为了解引起2017年10月甘肃省兰州市一起疱疹性咽峡炎(Herpangina,HA)暴发疫情的致病病原体及其病原学特征,对采集的患儿的37份咽拭子样本开展病毒分离培养,然后对分离到的毒株采取一代测序获取VP1区全长核苷酸序列,根据分子定型的方法...为了解引起2017年10月甘肃省兰州市一起疱疹性咽峡炎(Herpangina,HA)暴发疫情的致病病原体及其病原学特征,对采集的患儿的37份咽拭子样本开展病毒分离培养,然后对分离到的毒株采取一代测序获取VP1区全长核苷酸序列,根据分子定型的方法进行肠道病毒型别鉴定。根据VP1区核苷酸差异选择3个毒株进行二代测序,获取全基因组序列。使用MEGA软件(版本号11.0)进行病毒的核苷酸,氨基酸相似性分析,VP1区的氨基酸突变位点分析,并构建系统发育树,使用Simplot(版本号3.5.1)软件进行病毒重组分析。本次疫情中分离到的毒株鉴定为柯萨奇病毒A2型(Coxsackievirus A 2, CV-A2),分子分型显示属于D基因型,且VP1区第102位氨基酸发生了丙氨酸到甘氨酸(A102G)的突变,与省内2014-2015年分离到的两株CV-A2毒株在该位置的突变不同。重组分析发现,病毒在P2区和P3区发生了以CV-A4为主的重组事件。本次疫情的致病病原体CV-A2存在着VP1区氨基酸A102G的特征性变异及P2区和P3区的重组,需要加强对肠道病毒的变异和重组的监测和研究,以提升预警能力。展开更多
The aim of this study is to explore the genomic molecular organization and genogroup of human nomvirus from infected infants in Guangzhou of China. Primers were designed according to the genomic sequence of norovims i...The aim of this study is to explore the genomic molecular organization and genogroup of human nomvirus from infected infants in Guangzhou of China. Primers were designed according to the genomic sequence of norovims in the GenBank, and the nomvirus genome was amplified by RT-PCR. The PCR- products were cloned into T vector and sequenced, and the genomic nucleotide sequences were analyzed with the programs CLUSTAL W/X, DNASTAR and RAT （Recombination Analysis Tool）. The NVgz01 strain genome is 7558 bp in length and encodes three open reading frames （GenBank accession No. is DQ369797）. The genomic sequences of NVgz01 were compared with those of nomvirus in GenBank, which revealed that the homology with genogroup Ⅱ ranges between 76%-90%, and genogroup Ⅰ between 43%-44%. The ORF1 region shared 94% and 88% identity with Mc37 and Famiington strains, respectively; the capsid region （ORF2） shared 65% and 94% identity with Mc37 and Farmington strains, respectively. Phylogenetic trees were reconstructed by the neighbor-joining method. Comparative complete sequence analysis of the NVgz01 with reported human norovirus genomic sequences revealed that this isolate belongs to genogroup Ⅱ . The ORF1 and ORF2 regions shared different identity with Mc37 and Fannington strains, suggesting NVgz01 could be a recombinant virus.展开更多
目的描述一株新鉴定的流行重组型HIV-1 CRF103_01B在北京的检出情况,并分析其近全长基因组(near full-length genome,NFLG)遗传特征。方法对2017—2020年北京新诊断或初始抗病毒治疗病例进行HIV-1基因型耐药监测,发现5例疑似感染CRF103_...目的描述一株新鉴定的流行重组型HIV-1 CRF103_01B在北京的检出情况,并分析其近全长基因组(near full-length genome,NFLG)遗传特征。方法对2017—2020年北京新诊断或初始抗病毒治疗病例进行HIV-1基因型耐药监测,发现5例疑似感染CRF103_01B毒株。通过逆转录、近末端稀释法,分两段巢式PCR扩增HIV-1 NFLG。获得的序列与分型参考序列进行基因比对,使用MEGA11软件构建邻接法(neighbor-joining,NJ)系统进化树。用SimPlot 3.5软件分析确定基因重组断点,绘制基因组结构图。基于亲本毒株同源NFLG序列比对,挑选较长的重组片段构建NJ进化树,判断可能的亲本毒株来源。用斯坦福大学HIV耐药数据库解析基因型耐药特征。结果共获得5条CRF103_01B NFLG序列,其中4例经男男性行为(men who have sex with men,MSM)感染,1例为女性。与从河北检出的4条CRF103_01B NFLG序列合并分析其重组特征:在CRF01_AE骨架上,gag、pol和nef-3'-LTR基因片段被B亚型重组替换[HXB2 nt 1111±19-1539±16,2531-4478±16(片段Ⅴ),9008±23-9615]。亲本来源分析显示,CRF103_01B的片段Ⅳ与Ⅴ分别与北京B亚型(BJMP3294B)和g5簇CRF01_AE序列(JX112804)聚集成大单系进化簇(bootstrap值分别为97%和100%)。9个CRF103_01B病例均携带非核苷类逆转录酶抑制剂类V106I突变。结论CRF103_01B毒株最可能起源于北京MSM人群,并在北京和河北等地的MSM人群中低水平持续流行,并经异性性途径向一般人群播散。需加强该毒株分子流行病学和耐药监测,关注疾病进展。展开更多
