bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sputum PCR-Single-Strand Conformational Polymorphism Test for Same-Day Detection of Pyrazinamide Resistance in Tuberculosis Patients
Authors: Sheen Patricia , Méndez Melissa , Gilman Robert H. , Peña Lizeth , Caviedes Luz , Zimic Mirko J. , Zhang Ying , Moore David A. J. , Evans Carlton A. 
Published: June 2009

ABSTRACT

Pyrazinamide is a first-line drug for treating tuberculosis, but pyrazinamide resistance testing is usually too slow to guide initial therapy, so some patients receive inappropriate therapy. We therefore aimed to optimize and evaluate a rapid molecular test for tuberculosis drug resistance to pyrazinamide. Tuberculosis PCR-single-strand conformational polymorphism (PCR-SSCP) was optimized to test for mutations causing pyrazinamide resistance directly from sputum samples and
Mycobacterium tuberculosis
isolates. The reliability of PCR-SSCP tests for sputum samples (
n
= 65) and
Mycobacterium tuberculosis
isolates (
n
= 185) from 147 patients was compared with four tests for pyrazinamide resistance: Bactec-460 automated culture, the Wayne biochemical test, DNA sequencing for
pncA
mutations, and traditional microbiological broth culture. PCR-SSCP provided interpretable results for 96% (46/48) of microscopy-positive sputum samples, 76% (13/17) of microscopy-negative sputum samples, and 100% of
Mycobacterium tuberculosis
isolates. There was 100% agreement between PCR-SSCP results from sputum samples and
Mycobacterium tuberculosis
isolates and 100% concordance between 50 blinded PCR-SSCP rereadings by three observers. PCR-SSCP agreement with the four other tests for pyrazinamide resistance varied from 89 to 97%. This was similar to how frequently the four other tests for pyrazinamide resistance agreed with each other: 90 to 94% for Bactec-460, 90 to 95% for Wayne, 92 to 95% for sequencing, and 91 to 95% for broth culture. PCR-SSCP took less than 24 hours and cost approximately $3 to $6, in contrast with the other assays, which took 3 to 14 weeks and cost $7 to $47. In conclusion, PCR-SSCP is a relatively reliable, rapid, and inexpensive test for pyrazinamide resistance that indicates which patients should receive pyrazinamide from the start of therapy, potentially preventing months of inappropriate treatment.
Detection and Quantification of Hepatitis C Virus (HCV) by MultiCode-RTx Real-Time PCR Targeting the HCV 3′ Untranslated Region
Authors: Mulligan Elizabeth K. , Germer J. J. , Arens Max Q. , D'Amore Krista L. , Di Bisceglie Adrian , Ledeboer Nathan A. , Moser Michael J. , Newman Andrew C. , O'Guin Andy K. , Olivo Paul D. , Podzorski Diane S. , Vaughan Kimberly A. , Yao Joseph D. , Elagin Slava A. , Johnson Scott C. 
Published: June 2009

ABSTRACT
A prototype, real-time reverse-transcription PCR assay, based on MultiCode-RTx technology, quantifying hepatitis C virus (HCV) RNA by targeting the HCV 3′ untranslated region demonstrated linearity over 7 logs, with a good correlation between the quantitative results of this assay and the results of two commercially available comparator assays for 466 clinical specimens comprising all six HCV genotypes.
In Vitro Effect of Ultrasound on Bacteria and Suggested Protocol for Sonication and Diagnosis of Prosthetic Infections
Authors: Monsen Tor , Lövgren Elisabeth , Widerström Micael , Wallinder Lars 
Published: June 2009

ABSTRACT

Sonication of implants has been shown to be a promising method for diagnosis of prosthetic infections due to its improved sensitivity, simplicity, and low cost. The aim of the present study was to evaluate the effects of ultrasound performed under different conditions regarding temperature, duration, and composition of sonication tubes on bacterial species often associated with prosthetic infections. We found that ultrasound had an inhibitory effect on bacteria, of which gram-negative bacteria, in particular
Escherichia coli
, were almost eradicated after 5 min of sonication at 35°C. Gram-positive bacteria were found to be resistant to the effect of ultrasound. Four factors were important for the inhibitory effect of sonication: the type of microorganism, the temperature of the sonication buffer, the duration of exposure to ultrasound (minutes), and the material and composition of the sonication tube in which sonication is performed. On the basis of the results from the present study, we propose a protocol for sonication and recovery of bacteria associated with biofilm on infected implants prior to conventional culture. From the present protocol, we recommend sonication for 7 min at 22°C at the maximum effect which permits survival of gram-negative bacteria.
First Report of Human Infection with
Salmonella enterica
Serovar Apapa Resulting from Exposure to a Pet Lizard
Authors: Cooke Fiona J. , De Pinna Elizabeth , Maguire Clare , Guha Simantee , Pickard Derek J. , Farrington Mark , Threlfall E. John 
Published: June 2009

