Introduction

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the family Bunyaviridae, genus Nairovirus. It is an enveloped virus with a negative-sense single stranded RNA genome consisting of one small (S), one medium (M) and one large segment (L) that respectively encode the viral nucleocapsid (N), the membrane glycoprotein precursor (GPC), and RNA-dependent RNA polymerase (L) proteins [1,2].

Crimean-Congo hemorrhagic fever is a mainly tick-borne zoonosis that causes sporadic cases and severe outbreaks of acute human disease with a mortality rate of 5-60% depending on the country [3,4]. The genus Hyalomma, particularly the H. marginatum of Ixodes ticks, is the principal vector of CCHFV, and its geographical distribution correlates with the occurrence of CCHF [4]. In addition to Hyalomma spp., the transmission of CCHFV has been associated with Rhipicephalus (Europe, South Russia), Boophilus (Bulgaria, Russia, Pakistan), Dermacentor (Europe), Ixodes spp. (Crimea, Moldavia, Bulgaria, Hungary) [5], Argas persicus (Uzbekistan) and Ornithodoros lahorensis (Iran) [6,7]. Various wild and domestic mammals act as hosts for feeding ticks that can be infected with the virus; the animal infection is generally asymptomatic.

The disease’s seasonal pattern (between spring and early autumn) reflects the high degree of tick activity during this period [8]. Other modes of transmission are direct contact with infected animal blood, viremic CCHF patients and nosocomial infection [9,10].

CCHFV is endemic in parts of Eurasia and Africa, and continues to spread. A number of publications have demonstrated the presence of the virus in about 18 countries in Africa, 11 in South Asia and the Middle East, and eight in eastern Europe [6,8,11].

The migration or transportation of tick-infested birds [4], movements of livestock [12], the environment, tick density, the number of host vertebrate animals [13], and climatic and agricultural changes have all played major roles in taking CCHFV into new areas [8]. The first outbreak of severe hemorrhagic fever associated with CCHFV in Europe (and the world) occurred in the Crimean peninsula (on the Ukrainian coast of the Black Sea) in 1944-45, when hundreds of Soviet soldiers were infected [14]. Other large outbreaks have subsequently been described in Bulgaria (since 1953) and in the regions of Astrakhan and Rostov in south-eastern European Russia [8]. Recent studies in Greece have shown that the seroprevalence of already circulating and new-entry forms has increased from 1.1% to 3.14% over the last 20 years [15]; most of the detected antibodies were against the AP92 strain (isolated in 1970s), which is genetically different from all of the other strains and seems to be less or non-pathogenic to humans. Since 2002, Turkey has reported an exponentially growing number of human cases, whereas seroprevalence has decreased in Bulgaria since the 1974 introduction of an immunisation programme targeting military personnel and healthcare workers [8]. Kosovo experienced a reactivation of natural foci and the re-emergence of CCHF during the 1990s because of dramatic changes in social, political, environmental and economic factors that may together have led to more suitable ecological conditions. There is a seroprevalence of about 24% in the general population living in endemic areas in Kosovo [8,16]. In 2001, there was an outbreak of eight confirmed CCHF cases in Albania at the same time as an epidemic in Kosovo. During 2002-2006, another 24 patients were found to have confirmed CCHF, many of whom lived in Kukës Prefecture, in north-eastern Albania, which shares a border with Kosova. Another outbreak in the same prefecture was reported in 2010, and a few cases were also detected in the south-eastern part of the country in Kolonja near the border with Greece. Recent epidemiological surveillance has found an overall national seroprevalence in Albania of 2.1%, with significant differences between districts ranging from 0.8 to 12% (Silvia Bino, personal communication, 2013).

Analyses of the viral S and L-RNA segments have led to the identification of seven major genetic clades that segregate on a geographical basis [1,17-22]: Africa 1 (also classified as genotype 7), Africa 2 (genotype 5), Africa 3 (genotype 3), Asia 1 (genotype 1), Asia 2 (genotype 2), Europe 1 (genotype 4) and Europe 2 (genotype 6). Analysis of the M segment revealed only six clades, with all of the Asian isolates in a single monophyletic group [23].

We phylogenetically analysed publicly available S region sequences of CCHFV isolated in different geographical areas at different times (including two recently characterized isolates obtained in Albania) in order to reconstruct the most probable places of origin and pathways of dispersion of the CCHFV clades, particularly concentrating on the strain causing the recent outbreaks in eastern Europe.

