Virus Research 185 (2014) 77–81
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Grass carp reovirus induces apoptosis and oxidative stress in grass carp (Ctenopharyngodon idellus) kidney cell line Rui Jia a,b,1 , Li-Ping Cao b,c,1 , Jin-Liang Du b,c , Ying-Juan Liu a , Jia-Hao Wang a , Galina Jeney d , Guo-Jun Yin a,b,c,∗ a
Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China c International Joint Research Laboratory for Fish Immunopharmacology, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China d Research Institute for Fisheries, Aquaculture and Irrigation, Anna Light 8, Szarvas 4440, Hungary b
a r t i c l e
i n f o
Article history: Received 6 January 2014 Received in revised form 17 March 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: Grass carp reovirus Oxidative stress Apoptosis Caspase
a b s t r a c t Grass carp hemorrhage is an acute contagious disease caused by grass carp reovirus (GCRV). The pathogenesis of GCRV and the relationship between GCRV and the host cells remain unclear. The aim of the present study was to investigate the relations among apoptosis, intracellular oxidative stress and virus replication in GCRV infected-cells. The results showed that GCRV induced activation of caspase proteases as early as 12 h, and reached maximum activities at 24 h or 48 h post-infection in a grass carp kidney cell line (CIK cells). Meanwhile, the levels of tumor necrosis factor (TNF-␣) and interleukin-1 (IL-1) also were increased in GCRV-infected CIK cells and showed a statistically significant difference from 24 h to 96 h post-infection. The infection of GCRV caused the destruction of entire monolayer and the death of host cells. Accompanied by the infection, a severe oxidative stress occurred, which led to extensive loss of antioxidants and formation of lipid peroxidation after 48 h post-infection. These data suggested that the apoptosis which was triggered at an early stage (12–24 h) in the viral infection cycle, might be independent of virus replication, while the oxidative stress induced by GCRV was mostly related to the virus replication. © 2014 Elsevier B.V. All rights reserved.
Grass carp (Ctenopharyngodon idella), a member of cyprinid, is one of the most important freshwater aquaculture species in Asia, especially in China. Recently, the increased incidence of various kinds of fish diseases has become one of the major obstacles for the further development of grass carp cultivation industry (Zhong et al., 2002). It is well known that hemorrhagic disease, caused by grass carp reovirus (GCRV), has been frequently reported and resulted in heavy losses to the aquaculture industry (Jun et al., 1997; Zhang et al., 2007). GCRV, a dsRNA virus, belongs to Group C of aquareovirus (AQRV) and is the most virulent strain in AQRVs (Yang et al., 2011). The virion consists of a double-layered capsid and a genome composed of 11 segments of double-stranded RNA,
∗ Corresponding author at: Wuxi Fisheries College, Nanjing Agricultural University, 9 Eastern Shanshui Road, Wuxi 214081, China. Tel.: +86 510 85551442; fax: +86 510 85551442. E-mail address: [email protected] (G.-J. Yin). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.virusres.2014.03.021 0168-1702/© 2014 Elsevier B.V. All rights reserved.
encoding 7 structural proteins (VP1–VP7) and 5 nonstructural proteins (NS16, 26, 31, 38 and 80) (Qian and Liqun, 2011). In 1984, the virus was first discovered in China (Chen and Jiang, 1984), so far, there have been a large amount of reports which mainly focused on virus structure, infection, rapid detection and vaccine development (Lu et al., 2011; Qiu et al., 2001; Zhang et al., 2010), and more than 10 strains of GCRV have been isolated (Tian et al., 2013). In vitro, the GCRV and grass carp kidney cell line (CIK cells) constantly served as infection model to study infection mechanism, fish antiviral innate immune system screening antiviral drugs and developing vaccine (Peng et al., 2012; Wang et al., 2013). Apoptosis is a highly genetically controlled type of cell death which is accompanied by the activation of a spate of intracellular proteases and endonucleases (Chawla-Sarkar et al., 2003). It plays a critical role in maintaining the homeostasis of multicellular organisms by specifically removing damaged, spent, or misplaced cells (Oyadomari et al., 2002). Caspases are cysteine proteases that play fundamental roles in the apoptotic responses of cells to different stimuli, and are divided into initiators (caspases-8, -9, -10 and
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Time (h) Fig. 1. Cell survival in CIK cells infected by GCRV and mock. GCRV, GCRV-104-infecte cell; Mock, mock-infected cell. The results are expressed as the percentage of the control cells (0 h). The data represent means ± SD (n = 4) (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the data for mock infected-cells).
