Viral Plaque Assay

Vero cells (ATCC CCL-81) were cultured in Eagle’s minimal essential medium (EMEM; Lonza) with 8 % FCS (PAA) and antibiotics. Huh7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) containing 8 % FCS, 2 mM l-glutamine (PAA), non-essential amino acids (PAA) and antibiotics. Vero E6 and Calu3/2B4 cells were cultured as described previously (Snijder et al., 2006 (link); Yoshikawa et al., 2010 (link)). Infection of Vero, Vero E6, Huh7 and Calu3/2B4 cells with MERS-CoV (strain EMC/2012; Zaki et al., 2012; van Boheemen et al., 2012 (link)) at high m.o.i. (m.o.i. of 5) was carried out in PBS containing 50 µg DEAE-dextran ml⁻¹ and 2 % FCS. Inoculations with a low dose (m.o.i. ≤ 0.05) of MERS-CoV or SARS-CoV (strain HKU-39849; Zeng et al., 2003 (link)) were carried out directly in EMEM containing 2 % FCS. Virus titrations by plaque assay were performed as described previously (van den Worm et al., 2012 (link)). All work with live MERS-CoV and SARS-CoV was performed inside biosafety cabinets in Biosafety Level 3 facilities at Leiden University Medical Center or Erasmus Medical Center.

All procedures involving human subjects were reviewed and approved by the National Institute for Occupational Safety and Health (NIOSH) and West Virginia University (WVU) Institutional Review Boards. Written informed consent was obtained from all study participants.
Volunteer subjects were recruited from patients presenting with influenza-like symptoms at the student health clinic of WVU in Morgantown, West Virginia, USA, during October and November of 2009. After providing informed consent, each subject was given a rapid influenza test (QuickVue Influenza A+B test, Quidel). The rapid test was used to provide an initial estimate of influenza case numbers; however, because the sensitivity of the test was reported to be low [23] (link), subjects were allowed to continue participating in the study regardless of the outcome. Two nasopharyngeal mucus swabs were collected for analysis by qPCR and viral plaque assay (VPA), the subject's oral temperature was taken, and the subject was asked to answer a brief health questionnaire.
Cough-generated aerosols from the volunteer subjects were collected using the cough aerosol particle collection system (Figure 3) similar to that described previously [24] . The system consisted of an ultrasonic spirometer (Easy One, NDD Medical Technologies) and a 10 liter piston-style spirometer (SensorMedics model 762609) modified to allow aerosol collection using a NIOSH two-stage cyclone aerosol sampler [6] (link) or an SKC BioSampler with a 5 ml collection vessel (#225-9593, SKC). The NIOSH sampler collected cough aerosol particles in a 15 ml centrifuge tube (stage 1; #35-2096, Falcon), a 1.5 ml centrifuge tube (stage 2; #02-681-339, Fisher Scientific) and a 37 mm polytetrafluoroethylene (PTFE) filter with 2 µm pores (#225-27-07, SKC). The NIOSH sampler conforms to the ACGIH/ISO criteria for respirable particle sampling [25] . The flow rate through each NIOSH sampler was set to 3.5 liters/minute with a flow calibrator (Model 4143, TSI) before use. The SKC BioSampler collects aerosols into 5 ml of universal transport media (UTM; Copan Diagnostics) at 12.5 liters/minute.
Before each collection, the system was purged and the piston spirometer was partially filled with 5 liters of clean dry air. The subject was then asked to sit in front of the system, inhale, exhale, inhale as deeply as possible, seal their mouth around the mouthpiece, and cough into the machine using as much of the air in their lungs as possible. After each cough, the system valve was closed and the cough-generated aerosol was collected using the aerosol sampler. This procedure was repeated twice for a total of three coughs from each subject.
After collection, the nasopharyngeal swabs were immersed in 1 ml UTM in a storage tube. For the NIOSH samplers, 1 ml of UTM was added to each sampler tube, while the sampler filters were immersed in 1 ml UTM in a 50 ml centrifuge tube. For the SKC sampler, the UTM collection media was removed from the sampler and placed a storage tube. All tubes were vortexed thoroughly. 500 µl of UTM was then drawn from each tube and mixed with 500 µl of Lysis/Binding Solution Concentrate (LBSC; Ambion) in fresh tubes. The tubes with the remaining UTM were stored overnight at 4°C, while the tubes with UTM and LBSC were stored overnight at −20°C. In some cases, UTM was not used; instead, 500 µl of LBSC was added directly to each tube, and the tubes were stored overnight at −20°C.
To extract the sample RNA, tubes containing samples in LBSC were thawed, carrier RNA (Ambion) was added to enhance RNA extraction and XenoRNA (Applied Biosystems) was added as a qPCR internal control. Total RNA was extracted as previously reported [6] (link) and immediately transcribed into cDNA using High Capacity RNA to cDNA Master Mix (Applied Biosystems).
Real-time quantitative PCR was performed with a Model 7500 Fast Real-Time PCR system (Applied Biosystems) using influenza A matrix-specific primers and probe (Spackman, 2002).
To determine the relative genome copy from seasonal influenza A-positive aerosol samples, a standard curve was generated from 10-fold serial dilutions of the influenza M1 matrix gene and analyzed alongside all qPCR reactions. All reactions were run in duplicate. A negative control without template was included in all real-time PCR reactions. Real-time PCR detection of the XenoRNA internal control was performed using the XenoRNA Control TaqMan Gene Expression Assay from the TaqMan Cells to C_t Control Kit (Applied Biosystems). The internal controls were amplified in all samples.
For the viral plaque assay (VPA), Madin Darby canine kidney (MDCK) cells (CCL-34) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were propagated and maintained in 75-cm² flasks (Corning CellBind Surface, Corning, NY). Growth medium for MDCK cells consisted of Eagle's minimal essential medium (EMEM, ATCC) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc, Logan, Utah), 0.4 units/ml penicillin (Invitrogen, Carlsbad, CA), and 0.4 µg/ml streptomycin (Invitrogen). Cells were incubated at 35°C in a humidified 5% CO₂ incubator until about 90% confluent. The VPA was performed by trypsinizing, washing and plating MDCK cells at a density of 2.0×10⁶ per well (CoStar 6-well tissue culture plate, Corning). Cells were incubated at 35°C in a humidified 5% CO₂ incubator overnight. Confluent cellular monolayers were next washed two times with PBS (Invitrogen) and treated with the clinical samples. Following 45 min of adsorption, virus-infected MDCK cells were washed with phosphate buffered saline (PBS, Gibco), overlaid with an agarose medium solution and incubated at 35°C in a humidified 5% CO₂ incubator for 48 h. Plaques were visually enumerated and plaque forming units (PFU)/ml were calculated.
Initially, VPA's were performed only on nasopharyngeal swabs and cough aerosols from subjects with positive rapid influenza tests. After a few days, our preliminary results indicated that the rapid tests had a lower-than-expected sensitivity, and we changed our methodology to perform VPA's on all samples.

