bluuebunny/crossref_metadata_2025_split huggingface dataset:
Colorimetric Assay for Antifungal Susceptibility Testing of
Aspergillus
Species
Authors: Meletiadis Joseph , Mouton Johan W. , Meis Jacques F. G. M. , Bouman Bianca A. , Donnelly J. Peter , Verweij Paul E. ,   EUROFUNG Network
Published: July 2002

ABSTRACT

A colorimetric assay for antifungal susceptibility testing of
Aspergillus
species (
Aspergillus fumigatus
,
Aspergillus flavus
,
Aspergillus terreus
,
Aspergillus nidulans
, and
Aspergillus ustus
) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 μg of XTT/ml with 25 μM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10
2
CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.
Quantification of Human Immunodeficiency Virus Type 1 Proviral DNA by Using TaqMan Technology
Authors: Zhao Yuqi , Yu Min , Miller Johann W. , Chen Mingzhong , Bremer Eric G. , Kabat William , Yogev Ram 
Published: July 2002

ABSTRACT
A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.
Endotoxin Stimulates Liver Macrophages To Release Mediators That Inhibit an Early Step in Hepadnavirus Replication
Authors: Klöcker Uta , Schultz Ursula , Schaller Heinz , Protzer Ulrike 
Published: July 2002

ABSTRACTHepadnaviruses are known to be sensitive to various extracellular mediators. Therefore, bacterial endotoxin, which induces the secretion of proinflammatory mediators in the liver, was studied for its effect on hepadnavirus infection in vitro using the duck hepatitis B virus (DHBV) model. In initial experiments, endotoxin was shown to inhibit DHBV replication in primary duck hepatocyte cultures prepared by standard collagenase perfusion. As a primary endotoxin target, hepatic nonparenchymal cells (NPC) contaminating primary hepatocyte cultures, and among these probably macrophages (Kupffer cells), were identified to secrete polypeptide mediators into the cell culture medium. When added during DHBV infection, these mediators elicited the principal antiviral effect in a dose-dependent fashion. On the molecular level, they inhibited accumulation of viral proteins as well as amplification of the nuclear extrachromosomal DHBV DNA templates. In hepatocytes with an established DHBV infection, DHBV protein and progeny virus production was inhibited while the levels of established nuclear DHBV DNA templates and viral transcripts remained unaffected. Finally, in hepatocytes infected with a replication-deficient recombinant DHBV-green fluorescent protein (GFP) virus, the endotoxin-induced mediators markedly reduced GFP expression from chimeric DHBV-GFP transcripts, indicating that the major effect is at a level of translation of viral RNAs. Taken together, the data obtained demonstrate that antiviral mediators, and among these the cytokines alpha interferon (IFN-α) and IFN-γ, are released from hepatic NPC, most probably liver macrophages, upon endotoxin stimulation; furthermore, these mediators act at a posttranscriptional step of hepadnavirus replication.
Enhancement of Primary and Secondary Cellular Immune Responses against Human Immunodeficiency Virus Type 1 Gag by Using DNA Expression Vectors That Target Gag Antigen to the Secretory Pathway
Authors: Qiu Jian-Tai , Liu Bindong , Tian Chunjuan , Pavlakis George N. , Yu Xiao-Fang 
Published: July 2002

ABSTRACTIn this study, we have investigated the influence of antigen targeting after DNA vaccination upon the induction of cellular immune responses against human immunodeficiency virus type 1 (HIV-1) Gag. In addition to the standard version of HIV-1 Gag, we constructed Gag expression vectors that encode a secreted (Sc-Gag) and a cytoplasmic (Cy-Gag) Gag molecule. Although all three HIV-1 Gag expression vectors induced detectable humoral and cellular immune responses, after intramuscular injection the DNA vector encoding the Sc-Gag generated the highest primary cytotoxic T-lymphocyte (CTL) and T-helper responses. Mice immunized with one of the HIV-1 Gag DNA vectors (but not with the control vector pcDNA3.1) developed a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag, and this response persisted for 125 days. The magnitude of the protection correlated with the levels of Gag-specific ex vivo CTL activity and the number of CD8+T cells producing gamma interferon. The DNA vector encoding the Sc-Gag induced higher levels of protection and greater secondary CTL responses than did the DNA vector encoding Cy-Gag.
The C-Terminal Proline-Rich Tail of Human Immunodeficiency Virus Type 2 Vpx Is Necessary for Nuclear Localization of the Viral Preintegration Complex in Nondividing Cells
Authors: Pancio Heather A. , Vander Heyden Nancy , Ratner Lee 
Published: July 2002

