bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections
Authors: Soetens Oriane , Vauloup-Fellous Christelle , Foulon Ina , Dubreuil Pascal , De Saeger Ben , Grangeot-Keros Liliane , Naessens Anne 
Published: January 2008

ABSTRACT

Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (
P
= 0.036).
Outbreak Caused by a Multidrug-Resistant
Klebsiella pneumoniae
Clone Carrying
bla
VIM-12
in a University Hospital
Authors: Tokatlidou Despina , Tsivitanidou Maria , Pournaras Spyros , Ikonomidis Alexandros , Tsakris Athanassios , Sofianou Danae 
Published: January 2008

ABSTRACT

From November 2006 to April 2007, nine nonrepetitive isolates of
Klebsiella pneumoniae
with reduced susceptibility or resistance to carbapenems were recovered from clinical specimens from separate patients hospitalized in a tertiary care hospital. The imipenem-EDTA synergy test was positive for all isolates. PCR, sequencing, and transferability experiments revealed the novel
bla
VIM-12
metallo-β-lactamase gene, which was plasmid mediated and located in a class 1 integron. Pulsed-field gel electrophoresis demonstrated a single macrorestriction pattern, indicating the clonal spread of VIM-12-producing
K. pneumoniae
.
Evaluation of Eight Different Bioinformatics Tools To Predict Viral Tropism in Different Human Immunodeficiency Virus Type 1 Subtypes
Authors: Garrido Carolina , Roulet Vanessa , Chueca Natalia , Poveda Eva , Aguilera Antonio , Skrabal Katharina , Zahonero Natalia , Carlos Silvia , García Federico , Faudon Jean Louis , Soriano Vincent , de Mendoza Carmen 
Published: January 2008

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) tropism can be assessed using phenotypic assays, but this is quite laborious, expensive, and time-consuming and can be made only in sophisticated laboratories. More accessible albeit reliable tools for testing of HIV-1 tropism are needed in view of the prompt introduction of CCR5 antagonists in clinical practice. Bioinformatics tools based on V3 sequences might help to predict HIV-1 tropism; however, most of these methods have been designed by taking only genetic information derived from HIV-1 subtype B into consideration. The aim of this study was to evaluate the performances of several genotypic tools to predict HIV-1 tropism in non-B subtypes, as data on this issue are scarce. Plasma samples were tested using a new phenotypic tropism assay (Phenoscript-tropism; Eurofins), and results were compared with estimates of coreceptor usage using eight different genotypic predictor softwares (Support Vector Machine [SVM], C4.5, C4.5 with positions 8 to 12 only, PART, Charge Rule, geno2pheno coreceptor, Position-Specific Scoring Matrix X4R5 [PSSM
X4R5
], and PSSM
sinsi
). A total of 150 samples were tested, with 115 belonging to patients infected with non-B subtypes and 35 drawn from subtype B-infected patients, which were taken as controls. When non-B subtypes were tested, the concordances between the results obtained using the phenotypic assay and distinct genotypic tools were as follows: 78.8% for SVM, 77.5% for C4.5, 82.5% for C4.5 with positions 8 to 12 only, 82.5% for PART, 82.5% for Charge Rule, 82.5% for PSSM
X4R5
, 83.8% for PSSM
sinsi
, and 71.3% for geno2pheno. When clade B viruses were tested, the best concordances were seen for PSSM
X4R5
(91.4%), PSSM
sinsi
(88.6%), and geno2pheno (88.6%). The sensitivity for detecting X4 variants was lower for non-B than for B viruses, especially in the case of PSSM
sinsi
(38.4% versus 100%, respectively), SVM
wetcat
(46% versus 100%, respectively), and PART (30% versus 90%, respectively). In summary, while inferences of HIV-1 coreceptor usage using genotypic tools seem to be reliable for clade B viruses, their performances are poor for non-B subtypes, in which they particularly fail to detect X4 variants.
Major Uropathogenic
Escherichia coli
Strain Isolated in the Northwest of England Identified by Multilocus Sequence Typing
Authors: Lau Siu Ha , Reddy Suganya , Cheesbrough John , Bolton Frederick J. , Willshaw Geraldine , Cheasty Tom , Fox Andrew J. , Upton Mathew 
Published: January 2008

