Research progress of gut microbiota and primary biliary cholangitis / 医学研究生学报

Primary biliary Cholangitis (PBC) is a cholestatic autoimmune liver disease with a prolonged course and poor prognosis. The intestinal micro-ecological system is composed primarily by intestinal mucosa and gut microbiota. The system is closely related to the liver in function and anatomy, mediates the gut-liver axis and participates in maintaining various physiological, immune and metabolic functions of the body. Analysis of the relationship between the gut microbiota and PBC may provide new treatment ideas for clinical treatment of PBC. This article mainly reviews the relationship between gut microbiota imbalance and disease, the interaction of gut microbiota and its metabolites with the liver, and the potential value of fecal microbiota transplantation in the treatment of PBC.

Gene chip technology used in the detection of HPV infection in esophageal cancer of Kazakh Chinese in Xinjiang Province / 华中科技大学学报（医学）（英德文版）

This study was aimed to screen human papillomavirus (HPV) types associated with esophageal squamous cell carcinoma of Kazakh in Xinjiang using the gene chip technique and study the clinical significance of this application. The DNAs were collected from esophageal squamous cell carcinoma tissues and healthy esophageal mucosa of Kazakh adults in Xinjiang, and amplified firstly using HPV MY09/11 and then using HPV G5+/6+ to screen positive HPV specimens. These positive specimens were further detected by the gene chip technique to screen highly pathogenic HPV types. After determination with nested PCR amplification with HPV MY09/11 and G5+/6+, the infection rate of HPV was 66.67% in the esophageal squamous cell carcinoma group and 12.12% in the healthy control group. By testing the positive HPV specimens from the esophageal squamous cell carcinoma group, the infection rate of HPV16 was 97.72% and the co-infection rate of HPV16 and HPV18 was 2.27%. HPV16 infection may be involved in the development of esophageal squamous cell carcinoma in Xinjiang Hazakh adults.

Gene chip technology used in the detection of HPV infection in esophageal cancer of Kazakh Chinese in Xinjiang Province / 华中科技大学学报（医学）（英德文版）

This study was aimed to screen human papillomavirus (HPV) types associated with esophageal squamous cell carcinoma of Kazakh in Xinjiang using the gene chip technique and study the clinical significance of this application. The DNAs were collected from esophageal squamous cell carcinoma tissues and healthy esophageal mucosa of Kazakh adults in Xinjiang, and amplified firstly using HPV MY09/11 and then using HPV G5+/6+ to screen positive HPV specimens. These positive specimens were further detected by the gene chip technique to screen highly pathogenic HPV types. After determination with nested PCR amplification with HPV MY09/11 and G5+/6+, the infection rate of HPV was 66.67% in the esophageal squamous cell carcinoma group and 12.12% in the healthy control group. By testing the positive HPV specimens from the esophageal squamous cell carcinoma group, the infection rate of HPV16 was 97.72% and the co-infection rate of HPV16 and HPV18 was 2.27%. HPV16 infection may be involved in the development of esophageal squamous cell carcinoma in Xinjiang Hazakh adults.

