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VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens

Biomedical and Environmental Sciences 2018年第4期31卷 272-279页

作者： ZHANG Yi ZHANG Chen LI Bo LI Yang HE Xiao Zhou LI Acher WU Wei DUAN Su Xia QIU Fang Zhou WANG Ji SHEN Xin Xin YANG Meng Jie LI De Xin ma Xue Jun Key Laboratory for Medical Virology National Health and Family Planning Commission National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention State Key Laboratory of Plant Genomics and National Center for Plant Gene Research Institute of Genetics and Developmental Biology Chinese Academy of Sciences Pediatric Research Institute Children's Hospital of Hebei Province

Objective Unbiased next generation sequencing（NGS） is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification（VSITA） approach using a set of non-ribosomal and virus-enriched octamers（V8） was developed and compared with traditionally used random hexamers（N6）. Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR（qP CR）. Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences（sense and antisense） were selected from a dataset of random 4,096（4~6） hexamers（N6）. Two random nucleotides were added to the 5＇ end of the selected hexamers, and 480（30 × 4~2） octamers（V8） were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

关键词： Virus next generation sequencing Non-ribosomal Virus targeted

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HBV病人血清中丁型肝炎抗原检测和临床应用

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中华实验和临床病毒学杂志 1993年第4期7卷 335-338页

作者：赵炳华马伟杭浙江医科大学附属第一医院，杭州 310003

从HBV标志物阳性肝炎病人血清中，共检出HDAg阳性14份（14/167，8.38%）。其中慢活肝、肝硬化、慢迁肝HDAg阳性率各为30.00%、26.67%和16.67%，重症肝炎1例也为HDAg阳性。另用国产和荷兰产包被抗-HD和HRP-抗HD试作HDAg抗原性差异比较，... 详细信息

从HBV标志物阳性肝炎病人血清中，共检出HDAg阳性14份（14/167，8.38%）。其中慢活肝、肝硬化、慢迁肝HDAg阳性率各为30.00%、26.67%和16.67%，重症肝炎1例也为HDAg阳性。另用国产和荷兰产包被抗-HD和HRP-抗HD试作HDAg抗原性差异比较，发现人血清HDAg、黑猩猩肝HDAg及工程菌产HDAg各有两种反应类型。

关键词：丁型肝炎病毒 HDAg 抗-HD 检测

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2012—2021年青藏高原牦牛源沙门菌流行病学调查

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中国动物传染病学报 2024年第1期32卷 202-206页

作者：班马泽郎李天娇周赛赛吴丹卢姊豪宋仁德罗晓林赵索南马进寿参木友石红梅索朗斯珠西藏农牧学院动物科学学院林芝860000 青海玉树藏族自治州畜牧兽医工作站玉树815400 四川省草原科学研究院成都610097 青海省海北畜牧兽医科学研究所海北812200 青海省大通种牛场大通810102 西藏农牧科学院草原研究所拉萨850000 甘南州畜牧工作站甘南747000

为了明确青藏高原地区牦牛感染沙门菌(SE)情况,该研究采用ELISA双抗体夹心法,对2012—2021年采集自西藏、青海、甘肃、四川1725份牦牛血清样品进行SE抗体检测。结果显示:1725份牦牛血清中检测出SE抗体总阳性率为23.54%(406/1725),西藏... 详细信息

为了明确青藏高原地区牦牛感染沙门菌(SE)情况,该研究采用ELISA双抗体夹心法,对2012—2021年采集自西藏、青海、甘肃、四川1725份牦牛血清样品进行SE抗体检测。结果显示:1725份牦牛血清中检测出SE抗体总阳性率为23.54%(406/1725),西藏地区牦牛SE阳性率与其他地区相比较高;2012—2021年青藏高原地区牦牛血清SE抗体年阳性率分别为7.35%、46.02%、15.31%、17.68%、8.79%、19.84%、6.04%、23.64%、14.81%、74.64%;牦牛在不同海拔感染SE的阳性率分别为13.67%(2900~3400 m)、14.61%(3400~3900 m)、28.62%(3900~4400 m)、34.72%(4400 m以上);不同性别牦牛感染SE平均阳性率分别为公(25.61%)、母(21.99%);4省牦牛感染SE阳性率分别为西藏(27.67%)、青海(13.50%)、甘肃(13.33%)、四川(1.89%)。本研究结果可为了解青藏高原牦牛SE病的流行病学情况,提供参考。