A new cell culture system expressing the entire HCV geuome has been established in vitro. To initiate transcription of HCV RNA, HeLa cells were trausfected with arecombinant plasmid containing full-length HCV cDNA geu...A new cell culture system expressing the entire HCV geuome has been established in vitro. To initiate transcription of HCV RNA, HeLa cells were trausfected with arecombinant plasmid containing full-length HCV cDNA geuome by using lipofectamiue 2000, followed by infection with recombinant vacciuia virus vTF7-3 containing the T7 RNA polymerase geue. Synthesis of positive-strand HCV RNA could be detected in the trausfected cells by RT-PCR. Western blot analysis revealed that HCV structural and nonstructural proteins were correctly processed. In trausfected HeLa cells 47 um virus-like particles were assembled, whichcould be recognized by auti-HCV E2 antibodies. The titer of HCV was 107 copies/mL in our cell culture system, which was significantly higher than that of infected patients' sera and that from all reported cell culture systems. Superuataut from trausfected HeLa cells were infectious to Huh7 cells and thetiter of HCV was 106 copies/mL. Moreover, negative-strand RNA of HCV in Huh7 cells could be detected by using strand-specific RT-PCR, which demonstrated that replication of HCV occurred in the permissive cell lines.展开更多
目的分析北京市献血人群筛查中发现的1例HIV-1独特型重组毒株的基因结构和重组特点。方法基于2018年北京市血液筛查时发现的1例HIV核酸阳性抗体阴性的样本,提取病毒RNA并逆转录为cDNA,用近末端稀释法分两段扩增病毒近全长基因组并测定...目的分析北京市献血人群筛查中发现的1例HIV-1独特型重组毒株的基因结构和重组特点。方法基于2018年北京市血液筛查时发现的1例HIV核酸阳性抗体阴性的样本,提取病毒RNA并逆转录为cDNA,用近末端稀释法分两段扩增病毒近全长基因组并测定序列。运用RIP、jpHMM和SimPlot3.5软件进行重组分析,同时采用MEGA7软件构建Neighbor-joining系统进化树对该毒株的同源关系进行分析。结果共获得长度为8792 bp的HIV-1近全长基因序列,分析表明该序列由CRF01_AE和CRF07_BC重组片段组成。与Los Alamos HIV Database(www.hiv.lanl.gov/content/index)中收录的全长序列相比该毒株具有3个独特的重组断点,分4个重组片段,分别是ICRF01_AE(790~6035,HXB2),IICRF07_BC(6036~8586,HXB2),IIICRF01_AE(8587~8828,HXB2)和IVCRF07_BC(8829~9411,HXB2)。其中ICRF01_AE区域属于CRF01_AE的C4簇;IICRF07_BC区域属于CRF07_BC的CRF07BC_N簇。结论1例参与献血的HIV核酸阳性而抗体阴性的感染者携带的毒株具有新型独特的重组模式,提示需要加强监测献血人群中HIV毒株的流行情况,为保障安全用血提供科学数据。展开更多
文摘为了解引起2017年10月甘肃省兰州市一起疱疹性咽峡炎(Herpangina,HA)暴发疫情的致病病原体及其病原学特征,对采集的患儿的37份咽拭子样本开展病毒分离培养,然后对分离到的毒株采取一代测序获取VP1区全长核苷酸序列,根据分子定型的方法进行肠道病毒型别鉴定。根据VP1区核苷酸差异选择3个毒株进行二代测序,获取全基因组序列。使用MEGA软件(版本号11.0)进行病毒的核苷酸,氨基酸相似性分析,VP1区的氨基酸突变位点分析,并构建系统发育树,使用Simplot(版本号3.5.1)软件进行病毒重组分析。本次疫情中分离到的毒株鉴定为柯萨奇病毒A2型(Coxsackievirus A 2, CV-A2),分子分型显示属于D基因型,且VP1区第102位氨基酸发生了丙氨酸到甘氨酸(A102G)的突变,与省内2014-2015年分离到的两株CV-A2毒株在该位置的突变不同。重组分析发现,病毒在P2区和P3区发生了以CV-A4为主的重组事件。本次疫情的致病病原体CV-A2存在着VP1区氨基酸A102G的特征性变异及P2区和P3区的重组,需要加强对肠道病毒的变异和重组的监测和研究,以提升预警能力。
文摘The aim of this study is to explore the genomic molecular organization and genogroup of human nomvirus from infected infants in Guangzhou of China. Primers were designed according to the genomic sequence of norovims in the GenBank, and the nomvirus genome was amplified by RT-PCR. The PCR- products were cloned into T vector and sequenced, and the genomic nucleotide sequences were analyzed with the programs CLUSTAL W/X, DNASTAR and RAT （Recombination Analysis Tool）. The NVgz01 strain genome is 7558 bp in length and encodes three open reading frames （GenBank accession No. is DQ369797）. The genomic sequences of NVgz01 were compared with those of nomvirus in GenBank, which revealed that the homology with genogroup Ⅱ ranges between 76%-90%, and genogroup Ⅰ between 43%-44%. The ORF1 region shared 94% and 88% identity with Mc37 and Famiington strains, respectively; the capsid region （ORF2） shared 65% and 94% identity with Mc37 and Farmington strains, respectively. Phylogenetic trees were reconstructed by the neighbor-joining method. Comparative complete sequence analysis of the NVgz01 with reported human norovirus genomic sequences revealed that this isolate belongs to genogroup Ⅱ . The ORF1 and ORF2 regions shared different identity with Mc37 and Fannington strains, suggesting NVgz01 could be a recombinant virus.
