bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology
Authors: Ntziora F. , Paraskevis D. , Haida C. , Magiorkinis E. , Manesis E. , Papatheodoridis G. , Manolakopoulos S. , Beloukas A. , Chryssoy S. , Magiorkinis G. , Sypsa V. , Hatzakis A. 
Published: June 2009

ABSTRACTMutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.
Emerging Genotypes of Human Respiratory Syncytial Virus Subgroup A among Patients in Japan
Authors: Shobugawa Yugo , Saito Reiko , Sano Yasuko , Zaraket Hassan , Suzuki Yasushi , Kumaki Akihiko , Dapat Isolde , Oguma Taeko , Yamaguchi Masahiro , Suzuki Hiroshi 
Published: June 2009

ABSTRACT
Human respiratory syncytial virus (HRSV) is a common etiological agent of acute lower respiratory tract disease in infants. We report the molecular epidemiology of HRSV in Niigata, Japan, over six successive seasons (from 2001 to 2007) and the emerging genotypes of HRSV subgroup A (HRSV-A) strains. A total of 488 HRSV samples were obtained from 1,103 screened cases in a pediatric clinic in Niigata. According to the phylogenetic analysis, among the PCR-positive samples, 338 HRSV-A strains clustered into the previously reported genotypes GA5 and GA7 and two novel genotypes, NA1 and NA2, which were genetically close to GA2 strains. One hundred fifty HRSV-B strains clustered into three genotypes, namely, GB3, SAB3, and BA, which has a 60-nucleotide insertion in the second hypervariable region of the G protein. The NA1 strains emerged first, in the 2004-2005 season, and subsequently, the NA2 strain emerged in the 2005-2006 season. Both strains caused large epidemics in the 2005-2006 and 2006-2007 seasons. The average age of children who were infected with NA2 strains was significantly higher than that of those infected with GA5 and the frequency of reinfection by NA2 was the highest among all genotypes, suggesting that this genotype possessed new antigenicity for evading past host immunity. This is the first paper to show a possible correlation between an emerging genotype, NA2, and large outbreaks of HRSV in Japan. Continuing studies to follow up the genetic changes and to clarify the mechanism of reinfection in HRSV are important steps to understand HRSV infections.
Detection and Genotyping of Human Papillomavirus in Self-Obtained Cervicovaginal Samples by Using the FTA Cartridge: New Possibilities for Cervical Cancer Screening
Authors: Lenselink Charlotte H. , de Bie Roosmarie P. , van Hamont Dennis , Bakkers Judith M. J. E. , Quint Wim G. V. , Massuger Leon F. A. G. , Bekkers Ruud L. M. , Melchers Willem J. G. 
Published: June 2009

ABSTRACT

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF
10
-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening.
Evaluation of GenoType Mycobacteria Direct Assay in Comparison with Gen-Probe
Mycobacterium tuberculosis
Amplified Direct Test and GenoType MTBDR
plus
for Direct Detection of
Mycobacterium tuberculosis
Complex in Clinical Samples
Authors: Neonakis Ioannis K. , Gitti Zoe , Baritaki Stavroula , Petinaki Efi , Baritaki Maria , Spandidos Demetrios A. 
Published: June 2009

ABSTRACT

Three molecular assays were evaluated for the direct detection of
Mycobacterium t
uberculosis
complex bacteria in 125 respiratory and 22 nonrespiratory samples. The overall sensitivities obtained were as follows: GenoType MTBDR
plus
, 97.9%; GenoType Mycobacteria Direct, 93.7%; Gen-Probe
Mycobacterium tuberculosis
Amplified Direct Test, 89.6%. The specificity of the assays used was 100%.
Evaluation of MeningoFinder, a Novel Multiplex Ligation-Dependent Probe Amplification Assay for Simultaneous Detection of Six Virus Species Causing Central Nervous System Infections
Authors: Wolffs P. F. G. , Vink C. , Keijdener J. , Habek B. , Reijans M. , Simons G. , Bruggeman C. A. , van den Brule A. J. C. 
Published: June 2009

ABSTRACT
A multiplex ligation-dependent probe amplification assay for simultaneous detection of six virus species was developed and tested on clinical cerebrospinal fluid (CSF) samples. The assay, termed MeningoFinder, showed an accordance of 97%, concordance of 96%, interlaboratory sensitivity of 90%, and interlaboratory specificity of 94% compared to PCRs.
Therapeutic Memory T Cells Require Costimulation for Effective Clearance of a Persistent Viral Infection
Authors: Garidou Lucile , Heydari Sara , Truong Phi , Brooks David G. , McGavern Dorian B. 
Published: June 2009

