Materials

AcMNPV, BmNPV, and Spodoptera frugiperda cell line 9 (sf9 cell line) were provided by Dr. Xiulian Sun of the Wuhan Institute of Virology, Chinese Academy of Sciences, Hubei, P. R. China. AIV was provided by the College of Animal Medicinal Science, Huazhong Agricultural University, Hubei, P.R. China. Sf9 cells were maintained at 28° C in Grace's medium (Gibco,  http://www.invitrogen.com) supplemented with 10% fetal bovine serum (Invitrogen,  http://www.invitrogen.com). RPMI 1640 medium was purchased from Gibco. Medium was supplemented with 10% fetal calf serum (Gibco). 1,1-diphenyl-2-picrylhydrazyl (DPPH), Concanavalin A, lipopolysaccharide (LPS), 3-[4, 5-dimethylthiazol]-2, 5- diphenyltetrazolium bromide, and DNFB were purchased from Sigma-Aldrich ( http://www.sigmaaldrich.com). All other chemicals and reagents used were of analytical reagent grade.

Experimental animals and treatment

Specific pathogen free Kunming mice (18–22 g) were provided and fed at the Experimental Animal Center of Hubei Provincial Academy of Preventive Medicine, Wuhan 430079, P. R. China. Institutional approval license numbers for animal production and use are SCXK (Hubei) 2003–0005 and SCXK (Hubei) 2003–0014, respectively. They were randomly separated into four groups of ten each, placed in cages that were located in a room maintained at 22 ± 1° C with a 12 h:12 L:D cycle, and offered sterile diets and water. The mice were pre-treated with PEF suspension at 50 (lowdose), 100 (middle-dose), and 200 (high-dose) mg/kg·bw (microgram per kilogram of mouse's body weight) by forcedly feeding using a stomach tube (intragingival). The pretreatments were made at the same time for 20 days. At the same time, the mice of the control group were treated with normal saline in equal volume. The body weights of the mice were measured daily. Cervical dislocation was used in euthanasia of the mice.

Preparation of the PEF from M. domestica larvae

The PEF was prepared from M. domestica larvae according to the method of Wang et al. (2007). Third-instar larvae were collected, washed with distilled water, frozen, and lyophilized. The lyophilized maggots were extracted with petroleum ether (bp 30∼60° C) in a Soxhlet apparatus for 50 hrs. The defatted extract was pulverized at a low temperature and treated using three sequences of cold (4° C) protein buffer (0.1 M citrate-Na₂HPO₄, 0.18 M NaCl dilution, pH adjusted to 7.0) for 0.5 hrs. After being centrifuged at 1800 g for 15 minutes at 4° C, the supernatant was transferred to a new container and acidified to pH 5.8 with 0.01 M HCl. The solution was fractionated at 65% saturation of (NH₄)₂SO₄, and the precipitate was dialyzed against a low-ionic-strength pH 7.5 buffer. The precipitate was recovered by centrifugation and dissolved in distilled water prior to dialysis. All the dialyzed extracts were centrifuged, and the supernatant was concentrated and lyophilized. The lyophilized supernatant was PEF. It was stored at -20 °C until needed.

Antiviral assays

Antiviral bioactivity analysis of PEF against AIV. lHemagglutinin was quantified based on a method described by Nayak and Reichl (2004). Serial double dilutions of the test samples (PEF, 100 µL) were made in round-bottomed, 96-well microtiter plates containing 100 µL PBS (pH 7.4). Each sample concentration was measured five times. 100 µL of red blood chicken cells (2 × 10⁷ red blood cells/mL) were added to each cell and incubated for 60–90 minutes at room temperature. The last dilution showing complete hemagglutination was taken as the end point and was recorded as HA (dilution) or log₂HA of 0.1 mL, respectively. Additionally, a parallel HA standard and a positive control using chicken cells in buffer were run to control the assay.

