Targeting Nipah virus replication: A fluorescence polarization assay for high-throughput modulators screening against the NTAIL-XD interaction.

Virtual screening targeting the conserved domain of the IAV M2 protein reveals the potential broad-spectrum anti-IAV activity of ajmaline.

Lack of respiratory droplet transmission of two recent human influenza A(H5N1) viruses in female ferrets.

Engineering an abiotic antibody mimic: Structural and molecular mechanisms for targeting, neutralizing, and point-of-care testing virus antigens.

FNDR-11124, a broad-spectrum small molecule inhibitor of viral RNA polymerase, restricts replication of SARS-CoV-2 and Influenza virus in vitro and in vivo.

Accurate serological tools are essential for monitoring the transmission of arboviruses with pandemic potential, yet cross-reactivity between closely related viruses hampers diagnostics and surveillance. Here, we develop a high-throughput multiplex serological assay to quantify antibody responses to 28 antigens from nine arboviruses (dengue, Zika, yellow fever, West Nile, Usutu, Japanese encephalitis, chikungunya (CHIKV), Mayaro (MAYV), and O'nyong-nyong virus) and apply it to over 4000 samples from epidemiologically distinct sites on four continents. We implement a flexible analytical method based on Bayesian finite mixture models and Receiver Operating Characteristic analysis to evaluate assay performance and define seropositivity thresholds. As a case study, we resolve cross-reactive and virus-specific responses for CHIKV and the emerging MAYV by combining competitive immunoassays with mathematical modelling of multiplex serological and epidemiological data. This approach yields cross-reactivity-adjusted estimates of local transmission dynamics, in agreement with existing epidemiological evidence, and reveals that CHIKV is more prone to induce cross-reactive antibody responses than MAYV. Our results demonstrate the power of combining multiplex serology with experimental validation and modelling to disentangle exposure histories in the face of serological cross-reactivity. This integrative approach holds promise for improving arbovirus surveillance, particularly in settings with overlapping transmission of multiple viruses and limited diagnostic capacity.

Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses continue to expand geographically and across mammalian hosts, raising concern about pandemic potential. The degree and specificity of pre-existing immunity in humans are key determinants of this risk. We analyzed hemagglutinin (HA)- and neuraminidase (NA)-specific antibody responses in 300 sera collected from adults in New York City. While HA directed binding antibodies to clade 2.3.4.4b H5 were low and hemagglutination-inhibiting antibodies were absent, we detected widespread binding and functional NA antibodies against N1 neuraminidases from clade 2.3.4.4b H5N1 viruses. Neuraminidase inhibition (NI) titers were highest against North American D1.1 genotype N1 viruses and correlated strongly with neutralizing activity, whereas HA-binding antibodies did not. An additional N-linked glycosylation site, as found in the NA of a human D1.1 isolate from British Columbia, reduced susceptibility to NI antibodies. Antibodies titer to N5 from H5N5 were low to minimal. These findings indicate that population-level immunity to clade 2.3.4.4b H5 viruses is dominated by NA-directed antibodies, with important implications for pandemic risk assessment.IMPORTANCEUnderstanding how pre-existing human immunity shapes susceptibility to emerging influenza viruses is central to pandemic preparedness. Here, we determined that human sera contain widespread, functional antibodies targeting H5N1 neuraminidase, which correlate with virus neutralization, whereas HA-directed responses are limited. We further show that acquisition of an NA glycosylation site reduces antibody inhibition, highlighting a potential pathway for immune evasion. These results identify neuraminidase-specific immunity as a major immunological barrier to severe H5N1 disease in humans and emphasize the need to incorporate NA antigenicity into influenza surveillance, risk assessment, and next-generation vaccine design.

Influenza viruses are significant causes of acute respiratory infections, often leading to severe health issues and mortality. These viruses undergo continuous mutations and genetic reassortments, resulting in annual epidemics and potential pandemics. The A(H3N2) strains exhibit high genetic and antigenic variability, that influence vaccine efficacy. This study aimed to assess the prevalence of influenza viruses, including A(H3N2) strains, in Kazakhstan during 2020-2025. The study used nasopharyngeal swab and serum samples obtained from patients. The presence of influenza virus antigens in nasopharyngeal swabs was analyzed using real-time polymerase chain reaction. The level of specific antibodies in the blood serum was determined using the hemagglutination inhibition reaction and enzyme-linked immunosorbent assay methods. Influenza A/H1N1, A/H3N2 and B viruses were diagnosed using real-time PCR. Antibodies to A/H1N1pdm09, A/H3N2 and B were detected in serological studies. Our studies revealed a trend toward seasonal patterns in influenza A viruses circulation. Therefore, it was established that the A/H3N2 strains dominated in Kazakhstan during the 2021-2022 and 2023-2024 epidemic seasons. The 2023-2024 strains belong to the specific genetic clade J.2 or 3C.2a1b.2a.2a.3a.1. These studies confirmed the role of influenza viruses in the etiology of respiratory infections and emphasized the need to continue monitoring their spread in Kazakhstan.

