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A Bioreactor Method to Generate High-titer, Genetically Stable, Clinical-isolate Human Cytomegalovirus.

Victoria R Saykally1, Luke I Rast1, Jeff Sasaki1

  • 1Gladstone Institute for Virology and Immunology, University of California, San Francisco, California, USA.

Bio-Protocol
|December 12, 2017
PubMed
Summary
This summary is machine-generated.

This study introduces a novel bioreactor method for scalable production of high-titer human cytomegalovirus (HCMV) clinical isolates. The continuous infection system significantly enhances virus yield and maintains genetic integrity, overcoming limitations in current research methods.

Keywords:
BioreactorClinical isolateContinuous infection cultureHuman cytomegalovirusMicrobeads (Microcarrier beads)Viral tropism

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Area of Science:

  • Virology
  • Biotechnology
  • Cell Biology

Background:

  • Human cytomegalovirus (HCMV) poses significant risks to transplant recipients and is a leading cause of congenital defects.
  • Current methods for producing wild-type HCMV clinical isolates are inefficient, labor-intensive, and result in low yields and genetic instability.
  • Scalable, high-titer production of genetically intact HCMV is crucial for vaccine and therapeutic research.

Purpose of the Study:

  • To develop an efficient and scalable bioreactor method for producing high-titer wild-type HCMV clinical isolates.
  • To maintain the genetic integrity of key viral tropism factors and the viral genome during production.
  • To improve upon conventional static-culture methods for HCMV stock generation.

Main Methods:

  • Utilized a bioreactor system employing continuous infection of retinal pigment epithelial (ARPE-19) cells immobilized on microcarrier beads.
  • Maintained end-stage infection by regularly adding uninfected ARPE-19 cells.
  • Produced concentrated TB40/E IE2-EYFP stocks at titers of 10^7-10^8 pfu/ml.

Main Results:

  • Achieved a 100-fold increase in virus production compared to conventional static-culture methods.
  • Reduced handling time by 90% and eliminated the need for daily cell passaging or trypsinization.
  • Successfully maintained the genetic integrity of viral tropism factors and the viral genome.

Conclusions:

  • The developed bioreactor method offers a highly efficient and scalable approach for producing high-titer HCMV clinical isolates.
  • This method overcomes critical barriers in HCMV research by providing genetically stable virus stocks with significantly reduced labor.
  • The continuous infection environment holds potential for monitoring infection dynamics and real-time tracking of viral evolution.