ABSTRACT

We present the first documented human case of
Salmonella enterica
serovar Apapa infection, isolated concurrently from a hospital inpatient and a pet lizard. The isolates were identical by biochemical profiling and pulsed-field gel electrophoresis. This rare serotype is known to be associated with reptiles. The current practice for avoiding reptile-associated infections is reviewed.
Cytoplasmic APOBEC3G Restricts Incoming Vif-Positive Human Immunodeficiency Virus Type 1 and Increases Two-Long Terminal Repeat Circle Formation in Activated T-Helper-Subtype Cells
Authors: Vetter Michael L. , D'Aquila Richard T. 
Published: June 2009

ABSTRACT

Cytoplasmic APOBEC3G has been reported to block wild-type human immunodeficiency virus type 1 (HIV-1) infection in some primary cells. It is not known whether cytoplasmic APOBEC3G has residual activity in activated T cells, even though virion-packaged APOBEC3G does restrict HIV-1 in activated T cells. Because we found that APOBEC3G expression is greater in activated CD4
+
T-helper type 1 (Th1) lymphocytes than in T-helper type 2 (Th2) lymphocytes, we hypothesized that residual target cell restriction of incoming Vif-positive virions that lack APOBEC3G, if present, would be greater in Th1 than Th2 lymphocytes. Infection of activated Th1 cells with APOBEC3-negative virions did result in decreased amounts of early and late reverse transcription products and integrated virus relative to infection of activated Th2 cells. Two-long terminal repeat (2-LTR) circles, which are formed in the nucleus when reverse transcripts do not integrate, were increased after APOBEC3-negative virus infection of activated Th1 cells relative to infection of activated Th2 cells. In contrast, 2-LTR circle forms were decreased after infection of APOBEC3G-negative cells with APOBEC3G-containing virions relative to APOBEC3G-negative virions and with Th1 cell-produced virions relative to Th2 cell-produced virions. Increasing APOBEC3G in Th2 cells and decreasing APOBEC3G in Th1 cells modulated the target cell phenotypes, indicating causation by APOBEC3G. The comparison between activated Th1 and Th2 cells indicates that cytoplasmic APOBEC3G in activated Th1 cells partially restricts reverse transcription and integration of incoming Vif-positive, APOBEC3G-negative HIV-1. The differing effects of cytoplasmic and virion-packaged APOBEC3G on 2-LTR circle formation indicate a difference in their antiviral mechanisms.
Cell Fusion-Induced Activation of Interferon-Stimulated Genes Is Not Required for Restriction of a Herpes Simplex Virus VP16/ICP0 Mutant in Heterokarya Formed between Permissive and Restrictive Cells
Authors: Hancock Meaghan H. , Mossman Karen L. , Smiley James R. 
Published: June 2009

ABSTRACT
Herpes simplex virus VP16 and ICP0 mutants replicate efficiently in U2OS osteosarcoma cells but are restricted in other cell types. We previously showed that the restrictive phenotype is dominant in a transient cell fusion assay, suggesting that U2OS cells lack an antiviral mechanism present in other cells. Recent data indicate that unscheduled membrane fusion events can activate the expression of interferon-stimulated genes (ISGs) in fibroblasts, raising the possibility that our earlier results were due to a fusion-induced antiviral state. However, we show here that the permissive phenotype is also extinguished following fusion with Vero cells in the absence of ISG induction.
Role of Nitric Oxide in the Pathogenesis of Encephalomyocarditis Virus-Induced Diabetes in Mice
Authors: Lee Young-Sun , Li Na , Shin Seungjin , Jun Hee-Sook 
Published: June 2009

