In total, 23 full-length genomic sequences of ZIKV were included in the genomic tree, representing two lineages related to the geographic distribution of ZIKV as described previously; namely, the African lineage and the Asian lineage (Figure 1) (Haddow et al., 2012; Enfissi et al., 2016b). The Asian lineage includes ZIKV strains derived mainly from countries in Asia and the Americas, and the African lineage is composed only of ZIKV strains derived from Africa (Figure 1). Hence, ZIKV evolution is associated with its geographic distribution, although the African and Asian lineages evolved separately.
Figure 1. A phylogenetic tree of ZIKV strains based on their full-length genome sequences. Two lineages were identified and designated as the African lineage and the Asian lineage. The strains that were assigned to different phylogenetic positions among the full-length genomic tree, E tree, and NS5 tree (Figure 2) are labeled by black solid circles (●).
Robust E and NS5 trees were constructed by the NJ and ML methods, based on the E and NS5 coding regions available in GenBank (Figure 2). The NJ and ML trees presented identical topological structures. Besides the two main lineages, one more lineage was found in both the E tree (Figure 2A) and the NS5 tree (Figure 2B). This extra lineage was not found in the full-length genome tree, probably because few full-length genomic sequences of ZIKV are available in GenBank besides these new lineage sequences. According to the geographic distributions of the strains clustering into each lineage, the three lineages were designated as the Asian/American lineage, African lineage 1, and African lineage 2 (Figure 2). African lineage 2 evolved separately and diverged early from the other two lineages (Figure 2). In the E tree, it was composed of strains from Senegal in 1998 and 2001 and one strain from Cote d'Ivoire in 1980 (ArA1465) (Figure 2A). In the NS5 tree, the strain from Cote d'Ivoire (ArA1465) clustered in African lineage 1 (Figure 2B). These results indicated that primitive strains might circulate in Senegal and Cote d'Ivoire. Moreover, other strains from Senegal and Cote d'Ivoire were included in African lineage 1 (Figure 2A and B). These results suggested that at least two distinct lineages of ZIKV strains have co-circulated in these two countries.
Figure 2. Phylogenetic trees of ZIKV strains based on their envelope protein (E) coding regions (A) and non-structural protein 5 (NS5) coding regions (B). Three lineages were identified in the E and NS5 trees, and were designated as the Asian/American lineage, African lineage 1 and African lineage 2 according to their geographic distributions. The strains present at different phylogenetic positions among the full-length genomic tree (Figure 1), E tree, and NS5 tree are labeled by black solid circles (●). Chile/2014 A and Chile/2014 B represent the compressions of two clusters of strains identified from the Easter Island of Chile.
The global ZIKV epidemic appears to have been increasing in terms of the number of infections and spreading to new areas in recent years. Until Feb 2016, 73 countries and territories distributed in Africa, Europe, Asia, the Americas, and Oceania have confirmed individual ZIKV cases and ZIKV epidemics according to data from the CDC and WHO surveillance networks and national public health authorities (Figure 3A, Table 1). Autochthonous outbreaks of ZIKV were confirmed in most countries within the area at risk of DENV epidemics, which also coincided with the areas of CHIKV outbreaks (Figure 3A, indicated in purple color). Imported ZIKA infection cases were confirmed in 24 countries and territories (Figure 3A, indicated in yellow color, and Table 1), since the patients all had a history of travel to ZIKV epidemic areas. ZIKV isolates and strains with available genomic information were identified from 20 countries and territories (Table 1, Figure 3A, indicated in red characters).
Figure 3. The spatial and temporal distributions of ZIKV lineages worldwide. (A) A world map illustrating the countries and territories that have reported confirmed ZIKV infections. Countries and territories with autochthonous ZIKV outbreaks are indicated in purple color, while those with confirmed imported cases are indicated in yellow color. Countries and territories that have reported ZIKV outbreaks and confirmed cases are labeled with their abbreviated names as shown in Table 1. Countries and territories that could be traced from the annotation of the complete or partial sequences of ZIKV strains deposited in GenBank are labeled in red characters. The areas at risk of DENV infection are delineated by the geographic limits between the northern and southern hemispheres indicated by the dash lines in dark red color. The countries and areas at risks of CHIKV infection are delineated by the dash lines in cyan color. (B) A summary of the numbers of ZIKV strains from different lineages found in different geographic areas. (C) The emergence of ZIKV lineages over time. The periods of time (marked Ⅰ and Ⅱ) of the African lineage 1 epidemics are indicated by a light cyan background color; that of the African lineage 2 epidemics by a lavender background color; and that of the Asian/American lineage epidemics by a vermilion background color. The annotations Asian/American, African 1, and African 2 denote the lineages of the same names.
Table 1. Summary of the countries and territories with a history of ZIKV disease and reported cases
The phylogenetic lineages from different geographic distributions were summarized based on the combined datasets of both E and NS5 sequences (Figure 3B). Viruses of the Asian/American lineage were circulating mainly in American and Asian countries as well as the Pacific Islands, while viruses of African lineages 1 and 2 were circulating in African countries. The prevalence of ZIKV lineages over time was also characterized based on the information of the strain sequences based on the combined datasets (Figure 3C). Outbreaks and epidemics of ZIKV strains of African lineage 1 were identified from 1947 to 1997 as well as in 2002 and 2007 (Figure 3C, light cyan pattern Ⅰ and Ⅱ), whereas only two Asian/American lineage strains was isolated, one from Malaysia in 1966 (Marchette et al., 1969) and the other from Yap Island in the Federated States of Micronesia in 2007 (Lanciotti et al., 2008). Then the African lineage 2 strains from Senegal were identified from 1998 to 2001 (Figure 3C, lavender pattern). Since 2010, ZIKA diseases were reported to be caused by strains of the Asian lineage (Figure 3C, vermilion pattern).
