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。