Cells

293T and MDCK cells were obtained from the American Type Culture Collection (ATCC) and were maintained either in Dulbecco’s minimal essential medium (DMEM) or in minimal essential medium (MEM; Gibco) supplemented with 10% fetal calf serum (HyClone) and penicillin–streptomycin (Gibco), respectively. Recombinant viruses were grown in 10-day-old specific-pathogen-free embryonated hen’s eggs (Charles River Laboratories) at 37 °C for 2 days. Human tracheobronchial epithelial cells (hTBE; Lonza) were differentiated by seeding them on 12-mm Transwell filters (pore size, 0.4 μm; Corning) that were coated with collagen type I from human placenta (Sigma-Aldrich) in 12-well plates (Corning). Cells were incubated with a 1:1 mixture of bronchial epithelial cell growth medium (BEGM; Lonza) and DMEM. When cells became confluent, liquid from the upper compartment was removed and cells were differentiated in an air–liquid interface for 4–6 weeks. Medium in the lower compartment was renewed every 48 h50.

Construction of plasmids

All eight viral genes of the influenza A/Shanghai/1/2013(H7N9) strain (SH/1; GISAID accession numbers EPI439486–EPI439494) were in vitro-synthesized by GeneArt Gene Synthesis (Invitrogen). Since the 5′ and 3′ noncoding regions of the SH/1 viral RNA segments had not been reported, the genes were constructed with noncoding regions corresponding to the consensus sequences of closely related influenza A viruses. The NA gene of influenza A/Anhui/1/2013(H7N9) (AH/1; EPI439509) was kindly supplied by Dr Richard Webby, St Jude Children's Hospital, Memphis, TN, USA. The viral gene segments were then subcloned into the ambisense expression vector pDZ51.

Rescue of recombinant chimeric influenza A viruses

Five recombinant influenza viruses—wild-type Shanghai/1 (rSH/1), a 7:1 reassortant between Shanghai/1 and the NA gene of Anhui/1 (rSH/1:AH/1-NA); 7:1 reassortants of A/Puerto Rico/8/34(H1N1) (PR/8) encoding the NA genes of either Shanghai/1 (rPR/8:SH/1-NA) or Anuhi/1 (rPR/8:AH/1-NA) and rPR/8:SH/1-HA/NA, a 6:2 reassortant between the PR/8 internal genes and the Shanghai/1-HA and -NA genes—were rescued from plasmid DNA52,53. The eight pDZ plasmids encoding the viral gene segments (1 μg each) were co-transfected into 293T cells with Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. At 24 h post-transfection, the virus-containing cell supernatant was inoculated by 29-gauge syringe into the allantoic cavities of 10-day-old embryonated chicken eggs, which were incubated at 37 °C for 2 days and then slowly cooled overnight at 4 °C. Allantoic fluid was collected by aseptically opening the allantoic cavity through the air sac and aspirating the allantoic fluid by pipette. The presence of virus in allantoic fluid was evaluated by haemagglutination of chicken red blood cells, and virus stock titres were determined by plaque assay of 10-fold dilutions of allantoic fluid in PBS on MDCK cells overlaid with 0.65% agar (Oxoid Ltd.) in MEM supplemented with 0.4% BSA, penicillin–streptomycin, 0.01% DEAE dextran and 1 μg ml−1 tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin54.

In vitro enzyme assays

Reassortants of PR/8 encoding either the Shanghai/1-NA (rPR/8:SH/1-NA) or the Anhui/1-NA (rPR/8:AH/1-NA) were used for all in vitro assays at biosafety level (BSL)-2. For both Michaelis–Menten and NA inhibitor resistance assays, reactions were performed in 33 mM 2-(N-morpholino) ethane-sulfonic acid (MES; Sigma-Aldrich) with 4 mM calcium chloride at pH 6.6 (MES buffer), with a fluorogenic NA substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma-Aldrich), in 96-well plates (Corning)14,55. Relative fluorescence units (RFU) produced by cleaved MUNANA were measured with the Synergy H1 Hybrid Multi-Mode Microplate Reader and Gen5 software (BioTek).

Determination of Michaelis–Menten parameters for H7N9 NAs

NA content of stock 7:1 reassortant viruses rPR/8:SH/1-NA and rPR/8:AH/1-NA were quantified by western blot of serial virus dilutions, using antisera raised in mice against an influenza A(H11N9) virus as a primary antibody. Band densitometry was performed with ImageJ (NIH), and viruses were normalized to equivalent NA content by dilution in MES buffer. In 96-well plates, the normalized viruses were added to 1.67-fold dilutions of MUNANA substrate (11 concentrations ranging from 6–1,000 μM) and a twelfth control well of MES buffer without MUNANA to measure background fluorescence. Plates were incubated at 37 °C in the microplate reader, and the Gen5 software recorded the RFUs produced by catalysed MUNANA substrate every 90 s for 40 min, at excitation and emission wavelengths of 360 and 448 nm, respectively. For each MUNANA concentration, the measured RFUs (minus background fluorescence) were plotted against time to calculate reaction velocity (in RFU s−1) by linear regression. Michaelis–Menten parameters K m and V max were determined by non-linear regression of the MUNANA concentration versus velocity plots in Prism 6 (GraphPad Software)14. Two independent experiments, with three replicates per experiment, were performed, and results are expressed as an average of all replicates±s.e.

