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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 TamuraNei 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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RESULTS AND DISCUSSION |
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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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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 TamuraNei 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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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 12741706, 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 23552677 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(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 21433143 nt E8), and the 5' termini (an alignment window of 1402071 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.
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ACKNOWLEDGEMENTS |
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Received 19 March 2004;
accepted 24 June 2004.