Outbreaks of influenza, caused by the influenza A virus (IAV), occur almost every year in various regions worldwide, seriously endangering human health. Studies have shown that host non-coding RNA is an important regulator of host–virus interactions in the process of IAV infection. In this paper, we comprehensively analyzed the research progress on host non-coding RNAs with regard to the regulation of IAV replication. According to the regulation mode of host non-coding RNAs, the signal pathways involved, and the specific target genes, we found that a large number of host non-coding RNAs directly targeted the PB1 and PB2 proteins of IAV. Nonstructural protein 1 and other key genes regulate the replication of IAV and indirectly participate in the regulation of the retinoic acid-induced gene I-like receptor signaling pathway, toll-like receptor signaling pathway, Janus kinase signal transducer and activator of transcription signaling pathway, and other major intracellular viral response signaling pathways to regulate the replication of IAV. Based on the above findings, we mapped the regulatory network of host non-coding RNAs in the innate immune response to the influenza virus. These findings will provide a more comprehensive understanding of the function and mechanism of host non-coding RNAs in the cellular anti-virus response as well as clues to the mechanism of cell–virus interactions and the discovery of antiviral drug targets.

Serum-free suspension culture technology for animal cells involves the division and proliferation of cells in serum-free medium as single cells or cell clusters within shaking flasks or bioreactors. This approach enables large-scale cell culture, enhances the yield and quality of biopharmaceuticals, reduces costs, and broadens the applications of animal cells. Serum-free suspension culture of adherent cells (e.g., Madin–Darby canine kidney (MDCK), Chinese hamster ovary (CHO), Vero, baby hamster kidney (BHK-21), and human embryonic kidney (HEK293) cells) has been successfully achieved through direct or indirect adaptation, medium optimization, and genetic engineering. Additionally, novel suspension cell lines, such as duck embryonic stem (EB66) and human retinoblastoma (PER.C6) cells, have been developed as potential new substrates for biopharmaceutical production. This review examines animal cell suspension culture technology and its applications in viral vaccines, recombinant proteins, and monoclonal antibodies, providing insights into the development and utilization of this important technology.

The post-translational modification and stability regulation of RIG-I play critical roles in promoting IFN-I production and maintaining immune homeostasis. In this study, we found that ubiquitin-specific peptidase 1 (USP1) promotes RIG-I protein stability through deubiquitination, which in turn enhances antiviral immunity through the production of inflammatory cytokines, and inhibits the replication of influenza virus in MDCK cells. In contrast, USP1 knockdown inhibited the deubiquitination of RIG-I, decreased the RIG-I protein level, and significantly increased the influenza virus titer. Meanwhile, inhibition of USP1 expression did not have a significant effect on the proliferation of MDCK cells, suggesting that USP1 could be used as a target gene to establish a vaccine-producing MDCK cell line. The above results provide a more comprehensive understanding of the function of USP1 and the antiviral response mechanism, and provide a theoretical and methodological basis for the screening of target genes for the artificial establishment of high-yield MDCK cell lines for vaccine production.

Background: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation—particularly the effect of lncRNA on influenza virus proliferation—is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. Results: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. Conclusions: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

The MDCK cell line is perceived as better than the embryos of hen eggs for the production of influenza vaccines, but the tumorigenicity of these cells is concerning. Epidermal growth factor receptor (EGFR) is likely to be a crucial target that contributes to the tumorigenicity of MDCK cells. In this study, EGFR-knockdown and EGFR-overexpression cell lines were established. EGFR’s influence on cell growth, migration, clonogenic ability, and flu virus susceptibility was evaluated in vitro, and its role in cell tumorigenicity was examined in nude mice. GST pull-down coupled with mass spectrometry (MS) and bioinformatics analysis identified EGFR-interacting proteins. The expression levels of these proteins, as well as those of PI3K–AKT- and MAPK–ERK-signaling-pathway-related molecules, were confirmed at both gene and protein levels. The result indicates that EGFR overexpression can enhance cell proliferation, migration, and clonal formation; EGFR knockdown could effectively curtail tumorigenesis and amplify the titers of influenza viruses in MDCK cells. An analysis of the underlying mechanism identified a total of 21 interacting proteins implicated in tumor formation, and among these, AKT1, CDK4, GNB2, and MAPK8 were confirmed at both gene and protein levels. EGFR can activate key factors of the PI3K–AKT signaling pathway, AKT and PI3K, and promote their phosphorylation levels. Consequently, we concluded that EGFR interacts with GNB2, facilitating transmembrane signal transduction, activating the PI3K–AKT signaling cascade, controlling cell cycle alterations, stimulating cell proliferation, and promoting tumorigenesis.