Doctor of Philosophy, Case Western Reserve University, 2025, Pathology

The COVID-19 pandemic claimed the lives of millions of people and affected communities worldwide. SARS-CoV-2, the virus that causes COVID-19, continues to be a global health concern, as does the inevitable threat of new viral outbreaks. We must therefore learn from this virus in the hope of better preparing for future pandemics. We investigated the SARS-CoV-2 accessory protein ORF3a and its roles in modulating host antiviral strategies, namely inflammatory signaling and autophagy regulation. ORF3a activates NF-κB signaling, which induces an inflammatory response in infected cells and can also prime certain cells for inflammasome assembly and subsequent cell death. We found that, unlike the homologous protein SARS-CoV ORF3a, SARS-CoV-2 ORF3a does not depend on its N-terminal TRAF-binding sequence to activate NF-κB. The ORF3a homologs thus affect NF-κB signaling through different mechanisms. Second, SARS-CoV- 2 ORF3a blocks autophagy by binding to the human protein VPS39, a member of the complex that facilitates membrane fusion between autophagic compartments. We discovered that the predicted β-propeller domain of VPS39 is critical to its interaction with ORF3a. Regulating autophagy is important for productive SARS-CoV-2 infection; disrupting the ORF3a:VPS39 interaction could therefore be a future strategy to hinder SARS-CoV-2 propagation.

Doctor of Philosophy, Case Western Reserve University, 0, Molecular Virology

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by joint destruction and systemic inflammation. Specifically, RA patients exhibit dysregulation of both innate and adaptive immunity, with key roles played by autoreactive T cells, B cells, and proinflammatory cytokines. Additionally, the immunosuppressive treatments used for RA can further impair immune function, increasing risks of infection. The COVID-19 pandemic was especially severe for RA patients, who showed higher rates of SARS-CoV-2 infection, hospitalization, and mortality compared to people without immune diseases. The immune response to SARS-CoV-2 involves both innate and adaptive components, with antibodies, memory B cells, and T cells all contributing to viral clearance and durable protection. However, RA patients demonstrated impaired vaccine responses, with lower antibody levels and higher breakthrough infection rates following COVID-19 vaccination. Aging further compounds these immune deficits through immunosenescence - the age-related decline in immune function. This involves reductions in naive T cells, impaired B cell responses, and chronic low-grade inflammation. The intersection of RA, COVID-19, and aging thus creates a multi-faceted issue of immune dysfunction. Vaccination strategies for this vulnerable population must account for these multiple layers of immune impairment. Approaches like higher vaccine doses, additional booster shots, or use of adjuvants may be needed to overcome diminished immune responses seen in elderly RA patients. Overall, a nuanced understanding of how RA, viral infections, vaccines, and aging interact is critical for optimizing protection of this group against COVID-19 and other infections.

