bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Papillomavirus Quantification in Urine and Cervical Samples by Using the Mx4000 and LightCycler General Real-Time PCR Systems
Authors: Payan Christopher , Ducancelle Alexandra , Aboubaker Mohamed H. , Caer Julien , Tapia Malena , Chauvin Amelie , Peyronnet Damien , Le Hen Elodie , Arab Zohra , Legrand Marie-Christine , Tran Adissa , Postec Edith , Tourmen Françoise , Avenel Martine , Malbois Chantal , De Brux Marie-Anne , Descamps Philippe , Lunel Francoise 
Published: January 2007

ABSTRACT

During the last decade, growing efforts have focused on human papillomavirus (HPV) detection using liquid hybridization, conventional PCR, and real-time PCR-based methods to increase the overall proportion of patients participating in cervical cancer screening procedures. We proposed a new general HPV DNA real-time PCR on the Mx4000 (Stratagene) and LightCycler (Roche Diagnostics) systems usable for both cervical scrape specimens and urine samples. A linear range was obtained from 5 DNA copies to 8 log
10
DNA copies/ml, and intra- and interassay variations were between 1.8 and 4%. Cervical carcinoma and HPV DNA screening was performed in 333 individual women referred for gynecological examination at the university hospitals of Angers and Brest and enrolled in the PapU study. Among cervical specimens (
n
= 333), 45% were positive for HPV DNA, with a mean viral load at 5.00 log/ml (± 1.73). Among urine samples (
n
= 177), 37% were positive with a significant 50-fold-lower mean viral load (3.77 ± 1.32 log/ml;
P
< 0.0001). Kappa agreement for HPV DNA between cervical and urine specimens was excellent (93%). Thus, we developed a highly sensitive and quantitative general HPV DNA real-time PCR method that allows mass screening of patients with HPV infection. The ongoing longitudinal and prospective multicenter PapU study should give us the opportunity to validate this method adapted to HPV DNA screening in urine samples in a larger population.
Use of Capillary Blood Samples as a New Approach for Diagnosis of Dengue Virus Infection
Authors: Matheus Séverine , Meynard Jean-Baptiste , Lacoste Vincent , Morvan Jacques , Deparis Xavier 
Published: January 2007

ABSTRACT
We evaluated the use of capillary blood samples stored on filter papers for diagnosis of dengue virus infection. Venous and capillary blood samples were collected from 130 patients suspected of having dengue fever. We compared the performances of standard reference methods using capillary blood samples absorbed onto filter papers versus venous blood samples. The resulting sensitivity, specificity, and positive predictive value of tests performed on filter paper compared to those performed on venous blood samples were 81.6% (62/76; 95% confidence interval [CI], 74.9% to 88.3%), 90.7% (49/54; 95% CI, 85.7% to 95.7%), and 92.5% (62/67; 95% CI, 86.2% to 98.8%), respectively. During the acute phase of dengue virus infection (day 1 to day 4), the tests performed on capillary blood samples had a sensitivity of 88.5% (95% CI, 82.0% to 95.0%) and a specificity of 93.8% (95% CI, 88.9% to 98.7%). During the convalescent phase of infection, this method allowed the viral serotype to be determined for 4 of 15 (27%) dengue virus-infected patients for whom virological diagnosis using venous samples was negative. Capillary blood samples could therefore be a good alternative for the diagnosis of dengue virus infection in tropical areas. Indeed, these samples are convenient for storage and transport without the need for a cold chain and simplify the collection of samples from children. Moreover, our results suggest that viral particles persist longer in capillary blood than in peripheral blood. Analysis of the viability of viral particles under these conditions may give new insights into the physiopathology of dengue virus infection and the transmission of dengue virus during outbreaks.
Evaluation of Combined General Primer-Mediated PCR Sequencing and Type-Specific PCR Strategies for Determination of Human Papillomavirus Genotypes in Cervical Cell Specimens
Authors: Fontaine Véronique , Mascaux Corinne , Weyn Christine , Bernis Aurore , Celio Nathalie , Lefèvre Philippe , Kaufman Leonard , Garbar Christian 
Published: January 2007

ABSTRACT
A strategy combining human papillomavirus general primer (mainly the PGMY primers)-directed PCR sequencing and type-specific PCR is presented. DNA samples were first tested in general primer-mediated PCR. The amplified fragments of positive samples after ethidium bromide-stained DNA gel analysis were further sequenced, and corresponding DNA samples were further analyzed by PCR using type-specific primers for human papillomavirus (HPV) types 16, 18, 31, and 52. The comparison of the results of 157 samples analyzed by this strategy in parallel with the Hybrid Capture 2 tests and with the HPV INNO-LiPA (Innogenetics line probe assay) shows that this method is suitable for HPV detection and genotyping in cervical cell samples. Although the PCR sequencing method is as sensitive as the HPV INNO-LiPA for HPV detection, our method allows the identification of a broader range of HPV types. In contrast, the HPV INNO-LiPA was less time-consuming and better identified coinfections.
Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis
Authors: Ward Katherine N. , Leong Hoe Nam , Thiruchelvam Anton D. , Atkinson Claire E. , Clark Duncan A. 
Published: January 2007

