bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluating the Orthopoxvirus Type I Interferon-Binding Molecule as a Vaccine Target in the Vaccinia Virus Intranasal Murine Challenge Model
Authors: Golden Joseph W. , Hooper Jay W. 
Published: September 2010

ABSTRACT
The biological threat imposed by orthopoxviruses warrants the development of safe and effective vaccines. We developed a candidate orthopoxvirus DNA-based vaccine, termed 4pox, which targets four viral structural components, A33, B5, A27, and L1. While this vaccine protects mice and nonhuman primates from lethal infections, we are interested in further enhancing its potency. One approach to enhance potency is to include additional orthopoxvirus immunogens. Here, we investigated whether vaccination with the vaccinia virus (VACV) interferon (IFN)-binding molecule (IBM) could protect BALB/c mice against lethal VACV challenge. We found that vaccination with this molecule failed to significantly protect mice from VACV when delivered alone. IBM modestly augmented protection when delivered together with the 4pox vaccine. All animals receiving the 4pox vaccine plus IBM lived, whereas only 70% of those receiving a single dose of 4pox vaccine survived. Mapping studies using truncated mutants revealed that vaccine-generated antibodies spanned the immunoglobulin superfamily domains 1 and 2 and, to a lesser extent, 3 of the IBM. These antibodies inhibited IBM cell binding and IFN neutralization activity, indicating that they were functionally active. This study shows that DNA vaccination with the VACV IBM results in a robust immune response but that this response does not significantly enhance protection in a high-dose challenge model.
Multilocus Sequence Typing Identifies Evidence for Recombination and Two Distinct Lineages of
Corynebacterium diphtheriae
Authors: Bolt Frances , Cassiday Pamela , Tondella Maria Lucia , DeZoysa Aruni , Efstratiou Androulla , Sing Andreas , Zasada Aleksandra , Bernard Kathryn , Guiso Nicole , Badell Edgar , Rosso Marie-Laure , Baldwin Adam , Dowson Christopher 
Published: September 2010

ABSTRACT

We describe the development of a multilocus sequence typing (MLST) scheme for
Corynebacterium diphtheriae
, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize
C. diphtheriae
, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic
C. diphtheriae
strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of
C. diphtheriae
examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.
High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in
Mycobacterium tuberculosis
Clinical Isolates
Authors: Choi Go Eun , Lee Sun Min , Yi Jongyoun , Hwang Sang Hyun , Kim Hyung Hoi , Lee Eun Yup , Cho Eun Hae , Kim Jee Hee , Kim Hwa-Jung , Chang Chulhun L. 
Published: September 2010

ABSTRACT

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in
Mycobacterium tuberculosis
in an accurate, affordable, and rapid manner. Two hundred seventeen
M. tuberculosis
clinical isolates of known resistance phenotype were used. Twenty-nine known
rpoB
mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed:
rpoB
-F/R (for codons 516 to 539 of
rpoB
),
rpoB
-516F/R (for codons 508 to 536 of
rpoB
),
katG
-F/R (for the codon 315 region of
katG
), and
inhA
-F/R (for the nucleotide substitution of C to T at position −15 of
inhA
). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of
rpoB
. Twenty-seven of 29 known
rpoB
mutants were detected. In HRM curve analysis of
katG
and
inhA
, 90 INH-R strains that harbored
katG
or
inhA
mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring
katG
or
inhA
mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in
M. tuberculosis
in a rapid, accurate, simple, and cost-effective manner.
Phylogeny and Identification of
Nocardia
Species on the Basis of Multilocus Sequence Analysis
Authors: McTaggart L. R. , Richardson S. E. , Witkowska M. , Zhang S. X. 
Published: September 2010

