Determination of H5N1 Avian Influenza Virus Persistence Following a 2024 Backyard Poultry Outbreak in Romania

In November 2024, an outbreak of highly pathogenic avian influenza (HPAI) H5N1 was confirmed in backyard poultry in Timiș County, Western Romania. The index cases involved chickens and domestic geese found dead with lesions characteristic of HPAI. Laboratory confirmation was achieved by real-time RT-qPCR targeting the matrix, H5, and N1 genes, followed by virus isolation in embryonated specific-pathogen-free eggs. Sequencing of the hemagglutinin cleavage site revealed the multi-basic motif PLREKRRKR/GLFG, consistent with a highly pathogenic phenotype. To investigate potential viral persistence, tracheal and cloacal swabs were collected from apparently healthy selected backyard poultry (chickens, geese, ducks and pheasants). RNA extraction and RT-qPCR, performed using protocols validated by the European Union Reference Laboratory for Avian Influenza, yielded negative results for all samples. Internal controls confirmed assay reliability, excluding the possibility of PCR inhibition. The investigation confirmed the occurrence of HPAI H5N1 in backyard poultry and demonstrated the absence of detectable viral persistence in surrounding flocks under the tested conditions. These findings highlight the importance of rapid molecular diagnostics, active surveillance, and strict biosecurity in limiting virus spread. Continued monitoring under the One Health framework is essential to mitigate the risk of avian influenza at the human-animal-environment interface.