Nipah virus (NiV) is a zoonotic paramyxovirus with pandemic potential, for which no licensed vaccines or therapeutics for human use are available. The nucleoside analog 4′-fluorouridine (4′-FlU, EIDD-2749) has broad activity against RNA viruses including in vitro activity against the Malaysia and Bangladesh clades of NiV (NiV-M and NiV-B, respectively). Here, we report the pharmacokinetic profile of orally administered 4′-FlU in Syrian Golden hamsters and its efficacy against NiV-B. 4’-FlU was orally bioavailable and provided sustained exposure of its bioactive anabolite 4’-FlU 5’-triphosphate (4′-FlU-TP) in the brain. A 7-day treatment course after NiV infection delayed time to death. Extending treatment to 21–28 days reduced viremia, incidence of lung disease, lung lesion severity, inflammation, and mortality. Lesions, viral antigen, or viral RNA could be detected in some survivors, suggesting persistent infections. Sequence analysis of NiV populations showed nonsynonymous single-nucleotide variants (SNVs) in some brain specimens from 4′-FlU-treated animals. Amino acid changes occurred in the nucleocapsid, the polymerase, or the phosphoprotein. It is currently unclear whether they reduce viral susceptibility to treatment or impact virulence but caused a slight increase in 4′-FlU potency compared to the genetic parent virus in vitro. However, none of these SNVs increased viral fitness in human brain astrocytes. This study established proof-of-concept for efficacious oral treatment of lethal NiV infection with a small-molecule nucleoside analog inhibitor in a relevant animal model.

As part of the framework of preparedness and response to influenza viruses with pandemic potential, the World Health Organization (WHO) has developed a protocol for the characterization of a series of first cases and contacts of an agent with pandemic potential (FFX). At the end of 2019, PAHO/WHO invited Colombia to be among the pilot countries for implementing this protocol. It was conducted by the National Institute of Health of Colombia. The WHO generic protocol was reviewed and adapted to Colombia. In its implementation, a series of 99 cases of COVID-19 and 360 total contacts (159 positive contacts) were evaluated between August 2020 and January 2021 in three municipalities of Colombia. Surveys were conducted, along with RT-PCR and antibody titer tests. Sociodemographic, clinical, and virological transmission conditions were analyzed to calculate the main epidemiological indicators of viral transmission using measures of central tendency and absolute and relative frequencies. A total of 258 cases were confirmed to be positive for COVID-19 for a secondary attack rate of 43%. The most common symptoms were cough, fever, headache, and odynophagia. The percentage of symptomatic contacts, clinical attack rate, incubation period, and serial interval were 34%, 16.2%, 4.6 days, and 4.7 days, respectively. The significant rate of infection and incubation periods among contacts was similar to those reported worldwide. The implementation of the WHO FFX protocol in a novel context allowed the country to test its capacity to implement global studies to determine early public health interventions. The study faced limitations due to the selection bias towards severe cases and challenges in case recruitment and contact tracing.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a lethal pathogen with pandemic potential. Clade A and B MERS-CoV viruses have caused outbreaks in the Middle East since 2012 when they initially spilled over from camels to humans. Clade C viruses, however, are only found in camels across Africa and the spillover potential of these viruses seems to be lower than for clade A/B strains but remains to be fully understood. Here, we report that clade C spikes are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes and that most clade C spikes induce reduced syncytium formation. Additionally, we demonstrate that several East African clade C strains are less able to utilize the TMPRSS2-mediated pathway for viral entry in both cell lines and primary nasal epithelial cultures. We map the molecular basis of this reduced TMPRSS2 usage to the N-terminal domain and subdomain 2 of East African clade C MERS-CoV. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may underlie the reduced replication of East African clade C strains in humans, while the reduced replication of West African strains remains to be further investigated. Altered protease usage may contribute to differential tropism of East African clade C strains and indicate geographically distinct selection pressures on spike between MERS-CoV strains circulating in camels.

