Virology Commons^™

Design Of A Tetracycline Operon Inducible System For The Control Of Vaccinia Virus Replication: Implications For Vaccine Development, Caitlin J. Hagen

Master's Theses

The use of vaccinia virus (VACV) as a vaccine resulted in the eradication of smallpox in 1979. Characteristics that contribute to the effectiveness of VACV as a vaccine and viral vector include its ability to elicit strong, long-lived humoral and cell-mediated immune responses as a live-replicating virus and to accept large inserts of DNA into its genome. However, adverse events associated with its use as the smallpox vaccine have constrained it from being more widely utilized in vaccines and therapies. We propose to improve the safety of VACV as a live-replicating vector by using elements of the tet operon to …

Functional Genomics Reveals An Essential And Specific Role For Stat1 In Protection Of The Central Nervous System Following Herpes Simplex Virus Corneal Infection, Tracy J. Pasieka, Cristian Cilloniz, Victoria S. Carter, Pamela Rosato, Michael G. Katze, David A. Leib

Dartmouth Scholarship

Innate immune deficiencies result in a spectrum of severe clinical outcomes following infection. In particular, there is a strong association between loss of the signal transducer and activator of transcription (Stat) pathway, breach of the blood-brain barrier (BBB), and virus-induced neuropathology. The gene signatures that characterize resistance, disease, and mortality in the virus-infected nervous system have not been defined. Herpes simplex virus type 1 (HSV-1) is commonly associated with encephalitis in humans, and humans and mice lacking Stat1 display increased susceptibility to HSV central nervous system (CNS) infections. In this study, two HSV-1 strains were used, KOS (wild type [WT]), …

In Vitro Selection Of Aptamers Against Avian Influenza Virus H5n1, Jingjing Zhao

Graduate Theses and Dissertations

Over $10 billion losses in the poultry industry were caused by avian influenza (AI) so far. Rapid and specific detection of avian influenza virus is urgently needed with the concerns over the outbreaks of highly pathogenic H5N1 influenza virus and cases of animal and human infection. Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule with good affinity. They show better thermal stability than antibodies. The goal of this research was to select DNA-aptamers as the specific recognition element of AI H5N1virus to be used in detection assays specific for field application. In this study, Systematic …

Host-Virus Interactions Of Infectious Laryngotracheitis Virus Infection In Cultured Cells, Jeong Yoon Lee

Graduate Theses and Dissertations

Infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) causes upper respiratory diseases in mainly chickens and exhibits 90-100% of high morbidity and up to 70% of mortality, resulting in huge economic losses in the poultry industry worldwide.

To study host-ILTV interactions, the changes in genome-wide gene expressions in response to wild-type and vaccine ILTV infections in primary chicken embryo lung cells were investigated using microarray analysis. Results provide crucial insights into host cell pathogenic and immunogenic responses against wild-type and vaccine ILTV infections. Using microarray method and Ingenuity Pathway Analysis (IPA) bioinformatics tool, 273 and 306 differentially expressed genes were identified …

Molecular Basis For Drug Resistance In Hiv-1 Protease, Akbar Ali, Rajintha M. Bandaranayake, Yufeng Cai, Nancy M. King, Madhavi Kolli, Seema Mittal, Jennifer E. Foulkes-Murzycki, Madhavi N. L. Nalam, Ellen A. Nalivaika, Aysegul Ozen, Moses Prabu-Jeyabalan, Kelly Thayer, Celia A. Schiffer

Celia A. Schiffer

HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All …

Dynamics Of Preferential Substrate Recognition In Hiv-1 Protease: Redefining The Substrate Envelope, Aysegul Ozen, Turkan Haliloglu, Celia Schiffer

Celia A. Schiffer

Human immunodeficiency virus type 1 (HIV-1) protease (PR) permits viral maturation by processing the gag and gag-pro-pol polyproteins. HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy but the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates consideration of drug resistance in novel drug design. Drug-resistant HIV-1 PR variants no longer inhibited efficiently, continue to hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we termed the substrate envelope. Earlier, we showed that resistance mutations arise where PIs protrude beyond the substrate …

Evaluating The Substrate-Envelope Hypothesis: Structural Analysis Of Novel Hiv-1 Protease Inhibitors Designed To Be Robust Against Drug Resistance, Madhavi Nalam, Akbar Ali, Michael Altman, G. S. Kiran Kumar Reddy, Sripriya Chellappan, Visvaldas Kairys, Aysegul Ozen, Hong Cao, Michael Gilson, Bruce Tidor, Tariq Rana, Celia Schiffer

Celia A. Schiffer

Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type …

Promise Of Advances In Simulation Methods For Protein Crystallography: Implicit Solvent Models, Time-Averaging Refinement, And Quantum Mechanical Modeling, Celia Schiffer, Jan Hermans

Celia A. Schiffer

No abstract provided.

