Comparative genomic analysis of pre-epidemic and epidemic Zika virus strains for virological factors potentially associated with the rapidly expanding epidemic

Zhu, Zheng; Chan, Jasper Fuk-Woo; Tee, Kah-Meng; Choi, Garnet Kwan-Yue; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Tse, Herman; Yuen, Kwok-Yung

The single-stranded RNA genomes of the ZIKV strains used in this study range from 10 675 to 10 808 nucleotides encoding 3423 amino acids. The G+C content is 50.94%-51.26%. Similar to other flaviviruses, the ZIKV genomes have two flanking UTRs (5ʹ-and 3ʹ-UTR) and a single long open reading frame encoding a polyprotein. A type I cap structure without internal ribosomal entry site is present at the 5ʹ end followed by the conserved dinucleotide 5ʹ-AG-3ʹ. The 3ʹ-end of the genome lacks a polyadenylate tail and terminates in a conserved 5ʹ-CU-3ʹ. The 5ʹ-and 3ʹ-UTRs are 107 and 429 nucleotides long, respectively. The 5ʹ-and 3ʹ-UTR sequences are conserved among ZIKV strains with nucleotide identities of ⩾ 83.6% and ⩾ 83.9%, respectively, but different from other flaviviruses including DENV, JEV, WNV, YFV, and TBEV (5ʹ-UTR, ⩽ 69.5%; 3ʹ-UTR, ⩽ 63.4%). The polyprotein is cleaved into ten structural and non-structural (NS) proteins. Similar to other flaviviruses, the coding region orders and NS protein motifs of ZIKV are arranged in the order of 5ʹ-Capsid (C)-preMembrane (prM)-Envelope (E)-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3ʹ. Cleavage at the N terminus of the signal sequence for NS4B generates a 23 amino acid peptide (the 2 K peptides at amino acid position 2243-2265). The complete polyprotein sequences of ZIKV have low similarity with those of other human-pathogenic flaviviruses (DENV-2, 58.1% to 58.9%; SPOV, 68.3% to 69.0% nucleotide similarity). The ten structural and NS proteins contain multiple transmembrane domains that determine their location on the cytoplasmic or luminal side of the endoplasmic reticulum after cleavage ( Figure 1 ). Most of the transmembrane domains are found in the NS2A, NS2B, NS4A, and NS4B proteins. This is compatible with the finding that the NS2A, NS2B, NS4A and NS4B proteins of other flaviviruses are mostly located within the endoplasmic reticular membrane bilayer, except for short regions between the transmembrane domains. 19 The other NS proteins include NS1, a putative protease/helicase (NS3), and a putative RNA-dependent RNA polymerase (RdRp; NS5), which are essential components or enzymes involved in viral replication. 19 Putative nuclear localization signals are found in C, NS1 and NS5 of both the pre-epidemic and epidemic strains, and NS3 of the prototype Uganda ZIKV strain ( Figure 2 ). The putative O-glycosylation and N-glycosylation sites are mostly conserved, except for some variations at C, prM, E, NS1 and NS5 for O-glycosylation sites, and at E, NS2A and NS5 for N-glycosylation sites, which may be due to previous intracranial passage of the pre-epidemic Uganda and Malaysia ZIKV strains in mice. 20 Amino acid substitutions Using all 24 available ZIKV genome sequences for analysis, the Ka/Ks ratios for the various coding regions were calculated (Table 2) . Overall, the Ka/Ks ratios in ZIKV genomes are low, with the highest being observed at C coding region (0.077), suggesting that all the genes in the ZIKV genome are likely under stabilizing selection. Comparison between the pre-epidemic Asian lineage (Malaysia, 1966) Phylogenetic relationship among all ZIKV strains Phylogenetic analysis of the ten putative structural and NS coding regions showed that the ZIKV strains were clustered into the African and the Asian lineages ( Figure 3 ). The epidemic ZIKV strains collected from the Pacific islands and South America clustered together with the Asian lineage strains. The complete polyprotein sequences of ZIKV w