ABSTRACT

The D variant of encephalomyocarditis virus (EMC-D virus) causes diabetes in mice by destroying pancreatic β cells. In mice infected with a low dose of EMC-D virus, macrophages play an important role in β-cell destruction by producing soluble mediators such as interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and nitric oxide (NO). To investigate the role of NO and inducible NO synthase (iNOS) in the development of diabetes in EMC-D virus-infected mice, we infected iNOS-deficient DBA/2 mice with EMC-D virus (2 × 10
2
PFU/mouse). Mean blood glucose levels in EMC-D virus-infected iNOS-deficient mice and wild-type mice were 205.5 and 466.7 mg/dl, respectively. Insulitis and macrophage infiltration were reduced in islets of iNOS-deficient mice compared with wild-type mice at 3 days after EMC-D virus infection. Apoptosis of β cells was decreased in iNOS-deficient mice, as evidenced by reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. There were no differences in mRNA expression of antiapoptotic molecules Bcl-2, Bcl-xL, Bcl-w, Mcl-1, cIAP-1, and cIAP-2 between wild-type and iNOS-deficient mice, whereas expression of proapoptotic Bax and Bak mRNAs was significantly decreased in iNOS-deficient mice. Expression of IL-1β and TNF-α mRNAs was significantly decreased in both islets and macrophages of iNOS-deficient mice compared with wild-type mice after EMC-D virus infection. Nuclear factor κB was less activated in macrophages of iNOS-deficient mice after virus infection. We conclude that NO plays an important role in the activation of macrophages and apoptosis of pancreatic β cells in EMC-D virus-infected mice and that deficient iNOS gene expression inhibits macrophage activation and β-cell apoptosis, contributing to prevention of EMC-D virus-induced diabetes.
Vaccinia Virus CD8
+
T-Cell Dominance Hierarchies Cannot Be Altered by Prior Immunization with Individual Peptides
Authors: Wang Yang , Flesch Inge E. A. , Tscharke David C. 
Published: June 2009

ABSTRACT

Heterologous prime-boost is a common vaccination strategy to elicit CD8
+
T cells (T
CD8+
), and vaccinia virus (VACV) has been widely used as a boosting vector. Studies with other viruses have suggested that priming may reduce responses to native epitopes in boosting vectors as well as improve responses to primed epitopes. We explored this possibility with a VACV model in mice and find that irrespective of an epitope's dominance, prior priming was able to double T
CD8+
responses. More surprisingly, and in contrast to findings for other viruses, responses to remaining epitopes were undisturbed, leaving the overall dominance hierarchy unchanged.
The Infectious Molecular Clone and Pseudotyped Virus Models of Human Immunodeficiency Virus Type 1 Exhibit Significant Differences in Virion Composition with Only Moderate Differences in Infectivity and Inhibition Sensitivity
Authors: Provine Nicholas M. , Puryear Wendy Blay , Wu Xueling , Overbaugh Julie , Haigwood Nancy L. 
Published: June 2009

ABSTRACT

Two frequently employed methods for generating well-characterized, genetically defined infectious human immunodeficiency virus type 1 in vitro include the use of infectious molecular clones (IMCs) and pseudoviruses (PVs) competent for single-round infection. We compared six matched pairs of IMCs and PVs. The relative amounts of Env incorporated and efficiency of cleavage differed substantially between the two systems. Altering the ratio of proviral genome and
env
expression plasmids can produce pseudovirions that are structurally more similar to the matched IMCs. Differences in Env incorporation and cleavage translated into moderate differences in assays infectivity and sensitivity to neutralizing antibodies and entry inhibitors.
Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir
Authors: Šašková Klára Grantz , Kožíšek Milan , Řezáčová Pavlína , Brynda Jiří , Yashina Tatyana , Kagan Ron M. , Konvalinka Jan 
Published: June 2009

ABSTRACT
Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2′ binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.
Humanized Mice Show Clinical Signs of Dengue Fever according to Infecting Virus Genotype
Authors: Mota Javier , Rico-Hesse Rebeca 
Published: June 2009

