Master of Science / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include diverse groups of viruses that cause a wide variety of diseases in humans and animals. Most of these viruses encode proteases that cleave the viral polyprotein into intermediate or mature functional proteins during virus replication. As these proteases play a critical role in virus replication, they represent an attractive target for the development of antiviral drugs. In this study, the main goal was to establish assay systems and characterize the enzymatic activity of related proteases from Norwalk virus (NV), poliovirus, and transmissible gastroenteritis virus (TGEV). These proteases share several common characteristics including a typical chymotrypsin-like fold, a Cys residue as a nucleophile in the catalytic triad (or dyad) composed of Cys, His and Glu (or Asp) residues, and a preference for a Glu or Gln residue at the P1 position on the substrate. We cloned and expressed proteases from these viruses and characterized their enzymatic activities using a fluorescence resonance energy transfer (FRET) assay using a specific FRET substrate corresponding to each viral protease. First, assay conditions of the FRET assay was optimized for each virus protease. Second, inhibition profiles of each virus protein were investigated using five commercially available standard protease inhibitors (chymostatin, leupeptin, antipain, TPCK, and TLCK). The inhibition studies showed that TPCK inhibited NV, poliovirus, and TGEV proteases with varying strength, and chymostatin inhibited only NV protease. All other inhibitors had little effects on the virus proteases. The established FRET assays should facilitate screening potential antivirals.

Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviruses are enveloped, rod shaped viruses with circular, double-stranded DNA genomes. These viruses infect arthropods, primarily in the order Lepidoptera, although members of this virus family also infect species of Diptera, Hymenoptera, and Crustacea. The majority of these viruses undergo a bi-phasic cycle with one phase defined by the production of a budded virus (BV) form, responsible for cell to cell transmission, and the other defined by the production of an occlusion-derived virus (ODV) form, responsible for host to host transmission. The proto-typical member of the Baculoviridae family is considered to be Autographa californicaM Nucleopolyhedrovirus (AcMNPV). Its 133,894 base pair genome is predicted to encode for 156 proteins, a large number of which are essential for virus replication.. In this current work, we have further characterized two viral proteins that are highly conserved among baculoviruses. The first of these is an ortholog of the fibroblast growth factor family of proteins with sequence homology to the Drosophila Branchless protein as well as the mammalian FGF- 9, -16 and -20 subfamily. Despite its high degree of conservation among baculoviruses, the viral fibroblast growth factor (vFGF) is considered a non-essential protein, although its deletion from the genome does affect the lethality of the virus when ingested per os. In our study, we were able to localize vFGF to the membrane of BV. Its presence on the envelope affected the ability of the virus particle to bind to both heparin in vitro and to the cell surface in vivo, and may play a role in the attachment phase prior to virus entry. We also characterized AcMNPV’s open reading frame 109 (Ac-orf109). Unlike vFGF, Ac-orf109 is essential for virus replication since its deletion results in a complete lack of BV production. Transmission electron microscopy of cells transfected with an Ac-orf109 deletion virus shows the full range of virus-associated structures including mature capsid formation but there appears to be a deficiency in capsid egress out of the nucleus. Furthermore, the ODV retained in the nucleus appear to lack microvesicular membranes, an essential component for host to host transmission of infection.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Stephen Higgs / The genetic composition of arboviruses is a critical determinant of viral infectivity and the capacity for virus dissemination in arthropod vectors. Due to concerns related to a hypothetical potential for loss of attenuation, the supression of vector infection and dissemination is a critical component for the rationale-based design of live-attenuated flavivirus vaccine candidates. The yellow fever virus (YFV) 17D vaccine virus is not only attenuated in vertebrates, but also has low infectivity for Aedes agypti mosquitoes and since it does not disseminate, it is not transmissible. Using a reverse genetics system, the mutations present in the envelope protein YFV 17D virus were characterized in Ae. aegypti to determine the role of mutations in limiting the viral infectivity and dissemination capacity. This knowledge would contribute to the rational design of live attenuated vaccines with the desirable phenotype of being nontransmissible by arthropod vectors. The upper lateral portion of the YFV 17D envelope (E) protein domain III (EDIII) habors the T380R mutation in the FG loop. Experiments demonstrated that the T380R mutation was associated with the viral infectivity phenotype for mosquitoes, but did not influence dissemination into the secondary tissues. The G52R mutation in the molecular hinge region that is located between E protein domains I (EDI) and II, significantly reduced viral infectivity for mosquitoes. In contrast, when cloned into the Asibi wildtype virus genetic backbone, the T173I mutation in the loop structure between the G0 and H0 β- strands did not attenuate viral infection and dissemination. The double mutant virus containing both the G52R and T173I mutations in the E protein, showed a similar attenuated reduced infectivity to the single G52R mutant. The M299I mutation in the linker region between EDI and EDIII resulted in a significantly lower viral infectivity at the initial phase of viral infection at 7 days post-infection in Ae. aegypti. In conclusion, the characterization on four mutations in the YFV 17D vaccine E protein have demonstrated three genetic loci, that can influence the process of YFV infection in Ae. aegypti. These results provide new knowledge and understanding which may have broad applications for the rationale design of safe flavivirus vaccines, via targeting genetic loci and introducing specific mutations that preclude infection of, and transmission by arthropod vectors.

