Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus

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1 VIROLOGICA SINICA 2016, 31 (2): DOI: /s RESEARCH ARTICLE Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus Shu Shen, Junming Shi, Jun Wang, Shuang Tang, Hualin Wang, Zhihong Hu, Fei Deng * State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan , China Recent outbreaks of Zika virus (ZIKV) infections in Oceania's islands and the Americas were characterized by high numbers of cases and the spread of the virus to new areas. To better understand the origin of ZIKV, its epidemic history was reviewed. Although the available records and information are limited, two major genetic lineages of ZIKV were identified in previous studies. However, in this study, three lineages were identified based on a phylogenetic analysis of all virus sequences from GenBank, including those of the envelope protein (E) and non-structural protein 5 (NS5) coding regions. The spatial and temporal distributions of the three identified ZIKV lineages and the recombination events and mechanisms underlying their divergence and evolution were further elaborated. The potential migration pathway of ZIKV was also characterized. Our findings revealed the central roles of two African countries, Senegal and Cote d'ivoire, in ZIKV evolution and genotypic divergence. Furthermore, our results suggested that the outbreaks in Asia and the Pacific islands originated from Africa. The results provide insights into the geographic origins of ZIKV outbreaks and the spread of the virus, and also contribute to a better understanding of ZIKV evolution, which is important for the prevention and control of ZIKV infections. KEYWORDS migration Flaviviridae; Zika virus (ZIKV); phylogenetic analysis; lineages; recombination; INTRODUCTION Received: 18 March 2016, Accepted: 21 April 2016, Published online: 26 April 2016 *Correspondence: Phone: , Fax: df@wh.iov.cn ORCID: X Zika virus (ZIKV) is an emerging pathogen, transmitted by Aedes species, that belongs to the genus Flavivirus of the family Flaviviridae. ZIKV-infected patients usually have symptoms characterized by fever, headache, rash, myalgia, arthralgia, and conjunctivitis, which are similar to the clinical symptoms caused by Dengue virus (DENV) or Chikungunya virus (CHIKV) infection. Thus, ZIKV could be easily misdiagnosed as Dengue (DEN) or Chikungunya (CHIK) fever (Haddow et al., 2012). A body of evidence has shown that ZIKV infection is associated with the neurological disorders microcephaly (Mlakar et al., 2016) and Guillain Barré syndrome (Oehler et al., 2014). Further, not only has the number of ZIKV cases increased in recent years, but the disease has spread to new regions that had no previous record of Zika disease. Thus, Zika disease has become a serious public health problem. The worldwide dispersal and transmission of ZIKV should be of great concern to researchers, and their study will contribute to the prevention and control of outbreaks and epidemics of Zika disease. ZIKV first emerged in Africa. It was first isolated in 1947 from a sentinel monkey in Uganda (Dick et al., 1952). The first ZIKV infection in human was identified in Uganda and Tanzania in 1952 (Smithburn, 1952). ZIKV was detected in humans, Aedes species and animals in African countries in the following decades (Macnamara, 1954; Simpson, 1964; Faye et al., 2014). Then, ZIKV spread to Southern Asia. It was isolated from Ae- 118 A PRIL 2016 VOLUME 31 ISSUE 2 Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2016

