Overnight Titration of Human Respiratory Syncytial Virus Using Quantitative Shell Vial Amplification

The ability to reliably titrate human respiratory syncytial virus (RSV) is important in the development and evaluation of new antiviral drugs and for the facile manipulation of this virus in clinical and basic research endeavors. Since the isolation of human RSV by Chanock et al. in 1957 (3), investigators have relied on endpoint dilution or conventional plaque assay methods for its titration. The latter is considered to be more accurate (10). In RSV plaque assays, plaques that can be macroscopically counted in 9 days under agar overlays (7) or in 13 days under a liquid overlay (8) give reliable and reproducible titrations of a stock aliquot of virus. More rapid systems have also been described, in which syncytia or plaques can be microscopically enumerated in tissue culture or under a semi-fluid overlay of methylcellulose (4) in as few as 3 days. A microassay using a 3–7-day incubation period has also been described (10). We report the development of a simple and rapid titration assay for RSV using a quantitative shell vial approach. This assay is easy to perform, and the titration results are available in 16 h. To prepare the viral stocks, RSV type B (RSV-B), of the family Paramyxoviridae (ATCC VR-1401), and HEp-2 laryngeal carcinoma cells were obtained from ATCC (Rockville, MD, USA). HEp-2 cells were maintained in RPMI 1640 Medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine and antibiotics (c-RPMI). RSV-B was used to inoculate 180-cm2 flasks containing semi-confluent (ca. 60%) monolayers of HEp-2 cells in 50 mL media. When cytopathic effect, determined by visual inspection of syncytia formation, reached approximately 80%, the supernatants were harvested by centrifugation at 800× g to pellet cellular debris, then flash-frozen in 1mL aliquots and stored at -80°C. Six different stock RSV aliquots were used for the experiments described. The shell vial titration assay uses 1dram shell vials containing round coverslips with confluent monolayers of HEp-2 cells (Viromed, Minneapolis, MN, USA). Monolayers were inspected for confluency before use. Serial twofold dilutions of the virus stock were prepared in c-RPMI (1:50 to 1:3200). Shell vials were inoculated with 200 μL of virus from each dilution, then centrifuged at 700× g at 22°C for 1 h. Aliquots of the same dilution were used in standard plaque assays (described below). One milliliter of maintenance medium was added, and the shell vials were incubated at 37°C for 16 h and then fixed with acetone for 20 min. Immunofluorescence staining was performed by 60-min incubations of mouse anti-RSV fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (CHEMICON International, Temecula, CA, USA). Stained coverslips were mounted onto slides with FA Mounting Fluid, pH 7.2 (Difco Laboratories, Detroit, MI, USA). Each coverslip was observed using fluorescence microscopy at 200× magnification, and the number of fluorescent cells per coverslip was determined. Each dilution was performed in triplicate. The RSV plaque titration assay is a standard plaque assay that was performed on the same RSV dilutions that were used to inoculate the shell vials. Each plaque assay was matched to a shell vial and inoculated with the same dilution of RSV. The assay was performed as described by Kisch and Johnson in 1963 (7). Briefly, HEp-2 cells seeded at 4 × 105 cells/mL in 6well tissue culture plates were allowed to grow for 48 h. The medium was removed, and 1 mL of the appropriate RSV dilution (twofold serial dilutions in c-RPMI from 1:50 to 1:3200) was added to the cell monolayer. The virus was allowed to adsorb for 1 h at 37°C, and the plates were tilted every 15 min to distribute the inoculum evenly over the surface. Three milliliters of 0.75% overlay medium (1.5% molten agarose added to an equal volume of prewarmed c-RPMI) were added to each well and swirled gently to mix. Plates were incubated at 37°C in 5% CO2 for 4 days, at which time a second (0.5%) overlay medium was added. Incubation was continued for 4 more days, then the cell monolayers were fixed in 10% formaldehyde, and the agarose plugs were removed and stained for 5 min with 0.03% aqueous methylene blue.