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"VARICELLA ZOSTER VIRUS OPEN READING FRAME 7 IS A SKIN AND NEURON TROPIC FACTOR"

by
Anca I. Selariu
Interdisciplinary Biomedical Sciences Program
B.Sc. 2006, Biochemistry, Montclair State Univ., Montclair, NJ
B. Sc. Philology, 2001, Transilvania Univ., Brasov, Romania



Thesis Advisor: Hua Zhu, Ph.D.
Associate Professor
Department of Microbiology and Molecular Genetics

Wednesday, January 18, 2012
12:00 P.M., ICPH, 1st Floor Auditorium


Abstract

Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates as hepes zoster (shingles) upon weakening of the immune system, erupting from sensory neurons and infecting surrounding skin tissue. According to the CDC (2008), over 95% of people are infected by VZV and one in three people will develop shingles during their lifetimes. The current varicella vaccine is highly attenuated in the skin, yet retains its neurovirulence and may reactivate and damage sensory neurons. Therefore, there is an imperative public health need for a safer neuro-attenuated vaccine to eliminate the risk of recurrent herpes zoster. The factors involved in neuronal invasion and establishment of latency are still elusive.
VZV is difficult study due to its highly cell-associated nature in cell culture and almost exclusive preference for human cells. In order to facilitate the study of VZV mutants, we created a p-Oka derived bacterial artificial chromosome to which we added GFP and luciferase markers for easy in vitro and in vivo detection of viral replication. We generated a library of VZV mutants by systematic deletion of each of the 70 unique VZV ORFs, which we first tested in MeWo cells to determine their requirement in viral replication. Thus we found 44 essential and 26 non-essential genes for viral replication in vitro. Among the latter category, we found 8 genes to be defective in MeWo, while the other 18 had phenotypes similar to that of the WT. The nonessential genes, most of which are likely to function as tissue tropism modulators, were first screened in human skin organ culture (SOC). We found 4 genes with significant growth defects in SOC (ORFs 7, 10, 14 and 47), of which ORF7 was newly characterized as skin-tropic. We verified the ex vivo screening result in the in vivo SCID mouse model with human skin implants, and found that 7D had a severely altered growth properties in this context.
Next, screening of non-essential VZV gene deletion mutants in differentiated neuroblastoma cells led to the identification of ORF7 as a viral protein involved in neurotropism. Further tests in ex vivo human fetal dorsal root ganglia (DRG) and in a severe combined immunodefficient mouse model (SCID DRG) confirmed that ORF7 is required for VZV neuroinvasion in human tissues. The ORF7 deletion mutant failed to replicate and did not cause any cytopathic changes in human DRG in vivo.
In order to determine whether ORF7 specificity was restricted to skin and neurons, we tested ORF7 in immune cells and tissues, which are known sites of replication for VZV during establishment of viremia. We found that 7D was not required for replication in either T-cells, dendritic cells, spleen organ cultures or SCID-thymus implants. Thus, ORF7 is a tropism factor involved only in skin and neuron spread, but not in immune cell tropism, indicating that 7D is likely to be useful as an immune booster vaccine.
Upon further scrutiny of the ORF7 characteristics, we determined that ORF7 is a virion component which localizes to the Golgi network in infected cells and this localization is dependent on the conserved Cysteine at position 9. Interestingly, although ORF7 is not required for productive infection, its deletion is associated with loss of polykaryon formation in epithelial cells in vitro, suggesting ORF7 involvement in a viral spread mechanism reliant on syncytia or fusion complex formation. We propose that ORF7 is a crucial component of a viral maturation and egress complex which is used by VZV for infection of skin and neurons.
The discovery of ORF7 as an important component of skin neuron tropism of VZV makes 7D, in conjunction with its potential to raise or boost immunity by infecting immune cells, a safer VZV vaccine candidate against both chickenpox and shingles. Moreover, 7D is genetically defined and easy to manipulate, and could also serve as a convenient vector for delivery of exogenous antigens.


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