目的对登革病毒-2(DENV-2)感染人脐静脉内皮细胞(HUVEC)的差异磷酸化蛋白质进行分析以及初步探究DENV-2感染的可能致病机制。方法实验设置DENV-2感染HUVEC组和HUVEC空白对照组,每组3个重复。收集细胞沉淀后,利用SDT裂解法获取蛋白质,通...目的对登革病毒-2(DENV-2)感染人脐静脉内皮细胞(HUVEC)的差异磷酸化蛋白质进行分析以及初步探究DENV-2感染的可能致病机制。方法实验设置DENV-2感染HUVEC组和HUVEC空白对照组,每组3个重复。收集细胞沉淀后,利用SDT裂解法获取蛋白质,通过串联质谱标签(TMT)法对磷酸化蛋白质进行定性与定量分析,对鉴定到的差异磷酸化蛋白质进行氨基酸保守基序分析、亚细胞定位分析、蛋白质结构域分析、GO富集分析、KEGG通路分析和蛋白质互作(PPI)等生物信息学分析。利用Western blot检测JUN、MAP2K2和AKT1磷酸化蛋白的表达。结果共检测到1385个差异蛋白质上的2918个修饰肽段,其中有1346个修饰肽段显著上调(FC>1.2,P<0.05),1572个修饰肽段显著下调(FC<0.83,P<0.05)。氨基酸保守基序分析共获得49个磷酸化保守基序。蛋白质结构域分析中富集到的差异磷酸化肽段最多是RNA识别基序、蛋白激酶结构域和PH结构域。通过亚细胞定位分析发现差异的修饰肽段主要定位在细胞核和细胞质中。GO富集和KEGG通路分析结果显示差异肽段主要在刺激反应的调节、含核碱基的小分子生物合成过程、吞噬体和白细胞跨内皮迁移处富集。对上调和下调的差异磷酸化蛋白分别进行蛋白质互作—KEGG联合分析,结果发现上调和下调的差异磷酸化蛋白质共富集到15条通路,且通过Western Blot检测与自噬途径相关的3个差异磷酸化蛋白质即JUN、MAP2K2和AKT1的表达,结果显示DENV-2组与空白组相比,p-JUN表达显著下调(1.48±0.01 vs 1.23±0.01,P<0.0001);p-AKT1表达显著上调(0.87±0.02 vs 1.01±0.01,P<0.001);p-MAP2K2表达显著上调(1.10±0.02 vs 1.29±0.05,P<0.01)。结论DENV-2感染HUVEC存在较多差异表达蛋白,p-JUN的下调、p-MAP2K2和pAKT1的上调提示3个能够调节自噬的磷酸化蛋白可能与DENV-2感染机制有关。展开更多
Objective: To describe the clinical manifestation of patient with severe dengue, to identify the serotypes and genotypes of dengue viruses(DENV) which concurrently infecting the patient, and to explore the possible re...Objective: To describe the clinical manifestation of patient with severe dengue, to identify the serotypes and genotypes of dengue viruses(DENV) which concurrently infecting the patient, and to explore the possible relationship of severe dengue with the concurrent infection of DENV. Methods: Dengue diagnosis was performed using NS1 antigen detection and Ig G/Ig M ELISA. Standard clinical and laboratory examinations were performed to obtain the clinical and hematological data. DENV concurrent infections were detected and confirmed using RT-PCR and DENV Envelope gene sequencing. Phylogenetic analyses were performed to determine the genotypes of the viruses. Results: The patient was classified as having severe dengue characterized by severe plasma leakage, hemorrhage, and organ damage involving lung, liver, and kidney. Concurrent infection of DENV serotype 2 and 3 was observed. The infecting DENV-2 virus was grouped into Cosmopolitan genotype while DENV-3 virus was classified into Genotype Ⅰ. Both viruses were closely related to isolates that were endemic in Jakarta. Viremia measurement was conducted and revealed a significantly higher virus titer of DENV-3 compared to DENV-2. Conclusions: The occurrence of multi-serotype DENV infections was presented in a patient with severe clinical manifestation in Indonesia. The hyperendemicity of dengue in Indonesia may contribute to the DENV concurrent infections cases and may underlie the severity of the disease.展开更多
The gene fragment coding for amino acids 281 to 395 of the E protein of DENV-2 (New Guinea C strain) was amplified by PCR, comprising Domain III (amino acids 295 to 395) of the E protein. The fragment was cloned into ...The gene fragment coding for amino acids 281 to 395 of the E protein of DENV-2 (New Guinea C strain) was amplified by PCR, comprising Domain III (amino acids 295 to 395) of the E protein. The fragment was cloned into pMD18-T vector and subcloned to expression vector pET-28a and pMAL-c2X. The recombinant plasmid pET-28a-D2EIII was transformed into E.coli BL21(DE3) and the pMAL-c2X-D2EIII was transformed into E.coli TB1. The induced recombinant proteins were purified by His-tag and MBP-tag affinity chromatography, respectively. The purified protein His-D2EIII was used to immunize rabbit three times at two-week intervals, the immunized rabbit produced high titer anti-His-D2EIII polyclonal antibody. The result of western blot indicated that the expressed fusion protein could react with the polyclonal antibody against Domain III of E protein.展开更多
