Given the global threat posed by H5N1 clade 2.3.4.4b avian influenza, rapid development of effective vaccines is imperative. We design an mRNA vaccine encoding hemagglutinin (HA) from A/Texas/37/2024, the first bovine-to-human strain. In murine models, both wild-type and cleavage-site-modified HA vaccines elicit robust and durable humoral immunity, along with a balanced Th1/Th2 response, conferring complete protection against lethal homologous viral challenge. The vaccine, along with the World Health Organization (WHO)-recommended candidate (A/Astrakhan/3212/2020), elicits cross-clade binding antibody responses and demonstrates improvement against specific clades at a 1 μg dose. Pre-existing H1 immunity does not diminish H5-specific immunogenicity. In avian species, the vaccine also provides full protection against lethal clades (2.3.4.4b and 2.3.4.4h). Formulated with another ionizable lipid, the vaccine elicits responses comparable to benchmark lipid nanoparticles (LNPs) and shows a favorable safety profile in rats. This work establishes a rapidly adaptable mRNA-LNP vaccine prototype for pandemic preparedness against evolving avian influenza threats.

The viral RNA-dependent RNA polymerase (RdRp) is a conserved and compelling target for pan-coronavirus antiviral development. To date, all US Food and Drug Administration (FDA)-approved RdRp inhibitors are nucleotide inhibitors (NIs), which are prone to drug resistance and show limited clinical efficacy. Developing alternative non-nucleotide inhibitors (NNIs) with high target specificity, structural diversity, and metabolic stability could yield more effective antivirals. Here, we identify an allosteric RdRp NNI, BAY-850, via computational screening. BAY-850 exhibits potent, broad-spectrum antiviral activity against multiple SARS-CoV-2 variants and other human coronaviruses. Mechanistic studies show that BAY-850 binds directly to a previously unrecognized allosteric site on nsp12, thereby fine-tuning the catalytic motif F loop out-conformation the orthosteric site. Importantly, therapeutic administration of BAY-850 significantly reduces viral loads in the nasal turbinates and lungs of SARS-CoV-2-infected K18-hACE2 mice. These findings validate a novel allosteric site in RdRp and support BAY-850 as a start-point for broad-spectrum anti-coronavirus development.

OBJECTIVES:Influenza virus (IV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are significant causes of hospitalization. Earlier studies showed more severe disease for SARS-CoV-2 infections, but many were compared with historical influenza cohorts and were conducted before the milder Omicron variant emerged. We aimed to compare the severity of SARS-CoV-2 infection with a concurrent cohort of IV infection among hospitalized patients after the emergence of the Omicron variant in Hong Kong SAR, China. METHODS:This is a target trial emulation study based on the public electronic health database in Hong Kong SAR, China. Adults aged ≥18 years who were hospitalized with laboratory-confirmed SARS-CoV-2 or IV infection between January 1, 2023, and January 31, 2024, were included. Pooled logistic regression was used to explore the risk of the composite outcome (30-day mortality, mechanical ventilation, intensive care unit [ICU] admission). RESULTS:A total of 47,956 cases were identified, including 32,523 SARS-CoV-2 mono-infections, 15,103 IV mono-infections, and 330 SARS-CoV-2/IV co-infections. Overall, the risks of composite outcome (hazard ratio [HR], 1.14; 95% confidence interval [CI], 1.06-1.23), 30-day mortality (HR, 1.20; 95% CI 1.09-1.32), and ICU admission (HR, 1.21; 95% CI 1.06-1.38) were higher for SARS-CoV-2 than IV infection. Subgroup analysis showed that male patients with SARS-CoV-2 were at higher risk of the composite outcome (HR, 1.19; 95% CI 1.08-1.31), 30-day mortality (HR, 1.26; 95% CI 1.11-1.44), and ICU admission (HR, 1.2; 95% CI 1.02-1.4) than male patients with IV. Post hoc analysis showed that SARS-CoV-2/IV co-infection was associated with an increased risk of 30-day mortality than SARS-CoV-2 mono-infection (HR, 2.11; 95% CI 1.58-2.81) or influenza mono-infection (HR, 3.45; 95% CI 2.19-5.42). CONCLUSION:SARS-CoV-2 Omicron infection was associated with more severe outcomes than IV mono-infection, and co-infection was more severe than mono-infection among hospitalized patients. Our findings have important implications for health care policies, including vaccine recommendations for SARS-CoV-2.

