Oncolytic viruses (OV) are a promising alternative cancer therapy due to their specificity and lack of debilitating side effects, such as those which typically accompany conventional therapeutics such as chemotherapy and radiation. Bovine herpesvirus type 1 (BHV-1) is an alphaherpesvirus with the ability to infect and kill multiple human tumor cell types. In comparison to other species-specific viruses, for which deficiencies in type I interferon signalling pathways dictates cellular sensitivity to infection, mutations in KRAS were found to correlate with high levels of BHV-1 replication. Interestingly, BHV-1 is able to induce cellular cytotoxicity in the absence of a productive infection. In contrast to current breast cancer (BC) treatments, which are largely based on receptor expression status, BHV-1 is able to infect and kill BC cells and breast cancer initiating cells (BCICs) from luminal and basal subtypes. Furthermore, BHV-1-infected BC cells are significantly diminished in their capacity to form tumors in vivo, suggesting that BHV-1 reduces the tumor forming capacity of BCICs. Combination therapy involving OVs has been used to exploit differences in the mechanism of tumor cell death elicited by individual treatments. Treatment with epigenetic modifiers such as 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, has been shown to increase the antitumor activity of OVs. Our data indicates that 5-Aza strongly synergises with BHV-1, increasing virus replication and cytotoxicity in vitro. In vivo, BHV-1 monotherapy did not significantly impact tumor growth or survival of CR bearing subcutaneous breast tumors; however, combination therapy with 5-Aza significantly decreased the number of secondary lesions compared to BHV-1 monotherapy. Overall, the data presented in this dissertation indicate that BHV-1 is a promising broad spectrum OV with a unique mechanism of tumor cell targeting, and the ability to infect and kill tumor cells independent of a productive infection. / Thesis / Doctor of Philosophy (Medical Science)

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Herpes simplex virus (HSV) is a clinically important virus, whose life cycle alternates between productive replication and latency. Infected cell protein 0 (ICP0) is generally believed to play a key role in determining the outcome of HSV infections. Synthesis of ICP0 promotes the productive replication of HSV, whereas absence of ICP0 renders HSV prone to establish latent infections. In the first part of the dissertation, I attempt to address the question how is ICP0 gene regulated. To tackle this question, we constructed recombinant HSV that encodes GFP-tagged ICP0 so that the regulation of ICP0 gene can be visualized in real time. Using this reagent, we found that ICP0 gene was subject to potent repression immediately following infection. Surprisingly, HSV's major transcriptional regulator, ICP4, was necessary and sufficient to repress ICP0 gene, and did so in an ICP4-binding-site dependent manner. Synthesis of ICP0 alleviated the ICP4-dependent repression of ICP0 gene. ICP4 co-immunoprecipitated with FLAG-tagged ICP0, thus, a physical interaction between ICP0 and ICP4 likely explains how ICP0 antagonizes ICP4's capacity to silence the ICP0 gene. Therefore, our findings suggest that ICP0 gene is differentially regulated by virus-encoded repressor ICP4 and virus-encoded antirepressor ICP0. In the second part of the dissertation, I attempt to address the question what function does ICP0 assume. Since the discovery of ICP0, the nuclear function of ICP0 has been the focal point of studies, whereas the cytoplasmic function of ICP0 is unknown. While pursuing our first study, we unexpectedly found that GFP-tagged ICP0 was predominantly localized to the cytoplasm during infections. Taking advantage of live-cell imaging, we found that ICP0 translocated from nucleus to cytoplasm during early phase of HSV infections, where it bundled and dispersed microtubules. Synthesis of ICP0 was proved to be necessary and sufficient to dismantle microtubules in HSV-infected and transfected cells. Therefore, our findings suggest ICP0 might play a previously unrecognized role in the cytoplasm through dismantling microtubule networks of the host cells. Furthermore, our study represents the first report showing a virus-encoded E3 ligase disrupts host cell microtubule networks, thus suggests a general function of many other viral E3 ligases.

Papova viruses are a group of non-enveloped icosahedral viruses which contain a double-stranded circular DNA genome in the supercoiled configuration. There are two subgroups, i.e., the papilloma and the polyoma viruses. The papilloma viruses are generally larger than the polyoma-viruses, having a genome of approximately 5 x 106 daltons compared with 3,3 x 106 daltons, and virions of approximately 55nm diameter as opposed to 41nm. The papilloma viruses generally produce benign epithelial proliferations in the host e.g., the human wart, and attempts to propagate these viruses in cells in culture have been unsuccessful. On the other hand, polyoma viruses can usually be propagated in tissue culture and do not appear to be associated with any widespread pathology in their natural hosts. Although there is no convincing evidence of polyoma viruses causing malignancies in their natural host, nonpermissive cells of other species may be transformed and these viruses therefore have oncogenic potential in particular laboratory animals. Polyoma . viruses infect eukaryotic cells, and investigation thereof should allow further elucidation of eukaryotic gene expression and regulation. Members of the polyoma group which have been extensively studied include polyoma virus itself, which infects mice, simian virus 40, (SV40),which infects rhesus monkey cells, and RKV which infects rabbits. Interest in this polyoma group of viruses has increased ever since 1965 when a new papovavirus strain, JC, was isolated from brain glial cells of a patient with progressive multifocal leukoencephalopathy (PML) and was thus the first polyomavirus infection of humans to be discovered. (ZuRhein and Chou, 1965). In 1971, an immunologically distinct polyomavirus, BK, was isolated from the urine of an immunocompromised recipient of a renal allograft (Gardner et al., 1971). Interest in these two viruses in particular has been compounded by their potential oncogenicity in humans, (see section 1.8).

