bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Leishmania spp. by Molecular Amplification and DNA Sequencing Analysis of a Fragment of rRNA Internal Transcribed Spacer 2
Authors: de Almeida Marcos E. , Steurer Francis J. , Koru Ozgur , Herwaldt Barbara L. , Pieniazek Norman J. , da Silva Alexandre J. 
Published: July 2011

ABSTRACT

Isoenzyme analysis of cultured parasites is the conventional approach for
Leishmania
species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed PCR generic primers to amplify a segment of the rRNA internal transcribed spacer 2 (ITS2) from multiple
Leishmania
species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR followed by DNA sequencing analysis) with those from the parasitologic approach (
in vitro
culture followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of eight
Leishmania
species were identified. The species results were concordant for all but two patients: for one patient, the results were
Leishmania
(
Viannia
)
guyanensis
by the molecular approach versus
L. (V.) braziliensis
by the parasitologic approach; for the other patient, the results were
L.
(
Leishmania
)
tropica
versus
L. (L.) major
, respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among
Leishmania
species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of
Leishmania
parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens.
Multiplex Real-Time PCR Melting Curve Assay To Detect Drug-Resistant Mutations of Mycobacterium tuberculosis
Authors: Luo Tao , Jiang Lili , Sun Weiming , Fu G. , Mei Jian , Gao Qian 
Published: July 2011

ABSTRACT

Early diagnosis of drug-resistant
Mycobacterium tuberculosis
is urgently needed to optimize treatment regimens and to prevent the transmission of resistant strains. Real-time PCR assays have been developed to detect drug resistance rapidly, but none of them have been widely applied due to their complexity, high cost, or requirement for advanced instruments. In this study, we developed a real-time PCR method based on melting curve analysis of dually labeled probes. Six probes targeting the
rpoB
81-bp core region,
katG315
, the
inhA
promoter, the
ahpC
promoter, and
embB306
were designed and validated with clinical isolates. First, 10 multidrug-resistant (MDR) strains with a wide mutation spectrum were used to analyze the melting temperature (

T
m

) deviations of different mutations by single real-time PCR. All mutations can be detected by significant

T
m

reductions compared to the wild type. Then, three duplex real-time PCRs, with two probes in each, were developed to detect mutations in 158 MDR isolates. Comparison of the results with the sequencing data showed that all mutations covered by the six probes were detected with 100% sensitivity and 100% specificity. Our method provided a new way to rapidly detect drug-resistant mutations in
M. tuberculosis.
Compared to other real-time PCR methods, we use fewer probes, which are labeled with the same fluorophore, guaranteeing that this assay can be used for detection in a single fluorescent channel or can be run on single-channel instruments. In conclusion, we have developed a widely applicable real-time PCR assay to detect drug-resistant mutations in
M. tuberculosis
.
Bacteriological and Epidemiological Characteristics of Enterotoxigenic Escherichia coli Isolated in Tokyo, Japan, between 1966 and 2009
Authors: Konishi Noriko , Obata Hiromi , Monma Chie , Nakama Akiko , Kai Akemi , Tsuji Takao 
Published: July 2011

ABSTRACT

Enterotoxigenic
Escherichia coli
(ETEC) caused 131 outbreaks in Tokyo, Japan, between 1966 and 2009. The major serogroups were O6, O27, O148, and O159. The incidence of serogroups O25 and O169 recently increased. Heat-stable enterotoxin (ST) subtyping revealed that
E. coli
of serogroups O6, O15, O25, and O159 possessed the STh gene, whereas those serotyped as O27 and O169 possessed the STp gene.
The L-Coding Region of the DA Strain of Theiler's Murine Encephalomyelitis Virus Causes Dysfunction and Death of Myelin-Synthesizing Cells
Authors: Ghadge G. D. , Wollmann R. , Baida G. , Traka M. , Roos R. P. 
Published: July 2011

ABSTRACT
The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce an early transient subclinical neuronal disease followed by a chronic progressive inflammatory demyelination, with persistence of the virus in the central nervous system (CNS) for the life of the mouse. Although TMEV-induced demyelinating disease (TMEV-IDD) is thought to be immune mediated, there is also evidence that supports a role for the virus in directly inducing demyelination. In order to clarify the function of DA virus genes, we generated a transgenic mouse that had tamoxifen-inducible expression of the DA L-coding region in oligodendrocytes (and Schwann cells), a cell type in which the virus is known to persist. Tamoxifen-treated young transgenic mice usually developed an acute progressive fatal paralysis, with abnormalities of the oligodendrocytes and Schwann cells and demyelination, but without significant lymphocytic infiltration; later treatment led to transient weakness with demyelination and persistent expression of the recombined transgene. These findings demonstrate that a high level of expression of DA L can cause the death of myelin-synthesizing cells and death of the mouse, while a lower level of L expression (which can persist) can lead to cellular dysfunction with survival. The results suggest that expression of DA L plays an important role in the pathogenesis of TMEV-IDD. Virus-induced infection and death of oligodendrocytes may play a part in the demyelination of other diseases in which an immune-mediated mechanism has been stressed, including multiple sclerosis.
Deletion of the Monkeypox Virus Inhibitor of Complement Enzymes Locus Impacts the Adaptive Immune Response to Monkeypox Virus in a Nonhuman Primate Model of Infection
Authors: Estep Ryan D. , Messaoudi Ilhem , O'Connor Megan A. , Li Helen , Sprague Jerald , Barron Alexander , Engelmann Flora , Yen Bonnie , Powers Michael F. , Jones John M. , Robinson Bridget A. , Orzechowska Beata U. , Manoharan Minsha , Legasse Alfred , Planer Shannon , Wilk Jennifer , Axthelm Michael K. , Wong Scott W. 
Published: July 2011