-12) and effectors (caspases-1, -3, -6 and -7) based on their place in the caspase cascade (O’Brien, 1998). Evidences for apoptotic cell death have been described in many viruses, independent of virus replication (Danthi et al., 2013; Gadaleta et al., 2002). The precise mechanism of virus-induced apoptosis is not yet fully understood. Various viruses have been shown to cause cells death by induction of apoptosis. Cells commit suicide as a direct response to viral infection prior to viral maturation, limiting the spread of progeny virus (Clarke et al., 2000). However, apoptosis of virus-infected cells can also increase viral dissemination while evading immune recognition, which benefit the virus (Chattopadhyay et al., 2010; DeWitte-Orr and Bols, 2007; Oberhaus et al., 1997). By contrast, some viruses encode genes that suppress apoptosis, including adenovirus, herpes simplex virus and Epstein–Barr virus (Ito et al., 2002). The purpose of the present study was to investigate the impact of GCRV infection on apoptosis and oxidative stress in grass kidney cell line (CIK cells), and the possible relations among apoptosis, intracellular oxidative stress and virus infection. CIK cells were maintained in L-15 medium (Sigma, USA) supplemented with 5% fetal bovine serum (FBS) (Hangzhou, China), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, USA). The cell monolayers were either mock-infected or infected with GCRV-104 (CCTCC NO: V201217, kindly provided by Dr. Fan Yuding, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, China) at a multiplicity of infection (MOI) of 1PFU/cell (TCID50 = 10−4.71 ), and analyzed over a time course 96 h post-infection. The cytotoxicity induced by GCRV was examined by WST-1 assay (Hinshaw et al., 1994; Jia et al., 2012). As shown in Fig. 1, CIK cells showed little loss in viability at 12 and 24 h post-infection. By 48 h infection, the viability of GCRV-infected cells showed a marked decrease compared to mock-infected cells. At 72 and 96 h post-infection, 35.47% and 58.73% of the GCRV-infected CIK cells were dead, respectively. It indicated that the death of infected cells mainly occurred during the later stage of infection (72–96 h) due to virus replication, and the results accorded with provious studies (Zou and Fang, 2000). For viral replication assay, the cells and supernatants infected with GCRV were collected, freeze-thawed twice, centrifuged and diluted from 10−1 to 10−9 in L-15 for TCID50 /ml determination, and viral titers (TCID50/ml) were calculated by the method described by Reed–Muench (Reed and Muench, 1938). As shown in Fig. 2, 72 h after infection the titers of GCRV were sharply increased, which suggested the progeny virions were producted in the later stage of infection (72–96 h) and sufficient viral progeny production led to cells lysis and released into culture medium.
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Time (h) Fig. 2. The titers of grass carp reovirus (GCRV) in grass carp kidney (CIK) cells in different time of infection. The data represent means ± SD (n = 4).
For apoptosis assay, activities of caspase-3, caspase-8 and caspase-9 were measured using commercialized activity kit (Beyotime Institute of Biotechnology, China) (Chen et al., 2008). In brief, CIK cells were infected with GCRV-104 for 0, 12, 24, 48, 72 and 96 h, and then, were collected and rinsed twice with PBS, the lysates were centrifuged at 20,000 × g for 10 min at 4 ◦ C, and supernatants were incubated for 2 h at 37 ◦ C with 10 l Ac-DEVD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA as the substrates for caspase-3, caspase-8, and caspase-9, respectively. The absorbance at 405 nm was read using a microplate reader (Bio-Rad, USA). Meanwhile, tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1) levels assay were quantified with a commercial fish ELISA kit (BD Biosciences, China) (Hong-quan et al., 2013) according to the manufacturer’s instructions. Briefly, the plate coated with diluted capture antibody (derived from common carp) were incubated over night at 4 ◦ C, and its specificity was determined by western blotting. The plate was blocked with 0.5% BSA for 1 h, then washed four times, added diluted standard TNF-␣, IL-1 and the samples (supernatant of infected cells), incubated for 1 h, washed four times and added diluted detection antibody. After incubation for 1 h, the plate was washed and added alkaline phosphatase. After 1 h, the plate was washed and added p-nitrophenyl phosphate (p-NPP) substrate solution. Finally, the absorbance at 450 nm was measured using micro plate reader. Although apoptosis can be triggered by several different stimuli, apoptotic pathways were mainly classified into two groups: the intrinsic pathway and the extrinsic pathway (Kumar, 2006). The common event in the end point of both the intrinsic and extrinsic is the activation of a set of cysteine proteases (caspases) (Liu et al., 2005). The extrinsic pathway originates at the plasma membrane following the engagement of a family of cytokine receptors, such as tumor necrosis factor receptor-1 (TNF-R1) by their cognate ligands (TNF-␣). Ligand/receptor binding induces the recruitment of several adapter proteins and proenzymes, and then activates caspases (caspase-8, and -10), finally, results in apoptosis and cell death (Guicciardi and Gores, 2005). The intrinsic pathway is triggered by different extracellular or intracellular signals, such as oxidative stress, results in activation of the initiator caspase-9. Caspase-9, in turn, activates caspase-3, major effector caspase, responsible for degradation of cellular substrates (Allen et al., 1997). It is reported that some virus-infected cells can synthesize and release cytokines, such as TNF-␣ and IL-1 (Wang et al., 2011). In this study, both of the cytokines displayed changes in different degrees at each time point (Fig. 3). The level of IL- was significantly elevated at 48 h of GCRV infection and reached its maximum value at 96 h (Fig. 3A). Similarly, the level of TNF-␣ was markedly up-regulated by GCRV infection at 12, 24, and 48 h, and showed a uptrend (Fig. 3B). TNF␣, as a key mediator of apoptosis (Lokensgard et al., 2001), recruits
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Time (h) Fig. 3. The levels of IL-1 and TNF-␣ in CIK cells infected by GCRV and mock. The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) (twoway ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 compared with the values for mock infected-cells).
and activates caspases-8 within a death-inducing signaling complex (Chen and Goeddel, 2002). Just as shown in Fig. 4, the activities of caspase-8 and caspase-9 were increased in GCRV-infected cells at and after 12 h, and maximal increase were observed at 48 h and 24 h post-infection, respectively, when compared with those of mock-infected cells. Caspase-8 and -9, major initiator caspases, are recruited to death receptors and activated by their own intrinsic autocatalytic activity, and then initiate a caspase cascade resulting in the activation of the effector caspases (Clarke and Tyler, 2003; Grossmann, 2002). These effector caspases are responsible for the characteristic morphological changes of apoptosis. It is well known that apoptosis is an important mechanism of virus induced cell death during reovirus infection in cell culture and in vivo (Danthi et al., 2013). In aquatic animals, chum salmon reovirus, a different aquareovirus, has been shown to induce cell death via caspasedependent apoptosis in salmon cell lines (DeWitte-Orr and Bols, 2007). In the present work, significant differences of caspase-3 proteases activity was also seen at 12 h and peak effect was observed at 24 h in GCRV-infected CIK cells. It is a major effector caspase in both the extrinsic and intrinsic apoptotic pathways. Based on the results, it is concluded that GCRV infection in CIK cells induced apoptosis through activation of initiator caspases (caspase-8 and -9) and the effector caspase-3 at early phase of infection. Therefore, the apoptosis was dependent on caspases activation via both the intrinsic and extrinsic pathway (Kominsky et al., 2002). It is suggested that the caspases activation were independent of virus replication (Danthi et al., 2013). Moreover, the activities of three caspases were decreased in GCRV infection after 72 h possible due to massive death of CIK cells. For oxidative stress assay, the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidant capacity (T-AOC) were determined as previously described by Peskin and Winterbourn (2000), Brigelius-Flohé (1999), Cakmak and Horst (1991) and Benzie and Strain (1996),
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Time (h) Fig. 4. The levels of caspase-3, -8 and -9 in CIK cells infected by GCRV and mock. The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) GCRV, (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the values for mock infected-cells).
respectively, and the results were expressed as units per milligram protein. Protein content of GCRV infected CIK cells was determined by the Bradford method, using bovine serum albumin as a standard (Bradford, 1976). Virus-induced pathogenesis is associated not only with apoptosis but with oxidative damage (Valyi-Nagy and Dermody, 2005). Oxidative stress, primarily due to increased generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), is a feature of many viral infections (Li et al., 2010; Valyi-Nagy and Dermody, 2005). The ROS and RNS deplete intracellular antioxidant compounds and consequently lead to cells death (Edens et al., 2008). Clearly, oxidation status of host is important for infection of virus. Just as showed here that the levels of antioxidant compounds (T-AOC, SOD, CAT, GPx) in CIK cells were significantly decreased at least 72 h following GCRV infection (Fig. 5). As for role of oxidative stress in the pathogenicity of virus, Lin et al. (2011)
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Fig. 6. Lipid peroxidation and total protein (TP) content of GCRV infected CIK cells, The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 compared with the data for mock infected-cells).
