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

Abstract

Less than 20 sporadic cases of human Zika virus (ZIKV) infection were reported in Africa and Asia before 2007, but large outbreaks involving up to 73% of the populations on the Pacific islands have started since 2007, and spread to the Americas in 2014. Moreover, the clinical manifestation of ZIKV infection has apparently changed, as evident by increasing reports of neurological complications, such as Guillain-Barré syndrome in adults and congenital anomalies in neonates. We comprehensively compared the genome sequences of pre-epidemic and epidemic ZIKV strains with complete genome or complete polyprotein sequences available in GenBank. Besides the reported phylogenetic clustering of the epidemic strains with the Asian lineage, we found that the topology of phylogenetic tree of all coding regions is the same except that of the non-structural 2B (NS2B) coding region. This finding was confirmed by bootscan analysis and multiple sequence alignment, which suggested the presence of a fragment of genetic recombination at NS2B with that of Spondweni virus. Moreover, the representative epidemic strain possesses one large bulge of nine bases instead of an external loop on the first stem-loop structure at the 3'-untranslated region just distal to the stop codon of the NS5 in the 1947 pre-epidemic prototype strain. Fifteen amino acid substitutions are found in the epidemic strains when compared with the pre-epidemic strains. As mutations in other flaviviruses can be associated with changes in virulence, replication efficiency, antigenic epitopes and host tropism, further studies would be important to ascertain the biological significance of these genomic changes.

Figure 1

Putative transmembrane domains in the Zika virus genome. Abbreviations: capsid, C; envelope, E; nonstructural, NS; pre-Membrane, prM.

Figure 2

Comparative genomic analysis of the representative pre-epidemic and epidemic Zika virus strains. The results represent all 24 Zika virus strains unless otherwise specified (i.e., nuclear localization signals, and O-glycosylation and N-glycosylations sites). For the polymorphic sites, analysis was performed for all 24 Zika virus strains, but only the results of the three representative pre-epidemic African (Uganda 1947), pre-epidemic Asian (Malaysia 1966) and the epidemic (Brazil, 2015) strains are shown here. (A) Capsid (C), pre-Membrane (prM) and envelope (E); (B) nonstructural (NS) 1, NS2A, NS2B and NS3; (C) NS4A, NS4B and NS5. Abbreviation: DEAD-like helicase superfamily, DEXDc.

Figure 2

Comparative genomic analysis of the representative pre-epidemic and epidemic Zika virus strains. The results represent all 24 Zika virus strains unless otherwise specified (i.e., nuclear localization signals, and O-glycosylation and N-glycosylations sites). For the polymorphic sites, analysis was performed for all 24 Zika virus strains, but only the results of the three representative pre-epidemic African (Uganda 1947), pre-epidemic Asian (Malaysia 1966) and the epidemic (Brazil, 2015) strains are shown here. (A) Capsid (C), pre-Membrane (prM) and envelope (E); (B) nonstructural (NS) 1, NS2A, NS2B and NS3; (C) NS4A, NS4B and NS5. Abbreviation: DEAD-like helicase superfamily, DEXDc.

Figure 3

Phylogenetic analysis of the ten structural and non-structural coding regions of 24 Zika virus strains, rooted by Spondweni virus, dengue virus serotype 2, and tick-borne encephalitis virus. The trees were constructed by the maximum likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. The bootstrap values were calculated from 500 trees. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The bootstrap values <60% are not shown. All Zika virus strains are labeled as follow: accession number/strain number/country/year. All strains represent Zika virus strains unless otherwise specified for Spondweni virus, dengue virus serotype 2 and tick-borne encephalitis virus. Abbreviation: nonstructural, NS.

Figure 4

Genome organization and bootscan analysis of the Zika virus genomes. (A) Bootscanning was conducted with Simplot version 3.5.1 on a gapless nucleotide alignment, which was generated with ClustalX with the genome sequences of the available Asian lineage Zika virus strains as the query sequences. (B) Multiple alignment of the recombination fragment in NS2B nucleotide sequences of Zika virus strains MR766/Uganda/1947 (NC_012532.1) and Natal RGN/Brazil/2015 (KU527068.1), and Spondweni virus (DQ859064.1). In the Zika virus strains, only the nucleotides differing from those in Spondweni virus are depicted. The nucleotides in the Zika virus strains are highlighted in blue or red. Abbreviations: capsid, C; envelope, E; nonstructural, NS; pre-Membrane, prM.

Figure 5

Schematic representations of the Zika virus genome RNA secondary structures. The short conserved 5′-ACAG-3′ sequences in the top loop of the sHP-3′ SL structure are indicated in red. Abbreviations: dumbbell, DB; open reading frame, sHP-3'-ORF; small hairpin 3'-stem-loop, sHP-3' SL; stem loop, SL; Y-shape stem-loop, SLA; untranslated region, UTR.

Figure 6

Terminal region genome sequences that are involved in 3′–5′ long distance RNA–RNA interactions. The three pairs of inverted complementary sequences that may mediate genome cyclization and allow the RdRp to reach the 3′ SL initiation site for RNA synthesis are enlarged. Abbreviations: dumbbell, DB; open reading frame, sHP-3' ORF; small hairpin 3'-stem-loop, sHP-3'-SL; stem Loop, SL; Y-shape stem-loop, SLA; untranslated region, UTR.