ABSTRACTPersistent viral infections are a major health concern worldwide. During persistent infection, overwhelming viral replication and the rapid loss of antiviral T-cell function can prevent immune-mediated clearance of the infection, and therapies to reanimate the immune response and purge persistent viruses have been largely unsuccessful. Adoptive immunotherapy using memory T cells is a highly successful therapeutic approach to eradicate a persistent viral infection. Understanding precisely how therapeutically administered memory T cells achieve clearance should improve our ability to terminate states of viral persistence in humans. Mice persistently infected from birth with lymphocytic choriomeningitis virus are tolerant to the pathogen at the T-cell level and thus provide an excellent model to evaluate immunotherapeutic regimens. Previously, we demonstrated that adoptively transferred memory T cells require recipient dendritic cells to effectively purge an established persistent viral infection. However, the mechanisms that reactivate and sustain memory T-cell responses during clearance of such an infection remain unclear. Here we establish that therapeutic memory T cells require CD80 and CD86 costimulatory signals to efficiently clear an established persistent viral infection in vivo. Early blockade of costimulatory pathways with CTLA-4-Fc decreased the secondary expansion of virus-specific CD8+and CD4+memory T cells as well as their ability to produce antiviral cytokines and purge the persistent infection. Late costimulation blockade also reduced virus-specific T-cell numbers, illustrating that sustained interactions with costimulatory molecules is required for efficient T-cell expansion. These findings indicate that antiviral memory T cells require costimulation to efficiently clear a persistent viral infection and that costimulatory pathways can be targeted to modulate the magnitude of an adoptive immunotherapeutic regimen.
Kaposi's Sarcoma-Associated Herpesvirus Protein LANA2 Disrupts PML Oncogenic Domains and Inhibits PML-Mediated Transcriptional Repression of the Survivin Gene
Authors: Marcos-Villar Laura , Lopitz-Otsoa Fernando , Gallego Pedro , Muñoz-Fontela Cesar , González-Santamaría José , Campagna Michela , Shou-Jiang Gao , Rodriguez Manuel S. , Rivas Carmen 
Published: June 2009

ABSTRACT
Infection by herpesviruses causes a dramatic disturbance of PML oncogenic domains (PODs) that has been suggested to be essential for viral lytic replication. Several proteins from Kaposi's sarcoma-associated herpesvirus (KSHV) have been tested as putative POD-disrupting factors with negative results. Here, we show that LANA2, a viral protein that is absolutely required for the viability and proliferation of KSHV-infected primary effusion lymphoma (PEL) cells, increases the levels of SUMO2-ubiquitin-modified PML and induces the disruption of PODs by a proteasome-mediated mechanism. In addition, we demonstrate that this disruption is largely dependent on both the integrity of a SUMO interaction motif in LANA2 and the lysine 160 from PML. Moreover, silencing of LANA2 expression in PEL cells by RNA interference led to an increase in the PML levels. Finally, we demonstrate that LANA2 relieves PML-mediated transcriptional repression of survivin, a protein that directly contributes to malignant progression of PEL. This represents the first example of inactivation of these important antiviral structures by KSHV.
Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation
Authors: Whitman Shannon D. , Smith Everett Clinton , Dutch Rebecca Ellis 
Published: June 2009

ABSTRACT
Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.
Proteolytic Activation of the Spike Protein at a Novel RRRR/S Motif Is Implicated in Furin-Dependent Entry, Syncytium Formation, and Infectivity of Coronavirus Infectious Bronchitis Virus in Cultured Cells
Authors: Yamada Yoshiyuki , Liu Ding Xiang 
Published: June 2009

ABSTRACT

The spike (S) protein of the coronavirus (CoV) infectious bronchitis virus (IBV) is cleaved into S1 and S2 subunits at the furin consensus motif RRFRR
537
/S in virus-infected cells. In this study, we observe that the S2 subunit of the IBV Beaudette strain is additionally cleaved at the second furin site (RRRR
690
/S) in cells expressing S constructs and in virus-infected cells. Detailed time course experiments showed that a peptide furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, blocked both viral entry and syncytium formation. Site-directed mutagenesis studies revealed that the S1/S2 cleavage by furin was not necessary for, but could promote, syncytium formation by and infectivity of IBV in Vero cells. In contrast, the second site is involved in the furin dependence of viral entry and syncytium formation. Mutations of the second site from furin-cleavable RRRR/S to non-furin-cleavable PRRRS and AAARS, respectively, abrogated the furin dependence of IBV entry. Instead, a yet-to-be-identified serine protease(s) was involved, as revealed by protease inhibitor studies. Furthermore, sequence analysis of CoV S proteins by multiple alignments showed conservation of an XXXR/S motif, cleavable by either furin or other trypsin-like proteases, at a position equivalent to the second IBV furin site. Taken together, these results suggest that proteolysis at a novel XXXR/S motif in the S2 subunit might be a common mechanism for the entry of CoV into cells.
A Structure-Guided Mutational Analysis of Simian Virus 40 Large T Antigen: Identification of Surface Residues Required for Viral Replication and Transformation
Authors: Ahuja Deepika , Rathi Abhilasha V. , Greer Amy E. , Chen Xiaojiang S. , Pipas James M. 
Published: June 2009

