Our analysis of HCV-2 from the Amsterdam area uncovered substantial viral genetic diversity, including 8 known HCV-2 subtypes and 10 previously unclassified subtype-like lineages, and revealed a complex phylogenetic pattern associated with geography and risk groups. As discussed below, these associations are most likely driven by a combination of social, demographic, and economic factors over both recent and historical timescales. The phylogenetic distribution of the previously unclassified lineages suggests that they represent separate subtypes; however, our data did not satisfy the complete criteria for assigning new subtypes (36
). More sampling and (complete) genome sequences will be required before these lineages can be definitively classified. To obtain a broad sample of HCV infections, we included patients from an academic hospital, an inner-city hospital, and the public health service, of which the latter specifically deals with groups at risk for sexually transmitted and blood-borne infections. Although this study includes all patients diagnosed with HCV-2 at these three large centers in Amsterdam during a 10-year time period, selection bias cannot entirely be excluded.
The majority of Amsterdam isolates belonged to the epidemic subtypes 2a, 2b, and 2c commonly found in western Europe (9
). During the 20th century, the growth of parenteral routes of transmission resulted in the worldwide exponential growth of HCV, including HCV subtypes 2a, 2b, and 2c (30
). Although the phylogenies of these subtypes revealed similar epidemiological profiles, the existence of “epidemic” transmission networks for HCV-2 is best illustrated by the HCV-2a phylogeny (). The tree contains two risk-group-specific HCV-2a lineages in Amsterdam, one associated with contaminated blood transfusion and one with IDU. The IDU cluster is larger than the transfusion cluster, genetically less diverse, and also comprises strains from Germany, France, and Spain interspersed among the Dutch samples. This is typical for a recent introduction of a single virus that rapidly spread to IDU communities in neighboring countries via a joint transmission network. Similar observations have been made for HCV subtype 4d, which entered southern European IDU populations as early as 1954 and spread efficiently northwards thereafter (6
). In contrast, transfusion clusters remain country specific. We also identified a second transfusion-related cluster containing isolates mostly from former Soviet republics. The territorial clustering of transfusion-related isolates is perhaps unsurprising given that blood banks are organized at the national level. However, we note that these risk group clades do not receive strong statistical support, most likely due to insufficient phylogenetic signal, and therefore we suggest they should be interpreted with caution.
In addition to the epidemic subtypes, we observed 5 known endemic HCV subtypes and 10 distinct unclassified HCV-2 lineages each affecting 1 to 12 individuals, together accounting for one-third of our isolates. Endemic HCV-2 lineages have been found at varying frequencies in other Western countries (10% of HCV-2 infections in Canada to 67% in southeast France [24
]). In contrast to the French studies, the vast majority of Amsterdam endemic isolates were from individuals born outside the Netherlands, in Africa, Asia, or Latin America, particularly Surinam and Indonesia. The high frequency of endemic strains from the last two countries is unsurprising given the long history of Dutch colonial rule there. Nevertheless, HCV-2 reveals a remarkably widespread geographic distribution, with a pronounced geographical structure. Statistical tests demonstrated that isolates from each individual location significantly group together on the phylogeny. While incomplete sampling may contribute to this pattern, it also reflects the global nature of human movement during the 500 years prior to 1900. And while migration of HCV lineages within continental Africa could have resulted from land travel or the gradual diffusion of viruses through local interaction, the only plausible route by which HCV lineages could have moved among continents before the 20th century is via the emergence and rise of global marine navigation for exploration and trade.
Our results demonstrate the existence of multiple HCV-2 “migrant clusters,” which represent lineages that moved at some point in the past from West Africa (where HCV-2 originated [20
]) to other areas. Among others, these lineages include HCV-2e and 2f to Indonesia; HCV-2i to Morocco, France, Vietnam, and Quebec (24
); HCV-2j to Venezuela (39
); HCV-2k to Martinique and France (21
); HCV-2m to Vietnam (26
); HCV-2r to Haiti and the Dominican Republic (24
); and numerous nonclassified diverse HCV-2 lineages to Surinam. Strikingly, multiple separate migrations of HCV occurred from Africa to various countries in the Caribbean. Even a relatively small territory such as Surinam received multiple independent viral introductions, each drawn from the broader pool of HCV-2 diversity in West Africa. The likely low prevalence of endemic HCV in Africa and its low infectivity prior to the age of modern medicine suggest that such pattern was likely generated by a process that involved the movement of substantial numbers of Africans.
