Efficient inhibition of foot-and-mouth disease virus replication in vitro by artificial microRNA targeting 3D polymerase.

Foot-and-mouth disease (FMD) is a devastating acute viral disease of livestock with cloven hooves. Among various therapeutic control measures, RNA interference (RNAi) is one of the methods being explored to inhibit FMD virus replication and spread. The RNAi is achieved by short hairpin RNAs or artificial microRNAs (amiRNAs). Utility of amiRNAs as antiviral, targeting conserved regions of the viral genome is gaining importance. However, delivery of miRNA in vivo is still a challenge. In this study, the efficacy of amiRNAs in preventing FMD virus replication in a permissive cell culture system was investigated, by generating stable cell lines expressing amiRNAs targeting three functional regions of the FMD virus (FMDV) genome (IRES, 3B3 and 3D). The results showed that amiRNA targeting 3D polymerase is relatively more efficient. However, expression of multiple microRNAs targeting the three regions did not exhibit additive effect. The data suggest that 3D specific miRNA is a potential valid strategy in developing novel antiviral measures against FMDV infection. Keywords: artificial microRNA; foot-and-mouth disease virus; virus inhibition.

Baculovirus mediated transduction: analysis of vesicular stomatitis virus glycoprotein pseudotyping.

The recombinant baculoviruses were constructed to investigate the necessity of VSV-G pseudotyping for mammalian cell transduction. The viruses were designed to express green fluorescent protein (GFP) gene under the control of cytomegalovirus promoter, with or without pseudotyping with VSV-G. VSV-G was placed under the control of polyhedrin promoter that is recognized by insect cells, allowing the formation of pseudotyped baculovirus. The study findings demonstrate that the pseudotyping of baculovirus significantly enhanced transduction efficiency compared to non-pseudotyped baculovirus, resulting in consequent distinction in the expression of GFP in mammalian cells. The results confirmed that pseudotyping is important for baculovirus mediated gene delivery. Further, when full-length VSV-G pseudotyping was compared with truncated VSV-G containing GED domain (G-stem of ectodomain in conjunction with the TM and CT domains of the glycoprotein), latter was relatively less efficient in transducing mammalian cells. This study demonstrated that pseudotyping with full-length VSV-G had better transduction efficiency in mammalian cells. However, at higher multiplicity of infection, both full-length and truncated VSV-G showed equivalent transduction. This study established the significance of pseudotyping of baculovirus with full-length VSV-G for efficient transduction of mammalian cells, utilizing the highly sensitive GFP marker system. These findings have significant implications in designing of baculovirus vector based antigen delivery for developing new generation vaccines.