Influence of the depth of retained denatured dermis on the survival rate of grafted skin in burn swine with deep partial-thickness burn / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the influence of the thickness of retained denatured dermis on the survival rate of grafted skin in swine with deep partial-thickness burn.</p><p><b>METHODS</b>Four deep partial-thickness wounds were reproduced respectively on both sides of spine in 7 Chinese domestic pigs. The wounds of 6 pigs were divided into 0.25, 0.50, 0.75, and 1.00 mm groups with 12 wounds in each group according to the random number table. Tangential excision and auto-skin grafting were performed. Before the tangential excision, 1 tissue specimen was harvested from the center of each remaining wound for the estimation of the depth of burn, and histological observation was done. After the tangential excision, 1 tissue specimen was harvested from the area near the center of each wound for the measurement of the depth of retained denatured dermis with histological examination. The 8 wounds of one pig were set as the control group, and the operation was done, and then they were treated with exposure treatment after biopsy specimens were taken with above-mentioned method. The general condition of wounds in 5 groups was observed from immediately after injury to post injury month (PIM) 3. On post injury day (PID) 7, the survival rate of grafted skin was observed in 0.25, 0.50, 0.75, and 1.00 mm groups. Wound healing time was recorded. At PIM 3, the specimens were harvested from the wounds of 5 groups, and their ultra microstructures were observed by transmission electron microscope. Data were processed with rank-sum test, one-way analysis of variance, and LSD test.</p><p><b>RESULTS</b>The depth of the burn tissue was (1.120 ± 0.211) mm. The depths of retained denatured dermis in 0.25, 0.50, 0.75, and 1.00 mm groups were respectively (0.830 ± 0.031), (0.701 ± 0.010), (0.382 ± 0.031), and (0.141 ± 0.040) mm. At PID 8, all grafted skin in 0.25 and 0.50 mm groups became necrotic; most grafted skin in 0.75 mm group was necrotic; most grafted skin in 1.00 mm group survived with only a few became necrotic and separated from the wounds. The scabs were gradually separated from the wounds of control group. On PID 15, the grafted skin which did not survive in 0.25, 0.50, and 0.75 mm groups was gradually separated from the wounds with exudate forming scab on the surface in varying degrees, while the wounds in 1.00 mm group were all healed, and the incidence of scabs formation was highest in control group. At PIM 3, scar contraction was found in 0.25, 0.50, 0.75 mm groups and control group, while no obvious scar was observed in 1.00 mm group. There were statistically significant differences in the survival rate of grafted skin in 0.25, 0.50, 0.75, and 1.00 mm groups (χ(2) = 19.421, P < 0.001). The survival rate was the highest in 1.00 mm group [70% (60%, 80%)], while the survival rate was 20% (0, 30%) in 0.75 mm group, and it was in both 0.25 and 0.50 mm groups with non-survival of all the grafted skin. There were statistically significant differences in the wound healing time among 5 groups (F = 41.450, P < 0.001). The wound healing time in 0.25 and 0.50 mm groups were respectively (18.2 ± 1.5), and (18.7 ± 2.3) d, not statistically significant different from that of control group [(18.4 ± 1.7) d, P values both above 0.05]. The wound healing time in 0.75 mm group [(14.9 ± 2.6) d] was significantly different from those of 0.25, 0.50 mm groups and control group (P values all below 0.01). The wound healing time in 1.00 mm group [(9.5 ± 1.2) d] was significantly shorter compared with that of the other 4 groups (P values all below 0.01). Before tangential excision, the zone of infiltration of the inflammatory cells was observed in the deep dermis of wounds in 5 groups. After tangential excision and before auto-skin grafting, the depth from the fault surface to the zone of infiltration of the inflammatory cells varied in 0.25, 0.50, 0.75, and 1.00 mm groups while more inflammatory cells were observed in control group. At PIM 3, many fibroblasts were observed in the dermis of wounds in 1.00 mm group with abundant rough endoplasmic reticulum and basically intact organelles.</p><p><b>CONCLUSIONS</b>Performing autologous skin grafting on deep partial-thickness burn, in which the depth of retained denatured dermis was 0.10 mm, may help regenerate dermal function and alleviate scar formation.</p>

Comparative analysis of Brucellosis epidemic between Shanxi province and the country / 中国地方病学杂志

Objective To analyze the Brucellosis incidence and to predict the trends of the disease in Shanxi province and the national in recent years,which could provide the reference for surveillance,prevention and control of the disease.Methods Brucellosis data which was reported monthly during January 2006 and December 2010 in Shanxi province and the data released by Chinese Center for Disease Control and Prevention during January 2005 and December 2010 were collected.Several indexes,such as the annual increasing number,the development rate,growth rate and other indicators were applied to compare Shanxi province with the national Brucellosis epidemic in recent years.What's more,the seasonal autoregressive integrated moving average model (ARIMA) was fitted respectively with the data of Brucellosis incident number reported monthly,so as to predict the prevalence status in the coming two years by verifying the fitting effect.Results Brucellosis prevalence of Shanxi province reached the peak in 2008,and the incidence number was 5397,which was 900 more than 2007.From the onset of decline after 2008,the prevalence decreased by 17.67％ (906/5128) in 2010.However,national incidence of Brucellosis kept increasing before 2009 and the prevalence increased rapidly from 2007 to 2008,and the growth rate reached 39.16％ (8442/21 560).Although the number of Brucellosis fell by 2041 cases in 2010 than in 2009,the rate of decline was only 5.14％(2041/37 734).The fastigium of Brucellosis was from May to July yearly whether Shanxi province or the country.The ARIMA models of Shanxi province and the nation were ARIMA [(1,0,1)(1,1,0)12] and ARIMA[(1,0,1)(0,1,1)12],respectively,according to the incidence numbers reported monthly.The fitting effect of models showed that the predicted values of the two models were both consistent with the actual situation and all predicted values fell within the 95％ confidence limits,which depicted that they both fitted well.The predicted values depict that the incidence of Brucellosis overall trend was basically stable in Shanxi province,while the numbers in the nation would increase in a small extent in 2011 and 2012.The fastigium of Brucellosis was still from May to July yearly.Conclusions Brucellosis control measures are effective in Shanxi province,incidence of Brucellosis declining.The ARIMA model could predict the number of Brucellosis well,which can provide a valuable reference for the predication and evaluation of Brucellosis epidemic in the future.

Blocking 1800 MHz mobile phone radiation-induced reactive oxygen species production and DNA damage in lens epithelial cells by noise magnetic fields / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate whether the exposure to the electromagnetic noise can block reactive oxygen species (ROS) production and DNA damage of lens epithelial cells induced by 1800 MHz mobile phone radiation.</p><p><b>METHODS</b>The DCFH-DA method and comet assay were used respectively to detect the intracellular ROS and DNA damage of cultured human lens epithelial cells induced by 4 W/kg 1800 MHz mobile phone radiation or/and 2 muT electromagnetic noise for 24 h intermittently.</p><p><b>RESULT</b>1800 MHz mobile phone radiation at 4 W/kg for 24 h increased intracellular ROS and DNA damage significantly (P<0.05). However, the ROS level and DNA damage of mobile phone radiation plus noise group were not significant enhanced (P>0.05) as compared to sham exposure group.</p><p><b>CONCLUSION</b>Electromagnetic noise can block intracellular ROS production and DNA damage of human lens epithelial cells induced by 1800 MHz mobile phone radiation.</p>

Effect of Skp2 antisense oligodeoxynucleotide on growth and proliferation of gastric carcinoma SGC-7901 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of S-phase kinase-associated protein 2 antisense oligodeoxynucleotide (Skp2 ASODN) on the growth and proliferation of gastric carcinoma SGC-7901 cells and its mechanism.</p><p><b>METHODS</b>The Skp2 oligodeoxynucleotides (ODNs) were embedded in cationic liposome Lipofectamine 2000 reagent and transfected into SGC-7901 cells. The cell growth and proliferation were observed with light microscopy and MTT assay. Cell cycle was measured by flow cytometry. The expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The expression levels of Skp2 protein and its substrate p27 protein were detected by Western blot.</p><p><b>RESULT</b>After treatment with Skp2 ASODN, the growth and proliferation of SGC-7901 cells were inhibited in a dose-dependent manner with a peak value at 48 h. The inhibition rate of 200 nmol/L group at 48 h was 42.4 % (P<0.01). In cell cycle study the percentage of S phase cells in 200 nmol/L group was significantly higher than that in normal control group (P<0.05). Both Skp2 mRNA and its protein levels in 200 nmol/L group were significantly lower than those in control group and in Skp2 nonsense oligodeoxynucleotide (Skp2 NSODN) group (P<0.05). However, p27 mRNA level remained unchanged although its protein level was significantly higher than that in control group and NSODN group (P<0.05).</p><p><b>CONCLUSION</b>Skp2 ASODN can inhibit the growth and proliferation of SGC-7901 cells, which may be mediated by interfering with ubiquitin-proteosome pathway and cell cycle regulation.</p>

Laboratory testing of specimens from patients with viral encephalitis from some regions of China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the viral etiology of viral encephalitis in China by detecting IgM antibody and viral RNA in the clinical samples of patients from some provinces of China by enzyme-linked immunosorbent assay and polymerase chain reaction.</p><p><b>METHOD</b>Serum and cerebrospinal fluid samples of 771 patients with viral encephalitis or meningitis were collected from six provinces of China and were stored at -20 degrees C or -70 degrees C. Enzyme-linked immunosorbent assays were used for detection of IgM antibody to Japanese encephalitis virus, coxsackievirus, echovirus, herpes simplex virus, measles virus, varicella-zoster virus, mumps virus, cytomegalovirus and Epstein-Barr virus. Polymerase chain reaction was applied for the detection of viral RNA of enteroviruses and seadornavirus.</p><p><b>RESULTS</b>IgM antibody was detected in 567 of 771 (73.5%) cases. The most common pathogen was Japanese encephalitis virus (47.0%, 362/771), followed by mumps virus (10.6%, 82/771), enteroviruses (8.8%, 68/771), herpes simplex virus (5.7%, 44/771), measles virus (0.4%, 3/771), varicella-zoster virus (0.4%, 3/771), Epstein-Barr virus (0.4%, 3/771), and cytomegalovirus (0.3%, 2/771). Enterovirus was positive in 8 cases, seadornavirus was negative in all the cases by PCR.</p><p><b>CONCLUSION</b>According to the study, Japanese encephalitis was the most important encephalitis in China. Mumps virus was another important pathogen. Enteroviruses and herpes simplex virus were also important pathogens.</p>

