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 ā€˜C’), 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.