ABSTRACT
Human immunodeficiency virus type 2 (HIV-2), like other lentiviruses, is capable of infecting nondividing T cells and macrophages. The present work shows that in HIV-2-infected cells, Vpx is necessary for efficient nuclear import of the preintegration complex. In agreement with this finding, the subcellular localization of a GFP-Vpx fusion protein was found to be predominantly nuclear. However, deletion of the proline-rich C-terminal 11 residues of Vpx resulted in a shift of the fusion protein to the cytoplasm. Furthermore, the same deletion in the context of the provirus resulted in a decrease in nuclear import of the preintegration complex and attenuated replication in macrophages.
A New Mutant Class, Made by Targeted Mutagenesis, of Phage PRD1 Reveals That Protein P5 Connects the Receptor Binding Protein to the Vertex
Authors: Bamford Jaana K. H. , Bamford Dennis H. 
Published: July 2002

ABSTRACT

Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene
V
revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5
−
particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.
Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1
Authors: Pierson Theodore , Hoffman Trevor L. , Blankson Joel , Finzi Diana , Chadwick Karen , Margolick Joseph B. , Buck Christopher , Siliciano Janet D. , Doms Robert W. , Siliciano Robert F. 
Published: July 2002

ABSTRACTLatently infected resting CD4+T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1envgenes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.
CD40 Ligand-Dependent Activation of Cytotoxic T Lymphocytes by Adeno-Associated Virus Vectors In Vivo: Role of Immature Dendritic Cells
Authors: Zhang Yi , Chirmule Narendra , Gao Guang-ping , Wilson James 
Published: July 2002

ABSTRACTRecombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding β-galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L−/−) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a β-galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZtransduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.
Yeast Three-Hybrid Screening of Rous Sarcoma Virus Mutants with Randomly Mutagenized Minimal Packaging Signals Reveals Regions Important for Gag Interactions
Authors: Lee Eun-Gyung , Linial Maxine L. 
Published: July 2002

ABSTRACT

We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define
cis
-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MΨ) made using PCR amplification after cotransformation with GagΔPR protein into yeast cells. Colonies with low β-galactosidase activity were analyzed to locate mutations in MΨ sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.
Human Cytomegalovirus Latency-Associated Protein pORF94 Is Dispensable for Productive and Latent Infection
Authors: White Kirsten Lofgren , Slobedman Barry , Mocarski Edward S. 
Published: July 2002

ABSTRACT

Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the
ie1/ie2
region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associated
ie1/ie2
region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.
High-Level Resistance to 3′-Azido-3′-Deoxythimidine due to a Deletion in the Reverse Transcriptase Gene of Human Immunodeficiency Virus Type 1
Authors: Imamichi Tomozumi , Sinha Tanima , Imamichi Hiromi , Zhang Yi-Ming , Metcalf Julie A. , Falloon Judith , Lane H. Clifford 
Published: July 2002

ABSTRACT
A variant of human immunodeficiency virus type 1 (HIV-1) possessing a deletion in the reverse transcriptase (RT) gene at codon 67 was identified in a patient who had failed combination antiretroviral therapy. This deletion initially emerged under the selective pressure of combination therapy with 3′-azido-3′-deoxythymidine (AZT) plus 2′,3′-dideoxyinosine. It has persisted for more than 3 years in association with the accumulation of a variety of other well-described drug resistance mutations and an uncharacterized mutation at RT codon 69 (T69G). Phenotypic studies demonstrated that the codon 67 deletion by itself had little effect on AZT sensitivity. However, in the context of the T69G mutation and three other mutations known to be associated with AZT resistance (K70R, T215F, and K219Q), this deletion led to a increase in AZT resistance from 8.5-fold to 445-fold. A further increase in resistance (up to 1,813-fold) was observed when two mutations associated with nonnucleoside RT inhibitor resistance (K103N and L74I) were added to the deletion T69G K70R T215F K219Q construct. Hence, these results establish that a deletion at RT codon 67 may be selected for in the presence of antiretroviral therapy and may lead to high-level resistance to AZT.
Mutational Analysis of the GB Virus B Internal Ribosome Entry Site
Authors: Rijnbrand René , Abell Geoffrey , Lemon Stanley M. 
Published: July 2002

