Evidence of segment reassortment in Crimean-Congo haemorrhagic fever virus

Roger Hewson1, Anatoly Gmyl2, Larissa Gmyl2, Svetlana E. Smirnova2, Galina Karganova2, Bushra Jamil3, Rumina Hasan3, John Chamberlain1 and Christopher Clegg1

1 Public Health Affairs, Centre for Applied Microbiology and Research, Health Protection Agency, Porton Down, Salisbury SP4 0JG, UK
2 Chumakov Institute of Poliomyelitis and Viral Encephalitides RAMS, Moscow, Russia
3 Departments of Medicine, Microbiology and Pathology, The Aga Khan University Medical Center, Karachi, Pakistan

Correspondence
Roger Hewson
roger.hewson{at}hpa.org.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The complete nucleotide sequences of the small (S) and medium (M) segments of three independent strains of Crimean-Congo haemorrhagic fever (CCHF) virus isolated in Uzbekistan, Iraq and Pakistan have been determined. Partial S and M segment sequences from two additional strains and partial large segment sequences from five strains of CCHF virus have also been obtained. These data have been compiled and compared with published full-length and partial sequences of other CCHF virus strains. Analysis of virus strains for which complete and partial S and M segment sequences are available reveals that the phylogenetic grouping of some strains differ between these two segments. Data provided in this report suggest that this discrepancy is not the result of recombination, but rather the consequence of reassortment events that have occurred in some virus lineages. Although described in other genera of the Bunyaviridae family, this is the first report of segment reassortment occurring in the Nairovirus genus.

Supplementary TREE-PUZZLES are available in JGV Online.

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AY223475, AJ538196, AJ538198, AY223476, AJ538197, AJ538199, AY573565, AY573567, AY573566, AY573568, AJ620685, AJ579312, AJ620682, AJ620683 and AJ620684.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Crimean-Congo haemorrhagic fever (CCHF) is a virulent human disease caused by a virus classified within the Nairovirus genus of the family Bunyaviridae. CCHF virus is the most widely distributed agent of severe haemorrhagic fever known. It is distributed over much of Asia, extending from the XinJiang region of China to the Middle East and Southern Russia and to focal endemic areas over much of Africa and parts of southern Europe. It has recently emerged as an important human pathogen, principally as a result of greater surveillance and increased awareness (Hoogstraal, 1979; Watts et al., 1988). CCHF virus is maintained in nature by cycles of asymptomatic infection between tick vectors and small mammals and livestock. Humans usually acquire the virus through a tick bite or from contact with infected blood, or other tissues from patients or livestock. Severe disease, manifest as haemorrhagic fever with frequently high mortality rate, only occurs in humans. Mortality rates correlate with mode of transmission, which for tick–human transmission is 10–50 % but up to 80 % in the case of nosocomial incidents (Swanepoel et al., 1987; Shepherd et al., 1985). CCHF virus is designated a hazard group four pathogen. Its ability to cause severe human disease has led to fears about its intentional use as a bioweapon. Consequently, it has been classified as a category A select biological agent and it is also listed by the Australia Group of states for the purpose of export control.

The genome of CCHF virus is composed of single-stranded RNA divided into three segments. It has been understood for some time that RNA viruses with segmented genomes have the capacity to reassort their segments into new genetically distinct viruses if the target cells are subject to dual infection. Indeed this ability is believed to play a key role in the evolution, pathogenesis and epidemiology of important pathogens such as influenza viruses, rotaviruses and arthropod-borne orbiviruses (Li et al., 2003; Iturriza-Gomara et al., 2001; Pierce et al., 1998). Within the Bunyaviridae family, reassortment has been demonstrated for members of the genera Orthobunyavirus (Pringle et al., 1984; Beaty et al., 1985; Uruidi & Bishop, 1992), Phlebovirus (Turell et al., 1990), Hantavirus (Henderson et al., 1995; Rodriguez et al., 1998; Klempa et al., 2003) and Tospovirus (Qiu et al., 1998), but not to date for Nairovirus.

