Virus Research 190 (2014) 17–24
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
In vitro inhibition of enterovirus 71 infection with a nickel ion/chitosan microcomposite Ya-Ching Lin ∗ , Chun-Hsiang Chang Department of Biotechnology, Fooyin University, 151 Jinxue Rd., Daliao Dist., Kaohsiung City 83102, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 7 March 2014 Received in revised form 29 May 2014 Accepted 25 June 2014 Available online 3 July 2014 Keywords: Enterovirus 71 Microcomposite Inhibition Infection
a b s t r a c t In this study, a new microcomposite composed of nickel ion and chitosan was prepared for the purpose of inhibiting enterovirus 71 (EV71) infections. A Ni-chitosan (NiCS) microcomposite and a chitosan microcomposite (CS) were applied to treat Vero cells either during or after EV71 virus adsorption. During a 72-h period of post-infection, the addition of the NiCS microcomposite during virus adsorption exhibited an inhibitory effect on EV71 infection. An excellent effect of over 100% average relative cell viability was obtained and no infection occurred when ≥300 l of NiCS microcomposite was added. However, the addition of NiCS microcomposite after virus adsorption revealed a reduced inhibitory effect. Conversely, the cells treated with CS microcomposite showed a high rate of cell death caused by EV71 infection. The inhibitory effects of NiCS microcomposite on EV71 infection revealed no appearance of CPE in the cells and no viral particle synthesis, and the presence of nickel ion bound to the VP1 protein of EV71 prevented the entry and uncoating of EV71. Our results indicate the potential inhibitory effects of NiCS microcomposite on enterovirus 71 infections. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Enterovirus 71 (EV71) is a member of the enterovirus genus of the family of Picornaviruses. This virus is associated with a broad spectrum of clinical syndromes, including various enanthems and exanthems, hemorrhagic conjunctivitis, myocarditis, and pericarditis, and central nervous system syndromes, such as aseptic meningitis, encephalitis, and poliomyelitis-like paralysis (Wu et al., 2010). Serious epidemics associated with EV71 infection have been increasingly reported on the Asian continent in the past 15 years (Solomon et al., 2010). EV71 has been suggested to circulate endemically in Asia-Pacific countries and may also expand to other regions in the future (Huang et al., 2011). Without an effective vaccine or antiviral treatment against EV71 (Wu et al., 2010), early diagnosis and aggressive supportive management of identified cases are the only treatment option (Thibaut et al., 2012). The development of anti-EV71 strategies for supportive and symptomatic care are primarily focused on target-based chemical design or compound screenings for inhibiting virus attachment, entry, and uncoating, such as targeting EV71
∗ Corresponding author. Tel.: +886 7 781151x6340; fax: +886 7 7862707. E-mail addresses: [email protected], [email protected] (Y.-C. Lin). http://dx.doi.org/10.1016/j.virusres.2014.06.012 0168-1702/© 2014 Elsevier B.V. All rights reserved.