Master of Science / Department of Biology / A. Lorena Passarelli / Cydia pomonella granulovirus (CpGV) is a member of the Baculoviridae family of viruses. The CpGV open reading frame 46 (CpGV-ORF46) predicts a 545 amino acid protein that shares homology with matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. In silico analyses revealed the presence of putative mmp genes in all species from the Betabaculovirus genus, while no mmps were identified in members of the Alphabaculovirus, Gammabaculovirus or Deltabaculovirus genera. Unlike most cellular MMPs, baculovirus MMPs do not have a propeptide domain, a domain involved in regulating MMP activation, or a hemopexin-like domain, which is necessary for substrate binding and specificity in many MMPs. However, Betabaculovirus MMPs do contain a predicted conserved zinc-binding motif (HEXGHXXGXXHS/T) within their catalytic domain. The function of CpGV-MMP and its effects on baculovirus replication in cultured cells and insect larvae were investigated. CpGV-MMP was expressed in and purified from Escherichia coli, and activity was measured using a generic MMP substrate in vitro. CpGV-MMP had in vitro activity and its activity was specifically inhibited by MMP inhibitors. To study the effects of CpGV-MMP on virus replication and dissemination, CpGV-MMP was expressed from Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of a strong and constitutive promoter, the Drosophila heat shock 70 protein promoter. Expression of CpGV-MMP did not affect virus replication in cultured cells. The effects of expressing CpGV-MMP from AcMNPV during larval infection were evaluated in the presence or absence of the AcMNPV chitinase and cathepsin genes. Insect bioassays showed that the absence of cathepsin resulted in a significant delay in larval time of death; however, this delay was compensated by expression of CpGV-MMP. In addition, larval time of death was accelerated when cathepsin, chitinase, and CpGV-MMP were all expressed. Finally, we determined the effects of CpGV-MMP on larvae melanization and liquefaction. CpGV-MMP was able to promote larvae melanization in the absence of cathepsin. CpGV-MMP, in the absence of cathepsin, was not able to promote larvae liquefaction. When chitinase was engineered to be secreted from cells, CpGV-MMP rescued liquefaction in the absence of cathepsin. In conclusion, CpGV-MMP is a functional MMP which can enhance larvae mortality with the presence of cathepsin. In addition, CpGV-MMP can promote larvae melanization; however, it can only promote liquefaction when chitinase is engineered to be secreted from cells.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include a diverse group of pathogens that cause a wide array of diseases that can range from sub-clinical to lethal. These viruses infect humans and mammals as well as a variety of other hosts. For their successful replication, viruses interact closely with host cells from the binding to the receptor to the exit as complete viral progenies. During the events, viruses are dependent on host factors for receptor bindings, genome synthesis, and trafficking of viral genome and proteins. Thus there have been major efforts on the studies of understanding the virus-host interactions in the field of virology. In my PhD program, I have studied the host factors that regulate the replication of viruses using porcine reproductive and respiratory syndrome virus (PRRSV) and hepatitis C virus (HCV). I found that modulation of either the viral receptor or cellular signaling pathways had pronounced effects in the replication of PRRSV or HCV respectively. Using PRRSV, I found that the modulation of the level of the putative receptor CD163 on cells with cytokines significantly influence virus replication, suggesting the importance of cytokine presence in environments to determine the replication and pathogenicity of PRRSV via receptor expression in vivo. With HCV, I found that the enhancement of the virus replication occurs through the activation of the epidermal growth factor receptor/extracellular signal-regulated kinase pathway by bile acids which are abundant in the liver where the virus targets in vivo. Furthermore, I found that the bile acid-mediated signaling pathway significantly inhibited the antiviral activities against HCV. These results indicate the importance of environmental factors such as bile acids and signaling pathways in the replication and pathogenicity of HCV in vivo.

