bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis
Authors: Chen Xiaoyou , Kong Fanrong , Wang Qinning , Li Chuanyou , Zhang Jianyuan , Gilbert Gwendolyn L. 
Published: August 2011

ABSTRACT

A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in
Mycobacterium tuberculosis
by targeting resistance-associated mutations in the
katG
,
mabA-inhA
promoter,
rpoB
, and
gyrA
genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical
M. tuberculosis
isolates was selected for development and evaluation of HRMA. PCR amplicons from the
katG
,
mabA-inhA
promoter,
rpoB
, and
gyrA
genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified
katG
and/or
mabA-inhA
promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates,
rpoB
mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and
gyrA
mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in
M. tuberculosis
which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.
High-Resolution Melting Analysis for Identification of the Cryptococcus neoformans-Cryptococcus gattii Complex
Authors: Gago Sara , Zaragoza Óscar , Cuesta Isabel , Rodríguez-Tudela Juan L. , Cuenca-Estrella Manuel , Buitrago María J. 
Published: August 2011

ABSTRACT

We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the
Cryptococcus
species complex (
Cryptococcus neoformans
var.
grubii
,
Cryptococcus neoformans
var.
neoformans
, and
Cryptococcus gattii
). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main
Cryptococcus
species.
A Comprehensive Evaluation of the Proficiency Testing Program for the HIV-1 BED Incidence Assay
Authors: Dobbs Trudy , Liu Xin , Anderson Rebecca , Nkengasong John , Parekh Bharat S. 
Published: August 2011

ABSTRACT

The HIV-1 BED incidence assay was developed at the Centers for Disease Control and Prevention and since 2005 has been available as a commercial kit for use in HIV-1 incidence surveillance. A BED-specific proficiency testing (PT) program was initiated in 2006 that included a panel of eight coded specimens (six unique and two duplicates) to participating laboratories. The number of participating laboratories increased from 12 to 38 from 2006 to 2009. Overall, 96.1% of the laboratories reported results, and 95.4% of those reporting achieved a 100% score. The observed mean normalized optical density (OD-n) values of all participants correlated well with the expected OD-n values for all specimens (
R
2
= 0.98) used for seven PT rounds. BED testing demonstrated high reproducibility among all laboratories, with an agreement of 99.3% (574/578) between initial and confirmatory classification and regression statistics of
R
2
= 0.96, slope = 1.022, and intercept = 0.0066. Reproducibility among duplicate specimens was very high during each PT round, with mean deviation of 1.8%. Analysis of controls and calibrator specimen for all 343 runs showed a coefficient of variation of ca. 20% for raw ODs in the dynamic range, which was reduced to <10% when the OD was normalized (OD-n). Most laboratories that failed the PT assessment had transcriptional errors, kit reagent problems, or specimen handling errors. Thus, the BED-specific PT program enabled us to track performance of different laboratories conducting the BED assay while identifying areas for improvements. This program will also serve as a template for future PT programs for new incidence assays as they become available.
Molecular Epidemiology of Aspergillus fumigatus: an In-Depth Genotypic Analysis of Isolates Involved in an Outbreak of Invasive Aspergillosis
Authors: Guinea Jesús , García de Viedma Darío , Peláez Teresa , Escribano Pilar , Muñoz Patricia , Meis Jacques F. , Klaassen Corné H. W. , Bouza Emilio 
Published: August 2011

ABSTRACT

We recently reported an outbreak of invasive aspergillosis in the major heart surgery unit of Hospital Gregorio Marañón, Madrid, Spain (T. Peláez, P. Muñoz, J. Guinea, M. Valerio, M. Giannella, C. H. W. Klaassen, and E. Bouza, Clin. Infect. Dis., in press).
Aspergillus fumigatus
was isolated from clinical samples from 10 patients admitted to the unit during the outbreak period (surgical wound invasive aspergillosis,
n
= 2; probable pulmonary invasive aspergillosis,
n
= 4; colonization,
n
= 4). In the study described here, we have studied the genotypic diversity of the
A. fumigatus
isolates found in the air and clinical samples. We used short tandem repeats of
A. fumigatus
(STR
Af
) typing to analyze the genotypes found in the 168 available
A. fumigatus
isolates collected from the clinical samples (
n
= 109) from the patients and from the environmental samples taken from the air of the unit (
n
= 59). The genotypic variability of
A. fumigatus
was higher in environmental than in clinical samples. Intrasample variability was also higher in environmental than in clinical samples: 2 or more different genotypes were found in 26% and 89% of clinical and environmental samples, respectively. We found matches between environmental and clinical isolates in 3 of the 10 patients: 1 patient with postsurgical invasive aspergillosis and 2 patients with probable pulmonary invasive aspergillosis. A total of 7 genotypes from 3 different patients and the air grouped together in 2 clusters. Clonally related genotypes and microvariants were detected in both clinical and environmental samples. STR
Af
typing proved to be a valuable tool for identifying the source of invasive aspergillosis outbreaks and for studying the genotypic diversity of clinical and environmental
A. fumigatus
isolates.
Bead-Based Multiplex Genotyping of 58 Cutaneous Human Papillomavirus Types
Authors: Michael K. M. , Forslund O. , Bacevskij O. , Waterboer T. , Bravo I. G. , Pawlita M. , Schmitt M. 
Published: August 2011

