adults aged 60 and older. All licensed vaccines rely on stabilized RSV pre-fusion F (pre-F) as the sole antigen. RSV vaccines targeted to the other key RSV surface protein, the G glycoprotein, have been slow to advance because of sequence diversity and a historical association with vaccine-enhanced disease in animal models of infection. The recent development of structure-guided subunit immunogens and immune-modulating adjuvants has renewed interest in RSV G, as the combination of both F and G glycoproteins appears to improve vaccine efficacy over either one individually. RSV G is extensively O-glycosylated, with two mucin-like regions. Methods: This study investigated the effects of manipulation of O-linked glycosylation on a recombinant RSV G vaccine antigen in an RSV/A2 challenge study in BALB/c mice. Conclusions: We found that restricting the O-linked glycosylation on a recombinant RSV G vaccine antigen enhances its immunogenicity and protective efficacy in BALB/c mice.

Understanding virus-surface interactions is essential for developing effective biosensors, diagnostic tools, and antiviral strategies. In this study, we present a systematic investigation of the adsorption and coadsorption behavior of 12 respiratory viruses, including influenza, RSV, coronaviruses, adenovirus, and metapneumoviruses, using surface-enhanced Raman scattering (SERS) on SiO2-coated silver nanorod array substrates. Both single viruses (SVs) and binary virus mixtures (2VMs) were analyzed in water and normal human saliva, and spectral data were modeled using a modified Brunauer-Emmett-Teller (BET) adsorption framework. Linear least-squares spectral decomposition enabled the extraction of adsorption coefficients that correlate with virus concentration and surface binding affinity. All viruses exhibited multilayer physisorption consistent with Type II isotherms, with the BET constant q varying substantially across virus types. Notably, 2VMs demonstrated a significantly enhanced adsorption behavior, often with q values 4-25 times greater than in SVs, indicating strong cooperative or competitive effects. Saliva modulated virus-surface interactions in virus-specific ways, emphasizing the complexity of adsorption dynamics in physiological environments. These findings highlight the limitations of single-virus calibration for quantitative detection in mixed-virus samples and underscore the need for mixture-aware analytical models in biosensing applications. This work provides a robust framework for mechanistic insight and quantitative modeling of virus adsorption relevant to real-world diagnostics and environmental monitoring.

Respiratory syncytial virus (RSV) is a significant cause of disease in the young and old. Recently, pre-fusion F protein vaccines for RSV have received food and drug administration (FDA) approval to protect adults aged 60 years and older; however, vaccines evaluated against RSV typically do not elicit complete or durable protective immunity. We previously showed that an RSV G protein central conserved domain (CCD) nanoparticle vaccine containing an S177Q mutation (NP-S177Q) induced favorable immunogenicity and RSV-neutralizing antibodies compared with RSV G protein vaccination alone in mice. Boosting BALB/c mice with NP-S177Q vaccines improved correlates of protection and reduced markers of immunopathology following RSV challenge. This study examined microparticle (MP) vaccines displaying the CCD with an RSV G S177Q mutation (MP-S177Q) adjuvanted with monophosphoryl lipid A (MPLA) in BALB/c mice. Our findings show that mice adjuvanted MP-S177Q vaccination develop effective viral neutralization compared with MP-WT and MP-S177Q vaccination and have improved bronchoalveolar Th1-type cytokine responses following the RSV challenge compared with MP-WT or vehicle-vaccinated mice. This study shows that a rationally mutated RSV G protein MP vaccine is safe, effective, and can advance precision RSV vaccines.

Respiratory viruses such as respiratory syncytial virus (RSV) annually cause respiratory illness, which may result in substantial disease and mortality in susceptible individuals. Viruses exploit host cell machinery for replication, which engages the mitogen-activated protein kinases (MAPK) pathway. The MAPK signaling pathways are triggered by pattern recognition receptors that recognize the pathogen, infection, or external stimuli, leading to the induction and regulation of immunity and inflammation. Probenecid, used to improve renal function by inhibiting the tubular reabsorption of uric acid, has been shown to have therapeutic efficacy in reducing inflammation and blocking viral replication by inhibiting components of the MAPK pathway that preclude virus replication. This review summarizes key molecular cascades in the host response to virus recognition, infection, and replication and how this can be altered by probenecid treatment.

