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"GENETIC ANALYSIS OF HUMAN CYTOMEGALOVIRUS GENES REQUIRED FOR VIRAL GROWTH IN VIVO"

by
Kalpana Dulal
Microbiology and Molecular Genetics program
M. Sc. 2005, University of Maryland Eastern Shore



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

Wednesday, June 13, 2012
10:00 A. M., ICPH 1st floor auditorium


Abstract

Human Cytomegalovirus (HCMV) is an opportunistic human pathogen that causes serious clinical illnesses under immune-suppressed conditions. The seroprevalence of the virus is up to 90% of the population depending upon the socioeconomic status. The clinical strains and the attenuated strains of the virus differ in their genome sequence and arrangement. The attenuated strains have lost a 15-kb region from their genome which renders them unable to grow in the severe combined immuno-deficient mouse with human tissue implants (the SCID-hu mouse model). The clinical strains harboring the 15-kb region grow well in this system. This observation was similar to the inability of the attenuated strains to cause symptomatic infection in healthy volunteers whereas a challenge clinical strain (Toledo) could infect and produce symptomatic viral infection. The 15-kb region encodes twenty-two open reading frames (ORFs) which have not been studied in vivo and only some have been characterized in vitro. In this study we genetically analyzed the 15-kb region from the Toledo strain in order to identify the genes that are crucial for virus growth in vivo. To efficiently monitor viral growth in vitro and in vivo, a luciferase reporter gene was inserted in the Bacterial Artificial Chromosome (BAC) clone of the virus genome to produce Toledoluc BAC. On the backbone of Toledouc BAC, 15-kb deletion (15-kbD) and 15-kb rescue (15-kbR) viruses were made and studied in the SCID-hu mouse using bioluminescence imaging technique to measure virus growth. A novel gene capture method was developed to make rescue clones for larger deletions such as the 15-kbD. As expected the 15-kbD virus did not grow in the mouse implant, whereas the 15-kbR grew like wild-type. The 15-kb region was then divided into four segments (1, 2, 3 and 4) and four mutant viruses, each with deletion of one segment, were made and tested in vivo. Deletion of two of the four segments (1 and 4), when deleted separately, showed severe growth defects indicating that these segments carry genes that are essential for virus growth in vivo. Deletion of one gene or a few genes together from these regions did not impair virus growth in vivo. This rather interesting result suggested a likelihood of functional redundancy among viral genes within these regions. Our results showed that more than one gene, located at two different loci of the 15-kb region, are necessary for virus growth in vivo. Based on our observations, we propose that the viral genes located in the 15-kb region work as a group to allow the virus to grow in the SCID-hu mouse and the genes are likely to functionally complement each other. Transcript analysis of genes located in region 4 showed the genes are transcribed as polycistronic transcripts which undergo splicing.


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