Preparation of H9N2 avian influenza virus and cell culture
Ten-day-old embryonating specific-pathogen-free (SPF) chicken eggs (Guangdong Dahuanong Animal Health Products Co. Ltd, Guangzhou, China) were inoculated with H9N2 virus (0.2mL/egg). Infected allantoic fluids were harvested 48h post-inoculation and concentrated with a 100K tangential flow filtration capsule (Pall Life Sciences) by centrifugation at 40,000rpm for 1h. The suspension was loaded onto a 30 to 60% (wt/wt) sucrose gradient and subjected to centrifugation at 26,000rpm at 4°C with a SW-40 Ti rotor (Beckman Instruments, Palo Alto, CA) for 3h using the slowest acceleration and braking rates. The viral pellets were washed and centrifuged at 40,000rpm for 1h at 4°C. Subsequently, the pellets were re-dissolved in 1mL of PBS, filtered through a 0.22 Millipore filter, and stored at −70°C .
CEF cultures were prepared from 10-day-old chicken embryos according to standard protocols [10, 11]. Dulbecco’s Modified Eagle Medium (DMEM; Gibco-Invitrogen) was used as the growth medium. In brief, embryo tissue was cut into pieces and diluted to 1 × 106 cells/mL Following filtration the cells were cultivated in a 5% CO2 incubator at 37°C for 48h.
Extraction and purification of APS
Powder ground APS obtained from South China Agricultural University (Guangzhou, China) was boiled in distilled water for 4h at 100°C. After filtration to remove debris, the filtrate was concentrated in a rotary evaporator. Protein was removed using the Savage method . Crude polysaccharide fractions were obtained by precipitation with three volumes of ethanol and desiccation in vacuo. The precipitate was re-dissolved in distilled water and loaded on a D101 macro-porous resin column (2.6cm × 60cm) to remove pigment. The effluent was collected and the polysaccharide fractions were quantitatively determined using a phenol-sulfuric acid assay  with glucose as the reference standard.
Determination of safety concentration of APS to CEF
The CEF safety concentration was determined as reported by Huang et al. . In brief, CEF monolayers in 96-well plates were exposed to Astragalus polysaccharide at a series of concentrations; conducted in replicates of four wells per concentration. After culturing for 72h at 38.5°C in a humidified atmosphere of 5% CO2, the supernatant was removed and 100μL Dimethyl sulfoxide (DMSO; Sigma, Kent, UK) added. The plates were shaken for 5min to ensure complete dissolution of the crystals. The absorbance at 570nm (A570) for each well was measured by a microtiter enzyme-linked immunosorbent assay reader (Model DG-3022, East China Vacuum Tube Manufacturer). The A570 value correlates to the number of live cells; the higher the value at A570 the greater the number of viable cells. The A570 values for APS treated CEFs were higher than for the corresponding cells of the control group. These results indicated that the polysaccharide was not cytotoxic to CEFs at the concentrations used. The corresponding concentrations of APS were considered as the maximum safety concentration for CEF.
Determination of optimal APS concentration for CEF growth
CEF confluent monolayers, prepared in sextuplicate in 96-well culture plates, were overlaid with serial doubling dilutions of APS in DMEM (2,500 to 4.833μg/mL), and cultured at 37°C in a humidified 5% CO2 incubator for 8h. The effect of APS concentration on CEF viability was determined by the MTT 3-(4,5-Dimethyithiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Briefly, the media was discarded, 20μL of MTT (Amresco Solon, OH; 5 ìg/mL) added to each well, and the plates incubated at 37°C in a humidified atmosphere of 5% CO2 for 4h. At the completion of the incubation, 10μL DMSO (Sigma) was added and the plates incubated at 37°C for 5min to ensure complete dissolution of the formazan crystals. Absorbance was measured at A570 using a microtiter ELISA reader (Molecular Devices Emax, CA, USA).
Antiviral activity was evaluated as reported by Huang et al. . In brief, serial two-fold dilutions of APS and H9N2 virus were added to monolayers of CEF cultures under the conditions detailed below.
Pre-addition of APS: APS solutions (100mL/well, four wells per concentration) were added to CEF monolayers prior to the addition of virus. Following incubation in APS for 2h at 38.5°C with 5% CO2, the supernatant was removed, the cells washed twice with Hanks’ solution, and the virus added.
Post-addition of APS: The viral solutions were added to CEF monolayers prior to the addition of APS. Following incubation with H9N2 for 2h at 38.5°C with 5% CO2, the viral inoculum was removed, the cells washed twice with Hanks’ solution, and APS solution added; four wells for each concentration.
Simultaneous addition of APS and H9N2 virus: APS and virus solutions were pre-incubated together for 4h at 4°C prior to addition to CEF monolayers; four wells per concentration.
All plates were cultured at 38.5°C in a 5% CO2 incubator. When complete cytopathic effect (CPE) was observed for the H9N2 control group (~ 72h), CEF viability was measured by MTT assay. The mean cellular A570 values were an indicator of antiviral activity. When the A570 value of the APS group was significantly higher than that of the virus control group, it demonstrated that the corresponding polysaccharide had significant activity.