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Time (h) Fig. 5. Antioxidative state in CIK cells infected by GCRV and mock. (A) SOD; (B) CAT; (C) GPx; (D) T-AOC. The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 compared with the data for mock infected-cells).
demonstrated previously that avian reovirus increased the levels of reactive oxygen species (ROS). The ROS depleted antioxidant compounds, resulting in failure of antioxidant system (Griendling and FitzGerald, 2003). This is similar to what we presented here that GCRV-104 infection considerably induced the oxidative injury in CIK cells after 48 h post-infection. It also indicated that oxidative stress might be one of the main factors of cells death in late stage of infection (72–96 h). Generally, lipid peroxidation is a well-established mechanism of cellular injury. It was reflected by levels of MDA, a decomposition product of lipid hydroperoxides (Radi et al., 1991). It has been shown that some viruses, such as avian reovirus, murine reovirus promoted cell damage by initiating lipid peroxidation (Lin et al., 2011; Valyi-Nagy et al., 1999). In this study, MDA levels in the GCRV-infected cells were significantly elevated from 24 to 72 h post-infection, indicating increased lipid peroxidation and oxidative stress in the CIK cells (Fig. 5A). The decrease of protein content was also observed (P < 0.05), but it appeared that protein content in the early phase after infection (0–48 h) has not changed (Fig. 6B). In summary, the present study demonstrated, for the first time, GCRV-104 infection induced apoptosis and intracellular oxidation damage in CIK cells, both of which were important factors in virus pathogenesis. Through these data, it is suggested that the trigger of apoptotic cell death might be associated with the TNF-␣ release. During the early phase of infection (0–24 h), GCRV-104 infection induced TNF-␣ release. The TNF-␣ bound its receptor (TNF-R1), activated the initiator caspase-8, and then stimulated downstream caspases such as caspase-3, consequently, resulted in apoptosis and cell death. Simultaneously, another apoptotic pathway (intrinsic pathway) was triggered by GCRV-104 infection and required mitochondrial involvement to amplify the apoptotic signal from death receptors. The evidence obtained allows us to conclude that
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apoptosis is triggered at an early stage in the viral infection cycle. Moreover, the activation of oxidative stress mostly took place during the later phase of infection (72–96 h) and it may be principally related to virus replication. Based on the results, it is speculated that the pathological basis of the lesions resulting from GCRV infection is relevant to apoptosis at an early phase of infection, while oxidative stress and virus replication are responsible for it at a later phase of infection. Acknowledgments This work was supported by Central Public-interest Scientific Institution Basal Research Fund of China (2013JBFM11,12), Jiangsu Science and Technology Department (BK2012535) and National Natural Science Foundation of China (31,202,002, 31200918). References Allen, D.L., Linderman, J., Roy, R.R., Bigbee, A.J., Grindeland, R.E., Mukku, V., Edgerton, V., 1997. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am. J. Physiol. Cell Physiol. 273 (2), C579–C587. Benzie, I.F., Strain, J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239 (1), 70–76. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1), 248–254. Brigelius-Flohé, R., 1999. Tissue-specific functions of individual glutathione peroxidases. Free Radic. Biol. Med. 27 (9), 951–965. Cakmak, I., Horst, W.J., 1991. Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol. Plantarum 83 (3), 463–468. Chattopadhyay, S., Marques, J.T., Yamashita, M., Peters, K.L., Smith, K., Desai, A., Williams, B.R., Sen, G.C., 2010. Viral apoptosis is induced by IRF-3-mediated activation of Bax. EMBO J. 29 (10), 1762–1773. Chawla-Sarkar, M., Lindner, D., Liu, Y.-F., Williams, B., Sen, G., Silverman, R., Borden, E., 2003. Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis. Apoptosis 8 (3), 237–249. Chen, G., Goeddel, D.V., 2002. TNF-R1 signaling: a beautiful pathway. Science 296 (5573), 1634–1635. Chen, H., Xing, B., Liu, X., Zhan, B., Zhou, J., Zhu, H., Chen, Z., 2008. Ozone oxidative preconditioning inhibits inflammation and apoptosis in a rat model of renal ischemia/reperfusion injury. Eur. J. Pharmacol. 