ABSTRACT
Simian virus 40 large T antigen (TAg) transforms cells in culture and induces tumors in rodents. Genetic studies suggest that TAg interaction with the chaperone hsp70 and tumor suppressors pRb and p53 may not be sufficient to elicit complete transformation of cells. In order to identify additional cellular factors important for transformation, we designed mutations on the solvent-exposed surface of TAg. We hypothesized that surface residues would interact directly with cellular targets and that the mutation of these residues might disrupt this interaction without perturbing TAg's global structure. Using structural data, we identified 61 amino acids on the surface of TAg. Each surface amino acid was changed to an alanine. Furthermore, five patches containing clusters of charged amino acids on the surface of TAg were identified. Within these patches, we selectively mutated three to four charged amino acids and thus generated five mutants (patch mutants 1 to 5). We observed that while patch mutants 3 and 4 induced foci in REF52 cells, patch mutants 1 and 2 were deficient in focus formation. We determined that the patch 1 mutant is defective in p53 binding, thus explaining its defect in transformation. The patch 2 mutant can interact with the Rb family members and p53 like wild-type TAg but is unable to transform cells, suggesting that it is defective for action on an unknown cellular target essential for transformation. Our results suggest that the histone acetyltransferase CBP/p300 is one of the potential targets affected by the mutations in patch 2.
Bluetongue Virus Targets Conventional Dendritic Cells in Skin Lymph
Authors: Hemati Behzad , Contreras Vanessa , Urien Céline , Bonneau Michel , Takamatsu Haru-Hisa , Mertens Peter P. C. , Bréard Emmanuel , Sailleau Corinne , Zientara Stéphan , Schwartz-Cornil Isabelle 
Published: June 2009

ABSTRACTBluetongue virus (BTV) is the etiological agent of bluetongue, a hemorrhagic disease of ruminants (particularly sheep), which causes important economic losses around the world. BTV is transmitted primarily via the bites of infected midges, which inject the virus into the ruminant's skin during blood feeding. The virus initially replicates in the draining lymph node and then disseminates to secondary organs where it induces edema, hemorrhages, and necrosis. In this study, we show that ovine conventional dendritic cells (cDCs) are the primary targets of BTV that contribute to the primary dissemination of BTV from the skin to draining lymph nodes. Lymph cDCs support BTV RNA and protein synthesis, as well as the production of infectious virus belonging to several different BTV serotypes, regardless of their level of attenuation. Afferent lymph cell subsets, other than cDCs, showed only marginal levels of BTV protein expression. BTV infection provoked a massive recruitment of cDCs to the sheep skin and afferent lymph, providing cellular targets for infection. Although BTV productively infects cDCs, no negative impact on their physiology was detected. Indeed, BTV infection and protein expression in cDCs enhanced their survival rate. Several serotypes of BTV stimulated the surface expression of the CD80 and CD86 costimulatory molecules on cDCs as well as the mRNA synthesis of cytokines involved in inflammation and immunity, i.e., interleukin-12 (IL-12), IL-1β, and IL-6. BTV-infected cDCs stimulated antigen-specific CD4 and CD8 proliferation as well as gamma interferon production. BTV initially targets cDCs while preserving their functional properties, reflecting the optimal adaptation of the virus to its host cells for its first spread.
Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E
Authors: Kiermayr Stefan , Stiasny Karin , Heinz Franz X. 
Published: June 2009

ABSTRACT
The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.
Brd4 Regulation of Papillomavirus Protein E2 Stability
Authors: Zheng Gang , Schweiger Michal-Ruth , Martinez-Noel Gustavo , Zheng Leon , Smith Jennifer A. , Harper J. Wade , Howley Peter M. 
Published: June 2009

ABSTRACT
The papillomavirus (PV) E2 protein is an important regulator of the viral life cycle. It has diverse roles in viral transcription, DNA replication, and genome maintenance. Our laboratory has previously identified the cellular bromodomain protein Brd4 as a key interacting partner of E2. Brd4 mediates the transcriptional activation function of E2 and plays an important role in viral genome maintenance in dividing cells. E2 interacts with the C-terminal domain (CTD) of Brd4, and the CTD functions in a dominant-negative manner through binding E2 and interfering with E2's interaction with the full-length Brd4 protein. Previous studies have shown that PV E2 proteins are short lived; however, the mechanisms regulating their stability and degradation have not yet been well established. In this study, we explored the role of Brd4 in the regulation of bovine PV 1 (BPV1) and human PV 16 (HPV16) E2 stability. Expression of the Brd4 CTD dramatically increases E2 levels. Both BPV1 E2 and HPV16 E2 are regulated by ubiquitylation, and Brd4 CTD expression blocks this ubiquitylation, thus stabilizing the E2 protein. Furthermore, we have identified the cullin-based E3 ligases and specifically cullin-3 as potential components of the ubiquitylation machinery that targets both BPV1 and HPV16 E2 for ubiquitylation. Expression of the Brd4 CTD blocks the interaction between E2 and the cullin-3 complex. In addition to Brd4's role in mediating E2 transcription and genome tethering activities, these data suggest a potential role for Brd4 in regulating E2 stability and protein levels within PV-infected cells.
gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue
Authors: Stoddard Earl , Ni Houping , Cannon Georgetta , Zhou Chunhui , Kallenbach Neville , Malamud Daniel , Weissman Drew 
Published: June 2009

ABSTRACTThe human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.