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Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing

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Biomedical and Environmental Sciences 2024年第3期37卷 294-302页

作者： ZHANG Ya Li SU Wen Zhe WANG Rui Chen LI Yan ZHANG Jun Feng LIU Sheng Hui HU Dan He XU Chong Xiao YIN Jia Yu YIN Qi Kai HE Ying LI Fan FU Shi Hong NIE Kai LIANG Guo Dong TAO Yong XU Song Tao MA Chao Feng WANG Huan Yu Shaanxi University of Chinese Medicine Xianyang 712000ShaanxiChina National key laboratory of Intelligent Tracking and Forecasting for Infectious Diseases National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Guangzhou Center for Disease Control and Prevention Guangzhou 510000GuangdongChina Shandong Center for Disease Control and Prevention Jinan 250000ShandongChina Department of Ophthalmology Beijing Chaoyang HospitalCapital Medical UniversityBeijing 100020China Xi'an Center for Disease Control and Prevention Xi’an 710054ShaanxiChina

Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.

关键词： Viral encephalitis Amplicon sequencing High-throughput sequencing Multipathogen detection

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Advancements in the Worldwide Detection of Severe Fever with Thrombocytopenia Syndrome Virus Infection from 2009 to 2023

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China CDC weekly 2023年第31期5卷 687-693页

作者： Lin Ai Wei Wang Zheng Teng Institute of microbiology laboratory Shanghai Municipal Center for Disease Control and PreventionShanghaiChina Institute of microbiology laboratory Shanghai Institute of Preventive MedicineShanghaiChina.

Severe fever with thrombocytopenia syndrome(SFTS)is a growing concern as an emerging tick-borne infectious disease originating from the SFTS virus(SFTSV),a recent addition to the Phlebovirus genus under the family of bunyaviruses.SFTS is typically identified by symptoms such as fever,thrombocytopenia,leukopenia,and gastrointestinal problems,accompanied by a potentially high case fatality rate.Thus,early and accurate diagnosis is essential for effective treatment and disease management.This review delves into the existing methodologies for SFTS detection,including pathogenic,molecular,and immunological technologies.

关键词： diagnosis fever Fever

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New insights from a plant immune receptor may help design of disease resistant crops

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Science China(Life Sciences) 2023年第1期66卷 194-196页

作者： Jie Zhang Jian-Min Zhou State key laboratory of Plant Genomics Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China State key laboratory of Plant Genomics Institute of Genetics and Developmental BiologyInnovation Academy for Seed DesignChinese Academy of SciencesBeijing 100101China cas Center for Excellence in Biotic Interactions University of Chinese Academy of SciencesBeijing 100049China

Receptor-like proteins(RLPs)are a class of cell surface immune receptors highly important for plant defense against numerous pathogens.How these receptors detect pathogen molecules to activate immune signaling has remained poorly understood.A recent study published in Nature uncovers how an RLP protein called Response to XEG1(RXEG1)is activated by a pathogen protein called XEG1.

关键词： insight crops immune

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Crystal structure of histidine-containing phosphocarrier protein from Thermoanaerobacter tengcongensis MB4 and the implications for thermostability

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Science China(Life Sciences) 2011年第6期54卷 513-519页

作者： FENG ChunYan GAO Feng LIU YiWei WANG GuiHua PENG Hao MA YanHe YAN JingHua GAO George Fu cas key laboratory of pathogenic microbiology and immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China Graduate University of Chinese Academy of Sciences Beijing 100049China Institute of Biophysics Chinese Academy of SciencesBeijing 100101China laboratory of Structural microbiology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China State key laboratory of Microbial Resources Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China Beijing Institutes of Life Science Chinese Academy of SciencesBeijing 100101China

Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms,the structural basis of protein thermostability is not yet fully understood.The histidine-containing phosphocarrier (HPr) protein,a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species,is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors.In this study,the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified.Crystal structure with good quality has been determined at 2.3 resolution,which provides a firm foundation for exploring the thermostable mechanism.However,it shows that the crystal structure is conserved and no clue can be obtained from this single structure.Furthermore,detailed comparison of sequence and structure with the homologs from mesoor thermophilic bacteria shows no obvious rule for thermostability,but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr.Thus,mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria.Using site-directed mutations and the circular dichroism method,thermostability is evaluated,and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses,indicating that mutations cause instability in the HPrs.Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability,and is critical in engineering efficient enzymes that can work at a high temperature.