Developing innovative vaccine platforms and delivery strategies to induce broad, protective immunity in the respiratory tract is crucial for preventing influenza infection and transmission in potential epidemics and pandemics. In this study, we used cell-derived extracellular vesicles (EVs) as a vaccine platform to display mosaic human and avian influenza hemagglutinins (HAs) concurrently, including HA1/HA9/HA12 or HA3/HA4/HA10, on the EV surfaces. Immunization with the mosaic HA-EV vaccine elicited cross-reactive antibodies against influenza HA stalks and viruses, robust virus-specific cellular immune responses, and a balanced Th1/Th2 immune profile. Notably, the EVs demonstrated a promising application as an effective mucosal vaccine strategy, as evidenced by enhanced HA stalk- and virus-specific IgA in mucosal tissues and complete protection against heterosubtypic reassortant H7N9 and H5N1 virus infections in mice via intranasal immunization. EV-based mosaic HA vaccines hold great promise for developing universal influenza vaccines that target a mucosal route.

Evaluating the pandemic potential of Zika virus in a changing global landscape.

Influenza viruses present an ongoing global health risk because they are always changing, which in turn results in the ineffectiveness of current strain-specific vaccines and leaves the world vulnerable to potential pandemics. The need for a universal influenza vaccine, designed to develop lasting broadly protective immunity against volatile influenza virus strains has led to advances in immunogen design. Nanotechnology, specifically self-assembled nanovaccines, offers a truly revolutionary "bottom-up" strategy to address this issue. Nanovaccines that spontaneously self-assemble into easily discernable pathogen-like nanoparticles, including protein cages (e.g., ferritin) and virus-like particles, provide densely displayed conserved influenza epitopes-such as hemagglutinin (HA) stalk, neuraminidase (NA), and M2 ectodomain (M2e)-in a multivalent array, greatly enhancing B-cell activation, initiated by extensive receptor crosslinking, and generating immune responses to a magnitude and breadth that is unattainable with soluble antigens. Moreover, self-assembled nanovaccines, often in adjuvant-free or self-adjuvanting formulations, not only induce durable and broad cross-protective humoral and cellular immunity but also offer protection from numerous heterosubtypic viral challenges. While significant hurdles remain in scaling the process to a manufacturing level and subsequently translating it into the clinic, self-assembling nanovaccines represent a paradigm shift in influenza prevention, providing a rational and promising pathway toward the development of a universal vaccine and a rapid response platform for future pandemics.

Infections by endemic viruses, and the vaccines used to control them, often provide cross-protection against related viruses, potentially altering the transmission dynamics and likelihood of emergence of new zoonotic viruses with pandemic potential. Here, we investigate how population immunity after the COVID-19 pandemic has impacted the likelihood of emergence of a novel sarbecovirus, termed SARS-CoV-X. To this end, we combined empirical cross-neutralisation data with mathematical modelling to identify key immunological and epidemiological factors shaping sarbecovirus emergence. We show that sera from individuals with different COVID-19 immunological histories contained cross-neutralising antibodies against the spike (S) protein of multiple zoonotic sarbecoviruses. Simulations parameterised by these data predict that the likelihood of emergence of a novel sarbecovirus has been reduced significantly by population cross-immunity, with outcomes determined by the extent of cross-protection and R0 of the novel virus. Preventative vaccination against SARS-CoV-X using available COVID-19 vaccines can help resist emergence even in the presence of co-circulating SARS-CoV-2. However, a theoretical vaccine with high specificity to SARS-CoV-2 can increase emergence probability by suppressing SARS-CoV-2 prevalence and, by extension, levels of natural cross-protection. Overall, SARS-CoV-2 circulation and vaccination have generated widespread immunity against related sarbecoviruses, creating an immunological barrier to novel sarbecovirus emergence in humans.

