1 State Research Center of Virology and Biotechnology Vector, Koltsovo, Novosibirsk Region 630559, Russia
2 D. I. Ivanovskii Institute of Virology, Russian Academy of Medical Sciences, Moscow 123098, Russia
3 Freie Universität Berlin, Fachbereich Humanmedizin, Institut für Infektionsmedizin, 12203 Berlin, Germany
4 University of Nevada at Reno, Reno, NV 89557, USA
Correspondence
Vladimir Petrov and Sergey Morzunov
petrovvs{at}vector.nsc.ru and sergey@unr.edu
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ABSTRACT |
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The nucleotide sequences determined in this report are available in GenBank under the accession nos AY049078AY049083, AY045562AY045567, AF481799AF481805 and AF362743AF362746.
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INTRODUCTION |
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During the last decade, a large number of wild-type CCHFV strains and cell culture-grown isolates have been characterized genetically based on the partial/complete nucleotide sequences of their S and/or M genome RNA segments (Marriott & Nuttall, 1992, 1996
; Schwarz et al., 1996
; Rodriguez et al., 1997
; Papa et al., 2002
). However, data available currently on the geographical distribution, genetic diversity and prevalence of CCHFV in different species of ticks are far from being complete. Despite the longstanding history of CCHF research in the former Soviet Union, only one CCHFV strain from Russia (strain Drosdov) has been characterized genetically. This strain was isolated originally in 1967 from a patient in the Astrakhan region of Russia (Butenko et al., 1968
). No further data on the genetic variability of CCHFV strains from Russia and other former Soviet countries have been available in literature, although several new partial S genome segment sequences became available recently through GenBank (accession nos AF432116AF432121).
The goal of this study was to evaluate the genetic variability of CCHFV in southern regions of European Russia, Kazakhstan, Tajikistan and Uzbekistan. The phylogeny of the CCHFV strains from Russia and Central Asia, which has been established in the current study, provides valuable clues and establishes a firm basis for the further investigation of the CCHFV phylogenetic history and virushost relationships in Eurasia.
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METHODS |
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Ticks of the species H. marginatum were collected in 2000 in the four southern administrative regions of European Russia (Stavropol, Volgograd, Astrakhan and Rostov). Ticks of the species Dermacentor niveus were collected in 2001 in the Sarysu and Moiunkum districts of Kazakhstan. The ticks were grouped in pools of 1015 each for antigen testing using an ELISA kit manufactured at the D. I. Ivanovskii Institute of Virology. After testing, antigen-positive pools were divided into aliquots, frozen and stored at -70 °C before being used for RT-PCR.
Samples from 15 suspect CCHF patients, including six fatal cases, were obtained during local outbreaks in the southern regions of European Russia between 2000 and 2001 and from 16 patients in Kazakhstan in 2000. To detect CCHFV in whole blood from patients, in autopsy tissues and/or blood from fatal cases, we used the ELISA kit mentioned above. Specific IgM antibody titres in the serum samples of recovered patients were determined by ELISA, as described previously (Lavrova & Navolokin, 1986). The samples from 16 patients from Kazakhstan were blood sera and were examined for virus presence by RT-PCR only.
RT-PCR and sequencing.
Total RNA was extracted using the RNeasy MiniKit (Qiagen) from whole blood, autopsy tissues or serum from patients, and tissue culture supernatant from virus isolates. RT-PCR was performed using the Access RT-PCR kit (Promega) in accordance with the manufacturer's instructions. The origins of laboratory and wild-type strains of CCHFV used for RT-PCR analysis and subsequent sequencing are given in Table 1.
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The complete copy of the CCHFV S RNA segment was obtained by RT-PCR using the primer pair U1 and L2. It was then cloned using the pGEM-T Vector System kit (Promega). Both strands of the cloned fragments were sequenced with primers U1, F3, U2, PS3, L2, PS4, L1 and R2 (Table 2). Additional PCRs were performed to determine the nucleotide sequences of the 5'- and 3'-terminal regions using primer pairs E-R2 and E-U4, respectively. These fragments were sequenced using primers E, L3, R2 in the former, and U4, E in the latter case. Primer E (end) contains nine nucleotides (underlined in Table 2
) that are complementary to the 3'-terminal regions of both viral and complementary viral RNAs.
Nucleotide sequence comparisons and phylogenetic analyses.