Over recent years, a large number of CCHF virus isolates have been characterized genetically by partial or complete nucleotide sequencing of their small (S) and medium (M) segments. Complete sequences of 20 S and 11 M segments from various strains of CCHF virus have been published. However, only nine strains have a complete sequence for both S and M segments. Phylogenetic analysis based on large collections of partial and complete sequences of the S segment has indicated the existence of several distinct lineages (Rodriguez et al., 1997; Drosten et al., 2002; Hewson et al., 2003). These data indicate that there is a strong geographical component in the pattern of relationships between virus isolates. In addition, there are links between distant geographical locations, which may originate from trade in livestock, or from long-distance carriage of virus by infected ticks during bird migration (Gonzalez-Scarano & Nathanson, 1996). Phylogenetic analysis of the M segment (Morikawa et al., 2002) shows that for the available sequence data, Chinese CCHF virus isolates are clustered into three distinct groups, one of which also includes the Nigerian isolate IbAr10200. Comparison with full-length S phylogenetic trees (Hewson et al., 2003) highlights an interesting anomaly in that the S segment of the isolate IbAr10200 appears in its own African phylogenetic group, which is quite distinct from Asian lineages.

In this present study we report (i) the complete nucleotide sequence of the S and M segments from two Middle Eastern CCHF viruses including one from Uzbekistan, (ii) the partial nucleotide sequence of the S and M segments of a Russian and Nigerian strain and (iii) the partial nucleotide sequence of five large (L) segments. Analysis of these sequences suggests a geographical correlation between the relationships of S and L, but not between M segments. Furthermore, sequence analysis of S and M segments, together with other published data (totalling 23 S segments and 14 M segments), provides additional examples of anomalies in phylogenetic grouping between full-length S and M segments from the same virus strains (12 strains in total). These anomalies persist if (i) partial sequences, and so more strains, are analysed and (ii) if several small regions along the S and M segments are analysed. A biological explanation for these unexpected phylogenetic results is the occurrence of reassortment of RNA segments between CCHF virus strains.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Viruses and cells.
CCHF virus strain Hodzha was isolated in 1967 from the blood of an ill patient from Uzbekistan at the Institute of Poliomyelitis and Viral Encephalitides, Moscow (Chumakov et al., 1968). It has been passed 53 times through suckling mice. Strains IbAr10200 and IbAn7620 were delivered to Moscow by B. Henderson and J. P. Woodall (East Africa Virus Research Institute in Entebbe, Uganda). The strain IbAr10200 was originally isolated from ticks from Sokoto, Nigeria in 1966 (Cousey et al., 1970). It has been passed 11 times through suckling mice. Strain IbAn7620 was originally isolated from goat's blood near Ibadan, Nigeria in 1965 (Cousey et al., 1970) and it has been passed 17 times through suckling mice. Strain K229-243 was isolated from a pool of ticks collected from the Astrakhan region of European Russia in 1984 (Smirnova & Karavanov, 1985) and it has been passed six times through suckling mice. Strain SR3 has not yet been isolated, but nucleic acid was collected in the year 2000 directly from the blood of a terminally-ill patient during a nosocomial CCHF outbreak at the Aga Khan University Hospital in Karachi, Pakistan (B. Jamil and others, unpublished). Strain Baghdad-12 was originally isolated from the blood of a terminally-ill patient from an outbreak of CCHF in Iraq in 1979 (Al-Tikriti et al., 1981). It has been passed 12 times through suckling mice and several times through Vero E6 cells. Baghdad-12 was propagated in Vero E6 cells before being used. Vero E6 cells were grown in L15 media supplemented with 5 % bovine serum at 37 °C in T75 culture flasks. Medium supplemented with 2 % serum was used for virus propagation. Virus propagation and passages were performed under high containment (CL4) conditions.

Viral RNA isolation and first strand synthesis.
Total RNA was isolated from infected monolayers or 10 % brain suspensions with standard TRI-reagent and chloroform methods (Sigma-Aldrich) according to the manufacturer's instructions. For strain SR3, viral RNA was isolated using a viral RNA mini kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using random hexamers and primers based on consensus sequence data available from CCHF virus M segments deposited in GenBank.

