Evidence for the circulation of genotype 4 Eurasian avian-like lineage swine H1N1 viruses in Korean swine farms using a newly developed one-step multiplex RT-qPCR

Background: Genotype 4 Eurasian avian-like lineage swine H1N1 viruses, which are reassortants containing the pandemic 2009 H1N1 virus lineage, triple reassortant lineage internal genes, and EA lineage external genes, have been reported in China since 2013. These have been predominant in pig populations since 2016 and have exhibited pandemic potential. Objectives: Examination of the circulation of G4 and its-related EA H1N1 viruses in Korea. Methods: We developed a one-step multiplex RT-qPCR that targeted M, HA1, and PB2 genes to detect G4 and its-related EA H1N1 viruses. We performed the analytical sensitivity and specificity of the multiplex RT-qPCR. The developed multiplex RT-qPCR was applied to swine respiratory samples between 2020 and 2022 in Korea. Results: Among the swine samples, a G4 EA H1N1 virus-related positive sample (6IP) was detected. Phylogenetic analysis based on all eight partial gene segments showed that 6IP contains nucleic acids of IAV segmented genes, which are closely related to G4 EA H1N1 viruses in HA, NA, PB2, NS, and NP genes. Conclusions: The developed multiplex RT-qPCR proved the sensitivity and specificity for the efficient surveillance of G4 and its-related EA H1N1 virus in clinical samples. Considering the phylogenetic analysis, additional screening for G4 EA H1N1 or its related viruses should be performed in future epidemiological studies.