ABSTRACT

We demonstrated that the infection of humanized NOD-
scid IL2r
γ
null
mice with different strains (representing the four genotypes) of dengue virus serotype 2 (DEN-2) can induce the development of human-like disease, including fever, viremia, erythema, and thrombocytopenia. Newborn mice were irradiated and received transplants by intrahepatic inoculation of human cord blood-derived hematopoietic progenitor cells (CD34
+
). After 6 weeks, mouse peripheral blood was tested by flow cytometry to determine levels of human lymphocytes (CD45
+
cells); rates of reconstitution ranged from 16 to 80% (median, 52%). Infection (with approximately 10
6
PFU, the equivalent of a mosquito bite) of these humanized mice with eight low-passage-number strains produced a high viremia extending to days 12 to 18 postinfection. We observed a significant decrease in platelets at day 10 in most of the mice and an increase in body temperature (fever) and erythema (rash) in comparison with humanized mice inoculated with cell culture medium only. Comparison of Southeast (SE) Asian and other genotype viruses (American, Indian, and West African) in this model showed significant differences in magnitude and duration of viremia and rash, with the SE Asian viruses always being highest. Indian genotype viruses produced lower viremias and less thrombocytopenia than the others, and West African (sylvatic) viruses produced the shortest periods of viremia and the lowest rash measurements. These results correlate with virulence and transmission differences described previously for primary human target cells and whole mosquitoes and may correlate with epidemiologic observations around the world. These characteristics make this mouse model ideal for the study of dengue pathogenesis and the evaluation of vaccine attenuation and antivirals.
Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain
Authors: Kassa Aemro , Madani Navid , Schön Arne , Haim Hillel , Finzi Andrés , Xiang Shi-Hua , Wang Liping , Princiotto Amy , Pancera Marie , Courter Joel , Smith Amos B. , Freire Ernesto , Kwong Peter D. , Sodroski Joseph 
Published: June 2009

ABSTRACTBinding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.
Identification of a Novel WxSLVK Motif in the N Terminus of Human Immunodeficiency Virus and Simian Immunodeficiency Virus Vif That Is Critical for APOBEC3G and APOBEC3F Neutralization
Authors: Dang Ying , Wang Xiaojun , Zhou Tao , York Ian A. , Zheng Yong-Hui 
Published: June 2009

ABSTRACT

The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a cullin 5-based E3 ubiquitin ligase with A3G and A3F and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A
14
DRMR
17
domain binds to A3F,
40
YRHHY
44
binds to A3G, and
69
YxxL
72
binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here, we show that K26, and to a lesser extent K22, is critical for A3G neutralization. K22 and K26 are part of a conserved
21
WxSLVK
26
(x represents N, K, or H) motif that is found in most primate lentiviruses and that shows species-specific variation. Both K22 and K26 in this motif regulated Vif specificity only for A3G, whereas the SLV residues regulated Vif specificity for both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus,
21
WxSLVK
26
is a novel functional domain that regulates Vif activity toward both A3F and A3G and is a potential drug target to inhibit Vif activity and block HIV-1 replication.
A Patch of Positively Charged Amino Acids Surrounding the Human Immunodeficiency Virus Type 1 Vif SLVx4Yx9Y Motif Influences Its Interaction with APOBEC3G
Authors: Chen Gongying , He Zhiwen , Wang Tao , Xu Rongzhen , Yu Xiao-Fang 
Published: June 2009

ABSTRACT

The amino-terminal region of the Vif molecule in human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) contains a conserved SLV/Ix4Yx9Y motif that was first described in 1992, but the importance of this motif for Vif function has not yet been examined. Our characterization of the amino acids surrounding this motif in HIV-1 Vif indicated that the region is critical for APOBEC3 suppression. In particular, amino acids K22, K26, Y30, and Y40 were found to be important for the Vif-induced degradation and suppression of cellular APOBEC3G (A3G). However, mutation of these residues had little effect on the Vif-mediated suppression of A3F, A3C, or A3DE, suggesting that these four residues are not important for Vif assembly with the Cul5 E3 ubiquitin ligase or protein folding in general. The LV portion of the Vif SLV/Ix4Yx9Y motif was found to be required for optimal suppression of A3F, A3C, or A3DE. Thus, the SLV/Ix4Yx9Y motif and surrounding amino acids represent an important functional domain in the Vif-mediated defense against APOBEC3. In particular, the positively charged K26 of HIV-1 Vif is invariably conserved within the SLV/Ix4Yx9Y motif of HIV/SIV Vif molecules and was the most critical residue for A3G inactivation. A patch of positively charged and hydrophilic residues (K
22
x
3
K
26
x
3
Y
30
x
9
YRHHY
44
) and a cluster of hydrophobic residues (V
55
xIPLx
4-5
LxΦx2YWxL
72
) were both involved in A3G binding and inactivation. These structural motifs in HIV-1 Vif represent attractive targets for the development of lead inhibitors to combat HIV infection.
Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells
Authors: Sherer Nathan M. , Swanson Chad M. , Papaioannou Stelios , Malim Michael H. 
Published: June 2009

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the
cis
-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4×CTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE- but not 4×CTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.