Complete L segment coding-region sequences of Crimean Congo ...

Arch Virol (2006) 151: 465–475 DOI 10.1007/s00705-005-0648-0

Complete L segment coding-region sequences of Crimean Congo hemorrhagic fever virus strains from the Russian Federation and Tajikistan J. D. Meissner1 , S. S. Seregin2 , S. V. Seregin2 , N. V. Yakimenko2 , O. I. Vyshemirskii2 , S. V. Netesov2 , and V. S. Petrov2 1 Department

of Microbiology, University of New Mexico, Albuquerque, New Mexico, U.S.A. 2 State Research Center of Virology and Biotechnology “VECTOR”, Koltsovo, Novosibirsk Region, Russia Received June 15, 2005; accepted August 25, 2005 c Springer-Verlag 2005 Published online September 30, 2005


Summary. The large (L) RNA segment of Crimean Congo hemorrhagic fever (CCHF) virus strain AST/TI30908, isolated from pooled Hyalomma marginatum ticks collected in 2002 from theAstrakhan region of European Russia, was amplified piecemeal using reverse-transcription/polymerase chain reaction, followed by direct sequencing of gel-purified amplicons. After removal of 5 and 3 primer-generated termini, the assembled AST/TI30908 L segment sequence is 12112 nucleotides long, with 41.3% G + C content, and is greater than 87% and 96% identical at the nucleotide and translated amino acid levels, respectively, to partial or full-length CCHF virus L segment sequences deposited in GenBank. A complete L segment coding-region sequence for CCHF virus strain TAJ/HU8966, isolated from a patient in Tajikistan in 1990, was determined in a similar fashion. This L segment (12133 nucleotides long, 41.1% G + C content) shares 88% nucleotide identity with the full-length strain Matin from Pakistan, and 97% nucleotide identity with a partial L segment sequence of strain Khodzha from Uzbekistan. Strain TAJ/HU8966 shares at least 96% identity at the translated amino acid level with all other CCHF virus L segment sequences. Although, for the most part, CCHF virus L polyprotein primary sequences are uniformly well conserved, a region of marked variability was identified in the N-terminal half of the RNA-dependent RNA polymerase. This region,

GenBank accession numbers for sequences reported here are AY675240, AY720893, and AY995158.

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approximately 50 amino acids in length, is flanked by previously-reported arenavirus and bunyavirus-conserved regions, and may prove useful in CCHF diagnosis and viral taxonomy. Introduction First reported from the Crimean peninsula of the former Soviet Union sixty years ago [5], the arthropod-borne Crimean Congo hemorrhagic fever (CCHF) virus is now recognized as endemic in many parts of the Eastern Hemisphere, established in a broad range through much of Africa, the Middle East, Eastern Europe, Central Asia, and western China [11, 39, 33–35, 15; for recent review see 36]. Transmission of CCHF virus occurs through bites of Ixodid (hard) ticks, or by direct contact with body fluids or tissues of infected ticks, small mammals, livestock (e.g., cattle, sheep, goats, ostriches) or humans [11, 2]. Because no vaccine is commercially available, the potentially dire consequences of a CCHF outbreak in the absence of early ribavirin treatment have improved little from its earliest descriptions. Clinically, infection can present as a severe hemorrhagic and toxic syndrome, with outbreak case-fatality rates of 30% or more still routinely reported [11, 30, 13, 27, 4, 12, 23, 31]. Studies of CCHF case-fatality rate differences between geographically and ecologically distinct regions are largely anecdotal [3]. Intuitively, regional differences in fatality rates could be ascribed to differences in strain pathogenicity, to differing levels of viral exposure within certain at-risk professions (e.g., established butchering practices may put workers in contact with larger amounts of virus-infected substrate for longer periods of time), and to availability and quality of supportive care, including the use of barrier precautions. Within the same region, historical differences in fatality rates could be related to alterations in any of these parameters: Increased attenuation of an endemic strain upon repeated passage has also been speculated to play a role [24]. Crimean-Congo hemorrhagic fever virus is a member of the genus Nairovirus, family Bunyaviridae. Similar to other bunyaviruses, CCHF virus is a spherical, enveloped virus with a single-stranded, tripartite RNA genome of negative polarity. The three minus-strand genomic RNA segments, named small (S), medium (M), and large (L) segments according to their molecular weights (size), all have terminal inverted repeat sequences essential for replication and packaging. Their respective plus-strand complements, each with a characteristically short 5 non-coding region (NCR), encode the nucleocapsid (N) protein, the precursor of two surface glycoproteins (G2 and G1), and a polyprotein which includes an RNA-dependent RNA polymerase (RdRp) component [6, 17, 29, 10, 14]. During the last decade, a number of CCHF virus strains have been genetically characterized using partial or complete S and/or M segment nucleotide sequences [see 9 for an updated list]. However, sequencing of the CCHF virus L segment has lagged behind, slowed by a lack of closely-related nairovirus sequence on which to base primer design for reverse-transcription and amplification, as well as the

