HYDROPATHY

We report the genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus (CCHFV). Two CCHFV strains isolated in Xinjiang Province, a region endemic for CCHF in northwestern China, were studied. These strains, designated BA66019 and BA8402, were isolated in 1965 and 1984 from a CCHF patient and Hyalomma ticks, respectively. Viral RNA was extracted from suckling mouse brains infected with these two strains, amplified, and sequenced. The full-length M RNA, consisting of 5.3 kb, was determined for both strains. The coding nucleotide sequences of the two strains differed from each other by 17.5% and from the reference CCHFV strain IbAr10200 by a mean of 22%, suggesting that the genus Nairovirus comprises a group of genetically highly diverse strains.
Crimean Congo hemorrhagic fever (CCHF), one of the most severe human viral diseases, has a death rate of up to 50%. CCHF is a public health problem in many regions of the world, including Africa, Middle East, southern and eastern Europe, and Western Asia (1-9). The causative agent, Crimean Congo hemorrhagic fever virus (CCHFV), is the type species of the genus Nairovirus in the Bunyaviridae family. The virus is transmitted to humans by the bite of Ixodid ticks (mostly of the Hyalomma genus) or by contact with blood or tissues from human patients or infected livestock. After an incubation period of 3 to 7 days, the patient has sudden onset of fever, chills, myalgia, headache, and rapidly evolving severe illness, followed by a hemorrhagic state with bleeding from the mucous membranes and petechiae, associated with thrombocytopenia and leukopenia (10). The highly pathogenic nature of the virus; the risk for spread from person to person through exposure to infected blood, respiratory secretions and excreta, resulting occasionally in serious nosocomial outbreaks; and the lack of an effective and safe therapy indicate the need for adequate guidelines for management of viral hemorrhagic fevers.
CCHFV, like all members of the genus, is a negative-stranded RNA virus with a tripartite genome consisting of a small (S), a medium (M), and a large (L) segment. The S RNA segment codes for the nucleocapsid (N) protein, and the M RNA segment codes for the glycoprotein precursor, resulting in the two envelope glycoproteins G1 and G2, while the L segment encodes the putative RNA-dependent polymerase. The first 8 to 13 nucleotide bases at the 3' ends of all three RNA segments have a sequence that is conserved in the viruses of this genus, with a complementary consensus sequence occurring at the 5' end; the ends of the segments are noncovalently linked so that the RNA occurs in a loosely bound circular configuration within the nucleocapsids (11). The M segment of nairoviruses is 30% to 50% larger than the M segments of members of other genera in the Bunyavidae family and has a potential coding capacity of up to 240 kDa of protein (12). CCHFV shares some common antigenic and genetic properties with Dugbe virus (DUGV), another member of the genus Nairovirus; some G1 epitopes may be conserved between the two viruses. Current knowledge of the molecular heterogeneity of CCHFV strains circulating in different parts of the world is very limited.
In China, the first CCHF cases were observed in 1965, when CCHFV strain BA66019 was isolated from a patient who lived in Xinjiang Province, an autonomous region in northwestern China which is the most CCHF-endemic area in the country. Another strain, BA8402, was isolated in 1984 from Hyalomma asiaticum ticks from the same region. The full-length S genome segment of these strains has been sequenced and analyzed (13). Over a 30-year period (1965 to 1994), 260 CCHF cases have been reported in China, 54 (21%) of them fatal. In 1997, an outbreak occurred in Honghai village (Bachu County, Xinjiang Province). During the 45-day outbreak period, 26 cases were reported, 5 of them fatal. Antibodies to CCHFV have been detected in humans and animals in the following provinces: Xinjiang, Qinghai, Sichuan, Yunnan, Anhui, Hennan, and Inner Mongolia (13).
Sequences of short S RNA CCHFV fragments from different parts of the world have shown considerable genetic differences (14-16). However, knowledge about the M RNA fragment is very limited, as no reports have been published and the only available complete M RNA sequence is that of the reference strain IbAr10200, isolated in 1970 from ticks in Nigeria (GenBank accession number U39455).
The M gene is critical for immunity and pathogenicity, as well as for vaccine development. To define the molecular variability among CCHFV strains, we determined the nucleotide sequences of the M RNA genome segment of two CCHFV strains (BA66019 and BA8402) isolated in China. We also compared their predicted amino acid sequences with the respective sequences of the reference strain.
Virus Strains and Methods
Two CCHFV strains were studied. The first, strain BA66019, was isolated in 1965 from an ill resident of Xinjiang Province. The second strain, BA8402, was isolated in 1984 from Hyalomma asiaticum ticks from the same geographic region. Both strains were propagated in Vero E6 cells.
RNA was extracted from suckling mouse brains infected with the two strains by using Trizol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. Complementary DNA was prepared with one gene-specific primer and random hexamers. We used 12 sets of primers to amplify 12 overlapping fragments of the whole M RNA of the two CCHFV strains. Primers were designed with the OLIGO primer analysis software (Molecular Biology Insights, Cascade, CO), first based on the known sequences of CCHFV strain IbAr10200 and later on the basis of the sequences determined. The amplified polymerase chain reaction (PCR) products were purified and sequenced in an ABI 373A Perkin Elmer fluorescent dye automated sequencer (Perkin Elmer/Applied Biosystems, Foster City, CA). Sequences were aligned by ClustalW and genetic distances were estimated by the Kimura 2-parameter method with DNAdist program from the PHYLIP package (17). A phylogenetic tree was constructed by using Seqboot, DNAdist, Fitch and Consence from the same software package. Hydrophobicity plots were done by the Kyte and Doolittle method (18).

Results
Twelve overlapping M segment PCR products were amplified and sequenced in both directions, covering a continuous region of the whole M RNA segment of two CCHFV isolates, BA66019 and BA8402; the M RNA sequences were submitted to GenBank and assigned accession numbers AF350448 and 350449, respectively. The overlapping sequences were identical.