Alignments and comparisons of the CCHFV nucleotide sequences were performed using CLUSTAL W (Thomson et al., 1994) and the LINEUP and PILEUP programs of the GCG software package (Genetic Computer Group). Methods used for reconstructing the phylogeny of the CCHFV strains under study included the maximum-parsimony (MP) method [supported by MEGA 2.1 (Kumar et al., 1993
), PAUP 3.1.1 (Swofford, 1991
) and PAUP* 4.0b10 (Swofford, 1998
)], the distance-based neighbour-joining (NJ) method (supported by PAUP* 4.0b10), quartet puzzling (supported by PAUP* 4.0b10) and the maximum-likelihood method (supported by PAUP* 4·0b10). All methods mentioned above were employed to analyse three original data sets (sequences amplified with the primer sets F2-R3/F3-R2, PS and entire S segment sequences) as well as the combined data set, which included concatenated sequences.
In MP analyses, phylogenetic trees were obtained by the heuristic or the branch-and-bound search methodology using either equal weighting of all changes or weighting of transversions over transitions. Several weighting schemes ranging from 2 : 1 to 6 : 1 (this represents estimated transition/transversion ratios within major clades and within smaller subclades, respectively) were employed. Gaps were treated alternatively either as missing data or as a fifth character state. NJ analysis employed uncorrected distances and several distance correction models supported by PAUP* 4.0b10. Bootstrap confidence limits were obtained by 1000 heuristic (MP) or Kimura 2-parameter (NJ) search repetitions. The CCHFV sequences published previously and used in the analysis are listed in the legend to Fig. 1.
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RESULTS |
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Phylogenetic relationships of the CCHFV strains from Russia and Central Asia
By using various methods for reconstructing phylogeny, CCHFV phylogenetic trees were obtained based on either individual (see Fig. 1) or concatenated (data not shown) sequences of two fragments of the N protein gene described above, as well as on the available complete S genome segment sequences (data not shown). All phylogenetic trees obtained displayed identical placement and topology of the major branches. Virus sequences derived from H. marginatum and CCHF patients from European Russia were closely related to each other (nucleotide sequence diversity 03·2 and 01·5 %, respectively, in the two fragments amplified) and formed a separate clade on the CCHFV phylogenetic tree (Fig. 1
). Within this clade, one can observe a tendency of the genetic variants to cluster geographically on a smaller scale, with nucleotide sequence diversity between the strains from the same locality not exceeding 0·5 %. However, no one of these smaller groups had significant bootstrap support. It is of particular interest that phylogenetic reconstructions based on the S segment fragment amplified with F2-R3/F3-R2 primers (the only sequence data available for the CCHFV strains from Kosovo) placed the Kosovo sequences firmly within the clade of Russian sequences (Fig. 1
). The S RNA sequences within the Russian clade differed considerably from the CCHFV strains from other regions (up to 20·5 % and up 16·7 % for the F2-R3/F3-R2 and PS-derived fragments, respectively).
The virus sequences from Asia formed two well-supported clades on the CCHFV phylogenetic tree (Fig. 1), although few of those were also present in two other clades that contained the virus sequences from Africa. The first clade of Asian sequences comprised CCFHV strains from China and Central Asia, while the second contained strains mostly from United Arab Emirates (UAE) and Pakistan (and the strain from Madagascar). Within the first clade, two well-supported subclades can be seen. The first subclade included CCHFV sequences from China and the TI10145 strain from Uzbekistan. Two human- and one D. niveus tick-originated CCHFV strains from Kazakhstan formed the second subclade, together with a human-derived strain HU8966 from Tajikistan. Nucleotide diversity within this subclade reached 3·2 or 2·1 %, respectively, in the case of the F2-R3/F3-R2 and PS fragments.
On the larger scale, one Russian and two Asian clades are grouped together with the first African clade (which also contained one human-derived sequence from UAE) and with the CCHFV strain from Uganda, although interrelations of those clades within this superclade were uncertain (Fig. 1). The second African clade, which contained sequences mostly from Senegal and Mauritania (and one sequence from Iran), formed a sister clade to the superclade mentioned above. Finally, strain AP92 from Greece appeared to be the most ancestral CCHFV strain.
Genetic characterization of the complete S genome segments of two CCHFV strains from Russia and Uzbekistan
Complete sequences of the S RNA segment were determined for one representative each of the European group and of the Asian group. The entire S segment sequences of strains STV/HU29223 (European Russia) and TI10145 (Uzbekistan) were found to be 1674 and 1672 nucleotides in length, respectively. When compared to other CCHFV strains, length differences were found only in the 5'- and 3'-terminal non-coding regions of the S segment. Like other CCHFV isolates, the initiating codon of the N protein gene is located at positions 5658 and the gene encodes 482 amino acids. The nucleotide and amino acid sequence divergence between STV/HU29223 and TI10145 appeared to be 10·3 and 3·5 %, respectively. The nucleotide sequence of STV/HU29223 differed from other CCHFV strains by 4·7 % (Drosdov, Russia), 18·9 % (Ap92, Greece), 10·4 % (8402, China), 10·2 % (HY13, China), 11·6 (JD206, Pakistan) and 14·9 % (10200, Nigeria), with the corresponding amino acid divergences being 0·6, 7·9, 4·1, 3·5, 3·5 and 4·6 %, respectively. The nucleotide sequence divergence of TI10145 from the CCHFV strains mentioned above was 12·8, 18·2, 3·8, 3·7, 10·2 and 13·2 %, with the corresponding amino acid divergences being 3·3, 7·7, 1·0, 0·6, 2·3 and 2·5 %, respectively. The S RNA sequence of the strain STV/HU29223 was most closely related to that of strain Drosdov and the sequence of TI10145 was most closely related to that of the Chinese strains.