ABSTRACT

Nocardia
species identification is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA and cellular fatty acid analysis to discriminate many species, and the unreliability of biochemical testing. Here,
Nocardia
species identification was achieved through multilocus sequence analysis (MLSA) of gyrase B of the β subunit of DNA topoisomerase (
gyrB
), 16S rRNA (16S), subunit A of SecA preprotein translocase (
secA1
), the 65-kDa heat shock protein (
hsp65
), and RNA polymerase (
rpoB
) applied to 190 clinical, 36 type, and 11 reference strains. Phylogenetic analysis resolved 30 sequence clusters with high (>85%) bootstrap support. Since most clusters contained a single type strain and the analysis corroborated current knowledge of
Nocardia
taxonomy, the sequence clusters were equated with species clusters and MLSA was deemed appropriate for species identification. By comparison, single-locus analysis was inadequate because it failed to resolve species clusters, partly due to the presence of foreign alleles in 22.1% of isolates. While MLSA identified the species of the majority (71.3%) of strains, it also identified clusters that may correspond to new species. The correlation of the identities by MLSA with those determined on the basis of microscopic examination, biochemical testing, and fatty acid analysis was 95%; however, MLSA was more discriminatory.
Nocardia cyriacigeorgica
(21.58%) and
N. farcinica
(14.74%) were the most frequently encountered species among clinical isolates. In summary, five-locus MLSA is a reliable method of elucidating taxonomic data to inform
Nocardia
species identification; however, three-locus (
gyrB
-16S-
secA1
) or four-locus (
gyrB
-16S-
secA1
-
hsp65
) MLSA was nearly as reliable, correctly identifying 98.5% and 99.5% of isolates, respectively, and would be more feasible for routine use in a clinical reference microbiology laboratory.
Rapid Differentiation of
Mycobacterium tuberculosis
and
M. bovis
by High-Resolution Melt Curve Analysis
Authors: Ereqat Suheir , Bar-Gal Gila Kahila , Nasereddin Abedelmajeed , Azmi Kifaya , Qaddomi Sharif E. , Greenblatt Charles L. , Spigelman Mark , Abdeen Ziad 
Published: September 2010

ABSTRACT

Identification and characterization of the
Mycobacterium tuberculosis
strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between
M. tuberculosis
and
Mycobacterium
bovis
based on one single-nucleotide polymorphism (SNP) in the
narGHJI
and
oxyR
genes and determination of
M. bovis
based on the region of differences 1 (RD1). This assay correctly identified the 7 tested
Mycobacterium
reference strains and 52 clinical samples with a sensitivity of 2 pg DNA. This assay will help in prescribing adequate treatment and monitoring disease dynamics.
Carbapenem Heteroresistance in VIM-1-Producing
Klebsiella pneumoniae
Isolates Belonging to the Same Clone: Consequences for Routine Susceptibility Testing
Authors: Tato M. , Morosini M. , García L. , Albertí S. , Coque M. T. , Cantón R. 
Published: September 2010

ABSTRACT

Susceptibility results with low reproducibility by the same or different methods have been observed for metallo-beta-lactamase (MBL)-producing
Enterobacteriaceae
. Eighteen VIM-1-producing
Klebsiella pneumoniae
isolates (one per patient) belonging to a single epidemic clone in our hospital (2005 to 2008) but with different susceptibilities to carbapenems were studied. Imipenem MICs ranged from 8 to >128 mg/liter by standard CLSI microdilution, from ≤1 to >8 mg/liter by the semiautomatic Wider system, and from 0.75 to >32 mg/liter by Etest. Meropenem MICs ranged from 0.5 to 128, ≤1 to >8, and 0.38 to >32 mg/liter, respectively. Ertapenem MICs by CLSI microdilution and Etest ranged from 1 to 64 and 0.75 to >32 mg/liter, respectively. The rates of essential agreement (±1 log
2
dilution) for imipenem and meropenem MICs between the Wider system and the reference microdilution method were 45% and 49%, respectively. Those between Etest and the reference microdilution method for imipenem, meropenem, and ertapenem MICs were 33%, 67%, and 84%. The rates of very major errors for the Wider system and Etest were 33% and 28% for imipenem and 25% and 75% for meropenem, respectively. Low MIC reproducibility was observed even when the same inoculum was used (differences up to 4-fold dilutions). Heteroresistance was suspected due to the presence of colonies in the Etest inhibition zone. It was confirmed by population analysis profiles of 4 isolates displaying different imipenem MICs, with the exception of an OmpK36-porin-deficient isolate that homogeneously expressed carbapenem resistance (MIC, >128 mg/liter). Low carbapenem MIC reproducibility could be due to the presence of resistant subpopulations and variable expression of the resistance mechanisms. Since carbapenem MICs are not good markers of MBL production, reliable and reproducible phenotypic methods are needed to detect the presence of this mechanism.
Unique Hepatitis B Virus Subgenotype in a Primitive Tribal Community in Eastern India
Authors: Ghosh Sumantra , Banerjee Priyanka , RoyChoudhury Arindam , Sarkar Sumanta , Ghosh Alip , Santra Amal , Banerjee Soma , Das Kausik , Dwibedi Bhagirathi , Kar Shantanu K. , Rao Vikas Gangadhar , Bhat Jyothi T. , Singh Neeru , Chowdhury Abhijit , Datta Simanti 
Published: September 2010

