Fig 1.
Schematic diagram of aMPV/A-EGFP recombinant virus construction.
This schematic illustrates the construction of recombinant avian metapneumovirus subgroup A (aMPV/A) strains with enhanced green fluorescent protein (EGFP) inserted into seven distinct intergenic regions of the viral genome. CMV = cytomegalovirus promoter; HDRZ = hepatitis delta virus ribozyme sequence. The recombinant viruses were designated as raMPV/A-NP-EGFP (EGFP inserted into the N-P intergenic region), raMPV/A-PM-EGFP (P-M), raMPV/A-MF-EGFP (M-F), raMPV/A-FM2-EGFP (F-M2), raMPV/A-M2SH-EGFP (M2-SH), raMPV/A-SHG-EGFP (SH-G), and raMPV/A-GL-EGFP (G-L), respectively.
Fig 2.
The growth curve of the aMPV/A-EGFP recombinant virus.
Growth kinetics analysis of the parental aMPV/A strain and seven EGFP-expressing recombinant aMPV/A strains in Vero cells. The x-axis represents hours post-inoculation (hpi) with the virus, and the y-axis indicates viral titers expressed as log₁₀ 50% tissue culture infective dose per milliliter (log₁₀ TCID₅₀/mL). Vero cells were inoculated with each virus at a multiplicity of infection (MOI) of 0.1, and viral titers were determined at 4-hour intervals using the Reed-Muench method. Data are presented as the mean values of at least three independent experiments. P < 0.05 indicates a statistically significant difference compared with other recombinant strains.
Fig 3.
Comparison of EGFP expression levels in aMPV/A-EGFP recombinant virus.
Detection of EGFP expression in Vero cells inoculated with seven aMPV/A-EGFP recombinant viruses at 48 hpi (MOI = 0.1). A–G represent fluorescence micrographs of Vero cells infected with raMPV/A-NP-EGFP (A), raMPV/A-PM-EGFP (B), raMPV/A-MF-EGFP (C), raMPV/A-FM2-EGFP (D), raMPV/A-M2SH-EGFP (E), raMPV/A-SHG-EGFP (F), and raMPV/A-GL-EGFP (G), respectively. Scale bar = 100 μm. H is the quantitative analysis of relative EGFP fluorescence intensity in infected cells; the x-axis shows different recombinant virus strains, and the y-axis represents relative fluorescence intensity (no unit). Fluorescence intensity was quantified using ImageJ software, and data are presented as the mean values of at least three independent experiments. P < 0.05 indicates a statistically significant difference in fluorescence intensity compared with other recombinant strains.
Fig 4.
Expression of EGFP in different generations of aMPV/A-PM-EGFP recombinant viruses.
Genetic stability analysis of raMPV/A-PM-EGFP (the optimal recombinant strain) after serial passage (P5, P10, P15, P20; passage 5, 10, 15, 20) in Vero cells, with parental aMPV/C as the negative control and chicken β-actin as the internal reference protein. A Indirect immunofluorescence assay (IFA) for the detection of aMPV-F protein expression in raMPV/A-PM-EGFP at different passages. Scale bar = 100 μm. B Western blotting (WB) analysis of aMPV-F protein and EGFP expression in serially passaged raMPV/A-PM-EGFP. Primary antibodies included a monoclonal antibody against aMPV-F, a specific antibody against EGFP, and a monoclonal antibody against chicken β-actin. Lanes are as follows: 1 = parental aMPV/C (negative control); 2 = raMPV/A-PM-EGFP P5; 3 = raMPV/A-PM-EGFP P10; 4 = raMPV/A-PM-EGFP P15; 5 = raMPV/A-PM-EGFP P20.