ETIDIOH - NEW

  Abstract Background :  Monitoring antigenic drift in human influenza A viruses is essential for vaccine strain selection and ensuring protection against circulating strains. Antigenic drift is traditionally assessed using ferret antisera , which provide monospecific responses , and human vaccinee sera , which reflect exposure to multiple antigens. In this study we evaluated the pig as an alternative source of antisera to study antigenic drift compared to immune responses in ferrets and humans. We included seasonal influenza A(H1N1pdm09) human viruses that had shown different antigenic characteristics when using ferret or human antisera.  Methods :  Pairs of pigs were inoculated with six human A(H1N1)pdm09 viruses circulating between 2019 and 2023, a period of marked antigenic drift. Pig and ferret antisera were analysed by hemagglutination inhibition (HI) and virus neutralization (VN) assays.  Results :  Pigs were successfully infected with all strains, s...

  Abstract Highly pathogenic avian influenza A(H5) viruses globally impact wild and domestic birds, and have caused severe infections in mammals, including humans , underscoring their pandemic potential . The antigenic evolution of the A(H5) haemagglutinin (HA) poses challenges for pandemic preparedness and vaccine design . Here the global antigenic evolution of the A(H5) HA was captured in a high-resolution antigenic map . The map was used to design immunogenic and antigenically central vaccine HA antigens, eliciting antibody responses that broadly cover the A(H5) antigenic space. In ferrets , a central antigen protected as well as homologous vaccines against heterologous infection with two antigenically distinct viruses. This work showcases the rational design of subtype-wide influenza A(H5) pre-pandemic vaccines and demonstrates the value of antigenic maps for the evaluation of vaccine-induced immune responses through antibody profiles. Source: Nature,  https://www.nature.c...

  Highlights •  UniFluVec, an H1N1pdm vaccine candidate , includes NS1 and NEP modifications to boost attenuation and immunity. •  UniFluVec protects ferrets from H5N1 , enhancing clearance, limiting lung damage, and ensuring 100 % survival after one dose. •  Replication-deficient UniFluVec shows cross-protection , supporting its potential as a pre-pandemic intranasal vaccine. Abstract Background The emergence of new influenza strains with unpredictable antigenic properties poses a significant vaccination challenge. The increasing incidence of human H5 infections underscores the urgent need for effective pre-pandemic vaccines. Methods The UniFluVec and UniFluVec-wtNS1 viruses were designed as H1N1pdm vaccine candidates . Both viruses contained a heterologous A/Singapore/1/57-like (H2N2) NEP gene , which served as an attenuation factor . UniFluVec additionally carried a truncated to 124 amino acids NS1 gene , and an insertion of conserved influenza sequences. UniFluVe...

  ABSTRACT Influenza A viruses continuously circulate among avian and swine species, posing a persistent threat to public health . The development of influenza candidate vaccine viruses (CVVs) plays a pivotal role in the global strategy for influenza pandemic preparedness . Safety-testing of CVVs for attenuation in ferrets represents a critical step that takes place prior to making these viruses available to vaccine manufacturers . Development of pathogenicity standards is needed to establish acceptable thresholds of disease so that CVV safety can be assessed without the need for comparison to the parental virus. To assess the capacity of diverse CVVs to cause pathogenesis in mammalian hosts , clinical and virological parameters were compiled from CVV assessments in ferrets conducted using consistent methods over approximately 20 years to identify disease parameters most reflective of attenuation compared to wild-type strains. These analyses revealed an overall reduction in ferret ...

  Abstract Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses have infected >1,000 herds of dairy cattle and hundreds of poultry flocks in the United States since the beginning of 2024. Seventy human cases have been reported during that period, mainly through occupational exposure . Although prior influenza A(H1N1)pdm09 virus infection has been shown to confer protection against influenza A(H5N1) clade 2.3.4.4b virus infection in the ferret model, it remains unclear if influenza vaccines , known to elicit a less potent and narrower cross-reactive immune response, can achieve a similar effect. In this article, we demonstrate that immunization with commercially available human seasonal influenza vaccines also confers partial protection against disease caused by H5N1 clade 2.3.4.4b virus in ferrets , which is partially associated with the presence of cross-reactive antibodies targeting H5N1 virus antigens. Source: US Centers for Disease Control and Prevention,  h...

  ABSTRACT The ferret model is widely used to study influenza A viruses (IAVs) isolated from multiple avian and mammalian species , as IAVs typically replicate in the respiratory tract of ferrets without the need for prior host adaptation . During standard IAV risk assessments , tissues are routinely collected from ferrets at a fixed time point post-inoculation to assess the capacity for systemic spread. Here, we describe a data set of virus titers in tissues collected from both respiratory tract and extrapulmonary sites 3 days post-inoculation from over 300 ferrets inoculated with more than 100 unique IAVs (inclusive of H1, H2, H3, H5, H7 , and H9 IAV subtypes, both mammalian and zoonotic origin ). All experiments were conducted by a single research group under a uniform experimental protocol , making it the largest well-controlled publicly available data set to date of discrete tissue titers reported on a per-ferret level. Analysis of these tissues revealed spatial variation in i...

  ABSTRACT Alterations in the PB2-627 domain of avian influenza virus (AIV) can potentially increase the risk of cross-host species infections in humans and mammals . Recently, there has been a rise in human cases of AIV infections without the presence of the known mammalian determinant PB2-E627K . Here, we identified a variant, PB2-627V , which has evolved in poultry and has contributed to the increase in human AIV infections . By screening global PB2 sequences , we discovered a new independent cluster of PB2-627V that emerged in the 2010s , prevalent in avian, mammalian, and human AIV isolates , including those of H9N2, H7N9, H3N8, 2.3.4.4b H5N1 , and other subtypes. We functionally assessed its host adaptation , fitness , and transmissibility across three subtypes of AIVs (H9N2, H7N9, and H3N8) in different host models . PB2-627V combines the viral properties of avian-like PB2-627E and human-like PB2-627K , facilitating AIVs to efficiently infect and replicate in chickens and mi...