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somewhat daunting size of the nairovirus L segment compared to other member of the family Bunyaviridae [18, 10]. Alignment of published or deposited sequences of two unrelated CCHF virus strain L segments, and partial sequence determinations of additional strains [GenBank accession number AY422208; 9, 10, 14] have facilitated subsequent CCHF virus L segment sequence determination. Renewed interest in CCHF virus in the Western Hemisphere relates to its designation as a Category A bioterrorism agent [http://www.bt.cdc.gov; 32]. An equally relevant public health concern in the Eastern Hemisphere is posed by the appearance of disease in places where CCHF has not been previously documented. These may be “natural” introductions of CCHF virus, due to importation of infected (or tick-infested) livestock or through migration of birds infested with infected ticks, or may simply be a result of long-established but unrecognized endemic foci, owing to a lack of appropriate laboratory facilities or viral reagents necessary for testing. Whatever the cause, improved CCHF diagnostics are crucial for determining the accuracy and source(s) of reported new occurrences of disease [12], or apparent increases in disease incidence [4]. In recent years, SRC-VB “Vector” has become involved in epidemiologic investigations of reported CCHF outbreaks in Commonwealth of Independent States (CIS) countries. Disease outbreaks, or the presence of virus or viral genetic or antigenic material, have been reported in the southern regions of European Russia, as well in the former Soviet republics of Moldova, Ukraine, Armenia, Azerbaijan, Georgia, and the Central Asian countries of Tajikistan, Turkmenistan, Uzbekistan, the Kyrgyz Republic, and Kazakhstan [11, 35, 38 and Russian references therein]. The benefits of genetic characterization at the stage of initial diagnosis cannot be over-emphasized – where sequence data are available, recent CCHF outbreaks or “mini-outbreaks” in Albania, Bulgaria, the current Serbia and Montenegro, and Turkey have all been attributed to European Russian-type strains of CCHF virus [7, 12, 24–26]. This finding is not surprising given the European distribution of Hyalomma tick species [11]. Despite the longstanding history of CCHF research in the former Soviet Union, scant information is available on the L segment sequences of CCHF virus strains present in the Russian Federation and other CIS countries [9]. Our goals in the current study were to establish full-length L segment sequences of CCHF virus laboratory isolates representative of strains circulating in European Russia and Central Asia, identify shared features among these strains, and determine the level of conservation of these features with previously-characterized African and Middle Eastern strains. Comparisons of obtained CCHF virus L segment ORF sequences with available partial and full-length sequences revealed three short regions of variability in the encoded L polyprotein, set against an otherwise static background of strict amino acid residue conservation or isofunctional amino acid residue substitutions. More detailed examination of one of these variable regions indicates it might serve as a practical substitute for full-length L segment sequences in phylogenetic analysis, and could potentially supplement partial or full-length

