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العنوان
Study of Intracellular Hepatitis C Virus Replication in Vitro \
المؤلف
Mosaad, Mariam Mounir Kamel.
هيئة الاعداد
باحث / مريم منير كامل مسعد
مشرف / أحمد بركات بركات
مشرف / ريهام محمد حسن
مشرف / مروة إبراهيم خليل
تاريخ النشر
2020.
عدد الصفحات
144 p. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
علم الأحياء الدقيقة
تاريخ الإجازة
1/1/2020
مكان الإجازة
جامعة عين شمس - كلية العلوم - الميكروبيولوجي
الفهرس
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Abstract

The scarcity of having a robust infectious in vitro model for studying HCV replication has hindered scientific research efforts to understand the pathogenesis and life cycle of the virus.
Recently, a significant HCV culture model was achieved by the subgenomic replicon which enabled consistent HCV replication in vitro. Previous teams were able to clone HCV genotype 2a JFH1 which then was transfected into Huh-7 cell line, this recorded the first attempt to enhance virions production and replication. However, there are several limitations for the subgenomic replicon system, first is the utilization of the rare clone of HCV genotype 2a JFH1 and second, is the use of cloned HCV genotype 1a H77-S which was identified to have some adaptive mutations.
Many other groups have studied HCV replication using positive serum to infect hepatocytes in vitro, using infected serum provides the cocktail which contains the essential factors serving as a nourishing environment for HCV virions biology, dynamics and life cycle.
The aim of the present study is to establish an invitro model to enhance the replication of the virus in human hepatoma cells using different detection methods and techniques via serum from HCV genotype 4a-infected patients as a source of infection.
Blood samples were collected from patients infected with HCV, serum was isolated and used as a source of infection for Huh 7.5 cells, viral loads ranged from 4x106 to 20x106, total collected samples were 13.
An infection protocol was established and optimized in order to be used for detection using real time PCR. Optimal inoculum for infection was found to lie between 100 and 200 µl, we were able to detect bright bands using this volume.
Depleting positive serum from IgG and Albumin did not have any observed effect on the viral replication capacity, ELISA was performed but results were under detectable limits. PCR results showed no significant difference.
On the protein level, we used NS3 antibodies as a biomarker for viral replication, immune staining results showed successful infection of cells using the abovementioned protocol.
Co-culture systems are commonly used to study interactions between different cell populations. It is well known to enhance infection of pathogens, some groups used coculture models in the study of gene therapies using Adeno Associated Viruses (AAV), however no one tried using coculture models to study HCV replication.
We developed a coculture model using Huh 7.5 and HepG2 cells and compared replication in the coculture model versus individual cell lines.
Coculture model increased the infectivity of the virus significantly compared to Huh 7.5 cells alone and HepG2 cells alone with a p-value (0.01) and 0.07 and the experiment was repeated 3 biological repeats.
We also observed viral toxicity to Huh 7.5 cells using high viral load inoculum while HepG2 cells and the coculture model tolerated the same inoculum.
In terms of incubation period, we compared 3 days and 7 days, we started to detect the viral particles after 3 days but 7 days was better in signal detection.
We anticipated from our results that both Huh 7.5 and HepG2 cell lines synergize to develop either complementary, additivity or stimulatory relationship. As Huh 7.5 lack the antiviral response induced by the Retinoic-Acid Inducible Gene I, which enhance viral entry but they can’t polarize in culture, on the other hand, HepG2 cells are known to polarize which is necessary for compartmentalization of HCV receptors, as well as they lack CD81 which is necessary for viral entry Pileri et al. (1998).
Although, we hypothesized a complementary effect for both Huh 7.5 and HepG2 cell lines, further studies are required to validate and confirm these findings both on the molecular and protein levels