receptor binding or viral capsid-binding molecules (Arita et al., 2008; Buontempo et al., 1997; Pevear et al., 1995; Weng et al., 2005; Yang et al., 2009), and inhibiting viral structural and nonstructural protein synthesis (Arita et al., 2008; Chen et al., 2009; De Palma et al., 2009). Modulating environment of the host and reducing the oxidative stress of EV71-infected cells by the epigallocatechin gallate (EGCG) is another strategy (Ho et al., 2009). The development of the EV71 vaccine is ongoing; more available anti-EV71 options for preventing or reducing EV71 infections and their associated diseases are necessary and should be exploited and evaluated. Among the current developing anti-EV71 strategies, few natural materials have been exploited. Chitosan is a natural polysaccharide of -(1,4)-d-glucosamin and -(1,4)-N-acetyl-d-glucosamin, produced by the deacetylation of chitin which is widely present in crab and prawn shells. Chitosan has various biologically important properties such as unique antibacterial activities (Kong et al., 2010; Rabea et al., 2003), biocompatibility (Cheng et al., 2003), and biodegradability, and is non-toxic (Muzzarelli et al., 1988). Nanotechnology development has enabled applying chitosan micro/nanoparticles to pharmaceutical purposes such as drug delivery for anti-cancer or anti-bacterial therapy (Gao et al., 2012; Sezer and Cevher, 2012) and vaccine delivery (Illum et al., 2001). For antimicrobial or antiviral activities, chitosan micro/nanoparticles
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Y.-C. Lin, C.-H. Chang / Virus Research 190 (2014) 17–24
are familiarly prepared for loading antimicrobial/viral agents and releasing these agents to the target destination (Huang et al., 2010; Koyama et al., 2009; Onishi et al., 2010; Portero et al., 2002). Studies of the antiviral activity of chitosan micro/nanoparticles are alternative trend. Composite chitosan materials or nanoparticle have been demonstrated to inhibit HSV (Marchetti et al., 1995) or HIV (Artan et al., 2010) infection in vitro through the adsorption process between chitosan composites and virus blocking viral entry. Moreover, nanoparticle/chitosan composites containing metals such as silver have an antiviral activity against H1N1 influenza A virus (Mori et al., 2013). Our previous study showed the promising application of nickel ion composite chitosan beads to EV71 adsorption and removal (Lin et al., 2012) from an aqueous phase. Nickel is a nutritionally essential trace metal for human. When too little or too much Ni (>0.5 g) is taken up, either deficiency or toxicity symptoms can occur, respectively (Cempel and Nikel, 2006). In vitro, nickel compounds have cytotoxicity and transforming capability in human cells, such as fibroblastic and epithelial cells in particular (Denkhaus and Salnikow, 2002). However, the toxic function of nickel is directly related to its ability to enter cells. Therefore, it is important to prevent the diffusion and uptake of nickel to cellular environment. In this study, we prepared a new micro-composite composed of nickel ion and chitosan. The chitosan composite were stable after autoclaving and there was no significantly released nickel ions (
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
In vitro inhibition of enterovirus 71 infection with a nickel ion/chitosan microcomposite Ya-Ching Lin ∗ , Chun-Hsiang Chang Department of Biotechnology, Fooyin University, 151 Jinxue Rd., Daliao Dist., Kaohsiung City 83102, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 7 March 2014 Received in revised form 29 May 2014 Accepted 25 June 2014 Available online 3 July 2014 Keywords: Enterovirus 71 Microcomposite Inhibition Infection
a b s t r a c t In this study, a new microcomposite composed of nickel ion and chitosan was prepared for the purpose of inhibiting enterovirus 71 (EV71) infections. A Ni-chitosan (NiCS) microcomposite and a chitosan microcomposite (CS) were applied to treat Vero cells either during or after EV71 virus adsorption. During a 72-h period of post-infection, the addition of the NiCS microcomposite during virus adsorption exhibited an inhibitory effect on EV71 infection. An excellent effect of over 100% average relative cell viability was obtained and no infection occurred when ≥300 l of NiCS microcomposite was added. However, the addition of NiCS microcomposite after virus adsorption revealed a reduced inhibitory effect. Conversely, the cells treated with CS microcomposite showed a high rate of cell death caused by EV71 infection. The inhibitory effects of NiCS microcomposite on EV71 infection revealed no appearance of CPE in the cells and no viral particle synthesis, and the presence of nickel ion bound to the VP1 protein of EV71 prevented the entry and uncoating of EV71. Our results indicate the potential inhibitory effects of NiCS microcomposite on enterovirus 71 infections. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Enterovirus 71 (EV71) is a member of the enterovirus genus of the family of Picornaviruses. This virus is associated with a broad spectrum of clinical syndromes, including various enanthems and exanthems, hemorrhagic conjunctivitis, myocarditis, and pericarditis, and central nervous system syndromes, such as aseptic meningitis, encephalitis, and poliomyelitis-like paralysis (Wu et al., 2010). Serious epidemics associated with EV71 infection have been increasingly reported on the Asian continent in the past 15 years (Solomon et al., 2010). EV71 has been suggested to circulate endemically in Asia-Pacific countries and may also expand to other regions in the future (Huang et al., 2011). Without an effective vaccine or antiviral treatment against EV71 (Wu et al., 2010), early diagnosis and aggressive supportive management of identified cases are the only treatment option (Thibaut et al., 2012). The development of anti-EV71 strategies for supportive and symptomatic care are primarily focused on target-based chemical design or compound screenings for inhibiting virus attachment, entry, and uncoating, such as targeting EV71
∗ Corresponding author. Tel.: +886 7 781151x6340; fax: +886 7 7862707. E-mail addresses: [email protected], [email protected] (Y.-C. Lin). http://dx.doi.org/10.1016/j.virusres.2014.06.012 0168-1702/© 2014 Elsevier B.V. All rights reserved.