Doctor of Philosophy, The Ohio State University, 2024, Veterinary Biosciences

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to substantial damage to all aspects of our society. Vaccination is the most effective strategy to prevent infectious diseases. Since the pandemic, several vaccine platforms, including inactivated, mRNA, viral vector-based, and protein subunit vaccines have been developed. While these vaccines have been highly effective in reducing severe disease and mortality, they still have several limitations, such as the inability to prevent virus infection and transmission, particularly against emerging variants of concern (VoCs), short duration of protection, failure to induce mucosal IgA antibodies and resident memory T cells in the respiratory tract, and high cost of production or distribution. Therefore, there is an urgent need to develop the next generation of intranasal COVID-19 vaccines that induce durable and broadly protective immunity, both in the airways and systemically. The combined MMR (measles/mumps/rubella) vaccine has been available in the United States since 1971 and is one of the safest and most effective human vaccines. The MMR vaccine contains attenuated strains of measles virus (MeV), mumps virus (MuV), and rubella virus and confers lifelong protection against these three viruses. Both MeV and MuV have been used as delivery platforms for experimental vaccines against highly pathogenic viruses. In this study, we have developed a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. MMS vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston, and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2], which are known for their high safety and long-lasting immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Viral pandemics are caused by viruses spilling over from animal reservoirs and subsequently adapting to efficiently infect, replicate, and spread in human hosts. Given that coronavirus and avian influenza virus outbreaks have occurred in recent years in geographic regions in which human deficiencies in the Interferon-Induced Transmembrane Protein 3 (IFITM3) antiviral protein are common, we investigated whether IFITM3 may play a role in interspecies virus infection and adaptation. We found that both IFITM3-deficient mice and human cells could be infected with low doses of avian influenza viruses that failed to infect WT counterparts, identifying a new role for IFITM3 in controlling the minimum infectious viral dose threshold. Additionally, we used a panel of 11 diverse avian influenza viruses and 3 swine viruses, 2 of which are known to have transmitted to humans, to infect human cells with or without IFITM3. Each animal virus showed increased infection of human cells lacking IFITM3 as compared to controls, even upon interferon treatment. Remarkably, influenza viruses passaged through Ifitm3-/- mice exhibited enhanced host adaptation, a result that was distinct from passaging in mice deficient for interferon signaling, which caused virus attenuation. Passaging of a SARS-CoV-2 beta strain and an Omicron BA.4 strain through WT or Ifitm3-/- mice both resulted in elimination of tissue culture adaptations prevalent in the parent stocks of these viruses. Further, virus passaged 20 times through Ifitm3-/- animals gained >1-log replicative advantage and ability to induce weight loss in WT mice while WT-passaged virus did not significantly change in its ability to replicate or induce weight loss. Mouse adaptation of both influenza virus and SARS-CoV-2 was associated with discrete changes in the viral genomes resulting in amino acid substitutions, suggesting that enhanced virus replication in the absence of IFITM3 may facilitate adaptive mutations. Our data demonstra (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Comparative Biomedical Sciences

RNAs are not merely copies of DNA; they are complex, functional macromolecules characterized by a range of post-transcriptional modifications, including 3' polyadenylation, splicing, and chemical alterations. HIV-1 and other RNA viruses utilize every aspect of their densely packed RNA genomes to optimize their life cycles. Despite the potential significance of studying HIV-1 RNA, challenges arise from the inherent fragility of RNA and the technical difficulties associated with reverse transcription (RT), cDNA amplification, and the sequencing of long RNA/DNA molecules. Currently, no FDA-approved drugs specifically target the production, function, or packaging of HIV-1 RNA, primarily due to a limited understanding of RNA biology. To address these challenges, we developed efficient RNA sequencing technologies for comprehensive studies of RNA viruses, which can aid in identifying novel cellular and virus-specific factors and pathways essential for virus survival. Using these optimized sequencing techniques to read full-length RNAs, we uncovered a surprisingly simple epigenetic landscape in HIV-1 RNAs, marked by three major site-specific m6A modifications at the 3' end. Previous studies on m6A's roles in regulating HIV-1 replication have often overlooked site-specific effects, relying instead on system-wide manipulations of cellular m6A effectors and assuming m6A primarily cis-regulates RNA function. To explore the HIV-1-specific mechanisms of m6A modifications, we employed novel Oxford Nanopore Technologies (ONT) direct RNA sequencing (DRS) alongside various molecular biology and bioinformatics approaches to explore the regulatory mechanisms of site-specific m6A modifications on HIV-1 RNA stability, export, packaging efficiency, and decay. Our study revealed novel viral-specific regulatory dynamics, driven by m6As through both direct cis-regulation and HIV-1-specific trans-regulation involving the viral proteins, Rev and Gag. Whole-transcriptome profiling and isoform- (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Comparative Biomedical Sciences

More than 80 million people have been infected with human immunodeficiency virus type-1 (HIV-1), with approximately 38 million people currently living with the virus, according to UNAIDS (2021). Although antiretroviral therapy (ART) is available, the persistence of the virus within the viral reservoir remains a significant obstacle in developing more effective HIV-1 treatments. Recent advancements in technology have allowed studies to reveal that RNA modifications in HIV-1 play key roles in the biological processes, offering deeper insights into the disease. HIV-1 exploits all aspects of RNA, a versatile macromolecule that undergoes various post-transcriptional modifications. Despite recent studies highlighting the importance of chemical modifications on RNA, the evolutionary advantages of these modifications for HIV-1 are still not well understood. Most research has only provided low-resolution, population-averaged data on these modifications, often overlooking site-specific details and intra-RNA variability. In this study, we introduce multiplex reverse-transcription (mRT) and read-level binary-classification (m6Arp) techniques that enable full-length, single-molecule analysis of HIV-1 RNAs using nanopore direct RNA sequencing (DRS). Our analysis uncovered an unexpectedly simple modification landscape for HIV-1, identifying three dominant N6-methyladenosine (m6A) modifications near the 3' end. These m6As play critical roles in maintaining proper RNA splicing and translation and are much more densely present in mRNAs compared to genomic RNAs. HIV-1 produces various RNA subspecies with distinct patterns of m6As, demonstrating that each of the three m6As can independently regulate RNA splicing. The functional redundancy and intra-RNA diversity of these m6As help the virus reduce the impact of mutations that may eliminate a major m6A modification. This single-RNA-level study unveils novel RNA strategies employed by HIV-1 that contribute to the virus's stability and ad (open full item for complete abstract)