Airborne diseases transmitted through tiny respiratory droplets such as the Coronavirus disease can not only rapidly infect others but also worsen the symptoms in already affected individuals. People with COVID-19 are more likely to develop severe acute respiratory syndrome or SARS through aspiration pneumonia, which refers to when some virus-laden droplets are inhaled into the airway and lungs. This project aims to study droplet behavior in the upper airway in order to investigate methods to reduce the risk of infected droplets entering the upper airway in a patient. The project involves the design and development of a cough emulator that can simulate a human cough accurately, build a physical model of the upper airway using a material similar in texture to the human windpipe, and measure and track the generated particles as they transverse through the upper airway and exit the mouth. The criteria needed to be met to design, manufacture, and evaluate a cough emulator reproducing a human-like cough include the volume, pressure, and flow rate of a cough. To evaluate the validity and accuracy of the device, the number, size, and spread of cough droplets are compared to that of a real cough. The upper airway is fabricated using Elastic 50A resin due to its flexible and durable properties, and texture similarity to the tissue of the human trachea. In addition, particles are tracked in the upper airway using a Charged Couple Device (CCD) Camera.

The COVID-19 outbreak highlighted the important role that diagnostic tests play in the healthcare system. To reduce the impact of infectious disease outbreaks, the development of rapid and cost-effective point-of-care-tests (POCTs) is crucial. With the dissemination of the Monkeypox (Mpox) virus, it became a necessity to produce POCTS that are inexpensive and easy to use. This work explored the construction of two colorimetric assays that aim to detect Mpox genetic signatures. One is based on the split-peroxidase-like deoxyribozyme probes (sPDz), while the other utilizes a cascade system of split RNA-cleaving deoxyribozyme (sDz) and peroxidase-like deoxyribozyme (PDz). Both rely on catalytic probes as well as a G-Quadruplex (G4) structure to facilitate the production of a color change in the presence of the genetic signatures of Mpox. The sPDz probes were initially tested with synthetic genomic fragments of Mpox and other Orthopoxviruses for selectivity purposes. The sPDz probes were then further optimized. The optimal sPDz probes and the sDz/PDz cascade system were tested with an amplified genome fragment of Mpox. The genome fragments were generated by using both symmetric and asymmetric polymerase chain reaction (PCR). With further optimization to increase the signal-to-background ratio, these probes may show promise as an assay that may have the potential to be incorporated to develop POCTs.

Respiratory syncytial virus (RSV) is a pathogen associated with lower respiratory tract infection, and is a common cause of infant hospitalization worldwide. Despite efforts to create safe and cost-effective RSV therapeutics, there remains no vaccine, and antiviral drugs have been developed with limited success. Among the 11 proteins coded by the negative-sense single-stranded RNA genome of RSV, the phosphoprotein (P) and nucleoprotein (N) aid in the formation of an RNA-dependent RNA polymerase (RdRp) complex, which is essential for RSV virulence. The specificities of the N-P binding interaction have been researched extensively, which has provided researchers with a novel target for an RSV therapeutic. In this study, a recombinant peptide mimetic (P220-241) containing the final 21 C-terminal amino acids of RSV P fused to Maltose-Binding Protein (MBP), and a cell-penetrating peptide (CPP), was purified for the purpose of targeting this interaction. In addition to successfully entering cells, the peptide was shown to inhibit both RSV subtype A and subtype B infection in vitro, with a percent inhibition (PI) of infection as high as 95% at 20 μM. Additionally, P220-241 did not inhibit infection of parainfluenza virus type 2 (PIV-2), indicating this inhibition was not an artifact of the peptide acting as a pathogen-associated molecular pattern (PAMP). A series of three different assays demonstrated that P220-241 does not appear to have any cytotoxic effects in vitro. Finally, using both glutathione S-transferase (GST) pull-downs and in vitro immunoprecipitations, we demonstrated that P220-241 is able to bind the N protein, while also preventing binding of full-length P protein. Taken together, this study provides the framework for a novel method of targeting RSV protein-protein interactions using chimeric cell-penetrating peptide mimetics. / Thesis / Master of Science (MSc)

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Eighty Four rabbits were studied over a period of 2 years for the effects of eye and head X-irradiation on their latent herres simplex ocular infection. Animals were infected intraocularly with herpes simplex virus and allowed to recover from the resultant keratitis. After the latency of the virus was established through daily culture of the eyes of the rabbits, the animals were irradiated. Irradiation was followed by periods of daily culture of both eyes. Eye irradiation of 200, 400, 1200, and 2890 roentgens produce no significant reactivation of the virus in the irradiated eye. On the other hand, head irradiation of 3000 roentgens produced recurrence of the virus in the eyes in a high percentage of the treated rabbits. This effect was reproduced repeatedly. Further experiments seemed to suggest that the virus was latent in the brain and could be reactivated in situ by X-irradiation of the brain. The infectious viral particles appeared to move preferentially to the site of initial infection, This effect may have importance in the fields of clinical medicine, radiation therapy and radiology, and in space travel safety parameters. Further research is planned. / 2031-01-01

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