Silica Nanowires-Filled Glass Microporous Sensor for the Ultrasensitive Detection of Deoxyribonucleic Acid.

DNA carries genetic information and can serve as an important biomarker for the early diagnosis and assessment of the disease prognosis. Here, we propose a bottom-up assembly method for a silica nanowire-filled glass microporous (SiNWs@GMP) sensor and develop a universal sensing platform for the ultrasensitive and specific detection of DNA. The three-dimensional network structure formed by SiNWs provides them with highly abundant and accessible binding sites, allowing for the immobilization of a large amount of capture probe DNA, thereby enabling more target DNA to hybridize with the capture probe DNA to improve detection performance. Therefore, the SiNWs@GMP sensor achieves ultrasensitive detection of target DNA. In the detection range of 1 aM to 100 fM, there is a good linear relationship between the decrease rate of current signal and the concentration of target DNA, and the detection limit is as low as 1 aM. The developed SiNWs@GMP sensor can distinguish target DNA sequences that are 1-, 3-, and 5-mismatched, and specifically recognize target DNA from complex mixed solution. Furthermore, based on this excellent selectivity and specificity, we validate the universality of this sensing strategy by detecting DNA (H1N1 and H5N1) sequences associated with the avian influenza virus. By replacing the types of nucleic acid aptamers, it is expected to achieve a wide range and low detection limit sensitive detection of various biological molecules. The results indicate that the developed universal sensing platform has ultrahigh sensitivity, excellent selectivity, stability, and acceptable reproducibility, demonstrating its potential application in DNA bioanalysis.

On-The-Spot Sampling and Detection of Viral Particles on Solid Surfaces Using a Sponge Virus Sensor Incorporated with Finger-Press Fluid Release.

This paper presents a sponge-based electrochemical sensor for rapid, on-site collection and analysis of infectious viruses on solid surfaces. The device utilizes a conducting porous sponge modified with graphene, graphene oxide, and specific antibodies. The sponge serves as a hydrophilic porous electrode capable of liquid collection and electrochemical measurements. The device operation involves spraying an aqueous solution on a target surface, swiping the misted surface using the sponge, discharging an electrolyte solution with a simple finger press, and performing in situ incubation and electrochemical measurements. By leveraging the water-absorbing ability of the biofunctionalized conducting sponge, the sensor can effectively collect and quantify virus particles from the surface. The portability of the device is enhanced by introducing a push-release feature that dispenses the liquid electrolyte from a miniature reservoir onto the sensor surface. This reservoir has sharp edges to rupture a liquid sealing film with a finger press. The ability of the device to sample and quantify viral particles is demonstrated by using influenza A virus as the model. The sensor provided a calculated limit of detection of 0.4 TCID50/mL for H1N1 virus, along with a practical concentration range from 1-106 TCID50/mL. Additionally, it achieves a 15% collection efficiency from single-run swiping on a tabletop surface. This versatile device allows for convenient on-site virus detection within minutes, eliminating the need for sample pretreatment and simplifying the entire sample collecting and measuring process. This device presents significant potential for rapid virus detection on solid surfaces.

Evaluation of disease severity and prediction of severe cases in children hospitalized with influenza A (H1N1) infection during the post-COVID-19 era: a multicenter retrospective study.

