ETIDIOH - NEW

  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...

  Abstract Oz virus (OZV), a member of the genus Thogotovirus in the family Orthomyxoviridae, is an emerging tick-borne virus reported in Japan . A fatal human case and seroepidemiological evidence of widespread exposure among wild animals and humans suggest its potential public health significance . However, no animal models suitable for pathogenic studies or evaluation of countermeasures are available for OZV. Here, we have established a lethal mouse model of OZV infection using cell-adapted virus and mice lacking type I interferon signaling (B6 Ifnar1 KO mice). OZV infection resulted in 100% mortality and was characterized by robust viral replication in the liver and spleen, severe hepatitis, and acute liver injury . Using this model, we also demonstrated that oral administration of T-705 , an antiviral drug widely used against RNA viruses, as well as immunization with an inactivated whole virus particle vaccine, protected B6 Ifnar1 KO mice from lethal OZV infection by mitigatin...

  ABSTRACT Vaccinia virus (VACV) confers cross-protective immunity against monkeypox virus (MPXV), the causative agent of mpox, and has therefore been extensively exploited as a preventive vaccine . VACV Tiantan strain (VTT) is a second-generation smallpox vaccine used in China in the last century, and there are consistent efforts to minimize its virulence and ensure its best safety for potential clinical applications. In this study, an attenuated VACV rVTT△C12K2△A45 was constructed by deletion of gene segments related to virulence genes , host range genes, immune regulatory genes, and other functional genes from the VTT genome by genetic engineering. Attenuation characteristics of rVTT△C12K2△A45 were confirmed by smaller plaque size, lower replication capacity in various mammalian cell lines along with tests for neurotoxicity in mice , and lesion formation on rabbit skin . Immunization in BALB/c mice with rVTT△C12K2△A45 induced both anti-MPXV and anti-VACV neutralizing antibodies ...

  Abstract Neuraminidase inhibitors (NAIs) and cap-dependent endonuclease inhibitors (CENIs) represent two classes of antiviral drugs recommended for early treatment of patients with seasonal influenza A virus (IAV) infections. However, only limited human data , particularly on combination antiviral treatment , are available to inform optimal dosing regimens against novel IAVs, including highly pathogenic avian influenza A(H5N1) virus , associated with severe disease . Clade 2.3.4.4b A( H5N1 ) viruses have caused outbreaks in avian and mammalian species worldwide , highlighting the need to assess antiviral drug efficacy against these strains. We challenged ferrets with a D1.1 genotype A(H5N1) virus and treated infected animals with the NAI oseltamivir phosphate (OST) and the CENI baloxavir acid (BXA), alone or in combination , with treatment onset commencing pre- or post-symptom onset (24- or 48-hours post-inoculation (p.i.), respectively). When administered pre- or post-illness on...

  Abstract The continuous evolution and global spread of highly pathogenic avian influenza (HPAI) H5N1 viruses , particularly clade 2.3.4.4b, pose major challenges for pandemic preparedness . This study evaluates a low-dose inactivated split-virus vaccine derived from H5N1 clade 2.3.4.4b, formulated with an Alum/CpG adjuvant , in a preclinical female mouse model . The vaccine induces strong humoral and cellular immunity , generating high titers of cross-reactive antibodies against diverse H5 hemagglutinin (HA) and across different N1 neuraminidase (NA) glycoproteins. The Alum/CpG adjuvant supports substantial antigen dose sparing and promotes a balanced Th1/Th2 profile. Functional assays show potent virus neutralization , neuraminidase inhibition , and antibody-dependent cellular cytotoxicity , alongside robust antigen-specific CD4+ and CD8+ T cell responses, efficient control of lung viral replication, and reduced lung inflammation . Vaccinated mice are fully protected from lethal...

  ABSTRACT The outbreak of highly pathogenic avian H5 influenza (HPAI) clade 2.3.4.4b in cattle has spread across the United States . Mice with pre-existing immunity to H1N1 virus or with a live-attenuated influenza vaccine showed protection against a lethal bovine-derived HPAI H5N1 viral challenge . Notably, ferrets with mixed immunity also demonstrated protection against a feline-derived H5N1 virus, independent of cross-reactive neutralization titers , but antibodies to whole virus were observed. To investigate protective factors, we conducted T cell epitope mapping using published H1N1 viral sequences and found high conservation of key T cell epitopes in the bovine HPAI H5N1 strain . Depletion of T cells in mice prior to and during primary H1N1 infection impacted cross-protective antibodies to H5N1 virus, with CD4 depletion increasing mortality and CD8 depletion mildly impacting morbidity upon H5N1 viral challenge. This underscores the need to investigate memory T cell responses...

  Abstract Ebola virus (EBOV) is the causative agent of Ebola disease (EBOD), a viral hemorrhagic fever with a notably high case fatality rate . Current treatments for EBOD are limited to monoclonal antibodies or two licensed viral vector vaccines , a recombinant vesicular stomatitis virus (rVSV)-vectored vaccine or an adenovirus and modified vaccinia Ankara regimen. However, comparisons of protection, efficacy, and durability with alternative nucleotide platforms remain understudied . Here, we evaluated the immunogenicity of an mRNA vaccine expressing the EBOV glycoprotein (GP) in parallel with rVSV- and DNA-based vaccine platforms . The mRNA EBOV-GP vaccine , formulated in lipid nanoparticles , elicited significantly higher levels of total IgG and neutralizing antibody titers compared to the rVSV-EBOV-GP vaccine. Linear antibody epitope analysis indicated a preference for targeting the mucin-like domain in EBOV-GP1 following rVSV-based vaccination, while the mRNA platform distinc...