MRE16-eGFP and TR339-eGFP virus stocks were generated from SINV infectious cDNA clones with inserts of enhanced GFP (eGFP) driven by the second sub-genomic promoter, using standard methods [35 (link),46 (link),47 (link)]. MRE16-eGFP was passed once in baby hamster kidney (BHK-21) cells and twice in Aedes albopictus clone C6/36 cells. TR339 5'2J-eGFP was passed once in BHK-21 cells and three times in C6/36 cells.
For mosquito feedings, all viruses were diluted as indicated in de-fibrinated sheep blood and provided at 37°C in a water jacketed artificial feeder with parafilm membrane. An aliquot of each SINV stock was titered by viral plaque assay on Vero cells and the other aliquots were stored at -80°C and diluted prior to feeding. TR339-eGFP blood-meals contained approximately 3.3 × 10⁸ pfu/ml and MRE16 meals contained 2.2 × 10⁷ pfu/ml. Representative mosquitoes from each group were selected for dissection and eGFP detection with an Olympus epi-fluorescence microscope to confirm midgut infection with each batch of virus. Viral titers from individual mosquitoes were determined by plaque assays of filtered mosquito homogenates as previously described [29 (link)].

BALB/c mice (four-week-old, female) were purchased from Japan SLC (Shizuoka, Japan). The mice were divided into three groups with nine animals in each group. Mice were anesthetized and inoculated intranasally (20 µL) of rD/OK or rD/OK-AL (1.0 × 10⁵ PFU), respectively. Organs containing nasal turbinates, the tracheas, and the lungs were collected after three and six days following inoculation and homogenized in phosphate-buffered saline (PBS) for virus titration via the plaque assay.