Content area
Full text
Received Sep 17, 2017; Revised Nov 25, 2017; Accepted Dec 4, 2017
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
1. Introduction
Human respiratory syncytial virus (RSV) is a human respiratory tract pathogen of the Mononegavirales in the Orthopneumovirus genus within the Pneumoviridae family [1]. The virus causes over 30 million acute lower respiratory tract illnesses annually in children and is also an important cause of infant pneumonia mortality worldwide [2]. There is currently no licensed vaccine available to prevent RSV infection, and anti-RSV treatments are far from being satisfying. RSV F monoclonal antibody of palivizumab is effective in preventive treatment for high-risk infants, but it is costly and has no definite role in therapy for established infection; ribavirin, the only licensed antiviral agent for the treatment of RSV infection, is not recommended to be used routinely unless in patients with severe low respiratory tract disease due to its efficacy and toxicity issues [3]. Therefore, developing novel anti-RSV drugs becomes very urgent.
Searching RSV inhibitors is generally performed by laborious, time-consuming, and expensive methods. For example, the methods in use include immunoplaque upon enzyme immunoassay, plaque upon cytopathic effect (CPE), quantitative reverse transcription polymerase chain reaction- (qRT-) PCR, and enzyme-linked immunosorbent assays (ELISA). In contrast, the application of recombinant RSV virus (rRSV) encoding reporter genes such as enhanced green fluorescent protein (EGFP, rRSV-EGFP) and luciferase (Luc) would be more practicable to screen RSV inhibitors, even available for high-throughput screening (HTS) when combined with microplates and automated plate readers [4–7].
Here, firstly, we described the construction of a recombinant RSV virus, rRSV-EGFP, based on reverse genetic techniques. The full-length RSV Long antigenomic cDNA carrying EGFP expression cassette, flanked by T7 promoter, HDV ribozyme and T7 terminator, was cloned and placed into cloning vector derived from pBR322. After the resulting recombinant plasmid and the helper plasmids encoding nucleoprotein (N), phosphoprotein (P), large protein (L), and M2 ORF protein 1 (M2-1), respectively, were cotransfected into BHK/T7-9 cells expressing the T7 RNA polymerase, the recombinant virus rRSV-EGFP was successfully rescued and characterized in vitro. Then the growing kinetic of the resulting rRSV-EGFP and its feasibility for selecting...