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ABSTRACT: Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
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ABSTRACT: Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
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You have isolated a new virus, determined it has a lipid envelope, and wish to characterize the mechanism of entry. Discuss the important research questions you would ask and the types of experiments you might use to address the questions. You have isolated a new virus, determined it has a lipid envelope, and wish to characterize the mechanism of entry. Discuss the important research questions you would ask and the types of experiments you might use to address the questions. Solution Answer: (There may be several questions related to the attachment and entry process, a few have been discussed) The first encounter between a virus and a target cell occurs at the plasma membrane. It is here that viruses first engage binding and entry receptors to initiate the infection process. Specific research questions on the mechanism of entry of this virus may be: 1. Adhesion and attachment: While normally non-specific in nature, different glycosphingolipids on the viral surface have been reported to confer cell specificity: the sialyllactose moiety on human immunodeficiency virus (HIV) membrane gangliosides, for instance, has been suggested to promote uptake of HIV into mature dendritic cells (mDCs). So, it can be asked whether the mechanism of entry is through specific glycosphingolipids and new roles of these glycosphingolipids? Experiments: Using liposomes to mimic the lipid composition and size of the virus, it can be demonstrate whether gangliosides are the key molecules that mediate liposome uptake. Liposome and VLP (virus like particle) capture assays on cultured cells, confocal microscopy and neuraminidase assay with VLP and LUV (large unilamellar vescicles) can be used for the investigation. 2. Lipid-mediated signalling for entry: Low-density lipoprotein receptors, negatively charged phospholipids and gangliosides have all been shown to assist entry of different viruses. Low-density lipoprotein receptors, also known as cholesterol receptors, are used by several members of the Flaviviridae family, including hepatitis C virus (HCV). The interaction is likely to occur through lipoproteins associated with the virion, such as cholesteryl esters and apolipoproteins (ApoB and ApoE) acquired during the assembly and budding of virus. So, it can be determined whether the LDL receptor acts as a receptor for this new virus and whether the endocytosis of the virus correlated with the level of LDL receptors?. Investigation of endocytosis of the virus by cultured cells in vitro can be demonstrated to have LDL receptors with the use of anti-LDL receptor antibody or DiI-LDL uptake. These cell lines can then be inoculated with a high titer virus positive serum. Intracellular viral RNA then can be detected by using ISH. To determine whether endocytosis of the virus correlated with the level of LDL receptor expression on cells, the well known modulatory effect of lipoproteins on the LDL receptor can be used to increase the number of LDL receptors (up-regulate) on cel.
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Dr Susan E Caldwell Publication Samples
Dr Susan E Caldwell Publication Samples
Clonal b cells in patients with hepatitis c virus–associated mixed
Clonal b cells in patients with hepatitis c virus–associated mixed
็Hepatitis C virus discovery to cure
็Hepatitis C virus discovery to cure
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