581 (3), 306–314. Chen, Y., Jiang, Y., 1984. Morphological and physico-chemical characterization of the hemorrhagic virus of grass carp. Kexue Tongbao 29 (6), 832–835. Clarke, P., Meintzer, S.M., Gibson, S., Widmann, C., Garrington, T.P., Johnson, G.L., Tyler, K.L., 2000. Reovirus-induced apoptosis is mediated by TRAIL. J. Virol. 74 (17), 8135–8139. Clarke, P., Tyler, K., 2003. Reovirus-induced apoptosis: a minireview. Apoptosis 8 (2), 141–150. Danthi, P., Holm, G., Stehle, T., Dermody, T., 2013. Reovirus Receptors, Cell Entry and Proapoptotic Signaling. In: Pöhlmann, S., Simmons, G. (Eds.), Viral Entry into Host Cells., Vol. 790. Springer, New York, pp. 42–71. DeWitte-Orr, S.J., Bols, N.C., 2007. Cytopathic effects of chum salmon reovirus to salmonid epithelial, fibroblast and macrophage cell lines. Virus Res. 126 (1), 159–171. Edens, F., Read-Snyder, J., Somody, R., Surai, P., Taylor-Pickard, J., 2008. Selenium modifies avian reovirus pathogenicity related to malabsorption syndrome. Curr. Adv. Selenium Res. Appl., 133. Gadaleta, P., Vacotto, M., Coulombié, F., 2002. Vesicular stomatitis virus induces apoptosis at early stages in the viral cycle and does not depend on virus replication. Virus Res. 86 (1), 87–92. Griendling, K.K., FitzGerald, G.A., 2003. Oxidative stress and cardiovascular injury part I: basic mechanisms and in vivo monitoring of ROS. Circulation 108 (16), 1912–1916. Grossmann, J., 2002. Molecular mechanisms of detachment-induced apoptosis – Anoikis. Apoptosis 7 (3), 247–260. Guicciardi, M., Gores, G., 2005. Apoptosis: a mechanism of acute and chronic liver injury. Gut 54 (7), 1024–1033. Hinshaw, V.S., Olsen, C.W., Dybdahl-Sissoko, N., Evans, D., 1994. Apoptosis: a mechanism of cell killing by influenza A and B viruses. J. Virol. 68 (6), 3667–3673. Hong-quan, W., Tang, D.-Y., Zha0, Y.-R., Wang, X.-N., Jin, B.-T., Zhang, K., Xiao, X.i.-Y., 2013. Effects of dietary Achyranthes bidentata po1ysaccharide (ABP) on immunity and antioxidant function of grass carp (Ctenopharyngodon idellus). Acta Hydrobiol. Sin. 37 (2), 351–357.
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Short communication
Grass carp reovirus induces apoptosis and oxidative stress in grass carp (Ctenopharyngodon idellus) kidney cell line Rui Jia a,b,1 , Li-Ping Cao b,c,1 , Jin-Liang Du b,c , Ying-Juan Liu a , Jia-Hao Wang a , Galina Jeney d , Guo-Jun Yin a,b,c,∗ a
Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China c International Joint Research Laboratory for Fish Immunopharmacology, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China d Research Institute for Fisheries, Aquaculture and Irrigation, Anna Light 8, Szarvas 4440, Hungary b
a r t i c l e
i n f o
Article history: Received 6 January 2014 Received in revised form 17 March 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: Grass carp reovirus Oxidative stress Apoptosis Caspase
a b s t r a c t Grass carp hemorrhage is an acute contagious disease caused by grass carp reovirus (GCRV). The pathogenesis of GCRV and the relationship between GCRV and the host cells remain unclear. The aim of the present study was to investigate the relations among apoptosis, intracellular oxidative stress and virus replication in GCRV infected-cells. The results showed that GCRV induced activation of caspase proteases as early as 12 h, and reached maximum activities at 24 h or 48 h post-infection in a grass carp kidney cell line (CIK cells). Meanwhile, the levels of tumor necrosis factor (TNF-␣) and interleukin-1 (IL-1) also were increased in GCRV-infected CIK cells and showed a statistically significant difference from 24 h to 96 h post-infection. The infection of GCRV caused the destruction of entire monolayer and the death of host cells. Accompanied by the infection, a severe oxidative stress occurred, which led to extensive loss of antioxidants and formation of lipid peroxidation after 48 h post-infection. These data suggested that the apoptosis which was triggered at an early stage (12–24 h) in the viral infection cycle, might be independent of virus replication, while the oxidative stress induced by GCRV was mostly related to the virus replication. © 2014 Elsevier B.V. All rights reserved.
