A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus （JEV） by immunofluorescence ass...A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus （JEV） by immunofluorescence assay （IFA） and reverse transcription polymerase chain reaction （RT-PCR）, and named SXBJ07. The complete nueleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein （E1-E411）.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
目的研究丙型肝炎病毒(HCV)基因组核酸在亚甲蓝光化学法(methelene blue photochemistry,MB-P)灭活病毒前后的变化,在全基因组水平分析MB-P对基因组各结构区段的降解作用。方法含HCV的血浆中加入终浓度为1.0μmol/L的MB,经约30000Lux强...目的研究丙型肝炎病毒(HCV)基因组核酸在亚甲蓝光化学法(methelene blue photochemistry,MB-P)灭活病毒前后的变化,在全基因组水平分析MB-P对基因组各结构区段的降解作用。方法含HCV的血浆中加入终浓度为1.0μmol/L的MB,经约30000Lux强度的荧光照射后,在不同作用时间点取样;将HCV全基因组序列分为互相重叠的8个区段,分别进行RT-PCR,分析基因组核酸的完整性;同时运用实时定量PCR(real time-PCR,RT-PCR)技术观察核酸降解的动力学变化。结果基因组各区段RT-PCR结果发现,经过不同的光照时间,HCV基因组各区段的稳定性不同,第2、4、5、6区段对MB-P作用较敏感,基因组5’端区段和3’端区段经MB-P作用后的稳定性高于基因组其它区段;RT-PCR结果显示,随着光照时间延长,可被检测到的病毒核酸拷贝数逐渐下降。结论MB-P灭活过程中HCV基因组核酸被降解,而且基因组不同区段对光化学作用的反应性不同,提示RNA降解可能是病毒灭活的重要机制;检测病毒核酸稳定性以监测病毒灭活具有一定临床实用价值。展开更多
Cellular microRNAs(miRNAs) have been shown to modulate HCV infection via directly acting on the viral genome or indirectly through targeting the virus-associated host factors. Recently we generated a comprehensive map...Cellular microRNAs(miRNAs) have been shown to modulate HCV infection via directly acting on the viral genome or indirectly through targeting the virus-associated host factors. Recently we generated a comprehensive map of HCV–miRNA interactions through genome-wide miRNA functional screens and transcriptomics analyses. Many previously unappreciated cellular miRNAs were identified to be involved in HCV infection, including miR-135a, a human cancerrelated miRNA. In the present study, we investigated the role of miR-135a in regulating HCV life cycle and showed that it preferentially enhances viral genome replication. Bioinformatics-based integrative analyses and subsequent functional assays revealed three antiviral host factors, including receptor interacting serine/threonine kinase 2(RIPK2), myeloid differentiation primary response 88(MYD88), and C-X-C motif chemokine ligand 12(CXCL12), as bona fide targets of miR-135a. These genes have been shown to inhibit HCV infection at the RNA replication stage. Our data demonstrated that repression of key host restriction factors mediated the proviral effect of miR-135a on HCV propagation. In addition,miR-135a hepatic abundance is upregulated by HCV infection in both cultured hepatocytes and human liver, likely mediating a more favorable environment for viral replication and possibly contributing to HCV-induced liver malignancy.These results provide novel insights into HCV–host interactions and unveil molecular pathways linking miRNA biology to HCV pathogenesis.展开更多
Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA ex...Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.展开更多
Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to ident...Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication.Methods:Ribonucleic acid (RNA) structural analogs X94,X12,and X12c were constructed to have identical conformation but 94%,12%,and 0% sequence identity,respectively,to the X region of HCV genotype 2a.Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction.Results:In replicon BB7 cells,a constitutive replication model,HCV RNA levels decreased to 55%,52%,53%,and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46,X-94,X-12,and X-12c,respectively (p<0.001 for all).In an HCV genotype 2a infection model,RNA analogs 5B-46,X-94,and X-12 in hepatic cells inhibited replication to 11%,9%,and 12%,respectively.Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a,the sequence per se,or antisense effects were unlikely to be involved.Conclusions:The data suggest that conformation of secondary structures in 3'-UTR of HCV RNA genome is required for HCV replication.Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.展开更多
基金supported by the National Natural Science Foundation of China (30771067)
文摘A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus （JEV） by immunofluorescence assay （IFA） and reverse transcription polymerase chain reaction （RT-PCR）, and named SXBJ07. The complete nueleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein （E1-E411）.
