Indiana University-Purdue University Indianapolis (IUPUI) / Enteric viruses are among the most common infectious human viruses worldwide, causing an estimated 10-15 million infections per year in the United States. Among enteric viruses, Coxsackievirus is commonly isolated and can lead to the development of meningitis, encephalitis, pancreatitis, and hepatitis. Furthermore, Coxsackievirus B3 is the primary cause of viral myocarditis and can lead to pleurodynia, with nearly 40,000 symptomatic cases reported in the United States each year. The enteroviral ssRNA genome contains a 5’ untranslated region (5’UTR) which consists of two structural components, the cloverleaf and the internal ribosome entry site (IRES), both shown to be integral to viral success. Additionally, the viral genome encodes four structural VP proteins as well as 11 non-structural proteins. Polymorphisms found within the CVB3 population have been linked to viral virulence. Here, we compare two CVB3 Nancy variants to elucidate the downstream effects observed in response to mutations found in the CVB3 genome. Implementing our novel oral inoculation model, we aimed to determine the impact mutations found in the 5’UTR and VP regions exert on viral pathogenesis. We also aimed to delineate the in vitro effects of the observed mutations. We investigated the role mutations found in the structural regions played in virus host cell attachment, in vitro cell viability, and replication. Our work has further confirmed the relevance and impact of mutations found in the VP region of the CVB3 genome.

Indiana University-Purdue University Indianapolis (IUPUI) / Enteric viruses are infectious human pathogens that initiate infection in the gastrointestinal tract. They follow a fecal-oral route of transmission and are spread by contamination of food, water, or contact between individuals. Furthermore, enteric viruses also cause significant morbidity, mortality, and economic burdens yearly. Coxsackievirus (CV) is commonly isolated among enteric viruses and is an etiological agent of hand, foot, and mouth disease, hemorrhagic conjunctivitis, and myocarditis. The virus predominantly infects infants and young children and accounts for 11% of the fatality rate in neonates. Despite CV’s impact on human health, there are no treatments or vaccines for CV infections. Using a mouse model to study a key CV, Coxsackievirus B3 (CVB3), our laboratory has found two critical factors that impact CVB3 replication and pathogenesis. First, we have demonstrated that intestinal bacteria enhance intestinal CVB3 replication. We found that certain specific bacteria (Salmonella enterica) and its cell wall components, like lipopolysaccharides (LPS), enhanced CVB3 stability and infectivity in vitro. Additionally, we found that particular constituents of LPS are required for stability to occur. These data suggest that specific bacteria may be integral in maintaining CVB3 infectivity in the intestine. Besides virus-microbiome interaction, CVB3 is also impacted by sex hormones. Using castrated mice models, we observed a sex bias to CVB3 infection, with male mice succumbing to CVB3-induced disease at an increased rate compared to female mice. Our data suggest that testosterone, a predominant male sex hormone, enhanced CVB3 intestinal replication and viral dissemination to organs in male and female mice, but lethality only in male mice. Moreover, testosterone also affected the immune response by reducing the activation of the CD8+ T cells. CD8+ T cells are required to clear the viral infection and are integral in vaccine development. In contrast, we found an enhanced CD8+ T cell response in female mice to CVB3 infection, suggesting a sex-dependent T cell response that may underlie the sex bias in disease. Overall, these data represent an essential advancement in the CV field and will help develop future therapeutics and aid in vaccine design to limit CV infections.