Grass carp (Ctenopharyngodon idella), a member of cyprinid, is one of the most important freshwater aquaculture species in Asia, especially in China. Recently, the increased incidence of various kinds of fish diseases has become one of the major obstacles for the further development of grass carp cultivation industry (Zhong et al., 2002). It is well known that hemorrhagic disease, caused by grass carp reovirus (GCRV), has been frequently reported and resulted in heavy losses to the aquaculture industry (Jun et al., 1997; Zhang et al., 2007). GCRV, a dsRNA virus, belongs to Group C of aquareovirus (AQRV) and is the most virulent strain in AQRVs (Yang et al., 2011). The virion consists of a double-layered capsid and a genome composed of 11 segments of double-stranded RNA,
∗ Corresponding author at: Wuxi Fisheries College, Nanjing Agricultural University, 9 Eastern Shanshui Road, Wuxi 214081, China. Tel.: +86 510 85551442; fax: +86 510 85551442. E-mail address: [email protected] (G.-J. Yin). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.virusres.2014.03.021 0168-1702/© 2014 Elsevier B.V. All rights reserved.
encoding 7 structural proteins (VP1–VP7) and 5 nonstructural proteins (NS16, 26, 31, 38 and 80) (Qian and Liqun, 2011). In 1984, the virus was first discovered in China (Chen and Jiang, 1984), so far, there have been a large amount of reports which mainly focused on virus structure, infection, rapid detection and vaccine development (Lu et al., 2011; Qiu et al., 2001; Zhang et al., 2010), and more than 10 strains of GCRV have been isolated (Tian et al., 2013). In vitro, the GCRV and grass carp kidney cell line (CIK cells) constantly served as infection model to study infection mechanism, fish antiviral innate immune system screening antiviral drugs and developing vaccine (Peng et al., 2012; Wang et al., 2013). Apoptosis is a highly genetically controlled type of cell death which is accompanied by the activation of a spate of intracellular proteases and endonucleases (Chawla-Sarkar et al., 2003). It plays a critical role in maintaining the homeostasis of multicellular organisms by specifically removing damaged, spent, or misplaced cells (Oyadomari et al., 2002). Caspases are cysteine proteases that play fundamental roles in the apoptotic responses of cells to different stimuli, and are divided into initiators (caspases-8, -9, -10 and
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Time (h) Fig. 1. Cell survival in CIK cells infected by GCRV and mock. GCRV, GCRV-104-infecte cell; Mock, mock-infected cell. The results are expressed as the percentage of the control cells (0 h). The data represent means ± SD (n = 4) (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the data for mock infected-cells).
-12) and effectors (caspases-1, -3, -6 and -7) based on their place in the caspase cascade (O’Brien, 1998). Evidences for apoptotic cell death have been described in many viruses, independent of virus replication (Danthi et al., 2013; Gadaleta et al., 2002). The precise mechanism of virus-induced apoptosis is not yet fully understood. Various viruses have been shown to cause cells death by induction of apoptosis. Cells commit suicide as a direct response to viral infection prior to viral maturation, limiting the spread of progeny virus (Clarke et al., 2000). However, apoptosis of virus-infected cells can also increase viral dissemination while evading immune recognition, which benefit the virus (Chattopadhyay et al., 2010; DeWitte-Orr and Bols, 2007; Oberhaus et al., 1997). By contrast, some viruses encode genes that suppress apoptosis, including adenovirus, herpes simplex virus and Epstein–Barr virus (Ito et al., 2002). The purpose of the present study was to investigate the impact of GCRV infection on apoptosis and oxidative stress in grass kidney cell line (CIK cells), and the possible relations among apoptosis, intracellular oxidative stress and virus infection. CIK cells were maintained in L-15 medium (Sigma, USA) supplemented with 5% fetal bovine serum (FBS) (Hangzhou, China), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, USA). The cell monolayers were either mock-infected or infected with GCRV-104 (CCTCC NO: V201217, kindly provided by Dr. Fan Yuding, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, China) at a multiplicity of infection (MOI) of 1PFU/cell (TCID50 = 10−4.71 ), and analyzed over a time course 96 h post-infection. The cytotoxicity induced by GCRV was examined by WST-1 assay (Hinshaw et al., 1994; Jia et al., 2012). As shown in Fig. 1, CIK cells showed little loss in viability at 12 and 24 h post-infection. By 48 h infection, the viability of GCRV-infected cells showed a marked decrease compared to mock-infected cells. At 72 and 96 h post-infection, 35.47% and 58.73% of the GCRV-infected CIK cells were dead, respectively. It indicated that the death of infected cells mainly occurred during the later stage of infection (72–96 h) due to virus replication, and the results accorded with provious studies (Zou and Fang, 2000). For viral replication assay, the cells and supernatants infected with GCRV were collected, freeze-thawed twice, centrifuged and diluted from 10−1 to 10−9 in L-15 for TCID50 /ml determination, and viral titers (TCID50/ml) were calculated by the method described by Reed–Muench (Reed and Muench, 1938). As shown in Fig. 2, 72 h after infection the titers of GCRV were sharply increased, which suggested the progeny virions were producted in the later stage of infection (72–96 h) and sufficient viral progeny production led to cells lysis and released into culture medium.
