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ABSTRACT As companion animals, dogs are susceptible to various subtypes of influenza A virus (IAV), with the H3N2 and H3N8 subtypes of canine influenza virus (CIV) stably circulating among canines . Compared to the H3N8 CIV, the H3N2 CIV is more widely prevalent in canine populations and demonstrates increased adaptability to mammals , potentially facilitating cross-species transmission . Therefore, a comprehensive elucidation of the mechanisms underlying H3N2 CIV adaptation to mammals is imperative. In this study, we serially passaged the GD14-WT strain in murine lungs , successfully establishing a lethal H3N2 CIV infection model . From this model, we isolated the lethal strain GD14-MA and identified the key lethal mutations PA(S184N) and PB2(E627K). Moreover, the GD14-ma[PA(S184N)+PB2(E627K)] strain exhibited markedly enhanced pathogenicity in dogs . Viral titers in lung tissues from infected dogs and mice showed that GD14-ma[PA(S184N)+PB2(E627K)] does not increase its pathogenicity ...

Abstract The highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in aquatic birds since 1997, causing outbreaks in domestic poultry and occasional human infections worldwide. Recently, the cross-species transmission of a new reassortant variant from clade 2.3.4.4b of H5N1 to cattle in the US has heightened concerns regarding the expansion of host range and potential human infection . As eradicating the H5N1 virus from its reservoir is impossible, it is essential to prepare for a potential pandemic caused by an H5N1 derivative. Utilizing a deleted-NS1 live attenuated influenza viral vector vaccine system (DelNS1 LAIV), a system we have previously used in the development of a COVID-19 vaccine, we have rapidly developed an intranasal vaccine for cattle H5N1 and related clade 2.3.4.4b strains, based on publicly available sequences. Our research demonstrates that a single intranasal immunization can provide effective protection against lethal challenges from HPAI cattle or ...

Abstract The coronavirus membrane protein (M) is the main organizer of coronavirus assembly. Here, we report on an M-targeting molecule , CIM-834, that blocks the assembly of SARS-CoV-2. CIM-834 was obtained through high-throughput phenotypic antiviral screening followed by medicinal-chemistry efforts and target elucidation. CIM-834 inhibits the replication of SARS-CoV-2 (including a broad panel of variants) and SARS-CoV. In SCID mice and Syrian hamsters intranasally infected with SARS-CoV-2, oral treatment reduced lung viral titres to nearly undetectable levels, even (as shown in mice) when treatment was delayed until 24 h before the end point. Treatment of infected hamsters prevented transmission to untreated sentinels. Transmission electron microscopy studies show that virion assembly is completely absent in cells treated with CIM-834. Single-particle cryo-electron microscopy reveals that CIM-834 binds and stabilizes the M protein in its short form, thereby preventing the conformati...

LETTER Transmission among mammals of bovine highly pathogenic avian influenza (HPAI) H5N1 viruses , which have caused outbreaks in US dairy cattle (1–3), has been demonstrated in ferrets by our group (4, 5) and the US Centers for Disease Control and Prevention (CDC) (6). These studies showed that these viruses can be transmitted among ferrets via respiratory droplets , albeit with lower efficiency than seasonal human influenza viruses. In contrast, bovine HPAI H5N1 viruses spread easily among ferrets through direct contact (3 of 3 [100%] ferrets) (6). Although ferrets are frequently used for influenza virus transmission (7–9) and vaccine efficacy (10, 11) studies, they demand considerable housing space and personnel and can be difficult to handle. Here, we investigated the transmissibility of the bovine HPAI H5N1 virus A/Texas/37/2024 (TX/37), which was 100% lethal in ferrets inoculated with as little as 10 plaque-forming units (PFUs) (5) by using a hamster model .  (...) Bovine HP...

Abstract In the past two decades, two pandemic respiratory viruses (H1N1 and SARS-CoV-2) have emerged via spillover from animal reservoirs . In an effort to avert future pandemics, surveillance studies aimed at identifying zoonotic viruses at high risk of spilling over into humans act to monitor the `viral chatter' at the animal-human interface . These studies are hampered, however, by the diversity of zoonotic viruses and the limited tools available to assess pandemic risk. Methods currently in use include the characterization of candidate viruses using in vitro laboratory assays and experimental transmission studies in animal models . However, transmission experiments yield relatively low-resolution outputs that are not immediately translatable to projections of viral dynamics at the level of a host population. To address this gap, we present an analytical framework to extend the use of measurements from experimental transmission studies to generate more quantitative risk assessm...