  Abstract 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 assessments. Specifically, we use within-host viral titer data from index and contact animals to estimate parameters relevant to tran...

  ABSTRACT Airborne transmissibility of avian influenza viruses (AIVs) in humans is considered an essential component of their pandemic risk . Although several viral factors regulating airborne transmission (AT) have been delineated, it is not known what, if any, responses at the respiratory epithelia are determinants of AIV AT. Using responses in the ferret nasal epithelium to a panel of H1N1 AIVs, we describe host responses that segregate with AT phenotypes . AIV infection upregulated interferon alpha and gamma responses and IL-6 JAK-STAT signaling and downregulated oxidative phosphorylation . Single-cell transcriptomics revealed that cellular genotoxic stress and NF-kB, interferon, and cell fate pathways differentiated host responses to AIVs with different transmissibilities. These responses culminated in greater AIV antigen-containing exudate and debris in the respiratory spaces of the nasal epithelium of ferrets inoculated with AT AIVs. More abundant CMPK2, SP100, and CXCL10 t...

Editor’s summary The vast majority of the human population has immunity to influenza A virus (IAV) by prior infection, vaccination, or both . However, protection is generally subtype-specific , and it is not clear whether prior infection against one subtype could confer protection against clade 2.3.4.4b H5N1 IAVs , which are currently circulating in birds and dairy cows . Here, Restori et al. demonstrated that prior infection with the 2009 pandemic H1N1 IAV was protective against subsequent direct infection with H5N1 IAV in ferrets. Moreover, prior immunity reduced susceptibility to infection by transmission from an infected donor ferret. These data suggest that prior immunity to IAV, especially to the 2009 pandemic H1N1 virus, may offer a degree of protection against H5N1 infection. —Courtney Malo Abstract Zoonotic infections with emerging influenza viruses occur in the context of population-wide immunity to seasonal strains . Because of the worldwide spread of highly pathogenic clade...

Abstract Sudan virus (SUDV) causes highly lethal outbreaks of hemorrhagic disease throughout Africa , but there has yet to be an approved vaccine or therapeutic to combat this public health threat. The most common route of natural exposure to filoviruses is through mucosal contact which greatly impacts initial viral replication. Historically, SUDV animal models used an intramuscular infection route . Here, we sought to further characterize an animal model using mucosal challenge routes and compared the impact that intramuscular, intranasal, or aerosol exposure had on SUDV pathogenicity in a ferret model . We determined that the route of infection did not significantly impact overall SUDV pathogenicity; only subtle changes were detected in magnitude of viremia and oral viral shedding. Additionally, we sought to determine if preexisting Lloviu virus (LLOV) immunity could protect ferrets from lethal SUDV infection. We found that the previous immunity elicited by LLOV infection was not suf...

Abstract In March 2024, clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses were first detected in U.S. dairy cattle . Similar viruses have since caused 70 zoonotic human infections . To assess changes to zoonotic potential , we characterized A( H5N1 ) clade 2.3.4.4b viruses isolated from cows’ milk and birds . Bovine-derived viruses are lethal in mice and ferrets and transmit to direct but not airborne contact ferrets. All viruses replicate in human bronchial epithelial cells despite preferentially binding avian virus-like receptors. The bovine-derived viruses remain susceptible to FDA-approved antivirals , and they are inhibited by sera from ferrets vaccinated with WHO-recommended candidate vaccine viruses (CVV) or human sera from clade 2.3.4.4c vaccinees. While 2.3.4.4b viruses induce severe disease in mammalian models , they retain many avian virus-like characteristics. Combined, we conclude that the risk of contemporary bovine-derived viruses to humans not in contact ...

Abstract Collection of systemic tissues from influenza A virus (IAV)-infected ferrets at a fixed timepoint post-inoculation represents a frequent component of risk assessment activities to assess the capacity of IAV to replicate systemically. However, few studies have evaluated how the frequency and magnitude of IAV replication at discrete tissues contribute to within-host phenotypic outcomes, limiting our ability to fully contextualize results from scheduled necropsy into risk assessment settings. Employing aggregated data from ferrets inoculated with > 100 unique IAV ( both human- and avian-origin viruses, spanning H1, H2, H3, H5, H7, and H9 subtypes ), we examined relationships between infectious virus detection in four discrete tissue types ( nasal turbinate, lung, brain, and olfactory bulb [BnOB]) to clinical outcomes of IAV-inoculated ferrets, and the utility of including these discrete tissue data as features in machine learning (ML) models. We found that addition of viral ti...

  Abstract Highly pathogenic avian influenza A viruses subtype H5N1 (HPAIV H5N1), subclade 2.3.4.4b infect multiple avian and mammalian species , posing a potential pandemic risk . Here we describe the outcomes of infection of ferrets with a HPAIV H5N1 virus, isolated from a European grey seal in 2023, compared with an older HPAIV H5N1 (A/Indonesia/05/2005). Overall, infection of ferrets with A/grey seal/Netherlands/302603/2023 caused more rapid mortality than infection of ferrets with A/Indonesia/05/2005. Animals developed severe pneumonia and irreversible hypothermia , associated with high levels of virus replication and histopathological changes in the respiratory tract and peripheral organs. As animal models for severe avian influenza virus infections in humans play a key role in the development of intervention strategies against these infections, these findings highlight the importance of using updated ferret models based on circulating virus strains. Source: Journal of Infect...

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 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 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...