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Eurasian Journal of Veterinary Sciences Eurasian Journal of Veterinary Sciences www.eurasianjvetsci.org http://ejvs.selcuk.edu.tr RESEARCH ARTICLE Determination of lipid peroxidation biomarkers in Vero cell line inoculated with Bovine Ephemeral Fever Virus Oguzhan Avci*, Sibel Yavru, Irmak Dik Received: 15.08.2014, Accepted: 17.09.2014 Selçuk Üniversitesi, Veteriner Fakültesi,*[email protected] Viroloji Anabilim Dalı, 42003, Konya, Türkiye Özet Abstract Avcı O, Yavru S, Dik I. Bovine Ephemeral Fever Virus in- Avci O, Yavru S, Dik I. Determination of lipid peroxidation okule edilen Vero hücre kültürlerinde lipid peroksidasyon biomarkers in Vero cell line inoculated with Bovine Ephem- eral Fever Virus. biomarkırlarının belirlenmesi. Eurasian J Vet Sci, 2014, 30, 4, 217-221 DOI: 10.15312/EurasianJVetSci.201447379 Amaç: - Aim: The aim of the present study was to determine of li- - pid peroxidation biomarkers in Vero cell line inoculated Bu çalışma Bovine Ephemeral Fever Virus (BEFV, Gen with Bovine Ephemeral Fever Virus (BEFV, Genbank No: bank No: GQ229452.1) inokule edilen Vero hücre kültürler inde lipid peroksidasyon biomarkırlarını belirlemek amacı ile yapıldı. GQ229452.1). Gereç ve Yöntem: BEFV inoküle edildikten sonra 4’er saat Materials and Methods: Cell supernatants were collected - 4 h/day for 5 days after BEFV inoculation. Superoxide dis- - mutase (SOD), catalase (CAT), glutathione peroxidase (GPX) lazara (CAT),ile 5 gün glutasyon boyunca peroksidaz hücre süpernatantları (GPX) enzimleri, toplandı. glutasyon Hüc enzymes, glutathione (GSH) and malondialdehyde (MDA) re süpernatantlarındaki süperoksid dismütaz (SOD), kata- values were analyzed from the test media by commercially available ELISA kits. In addition to this, cytopathogenic ef- (GSH) ve malondialdehit (MDA) miktarı ticari olarak te fects (CPE) of BEFV in cell culture were evaluated periodi- min edilen ELISA kitleri ile ölçüldü. Bunun yanında ayrıca cally by invert microscope. BEFV’nin meydana getirdiği sitopatojenik etki (CPE) invert mikroskop yardımı ile periyodik olarak değerlendirildi. Bulgular: - Results: In this research, CPE of BEFV was observed at 72 h post-inoculation. Maximum level of SOD was determined at Araştırmada BEFV’nin CPE’si inokulasyonu taki 56 h, while minimum levels of CAT and GPX were determined ben 72. saatte elde edildi. Maksimum SOD miktarı 56. saatte at 8 and 104 hours, respectively. Maximum GSH levels were tespit edilirken, CAT ve GPX minimum seviyeleri ise sırasıyla determined at 30, 60, 84 and 120 hours while minimum GSH 8. ve 104. saat olarak belirlendi. GSH miktarı sırasıyla 30., 60., concentrations were measured at 44, 92 and 112 hours. A 84. ve 120. saatlerde maksimum; 44., 92. ve 112. saatlerde- minimum seviyede ölçüldü. MDA miktarında ilk 8 saatte ani hours occurred. In addition, CAT, MDA and SOD levels de- tespitdüşüş edildi.meydana geldiği belirlendi. CAT, MDA ve SOD düzeyle creasedsudden beforedecrease developing of MDA levelBEFV-caused was observed CPE. in the first 8 rinin BEFV’in neden olduğu CPE oluşumundan önce azaldığı Öneri: - Conclusion: It is concluded that lipid peroxidation biomark- - ers can be useful in the pathogenesis of BEFV. It may prove Lipid peroksidasyon biyomarkırlarının BEFV'nin pa helpful in the design of future protect from decreasing of oxi- togenezinde yararlı olabileceği öngörülmektedir. BEFV en dative damage associated with BEFV infection. feksiyonunda oluşacak oksidatif hasarın azaltılması ile ilgili çalışmaların planlanmasında yardımcı olabilir. Anahtar kelimeler: BEFV, SOD, CAT, GPX, MDA Keywords: BEFV, SOD, CAT, GPX, MDA 217 Eurasian J Vet Sci, 2014, 30, 4, 217-221 Virus inoculation and oxidative stress Avci et al Introduction CAT is a common enzyme in all living cells that catalyzes the decomposition of hydrogen peroxide to water and Bovine Ephemeral Fever (BEF), caused by Bovine oxygen (Peng et al 2014). GPX is an important cytoplasmic Ephemeral Fever Virus (BEFV, order Mononegavirales, family Rhabdoviridae, genus Ephemerovirus), is a viral et al 2006). GSH is a tripeptide made of cysteine, glycine vector borne disease (Murray 1997), and virus causes acute andenzyme, glutamate. containing It is fouran important selenium antioxidantatoms in structure that prevents (Zhou infection, suddenly fever, anorexia, depression (Wang et al damage to important cellular components caused by free 2001), arthritis and immobility joint swelling (Mellor 1996), radicals. It plays a critical role in cellular antioxidant defense noncontagious, that affects both cattle and buffalo (Uren systems and protects the liver against reactive oxygen species (Mitchel and Russo 1987). proteins (G, N, P, M, L), and propagated in one-day old babyet al 1992).mice after BEFV intracerebral has a negative inoculation. sense