Antiviral bioactivity analysis of PEF against AcMNPV. The antiviral activity of PEF against AcMNPV was determined according to the method reported by Popham et al. (2004) and Wu et al. (2003). PEF (0.5 mg/mL, 1.0 mg/mL) was combined with Ac- MNPV budded virus obtained from the supernatants of AcMNPV-infected Sf9 cells at a ratio of 1:1 (v/v), gently mixed and incubated at 28° C for 1 hr. Grace's insect medium was used as a control in the absence of PEF. Virus titres of these incubations were determined by end-point dilution assay (Slavicek et al. 2001). Sf9 cells were seeded at 1 × 10⁵ cells mL^-1 in 96-well plates and allowed to attach for 1 hr. The cells were infected with dilutions of virus/PEF or virus/medium at dilutions of 10-1 to 10-8, and plates were incubated for 1 week at 28° C. The plate wells were then scored as being positive for virus infection if polyhedra were visible within two or more cells, or negative for viral infection, and the results were used to calculate the virus titre as the TCID₅₀ according to the method of Reed and Muench (1938). Each test was performed three times. Statistical comparisons were done with SPSS software ( http://www-01.ibm.com/software/analytics/spss/).

Antiviral bioactivity analysis of PEF against BmNPV. The antiviral activity of PEF against BmNPV was determined according to the method reported by Li et al. (2006) and Nakazawa et al. (2004). The BmNPV occlusion bodies (OB) were diluted to 2 × 10⁵ OBs/mL by buffer (0.15mol/L NaCl, 0.1 mol/L Tris-HCl, pH7.4). The BmNPV OB was mixed with PEF, and the volume was adjusted to 1 mL. The final concentration of PEF was 0.5 mg/mL, and the OB was 1 × 10⁵ OBs/mL in this mixture solution. These solutions were incubated in a water bath of 37° C for 4 hrs. Fresh mulberry, Morus L. (Rosales: Moraceae) leaves (4cm × 4cm) were daubed uniformly with these mixture solutions and dried at room temperature. Then, 50 thirdinstar silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), larvae were fed with those leaves. 1 × 10⁵ OBs/mL OB solution without PEF was taken as the control, and each test was performed three times. The larval mortality rate was investigated every 24 hrs.

Effects PEF on body weight

The body weights of the mice in four groups were measured daily.

Immunomodulatory activities of PEF

Effects on macrophage phagocytosis in mice by the carbon clearance method. Mice were injected via the tail vein with 0.1 mL of pre-warmed colloidal carbon suspension (Indian ink), which was diluted eight times with PBS (pH 7.4) before use. Blood samples were drawn from the retro-orbital plexus at 2 and 12 minutes. 25 µl of blood was dissolved in 2 mL 0.1% sodium carbonate, then the absorbance was measured at 660 nm (Tiwari et al. 2004). The phagocytic index, K, was calculated by the equation K= (lg OD₁-lg OD₂) / (t₂-t₁), where, OD₁ and OD₂ depict the optical densities at times t₁ and t₂, respectively.

DNFB-induced delayed-type hypersensitivity (DTH) reaction in mice. Mice were sensitized by placing 20 µl 0.5% 2, 4-dinitrofluorobenzene in acetone-olive oil (1:1) on the shaved abdominal skin of recipients. Five days later, 10 µl 0.2% DNFB solution was placed on the right ear. Forty-eight hours later, ear swelling was expressed as the difference between the weight of the left and right ear patches obtained from 8-mm punches (Feng et al. 2002). The punches were from random areas of the middle of the ear. Ear swelling rate was calculated according to the following equation: Ear swelling rate = [(weight of the right ear − weight of the left ear) / (weight of the left ear)] × 100%.

Preparation of spleen cell suspension and lymphocyte proliferation assay. Spleens were aseptically obtained from the mice and placed in complete RPMI 1640 medium. Then, a single-cell suspension was prepared by teasing the spleens apart and filtering it through steel mesh (200 mesh). Erythrocytes were lysed with Tris-buffered ammonium chloride (0.155 M NH₄Cl and 16.5 mM Tris, pH 7.2). The cells were washed twice and then resuspended in complete RPMI 1640 medium. The concentration of splenocytes was adjusted to 5 × l0⁶ cells/mL. Concanavalin A and lipopolysaccharide were used as mitogens, and the final concentration was 5 µg/mL. The cells were cultured for 48 hrs, and pulsed for the last 4 hrs with 10 µl per well of 3-[4, 5-dimethylthiazol]-2, 5-diphenyltetrazolium bromide (5 µg/mL). The cells were washed three times with RPMI- 1640 medium, and 200 µl dimethylsulfoxide was added into the wells of a 96-well, flatbottom, microwell plate. Each test was replicated in three wells. The absorbance (OD₅₇₀) was measured in a microplate reader (Bio-Rad, Model 680,  http://www.bio-rad.com), and the proliferation index was calculated (Ivanovska et al. 1996).