receptor binding or viral capsid-binding molecules (Arita et al., 2008; Buontempo et al., 1997; Pevear et al., 1995; Weng et al., 2005; Yang et al., 2009), and inhibiting viral structural and nonstructural protein synthesis (Arita et al., 2008; Chen et al., 2009; De Palma et al., 2009). Modulating environment of the host and reducing the oxidative stress of EV71-infected cells by the epigallocatechin gallate (EGCG) is another strategy (Ho et al., 2009). The development of the EV71 vaccine is ongoing; more available anti-EV71 options for preventing or reducing EV71 infections and their associated diseases are necessary and should be exploited and evaluated. Among the current developing anti-EV71 strategies, few natural materials have been exploited. Chitosan is a natural polysaccharide of -(1,4)-d-glucosamin and -(1,4)-N-acetyl-d-glucosamin, produced by the deacetylation of chitin which is widely present in crab and prawn shells. Chitosan has various biologically important properties such as unique antibacterial activities (Kong et al., 2010; Rabea et al., 2003), biocompatibility (Cheng et al., 2003), and biodegradability, and is non-toxic (Muzzarelli et al., 1988). Nanotechnology development has enabled applying chitosan micro/nanoparticles to pharmaceutical purposes such as drug delivery for anti-cancer or anti-bacterial therapy (Gao et al., 2012; Sezer and Cevher, 2012) and vaccine delivery (Illum et al., 2001). For antimicrobial or antiviral activities, chitosan micro/nanoparticles
18
Y.-C. Lin, C.-H. Chang / Virus Research 190 (2014) 17–24
are familiarly prepared for loading antimicrobial/viral agents and releasing these agents to the target destination (Huang et al., 2010; Koyama et al., 2009; Onishi et al., 2010; Portero et al., 2002). Studies of the antiviral activity of chitosan micro/nanoparticles are alternative trend. Composite chitosan materials or nanoparticle have been demonstrated to inhibit HSV (Marchetti et al., 1995) or HIV (Artan et al., 2010) infection in vitro through the adsorption process between chitosan composites and virus blocking viral entry. Moreover, nanoparticle/chitosan composites containing metals such as silver have an antiviral activity against H1N1 influenza A virus (Mori et al., 2013). Our previous study showed the promising application of nickel ion composite chitosan beads to EV71 adsorption and removal (Lin et al., 2012) from an aqueous phase. Nickel is a nutritionally essential trace metal for human. When too little or too much Ni (>0.5 g) is taken up, either deficiency or toxicity symptoms can occur, respectively (Cempel and Nikel, 2006). In vitro, nickel compounds have cytotoxicity and transforming capability in human cells, such as fibroblastic and epithelial cells in particular (Denkhaus and Salnikow, 2002). However, the toxic function of nickel is directly related to its ability to enter cells. Therefore, it is important to prevent the diffusion and uptake of nickel to cellular environment. In this study, we prepared a new micro-composite composed of nickel ion and chitosan. The chitosan composite were stable after autoclaving and there was no significantly released nickel ions (