ABSTRACT

A total of 88 uropathogenic
Escherichia coli
isolates, including 68 isolates from urine and 20 isolates from blood, were characterized by multilocus sequence typing (MLST). MLST has identified an important genetic lineage of
E. coli
, designated sequence type 131 (ST-131), represented by 52 of these isolates, 51 of which were resistant to extended-spectrum cephalosporins. ST-131 appears to be a drug-resistant uropathogenic strain of
E. coli
responsible for causing urinary tract infections and bacteremia and is widely disseminated among both community and hospital patients from different geographical areas in the northwest of England. Application of MLST has helped to define the population biology which may underpin the epidemiology of pathogenic
E. coli
strains. The portability of MLST allows the accurate monitoring of this antibiotic-resistant uropathogenic strain of
E. coli
and will enhance surveillance for this important group of organisms.
Candida nivariensis
, an Emerging Pathogenic Fungus with Multidrug Resistance to Antifungal Agents
Authors: Borman Andrew M. , Petch Rebecca , Linton Christopher J. , Palmer Michael D. , Bridge Paul D. , Johnson Elizabeth M. 
Published: January 2008

ABSTRACT

In 2005,
Candida nivariensis
, a yeast species genetically related to
Candida glabrata
, was described following its isolation from three patients in a single Spanish hospital. Between 2005 and 2006, 16 fungal isolates with phenotypic similarities to
C. nivariensis
were submitted to the United Kingdom Mycology Reference Laboratory for identification. The strains originated from various clinical specimens, including deep, usually sterile sites, from patients at 12 different hospitals in the United Kingdom. PCR amplification and sequencing of the D1D2 and internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal gene cassette confirmed that these isolates from the United Kingdom are genetically identical to
C. nivariensis
. Biochemically,
C. glabrata
and
C. nivariensis
are distinguished by their differential abilities to assimilate trehalose. However, in contrast to the original published findings, we found that
C. glabrata
isolates, but not
C. nivariensis
isolates, are capable of assimilating this substrate. Antifungal susceptibility tests revealed that
C. nivariensis
isolates are less susceptible than
C. glabrata
isolates to itraconazole, fluconazole, and voriconazole and to have significantly higher flucytosine MICs than
C. glabrata
strains. Finally,
C. nivariensis
could be rapidly distinguished from the other common pathogenic fungus species by pyrosequencing of the ITS2 region. In the light of these data, we believe that
C. nivariensis
should be regarded as a clinically important emerging pathogenic fungus.
Human Papillomavirus Type 16 Variant Analysis of E6, E7, and L1 Genes and Long Control Region in Biopsy Samples from Cervical Cancer Patients in North India
Authors: Pande Shailja , Jain Neeraj , Prusty Bhupesh K. , Bhambhani Suresh , Gupta Sanjay , Sharma Rajyashri , Batra Swaraj , Das Bhudev C. 
Published: January 2008

ABSTRACTHigh-risk human papillomaviruses (HPVs), particularly HPV types 16 and 18 (HPV-16 and HPV-18, respectively), play a cardinal role in the etiology of cervical cancer. The most prevalent type, HPV-16, shows intratypic sequence variants that are known to differ in oncogenic potential and geographic distribution. This study was designed to analyze sequence variations in E6, E7, and L1 genes and the LCR (for long control region) of HPV-16 in cervical cancer patients to identify the most prevalent and novel HPV-16 variants and to correlate them with the severity of the disease. Cervical biopsies from 60 HPV-16-positive cancer cases were analyzed by PCR and DNA sequencing. The most frequently observed variations were T350G (100%) in E6, T789C (87.5%) in E7, A6695C (54.5%) in L1, and G7521A (91.1%) in the LCR. In addition, only one novel variant (T527A) in E6 and four new variants each in L1 (A6667C, A6691G, C6906T, and A6924C) and in the LCR (C13T, A7636C, C7678T, and G7799A) were identified. While E7 was found to be highly conserved, the variant 350G of E6 was the most prevalent in all of the histopathological grades. The majority of LCR variants were found at the YY1 transcription factor binding sites. Interestingly, a complete absence of the Asian lineage and a high prevalence of European lineages in E6, E7, L1, and the LCR (85, 86.7, 67.7, and 63.3%, respectively) indicate a possible epidemiological linkage between Europe and India with regard to the dissemination of HPV-16 infections in India.
Homogeneous Amplification of Genital Human Alpha Papillomaviruses by PCR Using Novel Broad-Spectrum GP5+ and GP6+ Primers
Authors: Schmitt Markus , Dondog Bolormaa , Waterboer Tim , Pawlita Michael 
Published: January 2008

