Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Rift Valley fever virus (RVFV) is an enveloped, negative-sense, ssRNA virus with a tripartite genome that causes morbidity and mortality in both livestock and humans. Although RVFV is mainly circulating in mainland Africa, this arthropod-borne virus is a potential threat to the other parts of the world. No fully licensed vaccines for human or animal use in the U.S., and effective antiviral drugs have not been identified. As virulent RVFV strains are only handled in biosafety level (BSL) 3 or higher level facilities in the U.S., few laboratories have access to RVFV which limits antiviral development. However, it is crucial to develop effective antivirals to protect public and animal health. Animal models that reproduce Rift Valley fever are vital to identifying and developing antiviral compounds. The currently available attenuated RVFV strain, MP12, provides a BSL-2 challenge model virus for preliminary investigations of RVFV prior to using the virulent RVFV strains. All strains of RVFV have a highly conserved genome, indicating that antivirals or vaccines effective against any RVFV strain will most likely be effective for all RVFV strains. Therefore, we hypothesize that the MP12 is a suitable model virus that can be used for identification and evaluation of effective RVF antivirals. The first objective of this project was to establish a mouse model susceptible to MP12 infection. Based on the literature, we selected and screened six different strains of mice to test their susceptibilities to MP12. We found the STAT-1 knockout mice are the most susceptible to MP12 infection based on clinical symptoms, mortality, viremia, virus replication, histopathological, and immunochemical analyses. Importantly, these mice displayed acute-onset hepatitis and delayed-onset encephalitis similar to severe cases of human RVFV infection. Our second objective was to identify potential antiviral drugs in vitro. We developed and employed a cell-based assay using the recombinant MP12 virus expressing Renilla luciferase to screen a library of 727 small compounds purchased from National Institutes of Health. Of the compounds, 23 were identified and further tested for their inhibitory activities on the recombinant MP12 virus expressing green fluorescent protein. Further plaque reduction assays confirmed that two compounds inhibited replication of parental RVFV MP12 strain with limited cytotoxic effects. The 50% inhibitory concentrations using an MP12 multiplicity of infection (MOI) of 2 were 211.4 µM and 139.5 µM, respectively. Our third objective was to evaluate these two candidates, 6-azauridine and mitoxantrone, in vivo using our mouse model. After one-hour post MP12 infection via an intranasal route, treatment was given intranasally twice daily. Mice treated with placebo and 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurological signs, while mice treated with ribavirin (a known antiviral drug) or mitoxantrone showed delayed onset of disease. This result indicates that the mitoxantrone can improve the outcome of RVFV infection in our mouse model. The underlying mechanism of mitoxantrone to inhibit RVFV replication remains to be investigated. Our studies build the foundation for identification and development of antivirals against RVFV in a BSL-2 environment.

Oncolytic Viruses (OVs) are often attenuated to increase their safety profile, however this can lead to reduced efficacy in heterogeneous malignancies and result in resistance to OV therapy. Our group utilizes small molecule enhancers of OV therapy termed viral sensitizers. These small molecules have been shown to enhance the replication and spread of oncolytic rhabdovirus VSVΔ51 in vitro and prolong survival in tumour-bearing mice. In this study, we evaluate the ef-fect of these viral sensitizers on the innate antiviral response in order to identify the mechanism of action responsible for their viral-sensitizing properties. Our previous data suggest that VSe1 and its structural analogues affect the type I IFN antiviral response and have the potential to af-fect cellular redox homeostasis. We hypothesized that VSe1 and its structural analogues potenti-ate VSV∆51 activity by inhibiting the type I IFN response via redox-mediated dysregulation. In this study, we demonstrate that the viral sensitizers inhibit the nuclear translocation and transcrip-tional activity of NFκB, which in turn dampens the expression of antiviral cytokines IFN-, TNFα and IL-6. We also provide evidence supporting the possibility that the NFκB inhibition may be a result of the formation of ROS intermediates by the viral sensitizers, which leads to re-duced nuclear translocation of NFκB subunits, thereby preventing NFκB-mediated cytokine production. Overall, this work contributes to the identification of the mechanism of action of our viral sensitizers and highlights the finding that oncolytic VSV infection can be enhanced through redox-mediated modulation of the innate antiviral response.