Objectives: To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris(P. pastoris).Methods: A codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized...Objectives: To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris(P. pastoris).Methods: A codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.Results: The recombinant DENV-2 NS I protein was secreted as a protein with a molecular weight of ~45 kDa, and the optimal expression condition was achieved by induction with 2%(v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.Conclusions: The resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.展开更多
Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecif...Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecifc viruses,and a novel MDV was previously isolated from Ae.albopictus in Guangzhou.This study aims to determine the prevalence of MDVs in wild Ae.albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV.Methods The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae.albopictus in Guangzhou.The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12,24,48,72,96,and 120 h post infection(hpi)by indirect immunofuorescence assay(IFA)and real time quantitative PCR(RT-qPCR).The midgut infection rate(MIR),dissemination rate(DR),and salivary gland infection rate(SGIR)in various tissues of MDV-infected mosquitoes were detected and quantifed at 0,5,10,and 15 days post infection(dpi)by RT-PCR and RT-qPCR.The chi-square test evaluated dengue virus serotype 2(DENV-2)and Aedes aegypti densovirus(AaeDV)infection rates and related indices in mosquitoes,while Tukey’s LSD and t-tests compared viral titers in C6/36 cells and tissues over time.Results The results revealed a relatively wide distribution of MDVs in Aedes,Culex,and Anopheles mosquitoes in China and an over 68%positive rate.In vitro,signifcant reductions in DENV-2 titers in supernatant at 120 hpi,and an appar‑ent decrease in DENV-2-positive cells at 96 and 120 hpi were observed.In vivo,DENV-2 in the ovaries and salivary glands was frst detected at 10 dpi in both monoinfected and superinfected Ae.albopictus females,while MDV super‑infection with DENV-2 suppressed the salivary gland infection rate at 15 dpi.DENV-2 titer in the ovary and salivary glands of Ae.albopictus was reduced in superinfected mosquitoes at 15 dpi.Conclusions MDVs is widespread in natural mosquito populations,and replication of DENV-2 is suppressed in MDVinfected Ae.albopictus,thus reducing vector susceptibility to DENV-2.Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.展开更多
Dengue fever is a mosquito-borne viral disease spread in tropical and subtropical regions caused by the dengue virus(DENV).DENV causes a febrile illness,severe forms including hemorrhagic fevers and shock with fatalit...Dengue fever is a mosquito-borne viral disease spread in tropical and subtropical regions caused by the dengue virus(DENV).DENV causes a febrile illness,severe forms including hemorrhagic fevers and shock with fatalities in humans.DENV-2 is frequently associated with severe dengue infections and epidemics.DENV-2 strains from Guangdong,China,have not been characterized to compare the phylogenetics and pathogenicity of different DENV-2 subgenotype strains in both vitro and vivo.A total of 22 patients tested to be DENV-2 positive and were enrolled in this study,22 complete genomes were obtained by virus isolation and high-throughput sequencing.Phylogenetic and single amino polymorphism(SAP)analysis indicated that two major subgenotypes(A and C)of DENV-2 Cosmopolitan were prevalent in Guangdong in 2018.The apparent change of major subgenotypes of DENV-2 circulating in Guangdong indicated the diversity of DENV-2 strains,including endemic genotype and imported genotype.It alerted the risk of cross-border transmission of DENV.A significant difference in replication rate was observed in