文摘目的描述一株新鉴定的流行重组型HIV-1 CRF103_01B在北京的检出情况,并分析其近全长基因组(near full-length genome,NFLG)遗传特征。方法对2017—2020年北京新诊断或初始抗病毒治疗病例进行HIV-1基因型耐药监测,发现5例疑似感染CRF103_01B毒株。通过逆转录、近末端稀释法,分两段巢式PCR扩增HIV-1 NFLG。获得的序列与分型参考序列进行基因比对,使用MEGA11软件构建邻接法(neighbor-joining,NJ)系统进化树。用SimPlot 3.5软件分析确定基因重组断点,绘制基因组结构图。基于亲本毒株同源NFLG序列比对,挑选较长的重组片段构建NJ进化树,判断可能的亲本毒株来源。用斯坦福大学HIV耐药数据库解析基因型耐药特征。结果共获得5条CRF103_01B NFLG序列,其中4例经男男性行为(men who have sex with men,MSM)感染,1例为女性。与从河北检出的4条CRF103_01B NFLG序列合并分析其重组特征:在CRF01_AE骨架上,gag、pol和nef-3'-LTR基因片段被B亚型重组替换[HXB2 nt 1111±19-1539±16,2531-4478±16(片段Ⅴ),9008±23-9615]。亲本来源分析显示,CRF103_01B的片段Ⅳ与Ⅴ分别与北京B亚型(BJMP3294B)和g5簇CRF01_AE序列(JX112804)聚集成大单系进化簇(bootstrap值分别为97%和100%)。9个CRF103_01B病例均携带非核苷类逆转录酶抑制剂类V106I突变。结论CRF103_01B毒株最可能起源于北京MSM人群,并在北京和河北等地的MSM人群中低水平持续流行,并经异性性途径向一般人群播散。需加强该毒株分子流行病学和耐药监测,关注疾病进展。
文摘A new cell culture system expressing the entire HCV geuome has been established in vitro. To initiate transcription of HCV RNA, HeLa cells were trausfected with arecombinant plasmid containing full-length HCV cDNA geuome by using lipofectamiue 2000, followed by infection with recombinant vacciuia virus vTF7-3 containing the T7 RNA polymerase geue. Synthesis of positive-strand HCV RNA could be detected in the trausfected cells by RT-PCR. Western blot analysis revealed that HCV structural and nonstructural proteins were correctly processed. In trausfected HeLa cells 47 um virus-like particles were assembled, whichcould be recognized by auti-HCV E2 antibodies. The titer of HCV was 107 copies/mL in our cell culture system, which was significantly higher than that of infected patients' sera and that from all reported cell culture systems. Superuataut from trausfected HeLa cells were infectious to Huh7 cells and thetiter of HCV was 106 copies/mL. Moreover, negative-strand RNA of HCV in Huh7 cells could be detected by using strand-specific RT-PCR, which demonstrated that replication of HCV occurred in the permissive cell lines.
文摘目的分析北京市献血人群筛查中发现的1例HIV-1独特型重组毒株的基因结构和重组特点。方法基于2018年北京市血液筛查时发现的1例HIV核酸阳性抗体阴性的样本,提取病毒RNA并逆转录为cDNA,用近末端稀释法分两段扩增病毒近全长基因组并测定序列。运用RIP、jpHMM和SimPlot3.5软件进行重组分析,同时采用MEGA7软件构建Neighbor-joining系统进化树对该毒株的同源关系进行分析。结果共获得长度为8792 bp的HIV-1近全长基因序列,分析表明该序列由CRF01_AE和CRF07_BC重组片段组成。与Los Alamos HIV Database(www.hiv.lanl.gov/content/index)中收录的全长序列相比该毒株具有3个独特的重组断点,分4个重组片段,分别是ICRF01_AE(790~6035,HXB2),IICRF07_BC(6036~8586,HXB2),IIICRF01_AE(8587~8828,HXB2)和IVCRF07_BC(8829~9411,HXB2)。其中ICRF01_AE区域属于CRF01_AE的C4簇;IICRF07_BC区域属于CRF07_BC的CRF07BC_N簇。结论1例参与献血的HIV核酸阳性而抗体阴性的感染者携带的毒株具有新型独特的重组模式,提示需要加强监测献血人群中HIV毒株的流行情况,为保障安全用血提供科学数据。