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S- and/or M-segment sequences as a molecular signature distinguishing even closely-related CCHF virus strains. Materials and methods CCHF virus strains Three laboratory-adapted CCHF virus strains were used in this work. Strain AST/TI30908, isolated in 2002 from pooled Hyalomma marginatum ticks from the Astrakhan province of the Russian Federation (48.05◦ N and 46.35◦ E), was obtained from the collection of the D. I. Ivanovsky Institute of Virology of the Russian Academy of Medical Sciences in Moscow as total RNA from infected SW-13 cells. Strain STV/TI28981, isolated in 2000 from pooled Hyalomma ticks in the Stavropol territory (approximately 400 km southwest of Astrakhan) was also provided by the Ivanovsky Institute as total RNA from infected cells. Strain TAJ/HU8966, isolated in 1990 from a CCHF patient in Tajikistan (38.57◦ N and 68.78◦ E), was part of the collection of the State Research Center for Virology and Biotechnology (SRC VB) “Vector”. Strain AST/TI30908 underwent five passages in SW-13 cell culture, as did strain STV/TI28981. Strain TAJ/HU8966 had been subjected to 15 passages in suckling mouse brain and three passages in SW-13 cell culture. RNA extraction Total RNA was extracted using either the RNeasy MiniKit (QIAGENE GmbH, Germany) according to the enclosed instructions, or a classical phenol – chloroform extraction method [28]. Reverse transcriptase – polymerase chain reaction (RT-PCR) Primers design was performed with OLIGO (version 6.0) program (Borland International, USA), and was based on pairwise alignment of complete L RNA segment sequences from previously characterized strains (strain Matin from Pakistan and strain IbAr10200 from Nigeria) or strain-specific sequence information as it became available. The goal of primer selection and design was to produce overlapping amplicons of 350–700 nucleotides spanning the entire L segment, for use in subsequent direct sequencing. Both degenerate and sequencespecific primers were synthesized either on-site by the State Research Center of Virology and Biotechnology “VECTOR” (Koltsovo, Novosibirsk Region, Russian Federation) or obtained from Integrated DNA Technologies (Coralville, Iowa, USA). A complete list of the approximately 155 primers required for this project is available from the corresponding author on request. RT-PCR was performed using the Access RT-PCR kit (Promega, Madison, Wisconsin, USA), according to the manufacturer’s instructions. If a second round of amplification, either nested or hemi-nested, was required to generate visible product, we used Taq DNA polymerase (SibEnzyme, Novosibirsk, Russian Federation) and 2 percent of the reaction product obtained from the RT-PCR stage. All PCR reactions were performed using the DNA amplifier MiniCycler, model PTC-150, manufactured by MJ Research Company (Waltham, Massachusetts). Product purification PCR products were analyzed by electrophoresis in a 1% agarose gel. DNA bands of the expected size were excised and purified using the Q-Biogene GeneClean extraction kit (Carlsbad, California, USA) according to the enclosed instructions.

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Sequencing and sequence comparison Gel-purified PCR products were directly sequenced using a CEQ 2000 Dye Terminator Cycle Sequencing kit run on the CEQ 2000XL DNA Analysis System (both from Beckman Coulter, Fullerton, California, USA) according to the manufacturer’s instructions. All sequences were confirmed on both strands. Sequence assembly was performed using Contig Express from the analysis package of the Vector NTI suite, version 9.0 (InforMax Corporation, Bethesda, Maryland, USA). Comparisons of CCHF virus and other nairovirus and bunyavirus nucleotide and amino acid sequences were performed using the DNASTAR package of analysis software (DNASTAR, Inc., Madison, Wisconsin, USA). Complete L-segment sequences of CCHF virus strains Matin (GenBank AY422208), IbAr10200 (AY422209), the nairovirus strain Dugbe (U15018) and partial L-segment sequences of CCHF virus strains K229–243 (AJ620683), Baghdad-12 (AJ579312), and Khodzha (AJ620685) were used for comparison. Sequence deposition Nucleotide sequences determined in this study were assigned the following GenBank accession numbers: AY675240 (strain AST/TI30908), AY720893 (strain TAJ/HU8966), and AY995158 (strain STV/TI28981).

Results and discussion Strain AST/ TI30908 L segment sequence analysis The CCHF virus strain AST/TI30908 (referred to hereafter as 30908) L segment is 12112 nucleotides in length, excluding the 5 and 3 terminal (primer-generated) nucleotides, with a G + C content of 41.3%. Assuming the length of the terminal inverted repeat is conserved in strain 30908 as it is in other nairovirus L segment termini sequenced thus far [18, 14], the total length of the 30908 L segment would be 12150 nucleotides. Although strain 30908 full-length S- and M- segment sequences have not yet been determined, partial S- and M- segment sequencing was performed as part of this work. Strain 30908 S segment (nucleotide (nt) positions 1001–1280, numbering based on CCHF virus strain Drosdov full-length S segment sequence, GenBank Accession Number U88412) and M segment (nt positions 2597–2905, numbering based on CCHF virus strain VLG/TI29414 full-length M segment sequence, GenBank AY179961) sequences shared up to 99% and 97% nucleotide identity, respectively, with S- and M-segment sequences previously reported for CCHF virus strains circulating in the southern region of European Russia [37, 38, 31]. A 1057-nt partial L-segment sequence of CCHF virus strain STV/TI28981 (from nt positions 2051–3107, with numbering based on strain 30908), isolated from the Stavropol territory of Russia in 2000, shares 99% nucleotide identity with strain 30908. The close identity of strain 30908 with S-, M-, and L-segment sequences of other strains from European Russia indicates it is representative of CCHF virus strains circulating in this region, both now and in the past. As well, it indicates that strain 30908 is not a reassortant virus, as has been proposed for strain Matin [9] and may prove to the be the case for strain IbAr10200, since its S segment and