PCR amplification and sequencing.
Primers were synthesized based on consensus data from the CCHF virus sequences deposited in GenBank. For strain Hodzha, the M segment was amplified using high fidelity PCR with Pfu polymerase in three overlapping fragments with primers (i) ME1 (5'-GGTCGACTCTCAAAGAAACAC-3') and MH2 (5'-CTTAGGGTGATTCGACATTC-3'), (ii) MS4 (5'-CCATTATTGGGCAAGATGGC-3') and MH10 (5'-TACACACACTACGGCCTCTG-3') and (iii) MH4 (5'-GGATTAGATGTGAAAGACC-3') and MEU (5'-CACCGGTCGACTCTCAAAGA-3'). For the Middle Eastern strains Baghdad-12 and SR3, M segments were amplified in two overlapping fragments from RNA reverse transcribed with primers M-RT(F1) (5'-TCTCAAAGAAATACTTGCGGCACGTCAGTACG-3') and M-RT(F2) (5'-TCACCAGTTCAATCAGCACCCATTG-3') using superscript II (Invitrogen), and PCR primers M-PCR(R1) (5'-TCTCAAAGAAATACTTGCGGCACGTCAGTACG-3') and M-PCR (R2) (5'-AGTATGCACAAAACAAAAGTGGTGTTGATGAT-3'). The extremities of PCR products were amplified by either 5' or 3' RACE methods (Roche), purified on agarose gels and sequenced directly by dideoxy chain termination using the Promega fmol kit on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems). For the partial sequence of L segments the primers L-RT1 (5'-KTGTCRAYAYTGACAGAAACAC-3') and L-PCR1 (5-GAGCAGYTRRCTTGTTTRAA-3'), were used.

Sequence analysis.
The nucleotide sequence data obtained were manipulated and analysed using the Lasergene suite of programs (DNAStar). For phylogenetic analysis, sequences were aligned using the CLUSTAL W (Thompson et al., 1994) program at EBI Hinxton and output in PHYLIP format. To construct maximum-likelihood phylogenetic trees quartet puzzling was applied using the program TREE-PUZZLE at Institut Pasteur (Schmidt et al., 2002; Strimmer & von Haeseler, 1997). The Tamura–Nei model of substitution was adopted, as has been performed in other phylogenetic studies demonstrating reassortment (Klempa et al., 2003). Trees were drawn using the program TREEVIEW (Page, 1996). The values at the tree branches represent the PUZZLE support values. Only CCHF virus strains that had overlapping sequence data, which could be aligned for their respective S, M and L segments were used to build phylogenetic trees. Included in the analysis, with the three full-length M segments and five partial L segments sequenced in this work, were the previously published CCHF virus S and M segments detailed in Table 1.


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Table 1. Published sequence data for S, M and L segments of CCHF virus

 

   RESULTS AND DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
S, M and L segment sequences
We have established the complete nucleotide sequences of the S and M segments of CCHF virus strains Hodzha, Baghdad-12 and SR3. These data have been deposited in GenBank under the accession numbers AY223475, AJ538196 and AJ538198 for S segments and AY223476, AJ538197 and AJ538199 for M segments, respectively. We have also obtained partial nucleotide sequences from the S and M RNA segments of strains IbAn7620 and K229-243. These are deposited in GenBank under AY573565 and AY573567 for S segments and AY573566 and AY573568 for M segments, respectively. In addition, we have partially sequenced the L RNA segment from strains: Hodzha (Uzbekistan), Baghdad-12 (Iraq), IbAr10200 (Nigeria), K229-243 (South Russia) and IbAn7620 (Nigeria). These data have also been deposited in GenBank AJ620685, AJ579312, AJ620682, AJ620683 and AJ620684.

Pairwise comparison of S segment sequences
Pairwise comparisons were restricted to those strains of CCHF virus for which full-length S and M segments were available. Table 2 shows the percentage identities among the collection of full-length S segments compared over their entire sequence at the 5' non-coding region (NCR), open reading frame (ORF) and 3' NCR. The S segment of strain Hodzha exhibits high nucleotide sequence similarity to those of Chinese strains, but generally less similarity to other CCHF virus S segments. The S segment strain Baghdad-12 exhibits highest nucleotide and amino acid similarity to the Middle Eastern S segments, strains Matin and SR3, but lower similarity with other strains. The S segment of strain SR3 exhibits highest nucleotide and amino acid similarity to that of Matin, the other Pakistani strain. As reported previously (Drosten et al., 2002; Hewson et al., 2003), there appears to be a correlation between S segment sequence and the geographical location of the original virus isolation. In Table 2 exact matches in the 5' NCR between some Chinese strains and also between the Pakistani strains SR3 and Matin exemplify this relationship, which is also seen in the 3' NCR. Here Chinese strains show high co-identity, with one exact match (88166 and 75024) as do Middle Eastern strains, with the greatest similarity here being identified between SR3 and Matin.


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Table 2. Complete S segment nucleotide and amino acid identities of CCHF virus strains

The identity values were calculated in the program CLUSTAL W. The percentage differences for nucleotide (above the diagonal) and amino acid (below the diagonal) sequences are presented. Strain name abbreviations and accession numbers are given in Table 1.

