Patients
The study was conducted during three consecutive winter seasons (November 2017–April 2020) at the department of pediatrics at the Reinier de Graaf Hospital, Delft, The Netherlands. Hospitalized children aged between 0 and 2 years with laboratory-confirmed RSV-A or RSV-B infections were both eligible. However, during these three seasons, only RSV-B positive patients were detected in the study. Patients were excluded if they were older than 2 years, or if signed informed consent was not obtained. For practical reasons, immunocompromised patients and patients that were experiencing symptoms for more than a week prior to hospitalization were not included. Patients with other co-morbidities, with viral co-infections, or use of medication, including antivirals, were not excluded, but this information was documented together with other clinical data obtained from the patient’s medical record using a standardized case record form, specifically developed for this study. Patients were hospitalized in single-patient rooms with an additional bed for parents. All patient rooms had a ventilation rate of 2 air changes per hour (ACH) with a fresh air transportation rate of 100 m3/h. Temperature and relative humidity were recorded every five minutes during the whole air sampling period using EL-GFX-2 dataloggers (Lascar Electronics Inc.).
Sample collection
For the collection of RSV from the air, the six-stage Andersen cascade impactor (Thermo Scientific™) was used [18]. The cascade impactor operates at a flow rate of 28.3 L per minute (LPM) and collects droplets and aerosols according to size in six different stages: Stage 1 (> 7.0 μm), stage 2 (7.0–4.7 μm), stage 3 (4.7–3.3 μm), stage 4 (3.3–2.1 μm), stage 5 (2.1–1.1 μm) and stage 6 (1.1–0.65 μm). The air inlet of the cascade impactor was positioned approximately 1 m away from the patient’s head at a height of 109 cm. Air was sampled daily for 30 min, starting the day after the informed consent was obtained, until discharge or a maximum of five days. To prevent contamination of samples, air sampling was performed in the morning, prior to routine care that may involve aerosol generating procedures like nasopharyngeal aspiration. Droplets and aerosols were impacted onto collection plates filled with an in-house developed semi-solid gelatin layer, as previously described [19]. The gelatin layer was prepared from commercial gelatin sheets (10 mg/ml; Dr. Oetker) dissolved in virus transport medium (VTM). VTM consisted of Minimum Essential Medium (MEM) – Eagle with Hank’s BSS and 25 mM Hepes (Lonza), glycerol 99 % (Sigma Aldrich), lactalbumin hydrolysate (Sigma Aldrich), 10 MU polymyxin B sulphate (Sigma Aldrich), 5 MU nystatin (Sigma Aldrich), 50 mg/ml gentamicin (Gibco) and 100 IU/ml penicillin 100 µg/ml streptomycin mixture (Lonza). To avoid high dilution factors, each collection plate was first filled with 32 ml of 2 % agarose (Roche) as a bottom layer on which 9 ml of the semi-solid gelatin was pipetted. Subsequently, plates were stored at + 4 °C for a maximum of 4 days.
On the last day of air sampling, surface swabs were taken using Copan flocked swabs (Copan Diagnostics Inc.) from the datalogger and the bed rail on the side where the cascade impactor was located. From the first day of air sampling onwards, nasopharyngeal aspirates (for decongestion) were obtained from patients during routine clinical care if available, and otherwise aspirates or nose swabs were taken specifically for this study if consent was given by the parents. From each parent, nose or throat swabs (either Copan eSwab® or Copan flocked swabs (Copan Diagnostics Inc.)) were taken on the first and last day of air sampling, when possible, to estimate their contribution to viral shedding in the air.
Sample processing
The collection plates with gelatin from the cascade impactor were processed after sampling by adding 6 ml of prewarmed VTM, followed by a 30 min incubation at 37 °C to dissolve the gelatin layer and harvesting of the samples [19]. Nose and throat swabs of parents were collected in Amies (eSwab®; Copan Diagnostics Inc.) or universal transport medium (UTM; flocked swabs; Copan Diagnostics Inc.). VTM was added to the nasopharyngeal aspirates of patients and nose and throat samples of parents to reach a total volume of 6 ml, after which nasopharyngeal aspirates were centrifuged at 500 G for 5 min to remove cell debris. All samples were subsequently aliquoted and stored at + 4 °C for a maximum of 4 days until further analysis. An additional vial of each sample was stored at -80 °C. The surface swabs were collected in UTM (flocked swabs; Copan Diagnostics Inc.), and stored in 25 % sucrose at -80 °C. At the end of the study, the samples were thawed and RNA was extracted followed by qRT-PCR analysis.
Cells
Hep-2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza or Gibco) supplemented with 10 % fetal bovine serum (FBS) (Greiner or Atlanta Biologicals), 100 IU/ml penicillin 100 µg/ml streptomycin mixture (Lonza), 200 mM L-glutamine (Lonza), 1.5 mg/ml sodium bicarbonate (Lonza), 10 mM HEPES (Lonza) and 0.25 mg/ml fungizone (Invitrogen). The cells were maintained at 37 °C and 5 % CO2.
RNA extraction and qRT-PCR
RNA was extracted from the samples using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) and subjected to qRT-PCR analysis for the detection of RSV-B RNA, and in case of the rhinovirus (RV) co-infected patients also for RV RNA [20]. For this purpose, 20 µl of extracted virus RNA was amplified in a final volume of 30 µl, containing 7,5 µl 4xTaqMan Fast Virus 1-Step Master Mix (Life Technologies) and 1 µl Primer/Probe mixture [20]. Amplification was performed using the following protocol: 5 min 50 °C, 20 s. 95 °C, 45 cycles of 3 s. 95 °C and 31 s. 60 °C. For all samples, a cycle threshold (Ct) value of > 40 was considered negative.
Virus titration
RSV-B-positive samples were titrated on Hep-2 cells for the quantification of infectious virus, as defined by the culturability of RSV. Briefly, Hep-2 cells were grown to confluency in 96 well plates overnight. Subsequently, cells were spin-inoculated (15 min, 2000 rpm) with 100 µl of 10-fold serial dilutions of RSV-B positive samples and incubated at 37 °C, 5 % CO2. One hour after inoculation, cells were washed once and cultured in serum-reduced (2 %) Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 100 IU/ml penicillin 100 µg/ml streptomycin mixture (Lonza), 200 mM L-glutamine (Lonza), 1.5 mg/ml sodium bicarbonate (Lonza), 10 mM HEPES (Lonza) and 0.25 mg/ml fungizone (Invitrogen). After 7 days of incubation, positive samples were identified by immunofluorescence assays using a FITC labelled polyclonal antibody directed against RSV (Fisher Scientific). Infectious virus titers were calculated from four replicates as tissue culture infective dose (TCID50) by the Spearman-Karber method.