Huh7 Hepatocyte Cells Cultured in Human Serum are an Improved In Vitro Model for the Study of Two Populations of Hepatitis A Virus

• Author / Creator

  Chua, Tiing Tiing

• Hepatitis A Virus (HAV) belongs to the family of Picornaviridae, a group of viruses that are non-enveloped, single stranded, positive sense RNA viruses. In 2013, studies found not only the standard non-enveloped HAV, but also a novel HAV population, quasi-enveloped HAV (quasi-eHAV), in the samples from HAV-infected chimpanzees and humans. Quasi-eHAV has a lower density (1.06-1.10g/cm3) and is found only in serum. HAV has a higher density (1.22-1.28g/cm3) and is found exclusively in feces. The secretion pathway of these two HAV populations is yet to be elucidated. To clarify this question in vitro, a cell culture system representative of hepatocytes which can secrete the two HAV populations is required. This study focuses on the development of an in vitro system mimicking the polarized hepatocyte able to secrete two HAV populations. Huh7 (a human hepatocarcinoma cell line) was cultured in a dual-chamber system with DMEM supplemented with 2% human serum (HS cultured cells) instead of the typical 10% fetal bovine serum (FBS cultured cells). HS cultured Huh7 cells undergo growth arrest and become more differentiated. The proper polarization of Huh7 cells in HS media cultured in the dual-chamber system was confirmed by dextran diffusion studies. In HAV infection studies, we compared the density profiles of the samples collected from HAV-infected HS cells that grown either monolayer or in the dual-chamber system. By using isopycnic gradient ultracentrifugation, we identified HAV populations based on their buoyant density. We used TCID50 assays and neutralization assays to further characterize the HAV populations released from both sides of HS cells in the dual-chamber system. Our results showed that HAV-infected Huh7 cells in HS media that were grown as a monolayer secreted one HAV population, but HAV-infected Huh7 cells in HS media that were grown in the dual-chamber system secreted two HAV populations. HAV populations were secreted preferentially, either into the upper (apical) or lower (basolateral) compartment of the dual-chamber system. The two in vitro HAV populations shared similar characteristics to the two HAV populations found in vivo, with respect to their infectivity level and their ability to be neutralized by HAV specific antibodies. In short, Huh7 cells in HS media grown in the dual-chamber system were shown to be a suitable cell culture model capable of secreting two HAV populations, such a system can be used to study aspects of HAV biology inaccessible by standard tissue culture methods.

• Subjects / Keywords

  • Hepatitis A Virus
  • In Vitro
  • Cell Culture
  • HAV

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3J960R1J

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.