Cells and viruses.
Virus cultures were done in R-Mix and R-Mix Too cells in 96-well or shell vial format (Diagnostic Hybrids, Inc. [DHI], Athens, OH). The cells were cultured in a 35 to 37°C CO2 incubator. Sf9 cells (Invitrogen, Carlsbad, CA) were grown following the manufacturer's instructions. Influenza A viruses and other prototype respiratory virus strains used in experiments were from the DHI virus repository, and most of them were originally from the American Type Culture Collection (ATCC). The 2009 H1N1 A/California/07/2009 and A/Mexico/4108/2009 viruses were obtained from the CDC. Virus stocks were propagated in MDCK cells (ATCC, Manassas, VA).
Clinical isolates and specimens.
Clinical isolates were obtained from various laboratories, and viruses were propagated in MDCK cells for 1 to 3 passages. Forty-three clinical isolates positive for 2009 H1N1 virus by RT-PCR were obtained from the Charleston Area Medical Center Health System, Inc. (Charleston, WV). Forty-five clinical isolates acquired before the 2009 H1N1 outbreak were from the DHI virus repository and were confirmed to contain influenza A virus by use of a DFA Respiratory Virus Screening & ID kit (Diagnostic Hybrids, Inc., Athens, OH). One hundred twenty-five influenza A virus-positive frozen clinical specimens collected during the 2009 H1N1 outbreak were provided by Kaiser Permanente Hospital (North Hollywood, CA). Fifty nasopharyngeal swab specimens were collected in November 2009 during the 2009 H1N1 outbreak from local hospitals in southeast Ohio. Two-well slides were prepared for immunofluorescence staining within 24 h of specimen collection. A portion of the original specimens from clinical isolates and of clinical specimens was used for viral RNA isolation and rRT-PCR testing.
Generation of 2009 H1N1-specific MAbs.
BALB/c mice were immunized with β-propiolactone-inactivated 2009 H1N1 A/California/07/2009 virus. Hybridoma production was carried out by previously described methods (9
). Hybridoma supernatants were screened by IFA on both R-Mix monolayers infected with a mixture of H1N1 A/California/07/2009 and H1N1 A/Mexico/4108/2009 and R-Mix monolayers infected with a mixture of H3N2 strains. Positive hybridoma cells were expanded and then screened on R-Mix cell monolayers infected with 2009 H1N1 strains, four seasonal H1N1 strains, and four seasonal H3N2 strains. The hybridoma cells that were positive for both H1N1 A/California/07/2009 and H1N1 A/Mexico/4108/2009, without cross-reactivity to seasonal H1N1 strains and H3N2 strains, were selected for cloning by limiting dilution. The same screening strategy was applied to hybridoma clone selection. The candidate clones were expanded for MAb production. The MAbs were purified from hybridoma supernatants by protein G affinity chromatography, using fast-performance liquid chromatography (FPLC). The isotypes of the MAbs were determined using an isotyping enzyme-linked immunosorbent assay (ELISA) kit (Southern Biotech, Birmingham, AL).
Recombinant HA proteins.
Primers were designed to target the HA gene sequence of A/Mexico/4108/2009. The full-length HA gene was amplified by RT-PCR from RNA extracted from the A/Mexico/4108/2009 virus. SpHI and EcoRI restriction enzyme sites were inserted into the primer sequences for directional cloning into the baculovirus transfer vector pFastBac HT-A (Invitrogen, Carlsbad, CA). The 2009 H1N1 HA recombinant baculovirus was constructed using the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. To express the 2009 H1N1 full-length HA protein, Sf9 cells were infected with the recombinant baculovirus stock. The protein was purified under denaturing conditions, using a nickel affinity chromatography column (Qiagen, Valencia, CA).
A truncated 2009 H1N1 gene (HA1 subunit) was also synthesized with codons optimized for expression in Escherichia coli. Multiple overlapping complementary oligonucleotides which encompass the HA1 coding sequences were synthesized, and the gene was assembled by PCR and then amplified using outermost primers in a second PCR. The PCR product was cloned into the TA vector (Invitrogen, Carlsbad, CA), followed by DNA sequencing. The HA1 gene was then subcloned into the pQE expression vector (Qiagen, Valencia, CA), and the HA1 protein was purified using nickel affinity chromatography (Qiagen, Valencia, CA).