Doctor of Philosophy / Department of Division of Biology / Rollie J. Clem / Mosquitoes are carriers of a variety of harmful human pathogens, including viruses. In order to be successfully transmitted, a virus must evade mosquito immune responses. In this work, the innate immune role of apoptosis in mosquito-virus interactions was examined utilizing the disease vector Aedes aegypti and Sindbis virus. Ae. aegypti is the main vector for yellow fever and dengue virus, which result in over 100 million infections per year. Sindbis virus (Togaviridae) can be transmitted to vertebrates by Ae. aegypti in the laboratory. Sindbis is also well characterized molecularly, making it a good model system for understanding virus-vector interactions. Sindbis MRE-16 recombinant virus clones were utilized to express either an antiapoptotic or pro-apoptotic gene during virus replication. Mosquitoes were infected with recombinant virus clones during a blood meal or by intrathoracic injection. Midgut tissue and whole body samples were analyzed for virus infection and dissemination. Virus was also quantified in saliva and mosquito survival was assayed. Decreased infection in the midgut and delayed virus replication were observed in mosquitoes that were infected with virus expressing a pro-apoptotic gene. Infection with this virus clone also resulted in less virus in the saliva and reduced survival of infected mosquitoes. In addition, negative selection against pro-apoptotic gene expression during virus replication was observed. Collectively, these data suggest that apoptosis can serve as an antiviral defense in Ae. aegypti and may potentially be exploited to control virus transmission. An additional study included in this dissertation focused on zebrafish development and migration of somitic precursors from the tailbud. The tailbud consists of a population of stem cells at the posterior tip of the embryonic tail. The exit of these stem cells from the tailbud is required for the formation of tail somites. A novel double mutant was identified that lacked the t-box transcription factor spadetail and the BMP inhibitor chordin. Double mutants completely lacked somites and had an enlarged tailbud due to accumulation of stem cells that were unable to exit the tailbud. This study indicates the importance of BMP inhibition and spadetail expression in the proper exit of muscle precursors from the tailbud.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Kyeong-Ok Chang / Caliciviruses are important pathogens of humans and animals. Noroviruses are major causes of foodborne gastroenteritis cases, but their research is hindered due to the inability to grow human noroviruses in cell culture. Detailed studies on entry events of caliciviruses are lacking and may be crucial for development of cell culture models. We examined the entry events of caliciviruses using porcine enteric calicivirus (PEC), feline calicivirus (FCV) and murine norovirus-1 (MNV-1). PEC replication in LLC-PK cells requires bile acid in the medium, but the mechanism is not well understood. Our studies showed that bile acids are required in the early stage of virus replication, and while internalization of PEC is not dependent of them, they are required for endosomal escape and successful replication. Further examination on virus entry, we demonstrated that endosomal acidification and cathepsin L activity are essential in the replication of PEC, FCV and MNV-1. The results showed that inhibition of endosomal acidification or cathepsin L activity led to retention of viruses in the endosomes. Also we demonstrated that recombinant cathepsin L cleaved structural protein of PEC, FCV or MNV-1, which suggests that the enzyme may facilitate uncoating viruses in endosomes. In addition to bile acids, we found that a cold shock treatment during virus entry supported PEC replication by facilitating the endosomal escape. While PEC alone did not induce ceramide formation, bile acids or cold shock treatment induce ceramide formation on endosomes through activation acid sphingomyelinase (ASM), and this event was crucial for virus replication because inhibition of ASM blocked ceramide formation and significantly reduced PEC replication. Incubation of FCV or MNV-1 with cells led to ceramide formation during virus entry, and inhibition of ASM also significantly reduced their replication. Inhibition of ASM led to endosomal retention of PEC, FCV or MNV-1 during virus entry, which may be the reason for the reduction of viral replication. These studies revealed the important and common events during calicivirus entry for successful replication, including virus endosomal escape, cathepsin L activity and ASM/ceramide formation. This detailed information may provide clues for understanding the replication of fastidious caliciviruses and for potential therapeutic targets.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Ying Fang / The Porcine Disease Complex (PDC) results in major economic problems for swine producers. PDC outbreaks result in increased mortality, decreased feed efficiency, higher cull rates, prolonged days to market and increased treatment costs. This disease involves the interaction and participation of many multifactorial etiologies including both bacterial and viral organisms playing a role in disease initiation and progression. The most common viral pathogens associated with the PDC include porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV2) and swine influenza virus (swIV). The recent outbreak of porcine epidemic diarrhea virus (PEDV) in the US swine herd has made the PDC even more complicated. In aid of the prevention and control of the PDC, veterinarians and producers require fast and efficient