In vitro antiviral activity of neem (Azardirachta indica L.) bark extract against herpes simplex virus type-1 infection

Abstract

Herpes simplex virus type 1 (HSV-1) causes significant health problems from periodical skin and corneal lesions to encephalitis. We report here that an aqueous extract preparation from the barks of neem plant Azardirachta indica acts as a potent entry inhibitor against HSV-1 infection into natural target cells. The neem bark extract (NBE) significantly blocked HSV-1 entry into cells at concentrations ranging from 50 to 100 microg/ml. The blocking activity of NBE was observed when the extract was pre-incubated with the virus but not with the target cells, suggesting a direct antiHSV-1 property of the neem bark. Further, virions treated with NBE failed to bind the cells which implicate a role of NBE as an attachment step blocker. Cells treated with NBE also inhibited HSV-1 glycoprotein-mediated cell-cell fusion and polykaryocytes formation suggesting an additional role of NBE at the viral fusion step. These findings open a potential new avenue for the development of NBE as a novel antiherpetic microbicide.

Figure 1. Cytotoxicity of neem bark extract (NBE) from A. indica on Vero cells monlayers

Data are expressed as percent cytotoxicity (OD₅₇₀ test/control) ×100. The NBE concentration at or below 1000 μg/ml was not significantly toxic to cells.

Figure 2. Neem bark extract (NBE) significantly blocks herpes simplex virus type-1 (HSV-1) entry into Chinese hamster ovary (CHO-K1) cells expressing glycoprotein D (gD) receptors

(A). In this experiment, β-galactosidase-expressing recombinant virus HSV-1 (KOS) gL86 (25 pfu/cell) was pre-incubated for 90 min at room temperature with NBE at indicated concentrations or mock-incubated with 1 × phosphate buffer saline (PBS). Subsequently the virus was incubated with CHO-K1 cells expressing gD receptors: nectin-1, HVEM and 3-OST-3 expressing cells. After 6 hr, ONPG assay was performed. In this and other figures each value shown is the mean of three or more determinations (± SD). HSV-1 treated with 1 ×PBS was used as a control. (B). Evaluation of HSV-1 entry blocking activity of NBE into natural target cells. Naturally susceptible cells (HeLa, Vero and retinal pigment epithelial; RPE) were used in this experiment. The β-galactosidase-expressing recombinant virus HSV-1 (KOS) gL86 (25 pfu/cell) was pre-incubated for 90 min at room temperature with NBE at indicated concentrations or mock treated with 1 × phosphate buffer saline (PBS). The virus was then incubated with HeLa, Vero and RPE cells. After 6 hr, ONPG assay was performed.

Figure 3. Confirmation of entry inhibition by X-gal (1.0 mg/ml) staining

Infection of nectin-1-expressing CHO-K1, Vero and primary corneal fibroblasts (CF) with HSV-1 (KOS gL86 at 25 pfu/cell) virus pre-incubated (50 μg/ml) for 90 min with NBE (panels A1, B1, and C1) is shown (panels A1, B1and C1). Cells infected with identical dosage of virus incubated with 1 × PBS was used as control (panels A, B and C). Dark (blue) cells represent viral entry. Microscopy was performed using a 20 × objective on Zeiss Axiovert 100. The slide book version 3.0 was used for image processing.

Figure 4. HSV-1 entry blocking activity of NBE is not viral-strain specific

Different strains of HSV-1 (F, G, MP and 17 strains of HSV-1 at 25 pfu/cell) were either pre-incubated with 1 × PBS (control) or with NBE at indicated concentrations for 90 min at room temperature. The two pools of viruses were incubated on CHO Ig8 cells that express β-galactosidase upon viral entry. The viral entry blocking was measured by ONPG assay (panel A) or by X-gal assay (panel B) as previously described. In X-gal assay (panel B), F-strain of HSV-1 is shown.

