目的对北京治疗前耐药监测发现的具有遗传相似性的两株新发重组毒株进行前病毒HIV-1 DNA近全长基因组扩增和序列分析,以了解其遗传特征和重组模式。方法对北京佑安医院募集的2例新发重组毒株感染者,用广州海力特公司核酸提取和纯化试剂...目的对北京治疗前耐药监测发现的具有遗传相似性的两株新发重组毒株进行前病毒HIV-1 DNA近全长基因组扩增和序列分析,以了解其遗传特征和重组模式。方法对北京佑安医院募集的2例新发重组毒株感染者,用广州海力特公司核酸提取和纯化试剂从白细胞富集层提取前病毒HIV-1 DNA。运用SimPlot 3.5软件分析重组模式和断点,并用Mega 11构建分片段最大似然法进化树验证重组事件。结果从2个男男性行为(men who have sex with men,MSM)感染者扩增获得HIV-1近全长基因组序列。SimPlot重组断点分析显示BL6218-00和BL6017-01基因组均以CRF5501B为骨架,分别在gag和env基因区,或gag、pol、vpu/env、nef/3’LTR基因区,由CRF07BC重组而成。2条序列的CRF07BC片段均与来自MSM人群的CRF07BCN参考毒株序列聚集成单系进化簇。结论用前病毒DNA扩增方法从北京MSM人群发现了两株以CRF5501B为骨架且重组模式不同的独特重组型毒株,重组亲本CRF07BC均来自我国北方MSM人群。展开更多
Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients...Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.展开更多
Equine infectious anemia virus strain L (EIAV-L) is the parental virulent virus of equine infectious anemia donkey leukocyte attenuated vaccine (DLA EIAV). In this study, peripheral blood leukocytes(PBL) were collecte...Equine infectious anemia virus strain L (EIAV-L) is the parental virulent virus of equine infectious anemia donkey leukocyte attenuated vaccine (DLA EIAV). In this study, peripheral blood leukocytes(PBL) were collected from a horse infected with EIAV-L.The PBL DNAs were extracted.The EIAV-L proviral DNA was amplified in four parts covering the entire proviral genomic sequence by polymerase chain reaction (PCR). Each of the four parts was cloned into the plasmid pBluescript SK, and the recombinant plasmids were designated as p2.8, p2.4. p3.1, and p1.2 respectively. After identification with restriction digestion, the inserts within the four plasmids were sequenced. The complete nucleotide sequence of EIAV-L provirus was determined by analyzing each of the four parts and connecting them as a whole. The genome of EIAV-L is 8235 bp in length, and G + C content is 38%. The comparison analysis by the computer software DNASIS showed that the sequence of EIAV-L shares 98.4% and 96.9% identities with that of D-A EIAV and DLA EIAV respectively. The high homology between these strains showed that they were genetically related. The homology between EIAV-L and D-A EIAV is higher than that between EIAV-L and DLA EIAV, and this is consistent with the derivation progress of DLA EIAV. At both ends of EIAV-L provirus, there is an identical long terminal repeat (LTR) sequence of 316bp in length. The LTR consists of U3, R, and U5 regions. The genome of EIAV-L provirus has three long open reading frames(ORF) corresponding to gag, pol and env genes respectively. The gag gene is 1200bp and located at position 613-1912nt. The pol gene is 3402bp and located at position 1708-5109nt. There is a termination codon within the env dividing it into two parts, envl of 699bp (position 5305-6003nt)and env2 of 1827bp (position 6073-7899nt). The provirus has three additional small ORFs: S1, S2 and S3 with sizes of 153bp(position 5113-5265nt), 204bp(position 5279-5482nt)and 402bp(position 7245-7646nt) respectively.展开更多
文摘目的对北京治疗前耐药监测发现的具有遗传相似性的两株新发重组毒株进行前病毒HIV-1 DNA近全长基因组扩增和序列分析,以了解其遗传特征和重组模式。方法对北京佑安医院募集的2例新发重组毒株感染者,用广州海力特公司核酸提取和纯化试剂从白细胞富集层提取前病毒HIV-1 DNA。运用SimPlot 3.5软件分析重组模式和断点,并用Mega 11构建分片段最大似然法进化树验证重组事件。结果从2个男男性行为(men who have sex with men,MSM)感染者扩增获得HIV-1近全长基因组序列。SimPlot重组断点分析显示BL6218-00和BL6017-01基因组均以CRF5501B为骨架,分别在gag和env基因区,或gag、pol、vpu/env、nef/3’LTR基因区,由CRF07BC重组而成。2条序列的CRF07BC片段均与来自MSM人群的CRF07BCN参考毒株序列聚集成单系进化簇。结论用前病毒DNA扩增方法从北京MSM人群发现了两株以CRF5501B为骨架且重组模式不同的独特重组型毒株,重组亲本CRF07BC均来自我国北方MSM人群。
基金supported by grants from the State Key Laboratory of Infectious Disease Prevention and Control(2011SKLID102)the National Nature Science Foundation of China(81172733 and 81561128006)the 12th Five-Year National Science and Technology Major Project(2013ZX10001-006)
文摘Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.
基金by National Science Foundation of China(No.39470535).
文摘Equine infectious anemia virus strain L (EIAV-L) is the parental virulent virus of equine infectious anemia donkey leukocyte attenuated vaccine (DLA EIAV). In this study, peripheral blood leukocytes(PBL) were collected from a horse infected with EIAV-L.The PBL DNAs were extracted.The EIAV-L proviral DNA was amplified in four parts covering the entire proviral genomic sequence by polymerase chain reaction (PCR). Each of the four parts was cloned into the plasmid pBluescript SK, and the recombinant plasmids were designated as p2.8, p2.4. p3.1, and p1.2 respectively. After identification with restriction digestion, the inserts within the four plasmids were sequenced. The complete nucleotide sequence of EIAV-L provirus was determined by analyzing each of the four parts and connecting them as a whole. The genome of EIAV-L is 8235 bp in length, and G + C content is 38%. The comparison analysis by the computer software DNASIS showed that the sequence of EIAV-L shares 98.4% and 96.9% identities with that of D-A EIAV and DLA EIAV respectively. The high homology between these strains showed that they were genetically related. The homology between EIAV-L and D-A EIAV is higher than that between EIAV-L and DLA EIAV, and this is consistent with the derivation progress of DLA EIAV. At both ends of EIAV-L provirus, there is an identical long terminal repeat (LTR) sequence of 316bp in length. The LTR consists of U3, R, and U5 regions. The genome of EIAV-L provirus has three long open reading frames(ORF) corresponding to gag, pol and env genes respectively. The gag gene is 1200bp and located at position 613-1912nt. The pol gene is 3402bp and located at position 1708-5109nt. There is a termination codon within the env dividing it into two parts, envl of 699bp (position 5305-6003nt)and env2 of 1827bp (position 6073-7899nt). The provirus has three additional small ORFs: S1, S2 and S3 with sizes of 153bp(position 5113-5265nt), 204bp(position 5279-5482nt)and 402bp(position 7245-7646nt) respectively.
