Co-evolutionary patterns of variation in small and large RNA segments of Crimean-Congo hemorrhagic fever virus

John Chamberlain, Nicola Cook, Graham Lloyd, Valerie Mioulet, Howard Tolley and Roger Hewson

Novel and Dangerous Pathogens, Virology, Centre for Emergency Preparedness and Response, Health Protection Agency – Porton Down, Salisbury, Wilts SP4 0JG, UK

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


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The genus Nairovirus of the family Bunyaviridae includes the Crimean-Congo haemorrhagic fever (CCHF) species group. The species is predominated by the hazard-group 4 pathogens, from which the name and majority of strain entries are derived. Additionally, the species embraces hazard-group 2 viruses that are classified as members by antigenic cross-reactivity. CCHF viruses have a tripartite RNA genome consisting of large (L), medium (M) and small (S) segments. Here, the sequence characterization of previously undescribed L and S segments from novel strains originating in the Middle East and Africa is reported. Further scrutiny of this data with phylogenetic tools, in the context of other publicly available sequence information, reveals analogous grouping patterns between the L and S segments. These groups correlate with the geographical distribution of strain isolation and indicate that the L and S segments of CCHF viruses have evolved together.

The GenBank/EMBL/DDBJ accession numbers for the L segment sequences of strains Baghdad-12, Semunya, SPU4/81, SPU128/81/7 and IbAr10200 are AY947890, DQ076412, DQ076417, DQ076414 and AY947891, respectively; those for the complete S segment sequences of strains Semunya, SPU4/81 and SPU128/81/7 are DQ076413, DQ076416 and DQ076415, respectively.

Phylogenetic trees built from smaller sequence alignments that span the full length of the L segment are available as supplementary figures in JGV Online.


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Crimean haemorrhagic fever was first brought to medical attention in 1944, when it was recognized as an acute febrile illness, accompanied by severe bleeding in over 200 cases, in the Steppe region of western Crimea (Chumakov, 1945). The viral aetiology of this human disease was not elucidated until much later (Chumakov et al., 1968). As a consequence, it became evident that a similar disease in the Central Asian Republics (Casals et al., 1970), which had been known for many years, was caused by the same virus. With the resulting developments in diagnostic reagents and a proliferation in international study (Casals, 1969), it was soon recognized that these and other strains of Crimean haemorrhagic fever virus that were circulating in Asia and Russia were almost identical to viruses that caused Congo fever in Africa – a febrile illness predisposing to severe haemorrhage and causing high mortality. The first isolation of Congo virus was made in 1956 from a human in what is now Kisangani, Democratic Republic of the Congo (Woodall et al., 1967; Casals, 1969) and the name Crimean-Congo hemorrhagic fever virus (CCHF virus) has been used since the late 1970s. CCHF virus is a member of the genus Nairovirus in the family Bunyaviridae (Elliott et al., 2000). The genome is composed of three negative-sense, single-stranded RNA segments, designated small (S), medium (M) and large (L), that minimally encode the nucleocapsid (N), envelope glycoproteins (Gn and Gc) and RNA-dependent RNA polymerase (RdRp), respectively (Schmaljohn & Hooper, 2001). The virus is maintained in nature by a zoonotic cycle involving a variety of non-human vertebrate species, with animal-to-animal transmission mediated by ticks. Both the vertebrate hosts and tick vectors act as reservoirs of viral infection. Transmission to humans commonly occurs by a bite from, or contact through crushing with, an infected tick, or by contact with blood or tissues of infected animals. In addition to zoonotic transmission, CCHF virus can be spread from person to person and it has particular propensity to cause nosocomial outbreaks of disease with high mortality rates.