Figure 5. Pre-incubation of NBE with HSV-1 virions but not with the target cells significantly blocks HSV-1 entry

In this experiment, target CHO-K1 cell expressing HSV-1 gD receptor nectin-1 (panel A) or a natural target HeLa cells (panel B) were either pre-incubated for 90 min with 50 μg/ml NBE or treated with 1 × phosphate buffer saline (PBS as a control) before viral infection. In parallel experiment β-galactosidase-expressing recombinant virus HSV-1 (KOS) gL86 (25 pfu/cell) was similarly pre-incubated with NBE. The four sets of combinations (virus alone, no virus, pre-incubated virus with NBE and pre-incubated target cells with NBE) were used to infect CHO-K1 cells expressing nectin-1 (A) or naturally target HeLa cells (B) for 2 hrs at room temperature followed by washing of cells with PBS. After 4 hr, ONPG assay was performed. Each bar represents the value of three independent experiments.

Figure 6. NBE significantly inhibits HSV-1 binding to the target cells (A–C)

Green fluorescent protein (GFP) expressing HSV-1 (K26 GFP at 25 pfu/cell) was pre-incubated with NBE (50 μg/ml; represent as white bar) or with 1 × PBS (represent as black bar) for 90 min at room temperature. The mixture was allowed to incubate with CHO-K1 cells expressing gD receptors; nectin-1, HVEM and 3-OST-3 (panel A) or natural target cells; HeLa, Vero and RPE cells (panel B) for 1 hr at room temperature followed by a quick citrate buffer treatment to remove unbound viruses. The fluorescent output as a result of viral binding to the cells was recorded using Tecan spectrophotometer is presented. Each value shown is the mean of three or more determinations (± SD). GFP-expressing HSV-1 pre-incubated with 1 × PBS was used as a control. (C). HSV-1 binding to cells in presence of NBE was quantified using flow cytometry analysis. GFP expressing HSV-1 virions (25 pfu/cell) pre-incubated with NBE (50 μg/ml) or treated with 1 ×PBS were cold bound to HeLa cells at 4°C. Uninfected RPE cells were used as a negative control. Cells were examined by fluorescence-activated cell sorter (FACS) analysis after 35 min of incubation with GFP-expressing HSV-1. Note that the NBE-pretreatment to GFP virions significantly impaired the ability to bind the cells (Red). Mock treated GFP-HSV-1 virions were able to bind cells (Green). Uninfected HeLa cells were used as background control (blue).

Figure 7. NBE significantly impairs HSV-1 glycoproteins induced cell to cell fusion and polykaryocytes formation

(A). The “effector CHO-K1 cells” expressing HSV-1 glycoproteins (gB, gD, gH-gL) along with T7 plasmid were pre-incubated with 100 μg/ml NBE or 1 × PBS for 90 min. The two pools of effector cells (NBE treated and PBS treated) were mixed with CHO-K1 cells expressing luciferase gene along with specific gD receptors; nectin-1, HVEM and 3-OST-3. Membrane fusion as a means of viral spread was detected by monitoring the luciferase activity. Black bars and white bars represent 1× PBS treated and NBE treated cells respectively. Error bars represent standard deviations. * P< 0.05, one way ANOVA. (B). Microscopic visualization of polykaryocyte impairments by NBE. The effector CHO-K1 cells expressing four essential HSV-1 glycoproteins (gB, gD, gH-gL) were pre-incubated with either NBE or 1 × PBS for 90 min before they were co-cultured in 1:1 ratio for 24 hrs. The cells were fixed for 20 min. and then stained with Gimesa stain (Fluka) for 20 min. Shown are photographs of representative cells (Zeiss Axiovert 200) pictured under microscope at 40 × objective. The left panel shows cell fusion and polykaryocytes formation in the absence of NBE, the right panel shows significant inhibition of polykaryocytes formation in the presence of NBE. (C). In parallel polykaryocytes formation was also quantified by counting polykaryons (group of 15 cells). The panel shows number of polykaryons in mock-treated cells (black bars) versus NBE treated cells (white bars). The values shown were from one representative experiment performed in triplicate.