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Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses

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Journal of Integrative Agriculture 2017年第9期16卷 2055-2061页

作者： WANG Xiu-rong GU Lin-lin SHI Jian-zhong XU Hai-feng ZHANG Ying ZENG Xian-ying deng guo-hua LI Cheng-jun CHEN hua-lan State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin（HA） cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.

关键词： H7N9 highly pathogenic influenza virus real-time RT-PCR

Protective efficacy of an H5/H7 trivalent inactivated vaccine(H5-Re13,H5-Re14, and H7-Re4 strains) in chickens, ducks, and geese against newly detected H5N1, H5N6, H5N8, and H7N9 viruses

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Journal of Integrative Agriculture 2022年第7期21卷 2086-2094页

作者： ZENG Xian-ying HE Xin-wen MENG Fei MA Qi WANG Yan BAO Hong-mei LIU Yan-jing deng guo-hua SHI Jian-zhong LI Yan-bing TIAN guo-bin CHEN hua-lan State Key Laboratory of Veterinary Biotechnology National Poultry Laboratory Animal Resource CenterHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

Some H5 viruses isolated in poultry or wild birds between 2020 and 2021 were found to be antigenically different from the vaccine strains(H5-Re11 and H5-Re12) used in China. In this study, we generated three new recombinant vaccine seed viruses by using reverse genetics and used them for vaccine production. The vaccine strain H5-Re13 contains the hemagglutinin(HA) and neuraminidase(NA) genes of an H5 N6 virus that bears the clade 2.***.4.4 h HA gene, H5-Re14 contains the HA and NA genes of an H5 N8 virus that bears the clade 2.***.4.4 b HA gene, and H7-Re4 contains the HA and NA genes of H7 N9 virus detected in 2021. We evaluated the protective efficacy of the novel H5/H7 trivalent inactivated vaccine in chickens, ducks, and geese. The inactivated vaccine was immunogenic and induced substantial antibody responses in the birds tested. Three weeks after vaccination, chickens were challenged with five different viruses detected in 2020 and 2021: three viruses(an H5 N1 virus, an H5 N6 virus, and an H5 N8 virus) bearing the clade 2.***.4.4 b HA gene, an H5 N6 virus bearing the clade 2.***.4.4 h HA gene, and an H7 N9 virus. All of the control birds shed high titers of virus and died within 4 days post-challenge, whereas the vaccinated chickens were completely protected from these viruses. Similar protective efficacy against H5 viruses bearing the clade 2.***.4.4 h or 2.***.4.4 b HA gene was observed in ducks and geese. Our study indicates that the newly updated H5/H7 vaccine can provide solid protection against the H5 and H7 N9 viruses that are currently circulating in nature.

关键词： avian influenza H5/H7 trivalent vaccine H5-Re13 H5-Re14 H7-Re4 strains protective efficacy chickens ducks and geese

Protective efficacy of an H5/H7 trivalent inactivated vaccine produced from Re-11, Re-12, and H7-Re2 strains against challenge with different H5 and H7 viruses in chickens

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Journal of Integrative Agriculture 2020年第9期19卷 2294-2300页

作者： ZENG Xian-ying CHEN Xiao-han MA Shu-jie WU Jiao-jiao BAO Hong-mei PAN Shu-xin LIU Yan-jing deng guo-hua SHI Jian-zhong CHEN Pu-cheng JIANG Yong-ping LI Yan-bing HU Jing-lei LU Tong MAO Sheng-gang guo Xing-fu LIU Jing-li TIAN guo-bin CHEN hua-lan State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China Harbin Weike Biotechnology Development Company Harbin 150069P.R.China

We developed an H5/H7 trivalent inactivated vaccine by using Re-11, Re-12, and H7-Re2 vaccine seed viruses, which were generated by reverse genetics and derived their HA genes from A/duck/Guizhou/S4184/2017(H5N6) (DK/GZ/S4184/17) (a clade 2.***.4.4d virus), A/chicken/Liaoning/SD007/2017(H5N1) (CK/LN/SD007/17) (a clade 2.***.2.1d virus), and A/chicken/ Guangxi/SD098/2017(H7N9) (CK/GX/SD098/17), respectively. The protective efficacy of this novel vaccine and that of the recently used H5/H7 bivalent inactivated vaccine against different H5 and H7N9 viruses was evaluated in chickens. We found that the H5/H7 bivalent vaccine provided solid protection against the H7N9 virus CK/GX/SD098/17, but only 50–60% protection against different H5 viruses. In contrast, the novel H5/H7 trivalent vaccine provided complete protection against the H5 and H7 viruses tested. Our study underscores the importance of timely updating of vaccines for avian influenza control.