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M segment sequences group with different strains in pairwise comparisons and phylogenetic trees [21, 4, 31]. Equally important, the less than 100% nucleotide identity of strain 30908 with other European Russian strains confirms that strain 30908 and its fulllength L-segment sequence are authentic, and not a laboratory or artifactual contaminant resulting from a strain previously passaged (or genetic material previously amplified) in this laboratory. The L ORF in strain 30908 extends from nt positions 59–11896, and would encode a polyprotein of 3945 amino acid (aa) residues with a predicted molecular mass of 448 kDa. This L polyprotein contains the core catalytic module identified in RNA-dependent RNA polymerases (RdRps) from both positive- and negative-strand RNA viruses [22, 14], as well as an N-terminal cysteine-protease motif typical of the ovarian tumor (OTU) protein superfamily [16]. Predicted helicase, topoisomerase, gyrase, and cytoskeleton-related elements previously reported for the CCHF virus strain IbAr10200 L-segment sequence [10, 14] are also conserved. The locations of RdRp regions in the CCHF virus strain 30908 L polyprotein compared to another representative Bunyaviridae are shown in Fig. 1. Strain TAJ/HU8966 L segment sequence analysis The L segment sequence for CCHF virus strain TAJ/HU8966 (hereafter referred to as 8966) is 12133 nucleotides in length (after removal of the 5 and 3 primergenerated ends), with a G + C content of 41.1%. The strain 8966 L segment ORF sequence shares 89% nucleotide identity with the Matin strain, and 97% identity with a partial 3 terminal L segment sequence reported for strain Khodzha from Uzbekistan [9]. The full-length M segment sequence of strain 8966 has already been determined [31] as has the S segment sequence [partial sequence reported in 37, 38]. The close relationship of these S-, M-, and L- segment sequences with other isolates or with genetic material amplified from humans or ticks in the same area indicates that strain 8966 is an authentic representative of strains circulating in this part of the world. CCHF virus L segment sequence comparisons and variable region features Size differences in the full-length L segment of CCHF virus strains sequenced thus far are attributable to differences in the lengths of the 5 and 3 non-coding regions (NCRs), as the lengths of the polyprotein ORF are identical among strains. The strain 30908 L segment ORF shares 90% nt identity with strain IbAr10200 and 88% nt identity with strain Matin (IbAr10200 and Matin are 87% identical). When partial L segment sequences are included in the analysis, strains 30908 and K229–243 from Russia are 93% identical over 980 nucleotides [9]. At the translated amino acid level, all CCHF virus strains sequenced thus far are at least 96% identical with each other. Although the number of full-length CCHF virus L

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Fig. 1. Schematic alignment of L protein of hantavirus strain Andes R123, total length 2153 amino acids [20] with L polyprotein of CCHF virus strain 30908, total length 3945 amino acids. Blocks labeled 1 through 4 are recognized regions of amino acid conservation (with or without assigned roles) in segmented negative-strand virus RNA-dependent RNA polymerases (RdRps) – (pre)motifs A through E of the core polymerase module (region 3) are omitted for clarity. Designations, locations and lengths of RdRp regions/modules taken from previously published alignments [22, 19, 1, 14] corrected for amino acid position numbering errors in the latter report – The CCHF virus strain 30908 L segment ORF is equivalent in length to the IbAr10200 L segment ORF deposited by Kinsella et al. [14; see GenBank AY389361], but CCHF virus L segment amino acid positions in their paper are consistently off by 4. The block labeled “O” is an ovarian tumor (OTU) protein superfamily motif found in nairovirus L polyproteins„ which contains a cysteine protease-like Asp-Cys-His catalytic triad [16, 10, 14]. The block labeled V is a region of increased amino acid variability in nairovirus L polyproteins. Numbers adjacent to these two blocks are amino acid positions in the CCHF virus L polyprotein. Numbers in parentheses indicate corresponding nucleotide positions for all identified regions in the CCHF virus strain 30908 L segment (note that numbering for this segment starts after removal of the initial 5 terminal primer-generated nucleotides). The block labeled “?” is a hypothetical CCHF virus protein encoded by the antisense (viral) RNA of strain 30908 and other CCHF viruses, with bracketed numbers indicating the strain 30908 L segment sense strand sequence location of this conserved CCHF virus L segment antisense (viral) strand ORF