关键词：蛋白质折叠嗜热微生物热稳定性晶体结构组氨酸基因突变结构基础稳定性评价

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Tolerance of Mice to Lipopolysaccharide is Correlated with Inhibition of caspase-3-mediated Apoptosis in Mouse Liver Cells

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Acta Biochimica et Biophysica Sinica 2007年第2期39卷 96-100页

作者： Jie LUAN Bingrong ZHOU Hui DING and Zhongtian QI Department of microbiology,State key laboratory of Medical immunology,Second Military Medical University,Shanghai 200433,China Department of microbiology State Key Laboratory of Medical Immunology Second Military Medical UniversityShanghai 200433 China

Bacterial endotoxin lipopolysaccharide (LPS) often results in multiple organ failure.However,pre-exposure of mice to a sublethal dose of LPS renders the animal tolerant to a lethal dose of LPS.Thisstudy was designed to determine whether pre-exposure of a small dose of LPS was able to suppressapoptosis in mice when challenged with LPS in combination with D-galactosamine,and to investigate theexpression changes of the apoptosis-associated molecules.The results showed that a characteristic apoptoticDNA fragmentation existed in mouse livers of the LPS-naive group,but not in control groups;and the miceof the LPS-naive group were all dead after 2 d.However,in the LPS-tolerance groups,both the lethal rateand apoptotic DNA fragmentation were suppressed after the mice were challenged with LPS/D-galactosamine,and the protection against the lethality and apoptotic reaction could be maintained for up to 7 d.In thisperiod, significantly lower levels of caspase-3 and its mRNA appeared in LPS-tolerant groups compared tothose of the LPS-naive group (Pcaspase-3 activities gradually recovered as the observationwas prolonged.Our findings suggest that LPS tolerance could suppress apoptosis in mouse liver cells,andthe expression and activity of caspase-3 could be down-regulated.

关键词： lipopolysaccharide endotoxin tolerance apoptosis caspase-3

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CD58 expression of liver tissue in patients with chronic hepatitis B virus infection

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Chinese Medical Journal 2008年第6期121卷 557-560页

作者： WANG Ping QI Bao-tai CHEN Ping HE Lin-jing LI Jie JI Yu-qiang XIE Ming Department of pathogenic microbiology and immunology Medical School of Xi'an Jiaotong University Xi'an Shaanxi710061 China

Background Several kinds of intercellular adhesion molecules closely relate to hepatitis B. The complex of CD2 and CD58 plays an important role in enhancing the adhesion of T lymphocytes to target cells, hyperplasia and activation of T lymphocytes. In this study, we explored the relationship between the expression of CD58 in liver tissue and chronic hepatitus B infection. Methods We determined the expression of the CD58 molecule on the surface of hepatoc^es by using immunohistochemistry and the levels of serum HBV DNA from patients with HBV infection and from normal controls. The biochemical parameters of hepatic function were analyzed as well. Results CD58 expression in hepatocytes significantly increased with the severity progression of chronic HBV infection. The IOD levels （log10） of CD58 in the control, mild, moderate, and severe chronic HBV infection groups were 0, （7.20±4.64）×10^3, （25.63±7.41）×10^3 and （37.47±11.17）×10^3 respectively （P〈O.05 compared with the control group, respectively） Conclusion CD58 probably increases cell mediated immunity to eliminate hepatitis B virus and leads to damage of hepatocytes.

关键词： hepatitis B, chronic CD58 immunohistochemistry

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An octamer of enolase from Streptococcus suis

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Protein & Cell 2012年第10期3卷 769-780页

作者： Qiong Lu Hao Lu Jianxun Qi Guangwen Lu George F Gao cas key laboratory of pathogenic microbiology and immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China Graduate University Chinese Academy of SciencesBeijing 100049China Research Network of Immunity and Health(RNIH) Beijing Institutes of Life ScienceChinese Academy of SciencesBeijing 100101China laboratory of Protein Engineering and Vaccines Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjin 300308China

Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells.The enzyme can also locate on the cell surface and bind to plasminogen,via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells.The functions of the eukaryotic enzymes on the cell surface expression(including T cells,B cells,neutrophils,monocytoes,neuronal cells and epithelial cells)are not known.Streptococcus suis serotype 2(S.suis 2,SS2)is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome(STSS)never seen before in human sufferers.We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection.In this study,a 2.4-angstrom structure of the SS2 enolase is solved,revealing an octameric arrangement in the crystal.We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay.These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well.This is,to our knowledge,the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically,and the second octamer enolase structure in addition to that of Streptococcus pneumoniae.We also investigated the plasminogen binding property of the SS2 enzyme.

关键词： enolase octamer Streptococcus suis structure plasminogen binding

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Protein Lactylation and Metabolic Regulation of the Zoonotic Parasite Toxoplasma gondii

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Genomics, Proteomics & Bioinformatics 2023年第6期21卷 1163-1181页

作者： Deqi Yin Ning Jiang Chang Cheng Xiaoyu Sang Ying Feng Ran Chen Qijun Chen key laboratory of Livestock Infectious Diseases in Northeast China Ministry of EducationKey Laboratory of ZoonosisCollege of Animal Science and Veterinary MedicineShenyang Agricultural UniversityShenyang 110166China The Research Unit for pathogenic Mechanisms of Zoonotic Parasites Chinese Academy of Medical SciencesShenyang 110866China

