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Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in china

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Science china(Life Sciences) 2016年第6期59卷 615-621页

作者： Shuai Lu Yingzhu Chen Kun Qin Jianfang Zhou Yongliang Lou Wenjie Tan Institute of Medical Virology Wenzhou Medical University National Institute for Viral disease Control and Prevention Chinese Center for Disease Control and Prevention

To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in china, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs(r NPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCo V-BJ70 and CRCo V-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that r NPs of CCo V and CRCo V were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the r NP from CRCo V could cross-react with mice antisera against human coronavirus(HCo V-229 E, NL63, OC43, HKU1), while r NP of CCo V had cross-reactivity with only anti-sera against viruses belonging to the same group(HCo V-229 E and NL63). In summary, CCo V and CRCo V r NPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.

关键词： canine coronavirus canine respiratory coronavirus nucleocapsid protein expression cross-reactivity

Development of a PDRA Method for Detection of the D614G Mutation in COVID-19 Virus—Worldwide,2021

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china CDC weekly 2021年第21期3卷 448-453页

作者： Ziwei Chen Xinxin Shen Ji Wang Xiang Zhao Yuan Gao Ruiqin Zhang Jinrong Wang Leping Liu Xinmin Nie Xuejun Ma Department of Laboratory Medicine Third Xiangya HospitalCentral South UniversityChangshaHunanChina Department of NHC Key Laboratory of Medical Virology and Viral diseases National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijingChina Department of Blood Transfusion Third Xiangya HospitalCentral South UniversityChangshaHunanChina Center for Biosafety mega-Science Chinese Academy of SciencesWuhanHubeiChina.

ABSTRACT Background:COVID-19 infection is a major public health problem worldwide,and the D614G mutation enhances the infectivity of ***:A probe-directed recombinase amplification(PDRA)assay was discussed to detect the D614G mutation at 39℃for 30 *** sensitivity,specificity,and reproducibility of the PDRA were evaluated by D614 and G614 recombinant *** clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative *** sequencing was carried out in parallel for ***:With good reproducibility and specificity,the PDRA assay worked well with the concentration in the range of 103–107 copies/*** with direct sequencing as a reference,the recombinase-aided amplification(RAA)assay obtained 100%sensitivity and 100%specificity using clinical samples.

关键词： specificity PDR D61

Preliminary Study on Drug Susceptibility Profile and Resistance Mechanisms to Macrolides of Clinical Isolates of Non-tuberculous Mycobacteria from china

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Biomedical and Environmental Sciences 2018年第4期31卷 290-299页

作者： LI Fu LI Gui Lian PANG Hui LIU Hai Can XIAO Tong Yang LI Shuang Jun LUO Qiao JIANG Yi WANG Rui Bai WAN Kang Lin State Key Laboratory for infectious diseases Prevention and Control Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention Immunology Department Changzhi Medical College Key Laboratory of Laboratory Medicine Wenzhou Medical University

Objective Macrolide susceptibility and drug resistance mechanisms of clinical non-tuberculous mycobacteria（NTM） isolates were preliminarily investigated for more accurate diagnosis and treatment of the infection in china. Methods Four macrolides, including clarithromycin（CLAR）, azithromycin（AZM）, roxithromycin（ROX）, and erythromycin（ERY）, were used to test the drug susceptibility of 310 clinical NTM isolates from six provinces of china with the broth microdilution method. Two resistance mechanisms, 23 S r RNA and erm, were analyzed with nucleotide sequence analysis. Results Varied effectiveness of macrolides and species-specific resistance patterns were observed. Most Mycobacterium abscessus subsp. massiliense were susceptible and all M. fortuitum were highly resistant to macrolides. All the drugs, except for erythromycin, exhibited excellent activities against slow-growing mycobacteria, and drug resistance rates were below 22.2%. Only four highly resistant strains harbored 2,058/2,059 substitutions on rrl and none of other mutations were related to macrolide resistance. G2191 A and T2221 C on rrl were specific for the M. abscessus complex（MABC）. Seven sites, G2140 A, G2210 C, C2217 G, T2238 C, T2322 C, T2404 C, and A2406 G, were specifically carried by M. avium and M. intracellulare. Three sites, A2192 G, T2358 G, and A2636 G, were observed only in M. fortuitum and one site G2152 A was specific for M. gordonae. The genes erm（39） and erm（41） were detected in M. fortuitum and M. abscessus and inducible resistance was observed in relevant sequevar. Conclusion The susceptibility profile of macrolides against NTM was demonstrated. The well-known macrolide resistance mechanisms, 23 S r RNA and erm, failed to account for all resistant NTM isolates, and further studies are warranted to investigate macrolide resistance mechanisms in various NTM species.