segments available for comparison is small, and does not include representative sequences from all identified groups or clades [38, 8], multiple alignment of CCHF virus L polyproteins indicate 94% of aa residues are “invariant”. When additional L segment sequences become available, it seems likely that, characteristic of other Bunyaviridae [20], the CCHF viral L polyprotein will be conserved to the same extent as the viral nucleoprotein. Amino acid differences between strains are not randomly distributed throughout the L polyprotein, but instead cluster to some degree in three locations – the N-terminal 200 aa (despite this being the region which includes the OTU protein superfamily cysteine-protease motif), the C-terminal 200 aa, and an approximately 50-aa region between the previously-identified arenavirus and bunyavirusconserved RdRp regions 1 and 2 [22, 14] corresponding to nt positions 2346–2513 in strain 30908, as shown in Fig. 1. Interestingly, this latter region (Fig. 2) is where a large deletion in the CCHF virus L segment relative to the Dugbe virus L segment is found [10, 18]. These three regions, which comprise just over 10% of the total L

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Fig. 2. Nairovirus partial L polyprotein single-letter amino acid alignment, with amino acid positions based on CCHF virus translated L segment ORF numbering. A dot represents amino acid identity at that position with the IbAr10200 sequence. The sequence included in this alignment corresponds to strain 30908 L segment nucleotide positions 2240–2635, and demonstrates a region of variable amino acid identity in the nairovirus RdRp flanked by essentially “invariant” regions in CCHF virus RdRp sequences, one of which is the previouslyidentified arena- and bunyavirus conserved region 1, as noted [22]. The variable region is arbitrarily defined as extending from nt positions 2342–2488 in strain 30908 (corresponding to CCHF virus L polyprotein amino acid positions 762 to 810, although there is obviously reduced variability after amino acid position 784 in the sequences included in this alignment). The choice of presenting size differences in the variable region as “insertions” in the nairovirus strain Dugbe L segment is for the sake of convenience, they could just as likely represent deletions in the CCHF viral progenitor

polyprotein residues, account for more than 30% of the total variable aa positions in CCHF virus translated full-length L segments. An explanation of why a stretch of ∼50 aa between RdRp regions 1 and 2 is allowed to vary so markedly in nairoviruses while the flanking regions are strictly conserved, indeed when the overwhelming majority of amino acid residues in the CCHF L segment polyprotein ORF are strictly conserved, awaits RdRp structural analysis by crystallography or mutational analysis using reverse genetics. Whatever the reason, this ∼150 nt region might serve a practical role in CCHF virus diagnosis and phylogenetic analysis, in that a single primer set for reverse transcription and amplification could be designed to hybridize to the conserved flanking regions of all CCHF viruses, while the variable region sequence, provided it varies between even closely-related strains, could yield robust information for taxonomy and molecular epidemiology. Given the time and expense required to determine CCHF virus full-length L segment sequences, it seems more likely that partial sequence determinations will predominate, at least for the foreseeable future. Concerns about the advantages

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of the CCHF virus L segment variable region for genetic analysis compared to other regions of the L segment, or to partial or full-length S- or M-segment sequence determinations, can only be addressed by comparison of a larger number of sequences from both closely-related and distantly-related strains. In light of the recognized potential for nairovirus genomic segments to reassort [9], it may prove necessary to examine more than one CCHF virus genome segment to better characterize strains. Acknowledgments This work was supported by the Cooperative Threat Reduction program of the United States Defense Threat Reduction Agency (DTRA) as part of the International Science and Technology Center (ISTC) grant #1291-2p, and by the Ministry of Public Health of the Russian Federation. The assistance of V. Gutorov, V. A. Ternovoi and other staff members of the SCR-VB sequencing facility, and G. F. Sivolobova, who kindly reviewed the first draft of the manuscript, was greatly appreciated. We also wish to extend thanks to D. K. Lvov of the Ivanovsky Institute in Moscow, who provided inactivated total RNA of CCHF virus-infected cells, and to J. Smith from the United States Army Military Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland, for his generous gift of the SW-13 cell line.

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