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A GENETIC STUDY OF VARICELLA ZOSTER VIRUS USING A NOVEL LUCIFERASE BACTERIAL ARTIFICIAL CHROMOSOME SYSTEM

by
Zhen Zhang
Microbiology and Molecular Genetics

M.S. Fudan University, Shanghai, China 1999-2002

M.D. Fudan University, Shanghai, China 1992-1999

Thesis Advisor: Hua Zhu, Ph.D.

Associate Professor

Department: Microbiology and Molecular Genetics

ICPH 1st Floor Auditorium

Thursday, April 16, 2009
12:15 p.m.


Abstract

Varicella zoster virus (VZV) is a ubiquitous human alpha-herpesvirus which is the causative agent of chicken pox and shingles. The latter is associated with a significant incidence of post-herpetic neuralgia. Although an attenuated VZV vaccine is now widely used in children, whether this vaccine will have a significant impact on the development of shingles remains unclear. Therefore VZV still remains a significant public health concern. The knowledge of VZV replication and pathogenesis is limited mainly due to its highly cell-associated nature in cultured cells and the poor infectivity of purified VZV DNA. In order to circumvent these problems, the entire VZV (p-Oka) genome was cloned into a bacterial artificial chromosome (BAC) including a dual-reporter system (an eGFP and a firefly luciferase gene). Using PCR-based mutagenesis and the homologous recombination system in E.coli DY380 strain, each of its 70 unique ORFs were individually deleted and a collection of viral mutants were made and examined systemically in cultured MeWo cells and human fetal skin organ cultures (SOC). Viral growth kinetics analysis, based on bioluminescence quantification, showed that 44 ORFs are essential for viral replication in MeWos and 26 are non-essential. Among the latter group 8 ORFs deletion mutants showed discernable growth defect. Selected ORF deletion mutants were further tested in SCID-hu mice thymus/skin xenografts. Most interestingly, some ORFs were found to be required for viral replications in cultured human fetal skin organs, but not in MeWo cells, suggesting their potential roles as skin tropism factors. The global functional profiling of the entire viral genome further elucidated the replication and pathogenesis of VZV in order to improve the prevention and therapy of chickenpox and shingles.


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