Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia

Lyudmila Yashina1, Irina Petrova1, Sergei Seregin1, Oleg Vyshemirskii2, Dmitrii Lvov2, Valeriya Aristova2, Jens Kuhn3, Sergey Morzunov4, Valery Gutorov1, Irina Kuzina1, Georgii Tyunnikov1, Sergei Netesov1 and Vladimir Petrov1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hyalomma marginatum ticks (449 pools, 4787 ticks in total) collected in European Russia and Dermacentor niveus ticks (100 pools, 1100 ticks in total) collected in Kazakhstan were screened by ELISA for the presence of Crimean-Congo haemorrhagic fever virus (CCHFV). Virus antigen was found in 10·2 and 3·0 % of the pools, respectively. RT-PCR was used to recover partial sequences of the CCHFV small (S) genome segment from seven pools of antigen-positive H. marginatum ticks, one pool of D. niveus ticks, four CCFH cases and four laboratory virus strains. Additionally, the entire S genome segments of the CCHFV strains STV/HU29223 (isolated from a patient in European Russia) and TI10145 (isolated from H. asiaticum in Uzbekistan) were amplified, cloned and sequenced. Phylogenetic analysis placed all CCHFV sequences from Russia in a single, well-supported clade (nucleotide sequence diversity up to 3·2 %). Virus sequences from H. marginatum were closely related or identical to those recovered from patients in the same regions of southern Russia. Newly described CCHFV strains from Central Asian countries fell into two genetic lineages. The first lineage was novel and included closely related virus sequences from Kazakhstan and Tajikistan (nucleotide sequence diversity up to 3·2 %). In contrast, a newly described CCHFV strain from Uzbekistan, strain TI10145, clustered on the phylogenetic trees with strains from China.

The nucleotide sequences determined in this report are available in GenBank under the accession nos AY049078AY049083, AY045562AY045567, AF481799AF481805 and AF362743AF362746.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Crimean-Congo haemorrhagic fever virus (CCHFV) is the type species of the genus Nairovirus, family Bunyaviridae. It possesses a tripartite, negative-sense, single-stranded RNA genome encoding viral RNA polymerase (L segment), surface glycoproteins G1 and G2 (M segment) and a nucleocapsid (N) protein (S segment) (van Regenmortel et al., 2000). The virus causes Crimean-Congo haemorrhagic fever (CCHF) in humans. This disease is characterized by a haemorrhagic and toxic syndrome, with case fatality rates ranging from 10 to 50 % (Hoogstraal, 1979). The virus can be transmitted to humans by bites of Ixodid ticks or by contact with blood or tissues from human patients or viraemic livestock (Chumakov, 1963). The disease was first discovered in the Crimean region of Russia in the 1940s (for an overview, see Chumakov, 1945) and is now reported in many regions of the world: Africa, the Middle East, Europe and Asia (reviewed by Hoogstraal, 1979). In the territory of the former Soviet Union, disease outbreaks or the presence of the virus were reported in the southern regions of European Russia, in Mouldova, Ukraine and Transcaucasus, and in Central Asian countries, in Tajikistan, Turkmenistan, Uzbekistan, Kyrgyzstan and Kazakhstan (reviewed by Aristova et al., 2001). In Russia, a long period without reports of CCHF ended in 1999, when a total number of 40 CCHF cases was documented (Onishchenko et al., 2001). A total of 83 cases of CCHF and eight fatalities were reported in Russia in the year 2000 (Onishchenko, 2001). In the Stavropol region, human infection occurred mainly through tick bites. Cases of disease among medical staff that took care of patients were also reported. The clinical course of disease varied from mild fever with a haemorrhagic syndrome to severe forms with internal bleeding that, as a rule, were fatal. (Kolobuhina et al., 2001). In Kazakhstan, 33 CCHF cases and one fatality were reported in the year 2000 (Kazakov et al., 2001). The main route of infection was through tick bites or contact with viraemic ticks during the shearing of sheep. Moderate clinical forms of CCHF were dominant.

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 virus–host relationships in Eurasia.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sample collection and preparation.
Four laboratory strains of CCHFV from the collection of the D. I. Ivanovskii Institute of Virology of the Russian Academy of Medical Sciences (Moscow) were used. Strain TI10145, isolated in 1985 from Hyalomma asiaticum ticks (Uzbekistan), had undergone 36 passages in suckling mouse brains and another three passages in SW-13 cell cultures; strain HU8966, isolated in 1990 from a CCHF patient (Tajikistan), had been subjected to 15 passages in suckling mouse brains and one passage in a SW-13 cell culture; strain STV/HU29223, isolated in 2000 from a CCHF patient (Stavropol region of Russia), and strain ROS/TI28044, isolated in 2000 from H. marginatum ticks (Rostov region of Russia), went through one passage in a SW-13 cell culture.

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 10–15 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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Table 1. Strains of CCHFV used for RT-PCR and sequencing

 
All samples were tested by nested RT-PCR using two sets of primers. These primers allow the amplification of two regions of the nucleocapsid (N) protein gene encoded by the S segment of the CCHFV genome. F2-R3 and F3-R2 primers were described previously (Rodriguez et al., 1997). Primers PS1-PS2 and PS3-PS4 (PS set) were designed in this study by using the computer program OLIGO (Rychlik & Rhoads, 1989). Nucleotide sequences and positions of the primers are shown in Table 2.


