Mathematical modeling of in vitro replication dynamics for multiple highly pathogenic avian influenza clade 2.3.4.4 viruses in chicken and duck cells

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 ([Formula: see text]), generation time ([Formula: see text]), and basic reproduction number (R₀). 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.IMPORTANCEHighly 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.