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen with pandemic potential, yet no vaccines or antivirals are available. Capsid inhibitors, such as pleconaril, that target the hydrophobic canyon on the viral capsid protein VP1, exhibit potent antiviral activity but have a low barrier to resistance. Here, we report a targeted protein degradation strategy to overcome antiviral resistance by selectively degrading the capsid protein VP1. Through a structure-based rational design, we developed a series of pleconaril-based PROTACs that recruit the cereblon (CRBN) E3 ligase to degrade the capsid VP1 protein. We established a single-cycle replication assay integrating immunofluorescence and Western blot to quantify VP1 protein levels when compounds were added postviral entry. Linker and CRBN ligand optimization led to the identification of Jun15702, a first-in-class VP1 degrader that displays potent antiviral activity against multiple wild-type EV-D68 strains and retains submicromolar potency against recombinant pleconaril-resistant variant, rMO-VP1 F159 V. Significantly, Jun15702 displayed a higher genetic barrier to drug resistance than pleconaril. Mechanistic studies demonstrate that Jun15702 not only inhibits viral entry, as does pleconaril, but also induces CRBN-dependent degradation of VP1 when added post viral entry. Collectively, this work demonstrates targeted degradation of a viral structural protein as a viable strategy and introduces a new paradigm for overcoming drug resistance in antiviral discovery against enteroviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus are dangerous respiratory pathogens with high pandemic potential. Since 2021, these two viruses have been co-circulating, which implies additional risks of co-infection with both pathogens. Prophylactic vaccination is widely recognized as the most effective way to prevent COVID-19 and influenza and to reduce the severity of these diseases. This review analyzes recent data on the simultaneous circulation of influenza and SARS-CoV-2 viruses worldwide, including epidemiological data and the pathogenetic mechanisms of co-infection. Next, we focus on current approaches to simultaneous and combined vaccination against influenza and COVID-19. We outline the types of vaccines and summarize the available findings on the effectiveness and safety of co-vaccination. A comprehensive search was conducted using PubMed, Scopus, Web of Science, and ClinicalTrials to identify data relevant to SARS-CoV-2 and influenza co-circulation and dual vaccination. Influenza and SARS-CoV-2 cause similar symptoms, and co-infection can significantly enhance the risks of pneumonia and acute respiratory distress syndrome progressing with a poor outcome, especially among children and the elderly. A range of influenza and COVID-19 vaccines built on different technological platforms is currently available on the market, with proven effectiveness, immunogenicity, and safety. A co-vaccination approach is more convenient for patients and is associated with better response to treatment, while also improving vaccine coverage and compliance and offering significant resource savings for healthcare systems. The concurrent circulation of SARS-CoV-2 and influenza viruses presents a growing public health challenge. Simultaneous and combination vaccination strategies have emerged as effective tools to streamline immunization, enhance protection, and reduce healthcare burden. Future studies should elucidate the mechanisms of the exacerbation of respiratory disease caused by co-infection, as well as the optimal strategies for co-administering influenza and COVID-19 vaccines for long-term control of seasonal and potentially pandemic respiratory viruses.

Prodrugs of nucleotides with aminoacidyl esters as one of the two masking groups of the phosphate are successful molecular constructs for making nucleosidic antivirals bioavailable. Still, some of those '"ProTide" prodrugs have a strong bias to exert their antiviral activity in liver cells, with little activity in nonhepatic tissues. Here, we show that the alcohol residue of the alaninyl esters of two established antivirals has a strong effect on the activity against RNA viruses with pandemic potential. This was first shown for remdesivir (REM), for which a cycloheptyl residue gave 50% inhibition against four different viruses at ≤110nM concentration. With the cyclobutyl derivative of bemnifosbuvir (BEM), nanomolar EC50 values against dengue virus were measured in a range of cell lines, including cells with much lower metabolic activity than hepatocytes, without significant cytotoxicity up to 50µM. These findings show how easily activity can be improved and broadened across different tissues through seemingly minor changes in ProTide structure. Our results may instruct the design of new antivirals with broad activity against RNA viruses to increase pandemic preparedness.

Neurological Long COVID (n-LC) includes persistent cognitive and autonomic symptoms after SARS-CoV-2 infection. Prior studies of post-COVID conditions have described diverse humoral autoreactivity, but findings are heterogeneous, and it remains unclear whether n-LC is associated with a consistent CNS-directed humoral signature. We performed a cross-cohort case-control analysis to detect autoantibodies in cerebrospinal fluid (CSF) and serum from n-LC participants. In the Yale COVID Mind Study cohort, CSF from n-LC participants and from pre-pandemic and post-COVID asymptomatic controls was assessed by mouse brain immunofluorescence and proteome-wide phage immunoprecipitation sequencing (PhIP-Seq), with candidate reactivities evaluated by orthogonal assays and supervised modeling. In the Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (IDCRP EPICC) cohort, post-COVID sera collected prior to iPhone- or iPad-based cognitive screening were profiled by PhIP-Seq and compared between participants with and without cognitive impairment. CSF immunoreactivity on mouse brain tissue was observed in both n-LC and controls, with similar overall frequencies, although n-LC participants more often showed nuclear-predominant staining patterns. PhIP-Seq identified sparse, largely patient-specific peptide reactivities to nuclear and neuronal proteins in CSF and serum. Supervised models provided limited discrimination between cases and controls. Candidate autoantigens had limited disease specificity on orthogonal testing. EPICC serum autoantibody profiling similarly failed to distinguish individuals with and without cognitive impairment. Across cohorts and compartments, n-LC did not exhibit a shared autoantibody signature. These findings support the absence of a dominant, common CNS autoantibody-mediated mechanism in n-LC. Grants HU00012020067, HU00012120103, HU00011920111, R01NS125693, R01MH125737, R01AI157488 from Defense health program and NIH.

On the brink of emergence: an evolutionary approach to Influenza A virus H5N1 isolated from humans.