Class of 2016 Abstract / Objectives: To compare the efficacy of direct acting antiviral agents for the treatment of hepatitis C virus genotype 1. Our primary null hypothesis is there will be no significant difference in efficacy among the treatment regimens for hepatitis C virus, genotype 1. Methods: This meta-analysis study will use published literature identified from Embase and PubMed for phase II or III clinical trials evaluating direct acting antiviral drug regimens to treat adults with hepatitis C virus (HCV) genotype 1 infection. The primary outcome of interest is SVR at 12 weeks after treatment initiation. Data will be analyzed both descriptively as well as using Bayesian mixed treatment comparison methods. After extracting the outcome data from individual studies, the data will be analyzed using Winbugs version 1.4.3. Moreover, a random effects model and indirect/mix-treatment comparison will be used during the analysis. The random effects model accounts for both between-study and within-study variance, and is exempted from normality assumption, possessing a wider credible interval. All pair-wise odds ratios will be generated and treatment regimens will be ranked based on the likelihood of achieving SVR. Results: Overall, combinations containing sofosbuvir and ledipasvir were significantly better than all other treatments except for simeprevir (OR 0.52, 95% CI 0.28-1.00). On the other hand, daclatasvir containing regimens were non-inferior only to simeprevir (OR 0.69, 95% CI 0.35-1.31) and grazoprevir (OR 0.66, 95% CI 0.41-1.04) while being inferior to other treatments. Sofosbuvir with ledipasvir was ranked highest in terms of obtaining a sustained viral response, followed by ABT-450, grazoprevir, simeprevir, and daclatasvir respectively. In previously treated patients, sofosbuvir with ledipasvir again demonstrated the best efficacy with only grazoprevir and ABT-450 being non-inferior (OR 0.64, 95% CI 0.3368-1.212 and OR 0.73 95% CI 0.29-1.88 respectively). Sofosbuvir with ledipasvir was followed by grazoprevir, ABT-450, simeprevir, and daclatasvir containing regimens respectively. Finally, in treatment naïve patients, simeprevir containing regimens were non-inferior to all other treatment groups, including sofosbuvir regimens (OR 1.24, 95% CI 0.28-9.93). With the exception of simeprevir, sofosbuvir with ledipasvir demonstrated superiority over all treatments. Simeprevir regimens and sofosbuvir with ledipasvir regimens were followed by ABT-450. In treatment naive patients daclatasvir was found to be non-inferior to grazoprevir (OR 1.26, 95% CI 0.75-2.10). Treatment naive patients were the only group we analyzed in which daclatasvir was not the least effective regimen, with grazoprevir claiming the last position. Conclusions: Our results reject our null hypothesis that there will be no difference between different treatment regimens in HCV genotype 1 patients. Generally, the combination of sofosbuvir and ledipasvir appears to be the most effective, while daclatasvir appears to be the least.

Picornavirus proteins VP1 to VP3 are exposed on the surface of the virus particle whereas VP4 is internal and modified at its amino terminus by the addition of myristic acid (Chow et al., 1987; Paul et al., 1987). Myristic acid occupies a position in the core of mature poliovirus particles; it has been suggested that it may be important for particle integrity or in the localization of the capsid protein precursor on the hydrophobic membranes during virion assembly (Chow et al., 1987). To determine the function of the amino-terminal myristylation of VP4 in picornaviruses, and to establish whether competition for the acylation site is a possible approach to antiviral chemotherapy, the effect of fatty acids on virus replication has been examined. Some fatty acids are able to enter picornavirus-infected cells and compete for the myristylation site on VP4. Unexpectedly, it was found that short chain fatty acids also inhibit an early event in the replication of bovine enterovirus (BEV) at concentrations which have no detectable effect on cellular macromolecular synthesis and cloning. These findings indicate that fatty acids inhibit cell-mediated uncoating. Short chain fatty acids inhibit the replication of bovine enterovirus but are almost ineffective against poliovirus type 1, coxsackievirus B5, encephalomyocarditis virus and human rhinovirus lB. Lauric acid binds to bovine enterovirus, thereby stabilizing the virus particle to heat degradation. Fatty acid-bound virions attach to susceptible cells but fail to undergo cell-mediated uncoating. The inhibitory effect is reversible with chloroform and may result from a hydrophobic interaction between the fatty acid and a specific site on the virus particie.

