Inhibition of Japanese encephalitis virus entry into the cells by the envelope glycoprotein domain III (EDIII) and the loop3 peptide derived from EDIII.

Japanese encephalitis virus (JEV) infection is a major cause of acute viral encephalitis both in humans and animals. The domain III of virus envelope protein (EDIII) plays important roles in interacting with host cell receptors to facilitate virus entry. In this study, recombinant JEV EDIII was expressed and purified. The protein showed the ability to inhibit JEV infection in BHK-21 cells with 50% inhibition at a concentration of 25µg/ml. Based on NMR structure of JEV EDIII, we chose several loop peptides that were reported to be related to receptor binding to test their possible inhibitory activities on virus infection. Our in vitro experiments demonstrated that one of the loop peptides (loop3) can prevent JEV infection with 50% inhibition at concentration of 10µM by interfering in virus attachment to the cells. Our in vivo experiments on mice showed the loop3 was the most protective peptide when administered before virus challenge. Therefore, the loop3 peptide may be served as basis for the development of novel antiviral agents against Japanese encephalitis virus or other flaviviruses infection.

The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α.

CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.