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 detectio...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.展开更多
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 wa...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.展开更多
文摘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.
基金Supported by the Ministry of Scientific Research, Academy of Scientific Research and Technology, Medical Research Council Code: P5-MED-030-01 and US-Egypt joint project BIO7-002-011
基金Supported by A grant from National Research Center to M.Awady and grant from Yousef Jameel Science&Technology Research Center(YJ-STRC)at the American University in Cairo to H.Azzazy
文摘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.
基金Supported by the Ministry of Scientific Research, Academy of Scientific Research and Technology, Medical Research Council Code: P5-MED-030-01US-Egypt joint project BIO7-002-011