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

## TL;DR

A 2024 H5N1 avian influenza outbreak in Romania was contained quickly, with no evidence of virus persistence in nearby backyard birds.

## Contribution

The study confirms the absence of viral persistence in surrounding poultry after a localized H5N1 outbreak in Romania.

## Key findings

- H5N1 was confirmed in backyard poultry in Timiș County through genetic testing and virus isolation.
- No viral presence was detected in healthy birds from a nearby village, indicating successful containment.
- Rapid diagnostics and biosecurity measures were critical in preventing further spread.

## Abstract

In November 2024, a highly contagious bird disease called avian influenza (H5N1) was detected in backyard chickens and geese in western Romania. This virus often spreads quickly among birds and can cause sudden death. To confirm the cause, laboratory tests were carried out, including special genetic tests and virus isolation, which showed that the birds were infected with the highly dangerous form of the virus. Because such outbreaks can sometimes continue to spread silently, the study also tested apparently healthy chickens, ducks, geese, and pheasants in a nearby village. None of these birds carried the virus, suggesting that the outbreak was successfully contained and did not persist in the surrounding area. These results show the importance of quick testing, strict farm controls, and careful monitoring to stop further spread. They also highlight that small backyard flocks, when quickly managed, may not always lead to long-term virus circulation. This information is valuable for veterinarians, farmers, and public health authorities, as it supports rapid response strategies to protect both poultry health and human health.

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.