NKCA assay. Lymphocytes from each mouse were prepared as above. NKCA was determined by lactate-dehydrogenase release assay based on the method described by Zhang et al. (2004). The lymphocytes were washed and suspended in complete RPMI-1640 medium, counted, and diluted to 1.0 × 10⁶/mL. The amount of the lactate-dehydrogenase released from the lysed target cells was determined for measuring NKCA. Yac-1 cell line (murine Tlymphoma cell) was used as the target cell. Yac-1 was washed with complete RPMI-1640 medium, counted, and diluted to a concentration of 1.0 × 10⁵/mL with medium. The same volume of Yac-1 cells and lymphocytes were added into the wells of a 96-well, flat-bottom, microwell plate (the cell ratio of effector-totarget was 10:1). Each test was replicated in three wells. After a 2-hr incubation at 37° C in a humid atmosphere with 5% CO₂, the plate was centrifuged at 1000 rpm/min for 5 min. The supernatant from each well (100 µ1) was transferred into the corresponding wells of a 96-well, flat-bottom, microwell plate. Then, 100 µ1 of lactic acid dehydrogenase substrate mixture was added into each well. After 3 min, 50 µ1 cold medium was added into each well to stop reactions. Finally, the absorbances (OD₄₉₀) were measured in a microplate reader (Bio-Rad, Model 680). The percentage of NKCA was calculated by the equation NKCA = (E-S) / (M-S) × 100%, where E represents the experimental release of lactatedehydrogenase activity from target cells incubated in the presence of lymphocytes, M represents the maximum release of the lactatedehydrogenase lactatedehydrogenase activity determined by lysing the target cells with 1% of NP-40, and S is the spontaneous release of the lactatedehydrogenase activity from target cells incubated in the absence of lymphocytes.

Hemolytic activity assay. Hemolytic activity was determined based on the method described by Zhao et al. (2005). The test mice were immunized with sheep red blood cells on Day 0, and PEF was orally administered at the designed experimental dose from Day 0 to Day 5. On Day 6, the blood was obtained from each mouse, and the serum samples were obtained from clotted samples and diluted by saline up to 500 times. 1 mL of the serum was placed in a microcentrifuge tube. Subsequently, 0.5 mL of diluted sheep red blood cells and 1 mL of diluted guinea pig complement were added to the above tube. The tube was incubated in a water bath at 37° C for 10 minutes with occasional shaking to allow the lysis of target cells, and was followed by centrifugation. Next, 1 mL of supernatant was taken out and diluted with 3 mL distilled water and placed in a cuvette. The amount of hemoglobin released was measured by OD at 540 nm. To measure the control, the normal saline, instead of the serum, was added to the tube containing the complement and the target cells, indicating spontaneous and complement- mediated lysis of the target cells in the absence of hemoglobin. The complete control hemolysis was achieved by incubating the processed serum with 1% Tween 20 in saline. The hemolytic activity was calculated as percentage according to the formula: Hemolytic activity = [(mean A540 nm value of the serum samples - mean A540 nm value of the saline samples) / (mean A540 nm value of the Tween 20 samples - mean A540 nm value of the saline samples)] × 100%.

Serum samples from all the mice were obtained from clotted samples and serially diluted (1:80) in normal saline. Serum of mice 0.5 mL and 5% sheep red blood cells 0.5 mL were mixed with serum of guinea pig (1:5) 0.5 mL and normal saline 0.5 mL. HC₅₀ was calculated by A₅₄₀.

