Egyptian cobra (Naja haje haje) venom phospholipase A2: a promising antiviral agent with potent virucidal activity against simian rotavirus and bovine coronavirus

Abstract

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC₅₀ of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log₁₀ TCID₅₀ and 2.5 log₁₀ TCID₅₀, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.

Keywords: Antiviral activity; COVID-19; Cobra; Phospholipase A2; Rotavirus; Venom.

Fig. 1

Purification of phospholipase A2 (PLA2) from N. haje venom in two chromatographic steps. A Gel filtration of 120 mg of N. haje dissolved in 1 ml of 0.05 M Tris–HCl buffer, pH 7 was applied to a Sephacryl S-200 column (1.6 × 90 cm) and, B Ion exchange chromatography of 25 mg of Active PLA2 pool on CM-Sepharose column (0.6 × 10 cm). The sample was equilibrated and washed with the dissolved buffer and eluted with different molarities of KCl (0.05, 0.1, 0.15, 0.3, 0.6, 1 M) in the same buffer. The column fractions were collected at a flow rate of 60 ml/h. The fractions with PLA2 activity were pooled, dialyzed, freeze-dried, and stored as Nh-PLA2

Fig. 2

15% SDS-PAGE of the purified N. haje PLA2 (30 µg) under reducing conditions. The samples were: protein ladder ranging from 14.4 to 97 kDa (M), and purified N. haje PLA2 (P). After that, the purified PLA2 was electro-transferred onto nitrocellulose paper then incubated with agarose–RBCs–egg yolk substrate gel. The samples are N. haje venom (C), and N. haje PLA2 (P)

Fig. 3

IEF gel (5%) of 10 µg of purified PLA2 (P) and IEF marker (M) under non-reducing conditions. After that, the purified PLA2 (P) was electro-transferred onto nitrocellulose membrane followed by incubation with fortified agarose gel. A hemolytic band was observed

Fig. 4

Cytotoxicity and antiviral effects of phospholipase A2 (N. haje PLA2) by MTT assay. CC50 is the cytotoxic concentration, IC50 is the half maximal inhibitory concentration and TI is the therapeutic index. Data represent the mean ± SD values (n = 3). Comparison test were performed using the GraphPad Prism 8.0 software; p < 0.05, p < 0.01and p < 0.001 are expressed by *, ** and ***, respectively

Fig. 5

Antiviral effects of phospholipase A2 (N. haje PLA2) on BCoV infected MDBK cells and RV infected MA 104 cells by yield reduction assay (TCID₅₀/0.1 ml). The N. haje PLA2 concentration was used at 3.75 µg/ml. The significance of differences between treated and untreated groups was analyzed by two-sample assuming equal variances t test using the GraphPad Prism 8.0 software. Here, p < 0.05, p < 0.01and p < 0.001 are indicated by *, ** and ***, respectively