ABSTRACT

GB virus B
(GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5′ nontranslated GBV-B RNA (5′NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5′NTR (M. Honda, et al. RNA 2:955–968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5′ end by structural domain II, a location analogous to the 5′ limit of the IRES in both the HCV and pestivirus 5′NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3′ limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.
Recombinant Rinderpest Vaccines Expressing Membrane-Anchored Proteins as Genetic Markers: Evidence of Exclusion of Marker Protein from the Virus Envelope
Authors: Walsh Edmund P. , Baron Michael D. , Rennie Louise F. , Monaghan Paul , Anderson John , Barrett Thomas 
Published: July 2002

ABSTRACT
Rinderpest virus (RPV) causes a severe disease of cattle resulting in serious economic losses in parts of the developing world. Effective control and elimination of this disease require a genetically marked rinderpest vaccine that allows serological differentiation between animals that have been vaccinated against rinderpest and those which have recovered from natural infection. We have constructed two modified cDNA clones of the vaccine strain RNA genome of the virus, with the coding sequence of either a receptor site mutant form of the influenza virus hemagglutinin (HA) gene or a membrane-anchored form of the green fluorescent protein (GFP) gene (ANC-GFP), inserted as a potential genetic marker. Infectious recombinant virus was rescued in cell culture from both constructs. The RPVINS-HA and RPVANC-GFP viruses were designed to express either the HA or ANC-GFP protein on the surface of virus-infected cells with the aim of stimulating a strong humoral antibody response to the marker protein. In vitro studies showed that the marker proteins were expressed on the surface of virus-infected cells, although to different extents, but neither was incorporated into the envelope of the virus particles. RPVINS-HA- or RPVANC-GFP-vaccinated cattle produced normal levels of humoral anti-RPV antibodies and significant levels of anti-HA or anti-GFP antibodies, respectively. Both viruses were effective in stimulating protective immunity against RPV and antibody responses to the marker protein in all animals when tested in a cattle vaccination trial.
Reduced Susceptibility of Human Immunodeficiency Virus Type 1 (HIV-1) from Patients with Primary HIV Infection to Nonnucleoside Reverse Transcriptase Inhibitors Is Associated with Variation at Novel Amino Acid Sites
Authors: Brown Andrew J. Leigh , Precious Heather M. , Whitcomb Jeannette M. , Wong Joseph K. , Quigg Marlynne , Huang Wei , Daar Eric S. , D'Aquila Richard T. , Keiser Philip H. , Connick Elizabeth , Hellmann Nicholas S. , Petropoulos Christos J. , Richman Douglas D. , Little Susan J. 
Published: July 2002

ABSTRACT
Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.
Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein
Authors: Gardner Jason P. , Frolov Ilya , Perri Silvia , Ji Yaying , MacKichan Mary Lee , zur Megede Jan , Chen Minchao , Belli Barbara A. , Driver David A. , Sherrill Scott , Greer Catherine E. , Otten Gillis R. , Barnett Susan W. , Liu Margaret A. , Dubensky Thomas W. , Polo John M. 
Published: July 2002

ABSTRACTThe ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gagelicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.
The K-bZIP Protein from Kaposi's Sarcoma-Associated Herpesvirus Interacts with p53 and Represses Its Transcriptional Activity
Authors: Park Junsoo , Seo Taegun , Hwang Seungmin , Lee Daeyoup , Gwack Yousang , Choe Joonho 
Published: July 2002

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma. KSHV encodes K-bZIP (open reading frame K8), a protein that belongs to the basic region-leucine zipper (bZIP) family of transcription factors. Here we show that K-bZIP associates with the cellular transcription factor p53 directly in vitro and in vivo. This interaction requires the bZIP domain of K-bZIP and the carboxy-terminal region (amino acids 300 to 393) of p53. We also show that K-bZIP represses the transcriptional activity of p53 which is required for apoptosis of the host cell. These results imply that K-bZIP blocks p53-mediated host cell death through its interaction with p53.