 
Pairwise comparison of M segment sequences
The nucleotide sequences of the three complete M segments that were determined in this work have lengths of 5356, 5354 and 5369 nt for strains Hodzha, Baghdad-12 and SR3, respectively. Each of the deduced ORFs encode a protein of 1689 aa. Comparisons with other available M segment ORFs show that the N termini of the three additional M ORFs have high levels of diversity for about the first 250 residues, consistent with other reports (Morikawa et al., 2002; Sanchez et al., 2002). Table 3 shows the degree of nucleotide and deduced amino acid sequence identity between the collection of M segments. [The M segment from the Pakistani strain Matin was not quite full-length and in order to accommodate comparisons, only an equivalent length of other strains was considered. Hence, NCR* refers to termini truncated by 23 nt from the 5' NCR (cDNA sense) and 22 from the 3' NCR]. The M segments of the three strains Hodzha, Baghdad-12 and SR3 all resemble each other, and in addition show high levels of similarity to the Chinese strains 75025 and 7803, and the Nigerian isolate IbAr10200. Similar relationships could also be deduced from comparative analysis of the 5' and 3' NCR* termini of M segments (Table 3).


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Table 3. Complete M segment nucleotide and amino acid identities of CCHF virus strains

The identity values were calculated in the program CLUSTAL W. The percentage differences for nucleotide (above the diagonal) and amino acid (below the diagonal) sequences are presented. Strain name abbreviations and accession numbers are given in Table 1 (NCR* denotes terminal truncations of sequence data analysed, see text).

 
Whereas the S segments of all the Middle Eastern strains are closely related to one another, the M segment of the Pakistani strain Matin appears to show a greater similarity to the Chinese strains 66019, 8402 and 88166 than it does to the other Middle Eastern strains.

Pairwise comparison of partial L segment sequences
The partial L segment nucleotide sequences of five different strains of CCHF virus determined in this work correspond to the 5' end of the genomic RNA segment and include the complement of the stop codon of the polymerase gene. In each case approximately 1 kb of sequence data was obtained. The nucleotide and deduced amino acid sequence identities between the six strains: Nigeria IbAn7620, Nigeria IbAr10200, Iraq Baghdad-12, Uzbekistan Hodzha, South Russia K229-243 and the corresponding sequence region of the recently published L segment from strain Pakistan Matin, have high levels of similarity (data not shown). In particular, high levels of similarity between the two Middle Eastern strains Iraq Baghdad-12 and Pakistan Matin, and between the two Nigerian strains IbAr10200 and IbAn7620 are evident at the nucleotide level.

Phylogenetic trees
To construct maximum-likelihood phylogenetic trees quartet puzzling was applied, using the program TREE-PUZZLE (Strimmer & von Haeseler, 1997; Schmidt et al., 2002). The Tamura–Nei model of substitution was adopted. Trees were drawn using the programme TREEVIEW (Page, 1996). The values at the tree branches represent the PUZZLE support values (Fig. 1).




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Fig. 1. Only strains that have corresponding full-length S and M segments are highlighted (additional sequences are not highlighted). (a) Phylogenetic maximum likelihood tree encompassing as many different strains of CCHF virus as possible (62 strains) constructed from 143 nt alignment of S segments. Seven distinct groups are evident. The location of strains within the clusters represented by full-length and partial S sequences (supplementary data in JGV Online E1, E2) is conserved when additional strains are analysed. (b) Phylogenetic maximum likelihood tree from 322 nt alignment of M segments encompassing as many different strains of CCHF virus as possible (26 strains). The location of strains within the clusters represented by full-length and partial M sequences (supplementary data in JGV Online E3, E4) is conserved when additional strains are analysed. (c) Phylogenetic maximum likelihood tree from a 1000 nt alignment of available L segment sequence. Computed with CLUSTAL W and TREE-PUZZLE, values at the tree branches represent the PUZZLE support values.