BACKGROUND: The rebound of influenza A (H1N1) infection in post-COVID-19 era recently attracted enormous attention due the rapidly increased number of pediatric hospitalizations and the changed characteristics compared to classical H1N1 infection in pre-COVID-19 era. This study aimed to evaluate the clinical characteristics and severity of children hospitalized with H1N1 infection during post-COVID-19 period, and to construct a novel prediction model for severe H1N1 infection. METHODS: A total of 757 pediatric H1N1 inpatients from nine tertiary public hospitals in Yunnan and Shanghai, China, were retrospectively included, of which 431 patients diagnosed between February 2023 and July 2023 were divided into post-COVID-19 group, while the remaining 326 patients diagnosed between November 2018 and April 2019 were divided into pre-COVID-19 group. A 1:1 propensity-score matching (PSM) was adopted to balance demographic differences between pre- and post-COVID-19 groups, and then compared the severity across these two groups based on clinical and laboratory indicators. Additionally, a subgroup analysis in the original post-COVID-19 group (without PSM) was performed to investigate the independent risk factors for severe H1N1 infection in post-COIVD-19 era. Specifically, Least Absolute Shrinkage and Selection Operator (LASSO) regression was applied to select candidate predictors, and logistic regression was used to further identify independent risk factors, thus establishing a prediction model. Receiver operating characteristic (ROC) curve and calibration curve were utilized to assess discriminative capability and accuracy of the model, while decision curve analysis (DCA) was used to determine the clinical usefulness of the model. RESULTS: After PSM, the post-COVID-19 group showed longer fever duration, higher fever peak, more frequent cough and seizures, as well as higher levels of C-reactive protein (CRP), interleukin 6 (IL-6), IL-10, creatine kinase-MB (CK-MB) and fibrinogen, higher mechanical ventilation rate, longer length of hospital stay (LOS), as well as higher proportion of severe H1N1 infection (all P < 0.05), compared to the pre-COVID-19 group. Moreover, age, BMI, fever duration, leucocyte count, lymphocyte proportion, proportion of CD3+ T cells, tumor necrosis factor α (TNF-α), and IL-10 were confirmed to be independently associated with severe H1N1 infection in post-COVID-19 era. A prediction model integrating these above eight variables was established, and this model had good discrimination, accuracy, and clinical practicability. CONCLUSIONS: Pediatric H1N1 infection during post-COVID-19 era showed a higher overall disease severity than the classical H1N1 infection in pre-COVID-19 period. Meanwhile, cough and seizures were more prominent in children with H1N1 infection during post-COVID-19 era. Clinicians should be aware of these changes in such patients in clinical work. Furthermore, a simple and practical prediction model was constructed and internally validated here, which showed a good performance for predicting severe H1N1 infection in post-COVID-19 era.

Fatal Viral and Bacterial Septicemia in a Seventeen-Year-Old Woman with Immunodepressive Influenza A H1N1: An Autopsy Case.

Abstract: The Influenza A H1N1 subtype can present with a wide spectrum of severity, from mild symptoms of influenza to severe respiratory distress. The morbidity and mortality connected to influenza are mostly associated with secondary bacterial infections. The influenza syndrome alone can cause a massive release of cytokines with dysregulation of the immune system, and it can act in synergy with other bacteria which can enhance cytokines secretion. This article deals with a case of severe pneumonia of H1N1 in a 17-year-old woman with bacterial superinfection with Staphylococcus aureus characterized by a high level of interleukine-6 (105900 pg/mL) and the appearance of severe leukopenia with immuno-suppression, such that HIV infection and hematological diseases were included in the initial differential diagnosis. After death, the autopsy confirmed the presence of severe pneumonia, in addition to an hepatic steatosis in absence of other risk factors. This case reports the rapid and lethal course of influenza A /H1N1 in a young and healthy subject without comorbidities, in an age group in which mortality is about 0.3 deaths per 100,000. The case underlines the importance of quickly diagnosis of viral infections and the differential diagnoses with other immunosuppressive diseases, which can be fatal even in adolescent and healthy subjects.

Supplementation of seasonal vaccine with multi-subtype neuraminidase and M2 ectodomain virus-like particle improves protection against homologous and heterologous influenza viruses in aged mice.

The conventional inactivated split seasonal influenza vaccine offers low efficacy, particularly in the elderly and against antigenic variants. Here, to improve the efficacy of seasonal vaccination for the elderly population, we tested whether supplementing seasonal bivalent (H1N1 + H3N2) split (S) vaccine with M2 ectodomain repeat and multi-subtype consensus neuraminidase (NA) proteins (N1 NA + N2 NA + flu B NA) on a virus-like particle (NA-M2e) would induce enhanced cross-protection against different influenza viruses in aged mice. Immunization with split vaccine plus NA-M2e (S + NA-M2e) increased vaccine-specific IgG antibodies towards T-helper type 1 responses and hemagglutination inhibition titers. Aged mice with NA-M2e supplemented vaccination were protected against homologous and heterologous viruses at higher efficacies, as evidenced by preventing weight loss, lowering lung viral loads, inducing broadly cross-protective humoral immunity, and IFN-γ+ CD4 and CD8 T cell responses than those with seasonal vaccine. Overall, this study supports a new strategy of NA-M2e supplemented vaccination to enhance protection against homologous and antigenically different viruses in the elderly.

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children.