The purified full-length HA (A/Mexico/4108/2009) recombinant protein expressed in Sf9 cells and the 2009 H1N1 HA1 subunit recombinant protein expressed in E. coli were separated by SDS-PAGE. The separated proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes. The blotted membranes were blocked with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 5% skim milk and then incubated with 5 μg/ml of each 2009 H1N1 candidate MAb. The MAbs that bound to the membrane were detected by anti-mouse IgG-horseradish peroxidase (HRP) conjugate, followed by development with a WesternC chemiluminescence detection kit (Bio-Rad, Hercules, CA).
Multiwell plates (EIA/RIA; Costar, Lowell, MA) were coated with purified recombinant 2009 H1N1 HA1 subunit protein at 10 μg/ml in coating buffer (0.1 M NaHCO3, pH 9.5). Plates were blocked with PBS containing 1% bovine serum albumin (BSA). After washing of the plates, the 2009 H1N1 candidate MAbs were added in duplicate at 5 μg/ml in PBS-0.1% Tween 20. After 1 h of incubation at room temperature, the binding of the MAbs to the immobilized HA1 protein was detected by anti-mouse IgG-HRP conjugate, followed by color development with BM Blue POD substrate (Roche, Mannheim, Germany). Optical density was read at 450 nm with a multiwell spectrophotometer (Molecular Devices, Sunnyvale, CA).
Clinical isolates and frozen specimens were inoculated into 96-well R-Mix plates or R-Mix Too shell vials (Diagnostic Hybrids, Inc., Athens, OH). The cells were centrifuged at 700 × g for 60 min and incubated overnight in a 35 to 37°C humidified 5.0% CO2 incubator. The cell monolayers were fixed with acetone for 10 min and then incubated with the 2009 H1N1-specific MAbs at 35°C to 37°C for 30 min. After washing of the cells with PBS, anti-mouse IgG-fluorescein isothiocyanate (FITC) was added, and the cells were incubated for another 15 to 30 min. Stained cells were washed with PBS and preserved with mounting fluid. Cells were examined at a magnification of ×200, using a Nikon TS100 microscope with an FITC wide-pass filter set and a 50-W mercury lamp to detect the presence of fluorescently stained cells. For direct specimen testing, cell suspensions were spotted onto each well of a 2-well slide, dried on a 37°C warming plate, and fixed with acetone. Duplicate slides were prepared for each clinical specimen, and two qualified lab technicians viewed the slides independently and without knowledge of the CDC rRT-PCR results.
Cells inoculated with virus isolates or frozen specimen and direct specimen slides were stained with D3 Ultra influenza A virus identification reagent (Diagnostic Hybrids, Inc., Athens, OH) according to the instructions in the product insert.
RNAs were isolated from clinical specimens by use of a QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA) according to the instructions in the product insert. Briefly, 140 μl of supernatant from clinical specimens or viral isolates was used for RNA isolation. RNA was eluted in a final volume of 60 μl. Five microliters was used for each rRT-PCR, following the CDC protocol for the detection of the 2009 H1N1 subtype (16
). As recommended by the CDC, all reactions used an Invitrogen Superscript III Platinum one-step quantitative kit and a Stratagene QPCR instrument. All RT-PCR runs included no-template controls (water) and positive-template controls (2009 H1N1 Mexico viral RNA). According to the CDC protocol, a sample was considered presumptively positive for 2009 H1N1 influenza A virus if both the influenza A virus (InfA) primers that target the matrix gene and the respective subtype-specific primers recognizing either the swine influenza A virus (swInfA) nucleocapsid gene or the H1N1 swine influenza A virus (swH1) hemagglutinin gene had amplification curves which crossed the threshold (CT
values) within 40 cycles.
The four presumptively positive specimens that failed to produce CT values with swH1 primers in the CDC rRT-PCR were amplified using either the CDC swH1 primers or the WHO sequencing primers. PCR amplicons derived from clinical specimens J769, J773, H464, and H465 were cloned using a TOPO T/A cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Sequencing was performed at the Ohio University Genomics Facility (Athens, OH). Sequence analysis was performed using Clone Manager Suite 7 (SciEd Central, Durham, NC).