RNA, Although five structural bovine It has been reported that free oxygen radicals can be increased kidney, hamster lung, Aedes albopictus (St. George 1985), by aging (Giacomoni et al 2000, Halliwell and Whiteman Baby Hamster Kidney-21 (Nandi and Negi 1999) and Vero 2004), and oxidative stress can occur in cancer, diabetes propagation in vitro. (Halliwell 2001, Andersen 2004, Lin and Beal 2006), and (Yeruham et al 2010) cell cultures can be preferred for virus viral(Zherebitskaya infections (Schwarzet al 2009), 1996, neurodegenerativeIsrael and Gougerot-Pocidalo disorder Oxidative stress can be determinate in all cells 1997, Peterhans 1997). Infectious diseases can be sources during physiologic events for vitality or harbinger of of free oxygen radicals (Maksimenko and Vavaev 2012). It is physiopathology damage. Unpaired electron in atomic or molecular structure is called free radical. Free oxygen radicals change in viral infections (Lutsenko 2013, Stukelj et al 2013), andlikely oxidative that antioxidant stress can enzyme occurs levels (Raju haset al shown 2000, significantCrist et al in 1954 (Turrens 1991). Oxidant structures are produced duringon biological various systems metabolic firstly events reported in organism. by Gershman Autoxidation et al. of polyunsaturated fatty acids (PUFAs) has been realizable It2013, was Yahya hypothesized et al 2013, that Fraisier oxidative et al stress2014). and free radicals with oxygen. Malondialdehyde (MDA) is formed as a result of can be formed in permanent Vero cell which inoculated with lipid peroxidation. MDA, is a dialdehyde, contains 3-carbon BEFV. The purpose of the present study was to determine and carbonyl group at C1 and C3. It can be learned the and compare the lipid peroxidation markers in Vero cell severity of lipid peroxidation by measurement of MDA levels inoculated with BEFV. (Fernandez et al 1997, Aydin et al 2009, Aydin et al 2010). Infectious diseases are reported to be a source of free oxygen Material and Methods radicals (Maksimenko and Vavaev 2012). Oxidative stress in viral infections was reported in the studies (Raju et al 2000, Cell culture and virus SuperoxideCrist et al 2013, dismutase Yahya et (SOD), al 2013, catalase Fraisier (CAT), et al 2014). glutathione supplied from Virology Department, Faculty of Veterinary, peroxidase (GPX) enzymes, and glutathione (GSH) play UniversityVero cell line of Selcuk.and BEFV Cells (Genbank were cultivated No: GQ229452.1) in 25 cm2 were as antioxidants in cells (Guemouri et al 1991, Nguyen et al flasks (Guemori et al 1991), provides conversion of superoxide heat-inactivated(Corning, USA) fetalwith calfDulbecco's serum (FCS, Modified Biological Eagle Industries, Medium to2004). hydrogen SOD, firstly peroxide defined and by oxygen McCord (Jung and Fridovichet al 2003). in 1969 The (DMEM, Biological Industries, Israel) supplemented with 5% Israel), and 1% antibiotics (10000 IU/mL penicillin G, 10 mg/ Figure 1. CPE after 72.h post-inoculation (X40). Figure 2. CPE after 120.h post-inoculation (X40). 218 Eurasian J Vet Sci, 2014, 30, 4, 217-221 Virus inoculation and oxidative stress Avci et al 2 incubator. A total of mL streptomycin, and 25µ/mL amphotericin B; Biological50 (0.5 mL)Industries, of the BEFVIsrael) was at 37°Cinoculated and 5% by COnon-adsorption method medium30 flasks werewere usedcollected during at experiment. 4 h intervals After for 100 5 TCIDdays. The supernatants were centrifuged for 10 min at 5000 g (+4°C) and stored at -80°C until use. ELISA Analyses Levels of SOD (Cayman, USA), CAT (Cayman, USA), GPX (Cayman, USA), GSH (OxisResearch, USA) and MDA Graphic 1. Periodic SOD values after BEFV inoculation. (OxisResearch, USA) in the supernatants of the Vero cell cultures were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The tests were performed as per the manufacturer’s instructions. The plates were then read on an automatic micro plate reader (Rayto RT 2100C, China). This study protocol has been approved by ethics committee of Veterinary Medicine of Selcuk University (Report No: 2014/02). Results Cytopathogenic effects of BEFV on Vero cell line after 72 VirusGraphic ino 2.culation Periodic and CAT oxidative values afterstress BEFV inoculation. and Avci 120 et alh post-inoculation were shown in Figure 1 and 2, respectively. SOD, CAT, GPX, GSH and MDA concentrations of BEFV were shown in Graphics 1, 2, 3, 4 and 5, respectively. 45 40 nmol/ml 35 Discussion 30 25 It has been recognized that oxidative stress plays an 20 15 important role in diverse viral infections (Valyi-Nagy and 10 Dermody 2005, Hahn et al 2008, Williams et al 2010). 5 0 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 In the current research, BEFV changed oxidative status Time (h) antioxidants including SOD (Graphic 1),
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