Zika virus
ZIKV is an arbovirus that consists of an envelope, positive-sense,
single-stranded RNA. Its genome encodes three structural proteins
(capsid, precursor of membrane and envelope) and seven non-structural
proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) responsible for RNA
replication[69].
Aliota and colleagues introduced a string of eight degenerate codons
where the third nucleotide of each codon is degenerated into a region of
the NS2A protein to generate as many synonymous mutations as possible
(Figure 5). This barcoded Zika virus was named
ZIKV-BC-1.0[26].
A multiplex-PCR approach was applied to sequence the entire coding
genome of the ZIKV-BC-1.0 library. Compared with its original strain,
ZIKV-IC, three single nucleotide mutations (site 1964, 8488 and 9581)
were detected outside of the barcode region in
ZIKV-BC-1.0[26].
Aliota and colleagues further used three different approaches to define
trustable barcodes referred to as authentic barcodes in their study.
Among these three approaches, the third approach (Approach āCā), with
which the authors acquired the highest threshold frequency (0.57%) was
placed to be the minimum threshold to identify authentic barcodes. By
using this threshold, 20 sequences in the sequencing output list were
considered to be
authentic[26].
Intriguingly, the first three barcodes (Zika_BC01, Zika_BC02 and
Zika_BC03) were more dominant than the rest of 17 barcodes when limited
input viral RNA was used for
amplification[26];
however at these stage it is still quite challenging to explain whether
barcode heterogeneity comes from a potential bias introduced by a
barcode itself or the evolutionary selection. The authors also claimed
that the absence of barcodes in sequencing reads could be due to the
fact that either barcodes were not present in certain biological samples
or the copy number of the barcodes were not sufficient to be detected by
sequencing, meaning that the presence of each barcode in ZIKV-infected
animals was not homogeneous. Viral infectivity and replication of
ZIKV-BC-1.0 showed no difference in vitro compared with Zika
infectious clone-derived- and wild-type
viruses[26],
indicating that in vitro additional degenerate nucleotides in the
viral genome did not cause a significant effect on viral fitness.In vivo three rhesus macaques were inoculated with ZIKV-BC-1.0 to
validate its replication capacity. Viruses were detectable in plasma one
day post inoculation (dpi) and viral load elevating between two and four
dpi was comparable to ZIKV-IC and wild-type
viruses[26],
indicating that viral infectivity and replication was not damaged by
molecular barcodes present in ZIKV-BC-1.0 in vivo , neither.