[Isolation and identification of arboviruses in Hebei Province].

BACKGROUND: To study the arboviruses carried by mosquitoes collected in Hebei Province. METHODS: Samples were collected from mosquito active sites and stored in liquid nitrogen till use. Pools of 20 to 30 mosquitoes were ground after sterilization, centrifugal supernant was inoculated onto C6/36 cell, cytopathic effect was observed for three sequential passages. Positive isolates were identified by IFA and RT-PCR. RESULTS: Totally 1310 mosquitoes were collected from two villages of She county, Hebei province. They were divided into 46 pools and ground respectively. Thirteen positive isolates were obtained. Two isolates reacted with alphaviral antibodies and were amplified by alphaviral primers, nucleotide sequence showed the highest homology (98%) to Getah virus (AY702913.1), so the two isolates were identified as Getah virus. CONCLUSION: Getah virus was isolated from mosquitoes in Hebei Province. This is the first report of isolating Getah virus from inland of China.

Liao ning virus, a new Chinese seadornavirus that replicates in transformed and embryonic mammalian cells.

Seadornaviruses are emerging arboviral pathogens from the south-east of Asia. The genus Seadornavirus contains two distinct species, Banna virus (BAV) isolated from humans with encephalitis and Kadipiro virus. BAV replicates within insect cells and mice but not in cultured mammalian cells. Here, the discovery of Liao ning virus (LNV), a new seadornavirus from the Aedes dorsalis mosquito, which was completely sequenced and was found to be related to BAV and Kadipiro virus, is reported. Two serotypes of LNV could be distinguished by a serum neutralization assay. According to amino acid identity with other seadornaviruses, and to criteria set by the ICTV for species delineation, LNV was identified as a member of a new species of virus. Its morphology was characterized by electron microscopy and found to be similar to that of BAV. LNV is the first reported seadornavirus that replicates in mammalian cells, leading to massive cytopathic effect in all transformed or embryonic cell lines tested. LNV- and BAV-infected mice producing a viraemia lasting for 5 days was followed by viral clearance. Mice infection generated virus quasi-species for LNV (the first reported observation for quasi-species in the family Reoviridae) but not for BAV. Challenge with BAV in mice immunized against BAV did not lead to productive infection. However, challenge with LNV in mice immunized against LNV was lethal with a new phase of viraemia and massive haemorrhage.

Yunnan orbivirus, a new orbivirus species isolated from Culex tritaeniorhynchus mosquitoes in China.

An orbivirus designated Yunnan orbivirus (YUOV) was isolated from Culex tritaeniorhynchus mosquitoes collected in the Yunnan province of China. Electron microscopy showed particles with typical orbivirus morphology. The YUOV genome was sequenced completely and compared with previously characterized orbivirus genomes. Significant identity scores were detected between proteins encoded by the segments (Seg-1 to Seg-10) of YUOV and those encoded by their homologues in insect-borne and tick-borne orbiviruses. Analysis of VP1 (Pol) and VP2 (T2, which correlates with the virus serogroup) indicated that YUOV is a new species of the genus Orbivirus that is unrelated to the other insect-borne orbiviruses. The replication of YUOV in mosquito cell lines was restricted to Aedes albopictus cells and the virus failed to replicate in mammalian cell lines. However, intraperitoneal injection of virus into naïve mice resulted in productive, non-lethal virus replication and viraemia. Infected mice developed serum neutralizing antibodies and were protected against a new infection challenge. Sequence analysis of clones from the segments encoding outer coat proteins (Seg-3 and Seg-6) of YUOV recovered from mouse blood did not show significant changes in the sequences. The availability of the complete genome sequence will facilitate the development of sequence-specific PCR assays for the study of YUOV epidemiology in the field.

Studies of coltivirus in China.

OBJECTIVE: The purpose of this article is to review the developments of studies of Coltivirus in China. DATA SOURCES: The data used in this review was obtained mainly from the studies of Coltivirus reported from 1990 to 2003 in China. STUDY SELECTION: Relevant articles on studies of Coltivirus in domestic and foreign literature were selected. DATA EXTRACTION: Data were maily extracted from the articles which are listed in the reference section of this review. RESULTS: Many Coltiviruses have been isolated not only from blood samples of patients with unknown fever or from cerebrospinal fluid of patients with encephalitis in Xishuangbanna area in Yunnan province, but also from mosquitoes collected in many areas in China. In some patients diagnosed as Japanese encephalitis or unknown fever, an increase of Coltivirus IgG antibody of fourfold, or more, has been detected using ELISA. Similarly, Coltivirus IgM antibody was positive in some patients with Japanese encephalitis or viral encephalitis. From most Chinese patients, except the northeastern, the isolates of Coltiviruses belong to subgroup B2, according to RT-PCR amplification of the ninth and twelfth segments of the isolates and sequence analysis of their amplicons. Some biological properties of Chinese Coltiviruses isolates are different from that of North American Coltiviruses. CONCLUSIONS: The isolates of Coltiviruses from Chinese patients are one of the common agents causing viral encephalitis and unknown fever in summer-autumn season. It might be an important public health problem due to its high isolation rate and wide distribution in China. Mosquito is the main transmission vector of the virus.

[Molecular biological identification of Batai virus isolated in China].