Post-acute sequelae of COVID-19 (PASC) poses a major health burden after SARS-CoV-2 infection. Although type 2 diabetes (T2D) is associated with PASC, the mechanism of T2D-mediated PASC in the lung remains elusive. Here, we found that people with T2D (PWT2D) exhibited significantly upregulated fibrosis-related genes in monocytes, which positively correlated with pulmonary fibrosis-related biomarkers up to 3 months after acute SARS-CoV-2 infection. Using db/db mice to model human T2D, we found consistently that SARS-CoV-2 infection resulted in upregulation of fibrosis-related genes in lung macrophages and persistent pulmonary fibrosis. Moreover, the macrophage-depletion demonstrated that pro-inflammatory macrophages in db/db mice were determinants for inducing pulmonary fibrosis post-infection. Importantly, the anti-T2D glucagon-like peptide-1 receptor agonist (GLP1-RA) reprogramed macrophage responses to SARS-CoV-2 by normalizing fibrosis-related genes, significantly reducing the pulmonary fibrosis in a glucose-independent manner. These findings demonstrated that SARS-CoV-2-induced proinflammatory macrophages are detrimental factors in T2D-mediated PASC, which can be prevented by GLP1-RA. IMPORTANCE:Some COVID-19 patients develop pulmonary post-acute sequelae of COVID-19 (PASC) with clinical symptoms lasting for years. Critically, the incidence of pulmonary PASC in PWT2D is four times higher than that in those without T2D. However, the immune mechanisms underlying pulmonary PASC in PWT2D remain poorly understood. Our findings demonstrate that SARS-CoV-2-induced proinflammatory macrophages are key drivers of PASC-associated pulmonary fibrosis. We further provide in vivo evidence that glucagon-like peptide-1 receptor agonists (GLP1-RAs) can reprogram pulmonary macrophages to prevent SARS-CoV-2-induced PASC in a T2D mouse model, with important implications for therapy in PWT2D.

Human metapneumovirus (hMPV) causes mild and self-limiting disease in adults. However, the risk factors for serious adverse outcomes following hMPV infection in adult patients without preexisting chronic airway diseases remain poorly understood. We conducted a territory-wide retrospective study on adult patients (aged ≥ 18 years) without chronic airway diseases hospitalized for hMPV infections between January 1, 2016 and June 30, 2023 in Hong Kong. We assessed the incidence and risk factors for in-patient mortality, severe respiratory failure (SRF), secondary bacterial pneumonia and acute kidney injury (AKI) were assessed. A total of 1552 eligible adult patients without chronic airway diseases hospitalized for hMPV infections were analyzed. Within the index admission, 92 (5.9%) patients died. Ischemic heart disease (IHD) was associated with increased risks of SRF [adjusted odds ratio (aOR) 2.00 (95% CI 1.48-2.71), p < 0.001]. IHD, heart failure (HF), and history of ischemic stroke were significant predictors for AKI [aOR 1.51 (95% CI 1.12-2.04), 2.87 (95% CI 2.14-3.85), and 1.47 (95% CI = 1.12-1.93), p = 0.007, < 0.001, and 0.005, respectively). Patients with end-stage kidney disease (ESKD) requiring renal replacement therapy (RRT) were at increased risk of in-patient mortality [aOR 6.36 (95% CI 2.34-17.26), p < 0.001] and SRF [aOR 8.80 (95% CI 3.84-20.16), p < 0.001]. The presence of cardiovascular diseases and ESKD requiring RRT is a strong predictor of severe in-hospital outcomes among adult patients without chronic airway diseases who are hospitalized for hMPV infections.

