In this study, a strain of Getah virus (GETV, GenBank: OM363683) was successfully rescued using a reverse genetics approach. Firstly, the optimized viral sequence was cloned into the low-copy plasmid pFK containing either the T7 or CMV promoter, named T7-GETV and CMV-GETV, respectively. Viral RNA was synthesized via in vitro transcription (IVT) using T7-GETV as a template, followed by RNA transfection into BHK-21 cells via liposome-mediated transfection. The viral titer was determined using 50% tissue culture infectious dose (TCID50) method and the expression of viral protein E1 was detected through western blot (WB), validating the successful rescue of GETV. Furthermore, the cytopathic effect (CPE) was observed via light microscope, and the growth characteristics of GETV were systematically analyzed using the TCID50 assay, real time fluorescent quantitative polymerase chain reaction (RT-qPCR), and WB. Additionally, based on the structure of GETV replicon, two replicative expression vectors were constructed using CMV-GETV as a template by retaining the non-structural protein genes and replacing the structural protein genes with the reporter gene mCherry or Renilla luciferase (Rluc), named pFK-GETV-mCherry and pFK-GETV-Rluc, respectively. Two control plasmids were also constructed by inserting only the reporter gene mCherry or Rluc downstream of the CMV promoter in the pFK plasmid, named pFK-mCherry and pFK-Rluc, respectively. Analysis with Image J software revealed that the average fluorescence intensity per cell of cells transfected with pFK-GETV-mCherry was approximately five times higher than that of cells transfected with pFK-mCherry. RT-qPCR results indicated that mCherry gene transcription level in cells transfected with pFK-GETV-mCherry was also approximately five times higher than that in cells transfected with pFK-mCherry, demonstrating high consistency between the two results. WB results indicated that mCherry protein expression level in cells transfected with pFK-GETV-mCherry was also higher than that in cells transfected with pFK-mCherry at the same time point. Renilla luciferase assay showed that Rluc expression efficiency in cells transfected with pFK-GETV-Rluc was about three orders of magnitude greater than that in cells transfected with pFK-Rluc. This study provides a robust reverse genetics tool for GETV research and demonstrates the ability of the GETV replicative expression vector to enhance exogenous gene expression, establishing a foundation for the development of replicative DNA vaccines.
YU Yong-Kang
,
CAO Heng
,
MANG Gang
. Construction of an infectious clone and a replicative expression vector of Getah virus[J]. Journal of Microbes and Infections, 2024
, 19(5)
: 267
-279
.
DOI: 10.3969/j.issn.1673-6184.2024.05.002
