Epidemiology and phylogenetic analysis of crimean-congo hemorrhagic fever viruses in xinjiang, china.

In 2004 and 2005, an epidemiological survey of Crimean-Congo hemorrhagic fever virus (CCHFV) was conducted in Xinjiang, China. A total of 5,629 serum samples of human and livestock were collected and tested for the CCHFV antibody, and 17,319 ticks were collected for viral identification. Reverse passive hemagglutination inhibition assays showed that the average prevalence of CCHFV antibody was 1.7% for the humans and 12.7% for the livestock. A relatively high antibody prevalence, ranging from 19.1% to 23.4%, was found in the livestock of the northwest, southwest, and northeast parts of the Tarim Basin. When the ticks were pooled to inoculate suckling mice, followed by reverse transcription-PCR (RT-PCR) to detect CCHFV RNA, the average RT-PCR-positive rates for Hyalomma asiaticum kozlovi and H. asiaticum asiaticum were 12.9% and 2.6%, respectively. A significant correlation was found between the antibody prevalence in the livestock and the CCHFV prevalence in H. asiaticum of the same geographic region. No CCHFV RNA was detected in Dermacentor nivenus, Rhipicephalus turanius, or Rhipicephalus sanguineus. A total of 27 partial S segments of CCHFVs were sequenced and used for phylogeny analysis. All but one Chinese isolate grouped into the Asia 1 clade, which contains the strains from Xinjiang and Uzbekistan, while the other strain, Fub90009, grouped with strains from the Middle East.

[Complete genome sequence of 84FLi, a Hantaan virus strain isolated from the liver of fetus aborted by a pregnant women with hemmorrhagic fever with renal syndrome].

OBJECTIVE: To study the complete genome sequence of the Chinese Hantaan virus vaccine strain 84FLi and learn about its molecular characters. METHODS: The virus strain 84FLi was isolated from the liver of a fetus aborted by pregnant women with hemorrhagic fever with renal syndrome. The cDNAs of L M and S segments were amplified fragment by fragment using RT-PCR. The purified PCR products were sequenced directly or cloned into pMD18-T vector and then sequenced. RESULTS: The complete genome of strain 84FLi was composed of L ( 6 533 bp) M (3 616 bp) and S (1 688 bp) coding 2151 1135 and 429 amino acids respectively. The entire sequence composition was 3830A 2050C 2510G and 3447T the GC and AT contents were 38.52% and 61.48%. Homology analysis showed that the homologies of 84 FLi S segment nucleotide sequences with strain RG9 (isolated in Guangzhou) and strain Chen4 (isolated in Anhui) were 99.6%. There were 83.7% 84.0% and 87.2% nucleotide sequences homology with the three segments of Hantaan virus foreign standard strain 76-118 while the amino acids sequences homology with those of 76-118 were 97.5% 96.0% and 97.9% respectively. CONCLUSION: The strain 84FLi is highly related to other Chinese Hantaan virus isolates and is in the same subtype with the other two Chinese isolates RG9 and Chen4.

[Molecular analysis on the S gene of three Crimean-Congo hemorrhagic fever virus strains in China].

OBJECTIVE: To compare the molecular characteristics of 3 Crimean-Congo hemorrhagic fever viruses(CCHFV) isolated in Xinjiang province. METHODS: YL05035, YT05099 and LT05146 were isolated in 2005 from Hyalomma ticks and viral RNA was extracted from suckling mouse brains infected with these three strains respectively. The polymerase chain reaction(PCR) products of S segments from the 3 strains of CCHFV were directly sequenced. RESULTS: The full-length'S RNA from the 3 strains of CCHFV all comprised 1673 nucleotides with ORF of them including 1449 nucleotides and encoding a protein which comprised 482 amino acids in a viral complementary sense. The sequences indicated that the three strains of CCHFV isolated from ticks in Xinjiang province were highly homologenic. Data from the phylogenetic analysis showed that the obtained sequences were identical. The homology between 3 strains of CCHFV was 99.5%. Their homologies compared with that of the other strains isolated from other region of Xinjiang were also high at nucleotide levels (92.7%-99.8%). The three strains which were clustered together with 7001 strain and 79121 strain (isolated from patient and rat in Xinjiang respectively) was only different by 2%-3%. The genetic difference from the prototype CCHFV Nigerian strain IBAR10200 was 13%. In comparison, the Nigerian CCHFV tick isolate was more divergent when compared with the reference China strains 66019 and with the three variants mentioned above. CONCLUSION: The CCHFV isolated from China comprised a group of genetically high conserved strains.

[Study on the molecular biology of hemorrhagic fever virus in Xinjiang].

OBJECTIVE: To explore the relationship between the structure and function at molecular level and the routes of transmission of Xinjiang hemorrhagic fever (XHF) virus. METHODS: S genes of five XHF virus strains were cloned, sequenced and compared with that of other Crimean-Congo hemorrhagic fever virus strains. RESULTS: It was found that S genes of the five viruses had 1,672 nuclei tides, while ORF of them including 1,449 nuclei tides and coded with a protein of 482 amino acid. The nucleotides homology of Chinese isolates (93.0%-99.5%) was obviously higher than that of any other S genes strains identified in other countries'. Phylogenetic tree showed that all Chinese isolates clustered into one branch and could be further divided into another three groups. CONCLUSION: The sequential difference of S genes was not totally related to the host, areas and time of the viruses isolated.

[Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

OBJECTIVE: To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. METHODS: Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. RESULTS: The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. CONCLUSIONS: This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

Genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus strains, China.

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.

[Detection of the expression of alpha3-integrin on hantavirus permissive cells].

BACKGROUND: To express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin. METHODS: The human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E. coli. The gene expression was confirmed by Western blot assay. Rabbit was inoculated with purified antigen to stimulate the antibody generation. The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis. The separated cell clones were confirmed by immunofluorescence and Western blot assay. RESULTS: The alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity. High titer antibody was generated. However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells. CONCLUSIONS: The results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.