ABSTRACT

The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log
10
copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log
10
copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (≥3.5 log
10
copies/ml) or whole blood (≥6.0 log
10
copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log
10
copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log
10
copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood.
Generic Detection of Coronaviruses and Differentiation at the Prototype Strain Level by Reverse Transcription-PCR and Nonfluorescent Low-Density Microarray
Authors: de Souza Luna Luciano Kleber , Heiser Volker , Regamey Nicolas , Panning Marcus , Drexler Jan Felix , Mulangu Sabue , Poon Leo , Baumgarte Sigrid , Haijema Bert Jan , Kaiser Laurent , Drosten Christian 
Published: January 2007

ABSTRACT
A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.
Expression of Rotavirus NSP4 Alters the Actin Network Organization through the Actin Remodeling Protein Cofilin
Authors: Berkova Zuzana , Crawford Sue E. , Blutt Sarah E. , Morris Andrew P. , Estes Mary K. 
Published: January 2007

ABSTRACT
Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.
The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis
Authors: Mach Michael , Osinski Karolina , Kropff Barbara , Schloetzer-Schrehardt Ursula , Krzyzaniak Magdalena , Britt William 
Published: January 2007

ABSTRACT
Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.
Role of the Coronavirus E Viroporin Protein Transmembrane Domain in Virus Assembly
Authors: Ye Ye , Hogue Brenda G. 
Published: January 2007

ABSTRACT
Coronavirus envelope (E) proteins are small (∼75- to 110-amino-acid) membrane proteins that have a short hydrophilic amino terminus, a relatively long hydrophobic membrane domain, and a long hydrophilic carboxy-terminal domain. The protein is a minor virion structural component that plays an important, not fully understood role in virus production. It was recently demonstrated that the protein forms ion channels. We investigated the importance of the hydrophobic domain of the mouse hepatitis coronavirus (MHV) A59 E protein. Alanine scanning insertion mutagenesis was used to examine the effect of disruption of the domain on virus production in the context of the virus genome by using a MHV A59 infectious clone. Mutant viruses exhibited smaller plaque phenotypes, and virus production was significantly crippled. Analysis of recovered viruses suggested that the structure of the presumed α-helical structure and positioning of polar hydrophilic residues within the predicted transmembrane domain are important for virus production. Generation of viruses with restored wild-type helical pitch resulted in increased virus production, but some exhibited decreased virus release. Viruses with the restored helical pitch were more sensitive to treatment with the ion channel inhibitor hexamethylene amiloride than were the more crippled parental viruses with the single alanine insertions, suggesting that disruption of the transmembrane domain affects the functional activity of the protein. Overall the results indicate that the transmembrane domain plays a crucial role during biogenesis of virions.
Contrasting Effects of Human, Canine, and Hybrid Adenovirus Vectors on the Phenotypical and Functional Maturation of Human Dendritic Cells: Implications for Clinical Efficacy
Authors: Perreau Matthieu , Mennechet Franck , Serratrice Nicolas , Glasgow Joel N. , Curiel David T. , Wodrich Harald , Kremer Eric J. 
Published: January 2007

ABSTRACTAntipathogen immune responses create a balance between immunity, tolerance, and immune evasion. However, during gene therapy most viral vectors are delivered in substantial doses and are incapable of expressing gene products that reduce the host's ability to detect transduced cells. Gene transfer efficacy is also modified by the in vivo transduction of dendritic cells (DC), which notably increases the immunogenicity of virions and vector-encoded genes. In this study, we evaluated parameters that are relevant to the use of canine adenovirus serotype 2 (CAV-2) vectors in the clinical setting by assaying their effect on human monocyte-derived DC (hMoDC). We compared CAV-2 to human adenovirus (HAd) vectors containing the wild-type virion, functional deletions in the penton base RGD motif, and the CAV-2 fiber knob. In contrast to the HAd type 5 (HAd5)-based vectors, CAV-2 poorly transduced hMoDC, provoked minimal upregulation of major histocompatibility complex class I/II and costimulatory molecules (CD40, CD80, and CD86), and induced negligible morphological changes indicative of DC maturation. Functional maturation assay results (e.g., reduced antigen uptake; tumor necrosis factor alpha, interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-10, IL-12, and IFN-α/β secretion; and stimulation of heterologous T-cell proliferation) were also significantly lower for CAV-2. Our data suggested that this was due, in part, to the use of an alternative receptor and a block in vesicular escape. Additionally, HAd5 vector-induced hMoDC maturation was independent of the aforementioned cytokines. Paradoxically, an HAd5/CAV-2 hybrid vector induced the greatest phenotypical and functional maturation of hMoDC. Our data suggest that CAV-2 and the HAd5/CAV-2 vector may be the antithesis ofAdenoviridaeimmunogenicity and that each may have specific clinical advantages.
Immunoglobulin A (IgA) Is a Natural Ligand of Hepatitis A Virus Cellular Receptor 1 (HAVCR1), and the Association of IgA with HAVCR1 Enhances Virus-Receptor Interactions
Authors: Tami Cecilia , Silberstein Erica , Manangeeswaran Mohanraj , Freeman Gordon J. , Umetsu Sarah E. , DeKruyff Rosemarie H. , Umetsu Dale T. , Kaplan Gerardo G. 
Published: January 2007

