Institute of Poliomyelitis and Viral Encephalitides RAMS, Moscow 142782, Russia
Correspondence
Alexander N. Lukashev
Alexander_lukashev{at}hotmail.com
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ABSTRACT |
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MAIN TEXT |
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Similar to other members of the family Bunyaviridae, CCHF virus possesses a tripartite, single-stranded RNA genome of negative sense that consists of large (L), medium (M), and small (S) segments of approximately 12100, 5050 and 1570 nt (Schmaljohn & Hooper, 2001). The S RNA segment encodes a nucleocapsid protein of 54 kDa. The M segment encodes a polypeptide, which is processed into two surface glycoproteins (Sanchez et al., 2002
). Complete sequences of the L segment emerged only last year (Honig et al., 2004
; Kinsella et al., 2004
). This part of the genome encodes a putative viral polymerase.
Recently, several attempts have been made to conduct phylogenetic analysis of CCHF virus strains from different regions of China and Europe, and several distinct phylogenetic groups have been identified based on sequence data from different parts of the S segment (Rodriguez et al., 1997; Papa et al., 2002a
, b
, c
; Yashina et al., 2003a
, b
; Chinikar et al., 2004
). Phylogenetic analysis of the M segment has also been performed and this work has divided the Chinese strains into three distinct groups, one of which also included the Nigerian isolate IbAr10200 (Morikawa et al., 2002
). Interestingly, the S segment of this isolate made up a separate African phylogenetic group. One recent paper reported convincing evidence of genome reassortment in CCHF virus (Hewson et al., 2004
). A comprehensive work on recombination in negative-strand RNA viruses was published in 2003, where patterns of sequence variation compatible with the action of recombination were found in CCHF virus (Chare et al., 2003
). In this work, I used a range of phylogenetic methods to provide clear proof on this issue.
All currently available CCHF virus sequences for S, M and L segments (Table 1) were aligned using CLUSTAL_X software (Thompson et al., 1997
) and screened for probable recombination using a range of methods. First, I checked the similarity plots produced by SimPlot version 2.5 software (Ray, 1999
) to obtain an overview of the situation. A number of the S segment sequences, which were equally similar all over the segment to their closest relative and did not carry a detectable mark of recombination, could be excluded to simplify the analysis. Most of the remaining sequences had similarity plots suggesting recombination (e.g. Fig. 1a
). Next, I performed bootscanning analysis (Salminen et al., 1995
), also implemented in SimPlot version 2.5. As higher numbers of sequences did not produce reliable bootstrap values in bootscanning, I further limited the analysis to seven sequences that bore the most obvious signs of recombination on similarity plots, namely, strains Ap92, Dak8194, Ug3010, IbAr10200, Drosdov, Matin and Ch88166. Next, I performed bootscanning among the selected strains. Bootscan analysis slides a window over an alignment and for each window plots the proportion of the bootstrap pseudoreplicates that support phylogenetic grouping of the query strain with the strains used for comparison. Bootstrap values over 70 % are generally considered significant. Whenever one reliable grouping changes for another, one may suggest a recombination event. Bootscanning analysis of the strains studied (Fig. 1be
) provided reasonably convincing evidence of phylogenetic conflict between different parts of the S segment.
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Similarity plots of 13 M segment sequences did not reveal any obvious cases of recombination. Next, bootscanning was carried out on all or selected sequences and provided inconclusive results. Only analysis with PDM (window size 200 or 500 nt) clearly supported recombination (Fig. 2d). One can think of many reasons for a virus to give a false recombination signal in one method, for example immune selection pressure or specific virushost interactions providing subtle convergent changes in different virus strains. No ultimate evidence, conflicting the phylogenies, could be obtained for the M segment; therefore, recombination in the M segment could neither be excluded nor proven with the currently available sequences.
Currently, there are only five L segment sequences available in GenBank. Interestingly, L segment sequences of strain IbAr10200, submitted independently by two different groups, [GenBank accession nos AY389361 (Kinsella et al., 2004) and AY389508 (Honig et al., 2004
)], did not match. Similarity plots here revealed an unusual situation: sequence AY389508 (the same as NC_005301) had a sharp drop in similarity at approximately nt 1100011200 compared with other L segment sequences, including another IbAr10200 sequence (AY389361) (Fig. 1f
). The same could be seen on the similarity plots for the translated protein sequences (not shown). Strain IbAr10200 differed from other CCHF virus strains by as much as 2832 % of its amino acid sequence, while other CCHF virus strains differ by only 25 % of amino acid sequence in this region, and strain IbAr10200 differed from other CCHF virus strains by 110 % of its amino acid sequence in other genome regions. In contrast, according to a BLAST search, this unusual region was still most similar to the CCHF virus. As it stands, a sequencing error cannot be excluded. Therefore, I will abstain from further discussion here. For other L segment sequences, similar to the M segment, PDM, but not other methods, provided evidence of recombination. Obviously, it is not possible at the moment to prove or rule out recombination in the L segment.
Analysis of the CCHF virus sequences has provided solid evidence of recombination in the S segment, although the situation is far less clear in the other genome segments. To my knowledge, this is the first analysis reporting a clear indication of recombination in tick-borne viruses and in Nairoviruses, although recombination has been previously described in Hantaviruses (Sibold et al., 1999). The possibility of recombination in CCHF virus was discussed recently by Chare et al. (2003)
, but no clear evidence was shown. Several important conclusions can be drawn from the current results. While data for the S segment strongly support recombination, all of the cases detected were phylogenetically ancient. Indeed, the S segment sequences discussed throughout the paper represent the most distant CCHF virus strains isolated from distant locations. With a tick-borne zoonosis such as CCHF, it is not possible to say even approximately when the described recombination occurred. It could well be decades or millennia ago, or could even have happened at the dawn of the virus species. Similarly, it is not possible to estimate the incidence of recombination in CCHF virus. However, only a few complete CCHF virus sequences are currently available, and evidence of recombination often comes as an avalanche with additional new sequences. In any case, the possibility of recombination should always be kept in mind while conducting phylogenetic studies on CCHF virus. In a number of previous works (Papa et al., 2002b
, c
; Yashina et al., 2003a
, b
), a fragment of S segment sequence often as short as 200250 nt was used, and some authors relied on such results to draw rather far-fetched conclusions. Such an approach does not seem to be very accurate in light of this report. Considering that there is also evidence of reassortment in CCHF virus (Hewson et al., 2004
), all three segments and the full sequence for the S segment should be used in a comprehensive molecular epidemiological study on CCHF virus, at least for the key strains.
The exchange of genetic material obviously requires the co-replication of two strains in one organism. The most suitable hosts for such co-infection events are tick vectors, where the virus can persist and undergo vertical transmission, which further increases the probability of recombination. Multiple recombination events involving strains from very different locations would therefore suggest that CCHF virus co-replication is not uncommon in nature. In consequence, this suggests the existence of a global reservoir of CCHF virus with local subreservoirs supporting extensive levels of virus circulation, which permits frequent co-infection. Such a global reservoir can be readily explained by the major role of migrant birds in CCHF epidemiology. Even though there has been no clear evidence of CCHF virus infection in migrant birds, they may translocate infected ticks to distant areas (Hoogstraal, 1979). From a practical point of view, the considerable incidence of recombination and reassortment in CCHF virus means that one should always expect the emergence of virus variants that differ genetically and serologically from common isolates in the area. These new variants can be poorly detectable by reliable serological tests and may evade protection by future vaccines.
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ACKNOWLEDGEMENTS |
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Received 15 February 2005;
accepted 19 April 2005.
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