关键词：青藏高原牦牛沙门菌流行病学

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Immuno-protective effect of tumor cell vaccine on Kunming mice bearing Ehrlich ascites tumor

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World Journal of Gastroenterology 1998年第5期4卷 41-45页

作者： ma Zheng, ZHOU Shao Juan, WU Kai Chun, PAN Bo Rong, QIAO Tai Dong, CHEN Bao Jun and FAN Dai Ming

AIM To evaluate the immunity of chemically modified tumor cell *** Tumor cell vaccines (TCV) were prepared by incubating the live Ehrlich ascites tumor cells with concanavalin A-mitomycin C (ConA-MMC), mitomycin C (MMC), concanavalin A-glutaraldehyde (ConA-Glu), glutaraldehyde (Glu), or paraformaldehyde (Para), respectively. The whole cell or soluble forms of the vaccines were administered intraperitoneally into Kunming mice once a week for three times prior to the intraperitoneal inoculation of a lethal dose of live tumor cells. A second challenge with live tumor cells was given four weeks later. Survival and antibody production of the mice were *** After the first challenge, the mice, received whole TCV of ConA-MMC, MMC (P＜0.01) and Glu (P＜0.05) promoted survival incidence than the controls. All the treated mice had the survival time prolonged. ConA-MMC vaccine treated mice had longer survival days than that of ConA-Glu ones (P＜0.05). For the soluble TCV immunized mice, those treated with vaccines of Para (P＜0.01), ConA-Para and ConA-Glu (P＜0.05) had longer survival periods compared with that of the controls. Following the second challenge, survival incidence of the mice received vaccines of ConA-MMC, MMC, ConA-Glu or Glu was significantly increased (P＜0.01). Moreover, all the treated mice had the survival time prolonged, and ConA-MMC vaccine treated mice had longer survival days than that of Para treated ones (P＜0.05). Antibodies against Ehrlich ascites tumor cells were found to be positive in sera of the mice treated with whole TCV of *** Ehrlich ascites tumor cells are immunogenic when treated with ConA-MMC, MMC, ConA-Glu, Glu or Para, which might act as safe and effective tumor vaccines with safety and effectiveness.

关键词： Ehrlich ascites tumor tumor cell vaccine Kunming mice antigen, neoplasm

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next-generation Sequencing Study of Pathogens in Serum from Patients with Febrile Jaundice in Sierra Leone

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Biomedical and Environmental Sciences 2019年第5期32卷 363-370页

作者： ZHANG Yi YE Fei XIA Lian Xu ZHU Ling Wei IDRISSA Laybohr Kamara HUANG Ke Qiang ZHANG Yong LIU Jun BRIma Kargbo WANG Ji LIANG Mi Fang SONG Jing Dong ma Xue Jun WU Gui Zhen National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206China Sierra Leone-China Friendship Biological Safety Laboratory FreetownSierra-Leone National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206China Chinese Center for Disease Control and Prevention Beijing 102206China Sierra Leone Ministry of Health and Sanitation Freetown Sierra-Leone Key Laboratory of Medical Virology National Health and Family Planning CommissionNational Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China

Objective People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus(YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice. Methods In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV‐negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice. Results The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus(89.58%), Epstein‐Barr virus(55.21%), hepatitis C virus(34.38%), rhinovirus(28.13%), hepatitis A virus(20.83%), coxsackievirus(10.42%), Ebola virus(8.33%), hepatitis E virus(8.33%), lyssavirus(4.17%), leptospirosis(4.17%), chikungunya virus(2.08%), Crimean‐Congo hemorrhagic fever virus(1.04%), and hepatitis B virus(1.04%). Conclusion The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.

