Luca Bordes

The introduction, spread, and subsequent detection of highly pathogenic avian influenza viruses (HPAIVs) in poultry are influenced by the virus replication fitness, transmission fitness, and virulence. Quantifying these fitness parameters is important for implementing surveillance and control measures for poultry. This study employs an avian in vitro model using primary chicken embryo fibroblast and duck embryo fibroblast to identify cellular correlates of fitness for a reference panel of eight dominant, highly pathogenic avian influenza clade 2.3.4.4 virus genotypes (H5N1, H5N8, and H5N6) isolated between 2014 and 2024. Because HPAIVs are pantropic in poultry, the use of primary fibroblasts could provide a robust systemic model to assess virus fitness in different poultry species. By integrating longitudinal infectious virus titers and cytopathogenicity data with a mathematical model, which delineates cell populations into susceptible, latent, infectious, and dead compartments, we disentangled “traditional” virological parameters such as peak virus titer and time to 50% cell death from mechanistic parameters, including the infecting time ( t i n f ), generation time ( t g e n ), and basic reproduction number ( R ₀ ). Our results confirm host-specific fitness characteristics consistent with in vivo observations. This study underscores the potential of integrating avian in vitro models with mathematical modeling to provide high-resolution characterization of genetically similar viruses, building toward rapid risk assessments of novel HPAIVs. IMPORTANCE: Highly pathogenic avian influenza (HPAI) outbreaks in poultry have resulted in the culling of millions of animals over the past decade. The introduction and spread of HPAI in poultry are driven by the ability of the virus to replicate, transmit, and cause disease, which varies among different poultry species. These viral fitness traits can change quickly through mutations or reassortment, leading to new variants with an altered risk of introduction and transmission. In this study, we integrated an in vitro and mathematical model to assess the viral fitness of genetically similar viruses and the risk they represent for two important poultry species. This approach offers a rapid and scalable alternative to traditional animal models, aligning with the 3R’s principle and supporting rapid assessment of the viral fitness of emerging virus variants for poultry. This approach can be easily expanded to other species, including humans, and is suitable to assess the viral fitness of all viral pathogens.

The continued circulation of H5 clade 2.3.4.4b highly pathogenic avian influenza virus (HPAIV) has caused extensive mortality in wild bird populations worldwide with increasing spillover to mammals. In 2022, H5 HPAIV emerged in Svalbard, Norway, with subsequent detections in wild birds, walruses, polar bears, and arctic foxes. To understand the population-level exposure among Svalbard arctic foxes, we analysed body fluids from carcasses trapped in 2006-2015 (n = 56), 2023-2024 (n = 112), and 2024-2025 (n = 94) for antibodies to H5 avian influenza (anti- H5), influenza A nucleoprotein (anti-NP), and neuraminidase subtypes using ELISAs, haemagglutination inhibition (HI), and a multiplex assay. Only three samples from 2006-2015 tested positive for anti-H5 and were interpreted as false positives. In 2023-2024, seropositivity for anti-H5 was high (95%), supported by a lower anti-NP seropositivity (85%) and antibody profiles consistent with mixed H5N1 (42%) and H5N5 (52%) exposure. In 2024-2025, anti-H5 and anti-NP seroprevalences remained high (83% and 57%), with H5N5 (87%) exposure predominating over H5N1 (3%). A subset of anti-H5-positive samples tested positive by HI (2023-2024: 30%; 2024-2025: 14%). Juveniles with exposure limited to the previous season had higher odds of anti-H5 seropositivity in 2023-2024 than in 2024-2025 (OR 5.5). Analysis of paired lung extracts from a subset of individuals (n = 63) yielded results concordant with body fluids, using an indirect anti-H5 ELISA adapted for carnivores. Our findings demonstrate widespread H5 virus exposure. Together with occasional reports of progression to fatal HPAI, this highlights the need for continued population monitoring to evaluate ecological consequences.

