The A(H5N1) avian flu is a highly contagious bird disease that has already infected millions of birds and is now appearing in some mammals.
It is spreading rapidly around the world, leading to mass culling of hens in poultry farms due to its highly contagious and deadly nature. This has resulted in significant disruptions to the global poultry industry, with examples including soaring egg prices in the US.
Avian flu is causing concern as it can cross the species barrier and infect mammals, including cows, cats and humans. Although it hasn’t spread between people, its ability to infect mammals raises public health concerns and calls for increased monitoring.
What technology is behind the new tests?
The JRC, in collaboration with the European Union Reference Laboratory (EURL) for Avian Flu and Newcastle disease, the Belgian Sciensano and the Italian Istituto Superiore di Sanità, developed two digital RT-PCR assays, one able to specifically detect the highly pathogenic influenza A(H5Nx) clade 2.3.4.4b viruses known for spreading across the globe and the second able to detect a broader range of influenza A viruses, including seasonal influenza. They can be used separately or in combination as a single diagnostic approach (duplex assay).
Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a laboratory technique used to detect the presence of a specific ribonucleic acid (RNA) in a sample. RT-PCR based assays can identify RNAs that carry the genetic material of specific viruses, including influenza viruses, such as the one causing bird flu. Detection of these RNAs in a clinical sample, indicate that the tested individual – human or animal – has been infected by the virus.
The newly developed digital RT-PCR tests are advanced and more sensitive than conventional RT-PCR versions. The digital tests are a high-precision way to detect and measure tiny amounts of the RNA genetic material of the virus even in complex matrices such as wastewater samples where more than a virus may co-exist.
The novelty of the digital RT-PCR assays lies in their ability to specifically distinguish between A(H5Nx) viruses of clade 2.3.4.4b and other A viruses, including seasonal human influenza, in a single test. Previously, distinguishing between these viruses was more cumbersome, expensive, and lengthy, often requiring multiple tests and sequencing.
The assays were swiftly developed using a specific computational workflow pioneered by the JRC and previously applied successfully for the development of SARS-CoV-2 assays:
Another example is the use of this computational workflow in the production of a certified reference material – a first of its kind in the EU – to improve the accuracy of COVID-19 tests.
For the new RT-PCR assays, the workflow analysed thousands of avian flu sequences to identify the most conserved and specific genetic elements unique to clade 2.3.4.4b, enabling precise and rapid differentiation in complex samples.
The new assays allow detecting and responding to outbreaks much more quickly and effectively. The tests are ready and available for use by all laboratories involved in the surveillance of avian influenza, including those involved in wastewater surveillance.
Policy context
The development of these tests supports Regulation (EU) 2022/2371 on serious cross-border threats to health, reaffirming the key role of surveillance systems in mitigating the negative impact of serious cross-border pathogenic threats.
In the animal health field, the work on the tests supports the implementation of the requirements for the surveillance of avian influenza in the EU that are laid down in Annex II to Delegated Regulation (EU) 2020/689.
The revised Urban Wastewater Treatment Directive, which entered into force on 1 January 2025, considers the influenza virus as a surveillance target.
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Novel (d)PCR assays for Influenza A(H5Nx) viruses clade 2.3.4.4b surveillance