关键词： Non-tuberculous mycobacteria Macrolide Drug resistance

Antarctic thermolabile uracil-DNA-glycosylase- supplemented multiple cross displacement amplification using a label-based nanoparticle lateral flow biosensor for the simultaneous detection of nucleic acid sequences and elimination of carryover contamination

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Nano Research 2018年第5期11卷 2632-2647页

作者： Yi Wang Hui Li Yan Wang Huaqing Xu Jianguo Xu Changyun Ye State Key Laboratory of infectious disease Prevention and Control National Institute for Communicable Disease Control and Prevention Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases Chinese Center for Disease Control and Prevention Beijing 102205 China Department of Microbiology Guizhou Medical University Guiyang 550004 China The Sixth People's Hospital of Zhengzhou Zhengzhou 450000 China

Here, we report a novel and universal methodology,termed ＂ntarctic thermolabile uracil-DNA-glycosylase （AUDG）-supplemented nucleic acid amplification techniques （NAAs） using a labeled-based nanoparticle lateral flow biosensor （LFB）＂（AUDG-NAAs-LFB）, which merges enzymatic （AUDG） digestion of contaminant amplicons with different nucleic acid amplification techniques （NAAs）, and uses a lateral flow biosensor （LFB） for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets （two amplicons generated from amplification of target sequences, and a chromatography control）, without the need for probe- hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification （MCDA）, which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA- LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof- of-concept method （AUDG-MCDA-LFB） can be easily reconfigured to detect various nudeic acid sequences by redesigning the specific MCDA primers.

关键词： Antarctic thermolabile uracil-DNA-glycosylase(AUDG) nucleic acid amplification techniques (NAAs) multiple cross displacement amplification (MCDA) lateral flow biosensor (LFB) limit of detection (LOD) human papillomaviruses(HPV)

Coxsackievirus B3 Infection Triggers Autophagy through 3 Pathways of Endoplasmic Reticulum Stress

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Biomedical and Environmental Sciences 2018年第12期31卷 867-875页

作者： LUO Xiao Nuan YAO Hai Lan SONG Juan SONG Qin Qin SHI Bing Tian XIA Dong HAN Jun State Key Laboratory of infectious disease Prevention and Control Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Molecular Immunology Laboratory Capital Institute of Pediatrics

Objective Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3(CVB3) infection induced autophagy through endoplasmic reticulum(ER) stress. Methods In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction(RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase(PERK) inhibitor, inositol-requiring protein-1(IRE1) inhibitor, or activating transcription factor-6(ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3(LC3). Results CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein(GFP)-LC3 punctuation and induced the conversion from LC3-Ⅰ to phosphatidylethanolamine-conjugated LC3-1(LC3-Ⅱ). CVB3 infection still decreased the expression of mammalian target of rapamycin(mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-Ⅱ to LC3-Ⅰ in CVB3-infected HeLa cells. Conclusion CVB3 infection induced autophagy through ER stress in HeL a cells, and PERK, IRE1, and ATF6 a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.

关键词： Coxsackievirus B3(CVB3) Autophagy Endoplasmic reticulum(ER) stress Unfolded protein response(UPR)

The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation

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Biomedical and Environmental Sciences 2022年第9期35卷 804-810页

作者： WANG Wen Jun FENG Shun Qiao HE Feng DU Hai Jun FENG Miao WANG Rui Fang MEI Guo Yong LIU Mi LIU Rong YAO Hai Lan HAN Jun State Key Laboratory of infectious disease Prevention and Control National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Department of Hematology Children’s Hospital of Capital Institute of PediatricsBeijing 100020China Department of Biochemistry&Immunology Capital Institute of PediatricsBeijing 100020China

Objective To detect the Epstein-Barr virus(EBV)viral load of children after hematopoietic stem cell transplantation(HSCT)using chip digital PCR(cdPCR).Methods The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 *** specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses(herpes simplex virus 1,herpes simplex virus 2,varicella zoster virus,human cytomegalovirus,human herpesvirus 6,and human herpesvirus 7).From May 2019 to September 2020,64 serum samples of children following HSCT were *** infection and the viral load of serum samples were detected by *** epidemiological characteristics of EBV infections were analyzed in HSCT *** The limit of detection of EBV cdPCR was 110 copies/mL,and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 *** result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain,and both were more sensitive than that of quantitative *** cdPCR,the incidence of EBV infection was 18.75%in 64 children after *** minimum EBV viral load was 140 copies/mL,and the maximum viral load was 3,209 copies/mL using *** average hospital stay of children with EBV infection(184±91 days)was longer than that of children without EBV infection(125±79 days),P=*** EBV cdPCR had good sensitivity and *** incidence of EBV infection was 18.75%in 64 children after HSCT from May 2019 to September *** cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.

