The D type influenza virus (Influenza D virus, IDV), a novel member of the influenza virus family, belongs to the family Orthomyxoviridae and the genus Deltainfluenzavirus. The IDV was first isolated from a pig displaying influenza-like symptoms in Oklahoma, USA, in April 2011(Hause et al., 2013). Subsequently, the influenza D virus was isolated from cattle in multiple states across the United States in 2013 and was officially designated as such by the International Committee on Taxonomy of Viruses (ICTV) in August 2016 (Yu et al., 2021). The IDV genome is comprised of seven segments of negative-sense single-stranded RNA (Hause et al., 2013), which encode the polymerase basic protein 1 (PB1), the polymerase basic protein 2(PB2), the polymerase acidic protein(P3), the unique surface glycoprotein hemagglutinin-esterase fusion (HEF), the nucleoprotein (NP), the matrix protein (M), and the non-structural protein (NS). Among these, the PB1 gene is regarded as a relatively conserved segment within the IDV genome and is frequently utilized as a target gene for molecular biology detection (Hause et al., 2013, Faccini et al., 2017, Kishimoto et al., 2017). Conversely, the HEF gene represents the most variable segment of the IDV genome, which is predominantly utilized for phylogenetic analysis. Currently, IDV strains can be classified into at least five distinct lineages based on the sequences of the HEF gene: D/OK, D/660, D/Yama2016, D/Yama2019, and D/CA2019(Martin et al., 2017, Yesilbag et al., 2022). The available data indicate that the D/OK, D/660 and D/CA2019 are the dominant IDV lineages mainly circulating in Europe and North America (Huang et al., 2021, Yu et al., 2021), while the D/Yama2016 and D/Yama2019 lineages are limited to Asian countries such as Japan, China, and Korea (Mekata et al., 2018, Hayakawa et al., 2020, Murakami et al., 2020, Yu et al., 2022, Lim et al., 2023). Among the five currently endemic IDV lineages, the D/OK lineage is so far the most frequently reported lineage worldwide (Yu et al., 2022). IDV has been successively detected in various animals, including cattle, pigs, goats, sheep, camels, horses, deer, and dogs (Jiang et al., 2014, Salem et al., 2017, Snoeck et al., 2018, Oliva et al., 2019, Guan et al., 2022, Sreenivasan et al., 2022, Trombetta et al., 2024). This has occurred in several countries, such as China, France, Japan, and Italy. Research indicates that cattle are the main natural host and viral reservoir for IDV (Hause et al., 2014), and IDV is one of the main pathogens of bovine respiratory disease syndrome (BRDC).

Rapid and effective IDV detection assays are crucial for preventing and controlling IDV infections. Currently, several conventional RT-PCR and real-time RT-PCR assays have been developed for this purpose (Hause et al., 2013, Faccini et al., 2017, Kishimoto et al., 2017). Although the assays mentioned above have been widely used for detecting IDV, there is a notable deficiency in the diversity of reference strains utilized for primer-probe design. This limitation fails to comprise all the strain sequences across the five lineages. Given the diversity and widespread nature of IDV strains, it is crucial to ensure that the developed assay effectively detects IDV strains from all lineages.

In the present study, we developed a RT-qPCR assay based on the PB1 gene for IDV strains of five different lineages. This assay was then used to detect 417 bovine nasal swab samples collected from 10 different regions of Hebei Province.