Three critically ill or fatal avian influenza A(H5N1) human infections have been reported in North America since November 2024. Notably, all were infected with genotype D1.1 instead of B3.13, the dominant genotype before November 2024. Here, we demonstrated that D1.1 could replicate to higher titers in human nasal and airway organoid–derived transwell monolayers from 6 donors. D1.1 exhibited a better binding to α2,3- and α2,6-linked sialic acid than B3.13. No significant differences in most inflammatory or antiviral cytokines/chemokines were observed. These observations suggest that D1.1 is better adapted to both the upper and lower human respiratory tract epithelium than B3.13.

We established robust protocols to generate physiological and functional alveolar organoids (nsoAlvO) from readily accessible and expandable nasal cell-derived organoids, and alveolar macrophages (monoAM) from peripheral blood monocytes. Through co-culture of nsoAlvO and monoAM, we generated organoid-macrophage assembloids, in which both components exhibited enhanced maturation. Comprehensive analyses, including immunostaining, functional assays, and single-cell RNA sequencing, demonstrated that the nsoAlvO and monoAM phenotypically and functionally resemble their native counterparts and engage in dynamic and extensive epithelial-macrophage communications. SSEA-1+ club cells were identified as the primary alveolar progenitor cells for nsoAlvO. Influenza virus infection in nsoAlvO revealed differential replicative fitness of H5N1 and H1N1 viruses, which recapitulate their authentic tropism in vivo. Notably, the addition of monoAM reduced H5N1 and H1N1 infection in nsoAlvO, suggesting a protective effect of alveolar macrophages against virus dissemination. These human alveolar organoids and organoid-macrophage assembloids provide universally accessible and physiologically relevant in vitro lung models for biomedical research and translational medicine.

BACKGROUND:There are few treatment options for patients with chronic hepatitis E unresponsive to ribavirin. Drug repurposing is required to identify new treatments. Molnupiravir, a nucleoside analogue, is approved for the treatment of coronavirus disease 2019 (COVID-19). This study evaluated the activity of molnupiravir against hepatitis E virus (HEV) in cell culture and animal models. METHODS:Cytotoxicity and antiviral efficacy of molnupiravir, ribavirin, and sofosbuvir were investigated using infectious cDNA clones and wild-type HEV isolates in PLC/PRF/5 cells and primary rat hepatocytes. Immunosuppressed rats were infected with HEV and treated with molnupiravir and ribavirin. The effectiveness of molnupiravir in clearing HEV in serum, feces, and liver tissue was compared with that of untreated and ribavirin-treated animals. Mutations arising in virus populations during treatment were assessed using next-generation sequencing. RESULTS:The antiviral effect of molnupiravir was comparable to that of ribavirin and superior to that of sofosbuvir against HEV strains in vitro, with decreased HEV RNA in supernatant (p<0.05) and cytoplasmic viral protein expression. No additive effect with sofosbuvir was observed. Rats (n=14 per group) treated with 400 mg/kg/d molnupiravir cleared viremia within 4 weeks of treatment, and 9/14 of these animals also cleared viral shedding in stool. Mean viremia and fecal viral loads were reduced compared with untreated and ribavirin-treated animals (p≤0.005). Partial effectiveness was apparent at the lower 250 mg/kg/d molnupiravir dose. Molnupiravir-treated rats had improved liver histology compared with control animals. Frequent transition mutations were observed in HEV from molnupiravir-treated animals. CONCLUSIONS:Molnupiravir limits HEV infection in cell culture and animal models. Molnupiravir could be an alternative for ribavirin-refractory chronic hepatitis E.