0
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Time (h) Fig. 2. The titers of grass carp reovirus (GCRV) in grass carp kidney (CIK) cells in different time of infection. The data represent means ± SD (n = 4).
For apoptosis assay, activities of caspase-3, caspase-8 and caspase-9 were measured using commercialized activity kit (Beyotime Institute of Biotechnology, China) (Chen et al., 2008). In brief, CIK cells were infected with GCRV-104 for 0, 12, 24, 48, 72 and 96 h, and then, were collected and rinsed twice with PBS, the lysates were centrifuged at 20,000 × g for 10 min at 4 ◦ C, and supernatants were incubated for 2 h at 37 ◦ C with 10 l Ac-DEVD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA as the substrates for caspase-3, caspase-8, and caspase-9, respectively. The absorbance at 405 nm was read using a microplate reader (Bio-Rad, USA). Meanwhile, tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1) levels assay were quantified with a commercial fish ELISA kit (BD Biosciences, China) (Hong-quan et al., 2013) according to the manufacturer’s instructions. Briefly, the plate coated with diluted capture antibody (derived from common carp) were incubated over night at 4 ◦ C, and its specificity was determined by western blotting. The plate was blocked with 0.5% BSA for 1 h, then washed four times, added diluted standard TNF-␣, IL-1 and the samples (supernatant of infected cells), incubated for 1 h, washed four times and added diluted detection antibody. After incubation for 1 h, the plate was washed and added alkaline phosphatase. After 1 h, the plate was washed and added p-nitrophenyl phosphate (p-NPP) substrate solution. Finally, the absorbance at 450 nm was measured using micro plate reader. Although apoptosis can be triggered by several different stimuli, apoptotic pathways were mainly classified into two groups: the intrinsic pathway and the extrinsic pathway (Kumar, 2006). The common event in the end point of both the intrinsic and extrinsic is the activation of a set of cysteine proteases (caspases) (Liu et al., 2005). The extrinsic pathway originates at the plasma membrane following the engagement of a family of cytokine receptors, such as tumor necrosis factor receptor-1 (TNF-R1) by their cognate ligands (TNF-␣). Ligand/receptor binding induces the recruitment of several adapter proteins and proenzymes, and then activates caspases (caspase-8, and -10), finally, results in apoptosis and cell death (Guicciardi and Gores, 2005). The intrinsic pathway is triggered by different extracellular or intracellular signals, such as oxidative stress, results in activation of the initiator caspase-9. Caspase-9, in turn, activates caspase-3, major effector caspase, responsible for degradation of cellular substrates (Allen et al., 1997). It is reported that some virus-infected cells can synthesize and release cytokines, such as TNF-␣ and IL-1 (Wang et al., 2011). In this study, both of the cytokines displayed changes in different degrees at each time point (Fig. 3). The level of IL- was significantly elevated at 48 h of GCRV infection and reached its maximum value at 96 h (Fig. 3A). Similarly, the level of TNF-␣ was markedly up-regulated by GCRV infection at 12, 24, and 48 h, and showed a uptrend (Fig. 3B). TNF␣, as a key mediator of apoptosis (Lokensgard et al., 2001), recruits
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Time (h) Fig. 3. The levels of IL-1 and TNF-␣ in CIK cells infected by GCRV and mock. The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) (twoway ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 compared with the values for mock infected-cells).