C6/36 between different DENV-2 strains,although growth kinetics comparison of different DENV-2 Cosmopolitan subgenotypes showed similar profiles.DENV-2 subgenotypes(A and C)replicated efficiently in IFNAR−/−C57BL/6 mice,and subgenotype A of Cosmopolitan infected mice showed increased weight loss and delayed viral clearance compared with the subgenotype C group.DENV-2 prevalent in Guangdong in 2018 showed apparent genetic and pathogenicity diversity in both vitro and vivo,indicating the necessity of molecular surveillance and exploration of the relationship between its pathogenicity and clinical characteristics.展开更多
文摘目的对登革病毒-2(DENV-2)感染人脐静脉内皮细胞(HUVEC)的差异磷酸化蛋白质进行分析以及初步探究DENV-2感染的可能致病机制。方法实验设置DENV-2感染HUVEC组和HUVEC空白对照组,每组3个重复。收集细胞沉淀后,利用SDT裂解法获取蛋白质,通过串联质谱标签(TMT)法对磷酸化蛋白质进行定性与定量分析,对鉴定到的差异磷酸化蛋白质进行氨基酸保守基序分析、亚细胞定位分析、蛋白质结构域分析、GO富集分析、KEGG通路分析和蛋白质互作(PPI)等生物信息学分析。利用Western blot检测JUN、MAP2K2和AKT1磷酸化蛋白的表达。结果共检测到1385个差异蛋白质上的2918个修饰肽段,其中有1346个修饰肽段显著上调(FC>1.2,P<0.05),1572个修饰肽段显著下调(FC<0.83,P<0.05)。氨基酸保守基序分析共获得49个磷酸化保守基序。蛋白质结构域分析中富集到的差异磷酸化肽段最多是RNA识别基序、蛋白激酶结构域和PH结构域。通过亚细胞定位分析发现差异的修饰肽段主要定位在细胞核和细胞质中。GO富集和KEGG通路分析结果显示差异肽段主要在刺激反应的调节、含核碱基的小分子生物合成过程、吞噬体和白细胞跨内皮迁移处富集。对上调和下调的差异磷酸化蛋白分别进行蛋白质互作—KEGG联合分析,结果发现上调和下调的差异磷酸化蛋白质共富集到15条通路,且通过Western Blot检测与自噬途径相关的3个差异磷酸化蛋白质即JUN、MAP2K2和AKT1的表达,结果显示DENV-2组与空白组相比,p-JUN表达显著下调(1.48±0.01 vs 1.23±0.01,P<0.0001);p-AKT1表达显著上调(0.87±0.02 vs 1.01±0.01,P<0.001);p-MAP2K2表达显著上调(1.10±0.02 vs 1.29±0.05,P<0.01)。结论DENV-2感染HUVEC存在较多差异表达蛋白,p-JUN的下调、p-MAP2K2和pAKT1的上调提示3个能够调节自噬的磷酸化蛋白可能与DENV-2感染机制有关。
文摘Objective: To describe the clinical manifestation of patient with severe dengue, to identify the serotypes and genotypes of dengue viruses(DENV) which concurrently infecting the patient, and to explore the possible relationship of severe dengue with the concurrent infection of DENV. Methods: Dengue diagnosis was performed using NS1 antigen detection and Ig G/Ig M ELISA. Standard clinical and laboratory examinations were performed to obtain the clinical and hematological data. DENV concurrent infections were detected and confirmed using RT-PCR and DENV Envelope gene sequencing. Phylogenetic analyses were performed to determine the genotypes of the viruses. Results: The patient was classified as having severe dengue characterized by severe plasma leakage, hemorrhage, and organ damage involving lung, liver, and kidney. Concurrent infection of DENV serotype 2 and 3 was observed. The infecting DENV-2 virus was grouped into Cosmopolitan genotype while DENV-3 virus was classified into Genotype Ⅰ. Both viruses were closely related to isolates that were endemic in Jakarta. Viremia measurement was conducted and revealed a significantly higher virus titer of DENV-3 compared to DENV-2. Conclusions: The occurrence of multi-serotype DENV infections was presented in a patient with severe clinical manifestation in Indonesia. The hyperendemicity of dengue in Indonesia may contribute to the DENV concurrent infections cases and may underlie the severity of the disease.
文摘The gene fragment coding for amino acids 281 to 395 of the E protein of DENV-2 (New Guinea C strain) was amplified by PCR, comprising Domain III (amino acids 295 to 395) of the E protein. The fragment was cloned into pMD18-T vector and subcloned to expression vector pET-28a and pMAL-c2X. The recombinant plasmid pET-28a-D2EIII was transformed into E.coli BL21(DE3) and the pMAL-c2X-D2EIII was transformed into E.coli TB1. The induced recombinant proteins were purified by His-tag and MBP-tag affinity chromatography, respectively. The purified protein His-D2EIII was used to immunize rabbit three times at two-week intervals, the immunized rabbit produced high titer anti-His-D2EIII polyclonal antibody. The result of western blot indicated that the expressed fusion protein could react with the polyclonal antibody against Domain III of E protein.
