Evidence for the circulation of genotype 4 Eurasian avian-like lineage swine H1N1 influenza A viruses on Korean swine farms, obtained using a newly developed one-step multiplex RT-qPCR assay.

Genotype 4 (G4) Eurasian avian-like lineage swine H1N1 influenza A viruses, which are reassortants containing sequences from 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. In this study, we developed a one-step multiplex RT-qPCR assay targeting the M, HA1, and PB2 genes to detect G4 and related EA H1N1 viruses, with detection limits of 1.5 × 101 copies/µL and 1.15 × 10-2 ng/µL for the purified PCR products and RNA templates, respectively. The specificity of the detection method was confirmed using various influenza virus subtypes. When the one-step multiplex RT-qPCR assay was applied to swine respiratory samples collected between 2020 and 2022 in Korea, a virus related to G4 EA H1N1 strains was detected. Phylogenetic analysis based on portions of all eight genome segments showed that the positive sample contained HA, NA, PB2, NS, and NP genes closely related to those of G4 EA H1N1 viruses, confirming the ability of our assay to accurately detect G4 EA H1N1 viruses in the field.

Innate antiviral responses against Shaan virus infection in HEK293, A549 and MARC-145 cells and limited role of viperin against Shaan virus replication.

The Shaan virus is a new paramyxovirus species recently isolated from an insectivorous bat. Therefore, its replication characteristics remain unclear. We used transcriptome analysis and molecular experiments to examine host cell responses in human A549, HEK293, and monkey MARC-145 cell lines infected with the Shaan virus (ShaV/B16-40). Transcriptome data showed that Shaan virus infection induced innate immune responses associated with defense mechanisms against viral infection in all infected host cells. In real-time RT-PCR, IFN-α, -ß and -λ1 were significantly upregulated in response to infection with Shaan virus in A549 and HEK-293 cells. However, the expression of IFN-α and -λ1 did not change in MARC-145 infected cells, while IFN-ß significantly increased compared to the control in all the infected cell lines. In DEG analysis, the viperin expression pattern by Shaan virus infection varied depending on the host cell types or their origins. Viperin was highly induced at the RNA level by Shaan virus infection, and viperin protein expression was detected by western blotting. Although viperin, an ISG, has broad inhibitory effects on a range of viral pathogens, viperin knockdown or knock-in in the infected cells indicated that this protein did not markedly affect Shaan virus replication. Interestingly, these effects were independent of CMPK2 expression, which is beneficial for the antiviral effects of viperin. Therefore, the present results suggest that Shaan virus might have a strategy to evade the antiviral effect of viperin or not be significantly affected by viperin.