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As obligatory intracellular parasites, viruses are in a constant battle with their host to establish infection. They can facilitate their propagation by modulating host immune or metabolic pathways. This modulation involves targeting various molecular factors such as microRNAs (miRNA), enzymes, or small molecules. Understanding how viruses alter the chemical makeup of a cell is crucial to identifying what pathways are being targeted, furthering our understanding of the virus life cycle, and may aid in identifying biomarkers of disease. Here, we examine host-virus interactions in the context of two viruses, hepatitis c virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). First, the modulation of serine hydrolases by a pro-viral microRNA, miRNA-122, is investigated using activity-based protein profiling (ABPP). This study identifies a downstream target of miRNA-122 that is differentially activated during HCV infection which can be targeted pharmacologically to reduce HCV infectivity. Second, we apply similar techniques to identify serine hydrolase changes associated with SARS-CoV-2 infection. Results point towards enrichment of endocannabinoid metabolism which may offer an alternative therapeutic avenue for combating SARS-CoV-2 infection. Together, the work presented in this thesis provides avenues for further investigation into miRNA-122 interactions during HCV infection and endocannabinoid metabolism in SARS-CoV-2 infection.

Hepatitis B virus (HBV) is a major etiologic agent of chronic liver disease (CLD) and hepatocellular carcinoma (HCC). The virally encoded X antigen, HBx, contributes importantly to the development of HCC through its trans-activating role in various signal transduction pathways. Pathways implicated in stem cell self-renewal also contribute to carcinogenesis. Thus, experiments were designed to test if HBx triggers malignant transformation by promoting properties that are characteristic of cancer stem cells (CSCs). To test this hypothesis, HBx expressing (HepG2X) and control (HepG2CAT) human cell lines were assayed for phenotypic and molecular characteristics of "stemness." Western blotting of protein extracts from HepG2X and HepG2CAT cells as well as immunohistochemical staining of HCC and adjacent liver tissue sections from HBV infected patients showed up-regulation of "stemness"-associated (EpCAM and beta-catenin) and "stemness" (Oct-4, Nanog, Klf-4) markers by HBx. Moreover, HBx stimulated cell migration and spheroid formation. HBx expression was also associated with depressed levels of E-cadherin and subsequent activation of beta-catenin and EpCAM. Results from ChIP-chip data performed previously in this lab suggest an associative link between HBx and the expression of epigenetic co-repressor, mSin3A, which is known to repress E-cadherin when complexed with histone deacetylases. Thus, experiments were also designed to test if HBx represses the E-cadherin gene (CDH1) through histone deacetylation by the mSin3A/HDAC complex. In HepG2X cells, decreased levels of E-cadherin and elevated levels of mSin3A were detected. Reciprocal immunoprecipitation with anti-HBx and anti-mSin3A demonstrated mutual binding. Further, HBx-mSin3A co-localization was showed by immunofluorescent staining. Chromatin immunoprecipitation revealed that HBx mediated the recruitment of the mSin3A/HDAC complex to the CDH1 promoter. HDAC inhibition by Trichostatin A treatment restored E-cadherin expression. Thus, HBx-associated epigenetic repression of E-cadherin and up-regulated expression of multiple "stemness" markers support the hypothesis that HBx contributes to hepatocarcinogenesis, at least in part, by promoting changes in gene expression that are characteristic of CSCs. This work is the first to propose that HBV promotes "stemness" in the pathogenesis of HCC. / Biology