Competition Between Ski And Creb-Binding Protein For Binding To Smad Proteins In Transforming Growth Factor-Beta Signaling, Weijun Chen, Suvana Lam, Hema Srinath, Celia Schiffer, William Royer, Kai Lin

Celia A. Schiffer

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, …

Mutation Patterns And Structural Correlates In Human Immunodeficiency Virus Type 1 Protease Following Different Protease Inhibitor Treatments, Thomas Wu, Celia Schiffer, Matthew Gonzales, Jonathan Taylor, Rami Kantor, Sunwen Chou, Dennis Israelski, Andrew Zolopa, W. Jeffrey Fessel, Robert Shafer

Celia A. Schiffer

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. …

Curling Of Flap Tips In Hiv-1 Protease As A Mechanism For Substrate Entry And Tolerance Of Drug Resistance, Walter Scott, Celia Schiffer

Celia A. Schiffer

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) protease is an essential viral protein that is a major drug target in the fight against Acquired Immune Deficiency Syndrome (AIDS). Access to the active site of this homodimeric enzyme is gained when two large flaps, one from each monomer, open. The flap movements are therefore central to the function of the enzyme, yet determining how these flaps move at an atomic level has not been experimentally possible.

RESULTS: In the present study, we observe the flaps of HIV-1 protease completely opening during a 10 ns solvated molecular dynamics simulation starting from …

Resilience To Resistance Of Hiv-1 Protease Inhibitors: Profile Of Darunavir, Eric Lefebvre, Celia A. Schiffer

Celia A. Schiffer

The current effectiveness of HAART in the management of HIV infection is compromised by the emergence of extensively cross-resistant strains of HIV-1, requiring a significant need for new therapeutic agents. Due to its crucial role in viral maturation and therefore HIV-1 replication and infectivity, the HIV-1 protease continues to be a major development target for antiretroviral therapy. However, new protease inhibitors must have higher thresholds to the development of resistance and cross-resistance. Research has demonstrated that the binding characteristics between a protease inhibitor and the active site of the HIV-1 protease are key factors in the development of resistance. More …

Structural Analysis Of Human Immunodeficiency Virus Type 1 Crf01_Ae Protease In Complex With The Substrate P1-P6., Rajintha Bandaranayake, Moses Prabu-Jeyabalan, Junko Kakizawa, Wataru Sugiura, Celia Schiffer

Celia A. Schiffer

The effect of amino acid variability between human immunodeficiency virus type 1 (HIV-1) clades on structure and the emergence of resistance mutations in HIV-1 protease has become an area of significant interest in recent years. We determined the first crystal structure of the HIV-1 CRF01_AE protease in complex with the p1-p6 substrate to a resolution of 2.8 A. Hydrogen bonding between the flap hinge and the protease core regions shows significant structural rearrangements in CRF01_AE protease compared to the clade B protease structure.

Species D Adenoviruses As Oncolytics Against B-Cell Cancers, Christopher Y. Chen, Julien S. Senac, Eric A. Weaver, Shannon M. May, Diane F. Jelinek, Philip Greipp, Thomas Witzig, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Purpose: Oncolytic viruses are self-amplifying anticancer agents that make use of the natural ability of viruses to kill cells. Adenovirus serotype 5 (Ad5) has been extensively tested against solid cancers, but less so against B-cell cancers because these cells do not generally express the coxsackie and adenoviral receptor (CAR). To determine whether other adenoviruses might have better potency, we "mined" the adenovirus virome of 55 serotypes for viruses that could kill B-cell cancers.

