Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

AIM: We designed two synthetic-core-specific peptides core 1 (C1) and core 2 (C2), and an E1-specific peptide (E1). We produced specific polyclonal antibodies againstthese peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes.METHODS: Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for 1 h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry.RESULTS: After 1 h of incubation, antibodies against C1,C2, and E1 detected HCV antigens on the surface of 27%,26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection.Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection.CONCLUSION: Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle.Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

瞄准：与丙肝的长期的复制建立一个房间文化系统病毒(HCV ) 染色体和病毒的抗原的表示在试管内。方法：HepG2 房间线被孵化与长期的丙肝从一个病人与浆液为它的危险性测试到 HCV。房间和上层清液在文化期间在各种各样的时间点被收获。文化上层清液为它感染天真的房间的能力被测试。存在减(反感觉) 在房间的核心和 E1 抗原的 RNA 海滨，和察觉被 RT-PCR 和免疫学的技术(流动血细胞计数和西方的污点) 分别地检验。结果：细胞内部的 HCV RNA 首先在 d 上被检测 3 在感染以后然后能一致地在至少三个月的一个时期上在房间和上层清液被检测。新鲜房间能从有教养的感染的房间感染上层清液。流动 cytometric 分析证明表面和在房子里使用的细胞内部的 HCV 抗原表示使 polyclonal 成为了抗体(反核心，和 anti-E1 ) 。西方的污点分析证明在分子量的产生免疫性的肽的簇的表示在一个月内在 31 和 45 kDa 之间延长了感染的房间的旧文化而这簇在 uninfected HepG2 房间是无法发现的。结论：HepG2 房间线产生 HCV 感染而且支持它的复制在试管内不仅。HCV 结构的蛋白质的表示能在感染的 HepG2 房间被检测。这些房间也能够流病毒的粒子进接着对 uninfected 房间变得传染的培养基。

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

AIM:To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus(HCV)internal ribosome entry sequences(IRES)on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS:HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance.IRES domainⅢ was cloned and 15 clones for each patient were sequenced.The obtained sequences were aligned with genotype 4a prototype using the ClustalW program and mutations scored.Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS:Analysis of RNA secondary structure revealed that insertions in domainⅢ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA,which may ultimately reduce binding affinity to ribosomal proteins.Insertion mutations in domainⅢwere statistically more prevalent in sustained viral response patients(SVR,n=14)as compared to breakthrough(BT,n=5)patients.CONCLUSION:The influence of mutations within domainⅢ on the response of HCV patients to combination therapy depends primarily on the position,but not the frequency,of these mutations within IRES domain Ⅲ.

Antibody to El peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

瞄准：对丙肝病毒(HCV ) 的 E1 区域分析抗体的抵销的活动。特定的 polyclonal 抗体与从 HCV 的 E1 区域被导出并且被显示高度在 HCV 之中被保存的合成的肽经由新西兰兔子的免疫被提起出版遗传型。方法：超 HCV E1 抗体与为 HCV RNA 积极的浆液样品在 4 度摄氏在夜里被孵化，与从 615 ～ 3.2 百万 IU/ mL 的病毒的负担。对待的重量的单位一为 90 min 与 HepG2 房间被孵化。由反 E1 抗体病毒的绑定和入口堵住进房间借助于 RT-PCR 和流动血细胞计数被测试。结果：用 FITC 染色的直接免疫结合了分析显示出的流动 cytometric 跟随的 E1 抗体在样品的减少的吝啬的荧光紧张与未经治疗的样品相比与 E1 抗体预先孵化。而且， 13 从 18 积极重量的单位一(72%) 显示出由 RT-PCR 检测了的传染性的完全的抑制。结论：在房子里生产 E1 抗体，块绑定和到在病毒绑定建议这 epitope 的参与的靶细胞的 HCV virion 感染的入口和入口。堵住病毒附件到人的房间的这些抗体的隔离作为治疗学的试剂有用。