Abstract
Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2–6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G418 screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID50 72 h later. The TCID50 titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID50 titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for influenza prevention and treatment.
Highlights
Influenza A virus (IAV) is a major cause of life-threatening respiratory tract diseases worldwide
Insert fragment of approximately 420 bp was separated by 2% agarose gel electrophoresis, which confirmed that the size of insert fragment was consistent with Mbd1-Mbd3 fusion gene and the correct orientation of the target insert in the eukaryotic expression vector pcDNA3.1(+)
Identification in Madin-Darby canine kidney (MDCK) cells. (A) Insert fragment of approximately 420 bp was separated by 2% agarose gel electrophoresis following digestion of pcDNA3.1(+)/mBD1-mBD3 with EcoR I and Xho I
Summary
Influenza A virus (IAV) is a major cause of life-threatening respiratory tract diseases worldwide. Β-defensins can modulate the host’s cell-mediated immunity via cytokine expression, providing an interface between innate and adaptive immune response [10]. Due to their unique mechanism of action, β-defensins are expected to be the ideal therapeutic agents mitigating the problem of acquired drug resistance [11]. Both human β-defensins and mouse β-defensins (mBD) all have been shown to be significant in airway and lung host defense [12,13]. A eukaryotic expression vector pcDNA3.1(+)/mBD1-mBD3, was constructed and its potential to inhibit IAV in MDCK cells and mice were investigated
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