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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Effect of Foot-and-Mouth Disease Virus 2B Viroporin on Expression and Extraction of Mammalian Cell Culture Produced Foot-and-Mouth Disease Virus-like Particles

To improve the production of foot-and-mouth disease (FMD) molecular vaccines, we sought to understand the effects of the FMD virus (FMDV) 2B viroporin in an experimental, plasmid-based, virus-like particle (VLP) vaccine. Inclusion of the FMDV viroporin 2B into the human Adenovirus 5 vectored FMD vaccine enhanced transgene expression despite independent 2B expression negatively affecting cell viability. Evaluating both wildtype 2B and mutants with disrupted viroporin activity, we confirmed that viroporin activity is detrimental to overall transgene expression when expressed independently. However, the incorporation of 2B into an FMD molecular vaccine construct containing a wildtype FMDV 3C protease, a viral encoded protease responsible for processing structural proteins, resulted in enhancement of transgene expression, validating previous observations. This benefit to transgene expression was negated when using the FMDV 3CL127P mutant, which has reduced processing of host cellular proteins, a reversion resulting from 2B viroporin activity. Inclusion of 2B into VLP production constructs also adversely impacted antigen extraction, a possible side effect of 2B-dependent rearrangement of cellular membranes. These results demonstrate that inclusion of 2B enhanced transgene expression when a wildtype 3C protease is present but was detrimental to transgene expression with the 3CL127P mutant. This has implications for future molecular FMD vaccine constructs, which may utilize mutant FMDV 3C proteases.

2B↗
DOE OSTI2021

Modeling hepatitis C virus kinetics during liver transplantation highlights the role of the liver in virus clearance

While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from five liver transplant patients throughout the anhepatic (absence of liver) phase and for 4 hr post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n = 3) or declined (n = 2) with t 1/2 ~1 hr. Immediately post-reperfusion, virus declined in a biphasic manner in four patients consisting of a rapid decline (t 1/2 = 5 min) followed by a slower decline (t 1/2 = 67 min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t 1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy

Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

NASA Discipline Regulatory Physiology↗
NASA NTRS2002

Epstein-Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART

Epstein-Barr virus (EBV) associated non-Hodgkin lymphoma is recognized as a complication of human immunodeficiency virus (HIV) infection. Little is known regarding the influence of highly active antiretroviral therapy (HAART) on the biology of EBV in this population. To characterize the EBV- and HIV-specific serological responses together with EBV DNA levels in a cohort of HIV-infected adults treated with HAART, a study was conducted to compare EBV and HIV serologies and EBV DNA copy number (DNAemia) over a 12-month period after the commencement of HAART. All patients were seropositive for EBV at baseline. Approximately 50% of patients had detectable EBV DNA at baseline, and 27/30 had detectable EBV DNA at some point over the follow-up period of 1 year. Changes in EBV DNA copy number over time for any individual were unpredictable. Significant increases in the levels of Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr early antigen (EA) antibodies were demonstrated in the 17 patients who had a good response to HAART. Of 29 patients with paired samples tested, four-fold or greater increases in titers were detected for EA in 12/29 (41%), for EBNA in 7/29 (24%), for VCA-IgG in 4/29 (14%); four-fold decreases in titers were detected in 2/29 (7%) for EA and 12/29 (41%) for EBNA. A significant decline in the titer of anti-HIV antibodies was also demonstrated. It was concluded that patients with advanced HIV infection who respond to HAART have an increase in their EBV specific antibodies and a decrease in their HIV-specific antibodies. For the cohort overall, there was a transient increase in EBV DNA levels that had declined by 12 months. Copyright 2002 Wiley-Liss, Inc.

NASA Discipline Regulatory Physiology↗
DOE OSTI2017

Nontargeted virus sequence discovery pipeline and virus clustering for metagenomic data

The analysis of large microbiome data sets holds great promise for the delineation of the biological and metabolic functioning of living organisms and their role in the environment. In the midst of this genomic puzzle, viruses, especially those that infect microbial communities, represent a major reservoir of genetic diversity with great impact on biogeochemical cycles and organismal health. Overcoming the limitations associated with virus detection directly from microbiomes can provide key insights into how ecosystem dynamics are modulated. Here, we present a computational protocol for accurate detection and grouping of viral sequences from microbiome samples. Our approach relies on an expanded and curated set of viral protein families used as bait to identify viral sequences directly from metagenomic assemblies. This protocol describes how to use the viral protein families catalog (~7 h) and recommended filters for the detection of viral contigs in metagenomic samples (~6 h), and it describes the specific parameters for a nucleotide-sequence-identity-based method of organizing the viral sequences into quasi-species taxonomic-level groups (~10 min).

Paez-Espino, David↗
DOE OSTI2011

The LANL hemorrhagic fever virus database, a new platform for analyzing biothreat viruses

Hemorrhagic fever viruses (HFVs) are a diverse set of over 80 viral species, found in 10 different genera comprising five different families: arena-, bunya-, flavi-, filo- and togaviridae. All these viruses are highly variable and evolve rapidly, making them elusive targets for the immune system and for vaccine and drug design. About 55 000 HFV sequences exist in the public domain today. A central website that provides annotated sequences and analysis tools will be helpful to HFV researchers worldwide. The HFV sequence database collects and stores sequence data and provides a user-friendly search interface and a large number of sequence analysis tools, following the model of the highly regarded and widely used Los Alamos HIV database [Kuiken, C., B. Korber, and R.W. Shafer, HIV sequence databases. AIDS Rev, 2003. 5: p. 52–61]. The database uses an algorithm that aligns each sequence to a species-wide reference sequence. The NCBI RefSeq database [Sayers et al. (2011) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res., 39, D38–D51.] is used for this; if a reference sequence is not available, a Blast search finds the best candidate. Using this method, sequences in each genus can be retrieved pre-aligned. The HFV website can be accessed via http://hfv.lanl.gov.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Evolution and Antigenic Advancement of N2 Neuraminidase of Swine Influenza A Viruses Circulating in the United States following Two Separate Introductions from Human Seasonal Viruses