Chronic infection with hepatitis B virus (HBV) is a primary risk factor for the development of hepatocellular carcinoma (HCC). HCC is the fifth most common cancer type worldwide with few treatment options. The hepatitis B encoded x antigen (HBx) plays a crucial role in the pathogenesis of HCC through several mechanisms. HBx alters signaling pathways shown to promote carcinogenesis and mediates epigenetic changes that silence tumor suppressor genes and activate host oncogenes. Short chain fatty acids (SCFAs) are made by selected gut bacteria with largely anti-inflammatory properties. They alter gene expression by functioning as histone deacetylase inhibitors (HDACi) and can bind to G protein coupled receptors (GCPR) to stimulate signaling pathways. Due to the documented anti-cancer properties of SCFAs, experiments were designed to test the hypothesis that SCFAs delay the development of HCC in HBx transgenic (HBxTg) mice. A diet of SCFAs was fed to HBxTg for three months prior to the expected appearance of dysplastic nodules and HCC. The results showed a statistically significant reduction in the number of dysplastic nodules as well as the presence and frequency of HCC. The effect of SCFAs on tumor growth was also evaluated in nude mice subcutaneously injected with human HCC cells. Tumor size in SCFA-treated mice was statistically smaller compared to the controls. The effect of SCFAs on cell viability of cancer and primary human hepatocytes was evaluated. SCFAs were shown to reduce cell viability in cancer cells only, with no effect on primary hepatocytes. Proteomics was performed on SCFA-treated compared to control livers from HBxTg to investigate changes on the molecular level that are associated with reduced preneoplastic and neoplastic nodule formation. Pathway analysis showed a decrease in important cancer-promoting pathways altered by HBx in HCC, including inflammation, oxidative stress, PI3K, VEGF, EGF, and Ras. These pathways are involved in biological processes central to carcinogenesis such as cell proliferation, survival, and angiogenesis. The ability of SCFAs to decrease these pathways has never been demonstrated. Further investigation confirmed that Ras activity was decreased in 12-month old livers treated with SCFAs. Taken together, these results show that SCFAs are capable of delaying the rate of tumor growth and tumor frequency in two mouse models of HBV-associated HCC, as well as reduce cell viability in cancer cells specifically. This data suggests that SCFAs may be a novel treatment option for HBV-associated HCC. / Biology

Background: Hepatitis C virus (HCV) is a widespread problem in the United States, but the disease's low screening rates mean that reported cases account for only a fraction of the population's antibody prevalence. In reality, chronic HCV is the most common chronic blood-borne infection in the country. While newer infections may be completely asymptomatic, the virus can lead to serious complications in the liver down the line, including hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Because HCV is a reportable disease, the Philadelphia Department of Public Health (PDPH) hepatitis surveillance registry has records of all of the positive tests performed in the city. Negative test results are not readily available, however, making it difficult to create an accurate picture of who is being tested for HCV. Our study used negative results collected from reference laboratories throughout the city to fill in many of the gaps and determine which neighborhoods were not sufficiently screening at-risk populations. Methods: Our dataset included approximately 100,000 individuals, a little over 90% of whom were HCV negative. Negative test results were obtained from Quest and Lab Corp, two of the major reference laboratories in the area that account for approximately 80% of all of the results of tests performed in Philadelphia. The screening data were then combined with records from the PDPH hepatitis surveillance registry. ArcGIS geographic information software was used to create maps out of neighborhood and census tract data, providing a visual representation of HCV screening distribution in Philadelphia. We also explored differences in demographic characteristics and testing facility information by test result. The data included anyone in the past 2 years who had undergone an antibody (Ab) or RNA test for HCV. Results: We found that census tract poverty rate was positively associated with HCV screening rate. Also, the majority of testing occurred in either hospital networks or private practices. There were significantly more males testing positive for HCV, despite the fact that many more females were tested overall. Similarly, the "baby boomer" age range (50-69) had the highest proportion of HCV positive test results, yet those aged 30-49 had the highest HCV testing rates. Conclusions: Negative test results for reportable diseases are rarely utilized, but can be extremely useful in identifying problem areas and focusing testing resources. Because so many people with HCV go undiagnosed, it is especially important that populations requiring additional attention be recognized and screened. / Epidemiology

Chronic Hepatitis B Virus (HBV) infection is a global health problem because of its connection to acute and chronic liver diseases as well as hepatocellular carcinoma (HCC). There is increasing evidence showing that HBV contributes to HCC due to persistently high levels of trans-activating protein---hepatitis B encoded x antigen (HBxAg). Studies have shown that the HBxAg affects and alters the activity of many different transcription factors and plays an essential role in several cytoplasmic signaling transduction pathways, such as Wnt signaling pathways. One of the upregulated genes, designated URG11, was found transactivated by HBxAg. URG11 could stimulate the ß-catenin promoter and hepatocellular growth and survival which suggest that URG11 may be a regulatory element in the ß-catenin signaling pathways. microRNA148a (miR148a) was identified from two miRNA microarrays as one of the up-regulated miRNAs in cells stably expressing HBxAg or over-expressing URG11. Moreover, the expression of miR148a was also elevated in HBV-mediated HCC patient tissue samples. To study the function of miR148a, HepG2 (hepatoblastoma) and Hep3B (hepatoma) cells stably expressing HBxAg or over-expressing URG11 were transduced by recombinant lentiviruses encoding anti-miR148a. anti-miR148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Further, introduction of anti-miR148a increased PTEN protein and mRNA expression, suggesting that PTEN was suppressed by miR148a. In addition, anti-miR148a blocked the stimulation of Akt signaling, resulting in decreased expression of ß-catenin. Thus, miR148a may play a central role in HBxAg/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type. / Biology