Probenecid has long been a versatile drug in pharmacological therapies, primarily known for blocking active tubular secretion in the kidney, affecting both endogenous substances like uric acid and exogenous ones like penicillin. Beyond its renal applications, probenecid has shown capabilities in crossing the blood–brain barrier and modulating the activity of various membrane channels and transporters. This compound has emerged as a potent antiviral agent, demonstrating efficacy against multiple viruses, including influenza, COVID-19, and RSV. Clinical trials with COVID-19 patients have confirmed its antiviral potential, sparking further investigation into its mechanisms of action. This study explores probenecid’s significant anti-inflammatory properties, focusing on its ability to inhibit inflammasome activation. Our study aims to unravel the anti-inflammatory effects of probenecid on the NLRP3 inflammasome and MAPK signaling pathways using murine macrophages as a relevant inflammation model. We reveal that probenecid treatment blocks JNK and ERK signaling without affecting p38 MAPK, suppressing NLRP3 inflammasome activation. Additionally, probenecid does not affect NFκB-directed protein expression, although it efficiently inhibits NLRP3 inflammasome outputs, e.g., IL-1β and pyroptosis. These results indicate probenecid’s potential therapeutic applications.

It is essential to understand the molecular mechanisms of influenza antiviral therapeutics to evaluate their efficacy. Virus plaque assays are commonly used to assess the antiviral effects of drugs on virus replication; however, this method is labor-intensive and can present challenges. We avoided this method by using a replication-competent influenza A virus (IAV) expressing a reporter fluorescent gene fused to the non-structural protein 1 (NS1) gene. The reporter IAV was detectable in normal human bronchoepithelial (NHBE) infected cells and offered an improved method to determine the therapeutic efficacy of the antiviral drugs probenecid and oseltamivir compared to a standard plaque assay. This method provides an excellent means for evaluating therapeutic approaches against IAV.

A useful and reproducible method to determine infectious respiratory syncytial virus (RSV) is to quantify virus plaques using immunostaining of infected Vero E6 cells. Immunostaining, in lieu of counterstaining, allows for accurate detection and quantification of RSV-specific plaques, avoiding spurious and nonspecific results. This method is important because RSV plaque morphology can be atypical in susceptible cell lines and often varies because it is a function of virus replication, cytopathic effect, and viral fitness. This protocol describes determining RSV titers using an immunostaining plaque assay on Vero E6 cells.

Multiple respiratory viruses can concurrently or sequentially infect the respiratory tract, making their identification crucial for diagnosis, treatment, and disease management. We present a label-free diagnostic platform integrating surface-enhanced Raman scattering (SERS) with deep learning for rapid, quantitative detection of respiratory virus coinfections. Using sensitive silica-coated silver nanorod array substrates, over 1.2 million SERS spectra are collected from 11 viruses, nine two-virus mixtures, and four three-virus mixtures at various concentrations in saliva. A deep learning model, MultiplexCR, is developed to simultaneously predict virus species and concentrations from SERS spectra. It achieves an impressive 98.6% accuracy in classifying virus coinfections and a mean absolute error of 0.028 for concentration regression. In blind tests, the model demonstrates consistent high accuracy and precise concentration predictions. This SERS-MultiplexCR platform completes the entire detection process in just 15 min, offering significant potential for rapid, point-of-care diagnostics in infection detection, as well as applications in food safety and environmental monitoring.

Concurrent or sequential infections by multiple respiratory viruses pose significant challenges for diagnosis, treatment, and disease management, necessitating accurate and rapid identification methods. Here, we introduce a label-free diagnostic platform that combines surface-enhanced Raman scattering (SERS) with deep learning to enable rapid, quantitative detection of respiratory virus co-infections. Utilizing highly sensitive silica-coated silver nanorod array substrates, we collected over 1.2 million SERS spectra from 11 viruses, nine two-virus mixtures, and four three-virus mixtures at varying concentrations in saliva. A novel deep learning model, MultiplexCR, was developed to simultaneously identify virus species and quantify their concentrations from the SERS spectra. The model achieved an exceptional classification accuracy of 98.6% for virus co-infections and a mean absolute error of 0.028 for concentration regression. In blind testing, MultiplexCR consistently demonstrated high accuracy and precise concentration predictions. This SERS-MultiplexCR platform performs the entire detection process within 15 minutes, highlighting its potential for rapid, point-of-care diagnostics in infection detection, as well as broader applications in food safety and environmental monitoring.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host–virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.