Abstract Ebola virus (EBOV) and Marburg virus (MARV) are zoonotic filoviruses that cause hemorrhagic fever in humans . Correlative data implicate bats as natural EBOV hosts , but neither a full-length genome nor an EBOV isolate has been found in any bats sampled. Here, we model filovirus infection in the Jamaican fruit bat (JFB), Artibeus jamaicensis, by inoculation with either EBOV or MARV through a combination of oral, intranasal, and subcutaneous routes . Infection with EBOV results in systemic virus replication and oral shedding of infectious virus. MARV replication is transient and does not shed. In vitro, JFB cells replicate EBOV more efficiently than MARV, and MARV infection induces innate antiviral responses that EBOV efficiently suppresses. Experiments using VSV pseudoparticles or replicating VSV expressing the EBOV or MARV glycoprotein demonstrate an advantage for EBOV entry and replication early , respectively, in JFB cells. Overall, this study describes filovirus species-sp...

Highlights The current review highlights the following major findings from the literature. 1.The genomic control of pathogenic properties of murine β-coronavirus. 2.Plausible mechanism of virus-induced neuroinflammatory demyelination and axonal loss. 3.Spike protein as a major determinant of MHV-induced neuropathogenesis. 4.The minimal essential motif in fusion peptide responsible for neuropathogenesis. 5.mCoV research sheds light on hCoV neuropathogenesis and helps design anti-virals. Abstract Coronaviruses have emerged as a significant challenge to human health . While earlier outbreaks of coronaviruses such as SARS-CoV and MERS-CoV posed serious threats , the recent SARS-CoV-2 pandemic has heightened interest in coronavirus research due to its pulmonary pathology , in addition to its neurological manifestations . In addition, the patients who have recovered from SARS-CoV-2 infection show long-term symptoms such as anosmia, brain fog and long COVID. A major hurdle in studying these v...

Abstract Rationale and Objectives :  Iron availability and metabolism are important in the pathogenesis of bacterial infections . More recently, links have been reported between iron and the severity of viral infections . In this study, we characterize a crucial relationship between iron metabolism and IAV infection and disease.  Methods :  Iron-related gene expression was assessed in human airway epithelial cells (AEC) infected with IAV. AECs were cultured with ferric iron, iron-loaded transferrin, or iron chelator, deferoxamine (DFO), prior to infection with IAV. Mice were placed on a high iron diet for 8 weeks prior to infection with IAV or treated with anti-transferrin receptor-1 (TFR1) antibody during IAV infection. The effects of iron modulation and depletion of TFR1-mediated responses on IAV infection were assessed.  Measurements and main results :  Iron-related gene expression and metabolism are altered systemically and in lung tissues and AECs during IA...

Abstract Testing approved antivirals against A(H5N1) influenza viruses circulating in peridomestic species, including dairy cows , is critical to public health and pre-pandemic planning . It cannot be tested in humans due to A(H5N1) disease severity. Here, in mice, we demonstrate that US FDA-approved baloxavir treatment mediates improved disease outcomes ( survival and viral dissemination ) over oseltamivir after lethal intranasal and ocular challenge with A(H5N1)-contaminated cow milk. Source: Nature Microbiology,  https://www.nature.com/articles/s41564-025-01961-5 ____

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 (106 infectious units) and monitoring transmission to contact animals sharing the same airspace . However, humans can be infected with a broad range of influenza virus doses. Therefore, we evaluated the relationship between virus 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 H3N2 pandemic 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 respiratory contacts that became infected, we calculated the transmissible dose 50% (TD50): the donor inoculation dose that resulted in transmission to ...

