Propagation of SARS-CoV-2 in Calu-3 Cells to Eliminate Mutations in the Furin Cleavage Site of Spike.

SARS-CoV-2 pathogenesis, vaccine, and therapeutic studies rely on the use of animals challenged with highly pathogenic virus stocks produced in cell cultures. Ideally, these virus stocks should be genetically and functionally similar to the original clinical isolate, retaining wild-type properties to be reliably used in animal model studies. It is well-established that SARS-CoV-2 isolates serially passaged on Vero cell lines accumulate mutations and deletions in the furin cleavage site; however, these can be eliminated when passaged on Calu-3 lung epithelial cell lines, as presented in this study. As numerous stocks of SARS-CoV-2 variants of concern are being grown in cell cultures with the intent for use in animal models, it is essential that propagation methods generate virus stocks that are pathogenic in vivo. Here, we found that the propagation of a B.1.351 SARS-CoV-2 stock on Calu-3 cells eliminated viruses that previously accumulated mutations in the furin cleavage site. Notably, there were alternative variants that accumulated at the same nucleotide positions in virus populations grown on Calu-3 cells at multiple independent facilities. When a Calu-3-derived B.1.351 virus stock was used to infect hamsters, the virus remained pathogenic and the Calu-3-specific variants persisted in the population. These results suggest that Calu-3-derived virus stocks are pathogenic but care should still be taken to evaluate virus stocks for newly arising mutations during propagation.

A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells.

An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

Reproducibility and validity of a handheld spirometer.

BACKGROUND: Handheld spirometers have several advantages over desktop spirometers, but worries persist regarding reproducibility and validity of data from handheld spirometers. We undertook an independent examination of the EasyOne handheld spirometer. METHODS: The laboratory testing included reproducibility and validity testing with a waveform generator. We used standard American Thoracic Society waveforms for in-line testing, calibration adaptor testing, and testing during compression of the mouthpiece. The clinical testing involved repeated tests with 24 spirometry-naïve volunteers and comparison to spirometry results from laboratory (volume-sensing dry rolling seal) spirometer. RESULTS: The EasyOne exceeded standard thresholds for acceptability with the American Thoracic Society waveforms. In-line testing yielded valid results from the EasyOne. Between the EasyOne and the reference spirometer readings the mean +/- SD difference was 0.03 +/- 0.23 L for forced vital capacity (FVC) and -0.06 +/- 0.09 L for forced expiratory volume in the first second (FEV(1)). The calibration adaptor showed no appreciable problems. Extreme compression of the mouthpiece reduced the measured values. In clinical testing the coefficients of variation and limits of agreement were, respectively, 3.3% and 0.24 L for FVC, 2.6% and 0.18 L for FEV(1), and 1.9% and 0.05 for the FEV(1)/FVC ratio. The EasyOne readings were lower than those from the reference spirometer; the differences were: -0.12 L for FVC, -0.17 L for FEV(1), and -0.02 for FEV(1)/FVC. The limits of agreement were within criteria for FVC but not for the FEV(1), possibly due to a training effect. CONCLUSION: The EasyOne spirometer yielded generally reproducible results that were generally valid, compared to the values from the laboratory spirometer. The use of the EasyOne in clinical, occupational, and research settings seems justified.

Exhaled nitric oxide measurement in workers in a microwave popcorn production plant.

Airways obstruction in microwave-popcorn workers has been attributed to inhalation of flavoring agents. Two former workers at a microwave-popcorn plant were found by lung biopsy to have bronchiolitis obliterans. The study's aim was to determine whether exhaled nitric oxide (FENO) levels were associated with exposure levels, respiratory symptoms, or airways obstruction. A questionnaire, spirometry, and FENO measurements were completed by 135 workers. The FENO levels of workers with high flavoring exposures (n = 107) were compared with those of workers with low exposures (n = 28) and healthy external controls (n = 31). FENO levels were significantly lower in the high-exposure group (p < 0.05). There is no indication that FENO is useful as a marker of lung injury in a flavoring-exposed worker population with a substantial lung disease burden, but the finding of low FENO in the high-exposure group should not be dismissed.

Induced sputum evaluation in microwave popcorn production workers.

OBJECTIVE: Severe airways obstruction and bronchiolitis obliterans have been reported in microwave popcorn production workers and attributed to inhalation of flavoring agents. We investigated whether exposure to flavoring agents is associated with airways inflammation in popcorn production workers. METHODS: Fifty-nine workers with high exposures and 22 patients with low exposures to flavoring vapors completed a questionnaire, spirometry, and sputum induction. Sputum cell counts were categorized as "high" if greater than (and "low" if less than or equal to) the median cell counts of a healthy external control group (n = 24). We compared high- and low-exposure groups as well as all workers with control subjects. RESULTS: Neutrophil concentrations in nonsmoking workers were significantly higher than those of the healthy nonsmoking control group (p < 0.05). The smoking-adjusted odds ratio for high neutrophil count (> 1.63 x 10(5)/mL) was 3.8 (95% confidence interval, 1.3 to 11.5) in the high-exposure group compared with the low-exposure group. Sputum interleukin-8 and eosinophil cationic protein levels were higher in high-exposure workers than in low-exposure workers (p < 0.05). For the worker group, mean values of FEV1 percentage of predicted and FEV1/FVC percentage of predicted were > 95%. There were no relationships between sputum characteristics and the presence of airways obstruction. CONCLUSIONS: High exposure to popcorn flavoring agents is associated with neutrophilic airway inflammation in popcorn production workers. These data provide further evidence that popcorn production workers face a significant occupational hazard through exposure to flavoring agents.

Nonlinear model for offline correction of pulmonary waveform generators.

Pulmonary waveform generators consisting of motor-driven piston pumps are frequently used to test respiratory-function equipment such as spirometers and peak expiratory flow (PEF) meters. Gas compression within these generators can produce significant distortion of the output flow-time profile. A nonlinear model of the generator was developed along with a method to compensate for gas compression when testing pulmonary function equipment. The model and correction procedure were tested on an Assess Full Range PEF meter and a Micro DiaryCard PEF meter. The tests were performed using the 26 American Thoracic Society standard flow-time waveforms as the target flow profiles. Without correction, the pump loaded with the higher resistance Assess meter resulted in ten waveforms having a mean square error (MSE) higher than 0.001 L2/s2. Correction of the pump for these ten waveforms resulted in a mean decrease in MSE of 87.0%. When loaded with the Micro DiaryCard meter, the uncorrected pump outputs included six waveforms with MSE higher than 0.001 L2/s2. Pump corrections for these six waveforms resulted in a mean decrease in MSE of 58.4%.