 
S segment phylogeny
The phylogeny of complete S segment nucleotide sequences from the collection of strains analysed (i.e. strains which have complementary full-length S and M segments) indicates that three major groups could be differentiated (supplementary data in JGV Online E1, E3). The Nigerian S segment from strain IbAr10200 is least similar to all other S segments and stands as an outgroup. The Pakistani strains (SR3 and Matin) form a well-supported monophyletic group with the other Middle Eastern strain Baghdad-12. Chinese strains and the Uzbekistan strain Hodzha also form a well-supported group, with strains 7001 and 79121 showing high similarity to each other. This phylogenetic grouping of S segments is maintained when the length of the analysed sequences is reduced so that a larger number of CCHF virus strains can be accommodated in the analysis. It is supported by a phylogenetic tree that focuses on a 438 nt region (nt 46–484, Baghdad-12) towards the 5' end of the genomic S segment (supplementary data in JGV Online E2), allowing an additional 12 strains to be analysed. This includes two strains: one from Russia (K229-243) and one from Nigeria (IbAn7620) for which M and L segment data are also available. As alignment windows decrease and more sequences are analysed, CCHF virus S segments fall into the seven different groups that correlate with their geographical location, two European, three African, one Middle Eastern and one Chinese/Far-Eastern. This grouping pattern is made more obvious when as many as possible S segments are aligned. This is represented in Fig. 1(a), a phylogenetic tree constructed from nt 1220–1363 (Baghdad-12), which accommodates 62 different strains. Furthermore, the location of strains within the clusters represented by full-length S sequences is conserved when additional strains are analysed.

M segment phylogeny
Interestingly, the phylogenetic grouping of the M segments is different to that observed for S segments. Discrepancies in the phylogenetic placement of strains are observed when phylogenetic trees based on full-length M segment (supplementary data in JGV Online E3) and corresponding full-length S segments (supplementary data in JGV Online E1) are compared. Distinct phylogenetic groups that were formed in the S segments by African, Middle Eastern and Chinese/Far-Eastern strains were not reproduced in the M segment phylogeny. A phylogenetic tree representing full-length M segments confirms three well-supported groups M1, M2 and M3. Comparisons between full-length M and S segments illustrates that the strain which shows the most distinctive differences in its phylogenetic placement is the Pakistani strain Matin. The S segment of strain Matin is closely related to the Middle-Eastern strains from Pakistan, including Baghdad-12 and a range of other Middle Eastern CCHF viruses (Fig. 1a). However, in the full-length M segment phylogeny, the M segment of the Matin strain resembles those of a group of Chinese strains identified as group M1 (strains 66019, 8402 and 8166, each of which have similar S segments that reside within the same Asia 2 cluster). The M segments of Chinese strains 7001 and 79121 (group M3) are notably divergent from the remaining collection of M segments, suggesting an earlier independent evolutionary route. By reducing the sequence analysis length, larger numbers of strains (partially sequenced in the M segment) can be used to build an M segment phylogenetic tree, more representative of the strains available. This result (supplementary data in JGV Online E4) focuses on a 432 nt alignment (nt 1274–1706, Baghdad-12) allowing an additional four M segments to be grouped; this also includes sequences from two strains (Russia, Astrakhan K229-243 and Nigeria, Ibadan IbAn7620) for which partial S and L segment data are available. A new group, denoted M4, is noted when additional strains are included, and these consist exclusively of strains isolated from Russia, while the other two additional strains segregate into the previously described group M2. The grouping patterns observed above are made more obvious when as many M segments as possible are aligned and used to construct a phylogenetic tree. This is shown in Fig. 1(b), which is a 322 nt alignment window representing nt 2355–2677 from the reference sequence Baghdad-12.

L segment phylogeny and geographical associations
The partial L sequences obtained from the five different strains of CCHF virus described earlier were aligned together with the published Matin strain and used to construct a maximum-likelihood phylogenetic tree with the corresponding region of the Dugbe L sequence. This tree is illustrated in Fig. 1(c), and shows the two Middle Eastern strains (Baghdad-12 and Matin) sharing the same well-supported phylogenetic group. Two African sequences (IbAr10200 and IbAn7620) also share the same well-supported phylogenetic group. While the number of L sequences available warrants caution in describing the phylogenetic grouping and inferring relationships between L, the well-supported groups in Fig. 1(c) also correlate with the geographical location of virus isolation, which has been observed for S, but not for M segments. For M segments, it may be possible that a previously existing geographical association has been obscured by rapid rates of evolution, imposed by immune pressure in mammals for example. Nevertheless, clear subtypes of M segment exist and the evolution of escape mutants with variations in M sequence cannot explain the discrepancies in M segment phylogenetic grouping alone.

Phylogenetic trees and reassortment
The discrepancy between the phylogenies generated from S and M segments is interesting. It might be explained by either recombination or reassortment events during the evolution of these strains. In order to test either of these hypotheses we focused on different regions of sequence alignment from the complete S and M segments, and constructed phylogenetic trees in an attempt to discover if phylogenies generated from these windows had significantly different topologies. Evidence of significant phylogenetic incongruence would then suggest recombination processes as the driving force behind the observed discrepancies.

