Influenza virus
In addition to HIV, Influenza virus A is another classic example that
the viruses have zoonotic origins with associated public health concern
of host adaptation, permanently establishing in the human
population[64].
Varble and
colleagues[24]first barcoded influenza A virus, with which the authors attempted to
understand the probability and stochastic nature resulting from
different transmission routes. Varble and
colleagues[24]employed molecular barcodes on the genome of a model influenza A virus
to track viral transmission. The challenge to insert molecular barcodes
in the genome of influenza A virus is because the eight single-stranded
RNA segments are all required for virion production; therefore there is
little space for molecular manipulation. Previously Varble and
colleagues[65]have chosen to engineer segment 8, encoding the nonstructural protein 1
(NS1) and the nuclear export protein (NEP, also referred to as NS2)
because of the shorter of two viral transcripts, facilitating to give
any genetic material. Since NS1 and NEP use different reading frames
with the overlap between the C-terminal transcript of NS1 and the
N-terminal transcript of NEP, the authors thus disrupted the endogenous
splice acceptor site and placed it after the stop codon of
NS1[65](Figure 4A). This modified segment 8 thus harbored a noncoding
intergenic region where a string of 22 nucleotides of a molecular
barcode was inserted (Figure 4A). Barcodes were amplified from the 30
arm of a shRNA
library[66],
along with 100 base pairs of common flanking sequence (Figure 4A). The
authors confirmed that inserted molecular barcodes did not affect viral
replication in human lung epithelial cells.
Russel and
colleagues[67]utilized three different types of barcodes, cell barcodes, UMI and viral
barcodes to distinguish every single mRNA transcript based on the
single-cell droplet-based
platform[67,68](Figure 4B). Cell barcodes were used to distinguish one cell from
another; UMIs incorporated in primers were designed to separate each
mRNA molecule during the process of reverse transcription, allowing to
quantify the number of molecules of each mRNA that have been captured
for each cell. The challenge here is how to separate viral- and cellular
mRNA generated in the same cell. The authors thus engineered the viral
genomic sequence where two synonymous mutations were given in the
vicinity of the 3’ end of each transcript (Figure 4B) for this
discrimination. The transcription of the viral hemagglutinin from the
viruses carrying these synonymous mutations was compatible with the
wild-type
viruses[67],
indicating that synonymous mutations did not affect viral fitness. The
authors successfully used these viral barcodes to distinguish the viral
transcripts carrying mutations from the transcripts produced by the
wild-type
viruses[67].
By employing this three-barcodes approach, the authors established a
threshold determining truly infected cells and quantitatively
investigated stochastic gene expression of influenza viruses among
individual infected
cells[67].