Cross-reactive antibodies with Fc receptor (FcR) effector functions may mitigate pandemic virus impact in the absence of neutralizing antibodies. In this exploratory study, we use serum from a randomized placebo-controlled trial of seasonal trivalent influenza vaccination in children (NCT00792051) conducted at the onset of the 2009 H1N1 pandemic (pH1N1) and monitored for infection. We found that seasonal vaccination increases pH1N1 specific antibodies and FcR effector functions. Furthermore, prospective baseline antibody profiles after seasonal vaccination, prior to pH1N1 infection, show that unvaccinated uninfected children have elevated ADCC effector function, FcγR3a and FcγR2a binding antibodies to multiple pH1N1 proteins, past seasonal and avian (H5, H7 and H9) strains. Whereas, children that became pH1N1 infected after seasonal vaccination have antibodies focussed to seasonal strains without FcR functions, and greater aggregated HA-specific profiles for IgM and IgG3. Modeling to predict infection susceptibility, ranked baseline hemagglutination antibody inhibition as the highest contributor to lack of pH1N1 infection, in combination with features that include pH1-IgG1, H1-stem responses and FcR binding to seasonal vaccine and pH1 proteins. Thus, seasonal vaccination can have benefits against pandemic influenza viruses, and some children already have broadly reactive antibodies with Fc potential without vaccination and may be considered 'elite influenza controllers'.

Immunogenicity and safety of concomitant administration of recombinant COVID-19 vaccine and quadrivalent inactivated influenza vaccine in Chinese adults: An open-label, randomized, controlled trial.

The immunogenicity and safety of the concomitant administration of recombinant COVID-19 vaccine and quadrivalent inactivated influenza vaccine (Split Virion) (QIIV) in Chinese adults are unclear. In this open-label, randomized controlled trial, participants aged ≥ 18 years were recruited. Eligible healthy adults were randomly assigned (1:1) to receive QIIV at the same time as the first dose of COVID-19 vaccine (simultaneous-group) or 14 days after the second dose of COVID-19 vaccine (non-simultaneous-group). The primary outcome was to compare the difference in immunogenicity of QIIV (H1N1, H3N2, Yamagata, and Victoria) between the two groups. A total of 299 participants were enrolled, 149 in the simultaneous-group and 150 in the non-simultaneous-group. There were no significant differences in geometric mean titer (GMT) [H1N1: 386.4 (95%CI: 299.2-499.0) vs. 497.4 (95%CI: 377.5-655.3); H3N2: 66.9 (95%CI: 56.1-79.8) vs. 81.4 (95%CI: 67.9-97.5); Yamagata: 95.6 (95%CI: 79.0-115.8) vs. 74.3 (95%CI: 58.6-94.0); and Victoria: 48.5 (95%CI: 37.6-62.6) vs. 65.8 (95%CI: 49.0-88.4)] and seroconversion rate (H1N1: 87.5% vs. 90.1%; H3N2: 58.1% vs. 62.0%; Yamagata: 75.0% vs. 64.5%; and Victoria: 55.1% vs. 62.8%) of QIIV antibodies between the simultaneous and non-simultaneous groups. For the seroprotection rate of QIIV antibodies, a higher seroprotection rate of Yamagata antibody was observed only in the simultaneous-group than in the non-simultaneous-group [86.0% vs. 76.0%, p = .040]. In addition, no significant difference in adverse events was observed between the two groups (14.2% vs. 23.5%, p = .053). In conclusion, no immune interference or safety concerns were found for concomitant administration of COVID-19 vaccine with QIIV in adults aged ≥ 18 years.

An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models.