Determination of H7N9 resistance to NA inhibitors

Serial dilutions of stock viruses rPR/8:SH/1-NA and rPR/8:AH/1-NA were incubated in a saturating concentration of MUNANA (400 μM) for 1 h at 37 °C to obtain the 50% effective virus concentration (EC 50 ), at which half the substrate was catalysed. Viruses were then diluted to the EC 50 in MES buffer for use in a fluorometric neuraminidase inhibition assay)14,55. Three NA inhibitors, in 10-fold dilutions, were evaluated: oseltamivir carboxylate (Toronto Research Chemicals, North York, ON; 1 mM–1 pM), peramivir (MedChemexpress, Princeton, NJ; 1 mM–1 pM), or zanamivir (GlaxoSmithKline, Philadelphia, PA; 0.1 mM–0.1 pM). In 96-well plates, normalized viruses were added to 10 drug dilutions plus 2 no-drug control wells to measure uninhibited NA activity. For each drug, the plate also contained a row of no-virus control wells, including the 10 drug dilutions mixed only with MES buffer, to measure background fluorescence. Plates were incubated at room temperature for 45 min, and then MUNANA (400 μM) was added to all wells, followed by a 1-h incubation at 37 °C. Substrate conversion was stopped by the addition of 140 mM sodium hydroxide in 83% ethanol, and RFUs of catalysed MUNANA substrate were measured at excitation and emission wavelengths of 360 and 448 nm on a microplate reader. After subtracting the background fluorescence (as measured in the no-virus control wells), the NA activity at each drug concentration was expressed as a percentage of uninhibited NA activity by dividing the RFUs produced at each of the 10 drug concentrations by the average of the RFUs produced in the two no-drug control wells. The IC 50 of each drug for the SH/1 and AH/1 NAs was determined by plotting the percent of uninhibited NA activity as a function of the log 10 of the drug concentration, and the data were fit to a dose–response curve by non-linear regression in Prism 6. A minimum of six replicates per virus/drug pair were performed, and results are expressed as an average of all replicates with a 95% confidence interval (95% CI).

Growth kinetics of recombinant viruses in hTBE cells

Differentiated hTBE cells were infected at an MOI of 0.01 in PBS with 0.3% BSA in an air–liquid interface. Cells were subsequently cultured with BEGM in the lower compartment and incubated at either 33 or 37 °C. Viral supernatants were collected by adding 100 μl of PBS with 0.3% BSA to each well (surface area: 100 mm2). Cells were incubated for 30 min, viral supernatants were collected and viral titres determined by plaque assay on MDCK cells.

Animals

All research studies involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of the Icahn School of Medicine at Mount Sinai and Rutgers, the State University of New Jersey. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council (8th Edition).

Female BALB/c mice (6–8-week-old) were purchased from Jackson Laboratories (Bar Harbor, ME). All viral infections of mice were performed in accordance with CDC and USDA guidelines in the Animal Biosafety Level (ABSL)-3+ facility at the Icahn School of Medicine at Mount Sinai. Procedures were performed in accordance with Animal Welfare Act guidelines and approved by the Mount Sinai Institutional Animal Care and Use and Institutional Biosafety Committees. Briefly, five groups of mice (n=5 per group) were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg kg−1) and xylazine (5 mg kg−1) and infected intranasally with one of four virus doses (101, 102, 103 or 104 PFU) in 50 μl of PBS with 0.3% BSA or mock-infected with 50 μl of PBS with 0.3% BSA. The mice were monitored daily for clinical signs of illness and for weight loss. Upon reaching 75% of initial body weight, animals were humanely euthanized by a lethal dose of ketamine and xylazine56. Kaplan–Meier survival curves were plotted in Prism 6 and compared by the log-rank test. The median mouse lethal dose (mLD 50 ) was calculated according to the method of Reed and Muench57, with 95% CI determined by the method of Thakur and Fezio58. Viral lung titres were determined by standard plaque assay on MDCK cells.