Combined effects of antisense TBRI eukaryotic expressing plasmid and antisense TIMP-1 eukaryotic expressing plasmid on rat liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To test the hypothesis that the introduction of antisense transforming growth factor beta receptor I (TBRI) plasmid and antisense tissue inhibitor of matrix metalloproteinase (TIMP-1) eukaryotic expressing plasmid into a rat liver fibrosis model may influence the progression of liver fibrosis.</p><p><b>METHODS</b>Fragments of TBRI cDNA and TIMP-1 cDNA were obtained by reverse transcription polymerase chain reaction (RT-PCR) and then amplified by nest PCR. pcDNA3.1(+)-antisense TBRI eukaryotic expressing plasmid was constructed by directional and inverted joins with the purified linear pcDNA3.1(+) and the purified fragment of TBRI, as well as, pcDNA3.1(+)-antisense TIMP-1 eukaryotic expressing plasmid. The recombinant was identified by restriction endonuclease digestion and DNA sequence analysis. The recombinant plasmids were encapsulated with Lipofectmine 2000, and then they were injected intraperitoneally into the liver fibrosis model rats. The protein expression of type I collagen was evaluated by immunohistochemistry. VG staining of liver slides of the rats was used for histopathological examination.</p><p><b>RESULTS</b>Compared with the empty plasmid control group and the disease control group, the deposition of type I collagen decreased in the three antisense treatment groups: antisense TBRI group (4.37+/-1.30) x 10(5), P less than 0.05; antisense TIMP-1 group (3.40+/-0.91) x 10(5), probability value less than 0.05; antisense TBRI + antisense TIMP-1 group (0.90+/-0.32) x 10(5), P less than 0.01; treatment control group (6.90+/-1.61) x 10(5); disease control group (7.34+/-1.68) x 10(5); and the normal control group (0.41+/-0.21) x 10(5)]. Significant differences in the pathological grades of fibrosis were found between the normal control group and the other five groups (P less than 0.05) and also between the disease control group and the three antisense treatment groups (antisense TBRI group P less than 0.05; antisense TIMP-1 group P less than 0.05; antisense TBRI + antisense TIMP-1 group P less than 0.01), but no difference was found between the empty plasmid control group and disease control group (P more than 0.05).</p><p><b>CONCLUSION</b>Both antisense TBRI eukaryotic expressing plasmid and antisense TIMP-1 eukaryotic expressing plasmid can inhibit the progress of liver fibrosis. A combined action can inhibit the progress of liver fibrosis more.</p>

Progress in mechanism of microcystin toxicity / 浙江大学学报·医学版

Microcystin is one of the monocyclic heptapeptides produced primarily by microcystis aeruginosa. Recent studies suggest that microcystin can induce cell apoptosis, as well as oxidative stress and mitochondrial alteration. Studies also indicate that Bcl-2 family and p53 may play an important role in the apoptosis induced by microcystin.