Chikungunya fever (CF) is a mosquito-borne viral disease caused by Chikungunya virus (CHIKV) that is being increasingly reported in previously non-endemic areas, including Guangdong, China. Despite the low mortality rate associated with CF, some patients may develop chronic joint pain that persists for months or years. Moreover, due to the similarities in symptomatology between CHIKV and other arboviruses, clinical diagnosis without laboratory confirmation is inaccurate. The CHIKV envelope protein E1, critical for viral entry and assembly, is a promising diagnostic and therapeutic target. In this study, we expressed the CHIKV E1 protein in Escherichia coli and developed 16 monoclonal antibodies (mAbs) against E1 to establish an enzyme immunoassay (EIA). The EIA specifically detects CHIKV E1 with no cross-reactivity to other major human-pathogenic arboviruses, including dengue virus (DENV). We evaluated its performance in serum samples of 84 CHIKV-infected patients, with qRT-PCR as the reference standard, and compared it with 93 healthy and 94 DENV-1-infected controls. The assay achieved a sensitivity of 94.05% (95% CI: 87.3–97.3) and a specificity of 98.4% (95% CI: 95.3–99.5). This E1 antigen-based EIA enables rapid and accurate diagnosis of CHIKV infection to facilitate the control of outbreaks of this emerging arbovirus.

H9N2 is currently the second most common avian influenza A virus subtype infecting humans. Monitoring viral phenotypic and genotypic adaptation to humans is crucial for risk assessment. Here, we compared the replication of an H9N2 human isolate collected in 2024 (A/HK/2346/2024) to a human isolate collected in 1999 (A/HK/1073/1999). In Madin Darby canine kidney (MDCK) cells, A/HK/2346/2024 and A/HK/1073/1999 replicated to 8 and 5 log10 plaque-forming units (PFU) per ml, respectively. In both human nasal and lung organoids, A/HK/2346/2024 replicated to 6 log10 PFU/ml, but A/HK/1073/1999 failed to replicate in either organoid. The infection rates of both ciliated and non-ciliated cells and the ratios of infected 2,6/2,3 cells were higher for A/HK/2346/2024 than A/HK/1073/1999. Apart from the mammalian adaptive substitutions that were present in the nasopharyngeal specimen collected on day 1 post-symptom onset (pso) (HA-D183N/D190 T/Q192R/Q226L; NA-del62-64; PB2-A588V/K702R; PB1-I368V; PA-K356R/S409N; M1-R95K), the mammalian-adaptive substitution PB2-D253N emerged de novo on day 7 pso. Analysis of all human (n = 96) and avian influenza (n = 14,762) H9N2 deposited at GISAID showed the dominance of several human-adaptive substitutions in H9N2 strains collected from humans in recent years. In summary, we demonstrated that a recent H9N2 virus is more adapted to humans, and is able to replicate to high titres in both upper and lower human respiratory tract which may confer higher person-to-person transmissibility and virulence. Our study underscores the importance of human organoid-based phenotypic monitoring and inter/intrahost genotypic monitoring for assessing the zoonotic risk of avian influenza viruses.

BACKGROUND:Influenza surveillance and drug resistance testing have always been central to clinical efforts. Therefore, researching the virus characteristics and antiviral drugs is essential. METHOD:The HA and NA activities were assessed in influenza strains, and mutations were identified through gene sequencing. The effects of oseltamivir, molnupiravir, and baloxavir treatments were evaluated in vitro. The effectiveness of molnupiravir monotherapy and its combination with baloxavir was also evaluated in a mouse model. Changes in body weight and lung tissue were examined, including pathological changes, virus replication, and inflammation levels. RESULTS:Forty-one seasonal influenza H1N1 strains from 2023 were used. The EC50 of oseltamivir was significantly increased compared to the 2009 reference strain. Correlation analysis showed that the increase in EC50 was related to the HA and NA activities. The antiviral effects of molnupiravir and baloxavir significantly inhibited virus replication; the combination treatment of molnupiravir/baloxavir showed more potent and synergistic inhibitory effects in vitro. In the mouse model, molnupiravir treatment effectively inhibited virus replication and lung inflammation, but the treatment did not improve weight loss or reduce mortality. With the molnupiravir/baloxavir treatment, viral replication was significantly inhibited and proved to be more effective than either monotherapy. The combination therapy also showed the lowest inflammatory response along with a higher survival rate. CONCLUSIONS:The increase in HA and NA activities of seasonal influenza reduced the efficacy of oseltamivir treatment, but the effectiveness of molnupiravir and baloxavir was retained. Combination therapy showed a significant antiviral effect, which provides a reference for the clinical treatment.