ABSTRACT
Cutaneous human papillomaviruses (HPVs) are a heterogeneous, nonmonophyletic assembly, comprising about 50 characterized types and at least 133 isolates putatively representing new types. Their natural history of infection and potential association with nonmelanoma skin cancer are not well understood. Several PCR systems have been developed that amplify a broad spectrum of cutaneous HPVs. However, amplicon genotyping by sequencing or reverse line blot assays are complex and not well suited for high-throughput analyses. We developed a novel multiplex cutaneous papillomavirus genotyping (McPG) assay for 38 defined and 20 putative cutaneous HPVs of the beta, gamma, mu, and nu genera. Viral DNA was amplified by the use of a modified single-tube nested “hanging-droplet” FAP PCR. The amplifiable papillomavirus (PV) spectrum was enlarged by the use of 9 outer and 13 inner primers. Biotinylated PCR products were hybridized to type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads and analyzed using Luminex technology. Analytical sensitivity was analyzed for 38 defined HPVs and was ≤100 genome copies for all types. Integrated β-globin primers allow for simultaneous DNA quality control. McPG is characterized by high reproducibility (κ= 0.84, 95% confidence interval = 0.79 to 0.88), good concordance with the original nested FAP PCR, followed by sequencing (70.2% complete or partial agreement) when 322 skin biopsy DNA samples were analyzed, and improved ability to detect multiple infections (on average 2.5 HPV types per HPV-positive sample compared to 1.7 HPV types with nested FAP-PCR). In conclusion, McPG is a powerful tool for genotyping multiple cutaneous HPVs in a high-throughput format and is thus suitable for large-scale epidemiological studies.
Evaluation of Two Magnetic-Bead-Based Viral Nucleic Acid Purification Kits and Three Real-Time Reverse Transcription-PCR Reagent Systems in Two TaqMan Assays for Equine Arteritis Virus Detection in Semen
Authors: Miszczak Fabien , Shuck Kathleen M. , Lu Zhengchun , Go Yun Young , Zhang Jianqiang , Sells Stephen , Vabret Astrid , Pronost Stéphane , Fortier Guillaume , Timoney Peter J. , Balasuriya Udeni B. R. 
Published: August 2011

ABSTRACTThis study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.
Extent of Antigenic Cross-Reactivity among Highly Pathogenic H5N1 Influenza Viruses
Authors: Ducatez Mariette F. , Cai Zhipeng , Peiris Malik , Guan Yi , Ye Zhiping , Wan Xiu-Feng , Webby Richard J. 
Published: August 2011

ABSTRACT
Highly pathogenic H5N1 avian influenza viruses emerged in 1996 and have since evolved so extensively that a single strain can no longer be used as a prepandemic vaccine or diagnostic reagent. We therefore sought to identify the H5N1 strains that may best serve as cross-reactive diagnostic reagents. We compared the cross-reactivity of 27 viruses of clades 0, 1, 2.1, 2.2, 2.3, and 4 and of four computationally designed ancestral H5N1 strains by hemagglutination inhibition (HI) and microneutralization (MN) assays. Antigenic cartography was used to analyze the large quantity of resulting data. Cartographs of HI titers with chicken red blood cells were similar to those of MN titers, but HI with horse red blood cells decreased antigenic distances among the H5N1 strains studied. Thus, HI with horse red blood cells seems to be the assay of choice for H5N1 diagnostics. Whereas clade 2.2 antigens were able to detect antibodies raised to most of the tested H5N1 viruses (and clade 2.2-specific antisera detected most of the H5N1 antigens), ancestral strain A exhibited the widest reactivity pattern and hence was the best candidate diagnostic reagent for broad detection of H5N1 strains.
Comparison of Multilocus Variable-Number Tandem-Repeat Analysis and Multilocus Sequence Typing for Differentiation of Hemolytic-Uremic Syndrome-Associated Escherichia coli (HUSEC) Collection Strains
Authors: Jenke Christian , Lindstedt Björn Arne , Harmsen Dag , Karch Helge , Brandal Lin Thorstensen , Mellmann Alexander 
Published: August 2011

