[Development of a real-time RT-PCR for detection of equine influenza virus].

This research involves the development of a diagnostic tool, a real-time reverse transcription polymerase chain reaction (RT-PCR), to quickly and accurately diagnose equine influenza viruses. In addition, the tool can characterize these viruses for epidemiological research.

Explanation of the Research

• The study revolves around the development of a real-time RT-PCR test, a modern diagnostic tool for the detection of equine influenza viruses (EIV), a common cause of respiratory illness in horses. The test is designed to specifically target a highly conserved region of the matrix gene, a part of the virus that doesn’t change much over time, enhancing its accuracy and reliability.
• Two additional RT-PCR methods were adapted for this study to allow for the amplification of large parts of the matrix and hemagglutinin (HA) genes. It aids in further characterizing the viruses for epidemiological research, which explores how diseases spread and affect populations.
• The primers and probe used in the RT-PCR showed very high homologies to (similarities with) both EIV and all influenza A viruses. These high homology rates make the assay (the test) potentially useful for diagnosing influenza in other species as well, not just horses.
• The diagnostic suitability of the assay was confirmed through its application to 20 equine, 11 porcine, and 2 avian samples, demonstrating its potential for cross-species application.
• High specificity for the influenza viruses was demonstrated both experimentally and by using software simulation, signifying the accuracy of the test in detecting and distinguishing influenza viruses.
• The analytical sensitivity of the assay was determined to be at the level of 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA. This level of sensitivity implies the assay’s ability to detect the virus even in circumstances where the virus level (viral shedding) is low.
• The amplified EIV sequences were grouped using phylogenetic analysis which allows the visualization of the evolutionary relationships between the viruses. This shows that all sequences fall within known lineages, further validating the test’s effectiveness.