2 Shu Shen et al des aegypti in Malaysia in 1966 (Marchette et al., 1969). The wide distribution of ZIKV in Indonesia, Malaysia, and Pakistan was further revealed by serological investigations (Kindhauser et al., 2016). Subsequently, ZIKV spread to countries in Oceania and the Americas. The first large outbreak of Zika disease in humans occurred on the Pacific island of Yap in the Federated States of Micronesia in 2007 (Lanciotti et al., 2008; Duffy et al., 2009). ZIKV was then identified from Suriname (Enfissi et al., 2016b) in South America, and on other Pacific Islands including French Polynesia (Berthet et al., 2014; Cao-Lormeau et al., 2014; Hancock et al., 2014), Easter Island of Chile (Tognarelli et al., 2015), the Cook Islands (Roth et al., 2014), and New Caledonia (Roth et al., 2014; Dupont-Rouzeyrol et al., 2015). Imported cases were also identified in countries of the Americas and Europe (Foy et al., 2011; Waehre et al., 2014; Zammarchi et al., 2015; Kindhauser et al., 2016). Until March 2016, a total of 10 imported infectious cases had been diagnosed in China. A brief review of ZIKV s history found that ZIKV migration seems to have followed the same path as CHIKV and DENV, which were first described in Africa and then spread to Asia before becoming distributed worldwide (Musso et al., 2015). Although the future is unpredictable, ZIKV has the potential to spread globally through the increasing international movement of humans, goods, and animals for travel and business, and could represent a major threat to human health. Investigating the migration pathways of ZIKV can help to shed light on the origins of ZIKV epidemics. Two major genetic lineages of ZIKV strains associated with its geographic distribution, namely the African and Asian lineages, have been characterized and limited ZIKV nucleic acid sequences are available (Haddow et al., 2012; Faye et al., 2014). The strains identified from recent epidemics in the Americas belonged to the Asian lineage (Enfissi et al., 2016b), suggesting that strains from Asia and the Americas are closely related. However, as only limited nucleic acid sequences of ZIKV strains are available in GenBank for phylogenetic analysis and other bio-informatics analyses (Haddow et al., 2012; Faye et al., 2014), knowledge of the genetic relationships of ZIKV strains and the geographic origins of ZIKV epidemics is limited. This study focuses on the phylogenetic relationships among ZIKV strains, the spatial and temporal distributions of ZIKV lineages, and the potential recombination and migration of ZIKV among different countries and territories. The results reveal the geographic origins and migration patterns of ZIKV epidemics, which will be further discussed. MATERIALS AND METHODS Sequence alignment and construction of phylogenetic trees ZIKV sequences deposited in GenBank until February 4, 2016 were used for phylogenetic analysis. Sequences were aligned by CLUSTAL W (MEGA 5.0). Phylogenetic trees were constructed for the full-length genome, the envelope protein (E) coding region, and the nonstructural protein 5 (NS5) coding region by MEGA 5.0 using both the neighbor-joining (NJ) and maximum-likelihood (ML) methods, and tested by the bootstrap method with 1000 replicates. The sequence of Spondweni virus (GenBank accession number: DQ859064) was included as the outgroup control. Recombination detection Recombination events were detected by the RDP software package using seven recombination detection methods (Martin et al., 2010) based on the concatenate sequences of E protein coding region and NS5 protein coding region. Recombination events that were significant by at least two methods (P < 0.05) and had an RDP recombination consensus score (RDPRCS) of more than 0.60 were considered to be confirmed events. Recombination events that were significant by at least two methods (P < 0.05) and had an RDPRCS score between were considered to be possible events. Events that did not meet these criteria were rejected. Virus migration pathway analysis The ZIKV spread pathway was analyzed using the MI- GRAPHYLA package (Wallace et al., 2007) based on the NS5 tree. The reliability of each migration event was evaluated using a Monte Carlo simulation process by randomizing the location of the leaf nodes while retaining the tree topologies. RESULTS Three lineages of ZIKV strains were identified by phylogenetic analysis 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. 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 APRIL 2016 VOLUME 31 ISSUE 2 119