With the lifting of COVID-19 non-pharmaceutical interventions, the resurgence of common viral respiratory infections was recorded in several countries worldwide. It facilitates viral co-infection, further burdens the already over-stretched healthcare systems. Racing to find co-infection-associated efficacy therapeutic agents need to be rapidly established. However, it has encountered numerous challenges that necessitate careful investigation. Here, we introduce a potential recombinant minibody-associated treatment, 3D8 single chain variable fragment (scFv), which has been developed as a broad-spectrum antiviral drug that acts via its nucleic acid catalytic and cell penetration abilities. In this research, we demonstrated that 3D8 scFv exerted antiviral activity simultaneously against both influenza A viruses (IAVs) and coronaviruses in three established co-infection models comprising two types of coronaviruses [beta coronavirus-human coronavirus OC43 (hCoV-OC43) and alpha coronavirus-porcine epidemic diarrhea virus (PEDV)] in Vero E6 cells, two IAVs [A/Puerto Rico/8/1934 H1N1 (H1N1/PR8) and A/X-31 (H3N2/X-31)] in MDCK cells, and a combination of coronavirus and IAV (hCoV-OC43 and adapted-H1N1) in Vero E6 cells by a statistically significant reduction in viral gene expression, proteins level, and approximately around 85%, 65%, and 80% of the progeny of 'hCoV-OC43-PEDV', 'H1N1/PR8-H3N2/X-31', and 'hCoV-OC43-adapted-H1N1', respectively, were decimated in the presence of 3D8 scFv. Taken together, we propose that 3D8 scFv is a promising broad-spectrum drug for treatment against RNA viruses in co-infection.

Long-term co-circulation of multiple influenza A viruses in pigs, Guangxi, China.

Influenza A viruses (IAVs) pose a persistent potential threat to human health because of the spillover from avian and swine infections. Extensive surveillance was performed in 12 cities of Guangxi, China, during 2018 and 2023. A total of 2540 samples (including 2353 nasal swabs and 187 lung tissues) were collected from 18 pig farms with outbreaks of respiratory disease. From these, 192 IAV-positive samples and 19 genomic sequences were obtained. We found that the H1 and H3 swine influenza A viruses (swIAVs) of multiple lineages and genotypes have continued to co-circulate during that time in this region. Genomic analysis revealed the Eurasian avian-like H1N1 swIAVs (G4) still remained predominant in pig populations. Strikingly, the novel multiple H3N2 genotypes were found to have been generated through the repeated introduction of the early H3N2 North American triple reassortant viruses (TR H3N2 lineage) that emerged in USA and Canada in 1998 and 2005, respectively. Notably, when the matrix gene segment derived from the H9N2 avian influenza virus was introduced into endemic swIAVs, this produced a novel quadruple reassortant H1N2 swIAV that could pose a potential risk for zoonotic infection.

Alveolar epithelial cells of lung fibrosis patients are susceptible to severe virus-induced injury.

Patients with pulmonary fibrosis (PF) often experience exacerbations of their disease, characterised by a rapid, severe deterioration in lung function that is associated with high mortality. Whilst the pathobiology of such exacerbations is poorly understood, virus infection is a trigger. The present study investigated virus-induced injury responses of alveolar and bronchial epithelial cells (AECs and BECs, respectively) from patients with PF and age-matched controls (Ctrls). Air-liquid interface (ALI) cultures of AECs, comprising type I and II pneumocytes or BECs were inoculated with influenza A virus (H1N1) at 0.1 multiplicity of infection (MOI). Levels of interleukin-6 (IL-6), IL-36γ and IL-1ß were elevated in cultures of AECs from PF patients (PF-AECs, n = 8-11), being markedly higher than Ctrl-AECs (n = 5-6), 48 h post inoculation (pi) (P<0.05); despite no difference in H1N1 RNA copy numbers 24 h pi. Furthermore, the virus-induced inflammatory responses of PF-AECs were greater than BECs (from either PF patients or controls), even though viral loads in the BECs were overall 2- to 3-fold higher than AECs. Baseline levels of the senescence and DNA damage markers, nuclear p21, p16 and H2AXγ were also significantly higher in PF-AECs than Ctrl-AECs and further elevated post-infection. Senescence induction using etoposide augmented virus-induced injuries in AECs (but not viral load), whereas selected senotherapeutics (rapamycin and mitoTEMPO) were protective. The present study provides evidence that senescence increases the susceptibility of AECs from PF patients to severe virus-induced injury and suggests targeting senescence may provide an alternative option to prevent or treat the exacerbations that worsen the underlying disease.

Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.

Pilot-scale production of a highly efficacious and stable monoglycosylated influenza split virus vaccine.