Crucially, in this study, we used a combined phylogeographic and molecular clock approach to directly test the previously proposed hypothesis (20
) that HCV was introduced to the Americas by the trans-Atlantic slave trade. If this hypothesis is correct, then the timing of viral introductions should coincide with the period of slave transportation as estimated from historical records. The transatlantic slave trade started gradually at the beginning of 16th century and ended in the latter half of the 19th century, during which it was responsible for the forced movement of an estimated 10.7 million people from Africa to the Americas (47
). We used evolutionary analysis to estimate the date of movement of each migrant lineage with appropriate confidence limits: the midpoint of these limits represents our best estimate of the date of viral introduction. Most of the estimated time windows for viral migrations fell within the 150 years between 1700 and 1850. This coincides with the historical period during which slave shipments reached their peak. Although trans-Atlantic slave trade continued for nearly 4 centuries, more than 80% (approximately 8.8 million people) of Africans ever transported were moved across the Atlantic during these 150 years (47
Phylogeographic analysis provides a second line of evidence supporting the trans-Atlantic slave trade hypothesis. All the HCV-2 trans-Atlantic “migrant clusters” (including those from Surinam) originated from the Benin-Ghana region of Africa (blue in ) while none originate from Senegambia (green in ). Present-day Ghana and Benin featured 30 European trading posts along their coast from the 15th to 19th centuries, compared to only 5 posts among the four countries of Senegambia (48
). Approximately 2.8 million slaves were moved to the New World from the Gold Coast and Bight of Benin (present-day Ghana, Benin, and Burkina Faso) between the 16th and 19th centuries, whereas ~600,000 slaves were transported from Senegambia during the same period. The former's relatively short span of coast was the embarkation point of more than a quarter of all people transported (47
The trans-Atlantic slave trade has been proposed as the driving force of intercontinental expansion of other viruses. A recent phylogenetic study suggests that West African hepatitis B virus (HBV) lineages were introduced to Haiti during the peak years of slave movement (1
). Similarly, recent studies of human T-cell leukemia virus type 1 (HTLV-1), which sometimes combine human and viral evidence, have indicated that Surinamese, Guyanan, Brazilian, Argentinian, and Peruvian HTLV-1 lineages grouped among West African lineages (3
Although the trans-Atlantic slave trade provides a plausible explanation for the movement of HCV-2 to the Americas, it leaves the long-term presence of endemic HCV-2 in Asia unexplained. European nations likely played a role in the global dissemination of HCV, additionally through trade and other forms of human migration among their colonies. An example is subtype 2e, which links an endemic HCV-2 lineage from the Benin-Ghana area to Indonesia. Dutch companies established multiple trading ports in this part of Africa and traded intensively with the largest Dutch colonial territory—Indonesia. Records also suggest the significant movement of African slaves from West Africa to the Dutch Cape colony and Asia by Dutch traders (27
). Our results also highlight secondary lineage movements among colonial territories sharing the same metropole. Subtypes 2e and 2f suggest viral movement in both directions between the former Dutch colonies of Surinam and Indonesia. Although the timing and direction of subtype 2e migration is uncertain, we provide evidence that HCV-2f moved from Surinam to Indonesia during the early 20th century. These more recent movements are consistent with the bidirectional migration of initially 2,600 Chinese migrant workers mainly from Java (Indonesia) between 1853 and 1874, and later 33,000 Javanese contract laborers from Indonesia to Surinam during 1890 to 1940, a direct consequence of the abolition of slavery in the latter by the Dutch government in 1862 (19
). About a quarter of these laborers returned to Indonesia at the end of their 5-year contract in the period between 1897 and 1956. People of Javanese descent still comprise 15 to 20% of the present-day Surinamese population (17
By combining demographic, historical, and viral genetic data, we have reconstructed the transmission history of HCV-2, which explains its worldwide distribution as well as the present-day pattern of diversity observed in Amsterdam. It appears that the colonial activities of European countries played a decisive role in the dissemination of HCV genotype 2, predominantly to the Americas, but also to former colonial territories in Asia. Interestingly, HCV subtype 2i (cluster C in ) falls within the Benin-Ghana region and solely contains isolates from francophone regions, including Morocco, France, Quebec, and Vietnam (former French Indochina). Although indicative of a role for the colonial activities of France in the dispersal of HCV-2i, our sample of “Dutch origin” is not sufficiently representative to address this hypothesis. The Benin-Ghana region of Africa, both a major embarkation area for vessels carrying slaves to the Americas and an important trading post along European-Asian trading routes, emerges as an important historical hub of global HCV-2 distribution. Since the global epidemic HCV subtypes 1a and 1b were estimated to originated from the United States (18
), we hypothesize here that trans-Atlantic slave trade was also responsible for the movement to the Americas of these genotype 1 endemic African lineages. Molecular clock dating of transatlantic movements of HCV from West Africa strongly supports our historical slave trade hypothesis. In addition to information on the global nature of human movement before 1900, our phylogenetic analysis of epidemic subtypes 2a, 2b, and 2c, together 67% of HCV-2 infections in our study, exposed both national and international transmission networks responsible for the emergence of HCV-2 in most Western countries during the 20th century, specifically injecting drug use and contaminated blood/blood products. Undiagnosed chronically HCV-infected individuals might benefit from an increased understanding of the epidemiological processes that generated the global pattern of HCV diversity seen today. Better understanding of the associations between risk factors, populations, and HCV subtypes might help target HCV prevention and screening campaigns and decrease the future burden of HCV-related liver disease.