3 Roles of African nations in Zika virus evolution and spread 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 ( ). 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 2B). These results suggested that at least two distinct lineages of ZIKV strains have co-circulated in these two countries. The spatial and temporal distributions of the epidemics caused by each ZIKV lineage. 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). 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 120 APRIL 2016 VOLUME 31 ISSUE 2 VIROLOGICA SINICA

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5 Roles of African nations in Zika virus evolution and spread 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. 122 APRIL 2016 VOLUME 31 ISSUE 2 VIROLOGICA SINICA

6 Shu Shen et al 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. 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 APRIL 2016 VOLUME 31 ISSUE 2 123

7 Roles of African nations in Zika virus evolution and spread Table 1. Summary of the countries and territories with a history of ZIKV disease and reported cases Country Abbreviation Year References Africa Burkina Faso* BF 1981 Faye et al., 2014 Cape Verde CV 2015 Kindhauser et al., Central African 1968, 1976, CF Republic* 1979, 1980 Saluzzo et al., 1981; Berthet et al., 2014 Cote d Ivoire* CI , 1999 Faye et al., 2014 Egypt EG 1954 Smithburn et al., 1954 Gabon* GA 1975, 2007 Jan et al., 1978; Grard et al., 2014 Nigeria* NG 1953, Macnamara, 1954; Moore et al., 1975; Kindhauser et al., 2016 Senegal* SN 1960s 1970s, 1990s 2000s Faye et al., 2014; Kindhauser et al., 2016 Tanzania TZ 1952 Smithburn, 1952 Uganda* UG 1947, 1952, 1963 Dick et al., 1952; Smithburn, 1952; Faye et al., 2014 Colombia CO 2015 Commonwealth of Puerto Rico Enserink, 2015; Kindhauser et al., PR Costa Rica CR 2016 Americas Barbados BB Bolivia BO Brazil* BR 2015 Waggoner and Pinsky, Canada * CA 2014, 2016 Fonseca et al., Chile mainland CL 2016 Kindhauser et al., Dominican Republic DO 2016 Curacao (Netherlands Antilles) Curacao(AN) Easter Island (Chile)* Easter Island (CL) 2014 Tognarelli et al., 2015 Ecuador EC El Salvador SV French Guiana and Martinique (France) GF and MQ Guatemala GT Guyana GY Haiti HT Honduras HN Jamaica JM Mexico* MX Nicaragua NI To be continued 124 APRIL 2016 VOLUME 31 ISSUE 2 VIROLOGICA SINICA

8 Shu Shen et al Continued Country Abbreviation Year References Panama PA 2015 Paraguay PY 2015 Saint Martin and Guadeloupe (France) Saint Martin and GP 2016 Suriname* SR 2015 United States of US America United States Virgin Islands Enfissi et al., 2016a november-2015-zika/en/ Duffy et al., 2009; Foy et al., microcephaly-usa/en/ VI (US) Venezuela VE 2015 Asia Cambodia* KH 2010 Heang et al., 2012 Guangdong (China) Guangdong (CN) India IN N/A Kindhauser et al., 2016 Indonesia ID 1977, 1981, 2012 Olson et al., 1981; Olson et al., 1983; Kindhauser et al., 2016 Israel IL Malaysia* MY 1966 Pond, 1963; Marchette et al., 1969; Kindhauser et al., 2016 Pakistan PK 1983 Darwish et al., 1983 Philippines* PH 2012 Alera et al., 2015 Taiwan (China) Taiwan (CN) Thailand* TH Pond, 1963; Buathong et al., 2015; Kindhauser et al., 2016 Vietnam VN N/A Pond, Europe Austria AT Denmark DK Finland FI Germany DE 2014, 2016 Tappe et al., Italy IT 2015 Zammarchi et al., 2015 Netherlands NL Norway * NO 2013 Waehre et al., 2014 Portugal PT 1972 Filipe et al., 1973 Spain ES _ html Sweden SE a-virus-sweden-case.html Switzerland CH United Kingdom UK APRIL 2016 VOLUME 31 ISSUE 2 125

9 Roles of African nations in Zika virus evolution and spread Continued Country Abbreviation Year References Cook Islands* CK 2014 Roth et al., 2014 Oceania American Samoa AS Australia AU 2015 Leung et al., 2015 Fiji FJ 2015 Kindhauser et al., 2016 French Polynesia* PF 2013 Baronti et al., 2014; Cao-Lormeau et al., 2014; Hancock et al., 2014 Marshall Islands MH New Caledonia* NC 2013, 2014 Roth et al., 2014; Dupont-Rouzeyrol et al., 2015 Samoa WS 2015, 2016 Kindhauser et al., Solomon Islands SB 2015 Kindhauser et al., 2016 Tonga TO Vanuatu VU 2015 Kindhauser et al., 2016 Yap Island (Micronesia) Yap Island(FM) 2007 Lanciotti et al., 2008; Duffy et al., 2009 Countries and territories where the imported cases were reported. *Countries and territories where the complete or partial ZIKV sequences were identified and deposited in GenBank. The year that ZIKV was detected in India is uncertain. It was only mentioned by a WHO research article found online (Kindhauser et al., 2016) that during the geographic distribution of ZIKV expanded to equatorial Asian countries, including India, Indonesia, Malaysia, and Pakistan. The year that ZIKV was detected in Vietnam is uncertain. The previous study (Pond, 1963) investigated the serological prevalence of antibodies against ZIKV among the population in Vietnam before (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). Recombination events in ZIKV indicated potential migration routes Three lineages were identified according to our phylogenetic analysis. However, two strains had different phylogenetic 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 ArD 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 Cote d 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 ArD (Table 2). Recombination hotspots were identified at nucleotide positions 9652, 9754, 10314, and (Table 2). The recombinants produced by recombination events 1 and 2, ArD 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 126 APRIL 2016 VOLUME 31 ISSUE 2 VIROLOGICA SINICA

10 Shu Shen et al Table 2. The recombination events of ZIKV detected by the RDP software package Event No. Recombinants (location/time/ lineage) Major parents (location/time/ lineage) Minor parents (location/time/ lineage) Methods RDPRCS Begin End ArD Senegal/2001/A1 ArA1456 Cote d Ivoire/1980/A1 ArD Senegal/2001/A1 ArA975 Cote d Ivoire/1999/A1 ArD30101 Senegal/1979/A1 ArB15076 Central African Republic/1980/A1 Note: A1, African lineage 1; A2, African lineage 2. ArD Senegal/1998/A2 ArD Senegal/1998/A2 ArD Senegal/2001/A1 GENECONV, MaxChi, RDP, SiScan, 3Seq, Chimaera SiScan, 3Seq, Chimaera Seq, MaxChi 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. 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. Analysis of the ZIKV migration pathway revealed important roles of Senegal and Cote d Ivoire in the spread of ZIKV 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 islands, 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 APRIL 2016 VOLUME 31 ISSUE 2 127

11 Roles of African nations in Zika virus evolution and spread 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. DISCUSSION Since the first identification and isolation of ZIKV in 1947 (Dick et al., 1952), records of ZIKV were sparse until the last decade (Kindhauser et al., 2016). Despite the limited numbers of ZIKV sequences available in GenBank, two major lineages of ZIKV, the African and Asian lineages, were characterized in several previous studies (Haddow et al., 2012; Faye et al., 2014). This current study also characterized these two major lineages by constructing a phylogenetic tree for the fulllength genome sequences of 23 ZIKV strains (Figure 1). However, in contrast to previous reports on ZIKV genetic relationships based on the limited number of sequences available (Haddow et al., 2012; Faye et al., 2014; Enfissi et al., 2016b), we found that, for the first time, three lineages of ZIKV could be clustered based on the robust E and NS5 trees. These three lineages were designated as the Asian/American lineage, African lineage 1, and African lineage 2, respectively, according to their geographic distributions. Because only partial sequences of the several strains clustered in the additional African lineage 2 were available, this cluster could not be identified by the previous phylogenetic analyses conducted using fewer ZIKV strains. From our phylogenetic analyses, it is noted that African lineage 2, which has mainly been isolated from Senegal, diverged earlier than the other two lineages, suggesting that the ancestral strains of ZIKV may derive from Senegal. Then, we found that at least two genotypes of ZIKV have co-circulated in Senegal. So, it seems highly probable that recombination between different genotypes has happened in Senegal to generate recombinants with new genotypes. Subsequently, our analysis of recombination events revealed that ZIKV strains from Senegal were more frequently involved in recombination events than other strains. Therefore, strains from Senegal played important roles in ZIKV evolution and divergence. Although it was not so much involved as Senegal, Cote d Ivoire is also an