关键词： Chip digital PCR Epstein-Barr virus Hematopoietic stem cell transplantation Quantitative PCR

Treating COVID-19 with Chloroquine

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Journal of Molecular Cell Biology 2020年第4期12卷 322-325页

作者： Mingxing Huang Tiantian Tang Pengfei Pang Man Li Ruolan Ma Jiahui Lu Jingxian Shu Yingying You Binghui Chen Jiabi Liang Zhongsi Hong Huili Chen Ling Kong Dajiang Qin Duanqing Pei Jinyu Xia Shanping jiang Hong Shan Department of infectious diseases The Fifth Affiliated HospitalSun Yat-sen University(SYSU)Zhuhai 519000China Guangdong Provincial Key Laboratory of Biomedical Imaging The Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China Department of Respiratory and Critical Care Medicine Sun Yat-sen Memorial HospitalSun Yat-sen UniversityGuangzhou 510120China Institute of Pulmonary diseases Sun Yat-sen UniversityGuangzhou 510120China Interventional Medical Center The Fifth Affiliated HospitalSun Yat-Sen UniversityZhuhai 519000China Research Center of Molecular Imaging The Fifth Affiliated HospitalSun Yat-Sen UniversityZhuhai 519000China Guangzhou Regenerative Medicine and Health Guangdong Laboratory Guangzhou 510700China Guangzhou Institutes of Biom edicine and Health Chinese Academy of SciencesGuangzhou 510530China Department of Pharmacy The Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China Department of Stomatology The Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China Department of Radiology The Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China University of Chinese Academy of Sciences Beijing 100049China

A novel coronavirus disease 2019(COVID-19)emerged around December 2019 in Wuhan,china and has spread rapidly worldwide(Lu et al.,2020).Until March 27,2020,the Chinese health authorities had reported 82082 confirmed COVID-19 cases in china with 3298 deaths and 381443 confirmed cases with 20787 deaths outside *** World Health Organization(WHO)named the virus SARS-CoV-2,which belongs to a distinct clade from the human severe acute respiratory syndrome CoV(SARS-CoV)and Middle East respiratory syndrome CoV(MERSCoV)(Zhu et al.,2020).

关键词： al. respiratory acute

Identification of Internal Ribosomal Entry Site inside Open Reading Frame of 14-3-3β Gene

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Biomedical and Environmental Sciences 2020年第4期33卷 273-276页

作者： SONG Qin Qin LUO Xiao Nuan CHI Miao Miao SONG Juan XIA Dong HAN Jun State Key Laboratory of infectious disease Prevention and Control Collaborative Innovation Center for Diagnosis and Treatment of Infectious DiseasesNational Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Center for Biosafety mega-Science Chinese Academy of ScienceWuhan 430071HubeiChina Department of Virology School of Public HealthShandong UniversityJinan 250012ShandongChina

Prion diseases are characterized by neurodegeneration and protein aggregation,which are caused by the accumulation of a misfolded and protease-resistant form of the cellular prion *** studies suggest that the accumulation of a misfolded alternate cellular protein disturbs quality control mechanisms,leading to endoplasmic reticulum(ER)stress involved in prion diseases[1-3].Prion infection activates the splicing of the unfolded protein response transcription factor *** mutant PrP associated with inherited.

关键词： Open folded alternate

Establishment of a Chinese street rabies virus library and its application for detecting neutralizing activity

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infectious diseases of Poverty 2018年第1期7卷 1217-1223页

作者： Peng-Cheng Yu Xiao-Yan Tao Li-Hua Wang Qing Tang Li-Yun Fan Shu-Xia Zhang Shu-Qing Liu Xue-Xin Lu Gui-Zhen Wu Wu-Yang Zhu Key Laboratory of Medical Virology Ministry of HealthNational Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China

Background:The injection of rabies immune globulin(RIG)is of the utmost importance in the management of category III exposures to rabies-suspect *** of the high cost and limited availability of existing RIG,one possible replacement for RIG is monoclonal antibodies(MAbs)against the rabies virus(RABV).Consequently,it is necessary to determine the neutralizing activity of the MAbs against rabies viruses,especially street rabies ***,the method to detect the neutralizing activity of MAbs against street rabies virus remains ***:To establish a method for detecting the neutralizing activity of MAbs against street rabies virus,we constructed a library consisting of 12 strains of street RABV from 11 provinces in *** this street RABV library and the Reed-Muench formula,we established a method for detecting the neutralizing titer of the *** reliability and repeatability of the method were evaluated by repeatedly measuring the neutralizing activity of a MAb and a post vaccination ***:A total of 12 strains of street RABV were chosen for inclusion in the street RABV library,which covered six Chinese lineages(china I-china VI)and grew to high titers in N2A cells(>105 FFD50/ml).On the basis of the library,we constructed the method to detect the neutralizing activity of the *** results of repeatedly measuring the MAbs and positive serum showed excellent reliability and repeatability of the method established in this ***:This study established a street RABV library reflecting the epidemiological features of Chinese rabies viruses,which provides a platform for detecting the neutralizing activity of MAbs against rabies viruses circulating in china.

关键词： Street RABV MAbs Neutralizing activity