Doctor of Philosophy, Miami University, 2024, Chemistry and Biochemistry

Pancreatic cancer remains a devastating disease with exceptionally poor prognoses due to inability to detect the disease when it is still surgically resectable and due to largely ineffective treatment modalities. Here, three engineered adenovirus vectors were developed to specifically target and suppress oncogenic activity of the HMGA1 architectural transcription factor that plays a crucial role in tumorigenesis, chemotherapy resistance and transformation of pancreatic cancer cells into pancreatic cancer stem cells. HMGA1 plays a similarly crucial role in many human cancers, including breast cancer. HMGA1 is an especially insidious oncoprotein because it is intrinsically disordered and therefore cannot be targeted by conventional small molecule drug therapy. Here, alternative adenoviral-mediated therapy approaches were designed and developed to suppress HMGA1 oncogenic activity in cancer cells and tested in MIA PaCa-2, PANC-1 and BxPC-3 human pancreatic cancer cell lines and in the ZR-75 breast cancer cell line. The engineered viruses characterized included: 1) a virus engineered to sequester overexpressed HMGA1 in cancer cells, 2) a virus engineered to express an artificial HMGA1 cis-antisense transcript and 3) a virus engineered to express an HMGA1-targeted shRNA transcript, the latter two viruses designed to suppress translation of HMGA1 mRNA into HMGA1 protein. Cancer cell characteristics after viral infection were evaluated using viability, toxicity, proliferation and wound healing assays and effects on HMGA1 mRNA transcript levels and HMGA1 protein levels were quantitatively evaluated. It was found that the virus designed to sequester HMGA1 proved more effective in suppressing HMGA1 oncogenic activity than the viruses designed to suppress HMGA1 translation.

Master of Science, The Ohio State University, 2024, Animal Sciences

Turkey arthritis reovirus (TARV) causes arthritic lameness affecting mostly market-age turkeys. Since 2011, TARV has caused significant economic losses in the turkey industry due to increased culling, reduced market weights, and decreased carcass quality, necessitating more effective control measures. Autogenous vaccine prevention strategies have been inefficacious partly due to a limited understanding of age-related susceptibility of turkeys to TARV. Moreover, nonpathogenic enteric reovirus are ubiquitously present in the intestinal tract, which might complicate the severity of turkey arthritis reovirus in commercial turkeys. Also, the interplay of enteric reovirus and arthritic reovirus during coinfection and how it alters the progression and severity of disease is not known. To begin addressing these questions, we investigated age-related host and gut microbiota responses to TARV infection in commercial turkeys derived from vaccinated breeder hens. Additional experiments investigated how coinfection of Specific Pathogen Free (SPF) turkeys with enteric reovirus and arthritic reovirus affects the progression and severity of disease. In our first study, poults with maternally derived antibodies were orally challenged with TARV O'Neil strain at 1-, 3-, and 7- weeks of age (WOA) and monitored for cloacal virus shedding, gastrocnemius tendon viral tropism, tendon inflammation, weight gain, and changes in gut microbiota. In the second study, SPF turkey poults were orally challenged with TERV MN1 at 1 day of age and TARV O'Neil at 8 days of age and euthanized at 1- and 4-weeks post-infection with TARV O'Neil. A transient TARV-induced weight gain reduction was evident in poults infected at 1- and 3- WOA during the first 3 weeks post-infection. Age-dependent variations in cloacal viral shedding, virus isolation from tendons, and tendon inflammation severity were also observed. There was significant dissimilarity in ileal and cecal bacterial communities between mock and (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Microbiology