important country related to ZIKV evolution. Co-circulation of ZIKV strains of different genotypes occurred in Cote d Ivoire, as African lineage 2, which showed an early divergence from other lineages, included one strain from Cote d Ivoire (ArA1465) in the E tree (Figure 2A) and other strains from Cote d Ivoire were clustered in African lineage 1 (Figure 2A and 2B). Moreover, strains from Cote d Ivoire were also involved in the recombination events. Taken together, our phylogenetic analysis revealed important phylogeographic roles of Senegal and Cote d Ivoire in ZIKV evolution and divergence. The migration of ZIKV was elaborated to illustrate the potential pathways for the spread of ZIKV and the geographic origins of recent ZIKV epidemics. Senegal and Cote d Ivoire were identified to be the countries that had most frequently exported and imported ZIKV strains. Furthermore, ZIKV migrated from Senegal to the Asian countries and Pacific islands where confirmed autochthonous cases were identified, suggesting Senegal as the geographic origin of all known ZIKV epidemics outside Africa. These results are supported by a previous report that illustrated the frequent movement of ZIKV between Senegal and Cote d Ivoire between 1920 and 1985, and mentioned that the ZIKV lineage from Africa spread to Malaysia around 1945, and to Micronesia around 1960 (Faye et al., 2014). ZIKV export from Senegal directly to Canada was also detected by our analysis using MIGRA- PHYLA. However, no autochthonous cases were confirmed in Canada and the patient had recently returned from Thailand (Fonseca et al., 2014). It is possible that the pathway from Senegal to Canada was identified by MIGRAPHYLA because the sequence of the strain PLCalZV that was imported to Canada is mostly similar to that of the strain SV0127/14 from Thailand, which had already imported ZIKV strains from Senegal (Figure 2B). The analyses of ZIKV recombination and migration were conducted according to the phylogenetic analysis in the current study, which are estimated based on the available sequences so far. So the amount of available ZIKV sequences may influence the results of the analyses. The human cases showed clinical manifestations of ZIKV infections that could be easily mistaken for DEN or CHIK fever. The latter two diseases emerged earlier and were much more commonly diagnosed than ZIKV infections in Africa (Faye et al., 2014). Based on their characterization of the history of ZIKV outbreaks and epidemics globally, Dr. Musso, and his colleagues reported that the spread of ZIKV seems to have followed the same path as DENV and CHIKV and that the potential for ZIKV epidemics might be overwhelming (Musso et al., 2015). The areas at risk of DENV and CHIKV transmission or with identified cases mostly matched with the areas of autochthonous ZIKV outbreaks (Figure 3A). Furthermore, the areas at risk of DENV and CHIKV are 128 APRIL 2016 VOLUME 31 ISSUE 2 VIROLOGICA SINICA

12 Shu Shen et al all located within the habitat areas of the Ae. Aegypti mosquito (Urdaneta-Marquez and Failloux, 2011), which is one of the principal arthropod vectors of ZIKV (Hayes, 2009), DENV, and CHIKV (World Health Organization, 2014; Weaver and Lecuit, 2015). Although wind-blown mosquitoes can migrate over the sea to distances of several hundred kilometers (Asahina, 1970), it is more likely that the migration of ZIKV over the great distances between continents was achieved as a result of increasing international travel and trade activities by which infected persons and animal hosts were transported to countries that were previously free from ZIKV (Duffy et al., 2009). Further, as the world s human population mostly gathers and lives in areas inhabited by Ae. Aegypti and other mosquitoes, the potential for the worldwide spread of ZIKV is likely to be still increasing. Therefore, it is important to investigate the relationship between ZIKV migration and the distributions and life cycles of mosquito vectors as well as international transport and trade activities. The information that can be provided by such studies will greatly help to prevent and control ZIKV outbreaks worldwide. ACKNOWLEDGMENTS This work