ETIDIOH - NEW

  ABSTRACT Respiratory viruses can infect hosts concurrently or sequentially, potentially influencing each other’s pathogenic trajectory . However, the underlying immune mechanisms governing these interactions remain poorly understood. In this study, we examined whether respiratory syncytial virus (RSV) infection modulates host susceptibility to subsequent SARS-CoV-2 infection using two murine models . We found that prior RSV infection conferred dose- and time-dependent heterologous protection against SARS-CoV-2 . Transcriptomic and immunological analyses revealed that RSV activated lung antigen-presenting cells (APCs) and SARS-CoV-2–reactive mucosal T cells by day 9 post-infection , with responses waning by 1 month . RSV also promoted expansion of pulmonary γδ T cells and upregulation of their metabolic pathways. Notably, RSV-infected TCRδ⁻/⁻ mice , which lack γδ T cells, exhibited diminished SARS-CoV-2–reactive mucosal T cell responses, elevated viral loads, and exacerbated lung ...

  Abstract Currently, there is limited knowledge on the impact of immunity to hemagglutinin (HA) and/or neuraminidase (NA) on the transmission of influenza viruses . Therefore, using intramuscular vaccination , intranasal vaccination , or infection with reassortant viruses , we induced immunity to each antigen alone or both antigens combined in ferrets . We then assessed transmission of the 2009 pandemic H1N1 virus from these ferrets to naïve respiratory contacts . For all strategies used to induce immunity, combined immunity to HA and NA resulted in the largest reductions in transmission . Moreover, immunity to HA and NA conferred additive rather than synergistic reductions in transmission. No escape variants emerged in our transmission studies, and logistical regression showed that the probability of transmission was less than 50% when viral titers in donors were reduced to 101.5 and 102 median tissue culture infectious dose per ml on days 1 and 3 postinfection, respectively. The...

  Abstract The ongoing outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the U.S. poses a significant public health threat . To date, 70 human cases have been confirmed in the United States , including two severe cases and one fatality . While suitable animal models are crucial for predicting the potential pandemic risk of newly emerging pathogens in humans, studies investigating contemporary HPAIV H5N1 transmission dynamics remain limited. Here, we investigate the pathogenicity and transmission efficiency of recent clade 2.3.4.4b H5N1 viruses isolated from a bovine, mountain lion, and a human case using Syrian hamsters . Intranasal inoculation results in productive virus replication in the respiratory tract and shedding for all three isolates . Transmission studies demonstrate limited efficiency via direct contact and airborne routes for all isolates. Although overall transmission is inefficient , the human H5N1 isolate demonstrates relatively greater con...

  Abstract Accumulating studies have identified the pivotal role of long non-coding RNAs (lncRNAs) in participating in host–virus interactions during virus infections . However, the regulatory roles of lncRNAs in influenza A virus (IAV) infection are still not fully elucidated . In this study, using high-throughput sequencing, we comprehensively compared the expression profiles of lncRNAs and mRNAs in mouse lungs infected either with the nonpathogenic parental (SDL124) H7N9 virus or its moderately pathogenic mouse-adapted (S8) variant . A total of 7636 significantly differentially expressed (SDE) lncRNAs were obtained in the S8-infected group compared to the mock group. As for the SDL124 group, 1042 SDE lncRNAs were identified. Subsequently, the mRNAs co-expressed with SDE lncRNAs were subjected to functional annotation and pathway enrichment analysis . The results indicated that the target mRNAs regulated by the S8 virus were mainly enriched in various immunological processes and ...

  Abstract Enteroviruses, which belong to the family Picornaviridae, cause hand, foot, and mouth disease (HFMD), respiratory symptoms, and severe neurological complications in children . Since vaccines cannot provide cross-protection against different serotypes of enteroviruses , the development of broad-spectrum anti-enteroviral drugs is imperative . The viral 3C protease (3Cpro), which is essential for polyprotein processing represents a validated target for therapeutic intervention . Importantly, enterovirus 3Cpro shares conserved structural and catalytic features with coronavirus main protease (Mpro, also known as 3C-like protease, 3CLpro), providing a rationale for cross-target inhibitor repurposing . Through targeted screening of peptidomimetic protease inhibitors, a clinical-stage SARS-CoV-2 Mpro inhibitor was identified as a potent inhibitor of enterovirus A71 (EV71) 3Cpro. Bofutrelvir displayed nanomolar antiviral activity in multiple cell lines and demonstrated broad-spec...