CoxsackievirusB3 (CVB3), a member of the Picornaviridae family is the causative agent of Virus-induced Myocarditis and Dilated Cardiomyopathy. The 5’UTR contains an Internal Ribosome Entry Site or IRES element that recruits ribosomes in a cap-independent manner. The ribosomes are recruited upstream of the AUG triplet at 591 (AUG591), also called as the cryptic AUG, after which they scan downstream for about 150 nucleotide, before initiating at the initiator AUG or AUG741. The 3’UTR of CVB3 is 99 nts long, highly structured RNA containing conserved domains, and is followed by a poly (A) tail of variable lengths. We have investigated possible involvement of host proteins which may interact with CVB3 IRES and influence its activity. We have demonstrated the role of Poly-pyrimidine tract binding protein (PTB) and established PTB as a bona-fide ITAF for CVB3, by characterizing the effect of partial silencing of PTB ex-vivo in HeLa cells. The IRES activity in BSC-1 cells, reported to have very low level of endogenous PTB, is found to be significantly low compared to that in HeLa cells. PTB is observed to interact with both the 5’ and 3’ UTR of CVB3, although with different affinities. Finer mapping of the interaction between PTB and the UTRs showed that the protein interacts with multiple regions of both UTRs. We have also shown the cis-acting effect of the CVB3-3’UTR on IRES mediated translation. The PTB contact points on the 3’UTRwas found to map to conserved regions, the deletion of which abrogates the 3’UTR mediated enhancement of the IRES activity. The possible role played by PTB in enhancing IRES activity by CVB3 3’UTR suggests that PTB protein might help in circularization of the CVB3 RNA by bridging the ends necessary for efficient translation of the viral RNA. In the second part, we have investigated possible role of some of the cis-acting element present in the CVB3 5’UTR RNA particularly the cryptic AUG. We have shown that mutation in cryptic AUG reduces the efficiency of translation mediated by the CVB3 IRES. Mutation in cryptic AUG moiety also reduces the interaction of mutant RNA with La protein. We have demonstrated that binding of 48S ribosomal complex with mutant IRES RNA was weaker compared to wt IRES RNA. We have investigated the possible alteration in secondary structure in the mutant RNA by chemical and enzymatic modification, which suggests that there is marginal alteration in the local structure due to mutation. It appears that integrity of cryptic AUG is important for efficient translation initiation by the CVB3 IRES. Results suggest that cryptic AUG plays a significant role in mediating internal initiation of translation of CVB3 RNA by mediating precise La binding and correct positioning of the 48S ribosomal complex. Finally, we have investigated the importance of a conserved hexa-nucleotide stretch in the apical loop within stem loop C (SLC, nt104-180), upstream of the ribosome landing site, on CVB3 IRES function. It has been already shown from our laboratory that the deletion at this apical loop resulted in significant decrease in IRES activity. This deletion mutant was shown to alter the secondary structure of the CVB3 5’UTR RNA. Here we have investigated the effect of point mutation in the apical loop SLC/c on CVB3 IRES activity by generating substitution mutation in the apical loop SLC/c in order to avoid possible alteration in secondary structure. Both the deletion or substitution mutation at this apical loop resulted in significant decrease in IRES activity. Both the mutant IRES RNAs (deletion and substitution mutant) failed to interact with certain trans-acting factors. Furthermore, expression of CVB3 2A protease significantly enhanced IRES activity of the wild type, but the effect was not so pronounced on the mutant IRESs. It is possible that the mutant RNAs were unable to interact with some trans-acting factors critical for enhanced IRES function. We have short-listed three proteins of approximate molecular mass of 56, 64 and 90 kDa, which showed reduced binding with mutant IRESs. By using RNA affinity column with biotinylated UTP labeled RNA we have purified couple of proteins and identified p64 as Cyto Keratin 1 protein by performing in-gel trypsin digestion followed by MALDI analysis. Overall, the results characterize the CVB3 IRES structurally and functionally, which could be useful in targeting critical RNA-protein interactions to develop candidate antiviral agent against Coxsackievirus infection.