关键词： avian influenza H5/H7 trivalent vaccine Re-11 Re-12 and H7-Re2 strains protective efficacy

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黄土隧道围岩的结构性变化特征分析

岩土工程学报 2008年第2期30卷 219-224页

作者：邓国华邵生俊西安理工大学岩土工程研究所陕西西安710048

在分析和修正已有结构性参数的基础上,首次提出了将结构性参数直接应用于工程实践的思路。研究了黄土隧道开挖后围岩结构性的变化规律。研究得出:围岩的结构性参数与径深和洞径密切相关;随着径深的增大,拱顶方向上围岩的结构性参数均呈... 详细信息

在分析和修正已有结构性参数的基础上,首次提出了将结构性参数直接应用于工程实践的思路。研究了黄土隧道开挖后围岩结构性的变化规律。研究得出:围岩的结构性参数与径深和洞径密切相关;随着径深的增大,拱顶方向上围岩的结构性参数均呈现出先减小后增大的规律,存在一个最小值。与开挖前原状土的结构性参数相比,围岩的结构性损失在距拱顶约1.7～2.5 m时达到最大。随着洞径的增大,开挖对围岩黄土的扰动增强,拱顶方向上围岩的结构性减弱,结构性参数的最小值变化不大,但最小值发生的位置逐渐远离拱顶。最后,提出了以围岩结构性损失最大为依据判断围岩松动圈的新方法。分析表明,黄土隧道围岩的松动圈在1.7～2.5 m变化。

关键词：黄土隧道结构性参数围岩松动圈

A prospective longitudinal study for latent tuberculosis infection in hospital in Beijing evaluating a T-cell-based assay health-care workers in a general

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Chinese Medical Journal 2013年第11期126卷 2039-2044页

作者： ZHANG Li-fan LIU Xiao-qing ZHANG Yao deng guo-hua Manish Pareek Ajit Lalvani Department of Infectious Diseases Peking Union Medical College Hospital Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100730 China Clinical Epidemiology Unit Peking Union Medical College Hospital Chinese Academy of Medical Sciences & Peking Union Medical College Beijing 100730 China

Background The health-care workers （HCWs） are at high risk of acquiring infection with Mycobacterium tuberculosis. The objectives of this study were to compare the performance of the *** and tuberculin skin test （TST） for latent tuberculosis infection （LTBI）, evaluate diagnostic concordance and risk factors for LTBI, and observe the progression to active tuberculosis （TB） disease among HCWs in a general hospital in Beijing. Methods The prospective cohort study enrolled HCWs in a tertiary general hospital in Beijing, China, to evaluate LTBI with *** and TST. The subjects were evaluated every 12 months during the 60-month follow-up. Results Of 101 participating HCWs, 96 and 101 had valid TST and *** results, respectively. Twenty-nine （28.7%, 95% confidence interval （C/）, 19.9%-37.5%） were defined as positive by *** and 53 （55.2%, 95% CI, 45.2%-64.9%） were defined as positive by TST （using a ≥10 mm cutoff）. An agreement between the two tests was poor （57.3%, K=0.18, 95% CI, 0.01%-0.52%）. In multJvariate analysis, direct exposure to sputum smear-positive TB patients was a significant risk factor for a positive *** （OR 5.76; 95% CI 1.38-24.00）. Pooled frequency of antigen- specific IFN-y secreting T-cells for subjects who reported direct contact with sputum smear-positive TB patients was significantly higher than that for participants without direct contact （P=0.045）. One of 20 participants with positive result of *** and TST developed active TB at 24-month *** *** is a more accurate, targeted method of diagnosing LTBI than TST.

关键词： T-SPOT.TB latent tuberculosis infection tuberculin skin test exposure follow-up study

Subtype Identification of Avian Influenza Virus on DNA Microarray

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Agricultural Sciences in China 2005年第9期4卷 700-706页

作者： WANG Xiu-rong YU Kang-zhen deng guo-hua SHI Rui LIU Li-ling QIAO Chuan-ling BAO Hong-mei KONG Xian-gang CHEN hua-lan Animal Influenza Laboratory of the Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology of Harbin VeterinaryResearch Institute of Chinese Academy of Agricultural Sciences Harbin 150001 P.R.China

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

关键词： Avian influenza virus DNA microarray Subtype identification

Epidemic of wild-origin H1NX avian influenza viruses in Anhui,China

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Infectious Diseases of Poverty 2017年第1期6卷 881-890页

作者： Ye Ge Qiu-Cheng Yao Xian-Fu Wang Zhi-Qiang Fan guo-hua deng Hong-Liang Chai hua-Lan Chen Yu-Ping hua College of Wildlife Resources Northeast Forestry UniversityHarbin 150040Heilongjiang ProvinceChina State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbinChina Natural Protection&Management Station of Forestry Department Centre of Anhui Province HefeiAnhui ProvinceChina School of Life Sciences Anqing Normal UniversityAnqingAnhui ProvinceChina

