ABSTRACT Title of dissertation: FUNCTIONAL STUDIES OF INFECTIOUS PANCREATIC NECROSIS VIRUS PROTEINS AND MECHANISM OF VIRUS-INDUCED APOPTOSIS

Title of dissertation: FUNCTIONAL STUDIES OF INFECTIOUS PANCREATIC NECROSIS VIRUS PROTEINS AND MECHANISM OF VIRUS-INDUCED APOPTOSIS Haichen Song, Doctor of Philosophy, 2003 Dissertation directed by: Professor Vikram N. Vakharia Center for Biosystems Research, and VA-MD Regional College of Veterinary Medicine Infectious pancreatic necrosis virus (IPNV) encodes a 12 or 15-kDa nonstructural protein, known as VP5. To study the function of VP5, we generated three recombinant viruses rNVI15, rNVI15-15K, and rNVI15-∆VP5, which could encode either 12-kDa VP5, 15-kDa VP5 or be deficient in VP5, respectively. VP5 was detected in rNVI15 and rNVI-15K infected cells but not in the cells infected with rNVI15-∆VP5. However, the opal stop codon at nucleotide position 427 in rNVI15 virus was read-through, giving rise to a 15-kDa VP5 that is expressed poorly than rNVI15-15K virus-infected cells. All three recombinant viruses show similar replication kinetics in both Chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells. Moreover, in Sp strains, IPNV segment A could encode a novel, putative 25-kDa protein from another ORF. This 25kDa protein could not be detected in virus-infected cells, however, we could recover a mutant virus lacking this ORF, indicating that it is not essential for virus replication. To assess the molecular basis of virus adaptation in the cell culture, virulent rNVI15 was serially passaged in CHSE cells nine times to obtain a tissue-culture adapted virus, rNVI15TC. Comparison of the deduced amino acid sequences showed only one amino acid substitution at position 221 (Ala → Thr) in VP2. However, this adaptation mutation is only acquired in CHSE cells but not in RTG-2 cells. Two chimeric viruses, rNVI15∆VP2 and rNVI15-15K∆VP2 were also generated, in which the residues at positions 217 and 247 in VP2 of the rNVI15 and rNVI15-15K viruses were replaced by the corresponding residues of an attenuated strain, Sp103. These two viruses have similar replication kinetics as Sp103, which replicates faster than rNVI15 in vitro, indicating that residues at positions 217 and 247 of VP2 may be the important markers for virus adaptation and attenuation in vitro. By generating a reassortant virus between rNVI1515K and Sp103, we also demonstrate that VP1 is not involved in virus cell adaptation. The signal pathways and nature of IPNV-induced apoptosis were investigated in RTG-2 cells. IPNV-induced apoptosis occurs at the late stage of viral life cycle. Caspase3 is activated during virus infection, and inhibition of caspase-3 could partially inhibit virus-induced apoptosis. Moreover, NF-κB activation is essential for IPNV-induced apoptosis, and it is involved in interferon-induced antiviral state. Both the NF-κB inhibitor and the antioxidant could inhibit NFκB activity and apoptosis induced by IPNV infection, but they do not affect viral replication. FUNCTIONAL STUDIES OF INFECTIOUS PANCREATIC NECROSIS VIRUS PROTEINS AND MECHANISM OF VIRUS-INDUCED APOPTOSIS