Comparison and characterization of construction strategies for Getah virus infectious clones with stable, high-level expression of reporter gene

Getah virus (GETV), a mosquito-borne arbovirus, has emerged as a significant threat to both animal health and public safety due to recent outbreaks and its potential for increased virulence.A comprehensive understanding of the molecular mechanisms underlying GETV pathogenesis is urgently needed to guide the development of effective prevention and therapeutic interventions. To this end, establishing a robust and efficient reverse genetics system for GETV is paramount. In this study, we rapidly constructed the full-length infectious clone of GETV based on the previously isolated attenuated strain GETV-BJ0304 using a seamless cloning method. Furthermore, we compared commonly used reporter gene integration strategies in alphavirus reverse genetics, specifically those utilizing the 2 A self-cleaving peptide and the subgenomic promoter. Experimental results confirmed that, for the backbone of attenuated strains, the insertion strategy employing the 2 A self-cleaving peptide to drive the reporter gene is highly efficient and stable. The recombinant reporter virus GETV-2 A/EGFP, constructed in this study, exhibits high infection efficiency and genetic stability, serving as a crucial technical platform for in-depth analysis of GETV replication and pathogenic mechanisms, as well as for exploring new strategies to assist the host in resisting GETV invasion.