was supported by the Science and Technology Basic Work Program (2013FY113500) from the Ministry of Science and Technology of China. COMPLIANCE WITH ETHICS GUIDELINES This article does not contain any studies with human or animal subjects performed by any of the authors. The authors declare no conflicts of interest. AUTHOR CONTRIBUTIONS SS and FD conceived of the study. JMS performed the ZIKV recombination detection and migration analyses. JW downloaded ZIKV sequences available in GenBank and generated the sequence datasets for phylogenetic analysis. ST collected all the information of ZIKV outbreaks and epidemics as listed in Table 1. SS constructed the phylogenetic trees, analyzed all the results, and wrote the first version of the manuscript. HLW, ZHH and FD checked and finalized the manuscript. REFERENCES Alera MT, Hermann L, Tac-An IA, Klungthong C, Rutvisuttinunt W, Manasatienkij W, Villa D, Thaisomboonsuk B, Velasco JM, Chinnawirotpisan P, Lago CB, Roque VG, Jr., Macareo LR, Srikiatkhachorn A, Fernandez S, Yoon IK Zika virus infection, Philippines, Emerg Infect Dis, 21: Asahina S Transoceanic flight of mosquitoes on the Northwest Pacific. Jpn J Med Sci Biol, 23: Baronti C, Piorkowski G, Charrel RN, Boubis L, Leparc-Goffart I, de Lamballerie X Complete coding sequence of zika virus from a French polynesia outbreak in Genome Announc, 2. Berthet N, Nakoune E, Kamgang B, Selekon B, Descorps-Declere S, Gessain A, Manuguerra JC, Kazanji M Molecular Characterization of Three Zika Flaviviruses Obtained from Sylvatic Mosquitoes in the Central African Republic. Vector Borne Zoonotic Dis, 14: Buathong R, Hermann L, Thaisomboonsuk B, Rutvisuttinunt W, Klungthong C, Chinnawirotpisan P, Manasatienkij W, Nisalak A, Fernandez S, Yoon IK, Akrasewi P, Plipat T Detection of Zika Virus Infection in Thailand, Am J Trop Med Hyg, 93: Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP, Sall AA, Musso D Zika virus, French polynesia, South pacific, Emerg Infect Dis, 20: Darwish MA, Hoogstraal H, Roberts TJ, Ahmed IP, Omar F A Sero-Epidemiological Survey for Certain Arboviruses (Togaviridae) in Pakistan. Trans R Soc Trop Med Hyg, 77: Dick GW, Kitchen SF, Haddow AJ Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg, 46: Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med, 360: Dupont-Rouzeyrol M, O'Connor O, Calvez E, Daures M, John M, Grangeon JP, Gourinat AC Co-infection with Zika and dengue viruses in 2 patients, New Caledonia, Emerg Infect Dis, 21: Enfissi A, Codrington J, Roosblad J, Kazanji M, Rousset D. 2016a. Zika virus genome from the Americas. Lancet, 387: Enfissi A, Codrington J, Roosblad J, Kazanji M, Rousset D. 2016b. Zika virus genome from the Americas. Lancet, 387: Enserink M INFECTIOUS DISEASES. An obscure mosquito-borne disease goes global. Science, 350: Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JV, Diallo M, Zanotto PM, Sall AA Molecular evolution of Zika virus during its emergence in the 20(th) century. PLoS Negl Trop Dis, 8: e2636. Filipe AR, Martins CM, Rocha H Laboratory infection with Zika virus after vaccination against yellow fever. Arch Gesamte Virusforsch, 43: Fonseca K, Meatherall B, Zarra D, Drebot M, MacDonald J, Pabbaraju K, Wong S, Webster P, Lindsay R, Tellier R First case of Zika virus infection in a returning Canadian traveler. Am J Trop Med Hyg, 91: Foy BD, Kobylinski KC, Chilson Foy JL, Blitvich BJ, Travassos da Rosa A, Haddow AD, Lanciotti RS, Tesh RB Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis, 17: Grard G, Caron M, Mombo IM, Nkoghe D, Mboui Ondo S, Jiolle D, Fontenille D, Paupy C, Leroy EM Zika virus in Gabon (Central Africa)--2007: a new threat from Aedes albopictus? PLoS Negl Trop Dis, 8: e2681. Haddow AD, Schuh AJ, Yasuda CY, Kasper MR, Heang V, Huy APRIL 2016 VOLUME 31 ISSUE 2 129

