Viruses and cells.
Madin-Darby Canine Kidney (MDCK) cells and chicken embryo fibroblasts (CEF) were maintained in Eagle's minimal essential medium (EMEM) containing 5% (vol/vol) newborn calf serum (NCS). Duck embryo fibroblasts (DEF) were maintained in EMEM containing unheated 5% (vol/vol) NCS according to the manufacturer's instructions. Normal human bronchial epithelial (NHBE) cells were obtained from Lonza (Walkersville, MD) and maintained in serum-free and hormone-supplemented bronchial epithelial growth medium (SABM; Cambrex) containing bovine pituitary extract (BPE) (30 μg/ml), hydrocortisone (0.5 μg/ml), human epidermal growth factor (hEGF) (0.5 ng/ml), epinephrine (0.5 μg/ml), transferrin (10 μg/ml), insulin (5 μg/ml), triiodothyronine (6.5 ng/ml), bovine serum albumin–fatty-acid free (BSA-FAF; 50 μg/ml), retinoic acid (RA) (0.1 ng/ml), gentamicin (30 μg/ml), and amphotericin B (15 ng/ml). All cells were incubated at 37°C with 5% CO2. All viruses used in this study were propagated in MDCK cells in MEM supplemented with 0.3% (vol/vol) BSA and were stored at −80°C until use. The viruses used in this study were influenza A/duck/Mongolia/301/01 (H3N2) virus, A/Kawasaki/173/01 (H1N1) virus, and A/duck/Vietnam/5001/04 (H5N1) virus. All experiments with live H5N1 viruses were performed in biosafety level 3 (BSL3) containment laboratories at the University of Tokyo (Tokyo, Japan), which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan.
Animals and experimental infection.
Five-week-old Japanese specific-pathogen-free quail (Nisseiken Co., Ltd., Tokyo, Japan; n = 3) were inoculated intranasally (i.n.) and intratracheally (i.t.) with 0.5 ml of allantoic fluid containing 108.7 50% egg infectious doses (EID50) of virus. They were euthanized 3 days postinfection, and their organs (nasal turbinate, trachea, lung, and colon) were collected. The virus was serially passaged in quail (three quail per passage) 19 times with 0.5 ml of 10% (wt/vol) pooled organ homogenate (i.e., nasal turbinate, trachea, lung, and colon) every 3 days. The titer of the virus in each organ was recorded as the median 50% tissue culture infective dose (TCID50), that is, the inverse of the dilution that resulted in the cytopathic effect (CPE) in 50% of wells, using MDCK cells.
Detection of Siaα2-3Gal and Siaα2-6Gal in quail tissues.
Nasal turbinates, trachea, lungs, and colon were collected, embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan), and frozen on a dry ice block. Sections of each tissue (8 μm) were cut with a cryostat (CM 3050S; Leica Microsystems, Nussloch, Germany), air dried, and fixed for 10 min with cold acetone before being lectin stained. To detect sialyloligosaccharides reactive with Siaα2-3Gal- or Siaα2-6Gal-specific lectins, we incubated the sections with 250 μl of either fluorescein-labeled Sambucus nigra lectin (1:100; FL-1301; Vector Laboratories, Burlingame, CA) or biotinylated Maackia amurensis lectin II (1:100 dilution; B-1265; Vector Laboratories) overnight at room temperature. After being washed three times with Tris-buffered saline (TBS) (pH 7.6), the sections were incubated with Alexa Fluor 594-conjugated streptavidin (1:100 dilution; P-11227; Molecular Probes, Inc., Eugene, OR) for 1.5 h at room temperature. The sections were then counterstained with 4′,6-diamino-2-phenylindole dihydrochloride (Cellestain DAPI solution; 340-07971; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). They were then washed three more times with TBS, covered with a coverslip, and observed under a fluorescence microscope (Eclipse TE300 with the fluorescence equipment mercury set; Nikon Co., Tokyo, Japan). Photographs were taken using a digital microscope camera (Olympus DP70; Olympus Optical Co., Ltd., Tokyo, Japan).
Isolation of viral RNA, reverse transcription (RT)-PCR, and sequence analysis.