ABSTRACT

Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (
n
= 4), or a recombinant MPXV-Zaire (
n
= 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication
in vivo
, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.
Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response
Authors: Carpenter John E. , Jackson Wallen , Benetti Luca , Grose Charles 
Published: July 2011

ABSTRACT
Autophagy is a recently recognized component of the life cycle of varicella-zoster virus (VZV). We have documented abundant autophagosome formation in skin vesicles (final site of virion assembly) from randomly selected cases of varicella and zoster. The fact that autophagy was an early event in the VZV replication cycle was documented by finding infected vesicle cells with the VZV IE62 protein confined to the nucleus. Next, we pursued studies in VZV-infected cultured cells to define whether autophagy was preceded by endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). First, we demonstrated that autophagosome formation in infected cells closely resembled that seen after treatment of cells with tunicamycin, a potent initiator of ER stress. Second, we demonstrated a marked expansion of ER size in both VZV-infected cells and cells transfected with the predominant VZV glycoprotein complex gE/gI. An enlarged ER is critical evidence of ER stress, which in turn is relieved by the UPR. To this end, we documented the UPR by detecting the alternatively spliced form of the XBP1 protein as well as CHOP (C/EBP homologous protein), both transcriptional activators of other UPR genes in an ER stress-dependent manner. Because VZV does not encode inhibitors of autophagy, the above results suggested that autophagy was a common event in VZV-infected cells and that it was provoked at least in part by ER stress secondary to overly abundant VZV glycoprotein biosynthesis, which led to UPR activation in an attempt to maintain cellular homeostasis.
Human Rhinovirus 2 Induces the Autophagic Pathway and Replicates More Efficiently in Autophagic Cells
Authors: Klein Kathryn A. , Jackson William T. 
Published: July 2011

ABSTRACT
Picornaviruses rearrange cellular membranes to form cytosolic replication sites. In the case of poliovirus and several other picornaviruses, these membranes are derived from subversion of the cellular autophagy pathway. We also reported observation of autophagosome-like structures during infection by two human rhinoviruses (HRVs), HRV-2 and HRV-14 (W. T. Jackson et al., PLoS Biol. 3:e156, 2005). Another group reported that HRV-2 does not induce autophagosomes or respond to changes in cellular autophagy (M. Brabec-Zaruba, U. Berka, D. Blaas, and R. Fuchs, J. Virol. 81:10815-10817, 2007). In this study, we tested HRV-2-infected cells for activation of autophagic signaling and changes in virus growth in response to changes in autophagy levels. Our data indicate that HRV-2 induces and subverts the autophagic machinery to promote its own replication.
High-Functional-Avidity Cytotoxic T Lymphocyte Responses to HLA-B-Restricted Gag-Derived Epitopes Associated with Relative HIV Control
Authors: Berger Christoph T. , Frahm Nicole , Price David A. , Mothe Beatriz , Ghebremichael Musie , Hartman Kari L. , Henry Leah M. , Brenchley Jason M. , Ruff Laura E. , Venturi Vanessa , Pereyra Florencia , Sidney John , Sette Alessandro , Douek Daniel C. , Walker Bruce D. , Kaufmann Daniel E. , Brander Christian 
Published: July 2011

ABSTRACTVirus-specific cytotoxic T lymphocytes (CTL) with high levels of functional avidity have been associated with viral clearance in hepatitis C virus infection and with enhanced antiviral protective immunity in animal models. However, the role of functional avidity as a determinant of HIV-specific CTL efficacy remains to be assessed. Here we measured the functional avidities of HIV-specific CTL responses targeting 20 different, optimally defined CTL epitopes restricted by 13 different HLA class I alleles in a cohort comprising 44 HIV controllers and 68 HIV noncontrollers. Responses restricted by HLA-B alleles and responses targeting epitopes located in HIV Gag exhibited significantly higher functional avidities than responses restricted by HLA-A or HLA-C molecules (P= 0.0003) or responses targeting epitopes outside Gag (P< 0.0001). The functional avidities of Gag-specific and HLA-B-restricted responses were higher in HIV controllers than in noncontrollers (P= 0.014 andP= 0.018) and were not restored in HIV noncontrollers initiating antiretroviral therapy. T-cell receptor (TCR) analyses revealed narrower TCR repertoires in higher-avidity CTL populations, which were dominated by public TCR sequences in HIV controllers. Together, these data link the presence of high-avidity Gag-specific and HLA-B-restricted CTL responses with viral suppressionin vivoand provide new insights into the immune parameters that mediate spontaneous control of HIV infection.
Green Fluorescent Protein Reporter System with Transcriptional Sequence Heterogeneity for Monitoring the Interferon Response
Authors: Mahmoud Linah , Al-Saif Maher , Amer Haitham M. , Sheikh Mustafa , Almajhdi Fahad N. , Khabar Khalid S. A. 
Published: July 2011

