Development of a Lentiviral-Based Expression System Targeting the Conserved Domain of the Rep Gene of Psittacine Beak and Feather Disease Virus

Abstract

Background: Psittacine beak and feather disease virus (PBFDV) causes severe immunosuppression and feather abnormalities in psittacine birds, and no effective antiviral treatments are currently available. RNA interference (RNAi) has emerged as a promising antiviral strategy; however, its evaluation requires a suitable avian cellular model expressing the viral target sequence. Objectives: Therefore, this proof-of-concept study aimed to construct a recombinant lentiviral vector expressing a conserved fragment of the PBFDV Rep gene and to establish a transduced chicken embryo kidney (CEK) cell population for future RNAi-based antiviral screening. Methods: A 286-bp conserved region of the PBFDV-Rep gene was inserted into the pCDH-CMV-MCS-EF1-cGFP-T2A-Puro plasmid and subsequently amplified in chemically competent Escherichia coli DH5α cells. Positive transformants were screened by colony PCR, and the recombinant construct was further confirmed by plasmid isolation and restriction enzyme digestion. All experiments were performed in three independent biological replicates unless otherwise stated. Lentiviral production was performed in three independent HEK293T transfections, whereas viral titration, CEK transduction, and RT-PCR analyses were performed using three biological replicates with technical replicate measurements. Lentiviral particles were produced in HEK293T cells and used to transduce CEK cells. Results: Competent DH5α cells enabled efficient plasmid transformation. Restriction digestion and gel purification verified correct insert cloning, and colony PCR confirmed the recombinant construct. Transfected HEK293T cells showed progressively increasing green fluorescent protein (GFP) expression. GFP-based quantification during viral titration demonstrated a transduction efficiency of approximately 95% in HEK293T cells, whereas GFP assessment after puromycin selection showed a transduction efficiency of approximately 95% in CEK cells at a multiplicity of infection (MOI) of 0.5. CEK cells exhibited GFP fluorescence, and qualitative RT-PCR confirmed transcript-level expression of the 286-bp PBFDV-Rep fragment. A specific 286-bp RT-PCR amplicon corresponding to the inserted PBFDV-Rep fragment was detected exclusively in transduced CEK cells. Conclusions: This study successfully generated a recombinant lentiviral vector expressing a conserved PBFDV-Rep transcript fragment and established a transduced CEK cell population exhibiting short-term transcript expression. Although no functional RNAi assay or protein-level validation was performed, this system provides a robust proof-of-concept platform for future RNAi-based antiviral screening and transcript-targeting studies for PBFDV.