Materials and Methods

Results

Phylogenetic analysis

Maximum likelihood analysis of the S segments showed that the isolates segregated into significant clades (Figure S3) corresponding to the seven known clades: Asia 1 Asia 2, Africa 1-3, and Europe 1 and 2. The clades were significantly supported by bootstrap values of ≥90%. As previously described, the Africa 1 clade included mainly (but not exclusively) isolates from western-Africa (Senegal and Mauritania); the Africa 2 clade isolates from central Africa (Uganda, Democratic Republic of the Congo); and the Africa 3 clade isolates from South Africa. The Asia 1 clade included isolates from the Middle East (Oman, Iraq, Pakistan, Afghanistan and Iran), and the Asia 2 clade isolates from the Far East and central Asia (China, Tajikistan, Uzbekistan). The Europe 1 clade included the majority of the eastern European isolates (Russia, Turkey and a single Greek strain), and contained a monophyletic group consisting of the three sequences from Kosovo and the two new Albanian isolates, whereas the Europe 2 clade isolates encompassed the prototype Greek strain AP92 and five Turkish isolates.

Evolutionary rate estimation and dated tree reconstruction.

Comparison of the coalescent models using the BF test showed that BSP (2lnBF constant vs BSP= 51.9) under the relaxed clock (2lnBF strict vs relaxed clock = 23.5) was the model that best fitted the data. A mean evolutionary rate of 2.96 x 10^-4 substitutions/site/year (credibility interval 1.6-4.7 x 10^-4 substitutions/site/year) was estimated under these conditions.

Figure 1 shows the maximum clade credibility (MCC) tree summarising all of the trees obtained during the MCMC search. All of the seven main clades corresponding to the viral genotypes were confirmed in the Bayesian tree, and supported by posterior probabilities of 1. There were also some highly significant subclades: two main groups in the Europe 1 clade separated the Russian isolates from most of the Turkish and all of the Balkan sequences (the last indicated by blue branches in Figure 1); and subclades in the Asia 2 clade separated the Chinese from the central Asian isolates. Asia 1 and 2 shared a common internal node with a high level of significance in the Bayesian tree (pp=0.99), but not in the ML tree (bootstrap value 0.59).

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Figure 1. The maximum clade credibility (MCC) tree of CCHFV S gene sequences.

The branches are coloured on the basis of the most probable location of the descendent nodes (A=Africa, AL=Albania, ASC=Central Asia, BU=Bulgaria, CH=China, G=Greece, KO=Kosovo, MO=Middle East, PA=Pakistan, T=Turkey). The numbers on the internal nodes indicate significant posterior probabilities (pp>0.8), and the scale at the bottom of the tree represents the number of years before the last sampling time (2010). The main geographical clades (genotypes) have been highlighted.

https://doi.org/10.1371/journal.pone.0079663.g001

Using the above evolutionary rate estimates, we calculated the mean tMRCA for the root and each internal node of the tree (Table 1). The mean tMRCA estimate for the tree root was 884.6 years ago (95%HPD 345-1522 years ago), thus suggesting that CCHFV has a remote origin. The Asian clades originated in the first decades of the 1800s, and a common ancestor of the two clades existed a mean 327 years ago (1683). The tMRCA estimates for the African clades were between 100 and 222 years ago, which suggests that these genotypes radiated between the late 1700s and the early 1900s. Finally, the Europe 1 clade had a mean tMRCA of 93 years ago, thus indicating its relatively recent emergence (mean estimate 1917), and the Europe 2 clade had a mean tMRCA of 201 years ago, corresponding to the first decade of the 1800s.

CLADE	Subclade	tMRCA1	CI tMRCA L2	CI tMRCA U3	Locality	State pp4
ROOT		884,6	345	1522	AFRICA	0,37
AFRICA 1		164,8	94	302	AFRICA	0,98
AFRICA 2		100	51	166	AFRICA	0,99
AFRICA 3		222,5	109	352	AFRICA	0,98
ASIA 1		164,8	79	261	MIDDLE EAST	0,57
	PAKISTAN	55,5	45	72	PAKISTAN	0,9
	MIDDLE EAST	127	56	204	MIDDLE EAST	0,68
ASIA 2		181,5	97	272	CHINA	0,55
	CHINA	137,29	81	205	CHINA	0,85
	CENTRAL ASIA	74,4	46	109	CENTRAL ASIA	0,93
EUROPE 1		93	57	138	RUSSIA	0,53
	TURKEY	58,5	36	86	TURKEY	0,5
	RUSSIA	71,3	49	99	RUSSIA	0,82
EUROPE 2		201	66	373	GREECE	0,35
ASIA 1+2		326,9	168	514	MIDDLE EAST	0,36

Table 1. Time of the most recent common ancestor (tMRCA) estimates.

¹tMRCA: Time of the most recent common ancestor

²CI tMRCA L: Lower credibility interval

³CI tMRCA U: Upper credibility interval

⁴pp: posterior probability

Time of the most recent common ancestor (tMRCA) estimates and credibility intervals (95%HPD) of the main clades observed in the MCC tree, with the corresponding years, most probable locations, and state posterior probabilities (pp).