In December 2025, highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype DI.2.1 virus was detected in a cat living on a dairy cattle farm. Milk and serum samples from the dairy cattle were tested for avian influenza virus. No viral RNA was detected; however, H5N1-specific antibodies were identified in serum samples from 34 (47.2%) of 72 lactating dairy cows and 24 (63.2%) of 38 youngstock. These demonstrate expansion of the mammalian host range of HPAI H5N1 in Europe.

We have previously shown that the calcium sensing receptor (CaSR) activates different G proteins and second messengers in single cells, and that GPCR and G protein-mediated ERK activity can be highly dynamic and heterogeneous at the single cell level. Here, we attempted to investigate the CaSR-Gi-ERK signaling pathway in single cells with previously characterized biosensors. We developed a strategy to simultaneously track G-protein and ERK activities in a single cell, using FRET-based and translocation sensors, a novel large-Stokes-shift green fluorescent protein, and an imaging setup with a single detector and a single wavelength for excitation. Extensive characterization and optimization to unmix the signals showed that the differences in dynamic range and type of read-out obtained from these biosensors made it very challenging to obtain robust FRET-measurements in the developed setup. When focusing solely on ERK, we found signs of possible ERK activity in response to calcium stimulation via CaSR, as well as milder changes in the absence of calcium treatment. Further optimization of the model, probes, and image processing will be necessary to develop a setup to robustly measure FRET and translocation simultaneously in single cells. We present the challenges we encountered and discuss future developments that can overcome them.

A marked increase in the incidence of mortality amongst wild mammals attributed to infection with highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses was observed in Europe in 2021. Neurological signs and high viral antigen levels in the brain of infected wild mammals indicate that the HPAI H5N1 virus causes severe disease in mammals, but serological analysis suggests that infections may be more widespread, implying that some mammals could experience mild disease. The clinical manifestation and transmissibility of HPAI H5N1 clade 2.3.4.4b viruses amongst mammals represent critical risk factors for potential zoonotic transmission to humans. This study examined the pathogenicity, viral tissue tropism and associated pathology of three HPAI H5N1 viruses in ferrets, which are a model for influenza A infection in humans. Ferrets were experimentally infected with an HPAI H5N1 poultry virus (genotype C) and two HPAI H5N1 viruses (genotype BA) isolated from a red fox, one of which carries the zoonotic PB2-E627K mutation. The red fox isolate, but not the poultry isolate, caused high morbidity, viral shedding and mortality in ferrets. Transmission to co-housed ferrets was investigated in a group setting for the virus carrying the PB2-E627K mutation and caused neurological signs accompanied by prominent viral antigen staining in recipient ferrets compared to directly inoculated ferrets. This study shows that HPAI H5N1 clade 2.3.4.4b viruses can infect mammals with varying pathogenicity and that mammal-to-mammal transmission can occur. This increases the zoonotic potential of the virus and highlights the need for enhanced surveillance in wild mammals for early detection of potential zoonotic threats.

The introduction and subsequent detection of highly pathogenic avian influenza (HPAI) in poultry is influenced by the virus replication fitness, transmission fitness, and virulence in poultry. These viral fitness parameters are important for implementing surveillance and control measures for poultry. This study investigates the potential application of an avian in vitro model using primary chicken embryo (CEF) and duck embryo fibroblasts (DEF) to identify the viral fitness for a reference panel of eight dominant HPAI clade 2.3.4.4 virus genotypes: four H5N1 viruses isolated between 2021 and 2024, as well as three H5N8 and one H5N6 virus isolated between 2014 and 2020. Infectious virus titre and cytopathogenicity were measured in the primary cell cultures over time and these data were analysed using a mathematical model which delineates cell populations into susceptible, latent, infectious, and dead compartments. In addition to obtaining “traditional” virological parameters such as peak virus replication and the time to 50% cell death, eight new parameters, key among those, the infecting time ( t _inf ), generation time ( t _gen ) and basic reproduction number (R ₀ ), were estimated using the mathematical model. Collectively, these parameters contribute to virus characterization, enhancing the resolution for comparing genetically similar viruses. This approach can allow for the evaluation of virus virulence, replication fitness, and, ideally, transmissibility fitness across different hosts. This study underscores the potential of integrating avian in vitro models with mathematical modeling and builds towards rapid risk assessments of novel HPAI viruses.