ABSTRACT

Multilocus variable-number tandem-repeat analysis (MLVA) was compared to multilocus sequence typing (MLST) to differentiate hemolytic uremic syndrome-associated enterohemorrhagic
Escherichia coli
strains. Although MLVA—like MLST—was highly discriminatory (index of diversity, 0.988 versus 0.984), a low level of concordance demonstrated the limited ability of MLVA to reflect long-term evolutionary events.
Myxoma Virus Induces Type I Interferon Production in Murine Plasmacytoid Dendritic Cells via a TLR9/MyD88-, IRF5/IRF7-, and IFNAR-Dependent Pathway
Authors: Dai Peihong , Cao Hua , Merghoub Taha , Avogadri Francesca , Wang Weiyi , Parikh Tanvi , Fang Chee-Mun , Pitha Paula M. , Fitzgerald Katherine A. , Rahman Masmudur M. , McFadden Grant , Hu Xiaoyu , Houghton Alan N. , Shuman Stewart , Deng Liang 
Published: August 2011

ABSTRACT

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the
Leporipoxvirus
genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-β, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.
Infection of Nonhost Species Dendritic CellsIn Vitrowith an Attenuated Myxoma Virus Induces Gene Expression That Predicts Its Efficacy as a Vaccine Vector
Authors: Top S. , Foulon E. , Pignolet B. , Deplanche M. , Caubet C. , Tasca C. , Bertagnoli S. , Meyer G. , Foucras G. 
Published: August 2011

ABSTRACTRecombinant myxoma virus (MYXV) can be produced without a loss of infectivity, and its highly specific host range makes it an ideal vaccine vector candidate, although careful examination of its interaction with the immune system is necessary. Similar to rabbit bone marrow-derived dendritic cells (BM-DCs), ovine dendritic cells can be infected by SG33, a MYXV vaccine strain, and support recombinant antigen expression. The frequency of infected cells in the nonhost was lower and the virus cycle was abortive in these cell types. Among BM-DC subpopulations, Langerhans cell-like DCs were preferentially infected at low multiplicities of infection. Interestingly, ovine BM-DCs remained susceptible to MYXV after maturation, although apoptosis occurred shortly after infection as a function of the virus titer. When gene expression was assessed in infected BM-DC cultures, type I interferon (IFN)-related and inflammatory genes were strongly upregulated. DC gene expression profiles were compared with the profiles produced by other poxviruses in interaction with DCs, but very few commonalities were found, although genes that were previously shown to predict vaccine efficacy were present. Collectively, these data support the idea that MYXV permits efficient priming of adaptive immune responses and should be considered a promising vaccine vector along with other poxviruses.
Rab5 and Class III Phosphoinositide 3-Kinase Vps34 Are Involved in Hepatitis C Virus NS4B-Induced Autophagy
Authors: Su Wen-Chi , Chao Ti-Chun , Huang Yih-Leh , Weng Shih-Che , Jeng King-Song , Lai Michael M. C. 
Published: August 2011

ABSTRACT
Autophagy has been shown to facilitate replication or production of hepatitis C virus (HCV); nevertheless, how HCV induces autophagy remains unclear. Here, we demonstrate that HCV nonstructural protein 4B (NS4B) alone can induce autophagy signaling; amino acid residues 1 to 190 of NS4B are sufficient for this induction. Further studies showed that the phosphorylation levels of S6K and 4E-BP1 were not altered, suggesting that the mTOR/S6 kinase pathway and mTOR/4E-BP1 pathway did not contribute to NS4B- or HCV-induced autophagy. Inhibition of Rab5 function by silencing Rab5 or overexpressing dominant-negative Rab5 mutant (S34N) resulted in significant reduction of NS4B- or HCV-induced autophagic vesicle formation. Moreover, the autophagy induction was impaired by inhibition of class III phosphoinositide 3-kinase (PI 3-kinase) Vps34 function. Finally, the coimmunoprecipitation assay indicated that NS4B formed a complex with Rab5 and Vps34, supporting the notion that Rab5 and Vps34 are involved in NS4B-induced autophagy. Taken together, these results not only reveal a novel role of NS4B in autophagy but also offer a clue to the mechanism of HCV-induced autophagy.
Adenovirus Type 5 Rupture of Lysosomes Leads to Cathepsin B-Dependent Mitochondrial Stress and Production of Reactive Oxygen Species
Authors: McGuire Kathleen A. , Barlan Arlene U. , Griffin Tina M. , Wiethoff Christopher M. 
Published: August 2011