  ABSTRACT Ferrets are widely used to model airborne transmission of influenza viruses in humans . Airborne transmission is evaluated by infecting donor ferrets with a high virus dose and monitoring transmission to contact animals sharing the same airspace . Humans can be infected with a broad range of influenza virus doses . Therefore, we evaluated the relationship between inoculation dose and transmission for two pandemic influenza viruses in ferrets . Donor ferrets were inoculated with 100 to 106 tissue culture infectious dose 50 (TCID50) of the 2009 pandemic H1N1 or 1968 pandemic H3N2 virus and were then paired with respiratory contacts . Using the proportion of donors that became infected across virus doses, we calculated the infectious dose 50 (ID50). Subsequently, by comparing the proportion of contacts that became infected, we calculated the transmissible dose 50% (TD50): the donor inoculation dose that resulted in transmission to 50% of contacts . For the 2009 pandemic H1N...

  Abstract The development of broadly protective and dose-sparing influenza vaccines remains a critical challenge , particularly for zoonotic H5N1 strains with pandemic potential . This study evaluates BECC470s, a synthetic TLR4 adjuvant , for its ability to enhance the immunogenicity and protective efficacy of recombinant H5 hemagglutinin (rHA) vaccination in murine models . BECC470s-adjuvanted rHA elicited robust IgG1/IgG2a antibody responses and complete survival following homologous 2004 H5N1 challenge in a prime–boost model . Although BECC470s broadened antibody binding to both variable HA head and conserved stalk domains by ELISA, functional neutralizing antibody responses were restricted to the matched 2004 H5N1 isolate, with no detectable neutralization of H5N1 viruses isolated in 2022 or 2024. These data indicate that BECC470s enhances the magnitude and apparent breadth of binding antibody responses while maintaining strain-specific neutralizing activity, supporting its po...

  Abstract Transmission of highly pathogenic avian influenza from H5 clade 2.3.4.4b has expanded in recent years to infect large populations of birds and mammals , heightening the risk of a human pandemic . Influenza viruses that are adapted to transmission in birds and a variety of mammals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively higher pH levels . Here, we combined five mutations in the H5 HA that increased its melting temperature and promoted stable closure of the HA trimer . Structural analysis by cryo–electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions while maintaining the local HA structure . We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than nonstabilized comparators. Epitope mapping of vaccine-elicited polyclonal antibody responses using negative-stain e...

  Abstract Dairy cattle have emerged as a prolific amplifying host for highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b and a new source for cross-species and zoonotic transmission . Independent introductions of H5N1 with unclear exposure routes have been reported in several dairy herds across the U.S. These events escalate the pandemic potential of HPAIV H5N1 as transmission within and between mammalian species present opportunities for mammalian adapted H5N1 viruses to emerge . Although more than 1000 herds have been infected, bovine H5N1 influenza virus pathogenesis, transmission, and evolution in dairy cattle remains not well characterized. Working with H5N1-infected lactating cattle in high containment has been a major challenge due to the required infrastructure and logistics associated with housing, husbandry, and waste management for this model. Thus, developing alternative bovine models that maintain biological relevance while reducing operational comple...

  Abstract For nearly 30 years, Goose/Guangdong-derived highly pathogenic avian influenza H5N1 viruses have posed significant risks to economic stability, food security, and public health . Virus evolution has resulted in various clades , including the panzootic subclade 2.3.4.4b, recognized for its pandemic potential . Here we present the potent in vitro activity of FNI9, a pan-influenza NA-inhibiting monoclonal antibody , against a range of pseudoparticles with NA spanning decades of H5N1 virus evolution . FNI9 also shows strong prophylactic protection in female mice against lethal challenges with H5N1 from clade 1 and 2.3.4.4b. Cryo-EM and molecular dynamics analysis reveal that FNI9 binds to 7 highly conserved H5N1 NA residues (R118, E119, D151, E228, E278, R293, and R368). In silico evolutionary escape profiling and machine learning predict low escapability, high fitness costs, and minimal spread likelihood for viral mutations that evade FNI9 binding. These findings support FN...