Background: The emergence of viral diseases has been the major threat to public health and social stability. A hundred years ago, 1918 Spanish flu (H1N1) pandemic spread worldwide, and about 3% ~ 5% of the world's population died from the flu-related illnesses. It is known as the deadliest catastrophic pandemics in human history. There have been five Public Health Emergency of International Concern (PHEIC) declarations over the past decade, including the 2014 Ebola outbreak in west Africa, the 2016 Zika outbreak and the ongoing COVID-19 pandemic. There is always a new strain of virus emerging on the horizon. We have urgent need to develop more broad-spectrum antivirals, which work effective against multiple viruses, for thwarting outbreaks in the future. Objective: Based on our previous experience in search of anti-HIV compounds from topical plants, we aimed to discover novel antiviral lead compounds from Justicia plants collected in Hong Kong. Further, structure modification of the natural compounds can lead to optimization of their drug properties for further development as drug candidates. To determine the antiviral targets of the lead compounds will further provide insights to elucidate the mechanism of actions. The present studies are to discover the antiviral lead compounds from Justicia plants, to analyze the structure-activity relationship of the modified structures, to identify the molecular targets of the lead compounds as antiviral agents against the multiple viruses. Methodology: Four common Justicia plants were collected in Hong Kong. The plant extracts and compounds isolated from the plants were explored for their antiviral activities via our established "One-Stone-Two-Birds" antiviral assay. Time-of-addition experiments were performed to determine the target stages of the antiviral compounds on the viral replication. Computational techniques (3D-QSAR and in silico pharmacokinetics evaluation) were employed to elucidate the structure-activity relationship of the compounds and thereby optimize their structures to enhance the antiviral activity. Comprehensive activity-based protein profiling (ABPP) of biotin-linked compounds using SWATH-MS technique was performed to identify the protein target(s) of the lead compounds in an unbiased manner. The role of the molecular target in viral replication was further verified by mRNA knockdown using siRNA. Result: The extracts of Justicia procumbens and Justicia championii showed potent antiviral effects with low cytotoxicity among the collected Justicia plants. By correlating the antiviral activity with their HPLC-UV profiles, arylnaphthalene lignans (ANLs) were determined as the principle active components. Among the isolated compounds from J. procumbens, diphyllin exhibited strong antiviral activities against VSV/HIV, H5N1/HIV and EBOV/HIV pseudoviruses with EC50 values ranging from 30-100nM. In time-of-addition experiments, diphyllin mainly acts on the entry stage of the viral infection. Considering the broad-spectrum antiviral properties and antiviral mechanism together, diphyllin is probably a host-targeting antiviral agent. In a subsequent lead optimization, a reliable and predictive 3D-QSAR was established from 25 synthesized ANLs. Compound 31 was found as the most potent antiviral agent based on the 3D-QSAR model. It showed 70 times more potent antiviral activity than the parent diphyllin, with retained broad-spectrum antiviral properties and improved predicted ADMET properties. In addition, comprehensive ABPP analysis of the biotin-linked diphyllin was employed for the target identification of the ANL compounds. Total 2343 proteins were captured by the ABPP probes. By quantitative analysis, the protein TFAM showed significant affinity to the diphyllin-based ABPP probes. The viral susceptibility of TFAM-deficient cells was shown to be reduced in the subsequent validation. We thus determined TFAM as the potential antiviral drug target of the ANL compounds against a broad spectrum of viruses.

Background: Natural products have been a rich source for the discovery of lead compounds in modern drug discovery. 6,7'-Cyclolignans are a class of secondary metabolites which are widely distributed in more than 20 families. This important class of lignans continue to attract the interest of the pharmaceutical industry owing to their remarkable biological benefits, particularly for their anti-tumor and antiviral properties. Arylnaphthalene lignans (ANLs) belong to 6,7'-cyclolignans which contain a 2,3-dimethyl-1-phenyl-naphthalene core structure. ANLs are widely distributed in plants. Justicia cf. patentiflora was identified as an anti-HIV (human immunodeficiency virus) plant lead through the screening of more than 3,500 plant extracts. Bioassay-directed fractionation of the methanol extract of the stems and barks of this plant has led to the isolation of three ANL glycoside compounds, which displayed potent inhibitory activity against broad HIV clinical strains with EC50 values in the range of 14-37 nM [Zidovudine (AZT): 77-95 nM]. They also showed significant inhibitory effects against drug-resistance HIV strains. Thus, the ANL glycosides have high potential as lead molecules for the development of new therapeutic drugs for HIV/AIDS. Objectives: 1) To establish an efficient route for the total synthesis of ANL compounds and to synthesize a library of ANL compounds through the established total synthetic approach; 2) To evaluate the cytotoxicities and anti-HIV activities of the synthesized ANLs; 3) To elucidate the