Background: Newcastle disease virus (NDV) is an important virus for humans. It is highly lethal in fowl and is newly identified as an oncolytic virus for cancer treatment. In vivo, NDV induces spleen, thymus, bursa, and mesenteric gland cell apoptosis, thereby causing immunosuppression. In vitro , NDV can induce apoptosis by caspase-dependent pathways including the mitochondria-mediated pathway and death receptor-mediated pathway.
Methods: In this study, the major materials were baby hamster kidney (BHK-21) cells, NDV virus ( Miyadera strain; 10 3.8 TCID 50 /μl), and pan-caspase inhibiter Z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-fmk). All of the experiments used a viral infection MOI of 1. Apoptosis was confirmed using DNA fragmentation and TUNEL assay. Finally, the apoptosis-independent pathway was confirmed by western blot analysis and immunofluorescence.
Results: In this study, we differentiate between the caspase group and caspase inhibition group (added 100 µM pan-caspase inhibitor [z-VAD-fmk]) in BHK-21 cells treated with NDV at 0, 12, and 24 h. In the DNA fragmentation and TUNEL assays, the apoptosis appears at 12 h and apoptosis increases over time, regardless of caspase or not. In protein level determination, the antiapoptotic protein Bcl-2 decreased over time, which is the opposite of how the proapoptotic proteins Bax, cytochrome C, Mst3, and AIF behaved. Further, using western blot and immunofluorescent staining, we checked the AIF and Endo G translocation from the mitochondria (cytoplasmic) to the nucleus. In addition to apoptosis, we found that NDV treatment of BHK-21 cells decreased actin, regardless of caspase. The actin always decreased in the NDV-treated BHK-21 cells at 12 and 24 h.
Conclusions: NDV-mediated BHK-21 cell apoptosis mechanisms involve complex pathway; when in the normal state, the caspase-dependent pathway is main apoptosis pathway; when the caspase is suppressed, the BHK-21 can switch on the caspase-independent pathway by AIF, Endo G, or Mst3 to allow apoptosis to continue.