Respiratory pathogens entail many bacteria and viruses that cause upper or lower respiratory tract diseases. The severity and transmissibility of respiratory pathogens vary widely, with some agents able to cause large outbreaks of severe diseases and even pandemics. Two examples of such pathogens that have recently gained global attention are SARS-CoV2, a newly emerging zoonotic coronavirus that caused the COVID-19 pandemic, and Bordetella pertussis, the causative agent of whooping cough, recently designated as an emerging pathogen by the National Institute of Allergy and Infectious Diseases (NIAID). Vaccines have been one of the most important measures to counteract respiratory pathogens. Multiple elements are involved in vaccine design to maximize the benefits and minimize the potential side effects. That includes the use of adjuvants that induce a strong favorable immune response, selecting the right antigens that are likely to be protective and also selecting the route of immunization that would induce immune response at the mucosal entry sites. I addressed all this strategies in my thesis. The adjuvant Bordetella colonization factor A (BcfA) is an outer membrane protein derived from Bordetella bronchiseptica. Previously, in our lab, we combined BcfA and Alum in a subunit vaccine formula against B. pertussis, and the vaccine led to better protection and a TH1/TH17 polarized immune profile. However, the molecular mechanisms underlying this phenotype still need to be elucidated. In the first part, I investigated the mechanism of action of BcfA. Initial pattern recognition receptors (PRR) screening identified TLR2 and TLR4 as potential BcfA receptors. Ex vivo analysis using murine bone marrow-derived dendritic cells (BMDCs) further confirmed that finding as it showed the ability of BcfA to induce the expression of costimulatory molecules and the secretion of innate cytokines, it also indicated a greater dependence of B (open full item for complete abstract)

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2024, Biological Sciences

West Nile virus (WNV) is an arbo-Orthoflavivirus belonging to the Japanese Encephalitis Complex that was first discovered in a febrile woman of the Uganda district in Africa. Following its initial discovery WNV has been detected on every major continent, except Antarctica, officially being detected in the Western hemisphere in the fall of 1999 in the Queens District of New York City. Since entering the western hemisphere 25 years ago, WNV has been reported in over 300 different avian species. In addition to avian cases, WNV has been detected in humans within all 50 states totaling 58,981 cases (both neuroinvasive and non-neuroinvasive) and 2,776 deaths. An RT-PCR protocol was developed and optimized to reproducibly generate cDNA for sequence analysis. Forty cDNA sequences were assembled from RT-PCR products of WNV origin, and the corresponding amino acid sequences predicted. Sequence variations at the nucleotide level were evident as 173 polymorphic sites; when translated these polymorphisms resulted in 18 polymorphic amino acid residues relative to the original New York reference strain. There were 42 total (36 different sites) instances where nucleotides present were ambiguous, suggesting that some of the samples analyzed contained material from more than one virion. Predicted polypeptide sequences indicated high conservation of specific envelop protein regions. These conserved regions were concentrated in envelope domain II, a region recognized to be essential for productive infection of host cells. My results suggest the number of WNV genomes recovered for analysis varied relative to local weather conditions during the 2018 collection season. Together these observations support the contention that environmental conditions have the potential to select variant genotypes of WNV.

Doctor of Philosophy, The Ohio State University, 2024, Microbiology

Microbes are engines of ocean biogeochemical processes. Viruses influence and shape microbial communities via lysis, horizontal gene transfer, and metabolic reprogramming. Viral lysis facilitates the export of carbon from the surface into the deep ocean via aggregates of sinking particles. In fact, they outperform prokaryotes and eukaryotes as the strong predictor for carbon fluxes in the oligotrophic ocean. Viruses also impact the gene flow of their hosts, and the genes transferred from virus-host interactions can be fixed in viral genomes. Viruses are known to carry and express host-derived auxiliary metabolic genes (AMGs) that directly reprogram metabolisms within virus-infected cells, termed virocells. However, viral communities are poorly characterized in the oligotrophic ocean, and their AMG-driven metabolic reprogramming lacks systematic descriptions from the global oceans. The Sargasso Sea is highly stratified and nutrient-depleted each year in the summer months. This seasonal pattern makes the Sargasso Sea one of the ideal model ecosystems to study oligotrophic oceans. In the Sargasso Sea, abundance of viral-like particles has seasonal and depth-associated structuring patterns. Here, to better survey the Sargasso Sea viruses, we apply sequencing approaches to characterize viral communities via metagenomics and uncover their biogeographical and ecological structures locally and globally in the ocean. As described in Chapter 2, comparison with global viral metagenomics revealed that Sargasso Sea viruses were similar across warm oligotrophic oceanic regions but not represented globally. They form discrete populations in the viral and cellular fractions at the viral maximum (80m) and mesopelagic (200m) depths. Inclusion of long-read data captured 1,257 viral genomes in addition to the 1,044 viral genomes derived from short-read assemblies, resulting in the identification of ecologically important and microdiverse viral genomes. Having established lo (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2024, Veterinary Preventive Medicine