Der Großteil MHC Klasse I restringierter Epitope wird bei der Proteindegradation durch das Ubiquitin Proteasom System (UPS) generiert. In der vorliegenden Arbeit wurde die Rolle des UPS in der Antigenpräsenation in einer Coxsackievirus B3 (CVB3) induzierten akuten und chronischen Myokarditis untersucht. Für in vitro Degradationsexperimente mit isolierten 20S Proteasomen wurden CVB3 Polypeptide synthetisiert und die Degradationsprodukte massenspektrometrisch analysiert. Eine erhöhte Substratumsatzrate und eine Verschiebung von Schnittpräferenzen durch Immunoproteasomen oder unter dem Einfluss von PA28 führten zu einer verbesserten Generierung immunrelevanter CVB3 Fragmente. Inflammatorische Kardiomyopathien können in Mäusen durch eine CVB3 Infektion ausgelöst werden. Resistente Stämme (C57BL/6) eliminieren das Virus vollständig, in anfälligen Mäusen (A.BY/SnJ) erfolgt keine vollständige Elimination. In Herzen gesunder Mäuse werden vorwiegend konstitutive 20S Proteasomen exprimiert. Eine myokardiale Entzündung, ausgelöst durch eine CVB3 Infektion, führte in den Herzen beider Mausstämme zu der Bildung von Immunoproteasomen, was zu einer gesteigerten Generierung immunrelevanter CVB3 Fragmente führte. Die größte Menge immunrelevanter Fragmente wurden durch Proteasomen gebildet, die am Tag vier aus den Herzen akut erkrankender C57BL/6 Mäuse und am Tag acht aus chronisch erkrankenden A.BY/SnJ Mäusen isoliert wurden. Dies korrelierte mit der Inkorporation von Immunountereinheiten in de novo assemblierende Proteasomen und einer unterschiedlichen Interferon (IFN) Typ I Kinetik. In Geweben lymphatischen Ursprungs hingegen waren Zusammensetzung und proteolytische Aktivität der Proteasomen im Verlauf der Infektion in beiden Mausstämmen unverändert. Die vorliegende Arbeit unterstreicht die Bedeutung einer zeitlich optimalen IFN Sekretion an der Infektionsstelle, die zu der Anpassung des UPS an die inflammatorischen Bedingungen führt. / The recognition of viral antigens bound to major histocompatibility complex (MHC) class I molecules by CD8+ T cells is crucial for virus elimination. Most MHC class I restricted antigenic peptides are produced by the Ubiquitin Proteasome System (UPS). In the present study, the impact of the UPS in antigen presentation during Coxsackievirus B3 (CVB3) induced acute and chronic myocarditis has been investigated. To examine whether the proteasome is involved in the generation of MHC class I ligands derived from the CVB3 polyprotein, polypeptides were synthesized for in vitro processing by 20S proteasomes. Mass spectrometry analysis demonstrated an enhanced generation of immunorelevant CVB3 fragments due to an increased substrate degradation rate and altered cleavage site preferences by immunoproteasomes or in the presence of PA28. Murine models of CVB3 induced myocarditis mimic human disease pattern with diverse outcomes. Permissive mice (A.BY/SnJ) develop chronic myocarditis with cardiac CVB3 persistence whereas resistant mice (C57BL/6) recover and eliminate the virus after acute infection. Constitutive 20S proteasomes are mainly expressed in hearts of healthy mice. Myocardial inflammation, caused by a CVB3 infection, resulted in immunoproteasome formation in hearts of both, resistant C57BL/6 and susceptible A.BY/SnJ mice, and was correlated with enhanced generation of immunorelevant CVB3 peptides. In concurrence with distinctive type I interferon kinetics, immunoproteasome formation and improved epitope generation peaked on day 4 post infection in resistant mice, and was delayed in susceptible mice. No alterations were observed in assembly and proteolytic activity of 20S proteasomes in lymphatic tissues during CVB3 infection, independent from mouse strain. The results emphasise the impact of a rapid adjustment of the UPS to viral infection due to early secretion of type I interferon at site of infection.

