Infectious Haematopoietic Necrosis Virus-Induced Cell Death in Fish Cell Culture

Abstract

Cell death development was investigated in the epithelioma papulosum cyprini (EPC) cell line after infection with infectious haematopoietic necrosis virus (IHNV). Cytopathic effect (CPE) was evaluated, immunohistochemistry for virus localisation and the development of cell death were studied. Modes of cell death were characterised by staining with TdT-mediated dUTP nick end labelling (TUNEL), and with Annexin V and propidium iodide.CPE was evaluated as 25%, 50%, 75% and 100% at the virus culture infective concentration doses (TCID50/0.1 ml) of 1. 25, 6.25, 31.25 and 156.25, respectively, and no CPE was found at the concentration lower than 0.25 TCID50/0.1ml. At the PBS:virus concentration 0.25 TCID50/0.1 ml only a few cells were found anti IHNV antibody positive, and no CPE was observed. In the range of virus concentrations from 6.25 to 156.25 TCID50/0.1 ml all cells were positive. The level of positive cells using the TUNEL technique increased 30% and 55% after the increasing the CPE up to 50% and 75%, respectively. At the higher levels of CPE, all the remaining cells were positive.Infected cells exhibited features of early stage of apoptosis as demonstrated reactivity with annexin V. Apoptotic cells expose external phosphatidylserine residues while preserving membrane integrity. Red fluorescence with propidium iodide is associated with increased levels of necrosis and appeared in cells simultaneously with FITC marker and also alone in vacuolated and enlarged necrotic cells. Apoptosis appears before the pathological changes of necrosis and it is correlated to the spread of infection. The relevance of the current view that IHNV infection includes at least two modes of cell death: apoptosis and necrosis, is discussed. Epithelioma papulosum cyprini, cell death, necrosis, immunohistochemistry, annexin V, TUNELCellular death can occur either by accident, referred to as necrosis, or by design variously described as physiological cell death, programmed cell death or apoptosis (Bowen et al. 1996). Apoptosis presents a range of morphological symptoms including cell shrinkage, chromatin margination followed by DNA fragmentation and a florid break-up of the cell into spherical apoptotic bodies (Wyllie 1981). It has been shown that apoptosis can be induced by genetic means (Ellis et al. 1991), but can also be induced by non-genetic means (Arends and Wyllie 1991). Necrotic cell death appears to be induced under extreme conditions such as ischaemia, hypoxia, toxin, hyperthermia. The subsequent morphological changes encompass oedema following calcium overload, dilatation of the endoplasmic reticulum, polysome disaggregation, and mitochondrial swelling. The chromatin condenses, and clumps are formed at the nuclear periphery (Bowen et al. 1996). Necrosis refers to the post mortem changes that occur following the death of the cell (Trump and Berezesky 1998).A range of approaches are available to demonstrate the apoptosis of cells in living tissue or cell suspensions. Immunocytochemical methods assaying DNA fragmentation (Trauth ...

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb

“…Infected RTgill-W1 monolayers in 6-well plates were evaluated daily using an inverted light microscope and compared with non-infected monolayers (L-15 medium only); CPE was categorized as described (Gregorc et al 2005) (Fig. 1).…”