Virus Research 141 (2009) 13–20 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis P.V. Ravindra ∗ , Ashok K. Tiwari, Barkha Ratta, Uttara Chaturvedi, Sudesh Kumar Palia, R.S. Chauhan 1 Molecular Biology Laboratory, Division of Animal Biotechnology, Indian Veterinary Research Institute, Izatnagar, 243122, UP, India article info Article history: Received 19 October 2008 Received in revised form 16 December 2008 Accepted 17 December 2008 Available online 17 January 2009 Keywords: Newcastle disease virus Cytopathic effect Apoptosis abstract The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant New- castle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Apoptosis is a physiological process that eliminates harmful and severely damaged cells and maintains tissue homeostasis in mul- ticellular organisms (Young et al., 1997). The process is defined based on changes in cellular morphology and biochemical fea- tures, including DNA fragmentation, cytoplasm vacuolation, plasma membrane blebbing, and apoptotic body formation (Kerr et al., 1972). The apoptotic process is executed mainly by a family of cys- teine proteases called caspases (Thornberry and Lazebnik, 1998). The apoptotic process is regulated by members that belong to the Bcl-2 protein family (including Bax and Bcl-2 proteins) and also by p53, a nuclear transcription factor (Reed et al., 1996; Liang and Clarke, 2001). The Bax protein promotes while Bcl-2 protein inhibits apoptosis. Therefore the ratio of Bax to Bcl-2 determines the fate of the cell (Oltavi et al., 1993). The p53 protein also pro- motes apoptosis in severely damaged cells (Yu et al., 2001). The process of apoptosis can be triggered by various intra- and extra- cellular signals including virus infections (Roulston et al., 1999). A number of viruses with the ability to induce apoptosis in infected cells have been identified (Shih et al., 2004; Nagaleekar et al., 2007; Suzuki et al., 2008). Additionally, it has been suggested that virus- induced apoptosis contributes to the cytopathic effect (CPE) in ∗ Corresponding author. Department of Cardio-thoracic Surgery, Biological Sci- ence Division, University of Chicago, Chicago, IL 60637, USA. E-mail address: raviravindra1@gmail.com (P.V. Ravindra). 1 CADRAD, Indian Veterinary Research Institute, Izatnagar 243122, India. infected cells (Duval et al., 2002; Parquet et al., 2002; Ruggieri et al., 2007). Newcastle disease virus (NDV) belongs to the Avulavirus genus of the Paramyxoviridae family (van Regenmortel et al., 2000). NDV causes a highly contagious and fatal ND in birds of various species world wide with huge economic impact (Alexander, 1997). The severity of the disease varies from mortality up to 100% to sub- clinical depending upon the virus strain, immune status, and environmental stress (Yusoff and Tan, 2001). The disease in chickens is characterized by respiratory distress, diarrhea and depression. In vitro, NDV replicates in the cytoplasm of infected cells and induces a specific CPE characterized by syncytia formation and subsequently by the appearance of cell death (Lamb and Kolakofsky, 1996). In the present study, several assays specific to apoptosis were used to determine whether NDV-induced CPE in vitro was caused by apoptosis. 2. Materials and methods 2.1. Virus and cell lines In this study, Vero adapted velogenic NDV that showed the haemagglutination titre of 1:64, mean death time (MDT) of 58 h in embryonated chicken eggs and the intravenous pathogenicity index (IVPI) of 2.12 in SPF chicken was used. The velogenicity of the virus was further confirmed based on the visceral lesions including the presence of haemorrhages in the proventriculus of birds which died at day 5 post-inoculation with virus. The virus stock that contained 10 7.7 TCID 50 was stored at -20 ◦ C until use. Vero cells were culti- 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.12.008