Dynamics of Nontypical Apoptotic Morphological Changes Visualized by Green Fluorescent Protein in Living Cells with Infectious Pancreatic Necrosis Virus Infection

Abstract

Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 μm according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.

Liver involvement in post‐smolt Atlantic salmon, Salmo salar L., infected with infectious pancreatic necrosis virus (IPNV): a retrospective histopathological study

“…Severely affected livers in the current study showed apoptosis could be combined with a spreading necrosis and loss of structure throughout large areas of the tissue. The induction of apoptosis by IPNV has been investigated in vitro in fish cell lines (Hong, Lin, Hsu & Wu 1998a,b; Hong, Hsu & Wu 1999b; Hong, Lin, Yang, Hsu & Wu 1999a; Hong & Wu 2002; Chen et al. 2009), in vivo through experimental studies in salmon by Santi et al.…”

Aquatic birnavirus induces apoptosis through activated caspase‐8 and ‐3 in a zebrafish cell line

“…Infection of CHSE-214 with IPNV isolate E1-S upregulates the host survival factor Mcl-1, which may enhance viral progeny production and may block caspase activity, but details are still lacking. This phenomenon may explain how convergent viral evolution ablates the host response by targeting strategic points in specific defence pathways and thus inhibits or delays protective mechanisms until sufficient viral yields have been produced (Hong et al , 1999a. Why VP5 has lost its protein function and how apoptosis responses are regulated in the host cell in the early virus replication cycle needs further investigation.…”

Knockdown of zebrafish YY1a can downregulate the phosphatidylserine (PS) receptor expression, leading to induce the abnormal brain and heart development

“…Embryos at the one- or two-cell stage were injected with YY1-MO (25 ng) or control-MO (25 ng) or YY1-MO (25 ng) plus PSR mRNA (1 ng), harvested at 18.5 hpf, fixed with 4 % paraformaldehyde in PBS (pH 7.4) at room temperature for 30 min, stained with acridine orange (1 μg.ml −1 ; 3–5 min), washed twice in PBS, and evaluated under a fluorescence microscope (using a 488-nm filter for excitation and a 515-nm long-pass filter for detection) [ 44 ]. For the TdT-dUTP labeling step, the embryos were fixed in paraformaldehyde at the end of the incubation period (12 and 24 hpf), dechorionated, incubated in blocking solution (0.1 % H 2 O 2 in methanol, 30 min, room temperature), rinsed with PBS, incubated on ice in 0.1 % Triton X-100 in 0.1 % sodium citrate for 30 min to increase permeability, rinsed twice with PBS, treated with TUNEL reaction mixture (50 μl; in-situ cell death-detection Kit, Boehringer Mannheim), incubated in a humidified chamber for 60 min at 37 °C, and evaluated for fluorescence-positive apoptotic cells under a fluorescence microscope equipped with a spot II CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).…”

“…Standard western-blot analysis was conducted using human anti-YY1 Mab (Cascade Bioscience, Winchester, MA), anti-zebrafish PSR N-terminus polyAb (self-made), and mouse anti-actin Mab (Chemicon, Temecula, CA). PSR was visualized using horseradish peroxidase-conjugated anti-mouse immunoglobulin (IgG) and the ECL detection kit (Amersham Pharmacia Biotech, Denmark) [ 44 ].…”