Published descriptions of major epidemics and outbreaks of CCHF have been reviewed extensively in the past (Hoogstraal, 1979; Watts et al., 1988). These reports illustrate the very wide distribution of CCHF virus. This distribution stretches 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 south-eastern Europe. Thus, CCHF virus is the most widely distributed agent of severe haemorrhagic fever known. Classic serological methods have been important in determining this distribution; however, these assays do not readily differentiate between alternative strains of CCHF viruses. In order to characterize viral strains in more detail and facilitate a global epidemiological study, molecular methods based on partial sequence data of the S segment have been used to identify certain S segment genotypes (Rodriguez et al., 1997; Drosten et al., 2002; Hewson et al., 2004a). These genotypes show a strong relationship to the geographical area of parent virus isolation, hence the terminology Asia 1, Asia 2, Europe 1, etc. Furthermore, these studies also show that similar genotypes are found in distant geographical locations, supporting the idea that virus or infected ticks may be carried over long distances during bird migration (Gonzalez-Scarano & Nathanson, 1996). Anthropogenic factors, such as the trade in livestock, may also have played a role in dispersing CCHF viruses. These factors support a global and dynamic reservoir of CCHF virus that enables viruses from different regions to blend with others through reassortment and, conceivably, recombination (Chare et al., 2003). These mechanisms, together with the likely contact and infection of new hosts, provide a foundation for the appearance of new CCHF disease and the emergence of new viruses (Holmes, 2004). This highlights the importance of molecular surveillance to monitor and track the natural fluxes of CCHF virus.

Current evidence of reassortment (Hewson et al., 2004b) points to the exchange of M segments between viruses in mixed infections. The role played by the S and L segments in reassortment is unclear. However, due to the ostensibly strong interrelationship between N and RdRp, including the non-random nature of segment reassortment in other bunyaviruses (Pringle et al., 1984), we hypothesized that closely related S and L segments have a co-requirement that, in order to produce viable virus, obliges them to stay together when confronted with reassortment opportunities with other CCHF viruses. To test this hypothesis, we have sequenced and compiled data on the S and L segments of a range of CCHF viruses and used phylogenetic tools to look for patterns of co-evolution or reassortment among these segments.

Whilst a handful of complete S segment sequences exist for different strains of CCHF virus (Marriott & Nuttall, 1992; M. D. Parker, P. J. Glass, G. B. Jennings, R. Lofts, J. F. Smith, M. M. Miller, K. W. Spik & R. Schoepp, GenBank accession no. U39455), such data on L segments have lagged behind because of the technical difficulties in working with them. Nevertheless, sequence data on the L segment of the IbAr10200 strain in particular have recently become available (Honig et al., 2004; Kinsella et al., 2004; J. D. Meissner, S. T. Nichol & S. C. St Jeor, GenBank accession no. AY422209). Here, we report the complete nucleotide sequences of the L segments of: (i) a Middle-Eastern CCHF virus strain isolated in Iraq (Baghdad-12); (ii) an African strain isolated in Uganda (Semunya); (iii) a strain isolated from one of the earliest reported outbreaks of CCHF in South Africa, (SPU4/81); and (iv) a South African tick strain (SPU128/81/7). In addition, we also report the complete S segment sequences of strains Semunya, SPU4/81 and SPU128/81/7. The Baghdad-12 strain was originally isolated via passage in suckling mice from the blood of a terminally ill patient from an outbreak of CCHF in Iraq in 1979 (Al-Tikriti et al., 1981) and stored at Porton Down, UK. Strain Semunya was isolated via passage in suckling mice from the blood of a febrile patient (Woodall et al., 1967). Strain SPU4/81 was isolated via passage in suckling mice from the blood of a febrile patient in 1981 (Swanepoel et al., 1983). Strain SPU128/81/7 was isolated via passage in suclking mice from a pool of crushed ticks (genus Hyalomma) collected from eland antelope in 1981 (Swanepoel et al., 1983). Viruses were grown in Vero cells maintained on Leibowitz L15 medium supplemented with 2 % serum and collected after 10–15 days at Advisory Committee on Dangerous Pathogens containment level 4. Virus in the medium supernatant was harvested by the addition of Tri reagent LS (Sigma). Samples were then removed from high containment, extracted with 1-bromochloropropane and centrifuged at high speed, and the aqueous phase was precipitated with propan-2-ol. RNA pellets were washed in 75 % ethanol and resuspended in DEPC-treated water. Viral RNA was then used as the basic template in RT-PCR steps, using a range of different primers. PCR products were sequenced by using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems); after unincorporated dye was removed with DyeEx columns (Qiagen), samples were sequenced via capillary electrophoresis by using an ABI 3100 genetic analyser (Applied Biosystems). Several alternative sets of RT-PCRs were carried out and the genomic ends of segments were determined by RACE (rapid amplification of cDNA ends) methods.