BACKGROUND: To study the molecular characteristics of YN92-4 strain isolated from mosquitoes in Yunnan Province and define its classification. METHODS: The S segment of YN92-4 strain was amplified and sequenced by 2 different sets of primers. The phylogenic tree of S fragment was constructed by Phylip bio-software. The amino acid sequences of N and NSs proteins were also studied. RESULTS: YN92-4 strain could be amplified by 2 sets of primers respectively, S segment showed a highest homology with Batai virus (X73464), reached 96.4%, the homology of protein N and NSs amio-acid sequence with Batai virus was 99.1% and 98% respectively. CONCLUSION: The YN92-4 strain belongs to Batai virus, this is the first report of molecular biological identification of Batai virus in China.

[Investigation of Arboviruses in Lancang river down-stream area in Yunnan province].

OBJECTIVE: To investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province. METHODS: Mosquitoes were collected from Lancang river downstream area (including Lancang county and Simao city) during summer in 1998 and stored in liquid nitrogen after classification. The mosquito pools were homogenized and centrifuged, then the supernatant was inoculated into C6/36 cells for virus isolation. New isolates were identified by neutralization test(NT), ELISA, immunofluorescence assay(IFA) and polyacrylamid gel electrophoresis(PAGE). RESULTS: Totally 22 isolates of arbovirus were obtained from 233 mosquito pools by inoculation of C6/36 cells and positive rate of the isolation was 9.4%. Ten strains were resistant to both ether and 5 prime-IDU. So they were non-enveloped double-stranded (ds) RNA virus. Twelve segmented RNAs were shown by PAGE and PAGE profiles from the ten strains were 6-6 with minor variation. The isolates can be neutralized by immunized mouse ascites fluid of BJ95-75 strains of coltivirus by NT, and reacted with monoclonal antibody against BJ95-75 by ELISA. These viruses were identified as coltivirus. Nine isolates were sensitive to ether and resistant to 5 prime-IDU. So they were non-enveloped RNA viruses. PAGE showed 10 segmented RNA, and they were identified to be orbiviruses. Three isolations were sensitive to ether. One of them can be neutralized with JEV A2 strain antibody by NT and was positive to the homologous antibody by IFA. It was identified being strain of JE virus. One strain(YN92-4) can be reacted with anti-bunyavirus group specific immune ascites fluid by both IFA and ElISA, but reacted neither with anti-alpha virus group, nor with anti-flavivirus group JE virus ascites fluid. The virions are spherical and about 87 nm in diameter with surface projections by negative staining. Conclusion Twenty-two isolates have been obtained from wild caught-mosquitoes of Lancang river down-stream area in Yunnan province. Among them ten, nine, one and one were identified as coltivirus, orbivirus, JE virus and bunyavirus, respectively. One is under identification. This is the first report on bunyavirus isolated from mosquitoes in China.

[Genotyping of the Chinese isolates of coltivirus].

OBJECTIVE: To classify the Chinese isolates of Coltiviruses. METHODS: Three sets of primers were selected among them two were specific to the 9th and 12th segments of subgroup B2, and one was for the 12th segment of subgroup B1-All the Chinese isolates of Coltivirus selected in the experiment were classified according to the lengths of different amplicons of the reverse transcriptase-polymerase Chain reaction (RT-PCR). The homogenicity of the nucleic acids of the isolates BJ95-75 and YN-6 was also compared with other Coltivirus strains belonging to subgroup B2. RESULTS: With the primers 12-854-S/12-B2-R, which were specific to the 12th segment of Coltivirus subgroup B2-850 bp amplicons were obtained from Beijing isolate BJ95-75 and all the Yunnan isolates such as YN-6, -67-1, -68-1, -69, -70-1, -70-2, -90, -92-2, -93 of Coltivirus 492 bp DNA fragments were also amplified from all of them with the segment 9th specific primers 9-JKT-S/9-JKT-R. However no positive results were obtained from Northeast isolates NE97-12, NE97-31 and control viruses YN-99(Orbivirus),YN-151-1(JEV) with the same two sets of primers. With 12-B1-S/12-B1R primers specific to the 12th segment of subgroup B1, no amplicons of right length were obtained from any of the Chinese isolates of Coltivirus and the control viruses. When compared the nucleic acid sequences of BJ95-75 and YN-6 with other Coltivirus strains such as Bannavirus, JKT6423, JKT6969, JKT7043, the amplicons from segment 12th of these two strains had more than 89.4% homology with the other strains, especially to the earlier Chinese isolate Bannavirus, the homolog was more then 98.9%. Nearly 96.5% and 99.2% of the nucleic acids of the amplicons from segment 9th of the two strains were being homologous to Bannavirus and about 84.0% to JKT6423, which had been classified into type B2a. But the maximal homogenicity was about 53% when compared with the other two coltivirus strains. JKT6969 and JKT7043 which had been classified into type B2b. CONCLUSION: Genotyping the recent Chinese isolates of coltivirus for the first time in our country. Most of the Chinese isolates belong to subgroup B2, more exactly type B2a. The Northeast isolates NE97-12 and NE97-31 were not correctly grouped with the available primers.

[Preparation and storage of coltivirus antigen and its application in detection of coltivirus antibodies in serum from patients].

OBJECTIVE: To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients. METHODS: The coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected. RESULTS: The highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE. CONCLUSIONS: Stable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.