structure activity relationship (SAR) of ANLs as a basis for the optimization of drug efficacy, improvement of pharmacokinetic properties as well as minimization of the toxicity of ANLs; 4) To synthesize potent anti-HIV ANL molecules with high selectivity. Methods: To achieve these objectives, a synthetic route for ANL was designed and a broad series of ANL derivatives were synthesized via modifications of rings A and B, as well as the functionalities at C-7. The synthesized compounds had been evaluated for their toxicity and antiviral activities. Cytotoxicity was determined using the SRB (Sulforhodamine B) assay, while anti-HIV activity was evaluated by utilizing the "One-Stone-Two-Birds" protocol. Results: We have accomplished the total synthesis of the key intermediate diphyllin with more than 20 g. Our modification of ANL derivatives focused on substitutions, additions and different configurations of the C-7 position, ring A and ring B. Specifically, the different structural components of the ANLs were systematically modified, resulting in the formation of six groups of compounds. A total of 72 ANL compounds with various functional groups were synthesized. Their structures have been confirmed by the MS and NMR spectral data. All the synthesized ANL compounds were purified to have purity ≥ 95%. The SAR of ANL compounds was revealed based on the analysis of the antiviral and cytotoxicity data of these synthetic analogues. After structural modification, all the modified derivatives based on rings A and B (groups 1 and 2) showed activity reduction in terms of both cytotoxic and anti-HIV activities. However, the modification of C-7 yielded divergent results, which included the groups of 3-6. Most compounds in groups 3-5 displayed comparable inhibitory effect with diphyllin (5). Group 6 represents the largest number of analogues among the six groups. In this group, the stereochemical properties and functionalization of the hydroxy groups on the sugar units are essential for their activities. Among these series of derivatives, compound 17b showed significant high potency of anti-HIV activity with an EC50 value of 2.6 nM and SI of 815. Conclusion: Using the synthesized diphyllin as the key intermediate, a compound library of 72 ANL derivatives was obtained. These compounds have been evaluated for their cytotoxicity and anti-HIV activity. Our bioactivity data revealed that the functionalization of the C-7 hydroxy group could significantly reduce the cytotoxicity and increase anti-HIV activity, while the modification on rings A and B would rather result in the reduction of both cytotoxicity and anti-HIV activity. Subsequently, novel diphyllin glycosides containing various sugar moieties were further synthesized. Several of these ANL analogues showed high anti-HIV activity with EC50 values in the nM range and low cytotoxicity (selective indices > 500). Future Perspective: This study clearly suggests ANLs as anti-HIV lead compounds and they have high potential to be developed as therapeutic drugs for the treatment of HIV. To further confirm the antiviral potential of ANLs, live HIV strains including some drug resistant strains should be further investigated. Although our data have shown that the ANL compounds are targeting the viral post-entry stages, their antiviral molecular targets are still unknown. However, since our SAR information has clearly revealed that the substitution of rings A and B are not involve in the antiviral activity for enhancing ANL compounds, the carbon positions in these rings may be explored to link a biotin unit, which can be used as a viable approach to pull down the antiviral target proteins of ANLs. Once the target proteins are identified, molecular docking is then made possible for a rational synthetic design to fine tune the chemical structures of ANLs in order to improve their antiviral properties such as high antiviral activity, low toxicity and enhanced water solubility. A further step to advance ANLs as anti-HIV drugs is the investigation of their drug properties in in vivo studies including the assessment of their anti-HIV efficacy in the rhesus model as well as obtaining their pharmacokinetic and safety parameters. These studies will help to provide more evidence about the anti-viral properties of ANLs

Background: The emergence of viral diseases has been the major threat to public health and social stability. A hundred years ago, 1918 Spanish flu (H1N1) pandemic spread worldwide, and about 3% ~ 5% of the world's population died from the flu-related illnesses. It is known as the deadliest catastrophic pandemics in human history. There have been five Public Health Emergency of International Concern (PHEIC) declarations over the past decade, including the 2014 Ebola outbreak in west Africa, the 2016 Zika outbreak and the ongoing COVID-19 pandemic. There is always a new strain of virus emerging on the horizon. We have urgent need to develop more broad-spectrum antivirals, which work effective against multiple viruses, for thwarting outbreaks in the future. Objective: Based on our previous experience in search of anti-HIV compounds from topical plants, we aimed to discover novel antiviral lead compounds from Justicia plants collected in Hong Kong. Further, structure modification of the natural compounds can lead to optimization of their drug properties for further development as drug candidates. To determine the antiviral targets of the lead compounds will further provide insights to elucidate the mechanism of actions. The present studies