Background:As the natural hosts of avian influenza viruses(AIVs),aquatic and migratory birds provide a gene pool for genetic transfer among species and across species,forming transient“genome constellations.”This work describes the phylogenetic dynamics of H1NX based on the complete molecular characterization of eight genes of viruses that were collected from 2014 to 2015 in Anhui Province,***:Hemagglutination and hemagglutination inhibition tests were used to determine the hemagglutination(HA)activity of the HA *** entire genomes of the viruses were sequenced on an ABI PRISM 3500xl DNA *** sequences were genetically analysed to study their genetic evolution using DNASTAR and MEGA *** pathogenic effects of the viruses were evaluated using mouse infection ***:Seven strains of the H1 subtype avian influenza virus were *** analysis indicated natural recombination of the H1 influenza viruses between the Eurasian lineage and the North American *** genes had high sequence identity with A/bean goose/Korea/220/2011(H9N2),which is a typical case involving viral reassortment between the Eurasian lineage and the North American *** results of infection experiments in mice showed that the viruses could acquire the ability to multiply in mouse respiratory organs without ***:These findings suggest that continued surveillance of wild birds,particularly migratory birds,is important to provide early warning of possible H1 influenza epidemics and to understand the ecology of the virus.

关键词： H1N1 Anseriformes Epidemic Phylogenetic Pathogenic

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基于平面应变统一强度公式的结构性黄土填料挡墙地震被动土压力研究

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岩土力学 2019年第4期40卷 1255-1262,1290页

作者：邵生俊陈菲邓国华西安理工大学岩土工程研究所陕西西安710048 西安理工大学陕西省黄土力学与工程重点实验室陕西西安710048 西安黄土地下工程技术咨询有限公司陕西西安710021

基于结构性黄土不同含水率方形试样的三轴剪切试验,研究了非饱和黄土的残余强度效应和构度与强度参数的关系,引入两种效应修正了平面应变统一强度公式,分析了地震荷载作用下墙后土应力摩尔圆的变化规律,采用水平层分析法,建立了综合考... 详细信息

基于结构性黄土不同含水率方形试样的三轴剪切试验,研究了非饱和黄土的残余强度效应和构度与强度参数的关系,引入两种效应修正了平面应变统一强度公式,分析了地震荷载作用下墙后土应力摩尔圆的变化规律,采用水平层分析法,建立了综合考虑中主应力、土结构性、残余强度的黄土填料被动动土压力公式。研究表明:平面应变统一强度公式计算得到的峰值或残余黏聚力和摩擦角随统一强度参数b值增大而增大,结构性对黏聚力的增幅较大,而对摩擦角影响则不明显;结构性黄土的地震被动土压力系数随b值、构度指标的增大而增大,而随水平地震系数的增大而减小;结构性较大时残余强度计算的地震被动土压力系数较峰值强度大,结构性较小时,两者计算结果接近。

关键词：统一强度理论结构性残余强度地震被动土压力黄土

Blood collection procedures influence contamination rates in blood culture： a prospective study

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Chinese Medical Journal 2011年第23期124卷 4002-4006页

作者： GE Ying LIU Xiao-qing XU Ying-chun XU Shan YU Min-hong ZHANG Wei deng guo-hua Department of Infectious Diseases Peking Union Medical College Hospital Chinese Academy of Medical Sciences ＆ Peking Union Medical College Beijing 100730 China

Background Blood culture contamination is a significant adverse event. The aim of this project was to evaluate the efficacy of a strict blood collection procedure in reducing the blood culture contamination rate. Methods A prospectively controlled study was performed in two different medical areas in Peking Union Medical College Hospital （PUMCH） for 16 months （from May 2006 to September 2007）. In test group, a strict blood collection procedure was carried out by trained nurses with the veinpuncture sites were scrupulously disinfected with 2.5% tincture of iodine plus 70% alcohol. In control group, commonly used procedure in PUMCH was performed with 0.45% chlorhexidine acetate plus 0.2% iodine. Blood culture positive results for 4 target organisms （Coagulase-negafive staphylococci, Propionibacterium acnes, physicians from infectious department to （contamination）. Corynebacterium species and Bacillus determine whether a sample was true species） were further assessed by positive （pathogen） or false positive Results Total 9321 blood culture collections were analyzed. The blood culture contamination rate in test group was significantly lower than that in control group （5/3177 （0.16%） vs. 77/6144 （1.25%）; x2=13.382, P 〈0.001）. The most common contaminant was Coagulase-negative staphylococcus （76.83%）. The average cultural time during which contaminated samples became positive was longer than that for true pathogen samples （42.0 hours vs. 13.9 hours, P=-0.041）. Conclusion Using a strict blood collection procedure can significantly reduce blood culture contamination rate.

关键词： blood culture contamination rate disinfection blood collection procedure