The yearly epidemics and unpredictable outbreaks of influenza have raisedserious concernsglobally and led to prioritizing the development of an effective vaccine toprotectagainst newly emerging variants. Previously, we demonstrated that monoglycosylated influenza virus vaccines derived from A/California/7/2009 or an updated A/Brisbane/02/2018 (IVR-190) vaccine strain recommended by WHO are superior to fully glycosylated vaccines and could broadly protect against past and new coming H1N1 variants. However, whether such a monoglycosylated virus vaccine can be mass-produced to meet clinical demands and stable enough to provide consistent efficacy against H1N1 viruses remains unclear. Herein, we developed a platform for the pilot-scale production of the monoglycosylated split virus vaccine from the IVR-190 strain (IVR-190mg) with a robust and cost-effective manufacturing process. The critical parameters of inoculum dose, concentration of kifunensine, and optimized Endo H treatment process were comprehensively investigated. Several aims for preclinical studies of IVR-190mg were achieved, including [i] the execution of three engineering batch runs to validate lot-to-lot consistency, [ii] the establishment of IVR-190mg specifications to meet the acceptance criteria of a conventional influenza vaccine, [iii] an investigation of the stability profile of IVR-190mg, and completion of a safety evaluation by conducting an animal toxicology study. The toxicology study under GLP guidance found no systemic toxicity after rabbits were vaccinated with IVR-190mg. The serological data showed that IVR-190mg is highly immunogenic and effective in inducing a cross-strain protective level of antibody immune responses, including hemagglutination-inhibition titers, viral neutralization activity, and broad HA- and NA-inhibiting antibody titers against past and new H1N1 viruses. In conclusion, this study provides efficacy and safety profiles of IVR-190mg for further clinical study and shows that this vaccine without a glycan shield has great potential to be safe and protective against H1N1 variants.

Model analysis and data validation of structured prevention and control interruptions of emerging infectious diseases.

The design of optimized non-pharmaceutical interventions (NPIs) is critical to the effective control of emergent outbreaks of infectious diseases such as SARS, A/H1N1 and COVID-19 and to ensure that numbers of hospitalized cases do not exceed the carrying capacity of medical resources. To address this issue, we formulated a classic SIR model to include a close contact tracing strategy and structured prevention and control interruptions (SPCIs). The impact of the timing of SPCIs on the maximum number of non-isolated infected individuals and on the duration of an infectious disease outside quarantined areas (i.e. implementing a dynamic zero-case policy) were analyzed numerically and theoretically. These analyses revealed that to minimize the maximum number of non-isolated infected individuals, the optimal time to initiate SPCIs is when they can control the peak value of a second rebound of the epidemic to be equal to the first peak value. More individuals may be infected at the peak of the second wave with a stronger intervention during SPCIs. The longer the duration of the intervention and the stronger the contact tracing intensity during SPCIs, the more effective they are in shortening the duration of an infectious disease outside quarantined areas. The dynamic evolution of the number of isolated and non-isolated individuals, including two peaks and long tail patterns, have been confirmed by various real data sets of multiple-wave COVID-19 epidemics in China. Our results provide important theoretical support for the adjustment of NPI strategies in relation to a given carrying capacity of medical resources.

Long-term outcomes of survivors with influenza A H1N1 virus-induced severe pneumonia and ARDS: a single-center prospective cohort study.

Introduction: Systematic evaluation of long-term outcomes in survivors of H1N1 is still lacking. This study aimed to characterize long-term outcomes of severe H1N1-induced pneumonia and acute respiratory distress syndrome (ARDS). Method: This was a single-center, prospective, cohort study. Survivors were followed up for four times after discharge from intensive care unit (ICU) by lung high-resolution computed tomography (HRCT), pulmonary function assessment, 6-minute walk test (6MWT), and SF-36 instrument. Result: A total of 60 survivors of H1N1-induced pneumonia and ARDS were followed up for four times. The carbon monoxide at single breath (DLCO) of predicted values and the 6MWT results didn't continue improving after 3 months. Health-related quality of life didn't change during the 12 months after ICU discharge. Reticulation or interlobular septal thickening on HRCT did not begin to improve significantly until the 12-month follow-up. The DLCO of predicted values showed negative correlation with the severity degree of primary disease and reticulation or interlobular septal thickening, and a positive correlation with physical functioning. The DLCO of predicted values and reticulation or interlobular septal thickening both correlated with the highest tidal volume during mechanical ventilation. Levels of fibrogenic cytokines had a positive correlation with reticulation or interlobular septal thickening. Conclusion: The improvements in pulmonary function and exercise capacity, imaging, and health-related quality of life had different time phase and impact on each other during 12 months of follow-up. Long-term outcomes of pulmonary fibrosis might be related to the lung injury and excessive lung fibroproliferation at the early stage during ICU admission.