  Highlights •  MERS-CoV structural proteins and ORFs potently induce T cell responses in mice •  MERS-CoV-specific T cell epitope repertoires are identified in C57BL/6 and BALB/c mice •  Airway ORF4b208-CD4+ and ORF5167-CD8+ T cells are optimal effector T cells •  ORF4b208 and ORF5167-specific T cells protect mice against MERS-CoV infection Summary Since its initial emergence in 2012, MERS-CoV has remained endemic and a global health threat . While accessory proteins (ORFs) are known for immune evasion , their role in adaptive immunity is unexplored. This study systematically investigated T cell responses against MERS-CoV ORFs. We mapped epitope repertoires targeting structural proteins and ORFs in C57BL/6 and BALB/c mice , revealing that ORFs potently induced virus-specific T cells . Notably, ORF5 induced the dominant CD8+ T cell responses in BALB/c mice. Further analysis revealed that ORF4b208-specific CD4+ and ORF5167-specific CD8+ T cells in the respiratory...

  ABSTRACT In this study, the infection dynamics, replication, and pathogenicity of a recombinant virus containing a deletion of ORF6 (rWA1ΔORF6) on the backbone of the highly virulent SARS-CoV-2 WA1 virus (rWA1) were investigated and compared to the parental rWA1 virus. While both rWA1 and rWA1ΔORF6 viruses replicated efficiently in cultured cells , the rWA1ΔORF6 virus produced smaller plaques, suggesting reduced cell-to-cell spread. Luciferase reporter assays revealed immune-suppressing effects of ORF6 on interferon (IFN) and nuclear factor kappa B (NF-κB) signaling pathways. Pathogenesis assessment in cats revealed that animals inoculated with rWA1 were lethargic and presented with fever on days 2 and 4 post-infection (pi), whereas rWA1ΔORF6-inoculated animals developed subclinical infection . Additionally, animals inoculated with rWA1ΔORF6 presented reduced infectious virus shedding in nasal and oral secretions and broncho-alveolar lavage fluid when compared with the rWA1-inocu...

  Abstract Influenza A virus is a highly contagious respiratory pathogen, and its rapid and continuous adaptive mutations for immune escape have limited the efficacy of existing vaccines and antiviral drugs. Here, we report a multimechanism anti-influenza platform based on the conjugation of zanamivir (ZMV) with amantadine (Aman). Aman acts as a hydrophobic tag that promotes the degradation of neuraminidase and concurrently enhances the physicochemical properties of ZMV , leading to improved membrane permeability and a significantly prolonged half-life. Meanwhile, the ZMV moiety counteracts Aman-induced cytotoxic autophagy . The resulting conjugate, compound 7j , exhibits potent activity against a wide range of neuraminidase and M2 ion channel mutations . Notably, a single intravenous dose of 7j fully protected mice from a lethal H1N1 challenge . Our work demonstrates that the rational fusion of ZMV and Aman achieves synergistic multimechanistic antiviral activity with enhanced eff...

  ABSTRACT Dogs are considered mixing vessels for influenza viruses , posing a pandemic potential via viral reassortment . Our previous studies indicated that the avian-origin H3N2 canine influenza virus (A/canine/Zhejiang/1/2010, abbreviated C1) is virulent in canine and mice . Furthermore, we found that the HA and NA genes of C1 share a close genetic relationship with an H3N2 avian influenza virus (A/duck/Shanghai/06/2009, abbreviated D6), but they exhibit distinct pathogenicity . However, the understanding mechanisms remain unclear. In the present study, we explored the genetic determinants that contribute to the different pathogenicity between the C1 and D6. By using the reverse genetics approaches, we rescued several single-gene and position-substituted reassortant viruses based on the C1. The replication in Madin–Darby canine kidney cells and pathogenic trial in mice showed that the neuraminidase (NA) gene played a critical role in C1 virulence. Further analysis demonstrated ...

  Abstract Intercontinental spread of highly pathogenic avian influenza A(H5N1) viruses poses significant pandemic risks and necessitates strong protective countermeasures . We evaluated the therapeutic efficacy of the neuraminidase inhibitor oseltamivir , the polymerase inhibitors baloxavir and favipiravir , and an ion-channel blocker amantadine , against severe influenza A( H5N1 ) virus infection in female BALB/c mice . Baloxavir (≥10 mg/kg, 1 dose) fully protected mice from death , significantly reduced virus respiratory replication, and prevented neuroinvasion . Oseltamivir (≥100 mg/kg/day for 5 days) provided limited survival benefits , reduced lung titers but failed to prevent viral neuroinvasion . Favipiravir (≥100 mg/kg/day for 5 days) provided partial protection , although did not reduce viral titers in lungs and brain . Amantadine provided no benefits . Although all drugs inhibited A(H5N1) viruses in vitro, in vivo correlations did not extend beyond baloxavir . Our result...