Experimental Design: Fifteen adenoviruses selected to represent Ad species B, C, D, E, and F were tested in vitro against cell lines and primary patient B-cell cancers …

Molecular Characterization Of The Host Defense Activity Of The Barrier To Autointegration Factor Against Vaccinia Virus, Nouhou Ibrahim, April Wicklund, Matthew S. Wiebe

Nebraska Center for Virology: Faculty Publications

The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts …

Using Vesicular Stomatitis Virus Glycoprotein To Pseudotype Bacmam Viruses For Enhancement Of Transduction Efficiency, Lydia Sanchez

PCOM Biomedical Studies Student Scholarship

Baculovirus has been a common tool for protein production since the mid 1980s. The virus has an exclusive tropism only capable of infecting the larval stages of insects. Originally, baculovirus recombinant gene expression used Spodoptera frugiperda (Sf9) insect cells as the host cells for protein production. However, in 1995, it was discovered that baculoviruses could be modified to transduce mammalian cells with the insertion of a mammalian promoter and gene of interest. Due to its ease of generation and low biology safety hazards, this technology, known as BacMam (Baculovirus Mammalian) is popular in the research field for protein production and …

Critical Stages In Viral Replication - Entry, Gene Regulation And Egress, Matthew S. Miller

Electronic Thesis and Dissertation Repository

Despite the amazing biological diversity exhibited by viruses, their very existence relies upon their ability to overcome a set of common barriers. The nature of these barriers reflects the nature of viruses themselves. During their extracellular phase, viruses are metabolically inert obligate parasites. Upon encountering a host cell, productive infection necessitates that the virus successfully enter the cell, regulate the expression of its genes, and after assembling new progeny particles, egress such that the cycle of infection can continue. These three basic processes are not only attractive candidates for therapeutic intervention, but also reveal much about virus biology in the …

The Fecal Viral Flora Of Wild Rodents, Tung G. Phan, Beatrix Kapusinszky, Chunlin Wang, Robert K. Rose, Howard L. Lipton, Eric L. Delwart

Biological Sciences Faculty Publications

The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat) collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially …

Three-Dimensional Structure And Function Of The Paramecium Bursaria Chlorella Virus Capsid, Xinzheng Zhang, Ye Xiang, David Dunigan, Thomas Klose, Paul R. Chipman, James L. Van Etten, Michael G. Rossmann

Nebraska Center for Virology: Faculty Publications

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the …

Group Size And Nest Spacing Affect Buggy Creek Virus (Togaviridae: Alphavirus) Infection In Nestling House Sparrows, Valerie A. Brown, Charles R. Brown

Papers in Ornithology

The transmission of parasites and pathogens among vertebrates often depends on host population size, host species diversity, and the extent of crowding among potential hosts, but little is known about how these variables apply to most vector-borne pathogens such as the arboviruses (arthropod-borne viruses). Buggy Creek virus (BCRV; Togaviridae: Alphavirus) is an RNA arbovirus transmitted by the swallow bug (Oeciacus vicarius) to the cliff swallow (Petrochelidon pyrrhonota) and the introduced house sparrow (Passer domesticus) that has recently invaded swallow nesting colonies. The virus has little impact on cliff swallows, but house sparrows are seriously …

The Role Of Human Ubc9 During The Human Immunodeficiency Virus Replication Cycle, Christopher R. Bohl

School of Biological Sciences: Dissertations, Theses, and Student Research

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus and the causative agent of the acquired immune deficiency syndrome (AIDS) pandemic. The retrovirus replication cycle is divided into early infectious events, which involve the infection and integration of the viral DNA into target cell chromosomes; and late events, which involve the expression of viral genes and assembly of infectious virions. To complete the replication cycle, HIV-1 utilizes various cellular pathways.

We identified the Ubc9 E2 SUMO conjugating enzyme as a HIV-1 Gag interaction partner. When this interaction was disrupted in HIV-1 producer cells by Ubc9 siRNA, the virus that was …

Mechanisms Of Adenovirus-Mediated Autophagy, Erin White

Dissertations & Theses (Open Access)

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered …

Upregulation Of Reactive Oxygen Species During The Retrovirus Life Cycle And Their Roles In A Mutant Of Moloney Murine Leukemia Virus, Ts1-Mediated Neurodegeneration, Soo Jin Kim

Dissertations & Theses (Open Access)

Viral invasion of the central nervous system (CNS) and development of neurological symptoms is a characteristic of many retroviruses. The mechanism by which retrovirus infection causes neurological dysfunction has yet to be fully elucidated. Given the complexity of the retrovirus-mediated neuropathogenesis, studies using small animal models are extremely valuable. Our laboratory has used a mutant moloney murine leukemia retrovirus, ts1-mediated neurodegneration. We hypothesize that astrocytes play an important role in ts1-induced neurodegeneration since they are retroviral reservoirs and supporting cells for neurons. It has been shown that ts1 is able to infect astrocytes in vivo and in …