Swine influenza A viruses (IAVsw) are important causes of disease in pigs but also constitute a public health risk. IAVsw strains show remarkable differences in pathogenicity. We aimed to generate airway organoids from the porcine lower respiratory tract and use these to establish well-differentiated airway epithelial cell (WD-AEC) cultures grown at an air–liquid interface (ALI) for in vitro screening of IAVsw strain virulence. Epithelial cells were isolated from bronchus tissue of juvenile pigs, and airway organoids were cultured in an extracellular matrix in a culture medium containing human growth factors. Single-cell suspensions of these 3D organoids were seeded on Transwell filters and differentiated at ALI to form a pseudostratified epithelium containing ciliated cells, mucus-producing cells and tight junctions. Inoculation with a low dose of IAVsw in a low volume inoculum resulted in virus replication without requiring the addition of trypsin, and was quantified by the detection of viral genome loads in apical washes. Interestingly, inoculation of an H3N2 strain known to cause severe disease in pigs induced a greater reduction in trans-epithelial resistance and more damage to tight junctions than H1N2 or H1N1 strains associated with mild disease in pigs. We conclude that the porcine WD-AEC model is useful in assessing the virulence of IAVsw strains.

Swine influenza A viruses (IAVsw) are important causes of disease in pigs but also constitute a public health risk. IAVsw strains show remarkable differences in pathogenicity. We aimed to generate airway organoids from the porcine lower respiratory tract and use these to establish well-differentiated airway epithelial cell (WD-AEC) cultures grown at air-liquid interface (ALI) for in vitro screening of IAVsw strain virulence. Epithelial cells were isolated from bronchus tissue of juvenile pigs, and airway organoids were cultured in extracellular matrix in medium with human growth factors. Single cell suspensions of these 3D organoids were seeded on transwell filters and differentiated at ALI to form a pseudostratified epithelium containing ciliated cells, mucus-producing cells and tight junctions. Inoculation with a low dose of IAVsw in a low volume inoculum resulted in virus replication without requiring addition of trypsin, and was quantified by detection of viral genome loads in apical washes. Interestingly, inoculation of an H3N2 strain known to cause severe disease in pigs induced greater reduction in trans-epithelial resistance and more damage to tight junctions than H1N2 or H1N1 strains associated with mild disease in pigs. We conclude that the porcine WD-AEC model is useful to assess the virulence of IAVsw strains.

Multiple genotypes of highly pathogenic avian influenza (HPAI) H5 clade 2.3.4.4b viruses have caused epizootics in wild birds and poultry. The HPAI H5N1 genotype C virus caused a modest epizootic, whereas the occurrence of the HPAI H5N1 genotype AB virus in 2021 resulted in the largest avian influenza epizootic in Europe to date. Here we studied the pathogenicity of two HPAI H5N1 viruses by experimentally infecting chickens, Pekin ducks, Eurasian wigeons and Barnacle geese. Our study demonstrates that pathogenicity of the H5N1-2021-AB virus is lower in Pekin ducks, Eurasian wigeons and Barnacle geese compared to the H5N1-2020-C virus, whereas virus shedding was high for both viruses. After inoculation with H5N1-2021-C viral antigen expression was higher in the brain of Pekin ducks, Eurasian wigeons and Barnacle geese, which caused higher mortality compared to inoculation with H5N1-2021-AB virus. Subclinical infections occurred in Pekin ducks and Eurasian wigeons and mortality was reduced in Barnacle geese after inoculation with H5N1-2021-AB virus while H5N1-2020-C virus caused high morbidity and mortality in these species. This H5N1-2021-AB virus trait may have contributed to efficient spread of the virus in wild bird populations. Therefore, high mortality, virus shedding and long-lasting viral antigen expression found in Barnacle geese may have increased the risk for introduction into poultry.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.