13 Roles of African nations in Zika virus evolution and spread R, Guzman H, Tesh RB, Weaver SC Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage. PLoS Negl Trop Dis, 6: e1477. Hancock WT, Marfel M, Bel M Zika virus, French Polynesia, South Pacific, Emerg Infect Dis, 20: Hayes EB Zika virus outside Africa. Emerg Infect Dis, 15: Heang V, Yasuda CY, Sovann L, Haddow AD, Travassos da Rosa AP, Tesh RB, Kasper MR Zika virus infection, Cambodia, Emerg Infect Dis, 18: Jan C, Languillat G, Renaudet J, Robin Y A serological survey of arboviruses in Gabon. Bull Soc Pathol Exot Filiales, 71: (In French) Kindhauser MK, Allen T, Frank V, Santhana RS, Dye C Zika: the origin and spread of a mosquito-borne virus. Bull World Health Organ. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, Emerg Infect Dis, 14: Leung GH, Baird RW, Druce J, Anstey NM Zika Virus Infection in Australia Following a Monkey Bite in Indonesia. Southeast Asian J Trop Med Public Health, 46: Macnamara FN Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg, 48: Marchette NJ, Garcia R, Rudnick A Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. Am J Trop Med Hyg, 18: Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics, 26: Mlakar J, Korva M, Tul N, Popovic M, Poljsak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodusek V, Vizjak A, Pizem J, Petrovec M, Avsic Zupanc T Zika Virus Associated with Microcephaly. N Engl J Med. 374: Moore DL, Causey OR, Carey DE, Reddy S, Cooke AR, Akinkugbe FM, David-West TS, Kemp GE Arthropodborne viral infections of man in Nigeria, Ann Trop Med Parasitol, 69: Musso D, Cao-Lormeau VM, Gubler DJ Zika virus: following the path of dengue and chikungunya? Lancet, 386: Oehler E, Watrin L, Larre P, Leparc-Goffart I, Lastere S, Valour F, Baudouin L, Mallet H, Musso D, Ghawche F Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia, December Euro Surveill, 19. Olson JG, Ksiazek TG, Gubler DJ, Lubis SI, Simanjuntak G, Lee VH, Nalim S, Juslis K, See R A survey for arboviral antibodies in sera of humans and animals in Lombok, Republic of Indonesia. Ann Trop Med Parasitol, 77: Olson JG, Ksiazek TG, Suhandiman, Triwibowo Zika virus, a cause of fever in Central Java, Indonesia. Trans R Soc Trop Med Hyg, 75: Pond WL Arthropod-Borne Virus Antibodies in Sera from Residents of South-East Asia. Trans R Soc Trop Med Hyg, 57: Roth A, Mercier A, Lepers C, Hoy D, Duituturaga S, Benyon E, Guillaumot L, Souares Y Concurrent outbreaks of dengue, chikungunya and Zika virus infections - an unprecedented epidemic wave of mosquito-borne viruses in the Pacific Euro Surveill, 19. Saluzzo JF, Gonzalez JP, Herve JP, Georges AJ [Serological survey for the prevalence of certain arboviruses in the human population of the south-east area of Central African Republic]. Bull Soc Pathol Exot Filiales, 74: Simpson DI Zika Virus Infection in Man. Trans R Soc Trop Med Hyg, 58: Smithburn KC Neutralizing antibodies against certain recently isolated viruses in the sera of human beings residing in East Africa. J Immunol, 69: Smithburn KC, Taylor RM, Rizk F, Kader A Immunity to Certain Arthropod-Borne Viruses among Indigenous Residents of Egypt. Am J Trop Med Hyg, 3: Tappe D, Nachtigall S, Kapaun A, Schnitzler P, Gunther S, Schmidt-Chanasit J Acute Zika Virus Infection after Travel to Malaysian Borneo, September Emerg Infect Dis, 21: Tognarelli J, Ulloa S, Villagra E, Lagos J, Aguayo C, Fasce R, Parra B, Mora J, Becerra N, Lagos N, Vera L, Olivares B, Vilches M, Fernandez J A report on the outbreak of Zika virus on Easter Island, South Pacific, Arch Virol. 161: Urdaneta-Marquez L, Failloux AB Population genetic structure of Aedes aegypti, the principal vector of dengue viruses. Infect Genet Evol, 11: Waehre T, Maagard A, Tappe D, Cadar D, Schmidt-Chanasit J Zika virus infection after travel to Tahiti, December Emerg Infect Dis, 20: Waggoner JJ, Pinsky BA Zika Virus: Diagnostics for an Emerging Pandemic Threat. J Clin Microbiol, 54: Wallace RG, Hodac H, Lathrop RH, Fitch WM A statistical phylogeography of influenza A H5N1. Proc Natl Acad Sci U S A, 104: Weaver SC, Lecuit M Chikungunya virus and the global spread of a mosquito-borne disease. N Engl J Med, 372: World Health Organization A global brief on vector-borne diseases. Zammarchi L, Stella G, Mantella A, Bartolozzi D, Tappe D, Gunther S, Oestereich L, Cadar D, Munoz-Fontela C, Bartoloni A, Schmidt-Chanasit J Zika virus infections imported to Italy: clinical, immunological and virological findings, and public health implications. J Clin Virol, 63: APRIL 2016 VOLUME 31 ISSUE 2 VIROLOGICA SINICA