Viral RNA was extracted from virus in cell culture (MDCK) fluids by use of a commercial kit (Isogen LS; Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions and converted to cDNAs by using primers containing the consensus sequences of the 3-prime ends of the RNA segments for HA,d NA, and reverse transcriptase (Superscript III; Invitrogen). The resultant cDNA products were used to amplify the HA and NA genes by a standard PCR method (Proof Start DNA polymerase; Qiagen). The purified PCR products were ligated into the pCR-Blunt II-Topo vector (Invitrogen) and used to transform Top10 cells (Invitrogen). Positive clones were cultured in Luria broth containing 50 mg/liter kanamycin and incubated overnight at 37°C in a shaking incubator. The bacterial culture was then pelleted by centrifugation, and the plasmid DNA was extracted for sequencing with a MagExtractor plasmid system (Toyobo, Osaka, Japan). The HA and NA gene sequences were analyzed with an Applied Biosystems (Foster City, CA) 3100 Auto Sequencer by use of cycle-sequencing dye terminator chemistry (Perkin Elmer, Boston, MA). The HA and NA genes of at least five cDNA clones were sequenced, and the other major sequences were determined for each sample. Primer sequences are available upon request.
Receptor specificity analysis with sialylglycopolymers.
Glycopolymers composed of poly-α-l
-glutamic acid backbones with 5-N
-acetylneuraminic acid linked to galactose through either an α-2-3 or an α-2-6 bond (Neu5Acα2-3LacNAcb-pAP and Neu5Acα2-6LacNAcb-pAP) were chemoenzymatically synthesized as described elsewhere (40
). Virus suspension (200 HA units/ml) diluted in ice-cold PBS was used to coat 96-well polystyrene microplates (F96 Cert.Maxi Sorp Nunc-Immuno Plate; Nunc, Denmark), which were then incubated for 5 h at 4°C (on ice). As a control, wells without virus were also incubated. Unbound virus was removed by washing the wells three times with ice-cold PBS. The wells were then blocked by incubating them at 4°C overnight with 300 μl of PBS containing 0.001% Tween 20 (TPBS). The virus-coated wells were then washed a further three times with ice-cold PBS before 25 μl of horseradish peroxidase (HRP)-conjugated bovine fetuin (which possesses both Neu5Acα2-3Gal and Neu5Acα2-6Gal) diluted in TPBS (1:2,000) was added. Then, 25 μl of serially diluted sialylglycoconjugated polymers was added, and the plates were incubated at 4°C for 2 h. After being washed five times with ice-cold PBS, the plates were incubated with 100 μl of substrate solution (0.4 mg/ml of O
-phenylenediamine, 0.01% H2
in 50 mM citrate-phosphate buffer, pH 5.5) at room temperature for 10 to 20 min. To stop the reaction, 50 μl of 0.1 N H2
was added to each well. The extent of inhibition of fetuin binding to virions with sialylglycoconjugate polymers was determined by measuring the absorbance at 490 nm.
Glycan array analyses.
Viruses were grown in MDCK cells and ultracentrifuged at 25,000 rpm for 2 h at 4°C and then laid over a cushion of 25% sucrose in PBS. Virus stocks were aliquoted and stored at −80°C. Virus concentrations were determined by use of an HA assay with 0.5% (vol/vol) turkey red blood cells (RBCs). Custom microarray slides were printed for the Centers for Disease Control and Prevention (CDC) using the CFG glycan library (CDC version 1 slides; see Table S1 in the supplemental material for the glycans) as described previously (4
). Virus preparations were thawed and suspended in PBS supplemented with 3% (wt/vol) BSA to an HA titer of 128, established to be optimal for glycan array analyses. Virus suspensions were supplemented with 10 nM zanamivir, overlaid on the printed region of the slides, and then incubated with gentle agitation in a closed container for 1 h at 4°C. Unbound virus was then eluted with brief rinses in PBS. The slides were immediately incubated with a mouse anti-Aichi/68HA monoclonal antibody (30 min), a biotinylated anti-mouse-IgG antibody (30 min), and a streptavidin-Alexa Fluor 635 conjugate (30 min) (Invitrogen, Carlsbad, CA) with brief PBS washes between incubations. After the final PBS wash, the slides were briefly rinsed in deionized water, dried under a gentle stream of air, and heat treated at 75°C for 1 h prior to imaging. Fluorescence intensities were captured by using a ProScanArray HT (PerkinElmer, Waltham, MA). ImaGene 8 software (BioDiscovery, El Segundo, CA) was used for image analyses. Data were processed in MS Excel to group similar sialoglycans and to generate a simplified chart. Three independent experiments were performed for each virus.