  Abstract Here we investigated the pathogenesis and contact transmission of bovine- and human-derived highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype B3.13 viruses in mammalian models . Using reverse genetics , we rescued three naturally occurring viruses : rTX2/24 (bovine-derived), rTexas/37 and rMichigan/90 (both human-derived), and compared their infection dynamics , replication and pathogenicity with the wild-type bovine TX2/24 strain in vitro and in vivo . All four viruses demonstrated comparable replication kinetics in four mammalian cell lines. However, the rMichigan/90 strain exhibited significantly smaller plaques in bovine and human cells . In vivo studies showed that mice infected with any of the viruses succumbed to infection within 4-5 days ; however, mice infected with the rMichigan/90 virus exhibited slightly lower viral replication and shedding compared to the other strains. Similarly, as in the mouse experiments, in hamsters, all viruses indu...

  ABSTRACT Oropouche virus (OROV) is reemerging in the Americas, along with a growing threat to global public health . Recent outbreaks have witnessed the first reported fatalities , vertical transmissions , and intercontinental importations of OROV, underscoring its expanding risk . Despite this, no vaccines or specific therapeutics are available, and fundamental research on OROV vaccinology and antigenicity remains limited. Here, we show that co-expression of the M polyprotein and nucleocapsid protein (NP) drives the assembly of OROV virus-like particles (VLPs) with high immunogenicity . Using the prototype strain OROV/sloth/Brazil/PA-UG-BeAn19991/1960, we developed an mRNA vaccine, M/N-vac, encoding these VLPs . Immunization with M/N-vac in mice elicited robust OROV-specific IgG and pseudovirus-neutralizing antibodies that cross-reacted with a contemporary circulating strain, hOROV/Brazil/AM-UKY-AM0088/2024. The vaccine also induced a durable, antigen-specific Th1-biased cellula...

  Abstract Seasonal and pandemic influenza viruses are continuous threats to human health, requiring rapid development of vaccines to multiple evolving viral strains. RNA vaccine technologies have the adaptability and manufacturability to facilitate pandemic preparedness but have limited flexibility in their route of administration , reducing the ability to establish local protective immune responses such as respiratory mucosal immunity . Here, we describe monovalent and bivalent replicon vaccines against A/Vietnam/1203/2004 H5N1 and A/Anhui/PA-1/2013 H7N9. These replicon vaccines express either H5 or H7 hemagglutinin and are formulated with a nanostructured lipid carrier (NLC) that permits both intramuscular (IM) and intranasal (IN) dosing. In mice , IM vaccination established systemic humoral and cellular responses but no detectable mucosal response , while IN administration induced robust systemic and mucosal immunity . The replicon-NLC vaccines protected against morbidity and m...

  Abstract The emergence and broad circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in wild birds and its spillover into dairy cows with sustained transmission in this species pose a major risk to felines , which are highly susceptible and often succumb to the infection . Here, we developed a novel recombinant hemagglutinin H5-based vaccine and evaluated its safety, immunogenicity, and protective efficacy against HPAI H5N1 virus in domestic cats . Immunization of cats with H5-vaccine candidate elicited robust levels of neutralizing antibodies against H5N1 virus and protection against disease, mortality, and infection upon H5N1 virus challenge. The vaccine-elicited immunity significantly reduced virus shedding and viremia , limiting systemic spread and disease severity in immunized animals. Importantly, beyond protecting susceptible felids, vaccinating cats against the H5N1 virus could also reduce the risk of human exposure - underscoring the One Health impact of i...

  Abstract Influenza B viruses cause substantial respiratory disease and seasonal outbreaks. Despite decades of circulation in humans , only the B/Victoria lineage persisted after the COVID-19 pandemic. Continual evolution has generated hemagglutinin deletion variants at residues 162-164 that drive successive epidemics , yet their functional consequences remain poorly understood. Using integrated phylodynamics and reverse genetics , we show that Clade V1A.1 viruses carrying a two-amino acid deletion exhibit enhanced replication and increased virulence compared with ancestral viruses lacking deletions. The recently prevailing Clade V1A.3 , which harbors a three-amino acid deletion together with the K136E substitution, has completely displaced V1A.1 and causes more severe disease in mice . Both clades bound efficiently to alpha 2-3 and 2-6 sialylated glycans and exhibited broad tolerance to acidic pH and elevated temperatures . These findings reveal that specific combinations of HA d...

  Abstract Selection of advantageous mutations drives the emergence of dominant variants during seasonal influenza epidemics . However, within-host detection of such variants remains rare , limiting our understanding of how selection operates at the scale of individual hosts. In this study, we used a controlled human infection model to examine the within-host evolutionary dynamics in thirteen participants intranasally infected with a seasonal H3N2 influenza A virus . Although this clinical trial is ongoing , our work represents a pre-planned, interim, exploratory analysis. Results in this system were contrasted with those observed in a ferret model of infection. The inoculum, used in both humans and ferrets, carried standing diversity that enabled evaluation of variant trajectories during infection. Although the dynamics were variable among participants, in humans , the minor variants in the PA and NP gene segments tended to increase in frequency as infection progressed. Variant dy...