Matching Items (21)

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Display of Domain III from Dengue 2 Envelope Protein on HBsAg Virus-like Particles Vectored by Measles Virus

Description

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E)

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.

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Created

Date Created
  • 2014-05

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Coronavirus Envelope Protein Transmembrane Domain: Impact of Positive Charges on Virus-like Particle Assembly

Description

Coronaviruses are a significant group of viruses that cause enteric and respiratory infections in a variety of animals, including humans. Outbreaks of Severe Acute Respiratory Syndrome (SARS) and Middle Eastern

Coronaviruses are a significant group of viruses that cause enteric and respiratory infections in a variety of animals, including humans. Outbreaks of Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS) in the past 15 years has increased research into coronaviruses to gain an understanding of their structure and function so one day therapies and vaccines may be produced. These viruses have four main structural proteins: the spike, nucleocapsid, envelope, and membrane proteins. The envelope (E) protein is an integral membrane protein in the viral envelope that acts as a viroporin for transport of cations and plays an important role in pathogenesis and viral assembly. E contains a hydrophobic transmembrane domain with polar residues that is conserved across coronavirus species and may be significant to its function. This experiment looks at the possible role of one polar residue in assembly, the 15th residue glutamine, in the Mouse Hepatitis Virus (MHV) E protein. The glutamine 15 residue was mutated into positively charged residues lysine or arginine. Plasmids with these mutations were co-expressed with the membrane protein (M) gene to produce virus-like particles (VLPs). VLPs are produced when E and M are co-expressed together and model assembly of the coronavirus envelope, but they are not infectious as they do not contain the viral genome. Observing their production with the mutated E protein gives insight into the role the glutamine residue plays in assembly. The experiment showed that a changing glutamine 15 to positive charges does not appear to significantly affect the assembly of the VLPs, indicating that this specific residue may not have a large impact on viral assembly.

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Agent

Created

Date Created
  • 2017-05

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Stress Granule Formation in Poliovirus Infected Cells

Description

Stress granules are cytoplasmic foci that form in response to various types of cellular stress, including viral infection. They contain mRNA, translation initiation factors, the small ribosomal subunit, RNA binding

Stress granules are cytoplasmic foci that form in response to various types of cellular stress, including viral infection. They contain mRNA, translation initiation factors, the small ribosomal subunit, RNA binding proteins, and other unique components depending on the type of stress the cell is under. Stress granules are thought to store these components until the stress as passed at which time the mRNA resumes translation. They also have an active role in the cell's antiviral response and are required for efficient induction of the interferon pathway. There are many viruses that induce or interfere with stress granules, including poliovirus. Poliovirus is a positive sense RNA virus that is part of the Picornaviridae family. Stress granules in poliovirus infected cells differ from stress granules in cells undergoing other types of stress because they contain the RNA binding protein Sam68, their formation is dependent on RNA export by the Crm1 pathway, and they are induced by poliovirus cleavage of eIF4G and PABP. It was found previously that Sam68 is found in the stress granules of poliovirus infected HeLa cells but not in oxidative stress of heat shock induced stress granules. My research shows that this finding is true in other cell lines and thus represents a biologically significant finding. The Crm1 pathway exports snRNAs and some mRNAs, rRNAs, and proteins. To determine which of these classes of RNA is necessary for stress granule formation in poliovirus infected cells but not in cells undergoing other types of stress, plasmids with modified PHAX protein were used to isolate the snRNA export pathway. More work needs to be done to determine the impact of snRNA export on stress granule formation. This research could eventually help us better understand the cell's anti-viral response and have implications for how we treat viral infections.