Scavenging ability on DPPH radical

The scavenging effect of PEF on 1,1-diphenyl-2-picrylhydrazyl radical was studied, employing the modified method described earlier by Ai et al. (2008b). Briefly, 400 µMDPPH solution in methanol was prepared, and 3.0 mL of this solution was added to 1.0 mL test samples at different concentrations (0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 4.0 mg/mL). The reaction mixture was shaken well and incubated for 30 min at room temperature, and the absorbance of the resulting solution was read at 517 nm against a blank. IC₅₀ value is the effective concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from linear regression analysis. Ascorbic acid was used for comparison. Each test was replicated three times. The radical scavenging ability was calculated using the following equation:

Superoxide anion radical scavenging assay

The superoxide scavenging ability of sulfated PEF was assessed by the method of Xing et al. (2005). The reaction mixture, containing PEF (0.125, 0.25, 0.5, 1.0, 2.0 mg/mL), phenazine methosulfate (30 µM), nicotinamide adenine dinucleotide (338 µM), and nitro blue tetrazolium (72 µM) in phosphate buffer (0.1 M pH 7.4) was incubated at room temperature for 5 min and the absorbance was read at 560 nm against a blank. Ascorbic acid was used for comparison. Each test was replicated three times. The capability of scavenging to superoxide radical was calculated using the following equation:

Statistical analysis

Using SPSS, the significance of the differences between PEF treated groups and the control group was evaluated by Student's t-test at p < 0.05 and p < 0.01.

The antiviral bioactivity of PEF against AIV

Avian influenza virus H₉N₂ adsorbs to chicken red blood cells, resulting in hemagglutination. We investigated if PEF could interfere with the AIV adsorption to red blood cells that would result in inhibition of hemagglutiniation. As shown in Figure 1, PEF exhibited significant antiviral activity in the concentration range of 1.25mg/mL, 2.5 mg/mL, and 5.0 mg/mL.

The antiviral bioactivity of PEF against AcMNPV

Photomicrographs of normal and sf9 cells infected with A. californica multicapsid nucleopolyhedrovirus are shown in Figure 2. An obvious cytopathic effect of PEF on sf9 cells in the viral infection group was observed after 72 hrs. AcMNPV was released from infected cells, and a large number of nuclear polyhedra were observed within cell medium (Figure 2B). Normal sf9 cells had better cell growth, and a cytopathic effect was not observed in the control group (Figure 2A).

The effect of PDF on virus titer assayed on sf9 cells

The antiviral activity of PEF from the larvae of M. domestica was detected using a TCID₅₀ assay for virus titer. Incubation of PEF (1.0 and 0.5mg/mL) with AcMNPV in vitro at 20° C for 1 hr dramatically reduced the TCID₅₀ mL^-1 when dilutions were subsequently assayed on sf9 cells. The virus titre declined significantly from 2.0 × 10⁷ ± 0.42 × 10⁷ to 4.4 × 10⁵ ± 1.2 × 10⁵ (0.5mg/mL PEF) and 1.7 × 10⁵ ± 2.1 × 10⁴ (1.0mg/mL PEF) TCID₅₀ mL^-1 (p < 0.05), indicating the PEF from the larvae showed significant virucidal activity against AcMNPV (Figure 3).

The antiviral bioactivity of PEF against BmNPV

After infection of B. mori larvae with nuclear Bm polyhydrosis, pathological changes were observed in the infected group (Figure 4B). As shown in Figure 5, after treatment of BmNPV occlusion bodies in vitro with PEF (0.5 mg/mL) and injection into B. mori, it was found that larval morality decreased significantly. This result demonstrated that PEF could effectively inhibit the infectivity of the BmNPV occlusion bodies.

Immunomodulatory activities of PEF

Effects on macrophage phagocytosis using the carbon clearance method

The phagocytic index of all the treated groups increased significantly compared with the control group (p < 0.05, Table 1).

Table 1.

The effect of protein-enriched fraction (PEF) isolated from the larvae of housefly on body weight, phagocytic activity, 2, 4-dinitrofluorobenzene-induced ear swelling, and hemolytic activity in mice.