Anatid herpesvirus 1 CH virulent strain induces syncytium and apoptosis in duck embryo fibroblast cultures
Introduction
Anatid herpesvirus 1 (AHV-1) is currently grouped in the family Herpesviridae (Kaleta, 1990) and AHV-1 infection (Attanasio et al., 1980) variously known as duck plague (DP) or duck virus enteritis (DVE) is one of the most widespread and devastating diseases of waterfowls in the family Anatidae and has severely affected the waterfowl industry because relatively high mortality could be observed and a wide host range including domestic and wild ducks, geese, swans and other birds mainly in the family Anatidae, order Anseriformes throughout the world are susceptible (Sandhu and Shawky, 2003).
A cell normally contains only one nucleus. However, a multinucleate cell (syncytium) formation by the joining together of two or more cells has been previously reported in certain types of infections caused by viruses, which include paramyxovirus (Shortridge et al., 1980, Horvath et al., 1992), retrovirus (Hildreth and Orentas, 1989), and herpesvirus (Nazerian and Purchase, 1970, Hamdy et al., 1974, Turner et al., 1998) and syncytia are one of the major cytopathic effects induced by these viruses. In this study, we investigated whether the AHV-1 can induce syncytium in infected DEF to provide more information on its pathogenesis study.
Apoptosis also named programmed cell death (PCD) is a physiologically important process that eliminates redundant, damaged, or infected cells and is defined by typical changes in cellular morphology and biochemical features, including DNA fragmentation, chromatin condensation, and cellular breakdown into apoptotic bodies (Kerr et al., 1972). Apoptosis as an energy-dependent process of cell suicide without provoking an inflammatory response (Vaux and Strasser, 1996) can be triggered by diverse intracellular and extracellular signals including virus infections. An increasing number of viruses or viral gene products have been reported to induce apoptosis (Groux et al., 1992, Ito et al., 1997, Ruggieri et al., 2007, Sanfilippo et al., 2004, Lovato et al., 2003, Sadzot-Delvaux et al., 1995, Aleman et al., 2001, Yuan et al., 2007) both in vitro and in vivo, which includes herpesvirus and is one of the cytolytic properties of viral infections and the phenotype of CPE in vitro.
During the recent years, AHV-1 gene related studies (Jia et al., 2009, Chang et al., 2009, Zhao et al., 2008) have been frequently noticed. However, study on AHV-1 induced apoptosis was not often observed. Since apoptosis plays an important role (Yuan et al., 2007) in the pathogenesis of AHV-1 infection in vivo, in this paper proper modifications were made based on the preliminary findings (Guo et al., 2007) and further steps were taken as well to investigate the AHV-1 induced apoptosis in vitro, which we believe will help clarify the pathological characteristics of AHV-1.
Section snippets
Cell, virus and infection of DEF with AHV-1
Trypsinization of 9–11-day-old duck embryonated eggs was performed according to standard practice. The dispersed cells were dispensed into cell culture flasks and flat-bottomed six-well tissue culture plastic plates (Corning, Inc., New York, USA) with flying cover slips. Growth medium was Eagle's minimal essential medium that contained 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Maintenance medium had 3% fetal bovine serum added. Cells were maintained at 37 °C with 5%
Inverted microscopy observation
The AHV-1 infected and mock-infected cell cultures were examined regularly under inverted microscope for CPE observation and it was found that the AHV-1 infected DEF gradually developed the CPE including cell rounding and detachment at and after 24 h after inoculation. This consisted of scattered foci of plaque surrounded by rounded and refractile cells and the plaques were formed by cell detachment from the culture surface. On the cell monolayer, syncytia (Fig. 1) were observed as the other
Discussion
Syncytia as one of the main cytopathic effects, can be formed when cells are infected with certain types of viruses including retrovirus, herpesvirus, paramyxoviruses and coronavirus. Infection of chick kidney cell monolayers with avian paramyxoviruses could lead to CPE consisting of small discrete syncytia (Shortridge et al., 1980) containing 2–6 nuclei which were formed by cell fusion and were visible from about 24 h onwards. In duckling kidney cell cultures infected by Muscovy duck reovirus,
Acknowledgements
We gratefully acknowledge the support of the National Natural Science Foundation of China (grant no. 30771598), the Cultivation Fund of the Key Scientific and Technical Innovation Project, department of Education of Sichuan Province (grant no. 07ZZ028), China Postdoctoral Science Foundation (grant no. 20060391027), New Century Excellent Talents program in University (grant no. NCET-06-0818), Scientific and Technological Innovation Major Project Funds in University (grant no. 706050), Program
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These authors contributed equally to this work.