Viral Diagnostic Methods

Four lower respiratory tract samples, including bronchoalveolar-lavage fluid, were collected from patients with pneumonia of unknown cause who were identified in Wuhan on December 21, 2019, or later and who had been present at the Huanan Seafood Market close to the time of their clinical presentation. Seven bronchoalveolar-lavage fluid specimens were collected from patients in Beijing hospitals with pneumonia of known cause to serve as control samples. Extraction of nucleic acids from clinical samples (including uninfected cultures that served as negative controls) was performed with a High Pure Viral Nucleic Acid Kit, as described by the manufacturer (Roche). Extracted nucleic acid samples were tested for viruses and bacteria by polymerase chain reaction (PCR), using the RespiFinderSmart22kit (PathoFinder BV) and the LightCycler 480 real-time PCR system, in accordance with manufacturer instructions.12 Samples were analyzed for 22 pathogens (18 viruses and 4 bacteria) as detailed in the Supplementary Appendix. In addition, unbiased, high-throughput sequencing, described previously,13 was used to discover microbial sequences not identifiable by the means described above. A real-time reverse transcription PCR (RT-PCR) assay was used to detect viral RNA by targeting a consensus RdRp region of pan β-CoV, as described in the Supplementary Appendix.

Isolation of Virus

Bronchoalveolar-lavage fluid samples were collected in sterile cups to which virus transport medium was added. Samples were then centrifuged to remove cellular debris. The supernatant was inoculated on human airway epithelial cells,13 which had been obtained from airway specimens resected from patients undergoing surgery for lung cancer and were confirmed to be special-pathogen-free by NGS.14

Human airway epithelial cells were expanded on plastic substrate to generate passage-1 cells and were subsequently plated at a density of 2.5×105 cells per well on permeable Transwell-COL (12-mm diameter) supports. Human airway epithelial cell cultures were generated in an air–liquid interface for 4 to 6 weeks to form well-differentiated, polarized cultures resembling in vivo pseudostratified mucociliary epithelium.13

Prior to infection, apical surfaces of the human airway epithelial cells were washed three times with phosphate-buffered saline; 150 μl of supernatant from bronchoalveolar-lavage fluid samples was inoculated onto the apical surface of the cell cultures. After a 2-hour incubation at 37°C, unbound virus was removed by washing with 500 μl of phosphate-buffered saline for 10 minutes; human airway epithelial cells were maintained in an air–liquid interface incubated at 37°C with 5% carbon dioxide. Every 48 hours, 150 μl of phosphate-buffered saline was applied to the apical surfaces of the human airway epithelial cells, and after 10 minutes of incubation at 37°C the samples were harvested. Pseudostratified mucociliary epithelium cells were maintained in this environment; apical samples were passaged in a 1:3 diluted vial stock to new cells. The cells were monitored daily with light microscopy, for cytopathic effects, and with RT-PCR, for the presence of viral nucleic acid in the supernatant. After three passages, apical samples and human airway epithelial cells were prepared for transmission electron microscopy.

Transmission Electron Microscopy

Supernatant from human airway epithelial cell cultures that showed cytopathic effects was collected, inactivated with 2% paraformaldehyde for at least 2 hours, and ultracentrifuged to sediment virus particles. The enriched supernatant was negatively stained on film-coated grids for examination. Human airway epithelial cells showing cytopathic effects were collected and fixed with 2% paraformaldehyde–2.5% glutaraldehyde and were then fixed with 1% osmium tetroxide dehydrated with grade ethanol embedded with PON812 resin. Sections (80 nm) were cut from resin block and stained with uranyl acetate and lead citrate, separately. The negative stained grids and ultrathin sections were observed under transmission electron microscopy.

Viral Genome Sequencing

RNA extracted from bronchoalveolar-lavage fluid and culture supernatants was used as a template to clone and sequence the genome. We used a combination of Illumina sequencing and nanopore sequencing to characterize the virus genome. Sequence reads were assembled into contig maps (a set of overlapping DNA segments) with the use of CLC Genomics software, version 4.6.1 (CLC Bio). Specific primers were subsequently designed for PCR, and 5′- or 3′-RACE (rapid amplification of cDNA ends) was used to fill genome gaps from conventional Sanger sequencing. These PCR products were purified from gels and sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3130XL Genetic Analyzer, in accordance with the manufacturers’ instructions.

Multiple-sequence alignment of the 2019-nCoV and reference sequences was performed with the use of Muscle. Phylogenetic analysis of the complete genomes was performed with RAxML (13) with 1000 bootstrap replicates and a general time-reversible model used as the nucleotide substitution model.