The H7N9 AIVs including the HP-H7N9 AIV (A/Chicken/Huizhou/HZ-3/2016), the LP-H7N9 AIV (A/Chicken/Guangdong /G1/2013), the LP-H7N9 AIV (A/Chicken/Guangdong /SW154/2015) , A/Guangdong/GH0741/2013, and other subtype AIVs including A/Swine/Guangxi/NN1994/2013 (H1N1), A/Swine/Guangxi/NNXD/2016 (H3N2), A/Duck/Yunnan/YN-9/2016 (H5N6) and A/Chicken/Guangdong/V/2008 (H9N2) were provided by the BSL3 Laboratory at South China Agricultural University.
The H7N9 AIVs including A/Chicken/Jiangsu/JX148/2014, A/Chicken/Jiangsu/JT98/2014, A/Chicken/Jiangsu/WJ170/2014, A/Chicken/Jiangsu/TM103/2014, A/Chicken/Shandong/SDL101/2014, A/Chicken/Jiangsu/JT115/2015, A/Chicken/Jiangsu/XZ256/2015, A/Chicken/Zhejiang/JX158/2015, A/Chicken/Anhui/AH284/2015, A/Chicken/Jiangsu/RG126/2015, A/Chicken/Shandong/SD183/2016, A/Chicken/Jiangsu/JS11/2016, A/Chicken/Jiangsu/JT156/2016, A/Chicken/Liaoning/LN1/2016, A/Chicken/Guangdong/GD15/2016, A/Chicken/Zhejiang/ZJ19/2017, A/Chicken/Jiangsu/LY246/2017, A/Chicken/Jiangsu/0116/2017, A/Chicken/Jiangsu/JT186/2017 and A/Chicken/Guangdong/GD4/2017 were provided by the College of Veterinary Medicine, Yangzhou University.
The other avian viruses such as avian infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Marek’s disease virus (MDV), and avian infectious bursal disease virus (IBDV) were obtained from the Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, China.
Monoclonal antibodies production
McAbs against H7N9 were developed following a standard procedure. Six-weeks-old female BALB/c mice were immunized with the inactivated H7N9 AIV (A/Chicken/Huizhou/HZ-3/2016) purified by differential centrifugation at an immunization dose of 20 μg/mouse in Freund’s adjuvant twice with a 3-week interval followed by final immunization with 20 μg H7N9 antigen at 3 days before cell fusion. Splenocytes from the immunized mouse were fused with Sp2/0 myeloma cells, and the hybridoma cells were screened by immunoperoxidase monolayer assay (IPMA) and enzyme-linked immunosorbent assay (ELISA) and cloned by the limiting dilution method. The ascitic fluids from the positive hybridomas were produced in mice.
Screening antibodies specific for HA protein
McAbs against the HA protein of H7 subtype AIV were screened by ELISA. The HA proteins of different influenza virus subtypes (Table 1) diluted in carbonate buffer (CBS) at a concentration of 1 μg/mL were added into 96-well plates at 50 μL/well and incubated at 37 ºC for 2 h. After blocked with 5% skim milk at 37 ºC for 1 h; hybridoma supernatant of McAbs were added and incubated at 37 ºC for 30 min. The reactions were then detected by HRP-labled Goat anti-mouse IgG, and color was developed using TMB solution at room temperature for 10 min which was was stopped by stop solution. The OD450 value of each well was read with a microplate reader for statistical analysis .
Identification of antigen epitopes recognized by monoclonal antibodies
The peptide scanning technique was used to identify the epitope recognized by the McAbs. According to the H7N9 subtype avian influenza HA protein amino acid sequence (ARG44098.1), peptide was synthesized by GL Biochem Ltd (Shang hai, China). The peptide was coupled to the bovine serum albumin (BSA) carrier protein by Sulfo-SMCC, and the coupled peptide was spot-printed on the nitrocellulose membrane. The H7N9 positive serum was used as a positive control, and BSA was used as a negative control. The supernatant of McAbs 9B6 and 10A8 were used as primary antibody, and reactions were then detected by HRP-labled Goat anti-mouse IgG. Finally, ECL color reagent was used to detect the reactivity of McAbs and HA polypeptide.
Virus neutralization test
Neutralizing activities of McAbs were determined by HI assay and virus neutralization (VN) assays. Briefly, 10-fold serial dilutions of McAbs from 103 were mixed with 200 TCID50 virus and incubated for 2 h at 37 ºC. The mixture was then used to infect Madin-Daby canine kidney cells (MDCK) and incubated for 24 h at 37 ºC. Then the 100% VN titers of McAbs were determined by Reed-Muench. At the same time, 10-fold serial dilutions of McAbs were mixed with virus to determine HI titers, and HI titers ≥ 4 were considered positive.