ABSTRACT
The hepatitis A virus cellular receptor 1 (HAVCR1/TIM1), a member of the T-cell immunoglobulin mucin (TIM) family, is an important atopy susceptibility gene in humans. The exact natural function of HAVCR1/TIM1 and the inverse association between HAV infection and prevention of atopy are not well understood. To identify natural ligands of human HAVCR1/TIM1, we used an expression cloning strategy based on the binding of dog cells transfected with a human lymph node cDNA library to a HAVCR1/TIM1 Fc fusion protein. The transfected cells that bound to the human HAVCR1/TIM1 Fc contained cDNA of human immunoglobulin alpha 1 heavy (Igα1) and lambda light (Igλ) chain and secreted human IgA1λ antibody that bound to the cell surface. Cotransfection of the isolated Igα1 and Igλ cDNAs to naïve dog cells resulted in the secretion of IgA1λ that bound to HAVCR1/TIM1 Fc but not to a poliovirus receptor Fc fusion protein in a capture enzyme-linked immunosorbent assay. The interaction of HAVCR1/TIM1 with IgA was inhibited by monoclonal antibodies (MAbs) against Igα1 and Igλ, excess IgA1λ, or anti-HAVCR1/TIM1 MAb. IgA did not inhibit HAV infection of African green monkey cells, suggesting that the IgA and the virus binding sites are in different epitopes on HAVCR1/TIM1. IgA enhanced significantly the neutralization of HAV by HAVCR1/TIM1 Fc. Our results indicate that IgA1λ is a specific ligand of HAVCR1/TIM1 and that their association has a synergistic effect in virus-receptor interactions.
Frequency and Phenotype of JC Virus-Specific CD8+T Lymphocytes in the Peripheral Blood of Patients with Progressive Multifocal Leukoencephalopathy
Authors: Lima Marco A. , Marzocchetti Angela , Autissier Patrick , Tompkins Troy , Chen Yiping , Gordon Jennifer , Clifford David B. , Gandhi Rajesh T. , Venna Nagagopal , Berger Joseph R. , Koralnik Igor J. 
Published: January 2007

ABSTRACTJC virus (JCV)-specific CD8+cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+T cells specific for two A*0201-restricted JCV epitopes, VP1p36and VP1p100, and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+patients with PML (HIV+PML patients) classified as survivors. After magnetic preenrichment of CD8+T cells, epitope-specific cells ranged from 0.001% to 0.22% by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+T cells in healthy individuals, HIV+subjects, or HIV+PML patients. However, in one HIV+PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+and two HIV+PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1p100CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+individuals with PML.
5′-Proximal Hot Spot for an Inducible Positive-to-Negative-Strand Template Switch byCoronavirus RNA-Dependent RNA Polymerase
Authors: Wu Hung-Yi , Brian David A. 
Published: January 2007

ABSTRACT

Coronaviruses have a positive-strand RNA genome and replicate through the use of a
3′ nested set of subgenomic mRNAs each possessing a leader (65
to 90 nucleotides [nt] in length, depending on the viral species)
identical to and derived from the genomic leader. One widely supported
model for leader acquisition states that a template switch takes place
during the generation of negative-strand antileader-containing
templates used subsequently for subgenomic mRNA synthesis. In this
process, the switch is largely driven by canonical heptameric donor
sequences at intergenic sites on the genome that match an acceptor
sequence at the 3′ end of the genomic leader. With
experimentally placed 22-nt-long donor sequences within a bovine
coronavirus defective interfering (DI) RNA we have shown that matching
sites occurring anywhere within a 65-nt-wide 5′-proximal
genomic acceptor hot spot (nt 33 through 97) can be used for production
of templates for subgenomic mRNA synthesis from the DI RNA. Here we
report that with the same experimental approach, template switches can
be induced in
trans
from an internal site in the DI RNA to the
negative-strand antigenome of the helper virus. For these, a
3′-proximal 89-nt acceptor hot spot on the viral antigenome (nt
35 through 123), largely complementary to that described above, was
found. Molecules resulting from these switches were not templates for
subgenomic mRNA synthesis but, rather, ambisense chimeras potentially
exceeding the viral genome in length. The results suggest the existence
of a coronavirus 5′-proximal partially double-stranded template
switch-facilitating structure of discrete width that contains both the
viral genome and
antigenome.
Poxvirus-Encoded Gamma Interferon Binding Protein Dampens the Host Immune Response to Infection
Authors: Sakala Isaac G. , Chaudhri Geeta , Buller R. Mark , Nuara Anthony A. , Bai Hongdong , Chen Nanhai , Karupiah Gunasegaran 
Published: January 2007

ABSTRACTEctromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpΔ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects.