In vitro passaging of H7N9 virus strains
All experiments were strictly performed following the standard procedures of Biological Safety Level 1–3 Laboratory. Two H7N9 virus strains, namely A/Anhui/1/2013 (AH-H7N9) and A/Shanghai/2/2013 (SH-H7N9), were provided by the virus resource database, and were isolated from chicken embryos at the Chinese Center for Disease Control and Prevention (China CDC). Following treatment of chick embryos from embryonic (E) day E9 to E10 with these viruses, chick embryos were incubated at 35 °C for 72 h, and placed at 4 °C overnight. The first transfer generations of AH-H7N9 and SH-H7N9 (P0 strain) were collected from allantoic fluid components in chick embryos. Additionally, simultaneous measurement of infectious virus titer was carried out by 50% tissue culture infective dose (TCID50) for each virus strain. Madin-Darby canine kidney (MDCK) cells were selected as the infection model for the generation of each virus strain. Briefly, 8 × 105 MDCK cells were seeded in a 25 cm-flask containing DMEM with 10% FBS, 100 μL/mL penicillin and 100 μL/mL streptomycin (pH 7.2–7.4). MDCK cells were cultured at 37 °C in a humid environment containing 5% CO2 until 70% confluence, and washed with TPCK-trypsin (2 μg/mL)-containing DMEM for 3 times. Then, 10 μL of allantoic fluid containing the P0 strain was inoculated and cultured with MDCK cells as described above for 2 h. Afterweards, the medium was discarded, and TPCK-trypsin (2 μg/mL; 5 mL)-containing DMEM was added in cells, which were cultured for another 48 h. Finally, the cytopathic effect (CPE) of the virus was observed under an inverted microscope. Next, the cultures were centrifuged for 10 min and supernatants as well as cells were collected. Absolute quantification was performed for virus in titers in supernatants and cells. Determination of 50% tissue culture infective dose (TCID50) and whole-genome sequencing were performed for supernatants and cell viruses, respectively. During long-term serial passaging in the host, viruses from the supernatants were collected, and inoculation was performed as described above, with a constant inoculation amount of 8 × 104 copies. Routinely, cells were assessed and passaged every 48 h. Viruses in the culture supernatants were passaged repeatedly till the sixth generation in MDCK cells, suggesting the generation of mutant viruses (Additional file 1: Fig. 1). Using an identical passaging protocol, serial passaging was also carried out repeatedly in A549 cells till the third generation. The TCID50 was measured and calculated by the Reed-Muench method , as logTCID50 = log (dilution where % of infected cells above 50%) + distance ratio × log (dilution coefficient).
Absolute quantification of virus titers by real-time RT-PCR
Virus was extracted from a total amount of 100 μL cell culture supernatant with QIAGEN RNeasy Mini Kit. The extracted viral RNA was finally dissolved in 100 μL eluent. Then, a total amount of 1 μL viral RNA was fluorescently quantified by real-time PCR. Rapid and sensitive quantification of 1 μL viral RNA was carried out by continuous fluorescent monitoring of real-time PCR using Multiplex Real Time PCR with Influa H7N9 Detection Kit, whose standard procedures were provided by the National Influenza Center. AgPath-ID™ One-step RT-PCR (Life Technologies, AM1005) was performed with H7 labeled by FAM™ dye on an Applied Biosystems 7500 Fast Dx Real-Time PCR instrument at the conditions of 10 min at 45 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 45 s at 60 °C. Absolute amounts of viral RNA in were determined by the 7500 software v2.0 according to the HA standard curve.
Sequence analysis of viral RNAs
Primers for overlapping PCR were designed, and further used for amplification reactions with the Qiagen one step RT-PCR kit. The standard PCR procedure was as follows: 30 min at 50 °C, 15 min at 95 °C, 40 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min at 72 °C and 10 min at 72 °C, respectively. PCR products were collected, and all gene segments from each virus passage replicated in embryonated eggs were amplified by high fidelity PCR using QIAGEN One Step RT-PCR Kit in the supernatants of homogenized cells and the cell supernatants. The expression vector, pGEM-T Easy Vector (Promega, Madison, USA), was designed for the convenient cloning of the purified PCR-amplified RNAs. Five positive clones for each gene segment from individual virus subtype were randomly selected for sequencing (Invitrogen, Shanghai, China). Assembly and analysis of the sequences was performed with DNASTAR-Lasergene’s SeqMan software and NCBI-Blast.
Recombination of viral protein mutants
Gene sequences of the HA7 and NA9 proteins isolated from AH-H7N9 or SH-H7N9 were optimized according to host cell characteristics, and sub-cloned into the pCAGGS plasmid (Genewiz). Then, site-specific mutations introduced by viral passaging were selected for further constructing HA7 or NA9 mutant plasmids using the Quickchange Lighting Site-Directed Mutagenesis kit. Briefly, primers containing site-specific mutations were synthesized. The standard PCR procedure was as follows: 2 min at 95 °C, 18 cycles of 20 s at 95 °C, 10 s at 60 °C, 8 min at 68 °C, and 10 min at 68 °C. DH5α competent ™ cells were transfected with these PCR products, by adding 8 μL PCR product into 50 μL DH5α cells, keeping the mixture on ice for 30 min, followed by heat-shock at 42 °C for 90 s and immediate placement on ice for 2 min. Transfected cells were added into 400 μL LB media and cultured in a 37 °C shaker for 45–60 min. Then, 50–100 μL cultured cells were transferred into ampicillin (100 μg/mL)-containing LB plates overnight at 37 °C, and single clones were picked for gene sequencing. Colonies with site-specific mutation accuracy were further inoculated after a 1000-fold dilution onto ampicillin-containing LB plates and cultured overnight in a 37 °C shaker. Plasmid recovery was performed using Qiagen Endo free Plasmid Maxi Kit according to the instructions provided by the manufacturer.
First, plasmids expressing the HA7 or NA9 proteins were transfected into 293 T cells, respectively. Expression levels of the former protein were detected by Western blot, and the activity of the latter protein was determined with NA Activity Kit. Then, the HA7, NA9 and pNL-4.3-Luc-R-E plasmids were co-transfected into 293 T cells, thereby constructing a pseudo-type influenza virus. Briefly, 293 T cells were inoculated in 6-well plates, allowing rapidly spreading viral infection in cultured cells until 70% confluence. The cells were then washed with PBS, and added to a culture medium with low-serum Opti-MEM (Gibco). Virus packaging was performed with Lipofectamine™ 2000 Transfection Reagent Kit (Life Technologies) according to the manufacturer’s instructions. Briefly, 100 μL solution A was added to 500 ng of each pCAGGS-HA7 and pCAGGS-NA9 plasmids, and 1500 ng of pNL-4.3-Luc-R-E, with 3.5 μL of vectors comprising a strong viral promoter/enhancer sequence. Solution A was rotated for 1 s, and kept at room temperature for 2–5 min. Then, solution B was prepared by adding 10 μL effective transfection reagent into 100 μL plasmid containing the enhancer sequence, and rotated for 10 s. Next, solutions A and B were mixed and kept for 5–10 min, added to 600 μL of Opti-MEM medium. The mixture was slowly and carefully added into 6- well plates containing cultured 293 T cells, after discarding the medium. After 6 h of incubation, the supernatant was aspirated, fresh Opti-MEM containing TPCK-trypsin (Sigma) at 2 μg/mL was added. Supernatants containing the H7N9-pseudovirus were collected after 48 h of culture, filtered with a 0.45-μm filter (Millipore) and stored at − 80 °C. Since the pNL-4.3-Luc-R-E plasmid contained the luciferase gene as a marker, the activity of the packaged virus was determined with the Firefly Luciferase Reporter Gene Assay kit. Briefly, all H7N9 pseudoviruses were respectively incubated with MDCK cells cultured in serum free DMEM containing TPCK-trypsin (2 μg/mL) in 96 well plates (104 /well) for 2 h at 37 °C in an environment containing 5% CO2. Then, the medium was replaced with 10% FBS supplemented DMEM (2 μg/ml TPCK-trypsin) for another 48 h of culture. Luciferase activity was measured with luciferase assay regents (Promega, Madison, WI), NA activity was determined with a NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set (Applied Biosystems) and a Luminescence Counter (Promega) according to the manufacturer’s instructions.
