Viral persistence in colorectal cancer cells infected by Newcastle disease virus

Abstract

Background: Newcastle disease virus (NDV), a single-stranded RNA virus of the family Paramyxoviridae, is a candidate virotherapy agent in cancer treatment. Promising responses were observed in clinical studies. Despite its high potential, the possibility of the virus to develop a persistent form of infection in cancer cells has not been investigated. Occurrence of persistent infection by NDV in cancer cells may cause the cells to be less susceptible to the virus killing. This would give rise to a population of cancer cells that remains viable and resistant to treatment.

Results: During infection experiment in a series of colorectal cancer cell lines, we adventitiously observed a development of persistent infection by NDV in SW480 cells, but not in other cell lines tested. This cell population, designated as SW480P, showed resistancy towards NDV killing in a re-infection experiment. The SW480P cells retained NDV genome and produced virus progeny with reduced plaque forming ability.

Conclusion: These observations showed that NDV could develop persistent infection in cancer cells and this factor needs to be taken into consideration when using NDV in clinical settings.

Figure 1

Viability and morphology of CRC cancer cell lines following NDV infection. (A) Viability of CRC cancer cell lines following selected times intervals post-infection with NDV strain AF2240. At 96 hours post-infection, only less than 40% cells remained viable across all the cell lines. (B) The remaining cells in the infected SW480 continued to proliferate following media replacement. These cells (SW480P) retained similar morphological features to the parental SW480 population.

Figure 2

Susceptibility of SW480P cells to NDV-induced cytolysis. (A) Viability of re-infected SW480P cells compared to the parental SW480. The cells were re-infected with the similar MOI as the initial SW480 infection. (B) The infected SW480P cells did not show any obvious cytopathic effects (CPE) after 72 hpi while the parental SW480 displayed the typical pattern of CPE due to NDV infection.

Figure 3

Detection of NDV genes and proteins in the SW480P cells. (A) RT-PCR analysis on DNA samples of SW480 and SW480P cells infected and mock infected with NDV. A DNA band with a size of 1.7 kb appeared in selected samples. (B) Immunofluorescent analyses of the infected and control cells using a monoclonal antibody against the NP protein of NDV. A speckled pattern of green antigen staining was visible in the cytoplasm of cells.

Figure 4

Detection of infectious viral progenies secreted into cell culture media. (A) A plaque assay was performed using spent culture media of the mock-infected SW480P and control cells. Different plaque morphologies were visible in the different samples. (B) A graphical representation of the number of plaques of less than 1 mm or in the range of 1-4 mm is shown.

Figure 5

Infectivity of mNDV on HT29 cells. mNDV was infectious towards HT29 cells, however it resulted in less killing effect. The cells were infected using spent culture media from the mock-infected SW480P cells as described in the Materials and methods section.