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Agent

Created

Date Created
  • 2016-12

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Genetic Trends in the Vertical Transmission of the Clone-13 Variant of LCMV

Description

Among wild rodent populations, vertical transmission is believed to constitute the primary route of infection for Lymphocytic Choriomeningitis Virus (LCMV), a non-lytic arenavirus with both acute and chronic forms. When

Among wild rodent populations, vertical transmission is believed to constitute the primary route of infection for Lymphocytic Choriomeningitis Virus (LCMV), a non-lytic arenavirus with both acute and chronic forms. When carrier mice infected at birth with the acute Armstrong strain reproduce, they generate congenital carrier offspring containing a quasispecies of LCMV that includes Armstrong as well as its chronic Clone-13 variant. This study examined the genetic trends in the vertical transmission of LCMV from mothers infected perinatally with Clone-13. Viral isolates obtained from the serum of congenital carrier offspring were partially sequenced to reveal residue 260 in the glycoprotein-encoding region of their S segment, the site of a major amino acid change differentiating the chronic and acute strains. It was found that the phenylalanine-to-leucine mutation associated with Clone-13 was present in 100% of the isolates, strongly indicating that the offspring of Clone-13 carriers contain exclusively the chronic variant. This research has broad implications for the epidemiology of the virus, and, given the predominance of Armstrong in the wild, suggests that there must be a biological cost associated with Clone-13 infection in non-carriers.

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Agent

Created

Date Created
  • 2015-05

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Surveillance of Influenza Virus Spread on the Arizona State University Campus

Description

The 2017-2018 Influenza season was marked by the death of 80,000 Americans: the highest flu-related death toll in a decade. Further, the yearly economic toll to the US healthcare system

The 2017-2018 Influenza season was marked by the death of 80,000 Americans: the highest flu-related death toll in a decade. Further, the yearly economic toll to the US healthcare system and society is on the order of tens of billions of dollars. It is vital that we gain a better understanding of the dynamics of influenza transmission in order to prevent its spread. Viral DNA sequences examined using bioinformatics methods offer a rich framework with which to monitor the evolution and spread of influenza for public health surveillance. To better understand the influenza epidemic during the severe 2017-2018 season, we established a passive surveillance system at Arizona State University’s Tempe Campus Health Services beginning in January 2018. From this system, nasopharyngeal samples screening positive for influenza were collected. Using these samples, molecular DNA sequences will be generated using a combined multiplex RT-PCR and NGS approach. Phylogenetic analysis will be used to infer the severity and temporal course of the 2017-2018 influenza outbreak on campus as well as the 2018-2019 flu season. Through this surveillance system, we will gain knowledge of the dynamics of influenza spread in a university setting and will use this information to inform public health strategies.

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Agent

Created

Date Created
  • 2019-05

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Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source

Description

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency.

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Created

Date Created
  • 2017-06-27

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Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

Description

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1),

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

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Created

Date Created
  • 2014-08-20

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Merging single-shot XFEL diffraction data from inorganic nanoparticles: a new approach to size and orientation determination

Description

X-ray free-electron lasers (XFELs) provide new opportunities for structure determination of biomolecules, viruses and nanomaterials. With unprecedented peak brilliance and ultra-short pulse duration, XFELs can tolerate higher X-ray doses by

X-ray free-electron lasers (XFELs) provide new opportunities for structure determination of biomolecules, viruses and nanomaterials. With unprecedented peak brilliance and ultra-short pulse duration, XFELs can tolerate higher X-ray doses by exploiting the femtosecond-scale exposure time, and can thus go beyond the resolution limits achieved with conventional X-ray diffraction imaging techniques. Using XFELs, it is possible to collect scattering information from single particles at high resolution, however particle heterogeneity and unknown orientations complicate data merging in three-dimensional space. Using the Linac Coherent Light Source (LCLS), synthetic inorganic nanocrystals with a core–shell architecture were used as a model system for proof-of-principle coherent diffractive single-particle imaging experiments. To deal with the heterogeneity of the core–shell particles, new computational methods have been developed to extract the particle size and orientation from the scattering data to assist data merging. The size distribution agrees with that obtained by electron microscopy and the merged data support a model with a core–shell architecture.

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Agent

Created

Date Created
  • 2017-08-27

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Serial femtosecond X-ray diffraction of enveloped virus microcrystals

Description

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

Contributors

Created

Date Created
  • 2015-08-20

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Diffraction Data of Core-shell Nanoparticles from an X-ray Free Electron Laser

Description

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory,

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

Contributors

Created

Date Created
  • 2017-04-11