Thesis (MSc (Veterinary Tropical Diseases))--University of Pretoria, 2008. / Includes bibliographical references. Also available in print format.

Enteroviruses belong to the Picornaviridae family and are small icosahedral viruses with RNA genomes of positive polarity, containing a single open reading frame. They mostly cause mild or asymptomatic infections, but also a wide array of diseases including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis, myocarditis, hand-foot-and-mouth disease, hepatitis and respiratory diseases, ranging from severe infections to the common cold. The projects described in this thesis have been carried out through reverse genetic studies of Enterovirus B and Rhinovirus C.                   In Papers I and II, a cassette vector was used to study recombination and translation of the RNA genome. It was found that the non-structural coding region could replicate when combined with the structural protein-coding region of other viruses of the same species. Furthermore, the genome could be translated and replicated without the presence of the structural protein-coding region. Moreover, it was found that when two additional nucleotides were introduced, shifting the reading frame, the virus could revert to the original reading frame, restoring efficient replication. In Paper III, a vector containing the genome of echovirus 5 was altered to produce an authentic 5’end of the in vitro transcribed RNA, which increased efficiency of replication initiation 20 times. This result is important, as it may lead to more efficient oncolytic virotherapy. An authentic 5’end was further used in Paper IV, where replication of Rhinovirus C in cell lines was attempted. Although passaging of the virus was unsuccessful, the genome was replicated and cytopathic effect induced after transfection. The restriction of efficient replication was therefore hypothesized to lie in the attachment and entry stages of the replication cycle. In Paper V, a cytolytic virus was found to have almost 10 times larger impact on gene expression of the host cell than a non-cytolytic variant. Furthermore, the lytic virus was found to build up inside the host cell, while the non-cytolytic virus was efficiently released.                   As a whole, this thesis has contributed to a deeper understanding of replication of enteroviruses, which may prove important in development of novel vaccines, antiviral agents and oncolytic virotherapies.

Picornaviruses are responsible for some of the most common and debilitating diseases affecting humans and animals worldwide. The objectives of this dissertation research were (1) estimate phylogenetic relationships among 11 picornavirus genera and within three species: foot-and-mouth disease virus (FMDV: Aphthovirus) which afflicts cloven-hoofed animals and human rhinovirus A and B (HRV: Enterovirus) which cause the common cold; (2) better understand the impact recombination has on genomic organization and evolution; (3) characterize where positive and purifying selection has occurred in proteins and how selection has influenced phenotype. The dissertation includes four studies. The first chapter provides an overview of the evolutionary significance of recombination, its detection and estimation, and its effect on phylogenetic analysis in four biological systems: bacteria, viruses, mitochondria, and the human genome. Chapter two investigates the inter- and intra-serotypic relationships within FMDV by examining 12 genes. Gene sequences were analyzed to assess recombination breakpoint locations, genetic diversity, and natural selection in FMDV. Recombination breakpoints were located throughout the genome. Paraphyletic relationships among serotypes were not as prevalent as previously reported, suggesting that convergent evolution was prevalent. Purifying selection was the dominant evolutionary force influencing both genotype and phenotype. Chapter three examines inter- and intra-specific relationships of HRV using 11 genes. Similar hypotheses were tested as in chapter two. No recombination was detected and phylogenetic relationships among enteroviruses, HRV-A, and HRV-B remain unresolved. The evolution of HRV-A major serotypes appeared to be under extensive purifying selection, HRV-A minor serotypes under predominantly positive selection, and a nearly equal influence from both kinds of selection was evident for HRV-B serotypes. Chapter four examines phylogenetic relationships among genera using three conserved genes. The hypothesis of cospeciation between picornaviruses and their hosts was also tested. The deepest split in the family separated Hepatovirus, ‘Tremovirus’, Parechovirus, and seal picornavirus type 1 from the remainder of the family. Enterovirus and ‘Sapelovirus’ were sister taxa. Cardiovirus, ‘Senecavirus’, Aphthovirus, Erbovirus, Teschovirus, and Kobuvirus were derived from a common ancestor with Kobuvirus occupying a basal position relative to the other genera in this clade. My analyses suggest that picornaviruses have not cospeciated with their known hosts.

In the infection process, native poliovirus (160S) first converts to a cell-entry intermediate (135S) particle, which causes the externalization of capsid proteins VP4 and the N-terminus of VP1 (residues 1-53). The externalization of these entities is followed by release of the RNA genome, leaving an empty (80S) particle. Three antibodies were utilized to track the location of VP1 residues in different states of poliovirus by cryogenic electron microscopy (cryo-EM). "P1" antibody binds to N-terminal residues 24-40 of VP1. Three-dimensional reconstruction of 135S-P1 showed that P1 binds to a prominent capsid peak known as the "propeller tip". In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, ~60 Å distant, at the icosahedral twofold axes. Analysis of 80S-P1 reconstructions showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips; only at the twofold axes; or simultaneously at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions. "C3" antibody binds to 93-103 residues (BC loop) of VP1. The C3 epitope shifts outwards in radius by 4.5% and twists through 15° in the 160S-to-135S transition, but appears unchanged in the 135S-to-80S transition. In addition, binding of C3 to either 160S or 135S particles causes residues of the BC loop to move an estimated 5 (±2) Å, indicating flexibility. The flexibility of BC loop may play a role in cell-entry interactions. At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and the N-terminus of VP1 externalize reversibly. An antibody, binding to the residues 39-55 of VP1, was utilized to track the location of the N-terminus of VP1 in 160S particle in the "breathing" state. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered 135S particle, but the N-terminus of VP1 is located near the twofold axes, instead of the propeller tip as in 135S particles.