ABSTRACT
Human papillomavirus (HPV) DNA detection and typing are important for diagnosis and management of HPV-associated diseases. One of the most commonly used PCR methods, GP5+/6+, shows weaknesses in amplifying certain types. To circumvent this limitation, we developed and validated broad-spectrum primers targeting the GP5+/6+ region. The addition of eight upstream and two downstream BSGP5+/6+ (BS) primers improved amplification of plasmids of 14 genital HPV types 10- to 1,000-fold versus GP5+/6+ PCR without altering sensitivity for the 10 others. For these 24 types, an analytic sensitivity of ≤1,000 plasmid copies in the presence of 100 ng cellular DNA was obtained. Additionally, we integrated an internal β-globin PCR into both HPV PCR systems, allowing simultaneous DNA quality control without affecting the sensitivity of HPV detection. Furthermore, we describe five additional low-risk HPV probes used in multiplex HPV genotyping (MPG) for simultaneous identification of all 15 high-risk, 3 putative high-risk, and 9 low-risk HPV genotypes. The performance of BSGP5+/6+ multiplexed with β-globin primers was compared to that of standard GP5+/6+ with DNA from 1,112 cervical scrapings. There was 79% overall agreement (kappa = 0.816). BSGP5+/6+ was significantly more sensitive than GP5+/6+ for detection of HPV 30, 39, 42, 44, 51, 52, 53, 68, 73, and 82, detecting 212 additional HPV infections and increasing the proportion of multiple infections from 17.2 to 26.9% in cancer patients. In conclusion, BSGP5+/6+ multiplexed with β-globin PCR provides an improvement in type-specific amplification sensitivity and homogeneity compared to GP5+/6+ and offers simultaneous internal control of DNA quality. BSGP5+/6+-MPG, therefore, is suitable for epidemiologic and also diagnostic applications.
Comparison of Results Obtained with Amplicor HIV-1 DNA PCR Test Version 1.5 Using 100 versus 500 Microliters of Whole Blood
Authors: Piwowar-Manning Estelle , Lugalia Lebah , Kafufu Bosco , Jackson J. Brooks 
Published: January 2008

ABSTRACT
The Amplicor HIV-1 DNA PCR assay (Roche Diagnostics, Branchburg, NJ) requires 500 μl of whole blood for a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, and this amount is often difficult to obtain from infants. A comparison was performed using 100 and 500 μl of whole blood from infants less than 18 months of age. The concordance rate for HIV DNA PCR-negative and -positive samples was 100% for the two different volumes.
Kaposi's Sarcoma-Associated Herpesvirus
ori
-
Lyt
-Dependent DNA Replication: Involvement of Host Cellular Factors
Authors: Wang Yan , Li Hong , Tang Qiyi , Maul Gerd G. , Yuan Yan 
Published: January 2008

ABSTRACT

Herpesvirus lytic DNA replication requires both the
cis
-acting element, the origin, and
trans
-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (
ori
-
Lyt
) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with
ori
-
Lyt
by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV
ori
-
Lyt
. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to
ori
-
Lyt
through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on
ori
-
Lyt
. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked
ori
-
Lyt
-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased
ori
-
Lyt
-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.
Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6
Authors: Dugan Gillian E. , Hewitt Eric W. 
Published: January 2008

ABSTRACTThe human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.
Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination
Authors: Balamurugan Arumugam , Lewis Martha J. , Kitchen Christina M. R. , Robertson Michael N. , Shiver John W. , Daar Eric S. , Pitt Jacqueline , Ali Ayub , Ng Hwee L. , Currier Judith S. , Yang Otto O. 
Published: January 2008

ABSTRACTVaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1gagDNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences fromgag,pol, andnefconfirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while thegagDNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.
Influenza A Virus Strains Differ in Sensitivity to the Antiviral Action of Mx-GTPase
Authors: Dittmann Jan , Stertz Silke , Grimm Daniel , Steel John , García-Sastre Adolfo , Haller Otto , Kochs Georg 
Published: January 2008

ABSTRACT
Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 “Spanish flu” virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the “Spanish flu” virus that may contribute to the high virulence of this unusual pandemic strain.
Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion
Authors: Howard Megan W. , Travanty Emily A. , Jeffers Scott A. , Smith M. K. , Wennier Sonia T. , Thackray Larissa B. , Holmes Kathryn V. 
Published: January 2008

ABSTRACTThe severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Δ19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Δ19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Δ19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.
Alteration of B-Cell Subsets Enhances Neuroinvasion in Mouse Scrapie Infection
Authors: von Poser-Klein Christine , Flechsig Eckhard , Hoffmann Tanja , Schwarz Petra , Harms Harry , Bujdoso Raymond , Aguzzi Adriano , Klein Michael A. 
Published: January 2008

ABSTRACTAcquired forms of prion diseases or transmissible spongiform encephalopathies are believed to occur following peripheral exposure. Prions initially accumulate in the lymphoid system before spreading to the nervous system, but the underlying mechanisms for prion transfer between the two systems are still elusive. Here we show that ablation of the B-cell-specific transmembrane protein CD19, a coreceptor of the complement system, results in an acceleration of prion neuroinvasion. This appears to be due to an alteration of the follicular dendritic cell (FDC) network within the lymphoid tissue, thereby reducing the distance between FDCs and adjacent nerve fibers that mediate prion neuroinvasion.