关键词： Sierra Leone Febrile jaundice next‐generation sequencing Virus

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青海省75例重症甲型H1N1流感患者临床分析

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中国危重病急救医学 2010年第3期22卷 164-165页

作者：李华马如存青海省传染病医院急诊科西宁810000

目的了解青海省重症甲型H1N1流感患者的特点。方法回顾分析2009年9月4日至12月31日青海省传染病院收治的157例甲型H1Nl流感患者中75例重症、危重症的流行特点、临床症状及体征、辅助检查及治疗情况。结果10月14日开始出现首例重症患者，... 详细信息

目的了解青海省重症甲型H1N1流感患者的特点。方法回顾分析2009年9月4日至12月31日青海省传染病院收治的157例甲型H1Nl流感患者中75例重症、危重症的流行特点、临床症状及体征、辅助检查及治疗情况。结果10月14日开始出现首例重症患者，至12月31日共出现75例，占47．78％。75例患者中农牧区患者45例（占60．00％），城市患者30例；男48例，女27例；合并肺炎者53例（占70．67％）；有基础疾病者41例（占54．67％），有发热者65例（占86．67％），咳嗽、咯痰63例（占***％），呼吸困难61例（占81．33％）；以脑膜炎为主要表现者1例，以急性肺水肿、左心衰竭为主要表现者2例。外周血白细胞降低28例（占37．33％），心肌酶异常28例（占37．33％），血糖异常23例（占30．67％），凝血功能异常8例（占10．67％），血脂异常4例（占5．33％），C-反应蛋白增高51例（占68．00％）。48例给予奥司他韦、27例给予军科奥韦抗病毒，27例给予低分子肝素钠抗凝，22例给予甲泼尼龙治疗，33例行无创机械通气治疗者中10例改用有刨机械通气，首选二线抗生素并进行痰培养，对8例合并真菌感染者行抗真菌治疗。75例患者除1例死亡外，余均治愈。结论青海省重症高峰来得较早，重症比例高；甲型H1N1流感患者易合并肺部疾病，病情进展迅速，易致呼吸衰竭、弥散性血管内凝血、多器官功能衰竭。

关键词：青海流感病毒A型甲型H1N1流感重症呼吸衰竭

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The Mechanisms of SAA/TLR4 Inducing Angiogenesis in Rheumatoid Arthritis through neTs Formation

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Open Journal of Immunology 2022年第4期12卷 98-121页

作者： Wilfried Noel Sam Qiushuang Yan Yang Wang Xuguo Sun Wei Wei Jun ma Fang Zheng Department of Clinical Immunology School of Medical Laboratory Tianjin Medical University Tianjin China Department of Rheumatology General Hospital Tianjin Medical University Tianjin China Department of Health Statistics College of Public Health Tianjin Medical University Tianjin China

Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which angiogenesis represents a critical early event of synovial inflammation. The present study aimed to reveal the potential molecular mechanisms of SAA/TLR4 induction of angiogenesis through neTs in RA. materials and methods: Firstly, immunohistochemistry and immunofluorescence were used to determinate TLR4 and neTs expression in synovial tissue, respectively. ELISA was used to detect the content of SAA, MPO and ne in serum and synovial fluid of patients. DNA quantification was done by fluorescence. DNA fluorescence staining was used to compare neTs formation in RA and HC sera, and to investigate the mechanism of neTs formation induced by SAA stimulation. PicoGreen DNA testing was used to characterize the DNA in the supernatants. Also, DNA fluorescence staining to explore whether neTs formation induced by SAA was dependent or independent on NADPH oxidase pathway. MTT assay, Wound healing assay, Tube formation assay were performed to analyze human veins umbilical cells (HUVECs) proliferation, migration, and tube vessels formation, respectively under neTs or neTs + DNase stimulants. Results: Firstly, we demonstrated that TLR4 was predominantly and widely expressed in synovial tissues with elevated serum levels of SAA, compared to osteoarthritis (OA) patients, and the similar results were observed for neTs formation. Afterwards, in a series of in vitro experiments, we reported an increased MPO and ne levels, and a relatively decreased DNA level in the sera of RA patients. Set apart, the levels of MPO and ne in RA were correlated to the disease activity. Moreover, an increased spontaneous neTs formation was observed in RA patients, enhanced under SAA stimulation and regulated by TLR4 activation. And the total DNA expressed in RA patients was partly composed of neT-DNA. Also, SAA induced neTs formation dependent on NADPH pathway. Finally, our results indicated that extracted SAA-induced neTs promoted end

关键词： Angiogenesis neutrophil Extracellular Traps Rheumatoid Arthritis Serum Amyloid A Toll-Like Receptor 4

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Increased TIM-4 negatively correlates with IL-1β levels in type 2 diabetes

Increased TIM-4 negatively correlates with IL-1β levels in ...