One hundred and twenty, five-day-old SPF white male leghorn avian broilers, purchased from Guangdong Dahuanong Animal Health products Co. Ltd., were divided into four groups and maintained in four positive pressure isolators. Experimental groups contained three sample groups (30 chickens per group) and one control group (including vaccinated and unvaccinated).
At 7 d post hatch, three sample groups were hypodermically injected with APS at three different concentrations (5mg/kg, 10mg/kg and 20mg/kg) once a day for five successive days. At 12 d post-hatch, six chickens per group were euthanized by cervical dislocation and their blood immediately collected. Peripheral blood lymphocytes separated from the blood samples (5mL per chicken) were used for flow cytometry and real-time PCR. The immune organs were weighted for statistical analysis and immediately stored at −70°C. Simultaneously, nine chickens per group were challenged with 107.5EID50/0.2mL of H9N2 AIV. Additionally, 15 birds were taken from each group, with the exception of the control group, and were immunized subcutaneously with an inactivated oil-emulsion vaccine of H9N2 avian influenza virus (Guangdong Dahuanong Animal Health Products Co. Ltd.) at 13 and 20 d post-hatch. Antibody titers were measured at seven and 14 d post-immunization. The experimental procedures performed on chickens at 12 d post-hatch were repeated at 26 d post- hatch.
All animal experiments and husbandry involved in this study, and presented in this manuscript, were conducted in accordance with the guidelines of the South China Agricultural University Animal Care and Use Committee, which operates under the Animal Welfare Law and Regulations of the Department of Health and Human Services. The South China Agricultural University Animal Care and Use Committee approved all details of this study.
Three color flow cytometry
Blood samples were placed in Petri tubes with sodium heparin and intermixed with an equal volume of Hanks’ balanced salt solution (HBSS, pH 7.2 ~ 7.4). An aliquot was layered over 5mL LSM (lymphocyte separation medium) and centrifuged at 2,000rpm for 20min in a swing-bucket rotor. Peripheral blood lymphocytes were recovered from the LSM-HBSS interface and washed 3 times with PBS. A total of 2 × 106 lymphocytes inrpmI 1640 medium (Roswell Park Memorial Institute 1640 medium) containing 10% (vol/vol) BSA were incubated with 20μL anti-chicken CDS-FITC (SouthernBiotech) in the absence of light for 20min. Cell suspensions were washed with 500μL PBS, centrifuged at 1,200rpm for 10min, the supernatant discarded and the cells re-suspended in 500μL PBS . The cells were then analyzed using a FACSAria flow cytometer (version 6.1; BD Biosciences).
Measurement of antibody titer
The antibody titer was measured by the hemagglutination inhibition (HI) test . The HI test was a standard beta test, using 4 hemagglutinating units of antigen in 96-well plates, where the test serum had been diluted two-fold. HI endpoint titers were determined as the reciprocal of the highest serum dilution that produced complete inhibition of hemagglutination.
Blood samples (1.0mL/chicken) obtained from the main brachial vein were drawn into Eppendorf tubes and allowed to clot at 37°C for 2 h. Serum was separated by centrifugation and stored at −20°C for HI antibody determination. Briefly, two-fold serial dilutions (1:2 to 1:2,048) of heat inactivated (56°C for 30min) serum, was applied to a 96-well, V-shaped bottom microtiter plate containing 50μL/wellcmF-PBS, and 50μL/well H9N2 antigen (4 hemagglutination units); with the exception of the last row (controls). The antigen: serum mixture was incubated for 10min at 37°C, 50μL of a 1% rooster erythrocyte suspension added to each well and the plates re-incubated for 30min. Positive serum, negative serum, erythrocytes, and antigens were also included as controls. The highest dilution of serum causing complete inhibition of erythrocyte agglutination was considered the end point. The geometric mean titer was expressed as reciprocal log2 values of the highest dilution that displayed anti-H9N2-HI.
Total RNA was extracted from CEF using Trizol reagent (Takara Biotechnology, Dalian, China) for the detection of cytokines and H9N2 expression. The quantity of total RNA was measured by ultraviolet spectrophotometry and the optical density (OD) 260/280 determined to be between 1.8 and 2.0. The isolated RNA was digested with Recombinant DNase I (Takara Biotechnology, Dalian, China) at 37°C for 30min. Oneμg of total RNA was used for reverse transcription with ReverTra Ace® qPCR RT kit (Toyobo; Osaka, Japan) and amplifications were performed with 1μL cDNA in a total volume of 20μL SYBR Green Real-time PCR Master Mix (Roche; Mortlake, Australia); all reactions were performed with the Stratagene Mx3005P QPCR system (La Jolla, Ca) according to the manufacturer’s instruction. All reactions were completed in triplicate. Relative expression fold change was calculated by the 2△△Ct method, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as the endogenous reference gene to normalize the expression level of target gene. The primers used in the real-time PCR assay were listed in Table 1.
All data were presented as mean ± SEM. Comparisons between two groups were analyzed using unpaired Student’s T- tests. Comparisons among multiple groups were analyzed by ANOVA followed by post hoc analysis using the Tukey’s multiple comparison test. All statistical analysis was conducted using SPSS 17.0 software (SPSS, Chicago, IL, USA) and P < 0.05 was considered to be statistically significant. All experiments were performed at least three times.