are to discover the antiviral lead compounds from Justicia plants, to analyze the structure-activity relationship of the modified structures, to identify the molecular targets of the lead compounds as antiviral agents against the multiple viruses. Methodology: Four common Justicia plants were collected in Hong Kong. The plant extracts and compounds isolated from the plants were explored for their antiviral activities via our established "One-Stone-Two-Birds" antiviral assay. Time-of-addition experiments were performed to determine the target stages of the antiviral compounds on the viral replication. Computational techniques (3D-QSAR and in silico pharmacokinetics evaluation) were employed to elucidate the structure-activity relationship of the compounds and thereby optimize their structures to enhance the antiviral activity. Comprehensive activity-based protein profiling (ABPP) of biotin-linked compounds using SWATH-MS technique was performed to identify the protein target(s) of the lead compounds in an unbiased manner. The role of the molecular target in viral replication was further verified by mRNA knockdown using siRNA. Result: The extracts of Justicia procumbens and Justicia championii showed potent antiviral effects with low cytotoxicity among the collected Justicia plants. By correlating the antiviral activity with their HPLC-UV profiles, arylnaphthalene lignans (ANLs) were determined as the principle active components. Among the isolated compounds from J. procumbens, diphyllin exhibited strong antiviral activities against VSV/HIV, H5N1/HIV and EBOV/HIV pseudoviruses with EC50 values ranging from 30-100nM. In time-of-addition experiments, diphyllin mainly acts on the entry stage of the viral infection. Considering the broad-spectrum antiviral properties and antiviral mechanism together, diphyllin is probably a host-targeting antiviral agent. In a subsequent lead optimization, a reliable and predictive 3D-QSAR was established from 25 synthesized ANLs. Compound 31 was found as the most potent antiviral agent based on the 3D-QSAR model. It showed 70 times more potent antiviral activity than the parent diphyllin, with retained broad-spectrum antiviral properties and improved predicted ADMET properties. In addition, comprehensive ABPP analysis of the biotin-linked diphyllin was employed for the target identification of the ANL compounds. Total 2343 proteins were captured by the ABPP probes. By quantitative analysis, the protein TFAM showed significant affinity to the diphyllin-based ABPP probes. The viral susceptibility of TFAM-deficient cells was shown to be reduced in the subsequent validation. We thus determined TFAM as the potential antiviral drug target of the ANL compounds against a broad spectrum of viruses.

Discovered in 1969 in California, enterovirus 71 (EV-71) is a serious cause of disease in young children. It is one of the major causative agents of hand, food, and mouth disease (HFMD), and can produce neurological complications, such as meningitis, encephalitis, and an acute flaccid paralysis. For serious cases, the fatality rate can be up to 26%, almost exclusively in young children. While the virus was initially discovered in the United States, it was soon detected in the Eastern hemisphere, causing outbreaks in Europe and Asia. The largest outbreak occurred in Taiwan in 2008, with approximately 490,000 cases and 128 fatalities. However, despite the seriousness of EV-71, there are currently no approved antiviral treatments. Physicians rely on supportive care and the off-label use of a purified antibody mixture, intravenous immunoglobulin, for treatment. Part of the difficulty in developing antivirals for EV-71 is a lack of drug testing in animal models. Animal testing is a crucial step in drug development, determining which compounds will progress to clinical trials in humans. However, viruses that cause disease in humans do not necessarily cause disease or the same type of disease in animals. As such, viruses often need to be adapted before they can cause disease in their animal hosts. Adaption isn’t always successful and can result in a virus that produces disease that is unlike that seen in humans. Furthermore, some animal models can produce disease only under a strict set of conditions, such as newborn mice. Sometimes these animal model conditions may be impractical for testing potential treatments. At the Institute for Antiviral Research (IAR), we developed an animal model for EV-71 in four-week-old AG129 mice. AG129 mice lack the alpha, beta, and gamma interferon receptors, making them immunocompromised. Being immunocompromised, these mice are more susceptible to infection, including infection from human viruses. In our model, EV-71 infection produces neurological signs, including a rear-limb paralysis (similar to the paralysis seen children with EV-71). The virus is also lethal in these animals, which provides an observable and consistent baseline for evaluating potential drugs. We assessed twenty-four potential treatments in our EV-71 model. Two compounds, STF434 and STF1019, provided 30% and 87% protection against mortality. Intravenous immunoglobulin was also examined and found to be about 50% protective against mortality, depending on the dose and time of administration. Intravenous immunoglobulin also reduced inflammatory modulators (cytokines) in the brain and spinal cord. We consider this to be highly relevant, given that inflammation is a serious component of EV-71 infection.

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