BACKGROUND:HEV is an important cause of morbidity in solid organ transplant (SOT) recipients. However, the total burden of hepatitis E, including subclinical infections in this group, is not well defined. We compared hepatitis E exposures in SOT recipients to non-transplant controls. We also examined the prevalence of rat HEV (rHEV), an emerging hepatitis agent, in this population. METHODS:This study was conducted in the main SOT center in Hong Kong. Quantitative HEV IgG, RT-PCR, IgM, and IgG avidity assays were used to measure conventional HEV and rHEV exposures in 669 SOT recipients and 667 non-transplant hospitalized controls. Follow-up samples from a subset of SOT recipients were assessed to measure longitudinal HEV exposures. RESULTS:Age-adjusted HEV IgG seroprevalence in SOT recipients (236/669; 35.3%) was significantly higher than non-transplant controls (185/667; 27.7%; p=0.001). Across baseline and follow-up samples, 25 (3.7%) SOT recipients had viremia (n=3) or serological evidence (n=22) of recent hepatitis E. The latter had IgM positivity (n=5), IgG seroconversion (n=16), or a 5-fold increase in longitudinal HEV IgG concentrations (n=1). Chronic hepatitis occurred in all 3 viremic individuals, while transient hepatitis was observed in 10/22 (45.4%) SOT recipients with serological evidence of recent hepatitis E. rHEV IgG levels were similar between SOT recipients and controls (p=0.424), but 2 viremic infections in the SOT group were due to rHEV and both turned chronic. CONCLUSIONS:SOT recipients have higher hepatitis E seroprevalence than the non-transplant population. Increased exposure is driven by viremic infections and a significant burden of subclinical infections in Hong Kong. rHEV is an important cause of chronic hepatitis E in SOT recipients.

KP.3.1.1 became a dominant successor to JN.1 by the second half of 2024 but the intrinsic pathogenicity and virological feature of KP.3.1.1 remain incompletely understood. Here, we comprehensively evaluated the pathogenesis and characteristics of KP.3.1.1 in comparison to JN.1 and other JN.1-derived variants including JN.1.7, KP.2, and KP.3. The unique S31del mutation on KP.3.1.1 spike confers further evasion to the clinically authorized mAb Pemivibart and reduces convalescent serum neutralization efficiency. Structural analysis indicates that S31del induces novel glycosylation sites that facilitates evasion of neutralizing antibodies. We further reveal that S31del significantly enhances pseudovirus entry efficiency in all evaluated cell types including the human primary nasal epithelial cells. Nevertheless, the intrinsic pathogenicity of KP.3.1.1 is similar to JN.1 and KP.3, and higher than that of JN.1.7 and KP.2 in a male hamster model. Interestingly, the increased virus infectivity conferred by S31del in KP.3.1.1 spike is counterbalanced by the NSP10 S33C mutation. Overall, our study indicates that a single spike mutation can confer both enhanced immune escape and increased viral infectivity. The opposing effects of spike and non-spike mutations highlight the complex interplay of viral genomic elements in shaping their overall fitness, and reveal the high plasticity of coronavirus evolution.