L segment sequences have been deposited in GenBank under the accession numbers AY947890, DQ076412, DQ076417 and DQ076414 for strains Baghdad-12, Semunya, SPU4/81 and SPU128/81/7, respectively. We also sequenced the complete S segments of strains Semunya (GenBank accession no. DQ076413), SPU4/81 (DQ76416) and SPU128/81/7 (DQ076415). In addition, the L segment of a stock of strain IbAr10200, which had been collected directly from Entebbe in 1967 by Drs J. P. Woodall and D. I. Simpson, working at the East Africa Virus Research Institute (Casals, 1969; Causey et al., 1970) (D. I. Simpson, personal communication) and the University of Ibadan, Virus Research Laboratory, Nigeria (GenBank accession number AY947891), was also obtained. This was carried out because of some confusion in the exact L segment sequence of the IbAr10200 strain, even though each of these sequences (GenBank accession numbers AY389361, AY389508 and AY422209) was obtained from the same source of IbAr10200. The inclusion of the new sequence (GenBank accession no. AY947891), obtained from an alternative, low-passage source, now resolves the earlier uncertainty of the original sequences.

Substitutions in the complete nucleotide sequences of the L segments studied were distributed randomly. Sequences were relatively conserved and nucleotide variability showed a maximum divergence of 16·8 %. The majority of nucleotide variations occurred at the third codon position and most were silent and synonymous, the maximum deduced amino acid divergence being 5·7 % (Table 1). The sequence context of the new full-length L segments was found to include the same collections of conserved motifs that have been reported previously for the IbAr10200 strain (Honig et al., 2004; Kinsella et al., 2004). Details of the levels of diversity and patterns of substitution between S segments have been described previously (Drosten et al., 2002; Hewson et al., 2004a).


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Table 1. Percentage identities of L segment nucleotide sequences and encoded protein sequences

Percentage identities were determined by pairwise comparisons of the L segments and their encoded protein sequences. Numbers at the top right indicate nucleotide identities and those at the bottom left indicate amino acid identities.

 
Sequence data on CCHF virus S and L segments were then aligned by using CLUSTAL W (Thompson et al., 1994) at EBI Hinxton, UK. To construct maximum-likelihood phylogenetic trees, quartet puzzling was applied by using TREE-PUZZLE (Strimmer & von Haeseler, 1997; Schmidt et al., 2002). Although a surrogate to classical maximum likelihood, this software enables very rapid tree reconstruction and is often used in such studies (Klempa et al., 2003). Phylogenetic trees were drawn by using the program TREEVIEW (Page, 1996).

The phylogenetic relationship between the available full-length S segments from several CCHF virus strains is depicted in Fig. 1. This is in agreement with previous data (Drosten et al., 2002; Hewson et al., 2004a) and the inclusion of three extra strains (SPU4/81, SPU128/81/7 and Semunya) has reinforced the notion of seven distinct lineages of S segment; in particular, strain Semunya has allied with the only other strain from the Africa 2 group, Congo 3010. The specific lineages are: Asia 1, comprising strains from the Middle East (Baghdad-12, Matin, JD206 and SR3); Asia 2 strains from Central Asia (Uzbek/TI10145, 66019, 75024, 7803, 88166, 8402, HY13, 68031, Hodzha, 79121 and 7001); Europe 1, including strains from southern Russia and the Balkans (Bul/HU517, STV/HU29223, DROSDOV and ROS/TI28044); Europe 2, comprising a single strain from Greece (AP92); and three African lineages previously described as Africa 1 (DAK8194 from Senegal), Africa 2, comprising two segments of viruses isolated in the Democratic Republic of Congo and Uganda (Congo 3010 and Semunya), and Africa 3, comprising Nigerian and South African strains (IbAr10200, SPU4/81, SPU128/81/7 and SPU415-85).