Three lineages were identified according to our phylogenetic analysis. However, two strains had different phylog-enetic positions within lineages and clusters among the full-length genomic tree, the E tree, and the NS5 tree (Figure 1, Figure 2A and Figure 2B, black solid circles, and Supplementary Table S1). Strain ArD157995 from Senegal in 2001 was assigned to African lineage 1 in the three trees, but presented great changes of phylogenetic positions between the E and NS5 trees. It is mostly close to the strains from Senegal in 1997 in the E tree (Figure 2A) while it clusters with the strains from Coted'Ivoire in 1999 in the NS5 tree (Figure 2B). Although it could not be included in the full-length genomic tree due to insufficient sequence data, strain ArA1465 from Cote d'Ivoire in 1980 was assigned to African lineage 2 in the E tree and African lineage 1 in the NS5 tree. These results suggested that recombination events may have contributed to the phylogenetic differences among ZIKV strains.
The recombination events were evaluated by the RDP software package, which identified three recombination events within the NS5 coding region to generate three recombinants: ArD157995, ArA1456, and ArD158084 (Table 2). Recombination hotspots were identified at nucleotide positions 9652, 9754, 10314, and 10315 (Table 2). The recombinants produced by recombination events 1 and 2, ArD157995 and ArA1456, were identical to the two strains found by visual comparison (>Supplementary Table S1), which further suggested that recombination within NS5 might have resulted in the phylogenetic shifts of ZIKV. Furthermore, each recombinant had major and minor parents, and at least one parent of each recombinant was identified from a different country than the respective recombinant (Table 2). Two of the three recombinants were identified from Senegal, while the other derived from Cote d'Ivoire, and four of the six parents were from Senegal, while one was isolated from Cote d'Ivoire. This indicated that recombination is more likely to occur within the genomes of the ZIKV strains from different lineages co-circulating in Senegal and Cote d'Ivoire, and that migration was more likely to happen between these two countries than among others.
Methods RDPRCS Begin End 1 ArD157995
GENECONV, MaxChi, RDP, SiScan, 3Seq, Chimaera 0.682 9754 10314 2 ArA1456 Cote
SiScan, 3Seq, Chimaera 0.594 9672 10315 3 ArD158084
ArB15076 Central African
3Seq, MaxChi 0.685 9672 10315 Note: A1, African lineage 1; A2, African lineage 2.
Table 2. The recombination events of ZIKV detected by the RDP software package
Migration events and pathways among the ZIKV epidemic countries and territories were characterized (Figure 4). Migration events showing statistical significance (P < 0.01) included one from Brazil to Suriname, two from Cote d'Ivoire to Senegal, and one from Cote d'Ivoire to Uganda (Figure 4, red arrows). Possible migration events (0.01 < P < 0.05) were detected from Senegal as the exporting country, including single events to Nigeria, Malaysia, Philippines, Canada, Cambodia, Thailand, and the Pacific island s, and three events to Cote d'Ivoire (Figure 4, green arrows in dash lines). Therefore, first, Senegal and Cote d'Ivoire are the two countries that have most frequently imported and exported ZIKV, which is consistent with the results of recombination detection showing that the involved strains are mainly from these two countries (Table 2). We speculate that the exchange of ZIKV between Senegal and Cote d'Ivoire might have resulted in the co-circulation of viruses from distinct lineages and thus promoted the recombination of viruses to generate distinct genotypes. Second, Senegal was detected as the exporting country responsible for the possible migration of ZIKV to other countries outside Africa (Figure 4). Thus, Senegal may be the geographic origin of the ZIKV outbreaks outside Africa, which indicates the important role of Senegal in the spread of ZIKV to other countries throughout the history of ZIKV epidemics.
Figure 4. A world map showing the ZIKV migration events and pathways. Countries and areas from which ZIKV strains with complete or partial sequences are available in GenBank are labeled with their abbreviated names as defined in Table 1. The confirmed migration pathways(P < 0.01) from the exporting to the importing countries are indicated by red arrows. The possible migration pathways(0.01 < P < 0.05) are indicated by green arrows in dash lines. The frequency of migration events is illustrated by the thickness of the lines.
Three lineages of ZIKV strains were identified by phylogenetic analysis
The spatial and temporal distributions of the epidemics caused by each ZIKV lineage.
Recombination events in ZIKV indicated potential migration routes
Analysis of the ZIKV migration pathway revealed important roles of Senegal and Cote d'Ivoire in the spread of ZIKV
Strains Locations Time Lineages Full-length E gene NS5 gene ArD157995 Senegal 2001 A1 A1 A1 ArA1465 Cote d'Ivoire 1980 N/A A2 A1 Notes: A1, African lineage 1; A2, African lineage 2; N/A, not applicable.
Table Table S1. Strains of discrepancies among tree topology by visual comparison