ABSTRACT

The interferon (IFN) response is initiated by a variety of triggers, including viruses and foreign RNA, and involves several receptors and intracellular mediators. Although there are common
cis
-acting consensus sequences in the promoters of many genes stimulated during the IFN response, they exhibit core and context heterogeneity that may lead to differential transcriptional activity. We have developed and validated a live cell-based enhanced green fluorescent protein (EGFP) reporter system employing more than a hundred constructs containing multiple viruses and IFN response elements derived from a variety of promoters involved in immunity to viruses. Common and distinct response patterns were observed due to promoter heterogeneity in response to different stimuli, including IFN-α, TLR3-agonist double-stranded RNA, and several viruses. This information should serve as a resource in selecting specific reporters for sensing nonself ligands.
The Members of an Epstein-Barr Virus MicroRNA Cluster Cooperate To Transform B Lymphocytes
Authors: Feederle Regina , Haar Janina , Bernhardt Katharina , Linnstaedt Sarah D. , Bannert Helmut , Lips Helge , Cullen Bryan R. , Delecluse Henri-Jacques 
Published: July 2011

ABSTRACT

Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP). Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBV's oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes
in vitro
. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miR-BHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miR-BHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells.
Differential Effects of Viral Vectors on Migratory Afferent Lymph Dendritic CellsIn VitroPredict Enhanced ImmunogenicityIn Vivo
Authors: Cubillos-Zapata C. , Guzman E. , Turner A. , Gilbert S. C. , Prentice H. , Hope J. C. , Charleston B. 
Published: July 2011

ABSTRACTTargeting dendritic cells (DC) is key to driving effective immune responses. Lymphatic cannulation provides access to the heterogeneous populations of DC draining peripheral sites in rodents and ruminants. Afferent lymph DEC-205+CD11c+SIRPα+DC were preferentially infectedex vivowith three vaccine viral vectors: recombinant human replication-defective human adenovirus 5 (rhuAdV5), recombinant modified vaccinia virus Ankara (rMVA), and recombinant fowlpox virus (rFPV), all expressing green fluorescent protein (GFP). The rhuAdV5-infected cells remained viable, and peak GFP expression was observed 16 to 24 h posttransduction. Increasing the incubation period of DC with rhuAdV5 enhanced GFP expression. In contrast, DC infected with rMVA-GFP or rFPV-GFP became rapidly apoptotic and GFP expression peaked at 6 h postinfection. Delivery of foot-and-mouth disease virus (FMDV) A22antigen to DC by rhuAdV5-FMDV-A22ex vivoresulted in significantly greater CD4+T cell proliferation than did delivery by rFPV-FMDV-A22. Delivery of rhuAdV5-GFP in oil adjuvantin vivo, to enhance DC-vector contact, resulted in increased GFP expression in migrating DC compared to that with vector alone. Similarly, CD4+T cell responses were significantly enhanced when using rhuAdV5-FMDV-A22in adjuvant. Therefore, the interaction between viral vectors and afferent lymph DCex vivocan predict the outcome ofin vivoimmunization and provide a means of rapidly assessing the effects of vector modification.
Structure of a Baculovirus Sulfhydryl Oxidase, a Highly Divergent Member of the Erv Flavoenzyme Family
Authors: Hakim Motti , Mandelbaum Amitai , Fass Deborah 
Published: July 2011

ABSTRACT

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in
Autographa californica
multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.
The Novel Anticytomegalovirus Compound AIC246 (Letermovir) Inhibits Human Cytomegalovirus Replication through a Specific Antiviral Mechanism That Involves the Viral Terminase
Authors: Goldner Thomas , Hewlett Guy , Ettischer Nicole , Ruebsamen-Schaeff Helga , Zimmermann Holger , Lischka Peter 
Published: July 2011

ABSTRACTHuman cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activityin vitroandin vivoand currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.
A Dual Strategy for the Suppression of Host Antiviral Silencing: Two Distinct Suppressors for Viral Replication and Viral Movement Encoded by Potato Virus M
Authors: Senshu Hiroko , Yamaji Yasuyuki , Minato Nami , Shiraishi Takuya , Maejima Kensaku , Hashimoto Masayoshi , Miura Chihiro , Neriya Yutaro , Namba Shigetou 
Published: July 2011

ABSTRACT

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus
Carlavirus
. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus
Carlavirus
.