Correlation between gene polymorphisms of Wnt signalling pathway related components and risk of gastric carcinoma: a case-control study / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate correlation between polymorphisms of rs3755557 and rs1880481 located in glycogen synthase kinase 3beta gene and beta-catenin gene respectively, the products of which are components of Wnt signalling pathway, and risk of gastric carcinoma.</p><p><b>METHODS</b>PCR and denaturing high-performance liquid chromatography combining with DNA sequencing were used to analyse genotype polymorphism of rs3755557 and rs1880481 of the subjects including 26 patients of gastric carcinoma and 33 patients of chronic superficial gastritis.</p><p><b>RESULTS</b>Chi-square analysis revealed that there was no significant difference in the frequencies of alleles and genotypes of rs3755557 polymorphic site between gastric carcinoma group and control group. As to the rs1880481 polymorphic site, there was no significant difference in the frequencies of alleles between gastric carcinoma group and the corresponding control group. The frequency of heterozygous genotype in male control group was 68.18% and it was significantly higher than 26.67% in male gastric carcinoma group, OR=5.893, 95%CI: 1.377-25.226 (P=0.013); but the frequencies of AA genotype of the site in male control group and male gastric carcinoma group were 9.09% and 40.00% respectively. There was statistical significance, OR=6.667, 95% CI: 1.121-39.660 (P=0.025).</p><p><b>CONCLUSION</b>The above results suggest that the genotypes and alleles of rs3755557 site do not contribute to the risk of gastric carcinoma. Low level of the heterozygous genotype or high level of AA genotype of rs1880481 polymorphic site in male patients might cause a higher risk of developing gastric carcinoma.</p>

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>

Expression of P53 and Bax in rat liver induced by microcystin LR in vivo / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To examine the expression of P53, Bcl-2 and Bax proteins in rat liver after exposed to microcystin LR.</p><p><b>METHODS</b>SD rats received microsystin LR by gastric perfusion. The expression of P53, Bax and Bcl-2 in liver was detected by Western blot.</p><p><b>RESULTS</b>The expression of P53 and Bax in each treatment group increased significantly compared with that in control group(P<0.05), with the exception of 0.1 microg/kg LR exposure group. Moreover, with exposure levels increasing the expression of P53 and Bax increased gradually; while no changes of the expression of Bcl-2 were observed.</p><p><b>CONCLUSION</b>P53 and Bax may play important roles in microcystin LR induced apoptosis, but Bcl-2 seems not be involved in this process.</p>

Molecular biological survey of tick-born arboviruses in southern part of Xinjiang / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To disclose the species and distribution of tick-borne arboviruses in the southern part of Xinjiang.</p><p><b>METHOD</b>Totally 5045 ticks were collected from 36 collecting sites of 23 places in the southern Xinjiang, which were made into cDNA pools with pd(N)6 primer through RT-PCR method. Then PCR was used to detect viral nucleotide sequence from cDNA.</p><p><b>RESULTS</b>All 34 cDNAs showed negative to flavivirus and California serogroup virus primers; but nairovirus and primers derived from Xinjiang hemorrhagic fever virus had amplified and yielded some obvious bands corresponding to the nucleotide sequences of Xinjiang hemorrhagic fever virus. A phylogenetic analysis was done to the obtained partial sequences of L and S segments.</p><p><b>CONCLUSION</b>Nucleotide sequences of Neither flaviviruses nor California serogroup viruses were detected from the samples. However partial L segment sequence was first reported in China. Phylogenetic analysis of partial L and S segments disclosed the molecular characteristic of Xinjiang hemorrhagic fever virus.</p>

Detection of Sindbis virus-specific nucleic acid with SYBR GREEN I real time PCR assay / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To develop a rapid, specific and sensitive method for detecting Sindbis virus (SINV) with SYBR GREEN I real time PCR.</p><p><b>METHODS</b>Total RNA of strains of Sindbis virus and a related virus were extracted and reverse transcribed to cDNAs. With the cDNAs as template, the SYBR GREEN I real time PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Sindbis virus, and the sensitivity, specificity and reproducibility were evaluated.</p><p><b>RESULTS</b>For the PCR, 55 degrees C was chosen as the optimal anneal temperature and 0.5 micromol/L as the optimal primer concentration. Using this method, all the selected SINV were detected as positive, while the results of control arboviruses such as Geta virus, Japanese encephalitis virus (JEV), Batai virus, Seadornavirus Orbiviruses and synthesized WEEV cDNA were negative. With this system, 0.1 PFU/ml SINV cDNA could be detected; The sensitivity of this assay was about 100 times higher than standard RT-PCR. All the results were reproducible within two compatible tests, and the stability of the detection system was very good. The test results of simulated infection human serum samples showed that human serum had no obvious interference with this system. With this system, 6 of 151 clinical samples with unknown fever or encephalitis were determined as positive.</p><p><b>CONCLUSION</b>The developed SYBR GREEN I real time PCR assay for detecting Sindbis virus was highly sensitive, specific and showed a good reproducibility and stability. It is our belief that the present method can be further used in clinic sample to verify its stringency.</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>

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>