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DISCUSSION |
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Our results indicated that closely related CCHFV strains circulating in Russia in a confined area between the Volga and the Don rivers are genetically distinct from CCHFV strains characterized previously from the other parts of Eurasia (Fig. 1). Within the European (Russian) group of CCHFV strains, the percentage of nucleotide differences correlates with the geographical distance between the localities of their origin. However, CCHFV strains described recently in Kosovo [strains 9553/2001 and 9717/01, GenBank accession nos AF428144 and AF428145, respectively, and strain Kosovo (Drosten et al., 2002
)] were almost genetically indistinguishable from the Russian strains. These similarities were rather surprising, since the Balkan region is located over 2000 km away from the territory we surveyed. Thus, we assume that genetically related CCHFV strains could also be found in the in-between territories, in Bulgaria, Mouldova and Ukraine. CCHF cases associated with H. marginatum, the same arthropod virus carrier as that in the southern regions of Russia, have been reported in the past from these countries (Hoogstraal, 1979
). This fact leads us to speculate that the genetic clade of the closely related CCHF strains from southern Russia and Kosovo is associated specifically and co-speciate with the tick species mentioned above. Further research is required to substantiate this hypothesis. Such future studies could also improve our understanding of the geographical formation of CCHF foci, since, along with a long geographical reach of genetically related viruses (Russia and Kosovo), genetically different CCHFV strains were discovered in two adjacent territories, Kosovo and Greece (Drosten et al., 2002
; Marriott et al., 1994
). According to the co-speciation hypothesis, the differences might be due to the simple fact that in Greece, CCHFV is transmitted by a different tick species, Rhipicephalus bursa (Papadopoulos & Koptopoulos, 1980
). An alternative hypothesis might be the fact that Greece is separated from Kosovo by the Balkan Mountains. Thus, isolated foci containing genetically distinct virus strains might have emerged in this geographically confined area.
Our analysis places all CCHFV strains from Central Asia in the same single phylogenetic clade with the virus strains known previously from China. Within this clade, the clustering of the genetic variants can be seen, i.e. two subclades represent the CCHFV strains from Uzbekistan/China and from Kazakhstan/Tajikistan, respectively. Previously, it has been demonstrated that Dermacentor species are the most common ticks in the Moiunkum district of Kazakhstan (Karimov et al., 1988). In our current investigation, we demonstrated the identity of the partial nucleotide sequence of the wild-type virus strain isolated from a Kazakhstan CCHF patient with that of a strain found in D. niveus. However, the percentage of the CCHFV-infected ticks appears to be low in this region. Further studies are required to obtain firm proof that Dermacentor species is the reservoir-vector in this region.
Results obtained for CCHFV strains circulating in Central Asia cannot be explained by invoking geographical factors alone. Although Kazakhstan borders the Xinjiang Province of China directly, a genetically related virus was found not in China but in Tajikistan, which is 1000 km away from that location (Fig. 1). Geographically, the most remote CCHFV strains from Central Asia, TI10145 strain from Uzbekistan and strains 8204 and HY13 from northwestern China (over 1500 km distance), turned out to be genetically more closely related than geographically less remote strains from Uzbekistan and Tajikistan (approx. 900 km distance). Once again, it should be noted that the greatest genetic differences were observed between CCHFV strains isolated from different tick species: H. asiaticum (Uzbekistan and China) (Yu-Chen et al., 1985
) and D. niveus (Kazakhstan). Even greater genetic differences (over 9·2 % nucleotide sequence divergence) were discovered between the strains under study and a known strain from the adjacent geographical region in Asia, strain JD206 from Pakistan (isolated from H. anatolicum) (Begum et al., 1970
). Based on these data, we suggest that a long-term association with a particular tick species (and, indirectly, with certain vertebrate host species) plays at least as great a role in casting genetically distinct CCHFV strains as does a geographical factor. Further studies of the CCHFV genetic variants circulating in Asian countries separated by the Pamirs and Tien Shan mountains may help us to clarify further the tickhostvirus relations and the complex pathways of CCHF foci formation in Central Asia.
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ACKNOWLEDGEMENTS |
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Received 2 September 2002;
accepted 8 January 2003.