Viral hepatitis is primarily caused by five unrelated hepatotropic viruses, hepatitis A, B, C, D, and E. While hepatitis A through C are commonly recognized as causing significant liver disease by general public due to successful public health campaigns, most are not aware of hepatitis E. Despite being unheard of in the public, hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide with an estimated 20 million cases annually. Hepatotropic viruses are notoriously tricky, utilizing differing mechanisms to avoid detection and elimination by the host organism. While hepatitis B and C infections often produce few symptoms in the host while becoming chronic and spreading silently to new hosts, HEV utilizes a different strategy to continue circulating in its hosts. HEV's long incubation period and ability to self-resolve in many infected individuals coupled with animal reservoirs that show little disease upon infection allow HEV to transmit to humans through the food chain. Endemic human strains have similar strategies, circulating at low levels within the populace waiting for conditions associated with socio economic turmoil when sanitary conditions decrease allowing for massive outbreaks through contaminated water. This virological game of hide and seek ensures the continued survivability and transmission of the pathogen. While most otherwise healthy individuals will be able to self-resolve HEV infections, people with underlying comorbidities, immunocompromised individuals, and pregnant women in their third trimester are at much greater risk of succumbing to hepatitis E. There is still much work to be done to unravel the nuances of HEV's deadly hide-and-seek game so that humans may rid themselves of this malady. Recently, the new emerging zoonotic rat HEV has been a hot topic in the field of HEV. The first rodent-associated hepevirus was discovered in 2010 from fecal and liver specimens of rats in Germany. Based on sequence divergence, this (open full item for complete abstract)

Master of Science, The Ohio State University, 2024, Immunology and Microbial Pathogenesis

Influenza B virus circulates seasonally in humans and primarily affects young children, the elderly, and the immunocompromised with 290,000-650,000 influenza B related deaths annually. The cellular protein interferon induced transmembrane protein 3 (IFITM3) is known to restrict several virus infections by altering endosomal membrane properties in a manner that prevents virus-to-cell membrane fusion reactions. IFITM3 is particularly beneficial in limiting the severity of influenza A virus infection in humans and mice, though whether it inhibits influenza B virus infections of cells or pathogenesis in vivo is not well studied. We thus sought to investigate the role of IFITM3 in influenza B virus infections in human cells and using the mouse model. From my work using influenza B virus in vitro with IFITM3 deficient and IFITM3 overexpressing cell models, and in vivo with wild type and IFITM3 knockout mice, we conclude that IFITM3 restricts influenza B virus infections of cells and pathogenesis in vivo.

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Understanding the viral infection and lifecycle of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is important in improving disease outcomes and reducing viral prevalence. In our SFTSV study, we discovered specific cytokine profiles associated with the severity of clinical symptoms. We used single cell RNA sequencing (scRNAseq) on patient blood samples to identify a unique expansion of the B cell population in SFTSV-induced fatal cases which indicated that plasma B cells are a primary reservoir of SFSTV replication. These findings present a potential method of reducing the severity of SFTSV infection, especially in aged patients who are more susceptible to adverse outcomes. In our KSHV study, we developed a novel oral 3D infection model and demonstrated that KSHV can only infect exposed basal epithelial cells in the oral epithelia. We used scRNAseq to show that keratinocyte differentiation and cell death pathways were affected by KSHV infection, suggesting that epithelial differentiation could contribute to KSHV reactivation through changes in epigenetic regulation. In addition, we found a unique population of infected cells with limited early lytic gene expression and a unique gene expression profile, which we termed latent-2 cells. These findings demonstrate that our in vitro 3D epithelial ALI culture model should be a valuable tool to further understand oral KSHV infection for the development of future anti-viral therapeutics to block KSHV transmission.