ABSTRACT Recent avian-origin H3N8 influenza A virus (IAV) that have infected humans pose a potential public health concern . Alterations in the viral surface glycoprotein, hemagglutinin (HA), are typically required for IAVs to cross the species barrier for adaptation to a new host, but whether H3N8 has adapted to infect humans remains elusive. The observation of a degenerative codon in position 228 of HA in human H3N8 A/Henan/4-10/2022 protein sequence , which could be residue G or S, suggests a dynamic viral adaptation for human infection. Previously, we found this human-isolated virus has shown the ability to transmit between ferrets via respiratory droplets , with the HA-G228S substitution mutation emerging as a critical determinant for the airborne transmission of the virus in ferrets. Here, we investigated the receptor-binding properties of these two H3N8 HAs. Our results showed H3N8 HAs have dual receptor-binding properties with a preference for avian receptor binding , and G228S...

Abstract Reports of human infections with an influenza A(H5N1) clade 2.3.4.4b virus associated with outbreaks in dairy cows in the United States underscore the need to assess the potential cross-protection conferred by existing influenza immunity . We serologically evaluated ferrets previously infected with an influenza A(H1N1)pdm09 virus for cross-reactive antibodies and then challenged 3 months later with either highly pathogenic H5N1 clade 2.3.4.4b or low pathogenicity H7N9 virus . Our results showed that prior influenza A(H1N1)pdm09 virus infection more effectively reduced the replication and transmission of the H5N1 virus than did the H7N9 virus, a finding supported by the presence of group 1 hemagglutinin stalk and N1 neuraminidase antibodies in preimmune ferrets. Our findings suggest that prior influenza A(H1N1)pdm09 virus infection may confer some level of protection against influenza A(H5N1) clade 2.3.4.4.b virus. Source: US Centers for Disease Control and Prevention,  htt...

Abstract The emergence of highly pathogenic avian influenza A(H5N1) virus in dairy cattle herds across the United States in 2024 caused several human infections . Understanding the risk for spillover infections into humans is crucial for protecting public health. We investigated whether immunity from influenza A(H1N1)pdm09 (pH1N1) virus would provide protection from death and severe clinical disease among ferrets intranasally infected with H5N1 virus from dairy cows from the 2024 outbreak. We observed differential tissue tropism among pH1N1-immune ferrets. pH1N1-immune ferrets also had little H5N1 viral dissemination to organs outside the respiratory tract and much less H5N1 virus in nasal secretions and the respiratory tract than naive ferrets. In addition, ferrets with pH1N1 immunity produced antibodies that cross-reacted with H5N1 neuraminidase protein. Taken together, our results suggest that humans with immunity to human seasonal influenza viruses may experience milder disease fro...

Abstract The epizootic prion disease of cattle, bovine spongiform encephalopathy (BSE), caused variant Creutzfeldt-Jakob disease (vCJD) in humans following dietary exposure . Codon 129 polymorphism of the human prion protein gene (PRNP), encoding either methionine (M) or valine (V), dictates the propagation of distinct human prion strains and up to now all but one neuropathologically confirmed vCJD patients have had a 129MM genotype. Concordant with this genetic association, transgenic modelling has established that human PrP 129V is incompatible with the vCJD prion strain and that depending on codon 129 genotype, primary human infection with BSE prions may, in addition to vCJD, result in sporadic CJD-like or novel phenotypes. In 2016 we saw the first neuropathologically confirmed case of vCJD in a patient with a codon 129MV genotype . This patient’s neuropathology and molecular strain type were pathognomonic of vCJD but their clinical presentation and neuroradiological features were m...

Abstract Influenza A virus (IAV) non-canonical replication products can be bound by host pathogen sensors , such as retinoic acid-inducible gene I (RIG-I). However, innate immune activation is infrequent in cell culture infection, in particular by adapted strains. Moreover, it is not understood why non-canonical IAV RNAs activate RIG-I in a sequence- or RNA structure-dependent manner. We therefore hypothesized that multiple errors need to occur before influenza virus RNA synthesis activates innate immune signaling . To test this idea, we investigated whether RIG-I activation is stimulated by the non-canonical or aberrant transcription of mini viral RNAs (mvRNA), a <125 nt long RNA that is overexpressed in pandemic and highly pathogenic IAV infections . Using mvRNA sequences identified in tissue culture and ferret infections , we find that mvRNAs can cause non-canonical transcription termination through a truncated 5ʹ polyadenylation signal or a 5ʹ transient RNA structure that interr...