Mechanisms Of Adenovirus-Mediated Autophagy, Erin White

Dissertations & Theses (Open Access)

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered …

Identification Of Host Proteins Required For Vesicular Stomatitis Virus Infection, Debasis Panda

School of Veterinary and Biomedical Sciences: Dissertations, Theses, and Student Research

Viruses usurp host cell pathways for different stages of their infection. Understanding virus-host interaction will be invaluable to elucidate molecular mechanisms of virus infection and to identify drug targets. In order to identify such critical cellular genes in vesicular stomatitis virus (VSV, a model non-segmented negative strand RNA virus) infection, we developed a stable cell line constitutively expressing replication proteins of VSV. Attempts to establish a cell line replicating a sub-genomic replicon was not successful because of induction of interferon response by replication of viral genomic analog. Subsequently, we used siRNA technology and conducted a genome-wide siRNA screen in HeLa …

Regulation Of Bovine Herpesvirus 1 (Bhv-1) Productive Infection By Cellular Transcription Factors, Aspen M. Workman

School of Biological Sciences: Dissertations, Theses, and Student Research

Bovine herpesvirus 1 (BHV-1) is a significant viral pathogen of cattle responsible for a variety of disease conditions, including: conjunctivitis, pneumonia, genital disorders, abortions, and shipping fever, a serious upper respiratory tract infection. Following acute infection in mucosal epithelium, BHV-1 establishes a lifelong latent infection in sensory ganglionic neurons. During latency, transcription is restricted to the latency related (LR) gene. Elevated corticosteroid levels due to stress and/or immune suppression can initiate reactivation from latency, resulting in virus shedding and spread to susceptible cattle. Additionally, administration of dexamethasone (Dex), a synthetic corticosteroid, to calves latently infected with BHV-1 reproducibly leads to …

Effect Of The Infection With Porcine Reproductive And Respiratory Syndrome Virus On The Regulation Of Cytokines - Tumor Necrosis Factor-Alpha And Interleukin-10, Sakthivel Subramaniam

School of Veterinary and Biomedical Sciences: Dissertations, Theses, and Student Research

Porcine reproductive and respiratory syndrome virus (PRRSV) causes late-term abortion in sows and pneumonia in growing piglets. PRRSV evades the host immune response by several mechanisms, including the modulation of cytokine secretions in infected pigs, which is the subject of this dissertation. Particularly, PRRSV reduces the secretion of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) but increases the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). The latter effect, however, is PRRSV strain-specific. In this dissertation, we have examined mechanisms by which PRRSV regulates TNF-α and IL-10 expressions. The pathogenic strain FL12, derived from a PRRSV infectious clone, consistently suppressed TNF-α …

Combating Bioterrorism: Purification And Structural Analysis Of The E3 Proteins Of The Venezuelan Equine Encephalitis Virus And The Chikungunya Virus, Tyshia Gwin

The Journal of Purdue Undergraduate Research

Outbreaks of the Venezuelan Equine Encephalitis Virus (VEEV) and the Chikungunya Virus (CHIKV) continue to emerge in Central and South America, Africa, and Asia, but there are currently no vaccines or anti-virals for these viruses. Given their ease of transmission, debilitating symptoms, and genetic alterability, VEEV and CHIKV have great potential for development into biological weapons. Therefore, there is an urgent need to determine possible methods of treatment or prevention. One possibility of prevention lies in determining the structure and biological function of the E3 protein that plays a key role in the infectivity of the viruses. In this research, …

Mexican Axolotls ( Ambystoma Mexicanum ) Appear To Be Resistant To Ambystoma Tigrinum Virus (Atv), Crystal Paulson, Robert Visalli, Mark Jordan

Mark A. Jordan

No abstract provided.

Comparison Of Adenoviruses As Oncolytics And Cancer Vaccines In An Immunocompetent B Cell Lymphoma Model, Eric A. Weaver, Christopher Y. Chen, Shannon M. May, Mary E. Barry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

We have screened human adenoviruses (Ads) for oncolytic activity against a variety of mouse and hamster cell lines and have found a number that are susceptible to a variety of Ad serotypes. A20 lymphoma is derived from BALB/c mice and is susceptible to infection and killing by a variety of human Ads. A20 is also a suitable cancer vaccine model, because these cells express a unique immunoglobulin variable region that can be targeted by vaccination. To compare Ads as cancer vaccines versus Ads as oncolytics, A20 tumors were initiated in immunocompetent BALB/c mice. Mice immunized with first-generation Ad5 expressing the …