Master of Science, The Ohio State University, 2024, Animal Sciences

Bovine coronaviruses (BCoVs) are important members of the Betacoronavirus genus, Embecovirus subgenus in Orthocoronavirinae subfamily within the Coronaviridae family. They can cause enteric or/and respiratory diseases in cattle. However, the mechanisms for BCoV tissue tropism and pathogenesis are still unknown and could be due to interactions of viral, host, and environmental factors. There are limited studies to investigate whether co-infection with other bovine pathogens, environmental factors (such as season of the year) and host factors (such as beef vs dairy; age, such as pre- vs post-weaning; and the level of pre-existing BCoV antibodies) contribute to enteric or respiratory tropism. In our study, we isolated recent BCoV strains and compared them with historical strains to study the evolution of BCoVs. Bovine fecal samples were collected from dairy calves, dairy cows, and beef cattle in Georgia by Dr. Palomares Lab at the University of Georgia. Bovine fecal and nasal samples from asymptomatic veal calves from an Ohio farm were provided by Dr. Habing's Lab at The Ohio State University. We detected BCoVs from both nasal (n=10) and fecal samples (n=156) and isolated BCoV strains from enteric samples. The BCoV genomic sequences of five strains BC7, BC8, BC9, BC39, and BC47 were characterized, and they all belong to genogroup II by phylogenetic analyses. Sequence analyses were performed to compare the mutations between the historical and current viruses and between the viruses isolated from respiratory and enteric samples. We found that one pair of samples BC8 and BC18 from the same calf, but different collection sites (fecal and nasal sites), showed two amino acid differences in the spike (S) protein; We predicted the locations of these two amino acid residues in the 3D protein structures. Reverse genetics is a state-of-the-art technology to study viral factors, but until now no infectious clones have been generated for BCoVs, and this has hampered in-dept (open full item for complete abstract)

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus causing hemorrhagic fever with mortality rate ranging up to 30%. However, licensed vaccine or targeted therapy against SFTSV is yet to be developed. In this study, we developed a protein subunit vaccine employing 24-mer self-assembling ferritin (FT) nanoparticles as a platform to present the head region of the SFTSV Gn (GnH). The purified GnH-FT nanoparticles preserved structural integrity and induced robust humoral and cellular immunity against SFTSV Gn. Furthermore, aged ferrets that received immunization with GnH-FT nanoparticles exhibited complete protection from lethal SFTSV challenge and symptoms of body weight loss, viremia, fever, thrombocytopenia, leukopenia, and mortality. We also developed mRNA vaccines encoding GnH and GnH-FT, which potently induce immunity against SFTSV and provides full protection against lethal SFTSV infection in mouse model. Furthermore, we repurposed the GnH-FT nanoparticle to develop therapeutic antibody against SFTSV. We identified five unique antibodies specifically binding SFTSV, including two candidates with sub-nanomolar KD. Our study suggests two vaccine candidates and potential therapeutic antibodies against SFTSV.

PhD, University of Cincinnati, 2023, Medicine: Molecular Genetics, Biochemistry, & Microbiology

The human immunodeficiency virus (HIV) is the viral pathogen underlying the ongoing AIDS pandemic. Current estimates place the number of infected individuals worldwide at nearly 40 million people, and the number of infected is still growing every year. Although modern antiretroviral therapy (ART) regimens are effective at halting the progression of disease, there is still no cure or vaccine available. The only viral antigen present on the surface of particles is the HIV Envelope glycoprotein (Env), and its expression on the surface is tightly regulated. Surprisingly, Env is endocytosed rapidly upon reaching the plasma membrane (PM), requiring recycling pathways in order to return to the PM site of particle assembly. Understanding how Env navigates the endosomal recycling compartment (ERC) could lead to development of novel therapeutics and improved vaccine design. The mechanisms underlying Env trafficking to the site of assembly following endocytosis are poorly defined. The cytoplasmic tail (CT) of Env contains structural determinants required for incorporation in physiologically relevant cells. The CT contains a long unstructured region followed by three amphipathic helices, and mediates interactions with cellular trafficking machinery including clathrin, clathrin adaptor protein, retromer, and Rab11 family interacting protein-1C (FIP1C). We created a series of truncation and point mutants in the CT in order to assess their ability to direct Env trafficking and particle incorporation. We initially utilized a truncated form of FIP1C that collapses the ERC as a marker of Env trafficking to this compartment. Trafficking to the ERC required specific tryptophan-based motifs in the LLP3 region of the CT. Disruption of these motifs eliminated ERC trafficking and rendered Env defective for incorporation into particles. This work was then extended to examine the trafficking of Env to the tubular recycling endosome (TRE). This work identified for the first time a prominent a (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2023, Comparative and Veterinary Medicine