These results are presented and summarized in Fig. 2(a) for the S segment and Fig. 2(b) for the M segment. Phylogenetic trees were constructed for each end, including a central region of the S and M segments; thus only segments for which full-length sequences are available were included in the analysis.



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Fig. 2. Phylogenetic maximum likelihood trees constructed from alignment windows at the 3' end of the S and M segment. Computed with CLUSTAL W and TREE-PUZZLE, values at the tree branches represent the PUZZLE support values. From 300 nt alignment of 3' terminus of S segment (a), 704 nt alignment of 3' terminus of M segment (b), (corresponding to variable region of M ORF). Additional phylogenetic trees generated from the 5' ends and central regions of each segment (supplementary data in JGV Online) display the same topology and there is no evidence of phylogenetic incongruence.

 
Fig. 2(a) shows the phylogenetic trees constructed from the 3' terminus of the S RNA segments. In addition phylogenetic trees were also constructed from the central region (an alignment window of 500–1220 nt E7), and the 5' termini (an alignment window of 1–300 nt E5) (supplementary data in JGV Online). It is clear that the general topology of these trees is the same, i.e. all strains from the Middle East group to Asia 1, and all strains from China and the Far East group together in Asia 2. The Nigerian strain IbAr10200, is the only complete African sequence available, and stands in its own group in each tree.

Fig. 2(b) shows the phylogenetic trees constructed from the 3' terminus of the M RNA segments. In addition, phylogenetic trees were also constructed from the central region (an alignment window of 2143–3143 nt E8), and the 5' termini (an alignment window of 140–2071 nt E6), (supplementary data in JGV Online). The grouping pattern of the M segments is also preserved and there is no evidence of phylogenetic incongruence. For the strains included in this analysis the topology originally described in Fig. 1(a) and (b) has been maintained and the phylogenetic groups M1, M2 and M3 are all verified by significant puzzle support values. The results presented in Fig. 2 do not demonstrate phylogenetic incongruence between different parts of the same segments, leading to the inference that recombination within these segments has not occurred during the evolution of the strains analysed. It follows therefore that the discrepancy in phylogenetic grouping between full-length S and M segments may be explained by reassortment events. We have also been able to confirm these results by performing Bootscan analysis on S and M segments (data not shown) using the program SimPlot (Lole et al., 1999).

The best evidence of reassortment is provided by the Matin strain isolated from Pakistan. If we consider full-length sequences where we have ruled out recombination events, there appear to be three types of S segment (African, Middle East-Asia 1 and Far East-Asia 2) and three types of M segment (M1, M2 and M3). From the limited number of sequences and the geographical location of virus isolations, it is possible to conceive that the majority of circulating viruses in the Middle East are composed of S-Asia 1/M2; while in the Far East viruses contain the combinations S-Asia 2/M2, S-Asia 2/M1 and S-Asia 2/M3. The data provided by L sequence analysis (Fig. 1c) suggest an important geographical component in virus evolution. We infer that strain Matin (S-Asia 1/M1) is the result of reassortment between a typical Middle Eastern virus S-Asia 1/M2 and an S-Asia 2/M1 Far-Eastern virus. Other strains may also have arisen by segment reassortment, but due to the limited number of full-length sequences it is impossible to draw firm conclusions.

Conclusions
Although reassortment of genomic RNA segments has been described in other bunyaviruses both in nature (Bowen et al., 2001; Sall et al., 1999) and in vitro (Beaty et al., 1985; Chandler et al., 1991), this is the first evidence of genetic reassortment occurring in nairoviruses. Clearly, reassortment requires coreplication of two strains in one organism/cell. The most suitable hosts for such coinfections are ticks, where the virus persists for extended periods, and superinfection with a second strain is very likely (Nuttall et al., 1991). The reassortment events involving strains from widely separated geographical locations, which we have observed, suggest that virus coreplication may be quite common. It follows that there may be a global reservoir of CCHF virus, with local subreservoirs supporting high levels of virus circulation permitting frequent coinfection, in which migratory birds play a significant role in virus dispersion. A practical conclusion from our results is that all CCHF diagnostic approaches and potential vaccines should be tested against isolates from all parts of the world, regardless of the intended location of use.


   ACKNOWLEDGEMENTS
 
We thank Nicola Cook, Howard Tolley and Graham Lloyd, Special Pathogens HPA Porton Down. This work was supported by the Department of Health and the Wellcome Trust (project grant 061414).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 19 March 2004; accepted 24 June 2004.