An integrated approach combining surface-enhanced Raman spectroscopy (SERS) with a specialized deep learning algorithm to rapidly and accurately detect and quantify SARS-CoV-2 variants is developed based on an angiotensin-converting enzyme 2 (ACE2)-functionalized AgNR@SiO2 array SERS sensor. SERS spectra with concentrations of different variants were collected using a portable Raman system. After appropriate spectral preprocessing, a deep learning algorithm, CoVari, is developed to predict both the viral variant species and concentrations. Using a 10-fold cross-validation strategy, the model achieves an average accuracy of 99.9% in discriminating between different virus variants and R2 values larger than 0.98 for quantifying viral concentrations of the three viruses, demonstrating the high quality of the detection. The limit of detection of the ACE2 SERS sensor is determined to be 10.472, 11.882, and 21.591 PFU/mL for SARS-CoV-2, SARS-CoV-2 B1, and CoV-NL63, respectively. The feature importance of virus classification and concentration regression in the CoVari algorithm are calculated based on a permutation algorithm, which showed a clear correlation to the biochemical origins of the spectra or spectral changes. In an unknown specimen test, classification accuracy can achieve >90% for concentrations larger than 781 PFU/mL, and the predicted concentrations consistently align with actual values, highlighting the robustness of the proposed algorithm. Based on the CoVari architecture and the output vector, this algorithm can be generalized to predict both viral variant species and concentrations simultaneously for a broader range of viruses. These results demonstrate that the SERS + CoVari strategy has the potential for rapid and quantitative detection of virus variants and potentially point-of-care diagnostic platforms.

We examined the effect of probenecid in regulating the ERK and JNK downstream MAPK pathways affecting respiratory syncytial virus replication. Background: We have previously shown that probenecid inhibits RSV, influenza virus, and SARS-CoV-2 replication in vitro in preclinical animal models and in humans. In a Phase two randomized, placebo-controlled, single-blind, dose range-finding study using probenecid to treat non-hospitalized patients with symptomatic, mild-to-moderate COVID-19, we previously showed that a 1000 mg twice daily treatment for 5 days reduced the median time to viral clearance from 11 to 7 days, and a 500 mg twice daily treatment for 5 days reduced the time to viral clearance from 11 to 9 days more than the placebo. Methods: In this study, we sought to determine the mechanism of action of the probenecid inhibition of RSV replication in human respiratory epithelial (A549) cells. Results: We show that probenecid inhibits the RSV-induced phosphorylation of JNKs and ERKs and the downstream phosphorylation of c-jun, a component of the AP-1 transcription complex needed for virus replication. The inhibition of JNKs by probenecid reversed the repression of transcription factor HNF-4. Conclusion: The probenecid inhibition of JNK and ERK phosphorylation involves the MAPK pathway that precludes virus replication.

Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly infectious bird virus infecting primarily poultry, but unlike other AIs, H5N1 also infects mammals and transmits to humans with a case fatality rate above 40%. Similarly, H7N9 can infect humans, with a case fatality rate of over 40%. Since 1996, there have been several HPAI outbreaks affecting humans, emphasizing the need for safe and effective antivirals. We show that probenecid potently inhibits H5N1 and H7N9 replication in prophylactically or therapeutically treated A549 cells and normal human broncho-epithelial (NHBE) cells, and H5N1 replication in VeroE6 cells and mice.

Influenza viruses cause epidemics and can cause pandemics with substantial morbidity with some mortality every year. Seasonal influenza vaccines have incomplete effectiveness and elicit a narrow antibody response that often does not protect against mutations occurring in influenza viruses. Thus, various vaccine approaches have been investigated to improve safety and efficacy. Here, we evaluate an mRNA influenza vaccine encoding hemagglutinin (HA) proteins in a BALB/c mouse model. The results show that mRNA vaccination elicits neutralizing and serum antibodies to each influenza virus strain contained in the current quadrivalent vaccine that is designed to protect against four different influenza viruses including two influenza A viruses (IAV) and two influenza B (IBV), as well as several antigenically distinct influenza virus strains in both hemagglutination inhibition assay (HAI) and virus neutralization assays. The quadrivalent mRNA vaccines had antibody titers comparable to the antibodies elicited by the monovalent vaccines to each tested virus regardless of dosage following an mRNA booster vaccine. Mice vaccinated with mRNA encoding an H1 HA had decreased weight loss and decreased lung viral titers compared to mice not vaccinated with an mRNA encoding an H1 HA. Overall, this study demonstrates the efficacy of mRNA-based seasonal influenza vaccines are their potential to replace both the currently available split-inactivated, and live-attenuated seasonal influenza vaccines.