diagnostic tests for controlling the disease. In this study, we have generated recombinant nucleocapsid antigens to these viruses for use in a Luminex™ technology-based fluorescent microsphere immunoassay (FMIA). Utilizing these recombinant nucleocapsid antigens, the FMIA was developed to simultaneously detect antibodies in serum from animals infected with PEDV, PRRSV, SwIV and PCV2. The FMIA was developed based on testing experimentally derived standard positive and negative control sera, and the diagnostic specificity and sensitivity were compared to that generated from the classical enzyme-linked immunosorbent assay (ELISA) or hemagglutination inhibition (HI) test. Based on an evaluation of 4147 serum samples with known serostatus, the multiplex FMIAs reached greater than 97.5% sensitivity and 92.3 % specificity. Results showed that multiplexing did not affect the diagnostic sensitivity or specificity of each individual assay. This work provides a platform for the development of multiplex assays for detecting various swine pathogens simultaneously and aids in preventing and controlling the PDC.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Juergen A. Richt / Influenza A virus is an important respiratory pathogen with the potential to affect both humans and animals, thereby creating the conditions for public health disasters, especially during pandemic episodes. At present, two primary strategies to combat influenza are vaccination and antiviral drugs. Since influenza viruses mutate rapidly and constantly via antigenic drift and shift, vaccines can become quickly outdated; and resistance to antiviral drugs can readily result. Interferon alpha (IFN-[alpha]) plays an important role as a first line of innate antiviral immunity. To investigate the antiviral potential of exogenously applied IFN-[alpha] on the replication of different subtypes of influenza A viruses, three subtypes of influenza A virus, i.e. swine H3N2, pandemic H1N1 and avian H9N2 were chosen. Their replication kinetics in the presence of Alferon N (human Interferon alpha) on human epithelium (A549) cells and swine testis (ST) cells was evaluated. In these tests of the three subtypes of influenza A viruses, it was found that the replication ability of all three viruses was inhibited when ST cells were treated with Alferon for four hours before infection. The ability of Alferon to inhibit influenza A viruses replication was found to be dose-dependent. Similar results were obtained when A549 cells were used; however, pretreatment of A549 cells with Alferon for more than 16 hours was necessary before infection. Furthermore, the expression of some ISGs (Interferon stimulated genes) between ST and A549 cells was also investigated. The differences in response of the ISGs between the two cell lines provided an explanation of the disparity towards exogenous interferon treatment. In summary, these results demonstrated that Alferon N has the ability to inhibit replication of different subtypes of influenza A viruses in cell cultures. This study provides a foundation for future in vivo studies using exogenous IFN-[alpha] treatment as an alternative approach to combat influenza A virus infection.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Each year, over 500 million people are infected with mosquito-borne diseases, including malaria, yellow fever and dengue fever, which cause several million deaths, and long-term disability and suffering. This dissertation focused on the mosquito Aedes aegypti, a vector for dengue virus and yellow fever virus. Since Sindbis virus (SINV) is an arthropod-borne virus (arbovirus) that is vectored by A. aegypti and is well characterized at the molecular level, the SINV - A. aegypti model was used to determine whether apoptosis plays a role in the control of vector competency. In Chapter 2, the effects of inducing or inhibiting apoptosis on SINV replication were tested in mosquito cells. It was observed that recombinant SINVs expressing pro-apoptotic genes caused extensive apoptosis in mosquito cells, with decreased virus production after the cells underwent apoptosis. Infection of mosquito cells with SINV expressing the caspase inhibitor P35 inhibited actinomycin D-induced apoptosis, but had no observable effects on virus replication. This study was the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells. Chapter 3 examined the effects of silencing apoptosis regulatory genes on SINV replication and dissemination in A. aegypti. Genes which either positively or negatively regulate apoptosis were silenced by RNA interference in mosquitoes, which were then infected with a recombinant SINV expressing green fluorescent protein (GFP). Reciprocal effects were observed on both the occurrence and intensity of expression of GFP in various tissues. These results suggest that systemic apoptosis positively influences SINV replication in A. aegypti. This was the first direct study to explore the role of apoptosis in determining mosquito vector competence for arboviruses. Finally, in Chapter 4, the mechanisms of apoptosis were explored in A. aegypti. Overexpression of IAP antagonists caused extensive cell death in mosquito cells, while silencing the expression of IAP antagonists attenuated apoptosis. The results showed that the IAP binding motif (IBM) of IAP antagonists was critical for their binding to AeIAP1. The IAP antagonists released initiator and effector caspases from AeIAP1 by competing for the binding sites and caused caspase-dependent apoptosis. These findings imply that the mechanisms of IAP antagonists regulating apoptosis are conserved between mosquitoes and the model insect where apoptosis has been mainly studied, Drosophila melanogaster.