Anti-Poliovirus activity of Nerium oleander aqueous extract

Nerium oleander (NO), a member of the Apocynaceae family, is an ornamental plant. In this study, we evaluated the antiviral activity of hot and cold extract of NO against six different viruses such as herpes simplex virus type 1 (HSV-1), poliovirus type 1 (Sb-1), vesicular stomatitis virus (VSV), reovirus type-1 (Reo-1), human immunodeficiency virus type-1 (HIV-1), and yellow fever virus (YFV). Interestingly the results of plaque reduction assay demonstrated that both, hot extract and cold extract (breastin) of NO inhibited Sb-1 viral infection. In order to identify the mechanism, by which NO exerts its antiviral activity, the virucidal effect, the time of addition and the adsorption assay were carried out. Results demonstrated that NO exerts its effect after infection period, particularly during the first two hours post infection.

Nerium oleander (NO), a member of the Apocynaceae family, is an ornamental plant. In this study, we evaluated the antiviral activity of hot and cold extract of NO against six different viruses such as herpes simplex virus type 1 (HSV-1), poliovirus type 1 (Sb-1), vesicular stomatitis virus (VSV), reovirus type-1 (Reo-1), human immunodeficiency virus type-1 (HIV-1), and yellow fever virus (YFV). Interestingly the results of plaque reduction assay demonstrated that both, hot extract and cold extract (breastin) of NO inhibited Sb-1 viral infection. In order to identify the mechanism, by which NO exerts its antiviral activity, the virucidal effect, the time of addition and the adsorption assay were carried out. Results demonstrated that NO exerts its effect after infection period, particularly during the first two hours post infection.

Experimental Plant material
Nerium oleander was collected from Vally Shoab which is located on the road to Jordan valley and dead sea and is about 4 Km south of the city Salt, Jordan. The plant was taxonomically identified by Prof. D. Al-Eisawi based on anatomy and internal structure of organs. Voucher specimen (Ner-Tf) was deposited in the herbarium of the Biology Department, University of Jordan, Jordan.

Hot extract (NO1) preparation
Sterile freshly ground leaves (200 g) of NO were boiled in distilled water (100 ml) for 3h and subsequently cooled for more than 6 h. Then, the extract was filtered, and the filtrate was boiled again for 1 h and cooled for 4 h and filtered three times. The volume was adjusted to 350 ml to obtain a clear, light brown filtrate (pH 5.84). The filtrate was concentrated using a rotary evaporator (Ozel 1992).

Cold extract (NO2) preparation
Sterile, freshly ground leaves (200 g) of NO were soaked in distilled water (1000 ml) under sterile conditions for at least 8 h. The solution was filtered and the volume was adjusted to 350 ml 3 to obtain a clear, dark brown filtrate. The sterile filtrate was lyophilized under sterile condition Rashan 2010, Rashan 2011).

Cells and viruses
Cell lines were purchased from American Type Culture Collection (ATCC). The absence of mycoplasma contamination was checked periodically by the Hoechst staining method. Cell lines supporting the multiplication of viruses were the following: human CD4 + T-cells containing an Viruses were maintained in our laboratory and propagated in appropriate cell lines. The viruses were stored in small aliquots at -80 °C until use.

Antiviral assays
Extracts activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in exponentially growing MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01.
Extracts activity against YFV and Reo-1 was based on inhibition of virus-induced cytopathogenicity in BHK-21 cells acutely infected at an m.o.i. of 0.01. After a 3 or 4-days incubation at 37 °C, cell viability was determined by the MTT method, as described earlier (Carta et al. 2018). Extracts activity against Sb-1, VSV, HSV-1 and RSV was determined by plaque reduction assays as described earlier (Carta et al. 2018). Briefly, monolayer of Vero-76 cells was grown overnight on 24-well plate. The cells were then infected for 2 h with 250 μl of proper virus dilutions to give 50-100 PFU/well. Following removal of non-adsorbed virus, 500 μl of medium [D-MEM with L-glutamine and 4500 mg/l D-glucose, supplemented with 1% inactivated FBS] containing 0.75% methyl-cellulose, with serial dilutions of test extracts, were added. The overlayed medium was also added to non-treated wells as non infection controls. Cultures were incubated at 37°C for 2 (Sb-1 and VSV), 3 (HSV-1) or 5 days (RSV) and then fixed with PBS containing 50% ethanol and 0.8% crystal violet, washed and air-dried. Plaques in the control (no inhibitor) and experimental wells were then counted. Concentrations resulting in 50% inhibition (CC 50 or EC 50 ) were determined by linear regression analysis. For the purpose of calculating selectivity index (SI), CC 50 values were taken at day 2. Selective activities of the compounds were calculated as follows: Selectivity index (SI) = CC 50 in µg/ml/EC 50 in µg/ml.

Virucidal activity assay
A Sb-1 suspension containing 5×10 5 PFU/ml was incubated with or without different concentrations of extracts for 1 h at 4 or 37 °C. At the end of incubation, the residual infectivity was determined by plaque assay in Vero-76 cells after dilution below the inhibitory concentration.

Adsorption assay
Vero-76 cells grown in 24-well plate were pre-chilled at 4° C for 1 hour and then infected with Sb-1, at an m.o.i. of 5, in the presence or absence of NO extracts. Plates were incubated for 60 min at 4 °C. Medium containing non-adsorbed virus was then removed, cell monolayer was washed 5 twice with PBS and overlaid with fresh medium, incubated for 24 h and then examined by plaque counting (Visintini Jaime et al. 2013).

Effect of Time Addition of NO extracts on the Sb-1 Replication Cycle
The confluent monolayers of Vero-76 cells in 24-well tissue culture plates were infected for 1h at room temperature with Sb-1 dilutions to give a final m.o.i. of 5. After adsorption, the monolayers were washed three times with PBS and incubated with D-MEM medium with Lglutamine, supplemented with 1% inactivated FBS, 1mM sodium pyruvate and 0.025 g/l kanamycin. Monolayers were then treated with NO extracts (5 × EC 50 g/ml) or reference for 1 h during infection period (at -1 to 0) and at specific time point, 0 to 2, 2 to 4, 4 to 6 and 6 to 8 h post infection. After each incubation period, the monolayers were washed two times with PBS and incubated with fresh medium. Then, the monolayers were frozen at -80 °C at 12 h p.i. and the extracellular virus production was determined by plaque assay.