INTRODUCTION:Respiratory viruses are responsible for significant worldwide morbidity and mortality. While vaccines are highly effective at reducing the morbidity and mortality associated with viral infections, this protection is incomplete. It requires a high degree of compliance, which is hindered by vaccine hesitancy. To address these gaps, antiviral agents and therapeutics are crucial in combating diseases caused by respiratory viruses. Antiviral agents are broadly classified into two groups: 1) direct-acting antivirals (DAA) and 2) host-directed antivirals (HDA). AREAS COVERED:This review comprehensively examines Phase II FDA-approved antiviral drugs for influenza virus, SARS-CoV-2, and RSV as published in clinicaltrials.gov. It focuses on DAAs and various monoclonal antibodies (mAbs) that have been approved for the prevention and treatment of viral respiratory tract infections. EXPERT OPINION:Antiviral drugs being developed assess different mechanisms of action to combat viruses and other delivery routes (i.e. oral, inhalation, or parenteral). The associated clinical trials address the impact on disease while determining the appropriate dosage levels for further investigation in Phase III. A robust pipeline of agents is necessary to meet the global need for effective antiviral therapeutics.

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infection and causes significant morbidity and mortality. There is no specific antiviral drug to treat HMPV or vaccine to prevent HMPV. This study determined if probenecid, a host-targeting antiviral drug, had prophylactic (pre-virus) or therapeutic (post-virus) efficacy to inhibit HMPV replication in LLC-MK2 cells in vitro and in the lungs of BALB/c mice. This study showed that ≥0.5 μM probenecid significantly inhibited HMPV replication in vitro, and 2–200 mg/kg probenecid prophylaxis or treatment reduced HMPV replication in BALB/c mice.

This study presents an integrated approach combining surface-enhanced Raman spectroscopy (SERS) with a specialized deep learning algorithm, SFNet, to offer a rapid, accurate, and label-free alternative for COVID-19 diagnosis and viral load quantification. The SiO2-coated silver nanorod arrays are employed as the SERS substrates, fabricated using a reliable and effective glancing angle deposition technique. A dataset of 4800 SERS spectra from 120 positive and 120 negative inactivated clinical human nasopharyngeal swabs are collected directly on the SERS substrates without any labels. A SFNet algorithm is tailored to adapt to the unique spectral features inherent to SERS data, achieving a test accuracy of 98.5% and a blind test accuracy of 99.04%. Moreover, an optimized SFNet algorithm unveils the capability of estimating SARS-CoV-2 viral loads, accurately predicting the cycle threshold values (Ct values) of the three vital gene fragments with a root mean square error (RMSE) of 1.627 (1.3 for blind test). The methodology is substantiated using actual clinical specimens and completed in <15 min, thereby strengthening its real-world point-of-care applicability. This rapid and precise yet label-free modality competes favorably with classical reverse-transcription real-time polymerase chain reaction (RT-PCR) and marks an advancement in SERS-based sensor algorithms.

Mucosal vaccination appears to be suitable to protect against SARS-CoV-2 infection. In this study, we tested an intranasal mucosal vaccine candidate for COVID-19 that consisted of a cationic liposome containing a trimeric SARS-CoV-2 spike protein and CpG-ODNs, a Toll-like receptor 9 agonist, as an adjuvant. In vitro and in vivo experiments indicated the absence of toxicity following the intranasal administration of this vaccine formulation. First, we found that subcutaneous or intranasal vaccination protected hACE-2 transgenic mice from infection with the wild-type (Wuhan) SARS-CoV-2 strain, as shown by weight loss and mortality indicators. However, when compared with subcutaneous administration, the intranasal route was more effective in the pulmonary clearance of the virus and induced higher neutralizing antibodies and anti-S IgA titers. In addition, the intranasal vaccination afforded protection against gamma, delta, and omicron virus variants of concern. Furthermore, the intranasal vaccine formulation was superior to intramuscular vaccination with a recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 spike glycoprotein (Oxford/AstraZeneca) in terms of virus lung clearance and production of neutralizing antibodies in serum and bronchial alveolar lavage (BAL). Finally, the intranasal liposomal formulation boosted heterologous immunity induced by previous intramuscular vaccination with the Oxford/AstraZeneca vaccine, which was more robust than homologous immunity.