ABSTRACTIn response to viral infection, reactive oxygen species (ROS) mediate innate immune signaling or generate danger signals to activate immune cells. The mechanisms of virally induced ROS are poorly defined, however. We demonstrate that ROS are produced within minutes of adenovirus type 5 (Ad5) infection of macrophages and that oxidative stress supports Ad5-induced cytokine secretion. We show that short hairpin RNA (shRNA) knockdown of TLR9 has no effect on ROS production despite observed decreases in Ad-induced cytokine secretion. A major source of ROS in macrophages is NADPH oxidase. However, shRNA knockdown of the NADPH oxidase subunit NOX2 does not attenuate Ad-induced ROS. Induction of ROS is not observed in cells infected with a temperature-sensitive mutant of Ad2,ts1, which is defective in endosomal membrane penetration during cell entry. Further, Ad5, but notts1, induces the release of lysosomal cathepsin B into the cytoplasm of infected cells. In agreement with this finding, we observe a loss of mitochondrial membrane potential upon Ad infection which requires Ad endosomal membrane penetration and cathepsin B activity. Overexpression of Bcl-2 attenuates Ad5-induced ROS, further supporting the role for mitochondrial membrane destabilization as the source of ROS in response to Ad5 infection. Together, these data suggest that ROS produced in response to Ad5 infection depends on the virally induced endosomal membrane rupture to release lysosomal cathepsins. Furthermore, the release of cathepsins leads to mitochondrial membrane disruption and thus the release of ROS from the mitochondria.
Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function
Authors: Andrabi Shaida , Hwang Justin H. , Choe Jennifer Kean , Roberts Thomas M. , Schaffhausen Brian S. 
Published: August 2011

ABSTRACT
Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but murine polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not.
Structural Basis for Antiviral Inhibition of the Main Protease, 3C, from Human Enterovirus 93
Authors: Costenaro Lionel , Kaczmarska Zuzanna , Arnan Carme , Janowski Robert , Coutard Bruno , Solà Maria , Gorbalenya Alexander E. , Norder Heléne , Canard Bruno , Coll Miquel 
Published: August 2011

ABSTRACT

Members of the
Enterovirus
genus of the
Picornaviridae
family are abundant, with common human pathogens that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically processed into mature proteins at Gln↓Gly cleavage sites by the 3C protease (3C
pro
), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3C
pro
an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3C
pro
from an enterovirus B, EV-93, a recently identified pathogen, alone and in complex with the anti-HRV molecules compound 1 (AG7404) and rupintrivir (AG7088) at resolutions of 1.9, 1.3, and 1.5 Å, respectively. The EV-93 3C
pro
adopts a chymotrypsin-like fold with a canonically configured oxyanion hole and a substrate binding pocket similar to that of rhino-, coxsackie- and poliovirus 3C proteases. We show that compound 1 and rupintrivir are both active against EV-93 in infected cells and inhibit the proteolytic activity of EV-93 3C
pro
in vitro
. These results provide a framework for further structure-guided optimization of the tested compounds to produce antiviral drugs against a broad range of EV species.
Timing of the Components of the HIV Life Cycle in Productively Infected CD4
+
T Cells in a Population of HIV-Infected Individuals
Authors: Murray John M. , Kelleher Anthony D. , Cooper David A. 
Published: August 2011

ABSTRACT

We estimate the time required for HIV to complete separate stages of its infection cycle in productively infected CD4
+
T cells
in vivo
by comparing initial delays after administration of single antiretroviral drugs until HIV RNA reduction in peripheral blood. Data were obtained from monotherapy studies of eight antiretroviral drugs from all currently licensed HIV drug classes: CCR5 blockers (maraviroc), fusion inhibitors (enfuvirtide), nucleoside and nonnucleoside reverse transcriptase inhibitors (abacavir, tenofovir, and rilpivirine), integrase inhibitors (raltegravir), and protease inhibitors (ritonavir and nelfinavir). We find that HIV requires an average of 52 h between export of virions in one generation to export in the next, with most of this (33 h) taken up by reverse transcription. Reverse transcription
in vivo
was three times longer than
in vitro
and began soon after virion fusion, as we determined no difference in mean times for commencement of reverse transcription and virion fusion as calculated by timing of the effects for tenofovir and maraviroc. Approximately 7 h is required between HIV integration and virion production. First-phase HIV RNA decay (half-life of 17 h over all drugs) seemed to slow as the stage being inhibited by the drug was further from viral production. The mean estimated half-life of plasma virions was 5 min, significantly shorter than previous estimates.