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第十届全国免疫学学术大会

作者： Peiqing Zhao Hongxing Wang Xiaohong Liang Chunhong ma Lifen Gao Department of Immunology Shandong University School of Medicine Department of Central laboratory Zibo Central Hospital

Background: T-cell immunoglobulin- and mucin-domain-containing moleculel-4(TIM-4), a novel immune regulator, is selectively expressed on antigen presenting cells, especially on macrophages and mature dendritic cells. TIM-4 plays key roles in mutiple immune diseases. However, whether TIM-4 involves in type 2 diabetes mellitus(T2D) remains unknown so far. This study aimed to investigate the expression of TIM-4 in T2 D and addressed its significance in disease progression. Methods: Peripheral blood mononuclear cells(PBMCs) from T2 D patients and healthy subjects were isolated to measure the TIM-4 m RNA expression by real time PCR, and the sera were collected to detect interleukin-1 beta(IL-1β) and other clinical indicators. Results: The TIM-4 m RNA expression in PBMCs from T2 D patients were significantly increased compared to healthy controls. The levels of TIM-4 m RNA were positively related to hs CRP. However, TIM-4 m RNA levels were negatively correlated with IL-1β, indicating the potential role of TIM-4 in negatively regulating IL-1β production. We also found that TIM-4 m RNA negatively correlated with LDL and a tendency of negative relationship with Hb A1 ***: The present study indicates that TIM-4, at least partly, contributes to T2 D pathogenesis possibly by regulating IL-1β. TIM-4 may participate in the process of T2 D through regulating IL-1β secretion. In addition, the data suggest that TIM-4 may be a target for diagnosis and therapy of T2 D *** work was supported in part by grants from the National Nature Science Foundation of China(No. 81172862, 81371831), Research Fund for the Doctoral Program of Higher Education of China(RFDP)(20110131110034) and the Shandong Provincial Natural Science Foundation(No. ZR2010HM025).

关键词： TIM-4 T2D IL-1β

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丹参对持续癫痫幼鼠脑损伤后血清NSE影响的实验研究

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时珍国医国药 2007年第1期18卷 73-74页

作者：李伟马华中国人民武装警察部队广东医院广东广州510507

目的探讨丹参对持续癫痫幼鼠脑神经元损伤可能具有的保护作用及其机制。方法美解眠诱发健康幼龄大鼠癫痫持续状态发作。随机分组后分批处死。电镜观察海马神经元超微结构的改变,ELISA法测定血清NSE(神经元特异性烯醇化酶)浓度变化。结... 详细信息

目的探讨丹参对持续癫痫幼鼠脑神经元损伤可能具有的保护作用及其机制。方法美解眠诱发健康幼龄大鼠癫痫持续状态发作。随机分组后分批处死。电镜观察海马神经元超微结构的改变,ELISA法测定血清NSE(神经元特异性烯醇化酶)浓度变化。结果丹参治疗组幼鼠电镜下可见海马神经元较轻的超微结构病变和血清NSE浓度在发作后相同时点均低于持续癫痫组。结论丹参对持续癫痫幼鼠脑神经元损伤具有一定的保护作用;血清神经元特异性烯醇化酶与持续癫痫幼鼠神经元损伤变化趋势基本一致,能作为判断脑神经元损伤及其恢复的有效指标。

关键词：丹参惊厥性脑损伤神经元特异性烯醇化酶

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Cloning and expression of cDNAs from hepatitis E virus structural gene

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Hepatobiliary & Pancreatic Diseases International 2003年第3期2卷 387-390页

作者： Bing Ruan Edward Zumbika Shu-Ying Wang Yong Chen Yi-Lin ma Ya-Gang Chen Ke-Zhou Liu the First Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310003, China Hangzhou First People’s Hospital Zhejiang Academy of Medical Sciences First Affiliated Hospital Medical College of Zhejiang University Hangzhou 310003 China. my_bing@***

OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection. METHODS: A 492 base cDNA was collected from 5'-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E. coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E. RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100. CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

关键词： hepatitis E virus open reading frame cloning polymerase chain reaction western blot

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