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.
文摘目的研究丙型肝炎病毒(HCV)基因组核酸在亚甲蓝光化学法(methelene blue photochemistry,MB-P)灭活病毒前后的变化,在全基因组水平分析MB-P对基因组各结构区段的降解作用。方法含HCV的血浆中加入终浓度为1.0μmol/L的MB,经约30000Lux强度的荧光照射后,在不同作用时间点取样;将HCV全基因组序列分为互相重叠的8个区段,分别进行RT-PCR,分析基因组核酸的完整性;同时运用实时定量PCR(real time-PCR,RT-PCR)技术观察核酸降解的动力学变化。结果基因组各区段RT-PCR结果发现,经过不同的光照时间,HCV基因组各区段的稳定性不同,第2、4、5、6区段对MB-P作用较敏感,基因组5’端区段和3’端区段经MB-P作用后的稳定性高于基因组其它区段;RT-PCR结果显示,随着光照时间延长,可被检测到的病毒核酸拷贝数逐渐下降。结论MB-P灭活过程中HCV基因组核酸被降解,而且基因组不同区段对光化学作用的反应性不同,提示RNA降解可能是病毒灭活的重要机制;检测病毒核酸稳定性以监测病毒灭活具有一定临床实用价值。
基金supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health
文摘Cellular microRNAs(miRNAs) have been shown to modulate HCV infection via directly acting on the viral genome or indirectly through targeting the virus-associated host factors. Recently we generated a comprehensive map of HCV–miRNA interactions through genome-wide miRNA functional screens and transcriptomics analyses. Many previously unappreciated cellular miRNAs were identified to be involved in HCV infection, including miR-135a, a human cancerrelated miRNA. In the present study, we investigated the role of miR-135a in regulating HCV life cycle and showed that it preferentially enhances viral genome replication. Bioinformatics-based integrative analyses and subsequent functional assays revealed three antiviral host factors, including receptor interacting serine/threonine kinase 2(RIPK2), myeloid differentiation primary response 88(MYD88), and C-X-C motif chemokine ligand 12(CXCL12), as bona fide targets of miR-135a. These genes have been shown to inhibit HCV infection at the RNA replication stage. Our data demonstrated that repression of key host restriction factors mediated the proviral effect of miR-135a on HCV propagation. In addition,miR-135a hepatic abundance is upregulated by HCV infection in both cultured hepatocytes and human liver, likely mediating a more favorable environment for viral replication and possibly contributing to HCV-induced liver malignancy.These results provide novel insights into HCV–host interactions and unveil molecular pathways linking miRNA biology to HCV pathogenesis.
基金Project supported by the Exchange Scholarship Programs of the Landesregierung Schleswig-Holstein,Germany,for Jian-er WO
文摘Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
文摘Background and Aims:The noncoding regions in the 3'-untranslated region (UTR) of the hepatitis C virus (HCV)genome contain secondary structures that are important for replication.The aim of this study was to identify detailed conformational elements of the X-region involved in HCV replication.Methods:Ribonucleic acid (RNA) structural analogs X94,X12,and X12c were constructed to have identical conformation but 94%,12%,and 0% sequence identity,respectively,to the X region of HCV genotype 2a.Effects of structural analogs on replication of HCV genotypes 1b and 2a HCV RNA were studied by quantitative reverse transcriptase polymerase chain reaction.Results:In replicon BB7 cells,a constitutive replication model,HCV RNA levels decreased to 55%,52%,53%,and 54% after transfection with expression plasmids generating RNA structural analogs 5B-46,X-94,X-12,and X-12c,respectively (p<0.001 for all).In an HCV genotype 2a infection model,RNA analogs 5B-46,X-94,and X-12 in hepatic cells inhibited replication to 11%,9%,and 12%,respectively.Because the X-12 analog was only 12% identical to the corresponding sequence of HCV genotype 2a,the sequence per se,or antisense effects were unlikely to be involved.Conclusions:The data suggest that conformation of secondary structures in 3'-UTR of HCV RNA genome is required for HCV replication.Stable expression of RNA analogs predicted to have identical stem-loop structures might inhibit HCV infection of hepatocytes in liver and may represent a novel approach to design anti-HCV agents.
