bhk21  (ATCC)

Journal: Microbiology Spectrum

Article Title: The P protein T25M substitution is involved in the quasispecies and virulence of Newcastle disease virus

doi: 10.1128/spectrum.00639-25

Figure Lengend Snippet: Comparison of the fusion indices of the virulence of NDV-Spotted Dove strains after the 100th passage. This figure presents the results of the investigation of the effect of viral infection using the “Dove strain” virus and its 100th passage (Dove 100th ) on cell morphology, nuclear integrity, viral replication, and cell viability. ( A ) Morphological and nuclear changes were induced by the viruses; Dove strain: Parental NDV-Spotted Dove strain, and Dove 100th , one hundred passages in DF-1 cell (Mock, Dove strain, Dove 100th ) show bright field (BF), DAPI-stained nuclei (Nucleus), and merged images (Merge). The control cell shows a healthy, confluent cell monolayer with intact nuclei and displays the overall cell morphology showing normal cell appearance. Dove strain-infected cells may show morphological changes due to viral infection; cells appear less dense and elongated, with still-visible but slightly affected nuclei. But Dove 100th exhibits the most dramatic effect. Cells are sparse and morphologically altered. DAPI staining reveals fewer and more condensed nuclei, suggesting cell death or severe nuclear damage. ( B ) In vitro viral replication kinetics changes in DF-1 cells after viral infection. Cells were infected with each virus at an MOI of 0.01, fixed at 96 hpi. A line graph showing viral titers (log10 TCID 50 /mL) at different time points (24–96 hpi) for Dove strain (blue line) and Dove 100th (red line). The parental Dove strain shows increasing viral titers, indicating robust replication due to the morphology of cells; therefore, Dove 100th shows initially rising titers but significantly lower than the Dove strain, suggesting reduced replication capacity due to the reduction in cell number. ( C ) Cell enumeration at 96 hpi, a bar graph showing the average size of cell number (%) for three Mock (gray), Dove strain (blue), and Dove 100th (red). The Mock maintains nearly 100% cell number across all three times, Dove strain significantly reduces cell number, indicating CPEs, and Dove 100th leads to the most profound reduction in cell number (near 0%), suggesting severe cytotoxicity despite lower replication. Asterisks indicate statistical significance (*** P < 0.001), confirming that both Dove and Dove100th strains significantly affect cell survival, with Dove100th being the most cytotoxic. Dove strain induces moderate CPE and replicates effectively. Dove 100th, while less competent in replication, is highly cytotoxic, possibly due to accumulated mutations enhancing cell-killing ability. Passaging of the virus from the parental NDV Dove strain to its 100th passage may have selected for a strain with increased pathogenicity but compromised replication efficiency. These findings suggest a potential trade-off between replication and cytotoxicity in viral evolution through serial passaging. ( D ) The fusogenic activity (syncytium formation) of a viral Dove strain and its 100th passage (Dove 100th ) compared to a mock control infected in the BHK-21 cell at an MOI of 0.001 and fixed at 36 hpi. Mock: Cells show normal morphology, indicating no infection. Cells infected with the Dove strain exhibit some syncytium formation, and we observed some changes in cell morphology compared to the mock, indicating that the virus induces fusion, but in the cell infected with Dove 100th , markedly increased syncytium formation compared to the original Dove strain, suggesting that repeated passage has enhanced fusogenic capacity. ( E ) The plaques formed by the two virus preparations are shown: Differences in plaque size, number, and morphology indicated the differences in virus virulence, replication, or cell-to-cell spread in repeated passage. The 100th passage strain plaques are noticeably larger and more diffuse than those from the original Dove strain, indicating more extensive cell fusion and spread. ( F ) Quantification of syncytium size at 36 h: There is almost no syncytium formation in the control cell, so the average size is near 0%. Cells infected with the original strain cause some syncytium formation, with an average size that is significantly higher than the mock (indicated by the statistical significance, e.g., **** P < 0.0001). The passaged virus causes even larger syncytia, as shown by the larger average size compared to the original strain. The statistical significance (asterisks) indicates that these differences are not due to random chance and that the passaged virus has a more potent effect on inducing cell fusion.

Article Snippet: Chicken embryo fibroblasts (DF-1 cells) and Baby Hamster Kidney (BHK-21) cells were purchased from American Type Culture Collection (ATCC) and were maintained in our laboratory.

Techniques: Comparison, Infection, Virus, Staining, Control, In Vitro, Passaging, Activity Assay