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Phylogeographical analysis

As shown in Figure 1, the highest posterior probability for the location of the tree root was Africa (pp=0.4 vs pp= 0.19 for the Middle East, the second most probable location).

The most probable locations for the different clades are shown in Table 1. In particular, the MRCA shared by the two Asian clades was located in the Middle East (pp=0.36 vs 0.25 for Pakistan), as was that of the clade Asia 1 (pp=0.57 vs. pp=0.34 for Pakistan). On the contrary, the MRCA of the second Asian clade (Asia 2) was located in China (pp=0.55 vs pp=0.38 for central Asia). Interestingly, the MRCA of the Europe 1 clade was more probably located in Russia (pp=0.53 vs pp=0.22 for Turkey). The majority of the Turkish isolates segregated into a specific subclade that also included the isolates from Kosovo and Albania (pp=0.55). A Bulgarian isolate was at the outgroup of the Turkish MRCA. Finally, the Europe 2 clade most probably originated in Greece (pp=0.35 vs 0.23 for Turkey).

Bayesian phylogeographical analysis found a mean of 10.5 non-zero rates (95%HPD=10-12), and the rates with a BF of >3 were those between Africa and the Middle East (BF=48.9), the Middle East and Pakistan (BF=2518), China and Central Asia (BF=91), Russia and Turkey (BF=4.7), Turkey and Kosovo (BF=6.0), Kosovo and Albania (BF=12.1), and Bulgaria and Turkey (BF=16.4). The migration routes are shown in Figure 2.

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Figure 2. Significant non-zero CCHFV migration rates worldwide.

Rates supported by a BF of >3 are highlighted: the relative strength of the support is indicated by the colour of the lines (from dark red = weak to light red = strong). Dotted lines indicate non-significant linkages. The map was reconstructed using SPREAD (see Methods). The numbers indicate the mean estimated year in which the virus entered the area.

https://doi.org/10.1371/journal.pone.0079663.g002

The gene flow (migration) of CCHFV between different European areas was also investigated by analysing the European clade alone using a modified version of the Slatkin and Maddison method. The null hypothesis of panmixia (i.e. no population subdivision or the complete intermixing of sequences from different geographical areas) was tested using a bubblegram (Figure 3), and was rejected for Kosovo, Russia, Turkey and Bulgaria by the randomisation test (p = 0.0001).

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Figure 3. Phylogeographical mapping of CCHF S gene sequences .

The bubblegrams show the frequency of gene flows (migrations) to/from ten European countries (same code as that used in Figure 1) . The surface of each circle is proportional to the percentage of observed migrations in the ML genealogy. The migrations were inferred using a modified version of the Slatkin and Maddison algorithm.

https://doi.org/10.1371/journal.pone.0079663.g003

Almost 14.3% of gene flow was from Kosovo to Albania and from Turkey to Kosovo. Interestingly, there was a bidirectional gene flow between Turkey and Russia.

Discussion

Various authors have studied the genetic heterogeneity of CCHFV worldwide and found that the existing isolates can be classified into seven main geographical groups: three African, two Asian and two European [21]. As also shown by us, the three African clades include isolates mainly obtained in western (Africa 1), central (Africa 2) and southern Africa (Africa 3); the two Asian clades encompass isolates from the Middle East and Pakistan (Asia 1) or from China and central Asia (Asia 2). Most of the isolates causing recent outbreaks in eastern Europe are in the Europe 1 clade, whereas Europe 2 has largely divergent strains isolated from ticks in Greece (including the prototype strain AP92) [34] and Turkey [35] that are currently associated with sub-clinical or mild cases. The strong spatial structure of the genetic variability of CCHFV suggested it would be worth studying the migration of the clades phylogeographically in order to reconstruct the possible origin and dispersion pathways of the different viral strains. A study published two years ago [22] used a phylogeographical approach based on maximum parsimony, but new Bayesian methods have recently been developed that allow simultaneous estimates of evolutionary rates and migration routes [31], which are useful for reconstructing the past and recent epidemiological history of highly variable viruses [36-38]

In order to calculate the tMRCAs for the root and internal nodes of the phylogeographical tree using a relaxed molecular clock model, we estimated a mean evolutionary rate of 2.96x10^-4 for the viral genomic S segment. This suggests that, like the majority of other RNA viruses, CCHFV is characterised by a rapid evolution, at least at the level of the S fragment.

Our phylodynamic and phylogeographical reconstruction suggests that the known CCHFV clades shared a common ancestor that existed about one thousand years ago, most probably in Africa. A possible origin in Western Africa was suggested by our preliminary analyses [data not shown], but the extensive geographical intermixing of the three African clades and the relatively small number of African isolates available prevented us from reaching any more precise conclusions.