Two separate introductions of human seasonal N2 neuraminidase genes were sustained in U.S. swine since 1998 (N2-98) and 2002 (N2-02). Herein, we characterized the antigenic evolution of the N2 of swine influenza A virus (IAV) across 2 decades following each introduction. The N2-98 and N2-02 expanded in genetic diversity, with two statistically supported monophyletic clades within each lineage. To assess antigenic drift in swine N2 following the human-to-swine spillover events, we generated a panel of swine N2 antisera against representative N2 and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assay and antigenic cartography. The antigenic distance between swine and human N2 was smallest between human N2 circulating at the time of each introduction and the archetypal swine N2. However, sustained circulation and evolution in swine of the two N2 lineages resulted in significant antigenic drift, and the N2-98 and N2-02 swine N2 lineages were antigenically distinct. Although intralineage antigenic diversity was observed, the magnitude of antigenic drift did not consistently correlate with the observed genetic differences. These data represent the first quantification of the antigenic diversity of neuraminidase of IAV in swine and demonstrated significant antigenic drift from contemporary human seasonal strains as well as antigenic variation among N2 detected in swine. These data suggest that antigenic mismatch may occur between circulating swine IAV and vaccine strains. Consequently, consideration of the diversity of N2 in swine IAV for vaccine selection may likely result in more effective control and aid public health initiatives for pandemic preparedness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

CDR3α drives selection of the immunodominant Epstein Barr virus (EBV) BRLF1-specific CD8 T cell receptor repertoire in primary infection

The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1 109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCRαβ sequencing of tetramer sorted CD8 T cells ex vivo , we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCRα chain. For the first time, we identify a CDR3α (complementarity determining region 3 α) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCRβ chains within the same donor (median 4; range: 1–9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3α motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3α. TCR cloning and site-directed mutagenesis of the CDR3α lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3α and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCRα, as well as TCRβ, in understanding the CD8 T cell receptor repertoire.

Microbiology↗
DOE OSTI2023

Reverse-zoonoses of 2009 H1N1 pandemic influenza A viruses and evolution in United States swine results in viruses with zoonotic potential

The 2009 H1N1 pandemic (pdm09) lineage of influenza A virus (IAV) crosses interspecies barriers with frequent human-to-swine spillovers each year. These spillovers reassort and drift within swine populations, leading to genetically and antigenically novel IAV that represent a zoonotic threat. We quantified interspecies transmission of the pdm09 lineage, persistence in swine, and identified how evolution in swine impacted zoonotic risk. Human and swine pdm09 case counts between 2010 and 2020 were correlated and human pdm09 burden and circulation directly impacted the detection of pdm09 in pigs. However, there was a relative absence of pdm09 circulation in humans during the 2020–21 season that was not reflected in swine. During the 2020–21 season, most swine pdm09 detections originated from human-to-swine spillovers from the 2018–19 and 2019–20 seasons that persisted in swine. We identified contemporary swine pdm09 representatives of each persistent spillover and quantified cross-reactivity between human seasonal H1 vaccine strains and the swine strains using a panel of monovalent ferret antisera in hemagglutination inhibition (HI) assays. The swine pdm09s had variable antigenic reactivity to vaccine antisera, but each swine pdm09 clade exhibited significant reduction in cross-reactivity to one or more of the human seasonal vaccine strains. Further supporting zoonotic risk, we showed phylogenetic evidence for 17 swine-to-human transmission events of pdm09 from 2010 to 2021, 11 of which were not previously classified as variants, with each of the zoonotic cases associated with persistent circulation of pdm09 in pigs. These data demonstrate that reverse-zoonoses and evolution of pdm09 in swine results in viruses that are capable of zoonotic transmission and represent a potential pandemic threat.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Vesicular stomatitis virus N protein‐specific single‐domain antibody fragments inhibit replication

Abstract The transcription and replication machinery of negative‐stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single‐domain antibody fragments ( VHH s) that protect cells from a lytic infection with vesicular stomatitis virus ( VSV ) by targeting the viral nucleoprotein N. We define the binding sites for two such VHH s, 1004 and 1307, by X‐ray crystallography to better understand their inhibitory properties. We found that VHH 1307 competes with the polymerase cofactor P for binding and thus inhibits replication and mRNA transcription, while binding of VHH 1004 likely only affects genome replication. The functional relevance of these epitopes is confirmed by the isolation of escape mutants able to replicate in the presence of the inhibitory VHH s. The escape mutations allow identification of the binding site of a third VHH that presumably competes with P for binding at another site than 1307. Collectively, these binding sites uncover different features on the N protein surface that may be suitable for antiviral intervention.

Hanke, Leo↗
NASA NTRS2003

The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

NASA Discipline Plant Biology↗
DOE OSTI2013

P1-Substituted Symmetry-Based Human Immunodeficiency Virus Protease Inhibitors with Potent Antiviral Activity against Drug-Resistant Viruses

Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.

08 HYDROGEN↗
DOE OSTI2015

The vaccinia virus E6 protein influences virion protein localization during virus assembly

Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

High-speed fixed-target serial virus crystallography

Here, we report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.

59 BASIC BIOLOGICAL SCIENCES↗