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Table 2. Primers used for RT-PCR and/or sequencing

Nucleotide positions refer to those of the S genome segment of CCHFV strain C68031 (Marriott & Nuttall, 1992). The incorporated recognition site of endonuclease BamHI is highlighted in bold. Underlined nucleotides are complementary to the 3'-terminal parts of both viral and complementary viral RNAs.

 
Second-round PCR products were analysed by electrophoresis in a 1·5 % agarose gel. DNA bands of the expected size were excised and purified using the QIAEX II Gel Extraction kit (Qiagen) according to the enclosed instructions. DNA sequencing was performed with the ABI Prism BigDye Terminator kit (PE Applied Biosystems) and an automatic ABI Prism 310 genetic analyser.

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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Fig. 1. Phylogenetic relationship (a) and geographical distribution (b) of the CCHFV genotypes examined in the current study. Phylogenetic relationships inferred by MP analysis of a 220 nt fragment of the S RNA segment amplified with primers F2-R3 and F3-R2. MP analysis conducted using the heuristic search option and a 3 : 1 weighting of transversions over transitions generated 293 equally parsimonious trees. One representative tree is shown (a); all other trees displayed identical placement of all well-supported branches. The lengths of the horizontal branches are proportional to the nucleotide step differences. Vertical branches are for visual clarity only. Bootstrap values above 50 %, obtained from 1000 replicates of the analysis, are shown at the appropriate branch points. CCHFV strains are described as strain designation/country of origin (source). CCHFV S RNA segment sequences published previously (with GenBank accession numbers) used in the analysis are as follows: IbAr10200 (U88410); Drosdov, HY 13 and JD 206 (U88412U88414, respectively); UGANDA 3010 (U88416); C68031 (M86625), ARD 8194 (U15021), 66019 (AJ010648), 8402 (AJ010649), AP92 (U04958), HU9509853 (U75672), TI9538886 (U75673), HU9447547 (U75670), TI9538889 (U75669), HU9509854 (U75671), ARMG951 (U15024), ArB604 (U15092), RSA (U75675), ArD97268 (U15091), ArD39554 (U15089), 9553/2001 (AF428144), 9717/01 (AF428145), ARTEH193-3 (U15022), AND15786 (U15020) and HD49199 (U15023). Two outgroup taxa included Dugbe virus (strain KT 281/75, AF434165) and Hazara virus (strain JC280, M86624). Abbreviations used in (a): UAE, United Arab Emirates; abbreviations used in (b): 1, Rostov; 2, Volgograd; 3, Stavropol; 4, Astrakhan regions of Russia.

 

   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Screening of ticks and suspect human CCHF cases
A total of 4787 H. marginatum ticks from Russia and 1100 D. niveus ticks from Kazakhstan were examined. The presence of CCHFV was confirmed by antigen-ELISA in 46 of 449 (10·2 %) H. marginatum pools and in 3 of 100 D. niveus pools, respectively (Table 3). Antigen positivity in H. marginatum tick pools ranged from 6·5 to 12·3 % in four regions studied; in D. niveus ticks it did not exceed 3·0 %. Six antigen-positive tick pools and two CCHFV strains isolated from ticks were tested by nested RT-PCR. All eight samples examined produced bands of the expected size (260 nt) with the F2-R3/F3-R2 primer set and seven of the eight samples produced bands of the expected size (505 nt) with the PS primer set.


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Table 3. Results of CCHFV antigen and genome detection in samples from patients and ticks collected in Russia and Kazakhstan

For tick samples, results are given as number positive/number of pools tested/number of ticks tested, whereas those for human samples are given as number positive/number of cases tested (including number of fatal cases tested). ND, Not done.

 
Samples from 15 patients with suspected CCHF from European Russia were tested by antigen-ELISA (Table 3). The presence of CCHFV was detected in 5 of 15 samples tested. All antigen-positive samples had been obtained from fatal cases. Attempts to detect antigen in blood failed in all nine patients who recovered. However, on days 10–14 post-onset of disease, specific IgM antibodies were found with titres of 1 : 1000. Two samples were chosen for RT-PCR analysis, one from a deceased patient and another from a recovered patient. Moreover, two laboratory virus strains isolated from patients were analysed. Bands of the expected size were obtained with the F2-R3/F3-R2 and/or PS primer sets. RT-PCR analysis of the 16 sera from Kazakhstan revealed two positive samples with both primer sets.

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 0–3·2 and 0–1·5 %, 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 56–58 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.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This report presents the first data on the genetic variability of CCHFV strains circulating in the southern territories of European Russia and Central Asian countries (Kazakhstan, Tajikistan and Uzbekistan). Although the majority of our data are based on partial nucleotide sequences of CCHFV S RNA segments, the complete sequences of the S RNA segment of two representative virus strains (STV/HU29223 from southern Russia and TI10145 from Uzbekistan) were determined as well.

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 tick–host–virus relations and the complex pathways of CCHF foci formation in Central Asia.


   ACKNOWLEDGEMENTS
 
This work was supported by the Cooperative Threat Reduction programme of the US Defense Threat Reduction Agency (DTRA) through the International Science and Technology Center (grant #1291-2p) and by the Ministry of Public Health of the Russian Federation. The authors wish to thank the US collaborators under this ISTC project, Drs Stephen St Jeor and Yi-Wei Tang for productive discussions of research data. We also wish to thank our colleagues S. V. Kazakov, K. S. Ospanov and S. Karimov for providing the samples from Kazakhstan and Dr Jonathan Smith at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) for kindly providing the SW-13 cells.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 2 September 2002; accepted 8 January 2003.