Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Human enteric caliciviruses are a leading cause of viral gastroenteritis in humans of all ages worldwide. However, most attempts to grow human enteric caliciviruses in routine cell cultures have failed. This has hampered research on pathogenesis, immunity, virus inactivation, and the development of antivirals and vaccines. Porcine sapovirus (PoSaV) Cowden strain causes diarrhea in pigs, and is one of only a few culturable enteric caliciviruses. The PoSaV Cowden strain was adapted to a porcine kidney cell line, LLC-PK, after serially passaging in gnotobiotic pigs and in primary porcine kidney cells. Compared with the wild-type (WT) PoSaV Cowden strain, the tissue culture-adapted (TC) PoSaV has eight conserved amino acid substitutions: two in the RNA-dependent RNA polymerase (RdRp) region and six in the capsid protein (VP1) region. By using a reverse genetics system for the TC Cowden strain, pCV4A, four (178, 289, 324, and 328) amino acid substitutions in the VP1, but not the substitutions in the RdRp region, were identified to be critical for the cell culture adaptation of PoSaV Cowden strain. Two (291 and 295) substitutions in the VP1 enhanced virus replication in vitro, but reduced virus replication in vivo. In addition, the 291 and 295 revertants induced higher serum and mucosal antibody responses than TC PoSaV Cowden strain. Three dimensional (3D) structural analysis of the VP1 showed that residue 178 was located in the shell domain near the dimer-dimer interface, which may affect VP1 assembly and oligomerization; residues 289, 291, 324, and 328 were located at the protruding subdomain 2 (P2), which may influence virus binding to the cellular receptors; residue 295 was located at the interface of two monomeric VP1 proteins, which may influence VP1 dimerization. Next, these findings were tested using other Cowden-like genogroup III PoSaVs. The complete genomes of two Cowden-like PoSaV strains, WT JJ259 and WT LL14 were sequenced and compared with those of Cowden (open full item for complete abstract)

Committee: Linda Saif Ph.D. (Advisor); Qiuhong Wang Ph.D. (Advisor); Gireesh Rajashekara Ph.D. (Committee Member); Armando Hoet Ph.D. (Committee Member) Subjects: Molecular Biology; Pathology; Virology

Doctor of Philosophy, Case Western Reserve University, 2008, Molecular Biology and Microbiology

In the advent of the promise for HIV-1 entry inhibitors to treat patients with drug resistance to available antiretroviral therapy, there has been a surge in studies related to HIV-1 entry. It is clear that the intrinsic susceptibility of primary virus isolates to entry inhibitors varies, which indicates a greater probability for intrinsic resistance to this class of drugs. The studies presented here provide tools and insights into the impact of the envelope (env) gene on HIV-1 replicative capacity, how that capacity influences the emergence of drug resistance, and how easily drug resistance is selected in vivo to a microbicide entry inhibitor. The fitness of a virus is a marker of its replication capability in a given environment. The specific impact of HIV-1 entry on the overall fitness of a virus was tested by cloning a region of the env gene into a common backbone and comparing the resultant recombinant fitness to that of the wildtype virus. The env gene was sufficient to determine the overall fitness of these viruses, which also correlated with sensitivity to entry inhibitors. Specifically, the binding avidity of a virus to the host cell coreceptor contributed the most to fitness. PSC-RANTES is an entry inhibitor that acts on the coreceptor CCR5 to block HIV-1 binding and down-regulate coreceptor expression. When used as a microbicide in a rhesus macaque model, PSC-RANTES failed to block transmission of SHIV¬¬¬SF162 at high doses in some animals. Two mutations were identified in isolates from one of these animals and were cloned into a common HIV-1 backbone to create a chimeric virus. The chimeric virus exhibited resistance to PSC-RANTES and an increase in fitness over that of the wildtype virus used to infect the rhesus macaques. This study showed for the first time the selection of drug resistant viruses to a microbicide in an HIV-1 animal model system. Lastly, a cloning strategy was developed to quickly create replication-competent, fully infectious HIV-1 ch (open full item for complete abstract)

Committee: Eric Arts (Advisor) Subjects: