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. 2024 Feb 7;20(2):e1011928. doi: 10.1371/journal.ppat.1011928

Fig 6. The replication ability of the recombinant viruses rHPRS103-N123I and rHPRS103 in vivo and in vitro.

Fig 6

(A) The schematic diagram shows the construction of the recombinant virus, rHPRS103-N123I. Using rHPRS103 as the backbone, the Asn123 of rHPRS103 is mutated to Ile123 for constructing the recombinant virus rHPRS103-N123I. (B) Western blotting. The recombinant viruses rHPRS103-N123I and rHPRS103 are detected with the 2E5 monoclonal antibody, rHPRS103 as reference. (C) IFA assay. DF-1 cells are infected with recombinant viruses, rHPRS103-N123I and rHPRS103, for 72 h, and then detected with the 4A3 monoclonal antibody and analyzed using a fluorescence microscope, rHPRS103 as reference. Scale bar: 400 μm. (D and E) Binding capacity between the rHPRS103-N123I and rHPRS103 with chNHE1 expressed on the cell membrane of transfected 293T cells is detected by RT-qPCR (D) and FCAS assays (E). (F) The replication ability of rHPRS103-N123I and rHPRS103 is detected using TCID50 in vitro. DF1 cells are incubated with rHPRS103-N123I and rHPRS103 at an MOI of 0.01, and then harvested and quantified at 1, 2, 3, 4, 5, 6, and 7 dpi. (G) The replication ability of rHPRS103-N123I and rHPRS103 is detected using an RT assay. DF1 cells are incubated with rHPRS103-N123I and rHPRS103 at an MOI of 0.01, and then harvested and quantified 1, 2, 3, 4, 5, 6, and 7 dpi. (H and I) One-day-old SPF chickens were injected intraabdominally with rHPRS103-N123I and rHPRS103 (n = 15, dose = 104 TCID50). (H) Viral loads in whole-blood samples are collected and evaluated using real-time SYBR qPCR mix PCR at different time points. (I) Cloaca swabs are collected and evaluated by ELISA at different time points. The error bars represent the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence; TCID50, 50% tissue culture infective dose; dpi, days post-inoculation; MOI, multiplicity of infection; SPF, specific pathogen-free; qPCR, quantitative polymerase chain reaction; SD, standard deviation.