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. 2008 Mar;132(1-2):76-85.
doi: 10.1016/j.virusres.2007.10.017. Epub 2007 Dec 3.

Cytoplasmic vacuolization responses to cytopathic bovine viral diarrhoea virus

Alexander V Birk  1 , Edward J Dubovi, Leona Cohen-Gould, Ruben Donis, Hazel H Szeto
Affiliations

Cytoplasmic vacuolization responses to cytopathic bovine viral diarrhoea virus

Alexander V Birk et al. Virus Res. 2008 Mar.

Abstract

Bovine Viral Diarrhea Virus (BVDV) is a positive sense, single-stranded RNA virus which exhibits two biotypes in standard cell culture systems. The cytopathic strains of this virus (cpBVDV) induce dramatic cytoplasmic vacuolization in cell cultures, while infection with the non-cytopathic (NCP-BVDV) strains produces no overt changes in the host cells. Our results show that extensive cytoplasmic vacuolization is the earliest morphological change in response to cpBVDV infection in MDBK cells. Cells with extensive vacuolization showed no co-existing chromatin condensation, caspase activation, or loss of membrane integrity. In addition, the caspase inhibitor (zVAD-fmk), although improving cell viability of infected cells from 6.7+/-2.2% to 18.8+/-2.2%, did not prevent vacuolization. On the ultrastructural level, the virus-induced cytoplasmic vacuoles are single membrane structures containing organelles and cellular debris, which appear capable of fusing with other vacuoles and engulfing surrounding cytoplasmic materials. LysoTracker Red which marks lysosomes did not stain the virus-induced cytoplasmic vacuoles. In addition, this lysosomal dye could be observed in the cytoplasm of vacuolized cells, suggesting a lysosomal abnormality. Our data demonstrate that cpBVDV induced a novel cell death pathway in MDBK cells that is primarily associated with lysosomal dysfunction and the formation of phagocytic cytoplasmic vacuoles, and this mode of cell death is different from apoptosis and necrosis.

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Figures

Fig. 1
Fig. 1
cpBVDV infection induces cytoplasmic vacuolization in MDBK cells in a time-dependent manner. (A) Microscopic analysis of a representative control, cpBVDV, and ncpBVDV-infected MDBK cells at 24 h post-infection. Arrows indicate representative vacuolized cells in slides 24 h post-infection. (B) Vacuolized cells in mock-infected (▴) and cpBVDV-infected (*) samples were analyzed and counted as described in Section 2. Error bars indicate standard deviation for each time point and specified condition (n = 3).
Fig. 2
Fig. 2
Cytoplasmic vacuolization in MDBK cells expressing NS3 viral protein. Microscopic analysis of a representative control and cpBVDV-infected MDBK cells at 24 h post-infection, stained for NS3 viral protein. Arrows indicate representative vacuolized cells in slides 24 h post-infection.
Fig. 3
Fig. 3
cpBVDV-induced cytoplasmic vacuolization is not associated with loss of plasma membrane integrity. (A) Infected cells (24 h post-infection) were stained with propidium iodide (PI) and analyzed using fluorescent microscopy. Vacuolated areas (arrows) show no sign of PI localization in nuclei. (B) Infected cells (24 h post-infection) were treated with 0.1 mM DEPC for 10 min and stained with propidium iodide and analyzed using fluorescent microscopy, as described in Section 2. Arrows indicated vacuolated cells.
Fig. 4
Fig. 4
Cytoplasmic vacuolization and apoptotic responses in cpBVDV-infected MDBK cells. Control and cpBVDV-infected cells were stained with Hoechst and FLICA dyes, for chromatin condensation and caspases activation, respectively, at 24 h post-infection. Vacuolized and dye-stained cells, were then analyzed and counted. Error bars indicate standard deviation for each specified analysis (n = 3).
Fig. 5
Fig. 5
Inhibition of caspase activation in infected cells partially prevents cpBVDV-induced cell death but does not block cytoplasmic vacuolization. (A) Light microscopy of infected MDBK cell (16 h post-infection) demonstrates that cpBVDV-induced cytoplasmic vacuolization is not affected by treatment with 100 μM zVAD-fmk. (B) Effect of zVAD-fmk (100 μM) on cell viability in cpBVDV infected cells.
Fig. 6
Fig. 6
Cytoplasmic vacuolization in cpBVDV-infected cells is not associated with apoptotic markers. (A) Microscopic analysis of Hoechst dye staining in cpBVDV-infected MDBK cells 48 h post-infection demonstrates uniformly low intensity stained nuclei in vacuolized cells and brightly stained nuclei in detached cells. Left panel correspond to light microscopy of representative cells and right represents fluorescent microscopy of the same cells. (B) Electron microscopy of infected MDBK cell (16 h post-infection) demonstrates the absence of chromatin condensation in nuclei of highly vacuolized cells. (C) Microscopic analysis of FLICA dye staining in cpBVDV-infected MDBK cells 24 h post-infection. Light microscopic and fluorescent microscopic images for FLICA staining were superimposed demonstrating no staining of vacuolized cells (dashed arrows) and brightly stained apoptotic cells (solid arrows, filled arrow heads).
Fig. 7
Fig. 7
(A) Electron micrographic analysis of representative control, ncpBVDV and cpBVDV-infected MDBK cell 24 h post-infection. Large vacuoles (V), encapsulated by single membranes and containing cellular debris, are found only in the cytoplasm of infected cells. Mitochondria and nuclei appear to be normal in infected cells. (B) Ultrastructural analysis of representative cytoplasmic vacuoles found in infected cells. Large encapsulated vacuoles were found to fuse with each other, forming larger vacuoles (left panel). Some of cytoplasmic autophagic vacuoles were found to be in a process of engulfing cytoplasmic material (as indicated by arrows) (right panel).
Fig. 8
Fig. 8
Cytoplasmic vacuolization induced by cpBVDV is insensitive to treatment with wortmannin. (A) Light microscopy of infected MDBK cell (16 h post-infection) demonstrates that cpBVDV-induced cytoplasmic vacuolization is not affected by treatment with 10 μM wortmannin. (B) Wortmannin (0.1–10 μM) had no effect on decrease in cell viability mediated by cpBVDV infection.
Fig. 9
Fig. 9
Microscopic analysis of LysoTracker Red fluorescence in cpBVDV-infected MDBK cells, demonstrating bright fluorescence in morphologically normal cells and depletion of fluorescence in vacuolized cells (arrow).
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