LETTER In March 2024, highly pathogenic avian influenza (HPAI) H5N1 of the clade 2.3.4.4b was detected in dairy cows in Texas and has since been detected in several other U.S. states (1). Virus has been detected within cow’s milk , indicating that the mammary epithelium may support viral replication (2). Virus has also been detected on milking machines , leading to a hypothesis that influenza is spreading through fomites from udder to udder instead of the intranasal route (3, 4). There have been studies using cows to better understand mammary infections; however, the cow model is costly and limited (1, 5). We sought to establish a model for intramammary infections of H5N1 and H1N1 influenza virus in mice . (...) Source: Journal of Virology,  https://journals.asm.org/doi/full/10.1128/jvi.01927-24?af=R _____

Abstract Influenza virus pandemics and seasonal epidemics have claimed countless lives. Recurrent zoonotic spillovers of influenza viruses with pandemic potential underscore the need for effective countermeasures . In this study, we show that pre-exposure prophylaxis with broadly neutralizing antibody (bnAb) MEDI8852 is highly effective in protecting cynomolgus macaques from severe disease caused by aerosolized highly pathogenic avian influenza H5N1 virus infection. Protection was antibody dose–dependent yet independent of Fc-mediated effector functions at the dose tested. Macaques receiving MEDI8852 at 10 milligrams per kilogram or higher had negligible impairment of respiratory function after infection, whereas control animals were not protected from severe disease and fatality. Given the breadth of MEDI8852 and other bnAbs, we anticipate that protection from unforeseen pandemic influenza A viruses is achievable. Source: Science,  https://www.science.org/doi/10.1126/science.ado64...

Abstract Viral infections are characterized by dispersal from an initial site to secondary locations within the host. How the resultant spatial heterogeneity shapes within-host genetic diversity and viral evolutionary pathways is poorly understood. Here, we show that virus dispersal within and between the nasal cavity and trachea maintains diversity and is therefore conducive to adaptive evolution , whereas dispersal to the lungs gives rise to population heterogeneity . We infected ferrets either intranasally or by aerosol with a barcoded influenza A/California/07/2009 (H1N1) virus . At 1, 2, or 4 days postinfection, dispersal was assessed by collecting 52 samples from throughout the respiratory tract of each animal. Irrespective of inoculation route, barcode compositions across the nasal turbinates and trachea were similar and highly diverse, revealing little constraint on the establishment of infection in the nasal cavity and descent through the trachea. Conversely, infection of the ...

Abstract Pigs serve as a mixing vessel for influenza viruses and can independently promote the emergence of pandemic strains in humans. During our surveillance of pig populations from 2021 to 2023 in China , 11 H1 subtype swine influenza viruses (SIVs) were isolated. All viruses were reassortants , possessing internal genes of identical origins ( PB2, PB1, PA, NP, M : pdm09/H1N1 origin, NS : North American triple reassortant origin). The H1N1 isolates were all the dominant G4 EA H1N1 viruses in China. Two H1N2 isolates carried early human pdm09/H1N1 HA genes, suggesting a possible pig-to-human transmission route. Mutations that dictate host range specificity were identified in all isolates, a phenomenon which may enhance the affinity to human receptors. These H1 subtype viruses effectively replicated both in vivo and in vitro without prior adaptation and exhibited different pathogenicity and growth characteristics. Some of the H1 viruses were even found to cause lethal infections in mi...

Abstract H7N9 avian influenza virus (AIV) first emerged in February 2013 in China , and early isolates were all low pathogenic (LP). After circulation for a few years in live poultry markets of China , LP H7N9 AIVs evolved into a highly pathogenic (HP) form in late 2016. Deduced amino acid sequence analysis of hemagglutinin (HA) gene revealed that all HP H7N9 AIVs have obtained four-amino-acid insertion at position 339-342 (H7 numbering), making the cleavage site from a monobasic motif (LP AIVs) to a polybasic form (HP AIVs). Notably, the polybasic cleavage site motifs are diversified, of which PEVPKRKRTAR↓GLF motif is prevalent. To elucidate the reasons accounting for its dominance, recombinant H7N9 virus carrying PEVPKRKRTAR↓GLF (rJT157-2) motif was generated based on LP H7N9 virus A/chicken/Eastern China/JT157/2016 (JT157). Besides, another two viruses containing PEVPKGKRTAR↓GLF (rJT157-1) and PEIPKRKRTAR↓GLF (rJT157-3) cleavage site motifs were also constructed as comparisons. We f...