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Development of an in vitro Model System for Newcastle Disease Virus Persistence in Bladder Cancer Cells
Poster Title: Development of an in vitro Model System for Newcastle Disease Virus Persistence in Bladder Cancer Cells
Submitted on 03 Dec 2018
Author(s): Ahmad U1,, Chan SC2, Chau DM1, Chia SL5, Abdullah S1,3, Yusoff K5 & Veerakumarasivam A1,4*
Affiliations: 1Medical Genetics Laboratory, Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2School of Foundation Studies, Perdana University, MAEPS Building, MARDI Complex, Jalan MAEPS Perdana, 43400 Serdang, Selangor, Malaysia. 3Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.4Department of Biological Sciences, Sunway School of Science and Technology, Sunway University, Jalan Universiti, Bandar Sunway, 47500 Selangor, Malaysia. 5Virology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
This poster was presented at ASEAN Emerging Researchers Conference 2018
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Poster Information
Abstract: Background and Objectives: Newcastle disease virus (NDV) is an avian virus that selectively replicates and kills many different types of cancer cells and is being developed for cancer treatment. Our aim was to establish persistent infection in TCCSUP bladder cancer cells and characterise it. Methods: Persistently infected (PI) cells were developed by infecting TCCSUP bladder cancer cells with NDV at MOI of 0.1 in a T25 flask. After an hour of incubation at 37C for viral absorption, cells were washed with 1X PBS and washed again twice with MEM alpha to inactivate unabsorbed viruses. Cells were replenished with maintenance medium (MM) constituted with 2% FBS and incubated for a period of 5 days. Majority of the cells were detached after these post infection days with many signs of an acute lytic crisis leaving few surviving cells. Cells were washed twice with 1X PBS and complete growth medium were added and allowed to growth until subconfluent monolayer is formed. These subset of cells were re-infected three more times and characterized by microscopic; TEM and flow cytometric analyses, RT-PCR, and ELISA. Results: Cytopathic effect (CPE) were observed with progressive acute lysis crisis when TCCSUP cells were infected with NDV at low MOI. Small percentage of cells survived the viral infection at day 5 and were termed PI cells. A visual difference between uninfected TCCSUP and TCCSUPPi monolayer at 24 hrs was observed. TCCSUPPi did not show characteristic elongation and spreading observed in the mock infected TCCSUP. PCR indicated the presence of viral genome in the PI cells. Flow cytometric analysis demonstrated that 85% of the PI cells at different passages expressed GFP at 24 hrs when infected with rNDV-GFP. NDV particles were observed in some endosomes of the TCCSUPPi after TEM analysis. PI cells developed an anti-viral state by producing low level of IFN beta and this was reduced by addition of anti-IFN beta antibody to PI cells making the cells more sensitive to NDV re-infection. Conclusion: This study presents the successful development of NDV persistency of infection in TCCSUP bladder cancer cells in vitro. The infection of TCCSUP with NDV produced a lytic crisis in which majority of the cells were killed by day 5. Only small numbers of cells survived the infection. RT-PCR and TEM analyses revealed that TCCSUP cells are persistently infected with NDV AF2240. Whole transcriptome (RNA-seq) experiments are underway to elucidate the mechanism involved in development of persistence in TCCSUP cells and identify possible molecular network associated with persistence infection. Finding from this study will help in facilitating the use of NDV in treatment of bladder cancer in the clinic. Summary: Newcastle disease virus (NDV) is a negative-stranded RNA virus known as avian paramyxovirus type-1 (APMV1) that is being develop for cancer treatment. NDV has been known to develop viral persistency since early 1960s. However, the studies couldn’t explain the molecular mechanism associated with persistent infection.References: Alexander, D. J., Manvell, R. J., Lowings, J. P. et al., (1997). Avian Pathology: Journal of the W.V.P.A 26, 399–418.
Ahmad, U., Ahmed, I., KeongShafee, Y. Y., Abd Manan, N., & Othman, F. (2015).  BioMed research international, 2015.
Rangaswamy, U. S., Wang, W., Cheng, X., et al., (2017). Journal of Virology, 91(16), e00770-17.
Stojdl, D. F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N., & Bell, J. C. (2000). Nature medicine, 6(7), 821.
Chia, S. L., Yusoff, K., &, N. (2014).  Virology journal, 11(1), 91.
Chin L. C (2018). Master thesis. Universiti Putra Malaysia.
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