Six-week-old female Hartley strain guinea pigs, weighing 400–450 g, were obtained from Charles River Laboratories (Montréal, QC). All viral infections of guinea pigs were performed in accordance with CDC and USDA guidelines in the ABSL-3 facility at the Regional Biocontainment Laboratory at Rutgers, the State University of New Jersey. Procedures were performed in accordance with Animal Welfare Act guidelines and approved by the Rutgers Institutional Animal Care and Use and Institutional Biosafety Committees. Guinea pigs were anesthetized by intramuscular injection of a mixture of ketamine (30 mg kg−1) and xylazine (5 mg kg−1) prior to phlebotomy, virus inoculation, nasal lavage and humane euthanasia by CO2 inhalation followed by bilateral thoracotomy56.

Virus susceptibility to NA inhibitors in vivo

Female BALB/c mice (6–8-week-old) were infected intranasally with 100 PFU of either rSH/1 or rSH/1:AH/1-NA in a total volume of 50 μl. Starting 1 h post infection, groups of five mice were administered one of four preparations: (1) intranasal zanamivir (GlaxoSmithKline), 25 mg kg−1 in 50 μl of PBS, twice daily for 5 days; (2) oral oseltamivir phosphate (Roche), 50 mg kg−1 in 400 μl of distilled water, twice daily for 5 days; (3) intranasal PBS, 50 μl twice daily for 5 days; or (4) distilled water orally, 400 μl twice daily for 5 days. Mice were euthanized on day 5 post infection, and virus titres in lung tissues were determined by plaque assay on MDCK cells.

Airborne respiratory transmission experiments

Guinea pig transmission experiments were performed with rSH/1 and rSH/1:AH/1-NA, with ABSL-3 containment, in the Regional Biocontainment Laboratory at Rutgers, the State University of New Jersey. During the experiment, ambient temperatures in the facility ranged from 67–72°F (19–22 °C), with 42–48% relative humidity. Each virus was assessed in four pairs of guinea pigs, with one virus-inoculated and one virus-exposed animal per pair. On day 0, an inoculum of 104 PFU of either rSH/1 or rSH/1:AH/1-NA in 300 μl was administered intranasally to four animals per group, and infected guinea pigs were then sequestered from naive animals to prevent cross-contamination. At day 1 post-inoculation, each guinea pig was individually housed in a modified 31 × 31 × 19 cm polycarbonate cage (#4, Thoren Caging Systems, Hazleton, PA), from one side of which a 20 × 10 cm opening had been cut and replaced with a panel of 1.25-cm wire mesh. The cages of one virus-inoculated and one virus-exposed guinea pig were aligned in pairs so that the wire mesh sides were directly opposed, allowing air to flow freely between cages but precluding direct contact between animals. Each inoculated-exposed guinea pig pair’s cages were placed in an isolator cabinet in a negative-pressure containment unit (Model RVB243116US6; Allentown, Inc., Allentown, NJ). On days 2, 4, 6 and 8 post infection, nasal lavages were performed by instilling a total of 1 ml of PBS into the nostrils and allowing it to drain by gravity onto a sterile Petri dish. Samples were collected into 1.5-ml tubes, centrifuged to pellet debris, and stored at −80 °C. Virus in nasal lavage specimens were titrated by plaque assay of 10-fold dilutions of thawed lavage fluid in PBS on MDCK cells, as described above20.

Seroconversion was assessed by ELISA of pre- and post-infection serum samples from guinea pigs. In 96-well ELISA plates (4 HBX Immunoassay Plates, Immulon), 50 μl of purified 6:2 reassortant virus rPR/8:SH/1-HA/NA, at a concentration of 2 μg ml−1 in PBS, was applied to coat the well bottoms during an overnight incubation at 4 °C. Virus was removed, and plates were blocked with 5% non-fat milk in PBS for 1 h at room temperature. Plates were washed with PBS, and guinea pig sera (in fivefold dilutions between 1/30 and 1/3,750, in duplicate) were added to the plate and incubated for 1 h at room temperature. Plates were washed again with PBS and then incubated with 50 μl of a 1:2,500 dilution of alkaline phosphatase-linked anti-guinea pig secondary antibody (Abcam) for 1 h at room temperature. Plates were washed again with PBS, and 50 μl of p-nitrophenyl phosphate substrate (Invitrogen) was then added. After 30 min, the reaction was stopped with 50 μl of 0.75 N NaOH, and the plates were read at 405 nm in an ELISA plate reader (DTX 880 multimode detector; Beckman Coulter)59. The ELISA positivity cutoff value was determined from the pre-infection serum samples, using a 99.9% upper confidence limit, according to the method of Frey et al.60, and the endpoint titre was defined as the reciprocal of the highest serum dilution with OD 450 above the cutoff value.

Transmission events were determined to have occurred in exposed guinea pigs with infectious virus isolated from nasal lavage specimens by plaque assay and with H7N9-specific antibodies detected in serum by ELISA. Transmission efficiencies were calculated as the number of exposed guinea pigs infected by transmission from an inoculated partner animal, divided by the total number exposed, and were compared by two-tailed Fisher’s exact test in Prism 6.