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Fig. 1. Unrooted maximum-likelihood tree of CCHF virus S segment sequences constructed from nt 56–1506 (numbering according to the Baghdad-12 sequence), showing the maximum number of alternative S segment sequences. The phylogenetic tree was computed with CLUSTAL W and TREE-PUZZLE. Values at the tree branches represent satisfactory levels of support as calculated by using quartet puzzling implemented in the TREE-PUZZLE package. Phylogenetic relationships between S segments show that seven groups are extant. These are grouped into: Asia 1 (Baghdad-12, Matin, SR3 and JD206); Asia 2 (Chinese strains, including TADJ/HU8966 and Hodzha); Europe 1 (DROSDOV, ROS/TI28044, STV/HU29223 and Bul/HU517); Europe 2 (AP92); Africa 1 (DAK8194); Africa 2 (Congo 3010 and Semunya); and Africa 3 (IbAr10200, SPU415-85, SPU4/81 and SPU128/81/7). Building on previous data, the inclusion of three extra S segment sequences provided in this work (from strains SPU4/81, SPU128/81/7 and Semunya) has reinforced the previously observed grouping patterns. Bar, 0·1 nucleotide substitutions per site.

 
The phylogenetic relationships between full-length L segments is shown in Fig. 2 and shows a grouping pattern that correlates with the geographical location of strain isolation. Furthermore, these groups are consistent over several windows of sequence alignment that span the full-length data (available as supplementary data in JGV Online). Given this reliability over the entire sequence, it is possible to include partial L segment data from other strains in an L segment tree that encompasses more sequences (available as supplementary data in JGV Online). For the L segment, there are five different lineages or genotypes that, like S segments, have grouped according to their geographical location of isolation. Specifically, these are Asia 1 (comprising Baghdad-12 and Matin), Asia 2 (comprising TADJ/HU8966 and Hodzha), Europe 1 (comprising strains from southern Russia; 30908 and K229-243) and two groups from Africa, Africa 2 (composed of the Semunya strain) and Africa 3 (composed of strains IbAr10200, SPU4/81 and SPU128/81/7). From these data, it appears likely that the L segments also conform to the same grouping pattern as observed for S segments, although there are fewer L segment sequences. Inclusion of sequences from groups Europe 2 and Africa 1 would have been desirable, but this was not possible due to the lack of availability of these strains in this study. As more CCHF virus L segment data become available, it will be interesting to observe whether the current groupings will be expanded to a seven-lineage pattern, for example by completion of the Greek strain AP92 L segment sequence and the Senegal strain DAK8194 L segment sequence.



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Fig. 2. Unrooted maximum-likelihood tree of CCHF virus L segment sequences constructed from nt 76–11909 (numbering according to the Baghdad-12 sequence), showing the maximum number of alternative L segment sequences. The phylogenetic tree was computed with CLUSTAL W and TREE-PUZZLE. Values at the tree branches represent satisfactory levels of support as calculated by using quartet puzzling implemented in the TREE-PUZZLE package. Phylogenetic relationships between L segments permit sequences to be grouped into: Asia 1 (Baghdad-12 and Matin); Asia 2 (TADJ/HU8966); Europe 1 (30908); Africa 3 (IbAr10200 and IbAn10248, SPU4/81 and SPU128/81/7); and a new group composed of the L segment from strain Semunya. By analogy with the S phylogeny, this group has been termed Africa 2. Bar, 0·1 nucleotide substitutions per site.

 
By examining the phylogenies of L and S segments in this way, it is possible to test hypotheses about the behaviour of these segments in virus infections. Given the current data on the L and S segments of CCHF viruses, it appears that they have evolved together as partners and that, in mixed virus infections where reassortment is a possibility, partner L and S segments have a propensity to end up in the same particle in order to constitute a viable new virus. M segments seem to be more autonomous, as demonstrated previously (Hewson et al., 2004b). An extension of this premise would be that reassortment events between S segments or between L segments would only be viable if they occurred within groups, whereas reassortment between M segments would not be restricted to groups and could result in new virus types. It follows that, as CCHF viruses are dispersed and introduced into new areas in which they are already endemic, the emergence of new CCHF viruses would be the result of M segment reassortment.


   ACKNOWLEDGEMENTS
 
We thank John Meissner for his contribution to sequencing the L segment of strain SPU128/81/7 and Ruth Mitchell for critical reading of the manuscript. This work was supported by the UK Department of Health.


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Received 27 May 2005; accepted 25 August 2005.



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