The first report of Kadipiro virus isolation in China / 病毒学报

5 strains of virus isolated from Culex tritaeniorhynchus, Anopheles sinensis and Armigeres subalbatus, which caused cytopathic effect in C6/36 cells, had been obtained in the survey of arboviruses in Northwestern Yunnan Province. China. The virus particles displayed 70 nanometers diameter (n=7) with no envelope but spikes on the surfaces. RNA-PAGE of the genomes of the isolates showed 6-5-1 profile. A fragment of the 12th segment sequence was amplified by a pair of specific primers for Kadipiro virus strain JKT-7075 in RT-PCR. The full length of the 12th segment was 758 nucleotides, BLAST analysis revealed the highest identity was 90% to JKT-7075. Phylogenetic analysis demonstrated that the isolates appeared to be Kadipiro viruses (Family Reoviridae). It was the first report of kadipiro virus isolation in China.

Isolation and identification of Banna virus from mosquito for the first time in Inner Mongolia / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region.</p><p><b>METHODS</b>A strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar).</p><p><b>RESULTS</b>The new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%.</p><p><b>CONCLUSION</b>The virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.</p>

The study on vertical transmision of Nam Dinh Virus and Coltivirus group B from Culex quinque fasciatus in Can Tho Province

Background:\r\n', u'There are two virus known as Nam Dinh Virus, and Colti group B be found in Viet Nam. These viruses have appeared in the South, the Middle and the Highland. They haven\u2019t been reported in the Southern provinces and Can Thoas well. \r\n', u'Objectives: \r\n', u'To identify the circulation of Nam Dinh virus strain, and coltivirus group B strain in Can Tho, Southern Viet Nam, and their existence in nature.\r\n', u'Subjects and method: \r\n', u'Thirty-four mosquito samples (7, 453 individual mosquitoes) from Culex quinque faciatus and Culex pseudovishnui were collected in Can Tho provice, southern Vietnam 2005.\r\n', u'Isolatingviruses on Aedes albopictuc clone C6/36, Vero cells, and using PT- PCR and ELISA Sandwich for identification. \r\n', u'Results:\r\n', u'2 Nam Dinh virus strains, 2 coltivirus group B strains and 1 flavivirus strain (insect flavivirus) were isolated from Culex quinque faciatus, and no virus was isolated from Culex pseudovishnui.\r\n', u'Conclusion: \r\n', u'The identification of the transmission of Nam dinh Virus, and coltivirus group B in Can Tho province by isolating virus from Culex quinque faciatus has shown the evidence for natural vertical transmission of these viruses.\r\n', u'

Detection of Banna virus-specific nucleic acid with TaqMan RT-PCR assay / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus.</p><p><b>METHODS</b>Total RNA of strains of Banna virus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Banna virus. The sensitivity, specificity and the possibility of this assay to detect Banna virus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Banna virus culture medium supernatant were evaluated.</p><p><b>RESULTS</b>All the collected 12 Banna virus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Banna virus in 50 microl samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees C instead of 99 degrees C, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees C. There was no significant difference in the results of Banna virus detection between artificial human serum samples and positive control virus. Six were detected as positive for Banna virus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation.</p><p><b>CONCLUSION</b>The Banna virus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes screening.</p>

Studies of coltivirus in China / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>The purpose of this article is to review the developments of studies of Coltivirus in China.</p><p><b>DATA SOURCES</b>The data used in this review was obtained mainly from the studies of Coltivirus reported from 1990 to 2003 in China.</p><p><b>STUDY SELECTION</b>Relevant articles on studies of Coltivirus in domestic and foreign literature were selected.</p><p><b>DATA EXTRACTION</b>Data were maily extracted from the articles which are listed in the reference section of this review.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Genotyping of the Chinese isolates of coltivirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To classify the Chinese isolates of Coltiviruses.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Investigation of Arboviruses in Lancang river down-stream area in Yunnan province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p>

Preparation and storage of coltivirus antigen and its application in detection of coltivirus antibodies in serum from patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>