Porcine epidemic diarrhea Virus (PEDV) is a virus effecting swine. It is the cause of disease that manifests with symptoms ranging from depression, to severe dehydration and death. Young piglets are particularly susceptible to the virus, which can reach mortality rates of 100%. Presence of the virus on a swine farm can therefore cause severe economic losses. Treatments currently exist for PEDV, but are mostly generated from the virus itself. There has recently been renewed interest in a vaccine that is made from a different source, which might help eliminate some of the side effects of those that currently exist on the market. This project outlines three experiments performed in animals. During the first experiment, a structural protein from the Hepatitis B virus was genetically altered to include important structural portions of PEDV. This new protein is generated in E. coli and purified. After purification, the protein assembles into a virus-like particle (VLP). VLPs are structural proteins of existing viruses that are expressed and assembled to mimic the virus. By doing so, the immune system recognizes the protein as a potential threat, and launches a response in the form of antibodies. Manipulations of the VLPs as describe herein allow the new vaccine to generate antibodies toward other diseases such as PEDV. Although all five of the vaccines used in the first experiment were able to generate appropriate antibodies, only two of them were effective at preventing PEDV from entering susceptible cells (virus neutralization). A second experiment, with three newly designed vaccines was therefore performed. This experiment, like the first, was successful in producing antibodies to several of the included PEDV protein sections, but none were able to neutralize the virus. These results led to a third experiment, during which further design improvements were made to the basic vaccine structure in an attempt to increase the neutralization capabilities of the vaccines. The results from the third experiment indicated that several changes to the vaccine increased the immune response to the structural portions of PEDV, providing a better overall vaccine candidate. This also led to the conclusion that one specific sequence from PEDV has a better ability to neutralize the virus than the other sections. / PHD / Porcine epidemic diarrhea Virus (PEDV) is a virus effecting swine. It is the cause of disease that manifests with symptoms ranging from depression, to severe dehydration and death. Young piglets are particularly susceptible to the virus, which can reach mortality rates of 100%. Presence of the virus on a swine farm can therefore cause severe economic losses. Treatments currently exist for PEDV, but are mostly generated from the virus itself. There has recently been renewed interest in a vaccine that is made from a different source, which might help eliminate some of the side effects of those that currently exist on the market. This project outlines three experiments performed in animals. During the first experiment, a structural protein from the Hepatitis B virus was genetically altered to include important structural portions of PEDV. This new protein is generated in E. coli and purified. After purification, the protein assembles into a virus-like particle (VLP). VLPs are structural proteins of existing viruses that are expressed and assembled to mimic the virus. By doing so, the immune system recognizes the protein as a potential threat, and launches a response in the form of antibodies. Manipulations of the VLPs as describe herein allow the new vaccine to generate antibodies toward other diseases such as PEDV. Although all five of the vaccines used in the first experiment were able to generate appropriate antibodies, only two of them were effective at preventing PEDV from entering susceptible cells (virus neutralization). A second experiment, with three newly designed vaccines was therefore performed. This experiment, like the first, was successful in producing antibodies to several of the included PEDV protein sections, but none were able to neutralize the virus. These results led to a third experiment, during which further design improvements were made to the basic vaccine structure in an attempt to increase the neutralization capabilities of the vaccines. The results from the third experiment indicated that several changes to the vaccine increased the immune response to the structural portions of PEDV, providing a better overall vaccine candidate. This also led to the conclusion that one specific sequence from PEDV has a better ability to neutralize the virus than the other sections.

No description available.