  Highlights •  A swine influenza virus H3N2 subtype was isolated during epidemiological survey. •  It is a complex and novel reassortant , and acquired accumulation of adaptive mutations. •  Both rescue and parent strains demonstrated efficient replication in mammalian cells. •  Key residues of the H3N2 HA collectively enhance the binding preference for human-type receptor. •  The rescued H3N2 cause significant pulmonary pathological damage in mice. Abstract Pigs serve as key "mixing vessels" for influenza A viruses, playing a critical role in cross-species transmission , while the H3N2 subtype represents an important lineage within the swine influenza virus (SIV) family. In this study, a novel reassortant H3N2 SIV strain , designated A/Swine/Jiangsu/YZ07/2024 , was isolated from pigs exhibiting clinical symptoms in Northern Jiangsu , China during epidemiological survey . Genetic analysis revealed that the virus is a complex reassortant, with the internal ...

  Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a human pathogen in 2012 and causes ongoing sporadic infections and outbreak clusters . Despite case fatality rates (CFRs) of over 30% and considerable pandemic potential , a safe and efficacious vaccine has not been developed. Here we report the design, characterization, and preclinical evaluation of MERS-CoV antigens . Our lead candidate comprises a stabilized spike displayed on a self-assembling ferritin nanoparticle that can be produced from a high-expressing, stable cell pool . This vaccine elicits robust MERS-CoV pseudovirus and authentic virus neutralizing antibody titers in BALB/c mice. Immunization of male non-human primates (NHPs) with one dose of Alhydrogel-adjuvanted vaccine elicited a > 103 geometric mean titer of pseudovirus neutralizing antibodies that was boosted with a second dose. Sera from these NHPs exhibited cross-reactivity against spike-pseudotyped lentiviruses from MERS-C...

  Abstract Highly pathogenic avian influenza H5N1 virus has spread to over 1,080 dairy farms across 18 states in the United States, resulting in 41 human infections and posing serious risks to both animal and public health . To address these risks, a hemagglutinin-based mRNA–lipid nanoparticle vaccine was developed , and its safety, immunogenicity, and protective efficacy in high-yielding lactating dairy cows were evaluated. The vaccine was well tolerated, had no adverse effects on health or milk production , and induced strong antibody responses . Two weeks after the second immunization, all the immunized cattle were fully protected against a high-dose H5N1 virus challenge . Notably, two-thirds of the cattle were still completely protected even at the 19th week after the first vaccination , when their serum antibody levels were very low. These data demonstrate that the mRNA vaccine confers robust, lasting protection against H5N1 virus in lactating dairy cows, providing a foundatio...

  Abstract Recent A(H5N1) zoonotic cases linked to poultry and cattle in North America highlight the urgent need to assess the pandemic potential of emerging strains . Using male ferrets , we evaluate two B3.13 and two D1.1 genotype A(H5N1) viruses isolated from humans and observe fatal disease and varying capacities for direct contact transmission . To enhance pandemic risk assessment , we conduct aerosol sampling using cyclone BC251 and water condensation capture-based SPOT samplers and perform comparative analyses to include additional A(H5N1), A(H9N2), A(H7N9), and A(H1N1)pdm09 strains with known transmissibility profiles. Although none of the A(H5N1) strains transmit via the air, B3.13 viruses are detected at significantly higher levels compared to D1.1 strains . Here we show strong correlations between viral loads in nasal washes, airborne virus shedding, and transmissibility in ferrets , highlighting the value of these metrics for identifying zoonotic influenza viruses that ...

  Highlights •  IN-delivered ChAd-Texas vaccine elicits mucosal antibody and T cell responses •  IN-delivered ChAd-Texas vaccine protects against H5N1 in mice and hamsters •  IN delivery of ChAd-Texas vaccine confers greater protection than IM delivery •  ChAd-Texas induces H5N1 immunity in the setting of prior influenza immunity Summary The emergence of highly pathogenic avian H5N1 influenza viruses in dairy cows and humans has increased the potential for another pandemic . To address this risk, we developed chimpanzee adenoviral (ChAd)-vectored H5 hemagglutinin-targeted vaccines and tested their immunogenicity and efficacy in rodents . Immunization with ChAd-Texas (clade 2.3.4.4b) vaccine in mice elicits neutralizing antibody responses and confers protection against viral infection and mortality upon challenge with a human H5N1 isolate (A/Michigan/90/2024, clade 2.3.4.4b). Intranasal delivery of the ChAd-Texas vaccine elicits mucosal antibody and T cell respon...