Trace elements are essential for the host defence against infections, and during common infections, the balance of trace elements is changed in serum and tissues. Supplementation with selenium (Se), an essential trace element, is known to decrease the severity of coxsackievirus B3 (CVB3) infection in mice. Even the non-essential trace element arsenic (As) seems to influence the replication of some viruses. During the course of an acute CVB3 infection in mice, Se concentrations decreased in most tissues and were negatively correlated to viral load in our study. However, As concomitantly decreased in most tissues. As has previously been shown to interfere with the balance of essential trace elements. However, in the present study As supplementation in healthy mice resulted in minor effects on seven studied trace elements in serum and tissues. The effects of As supplementation were more pronounced in CVB3-infected mice, with an increase in As, but a decrease in Se in most tissues when compared with non-infected mice. As supplementation during CVB3 infection in mice decreased viral RNA concentrations in the brain (97%) and pancreas (75%), two of the target organs of this infection. In vitro experiments indicate that As caused an impaired virion assembly or release. In vivo, infection-induced expression of the host defence-associated genes nuclear factor κB (NFκB) and interferon γ (IFN-γ) were unaffected by As supplementation, except for an earlier increase in IFN-γ in the brain. In conclusion, a clinically relevant dose of As decreased the replication of CVB3 in vitro and in vivo. This antiviral effect in vivo was not related to changes in specific trace elements or in the host’s immune-mediated defence. Although the mechanism underlying the observed effect on viral replication remains to be further elucidated, As seems to be an intriguing trace element to study in the pursuit of new antiviral drugs.

With global freshwater supplies under pressure, viable water reuse methods are being examined to assist in improving water supplies. Municipal effluent is an ideal source for water reclamation as it is consistent in quality and quantity. The health aspects of water reuse have been identified as an issue of concern, in particular the potential presence of enteric viruses. Managed Aquifer Recharge (MAR) is a method that can aid water reclamation by recharging water such as treated effluent into a suitable aquifer. Research into the removal of pathogenic contaminants by natural processes within aquifers, namely the action of autochthonous bacteria, has led to the consideration that MAR could be used to assist in the removal of microbial pathogens. Pathogens have been demonstrated to be removed during residence in groundwater, but the presence of active autochthonous groundwater bacteria are required for significant removal rates to occur. The aim of this research was to investigate the interaction between autochthonous groundwater bacteria (AGB) and the enteroviruses Poliovirus type 1, Coxsackievirus B3 and Adenovirus B41. It was established that these viruses decrease in number in the presence of AGB but the mechanisms causing this decrease are poorly understood. Experiments were designed to examine how the individual AGB caused decay of the viruses. In this study AGB were isolated and tested for their ability in increase the decay of the viruses. It was determined that 27 % (17/63) of the isolated AGB influenced viral decay. The AGB isolates varied in their influence with only 3 out of 17 isolates being able to cause of the decay of both poliovirus and coxsackievirus. Similar variations in decay were observed for adenovirus. Decay times for all three viruses varied amongst the AGB and between the viruses. Experiments were undertaken to characterise the mechanism causing the antiviral activity of four groundwater isolates (1G, 3A, 4B and 9G) under varying conditions and treatments to give insight into the compounds or mechanisms responsible for viral decay. This would indicate whether compounds produced by the AGB responsible for viral decay were closely associated to bacterial cells (perhaps membrane bound), independent of metabolic activity, heat labile or were enzymatic in nature. The influence of enzyme inhibiters and heat treatment indicated that viral degradation is caused by compounds that are enzymatic in nature. As viral numbers were monitored by nucleic acid copy numbers rather than via infectivity assays, the viral protein coats must be the first step in degradation followed by the removal of the viral nucleic acid. This two step process would require both protease and nuclease enzymes to result in loss of viral numbers as measured by RT-PCR/PCR. Further characterisation and identification of these four bacterial isolates was also carried out. Three out of the four isolates were sequenced and analysed using partial 16S rRNA gene sequences to determine their phylogenetic relationships compared to related organisms. Isolate 3A was placed in the order Burkholderiales. Isolate 4B was placed in the family Xanthomonadaceae. Isolate 9G was placed in the family Rhizobiaceae. Isolate 1G was only partially sequenced and preliminary identification placed it in the phylum Bacteriodetes. Understanding of the processes carried out by AGB within an aquifer during MAR using reclaimed waters will aid in increasing the viability of this water reuse process. If important natural processes could be utilised to remediate any potential pathogens, the health concerns with reclaimed waters could be addressed and solved simply through prescribed retention times within the aquifer. Key species of AGB may even be utilised as markers to assess the suitability of an aquifer for MAR.