5' methylcytosine (m5C) refers to the addition of a methyl group onto the fifth carbon of the cytosine ring. Among more than 180 types of RNA modifications, m5C is one of the most prevalent modifications and is widely distributed in many RNA species. It plays important roles in RNA metabolism, nuclear export, and translation. The m5C methylation is catalyzed by m5C methyltransferase (MTases) including seven members of the NOL1/NOP2/SUN domain (NSUN) family (NSUN1-7) and DNA methyltransferase-like 2 (DNMT2). Currently, the potential role of these m5C MTases and RNA m5C methylation in innate immunity and virus infection remains poorly understood. To begin to understand the role of m5C MTase in virus infection, we depleted the NSUN2 from A549 cells and examined its impact on virus replication. We found that depletion of NSUN2 significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses including human respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), human metapneumovirus (hMPV), and Sendai virus (SeV), and herpes simplex virus (HSV). Importantly, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN-I) response upon NSUN2 depletion. To understand the mechanism by which NSUN2 modulates IFN-I response, we recapitulated the IFN-I signaling pathway and found that cytosolic RNA sensor RIG-I but not MDA5 is involved in IFN-I activation. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant non-coding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially upregulated, leading to an increased level in unshielded 7SL RNA in cytoplasm, which served as direct ligands for the RIG-I mediated IFN response. In NSUN2 de (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2023, Comparative Biomedical Sciences

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic deltaretrovirus estimated to infect 5-10 million individuals globally. Approximately 5-10% of infected people develop disease after a prolonged clinical latency period of up to several decades. Diseases caused by HTLV-1 include the highly aggressive CD4+ T-cell malignancy known as adult T-cell leukemia/lymphoma (ATL) and the debilitating neurodegenerative disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Unfortunately, limited therapeutic options are available, resulting in dismal prognoses for individuals with these diseases. Research efforts aimed at unraveling HTLV-1 pathobiology are crucial in identifying novel therapeutic targets and devising successful medical interventions. Chapter One reviews our current knowledge of HTLV-1 transmission, life cycle, and pathogenesis. Two viral genes, Tax and Hbz, are individually linked to oncogenic transformation and play an important role in HTLV-1 pathogenesis. Tax is essential for de novo infection and cellular immortalization while Hbz promotes proliferation and survival of infected cells in both its protein and mRNA forms. Hbz is uniquely encoded on the antisense strand of the proviral genome and is the only viral gene constitutively expressed in ATL patients. However, recent research suggests that sporadic and transient Tax expression is still required for survival of the HTLV-1 leukemic cells. Transcription of Tax and Hbz relies on promoter elements within the long terminal repeats (LTRs) at the 5′ and 3′ ends of the integrated genome. Consequently, regulation of HTLV-1 gene expression is a central feature in the viral life cycle and directly contributes to its pathogenic potential. In Chapter Two, we investigated interactions between viral and host proteins to enhance our understanding of HTLV-1 transcriptional regulation. Specifically, we identified a host transcription factor, Y-box binding protein-1 (YBX1), that (open full item for complete abstract)

Doctor of Philosophy, The Ohio State University, 2023, Molecular Genetics

Geminiviruses infect a wide variety of plants and encode very few viral genes. As such, they rely heavily upon host machinery to drive their replication and hijack host pathways to ensure a productive infection. In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but instead is directed to responsive promoters through interactions with host factors, most likely transcriptional activators or repressors. In the main chapter of this dissertation, we describe a plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb repressive complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, CLE mutation in Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest a model where TCP24 binds the CLE early in infection and directly or indirectly recruits PRC2, resulting in CP promoter repressio (open full item for complete abstract)