The biology of Toxoplasma gondii,the causative pathogen of one of the most widespread parasitic diseases(toxoplasmosis),remains poorly understood.Lactate,which is derived from glucose metabolism,is not only an energy source in a variety of organisms,including T.gondii,but also a regulatory molecule that participates in gene activation and protein function.Lysine lactylation(Kla)is a type of post-translational modifications(PTMs)that has been recently associated with chromatin remodeling;however,Kla of histone and non-histone proteins has not yet been studied in T.gondii.To examine the prevalence and function of lactylation in T.gondii parasites,we mapped the lactylome of proliferating tachyzoite cells and identified 1964 Kla sites on 955 proteins in the T.gondii RH strain.Lactylated proteins were distributed in multiple subcellular compartments and were closely related to a wide variety of biological processes,including mRNA splicing,glycolysis,aminoacyl-tRNA biosynthesis,RNA transport,and many signaling pathways.We also performed a chromatin immunoprecipitation sequencing(ChIP-seq)analysis using a lactylationspecific antibody and found that the histones H4K12la and H3K14la were enriched in the promoter and exon regions of T.gondii associated with microtubule-based movement and cell invasion.We further confirmed the delactylase activity of histone deacetylases TgHDAC2–4,and found that treatment with anti-histone acetyltransferase(TgMYST-A)antibodies profoundly reduced protein lactylation in T.gondii.This study offers the first dataset of the global lactylation proteome and provides a basis for further dissecting the functional biology of T.gondii.

关键词： Toxoplasmagondit Lactylation Proteinpost-translational modification Metabolism ChIP-seq

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Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human

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Science China(Life Sciences) 2013年第6期56卷 485-492页

作者： LI Jun YU XinFen PU XiaoYing XIE Li SUN YongXiang XIAO HaiXia WANG FenJuan DIN Hua WU Ying LIU Di ZHAO GuoQiu LIU Jun PAN JingCao Hangzhou Center for Disease Control and Prevention Xiaoshan Center for Disease Control and Prevention laboratory of Protein Engineering and Vaccine Tianjin Institute of Industrial BiotechnologyChinese Academy of Sciences cas key laboratory of pathogenic microbiology and immunology Institute of MicrobiologyChinese Academy of Sciences Network Information Center Institute of MicrobiologyChinese Academy of Sciences National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention

A novel H7N9 influenza A virus has been discovered as the causative identity of the emerging acute respiratory infection cases in Shanghai,China.This virus has also been identified in cases of infection in the neighboring area Hangzhou City in Zhejiang Province.In this study,epidemiologic,clinical,and virological data from three patients in Hangzhou who were confirmed to be infected by the novel H7N9 influenza A virus were collected and analyzed.Human respiratory specimens and chicken feces from a contacted free market were tested for influenza virus by real-time reverse transcription PCR(RT-PCR) and sequencing.The clinical features of the three cases were similar featured with high fever and severe respiratory symptoms;however,only one of the patients died.A certain degree of diversity was observed among the three Hangzhou viruses sequenced from human samples compared with other reported H7N9 influenza A viruses.The sequences of the novel avian-origin H7N9 influenza viruses from Hangzhou City contained important amino acid substitutions related to human adaptation.One of the Hangzhou viruses had gained a novel amino acid substitution(Q226I) in the receptor binding region of hemagglutinin.More importantly,the virus sequenced from the chicken feces had a 627E substitution in the PB2 protein instead of the mammalian-adapted 627K substitution that was found in the PB2 proteins from the Hangzhou viruses from the three patients.Therefore,the newly-emerging H7N9 virus might be under adaptation pressure that will help it "jump" from avian to human hosts.The significance of these substitutions needs further exploration,with both laboratory experiments and extensive field surveillance.

关键词： A型流感病毒病毒感染人类环境禽流感起源逆转录聚合酶链反应呼吸道症状连接

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Strengthening the functional research on the interaction between host genes and microbiota

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Science China(Life Sciences) 2020年第6期63卷 929-932页

作者： Jian Li Jun Wang Hong Wei Precision Medicine Insttiute The First Affiliated HospitalSun Yat-sen UniversityGuangzhou 510080China State key laboratory of Agricultural microbiology Key Laboratory of Agricultural Animal GeneticsBreedingand Reproduction of the Ministry of Education&Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural AffairsCollege of Animal Sciences and TechnologyHuazhong Agriculural UniversiyWiuhan 430070China cas key laboratory of pathogenic microbiology and Immwnology Institute of MicrobiologyChinese Academy of SciencesBejing 100049China

In recent years,many studies have focused on the interaction between host genes and microbiota and their mutual influence(Bonder et al.,2016;Goodrich et al.,2014;Rothschild et al.,2018;Wang et al.,2016;Zhang et al.,2019).Host genetic variation influences the composition of the microbial community,for instance in inflammatory bowel disease,host genetic susceptibilities induce pathological shifts in microbiome composition.

关键词： Strengthening the functional research on the interaction between host genes and microbiota

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