基金supported by grants from the National Key R&D Program of China(2018YFA0507103)The National Natural Science Foundation of China(31870719)+5 种基金Shenzhen STIC(JCYC20160331-115853521)Shenzhen STIC(JCYJ20170307-110657570)Shenzhen STIC(JCYJ20170817110434640)Shenzhen San-Ming Project(SZSM201809085)Chinese Postdoctoral Science Foundation Project(2018M641077)Ministry of Science and Technology of China(2018ZX09711003-003-004)~~
基金"Penelitian Unggulan Strategis Nasional 2013" under the contract number of 0400/I1/B04/SPK-WRRI/VI/2014,Ministry of Research,Technology,and Higher Education of Indonesia,for funding this work
文摘Objectives: To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris(P. pastoris).Methods: A codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.Results: The recombinant DENV-2 NS I protein was secreted as a protein with a molecular weight of ~45 kDa, and the optimal expression condition was achieved by induction with 2%(v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.Conclusions: The resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.
文摘Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecifc viruses,and a novel MDV was previously isolated from Ae.albopictus in Guangzhou.This study aims to determine the prevalence of MDVs in wild Ae.albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV.Methods The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae.albopictus in Guangzhou.The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12,24,48,72,96,and 120 h post infection(hpi)by indirect immunofuorescence assay(IFA)and real time quantitative PCR(RT-qPCR).The midgut infection rate(MIR),dissemination rate(DR),and salivary gland infection rate(SGIR)in various tissues of MDV-infected mosquitoes were detected and quantifed at 0,5,10,and 15 days post infection(dpi)by RT-PCR and RT-qPCR.The chi-square test evaluated dengue virus serotype 2(DENV-2)and Aedes aegypti densovirus(AaeDV)infection rates and related indices in mosquitoes,while Tukey’s LSD and t-tests compared viral titers in C6/36 cells and tissues over time.Results The results revealed a relatively wide distribution of MDVs in Aedes,Culex,and Anopheles mosquitoes in China and an over 68%positive rate.In vitro,signifcant reductions in DENV-2 titers in supernatant at 120 hpi,and an appar‑ent decrease in DENV-2-positive cells at 96 and 120 hpi were observed.In vivo,DENV-2 in the ovaries and salivary glands was frst detected at 10 dpi in both monoinfected and superinfected Ae.albopictus females,while MDV super‑infection with DENV-2 suppressed the salivary gland infection rate at 15 dpi.DENV-2 titer in the ovary and salivary glands of Ae.albopictus was reduced in superinfected mosquitoes at 15 dpi.Conclusions MDVs is widespread in natural mosquito populations,and replication of DENV-2 is suppressed in MDVinfected Ae.albopictus,thus reducing vector susceptibility to DENV-2.Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.
基金funded by grants from the National Key Research and Development Program of China(2020YFC1200100,2018YFC1200100,2018ZX10301403)the Guangdong Provincial Department of Science and Technology(2020A1515010911,201803040006,2019B030316028)+2 种基金National Natural Science Foundation of China(81772191,91842106 and 8181101118,32000658)National Key Technology R&D Program(2018YFC1311900)Guangzhou Medical University High-level University Innovation Team Training Program(Guangzhou Medical University released[2017]No.159).
文摘Dengue fever is a mosquito-borne viral disease spread in tropical and subtropical regions caused by the dengue virus(DENV).DENV causes a febrile illness,severe forms including hemorrhagic fevers and shock with fatalities in humans.DENV-2 is frequently associated with severe dengue infections and epidemics.DENV-2 strains from Guangdong,China,have not been characterized to compare the phylogenetics and pathogenicity of different DENV-2 subgenotype strains in both vitro and vivo.A total of 22 patients tested to be DENV-2 positive and were enrolled in this study,22 complete genomes were obtained by virus isolation and high-throughput sequencing.Phylogenetic and single amino polymorphism(SAP)analysis indicated that two major subgenotypes(A and C)of DENV-2 Cosmopolitan were prevalent in Guangdong in 2018.The apparent change of major subgenotypes of DENV-2 circulating in Guangdong indicated the diversity of DENV-2 strains,including endemic genotype and imported genotype.It alerted the risk of cross-border transmission of DENV.A significant difference in replication rate was observed in C6/36 between different DENV-2 strains,although growth kinetics comparison of different DENV-2 Cosmopolitan subgenotypes showed similar profiles.DENV-2 subgenotypes(A and C)replicated efficiently in IFNAR−/−C57BL/6 mice,and subgenotype A of Cosmopolitan infected mice showed increased weight loss and delayed viral clearance compared with the subgenotype C group.DENV-2 prevalent in Guangdong in 2018 showed apparent genetic and pathogenicity diversity in both vitro and vivo,indicating the necessity of molecular surveillance and exploration of the relationship between its pathogenicity and clinical characteristics.