No description available.

Das Proteasom ist ein multikatalytischer, ATP-abhängiger Enzymkomplex, der kurzlebige und regulatorische Proteine in der Zelle abbaut. Im Rahmen der Proteinqualitätskontrolle werden durch das Proteasom auch fehlerhaft synthetisierte bzw. falsch gefaltete oder chemisch geschädigte Proteine degradiert. Zellen hämatopoetischen Ursprungs exprimieren sogenannte Immunoproteasomen, die durch drei alternative katalytische Untereinheiten (LMP2, MECL-1 und LMP7) charakterisiert sind. Unter dem Einfluss von Interferonen kommt es auch in nicht-hämatopoetischen Zellen zur de novo Assemblierung von IP. Sie weisen im Vergleich zu Standardproteasomen einen erhöhten Substratumsatz sowie veränderte Schnittpräferenzen auf. Dadurch können Standard- und Immunoproteasomen verschiedene MHC Klasse I-restringierte antigene Peptide generieren. Die vorliegende Arbeit untersucht die Relevanz der LMP2- bzw. der LMP7- Untereinheit im Rahmen der Coxsackievirus B3 Myokarditis. LMP7-/- Mäuse zeigen eine suffiziente CD8+ T Zell Antwort, die zur vollständigen Viruselimination nach der akuten Entzündungsphase beiträgt. Die reguläre Expression pro-inflammatorischer Zytokine und antiviraler Signalwege sowie CVB3-spezifischer IgG-Antikörper spricht gegen eine spezielle Funktion von IP bei der Induktion einer effektiven Immunantwort in diesem Modell. Es konnte jedoch gezeigt werden, dass der verminderte Einbau aller IP-Untereinheiten in LMP7-defizienten Mäusen mit einer schweren Inflammation und Myokardschädigung einhergeht. Der verringerte Substratumsatz führt zur Akkumulation von polyubiquitinylierten, oxidativ geschädigten Proteinen sowie zur verstärkten Apoptose IP-defizienter Kardiomyozyten und inflammatorischer Zellen. / The standard proteasome is the major ATP-dependent multi-catalytic protein complex that is important for the proteolytic processing of short-lived and regulatory proteins. It also degrades exogenous or improperly synthesized, misfolded, and damaged proteins. Cells of hematopoietic origin predominantly express an alternative variant - the immunoproteasome, which is characterized by three specific catalytically active subunits (LMP2, MECL-1 and LMP7). In non-immune cells, these immunosubunits are also induced and incorporated into newly assembling IPs upon exposure to interferons. As compared to standard proteasomes, IPs display altered cleavage site preferences, resulting in the generation of a different spectrum of antigenic peptides for MHC class I presentation. The present thesis investigates the impact of LMP2- and LMP7 within the context of viral heart disease, making use of the well-established murine model of coxsackievirus B3 infection. LMP7-deficient mice demonstrate a potent CD8+ T cell capacity to control CVB3 infection, resulting in viral clearance after the acute stage of disease. The expression of pro-inflammatory cytokines, innate antiviral mediators, and CVB3-specific IgG antibodies argue against a specific role of IPs in the induction of an effective immune response against CVB3 infection. However, the impaired incorporation of all three immunosubunits in LMP7-deficient hearts coincides with severe inflammation and myocardial tissue damage. Exposure to IFN-γ gives rise to prolonged accumulation of oxidant-damaged, poly-ubiquitylated proteins in IP-deficient cardiomyocytes and inflammatory cells. Along with the restricted degradation of toxic protein aggregates, inflammatory cells and the adjacent myocardium are prone to increased apoptotic cell death.

No description available.