Severe A(H1N1)pdm09 influenza acute encephalopathy outbreak in children in Tuscany, Italy, December 2023 to January 2024.

A severe outbreak of influenza A(H1N1pdm09) infection in seven children (median age: 52 months) occurred between December 2023 and January 2024 in Tuscany, Italy. Clinical presentation ranged from milder encephalopathy to acute necrotizing encephalopathy (ANE) with coma and multiorgan failure; one child died. This report raises awareness for clinicians to identify and treat early acute encephalopathy caused by H1N1 influenza and serves as a reminder of severe presentations of influenza in young children and the importance of vaccination.

Myositis in H1N1 Infection Compounds to Myasthenic Crisis.

ABSTRACT: Infection is an important trigger of myasthenic crisis (MC), and those infections manifest with pneumonia and muscle involvement may result in more frequent MC. We report two myasthenia gravis (MG) patients with H1N1 infection, and highlight the reasons for deterioration. Two patients with MG had H1N1 infection. The diagnosis of MG was confirmed by neostigmine, repetitive nerve stimulation, and anti-acetylcholine receptor antibody tests. H1N1 was confirmed by nucleic acid detection study, and myositis by creatinine kinase. The patient with pneumonia and myositis had MC needing mechanical ventilation for 10 days, and the other patient without myositis did not have MC. They were treated with oseltamivir 75 mg twice daily for 5 days, and the patients with MC received ceftriaxone intravenously. Both the patients were on prednisolone and azathioprine, and none received prior H1N1 vaccination. The lady with MC with myositis was discharged on day 27 in wheelchair bound state, and the other one patient without myositis or MC was discharged on 6th day with full recovery. These patients highlight the need for evaluation for myositis along with pneumonia in the MG patients with H1N1 infection. Vaccination in MG patients on immunosuppression may be useful.

A subunit-based influenza/SARS-CoV-2 Omicron combined vaccine induced potent protective immunity in BALB/c mice.

Infection with influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant risk to human life, health, and the global economy. Vaccination is one of the most effective strategies in the fight against infectious viruses. In this study, we, for the first time, have evaluated the immunogenicity and protective effect of an influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with MF59 and administered to BALB/c mice. Results showed that the combined vaccine induced high levels of IgG, IgG1 , and IgG2a antibodies, as well as influenza A H1N1/California/2009 virus-specific hemagglutination-inhibiting antibodies in BALB/c mice. Moreover, this subunit combined vaccine induced high titers of neutralization antibodies against SARS-CoV-2 Omicron sublineage BA.5 pseudovirus and effectively reduced the viral load of authentic SARS-CoV-2 Omicron sublineage BA.5.2 in the cell culture supernatants. These results suggested that this subunit combined vaccine achieved protective effect against both H1N1 A/California/07/2009 strain and SARS-CoV-2 Omicron BA.5.2 variant. It is therefore expected that this study will establish the scientific foundation for the next-step development of combined vaccines against other strains or variants of IAV and SARS-CoV-2.

Development of a virus-based affinity ultrafiltration method for screening virus-surface-protein-targeted compounds from complex matrixes: Herbal medicines as a case study.

Herbal medicines (HMs) are one of the main sources for the development of lead antiviral compounds. However, due to the complex composition of HMs, the screening of active compounds within these is inefficient and requires a significant time investment. We report a novel and efficient virus-based screening method for antiviral active compounds in HMs. This method involves the centrifugal ultrafiltration of viruses, known as the virus-based affinity ultrafiltration method (VAUM). This method is suitable to identify virus specific active compounds from complex matrices such as HMs. The effectiveness of the VAUM was evaluated using influenza A virus (IAV) H1N1. Using this method, four compounds that bind to the surface protein of H1N1 were identified from dried fruits of Terminalia chebula (TC). Through competitive inhibition assays, the influenza surface protein, neuraminidase (NA), was identified as the target protein of these four TC-derived compounds. Three compounds were identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), and their anti-H1N1 activities were verified by examining the cytopathic effect (CPE) and by performing a virus yield reduction assay. Further mechanistic studies demonstrated that these three compounds directly bind to NA and inhibit its activity. In summary, we describe here a VAUM that we designed, one that can be used to accurately screen antiviral active compounds in HMs and also help improve the efficiency of screening antiviral drugs found in natural products.