Cytokine therapy, a non-antigen-specific strategy, has led to several FDA-approved drugs. Given the role of dysregulated cytokine expression in diseases such as COVID-19, accurate quantification is critical in both clinical and research settings. While antibody-based assays offer high sensitivity, their reliance on specific antibodies limits multiplexing and increases analytical complexity. Conversely, mass spectrometry methods like multiplexed reaction monitoring provide higher throughput but lack the sensitivity to detect physiological cytokine levels and the resolution to distinguish structural isomers. Thus, a new MS-based approach is needed that integrates high sensitivity with the ability to resolve structurally similar cytokines. We developed an ion mobility-mass spectrometry (IM-MS)-based parallel reaction monitoring (PRM) method to establish the first Cytokine Ion Mobility Peptide (CIMP) databank and enable high-throughput cytokine profiling in serum samples from COVID-19 patients. By introducing ion mobility as an additional gas-phase separation dimension alongside liquid chromatography, the method enhances analyte resolution based on structural differences, facilitating the separation of isomers within the ion mobility trap. The incorporation of ion mobility as a complementary separation parameter enables the distinction of homologous cytokines and structural isomers (e.g., IFNA1/IFNA2, IFNL1/IFNL3, and peptide isomers), which remains challenging for conventional antibody-based assays. The method achieved a limit of detection of 62.9 fmol/L and a limit of quantification of 210 fmol/L across 31 cytokines, demonstrating greater sensitivity than traditional multiple reaction monitoring (MRM) approaches and enabling quantification at physiological concentration levels, assuming comparable background signal across platforms. The IM-MS-PRM method offers a multiplexed, high-throughput, and adaptable platform that eliminates the need for multiple assays while delivering excellent reproducibility. It enables accurate and sensitive cytokine quantification from minimal volumes of COVID-19 patient serum. Combined with the CIMP databank, this approach allows precise differentiation between early and late severe COVID-19 cases, supporting improved diagnostic and therapeutic decision-making.

The global burden of influenza virus and rhinovirus, along with significant mortality and severe case reports, underscores the urgent need for new antivirals. Human defensins serve as the first line of defense against viruses; however, the antiviral activity of defensin peptides is often sensitive to salt, which affects their effectiveness. This study investigates a branched human-defensin peptide H30 (4H30) that can more effectively inhibit influenza virus and rhinovirus compared to the linear form of H30. Mechanistic studies reveal that 4H30 binds to influenza HA to aggregate the virus, thereby blocking viral entry. 4H30 can also cross-link H1N1 virus with cell surface glycosaminoglycans to prevent viral release. The dual-functional peptide 4H30 protects mice from the lethal challenge of the A(H1N1)pdm09 virus, demonstrating a high barrier to viral resistance after 15 viral-culture passages in the presence of 4H30. Notably, 4H30 interferes with the low-density lipoprotein receptor (LDLR) to impede the entry of minor group rhinovirus and significantly inhibits rhinovirus replication in RD cells, nasal organoids, and stem cell-derived cardiomyocytes. These findings suggest that the branched peptide 4H30, targeting both the virus and host, can more effectively inhibit influenza and rhinovirus than the linear H30, providing a new avenue for antiviral peptide development.

The efficacy of VIR-7831, a class 3 anti-SARS-CoV-2 monoclonal antibody (mAb), was demonstrated repeatedly in clinical trials; yet, reduced neutralization against Omicron variants in cell-line-based neutralization assays led to its withdrawal from clinical use. We developed organoid-based neutralization assays to measure mAb potency. We found that most class 3 mAbs, especially those not blocking receptor-binding domain-ACE2 binding, including VIR-7831, were substantially underestimated in cell-line-based assays. Nasal organoids adequately recapitulated the real-world effectiveness of VIR-7831 because of biologically relevant low ACE2 expression, and exclusively reproduced the in vivo protection of S2 mAbs due to the high TMPRSS2 expression, reminiscent of native human respiratory epithelial cells. Collectively, the robust organoid culture system and biologically relevant expression profiles of ACE2 and TMPRSS2 make nasal organoids present a correlate of in vivo protection of neutralizing mAbs exclusively. The organoid-based neutralization assays, superior to conventional cell-line-based assays, can recapitulate and predict the real-world efficacy of mAbs.