Generation of viruses by reverse genetics.
Reassortant viruses were generated by using the plasmid-based reverse-genetics system described previously (31
). Viral RNA was extracted from virus in cell culture (MDCK) fluids by use of a commercial kit (Isogen LS; Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions and converted to cDNAs by using primers containing the consensus sequences of the 3-prime ends of the RNA segments for HA, NA, and reverse transcriptase (Superscript III; Invitrogen). The resultant cDNA products were then used to amplify the HA and NA genes by a standard PCR method (Proof Start DNA polymerase; Qiagen). The cDNAs were cloned into a plasmid under the control of the human polymerase I promoter and the mouse RNA polymerase I terminator (referred to as PolI plasmids). Viruses possessing the HA and NA genes of plaque-purified parent virus or quail-passaged viruses (P19T clone 2 or clone 4), which have the internal genes of the A/whistling swan/Shimane/499/83 (H5N3) virus, were generated by using reverse genetics and are referred to as WT(HA,NA)-RG, P19Tcl2(HA,NA)-RG, and P19Tcl4(HA,NA)-RG. Viruses possessing the internal genes of the A/Yokohama/2017/03 (H3N2) virus were also generated for the growth curve in NHBE cells and are referred to as WT(HA,NA)-human-RG, P19Tcl2(HA,NA)-human-RG, and P19Tcl4(HA,NA)-human-RG. WT(HA)-P19Tcl2
(NA)-RG and WT(HA)-P19Tcl4
(NA)-RG were also generated for the virus elution assay.
Virus elution assay.
The ability of NA to elute virus bound to erythrocytes was assessed as follows. Fifty microliters of 2-fold dilutions of virus containing HA titers of 1:128 was incubated with 50 μl of 0.5% (vol/vol) chicken erythrocytes or 1% (vol/vol) guinea pig erythrocytes in microtiter plates at 4°C for 1 h. The microtiter plates were then stored at 37°C, and the reduction in HA titers was recorded periodically for 12 h. Calcium saline (6.8 mM CaCl2-154 mM NaCl in 20 mM borate buffer, pH 7.2) was used as a diluent.
Viral growth kinetics in cell culture.
DEF, CEF, MDCK cells, and NHBE cells were infected with virus at a multiplicity of infection (MOI) of 0.001. After adsorption for 1 h, medium containing virus was removed, and the cells were overlaid with EMEM containing 0.3% BSA and 0.5 μg/ml of trypsin for DEF, CEF, and MDCK cells or serum-free, hormone-supplemented SABM for NHBE cells. The plates were then incubated at 37°C in 5% CO2. At various times postinfection, the virus titers in the cell culture supernatant were determined by using plaque assays with MDCK cells.
Infection of human lung tissue with influenza A viruses.
Fresh, surgically removed normal human lung specimens containing bronchi and alveoli were cut into ~0.5-cm3 cubes, washed with culture medium (F-12K nutrient mixture with 15% fetal calf serum [FCS], l-glutamine, and antibiotics), and incubated with virus (~108 PFU/ml) at 37°C in the medium. Twelve hours postinfection, the tissue blocks were fixed in 10% neutral-buffered formalin and processed for routine paraffin embedding and immunohistochemical analysis with a mouse anti-influenza A virus nucleoprotein antibody. The reactions were visualized by using a two-step dextran polymer system (Dako) and 3,3′-diamino benzidine (DAB). These experiments were performed with tissues from three patients. Because the results for all three were comparable, the findings from only one set of specimens are shown.
Microarray data accession numbers.
The glycan microarray data presented here are available online through the Consortium for Functional Glycomics website (http://www.functionalglycomics.org
). Resource request identifiers are cfg_rRequest_1336/95 (parent virus), cfg_rRequest_1336/96 (P19T), cfg_rRequest_1336/97 (clone 2), and cfg_rRequest_1336/98 (clone 4).