<p>The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied <i>in vitro</i> in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.</p><p>It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which <i>in vivo</i> may increase a virus’s ability to persist in islets.</p><p>Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.</p><p>All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis <i>in vivo</i>.</p><p>Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.</p><p>To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells <i>in vivo</i>, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction <i>in vivo</i>.</p>

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound. It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets. Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect. All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo. Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study. To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

Polioviruses, Coxsackieviruses, and Echoviruses belonging to the Picornaviridae family of positive-stranded, non-enveloped viruses, are highly infectious and associated with a range of illnesses in children from minor febrile illness to severe, potentially fatal conditions (eg, aseptic meningitis, encephalitis, paralysis and myocarditis). The viruses encodes 11 viral proteins along with the transient set of intermediates unique to viral propagation. 3A, one of the non-structural proteins, plays a crucial role in viral replication by anchoring the replication complex to the membrane vesicle and by recruiting essential cellular factors to the site of replication. It is an 89 amino-acid longprotein, and consists of a soluble N-terminal region and a hydrophobicC-terminal region. The soluble region contains two amphipathic alphahelices that form a hairpin, which are flanked by unstructured regions.Since, Enteroviruses have limited coding capacity,viral protein interactions with cellular proteins and lipids are essentialin viral replication, translation, polyprotein processing andpathogenesis. Understanding these interactions is essential inunderstanding the molecular mechanisms associated pathogenesis, andidentifying drug targets. Our studies are aimed at identifying hostfactors interacting with 3A protein and their functional significance invirus replication.We have identified thepotential 3A-interacting cellular candidate proteins using pull-down followed by liquid chromatography associated mass spectrometry. Gene ontology analysis revealed asignificant enrichment in cellular pathways, functions, and proteindomains in comparison with the control. Further studies were focused on Sec12 (guanine nucleotideexchange factor), ACBD3 (acyl-CoA binding domain containing 3) andPhosphatidylinositol 4-kinase beta (PI4KIIIß) interactions with the 3Aprotein, and their significance in viral replication. Sec12 (GEF) initiates the COPII vesicle-mediated ER-to-Golgi membrane trafficking by recruiting and activating the small GTP binding protein Sar1A to the membrane, which further recruits Sec23/24, cargo and Sec13/31 coat proteins to form functional COPII vesicles.We demonstrated that Sec12 and 3A interact directly in the ER through their C-terminal hydrophobic regions in oligomerization independent manner, leading toreduced the level of recruitment of individual COPII components such as Sar1A, Sec24A, and Sec31A to the membranes, thereby inhibiting virus replication. But in infected cells, other viral proteins such as 2B and 2BC likely stabilize the membrane-recruited Sar1A to support the viral replication. The viral proteins, ACBD3, PI4KIIIß interacted and co-localized with the Echovirus 3A protein.Knockdown of Sec12 or PI4KIIIß and expression of 3A or DN-Sar1A inhibited Echovirus replication, unlike proteins which support the COPII vesicle mediated ER-to-Golgi trafficking.Our results collectively indicate Sec12 is a crucial component in the anterograde membrane trafficking and is a novel host factor in Echovirus replication.

Ljungan virus (LV) is a recently identified picornavirus of the genus Parechovirus. LV has been isolated from voles trapped in Sweden and also in the United States. LV infected small rodents may suffer from diabetes type 1 and type 2 like symptoms, myocarditis and encephalitis. LV has been proposed as a human pathogen, with indications of causing diabetes type 1, myocarditis and intrauterine fetal deaths. In this thesis, cell culture adapted LV strains were utilised for development and adaptation of several basic methodological protocols to study the LV biology, e.g. real time PCR, highly specific antibodies and a reverse genetics system. These methods allowed detailed studies of this virus and how it interacts with the host cell. The genomic 5'-end was identified and modelling showed unique secondary structure folding of this region. The LV encodes an aphthovirus-like 2A protein with a DvExNPGP motif. This motif was found to mediate primary cleavage of the LV polyprotein in vitro and is proposed to constitute the carboxy terminus of the structural protein VP1 in LV. Rabbit polyclonal antibodies generated against recombinant structural proteins were used to verify that the LV virion is composed of the structural proteins VP0, VP1 and VP3. Cell culture studies showed that LV replicates to low titer with an absent or delayed cell lysis. LV is proposed to be able to spread by a, for picornaviruses, not previously demonstrated direct cell-to-cell transmission. All results taken together suggest a maintenance strategy of LV including low amounts of the LV genome and persistently infected hosts. Stability studies showed that the LV virion not only maintain activity in acidic and alkaline environments but also exhibit resistance to the commonly used disinfectant Virkon®.The results presented in this thesis show that LV has several unique properties, not previously observed for a picornavirus.