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.
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.
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.
https://www.nejm.org/doi/full/10.1056/NEJMoa2001017
Extract