Preparation of colloidal gold and gold-labeled antibodies
Preparation of colloidal gold by trisodium citrate method . Briefly, 1 mL of 1% chloroauric acid and 99 mL of double distilled water was added into the erlenmeyer flask, stiring and heating, followed by the rapid addition of 1.6 mL of 1% trisodium citrate solution with rapid stirring. The reaction mixture was boiled until the color gradually changes from light yellow to deep red and no longer changes in color, with the above process taking about 20 min. The colloidal gold solution was cooled to room temperature. 12 McAbs were incubated with different pH colloidal gold solution for 30 min. The 10% BSA was added to the colloidal gold conjugation and incubated for 10 min. The mixture was then centrifuged at 13,000 rpm and 4 ºC for 30 min to remove any unbound antibody. The pellet was resuspended in boric acid buffer containing 1% BSA.
Selection of paired McAbs for the strip
Among the twelve positive clones, two H7-HA McAbs which showing higher binding affinity were selected to establish a rapid detective strip by sandwich Dot-blot. The sandwich Dot-blot was performed as following. Twelve capture antibodies was blotted on the nitrocellulose membrane (Table 2) at 37 °C for 30 min. After blocking the nitrocellulose membrane using phosphate buffered solution (PBS) containing 1% BSA, 200 μL per membrane of sample diluted in antigen dilution buffer were added and then incubated for 30 min. Then the membrane were rinsed five times with PBS containing 0.2% Tween 20. Twelve colloidal gold conjugated McAbs was added to twelve membranes with 50 μL every membrane, respectively. The pairing of two specific antibodies were selected by observing the color strength of the nitrocellulose membrane.
Preparation of the rapid detective strip
Two McAbs with good specificity were selected to develop the immunochromatographic strip. Briefly, the purified H7-HA specific McAbs were labeled with colloidal gold as conjugated mAb then dispensed on the fiberglass pads to generate conjugate pads. The conjugate pad was dried at 42°C for 50 min. On a 2.79-cm nitrocellulose membrane, the H7-HA specific McAbs and rabbit anti-mouse IgG antibody solutions were dispensed as test and control lines, respectively. The nitrocellulose membrane was dried at 45°C for 4 h. The fiberglass sample pad, conjugate pad, nitrocellulose membrane, and absorption pad were assembled on the support board sequentially, with 1-2 mm overlapping each other and cut into 2.79-mm pieces (CM 4000 cutter; Bio-Dot).
Broad reaction of the strip for H7 subtype AIVs
To evaluate the broad reaction of the rapid detective strip, H7N9 AIVs isolated from 2013 to 2017 were tested. 100 μL of each sample cotaining 105 TCID50 allantoic fluid was added to the sample pad of the test strip and incubated for 10 min at room temperature.
Specificity evalution of the rapid detective strip
To evaluate the specificity of the rapid detective strip, the H1, H3, H5, H7, H9 subtype influenza viruses and other avian viruses including NDV, MDV, IBV and IBDV were simultaneously detected. 100 μL of each sample cotaining 105 TCID50 virus was added to the sample pad of the test strip and incubated for 10 min at room temperature.
Sensitivity evalution of the Rapid rapid detective strip
Three viruses HZ-3, G1 and SW15154 of the H7 subtype were used to detect the sensitivity of the rapid detection strip. The virus was diluted 2 times with 0.01 M PBS from 2-1 to 2-15 and PBS was used as a negative control.
Stability evalution of the rapid detective strip
These strips were tested to determine their sensitivity in detecting the virus HZ-3 upon storage at room temperature for 6 months. The virus HZ-3 was diluted 2 times with 0.01 M PBS from 2-1 to 2-15 and PBS was used as a negative control.
Detecting tissue samples from experimentally infected chickens
Three-weeks-old SPF chickens purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd, were inoculated intranasally with 106 EID50 of H7N9 AIV (A/Chicken/Huizhou/HZ-3/2016) in a 0.2 mL volume (n = 6). In addition, the other two chickens were not inoculated with virus as negative control. After 60 hours, the virus-infected chickens began to die. In order to confirm H7N9 AIV infection, the tissues (brain, windpipe, heart, liver, spleen, lung, thymus, pancreas, bursa of fabricius and cecal tonsil) were dissected from each chicken, and these samples were tested using the H7 detection strip and RT-PCR, respectively.
Detection of simulated clinical swab samples
Tracheal swabs and cloacal swabs (n = 30) were collected from healthy poultry in Henan Province. Swab samples were collected in 2 mL PBS, and the virus allantoic fluids (HZ-3) were added into tracheal swabs and cloacal swabs to simulate clinical swab samples. The simulated clinical swab samples were 2-time-diluted from 2-8 to 2-12 to evaluate the rapid detection strip by HA test and strip test.