and activates caspases-8 within a death-inducing signaling complex (Chen and Goeddel, 2002). Just as shown in Fig. 4, the activities of caspase-8 and caspase-9 were increased in GCRV-infected cells at and after 12 h, and maximal increase were observed at 48 h and 24 h post-infection, respectively, when compared with those of mock-infected cells. Caspase-8 and -9, major initiator caspases, are recruited to death receptors and activated by their own intrinsic autocatalytic activity, and then initiate a caspase cascade resulting in the activation of the effector caspases (Clarke and Tyler, 2003; Grossmann, 2002). These effector caspases are responsible for the characteristic morphological changes of apoptosis. It is well known that apoptosis is an important mechanism of virus induced cell death during reovirus infection in cell culture and in vivo (Danthi et al., 2013). In aquatic animals, chum salmon reovirus, a different aquareovirus, has been shown to induce cell death via caspasedependent apoptosis in salmon cell lines (DeWitte-Orr and Bols, 2007). In the present work, significant differences of caspase-3 proteases activity was also seen at 12 h and peak effect was observed at 24 h in GCRV-infected CIK cells. It is a major effector caspase in both the extrinsic and intrinsic apoptotic pathways. Based on the results, it is concluded that GCRV infection in CIK cells induced apoptosis through activation of initiator caspases (caspase-8 and -9) and the effector caspase-3 at early phase of infection. Therefore, the apoptosis was dependent on caspases activation via both the intrinsic and extrinsic pathway (Kominsky et al., 2002). It is suggested that the caspases activation were independent of virus replication (Danthi et al., 2013). Moreover, the activities of three caspases were decreased in GCRV infection after 72 h possible due to massive death of CIK cells. For oxidative stress assay, the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidant capacity (T-AOC) were determined as previously described by Peskin and Winterbourn (2000), Brigelius-Flohé (1999), Cakmak and Horst (1991) and Benzie and Strain (1996),
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Time (h) Fig. 4. The levels of caspase-3, -8 and -9 in CIK cells infected by GCRV and mock. The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) GCRV, (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the values for mock infected-cells).
respectively, and the results were expressed as units per milligram protein. Protein content of GCRV infected CIK cells was determined by the Bradford method, using bovine serum albumin as a standard (Bradford, 1976). Virus-induced pathogenesis is associated not only with apoptosis but with oxidative damage (Valyi-Nagy and Dermody, 2005). Oxidative stress, primarily due to increased generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), is a feature of many viral infections (Li et al., 2010; Valyi-Nagy and Dermody, 2005). The ROS and RNS deplete intracellular antioxidant compounds and consequently lead to cells death (Edens et al., 2008). Clearly, oxidation status of host is important for infection of virus. Just as showed here that the levels of antioxidant compounds (T-AOC, SOD, CAT, GPx) in CIK cells were significantly decreased at least 72 h following GCRV infection (Fig. 5). As for role of oxidative stress in the pathogenicity of virus, Lin et al. (2011)
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Fig. 6. Lipid peroxidation and total protein (TP) content of GCRV infected CIK cells, The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 compared with the data for mock infected-cells).
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Time (h) Fig. 5. Antioxidative state in CIK cells infected by GCRV and mock. (A) SOD; (B) CAT; (C) GPx; (D) T-AOC. The cell monolayers were either mock-infected or infected with GCRV-104 at a multiplicity of infection (MOI) of 1PFU/cell. The data are expressed as means ± SD (n = 4) (two-way ANOVA with Bonferroni posttests, *p < 0.05, **p < 0.01 compared with the data for mock infected-cells).
demonstrated previously that avian reovirus increased the levels of reactive oxygen species (ROS). The ROS depleted antioxidant compounds, resulting in failure of antioxidant system (Griendling and FitzGerald, 2003). This is similar to what we presented here that GCRV-104 infection considerably induced the oxidative injury in CIK cells after 48 h post-infection. It also indicated that oxidative stress might be one of the main factors of cells death in late stage of infection (72–96 h). Generally, lipid peroxidation is a well-established mechanism of cellular injury. It was reflected by levels of MDA, a decomposition product of lipid hydroperoxides (Radi et al., 1991). It has been shown that some viruses, such as avian reovirus, murine reovirus promoted cell damage by initiating lipid peroxidation (Lin et al., 2011; Valyi-Nagy et al., 1999). In this study, MDA levels in the GCRV-infected cells were significantly elevated from 24 to 72 h post-infection, indicating increased lipid peroxidation and oxidative stress in the CIK cells (Fig. 5A). The decrease of protein content was also observed (P < 0.05), but it appeared that protein content in the early phase after infection (0–48 h) has not changed (Fig. 6B). In summary, the present study demonstrated, for the first time, GCRV-104 infection induced apoptosis and intracellular oxidation damage in CIK cells, both of which were important factors in virus pathogenesis. Through these data, it is suggested that the trigger of apoptotic cell death might be associated with the TNF-␣ release. During the early phase of infection (0–24 h), GCRV-104 infection induced TNF-␣ release. The TNF-␣ bound its receptor (TNF-R1), activated the initiator caspase-8, and then stimulated downstream caspases such as caspase-3, consequently, resulted in apoptosis and cell death. Simultaneously, another apoptotic pathway (intrinsic pathway) was triggered by GCRV-104 infection and required mitochondrial involvement to amplify the apoptotic signal from death receptors. The evidence obtained allows us to conclude that
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