Cultured cells were washed with PBS twice, lysed with lysis buffer (20 mM Tris, pH 8.0, 0.5% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, protease inhibitor cocktail), centrifuged at 15,000g for 15 min, and supernatants were collected for protein quantification by the Lowry’s method. After diluting the samples to 2 mg/mL, proteins were heat-denatured for 5 min, submitted to 4–10% SDS-PAGE, transferred onto a PVDF membrane, blocked with 1% bovine serum albumin, and incubated with H7 monoclonal antibody (1000; mouse anti-H7N9 hemagglutinin/HA antibody, Sino Biological, Beijing) at 4 °C overnight. The membrane was then incubated with anti-mouse IR dye 800-labeled IgG secondary antibody (Li-Cor, Lincoln, NE), measured at a recommended wavelength, and analyzed by the Odyssey software.
Interaction detection between the recombinant HA and receptors
HA quantification was first performed following the instructions provided by the manufacturer. Recombinant HAs were coated onto a polystyrene plate at 4 °C overnight. The AH-H7N9 hemagglutinin/HA protein (Sino Biological, Beijing) was used as the standard and serially diluted. The plate was then blocked with 5% bovine serum albumin (BSA) (Sigma) at 37 °C for 2 h. After washing, mouse anti-HA mAbs (1:1000) were added, and the plate was incubated at 37 °C for another hour. The plate was incubated with HRP-labeled secondary anti-mouse IgG (1:5000) (Sigma) at 37 °C for another hour following washing. OPD/H2O2 was then added; the chromogenic reaction was terminated by addition of H2SO4. The amounts of the HA protein were measured at 450/650 nm, using a standard curve.
A solid-phase binding assay with competing glycopolymers was performed for determining receptor affinity as described previously. Serially diluted HA proteins (threefold) from 200 μg/mL (3.4 μM) were allowed to incubate overnight at 4 °C in 96-well plates. BSA (5%) was used to block the plates for 2 h at 37 °C. Then, [3′-Sialyllactose-PAA-biotin, 3′SL] (GlycoTech, 01-038) or [6′-Sialyllactose-PAA-biotin, 6′SL] (GlycoTech, 01-039) was added to the wells at 200 ng/mL for 1 h at 37 °C, respectively. Horseradish peroxidase (HRP)-conjugated avidin (1:5000; Sigma) was added to wells for visual detection of the proteins by a chromogenic reaction. The extent and accuracy of binding of the recombinant HA by a given polysaccharide receptor was determined by absorbance at 450/650 nm. Data were analyzed by the GraphPad Prism 5.0 software.
Interaction detection between H7N9-pseudotyped viruses and receptors
Interaction detection between recombinant viruses and receptors was performed with the HIV-1 p24 Antigen ELISA kit (Keybiotech, Beijing), by assessing receptor-labeled 3′SL and 6′SL, according to the manufacturer’s instructions. Briefly, p24-antigen containing- and standard samples were added to p24 McAb-coated 96-well plates and incubated at 37 °C for 60 min. Following incubation, the plates were washed, and biotin-labeled p24 polyclonal antibody and HRP-labeled SA were added for further studies. After 5× washing, H2O2/DAB was added, and the reaction was terminated 15 min later. Virus quantification was carried out by detecting OD at 450/650 nm. Then, p24 protein levels were adjusted to 200 ng/mL after dilution of the virus, which was coated to 96-well plates as described above. The 3′SL and 6′SL were twofold serially diluted from 200 ng/mL, allowing the binding detection of HRP-SA by DAB and OD measurement, as described above for the solid-phase binding assay between HA and glycol- polymers.
The Student’s t-test was utilized to determine the statistical significance of differences between experimental groups. p < 0.05 was considered as statistical significant.