Abstract
H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.
Highlights
Swine influenza virus (SIV) is an orthomyxovirus with a short incubation period and will spread to the whole herd soon
This study aims to reveal the expression profiles of four RNAs in the process of 3D4/21 cells against SIV infection for the first time, which could provide a basis for studying transcriptional regulation of H1N1 and H3N2 infecting 3D4/21 cells and provide a theoretical basis for further study of its possible molecular mechanism
After Madin-Daby canine kidney cells (MDCK) cells were infected by H1N1 and H3N2, the cytopathic effect (CPE) was calculated separately and the corresponding 50% tissue culture infective dose (TCID50) was calculated according to the Reed-Muench method [12]
Summary
Swine influenza virus (SIV) is an orthomyxovirus with a short incubation period and will spread to the whole herd soon. The infection rate of SIV can be as high as 100% and it can cause concurrent or secondary infections with porcine pleuropneumonia, swine streptococcus disease and porcine reproductive and respiratory syndrome, which makes the condition more complicated and worse and leads to a sharp increase in mortality in the pig herd [1,2]. Pigs are often regarded as “mixing vessels” of influenza A, which can help to change and develop disease strains and spread them to other mammals, such as humans [3]. A recent report pointed out that a new SIV strain (G4), similar to the 2009 pandemic virus, could bind to human receptors and produce higher titers of progeny viruses [4]. The molecular mechanism and regulatory network of SIV infection in host are still unclear. Revealing the molecular mechanism of pigs against influenza virus infection from the genetic nature can provide important guidance and basis for improving the disease resistance of pigs through molecular breeding in the future
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