In our reconstruction, CCHFV left Africa in the second half of the XVII century, reached the Middle East, and then dispersed in two directions in the early XIX century to form the two Asian clades: one spreading in Iran and Pakistan, and the second in China and central Asia (Uzbekistan, Tajikistan). The virus therefore originally spread in an eastward direction to the Middle East and south-east Asia, crossing an area with a constant presence of CCHF susceptible species and the Himalayan mountains as a barrier to natural dispersion. It has recently been speculated that pathogens spread along the Eurasian ruminant route, as in the cases of foot-and-mouth disease [39,40] and Rinderpest [41].

Two highly divergent CCHFV strains entered Europe on at least two occasions: the first in the early 1800s (when a less or non-pathogenic virus confined to the animal/vector population reached Greece and Turkey), and the second in the first decades of the XX century, when a more pathogenic strain caused human outbreaks in eastern Europe until recently. In our phylogeographical reconstruction, the most probable location of the MRCA of this European clade 1 was Russia, which suggests that this was the gateway through which genotype 4 CCHFV entered Europe in the early 1900s. This partially conflicts with previous findings [22] suggesting that Turkey was the origin of genotype 4, although our analysis of significant migration rates confirmed the central role of Turkey in spreading the virus throughout Europe. Interestingly, the maximum parsimony analysis showed in- and outflows to and from Turkey and Russia, which suggests a continuous exchange between neighboring areas in the region of the Black Sea (possibly justifying the partial discrepancies between studies), and a complex web of viral introductions/transmissions from Turkey to Kosovo, and from Kosovo to Albania.

A recent study [42] has suggested the importance of anthropogenic factors in the dispersion of tick-borne encephalitis virus (TBEV), and provided evidence that its temporal and spatial distribution was related to the first land road into Siberia and the trans-Siberian railway. It is interesting to note that the entire area between the Black and Caspian Seas in the time span estimated for the origin of CCHFV (the first decades of the XX century) was the theatre of a number of wars (i.e. the Crimean war, World War II, and the Civil war) and mass deportations. The virus later spread to Turkey, where it probably circulated in the wildlife population for a long time (as previously postulated by [22 ]) as the first human cases of hemorrhagic fever were reported in 2002 [8].

In our reconstruction, the virus spread from Turkey to the Balkans, reaching Kosovo in the 1990s and Albania in the last decade. It is possible to hypothesise that the main cause of its dispersion through eastern Europe was the movement of wild and domestic ungulates carrying infected ticks, although outbreaks of CCHFV infection in South Africa have been associated with the passive transportation of infected ticks by birds [9,43], and the same has recently been suggested as introducing the virus to Spain [13]. Although the region between Black and Caspian Seas is one of the major migratory routes for birds crossing the African-Eurasian flyways (http://www.birdlife.org/), movements from East to West are extremely rare [44].

Turkey has one of the largest ruminant populations in Europe and the Middle East, and witnesses the movement of large and small ruminants for breeding, transhumance (within and across its borders), slaughter and import/export. The main direction of the flow is from neighboring countries such as Russia and Iran [45] and the regions of eastern and central Anatolia to the West. Movements take place throughout the year, but especially before and during religious festivals, when there is an increase in the export of small ruminants [46]. This large-scale movement of livestock over a short period of time has been previously associated with CCHF, Rift Valley fever and peste-des-petits ruminants [47]. Moreover, cross-border transhumance occurs on the Russian border [39], and could explain the results of the maximum parsimony analysis of in- and outflows between Turkey and Russia.

The currently used methods of phylogeographical reconstruction are inherently limited by the availability of sample locations and the numbers of isolates at each location. The sensitivity test performed in this study (which suggested that sampling frequencies had little impact on the root location) cannot exclude the influence of unsampled locations. Nevertheless, the analysed data set included all of the sequences with a known sampling location and year that were available in public databases at the time the study began.

In particular, the scarcity of sequences from Bulgaria prevented us from fully clarifying the country’s role in disseminating the infection. Bulgarian Thrace and Thrace as a whole (a geographical area covering south-eastern Bulgaria, north-eastern Greece and the European part of Turkey) is a high-risk zone for the cross-border spread of animal infectious diseases, as has recently been reported in the case of outbreaks of foot-and-mouth disease in Bulgaria and Greece close to the Turkish border [46]. Moreover, the emergence of human CCHF cases in Thrace over a limited period of time and encompassing three different countries -Turkey [48], Bulgaria and Greece [49,50]- confirm the key role of this area in the spread of infectious diseases.

The findings of this study indicate that continuous surveillance of the CCHF epidemic in Turkey and the entire Thracian area may be very important for monitoring and predicting future CCHF outbreaks in the Balkans.

Supporting Information