Identification of an optimal exogenous gene insertion site (P–M) and establishment of a reverse genetics system for aMPV/A

Abstract

Avian metapneumovirus subgroup A (aMPV/A) causes widespread infections in commercial poultry worldwide. The development of highly efficient viral vector vaccines is constrained by multiple factors, among which the lack of a mature reverse genetics system and the uncertainty regarding the optimal insertion site for foreign genes represent one of the core technical bottlenecks. Here, we constructed an aMPV/A reverse genetics system based on the pBR322 vector and inserted EGFP into seven of its intergenic regions to generate recombinant viruses. Viral replication, exogenous gene expression and genetic stability were systematically assessed via multiple assays. The results demonstrated that the expression level at the P‑M locus after EGFP insertion was approximately 1.2‑fold higher than that of other recombinant viruses. In addition, there was no significant difference in viral titer, and good genetic stability was observed over 20 generations. This study establishes a stable and efficient reverse genetics system for aMPV/A, which provides a critical technical platform for aMPV/A molecular mechanism research and novel recombinant vector vaccine development, with important practical value for global aMPV prevention and control.