ABSTRACT
Hepatitis B virus (HBV) strains isolated from members of the primitive Paharia ethnic community of Eastern India were studied to gain insight into the genetic diversity and evolution of the virus. The Paharia tribe has remained quite separate from the rest of the Indians and differs culturally, genetically, and linguistically from the mainstream East Indian population, whose HBV strains were previously characterized. Full-length HBV DNA was PCR amplified, cloned, and sequenced. Phylogenetic relationships between the tribal sequences and reference sequences from the mainstream population were assessed, and divergence times of subgenotypes of HBV genotype D were estimated. HBV was found in 2% of the Paharias participating in the study. A predominance of hepatitis B e antigen-negative infection (73%) was observed among the Paharias, and the genome sequences of the HBV strains exhibited relative homogeneity, with a very low prevalence of mutations. The novel feature of Paharia HBV was the exclusive presence of the D5 subgenotype, which was recently identified in Eastern India. Analysis of the four open reading frames (ORFs) of these tribal HBV D5 sequences and comparison with previously reported D1 to D7 sequences enabled the identification of 27 specific amino acid residues, including 6 unique ones, that could be considered D5 signatures. The estimated divergence times among subgenotypes D1 to D5 suggest that D5 was the first to diverge and hence is the most ancient of the D subgenotypes. The presence of a specific, ancient subgenotype of HBV within an ethnically primitive, endogamous population highlights the importance of studies of HBV genetics in well-separated human populations to understand viral transmission between communities and genome evolution.
Detection of Oseltamivir Resistance during Treatment of 2009 H1N1 Influenza Virus Infection in Immunocompromised Patients: Utility of Cycle Threshold Values of Qualitative Real-Time Reverse Transcriptase PCR
Authors: Mehta Tejal , McGrath Eric , Bheemreddy Suchitha , Salimnia Hossein , Abdel-Haq Nahed , Ang Jocelyn Y. , Lum Lawrence , Chandrasekar Pranatharthi , Alangaden George J. 
Published: September 2010

ABSTRACT
Two immunocompromised patients with 2009 H1N1 influenza pneumonia had viral shedding for over 5 weeks despite therapy with oseltamivir. Declining or persistently low cycle threshold values noted on serial qualitative real-time reverse transcriptase PCR (rRT-PCR) of respiratory specimens implied increasing viral load and probable drug resistance. Oseltamivir resistance was later confirmed by pyrosequencing.
Better Detection of
Staphylococcus aureus
Nasal Carriage by Use of Nylon Flocked Swabs
Authors: Verhoeven Paul , Grattard Florence , Carricajo Anne , Pozzetto Bruno , Berthelot Philippe 
Published: September 2010

ABSTRACT

Flocked swabs (Copan) were compared to rayon swabs (Copan) for the nasal detection of
Staphylococcus aureus
in 90 healthy volunteers sampled sequentially during a 5-week period. The use of flocked swabs improved the number of nasal carriers (
P
= 0.026), the number of positive specimens (
P
= 0.01), and the quantity of bacteria in positive samples (
P
= 0.004).
Regulation of Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Receptor Protein (TWEAKR) Expression by Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Prevents TWEAK-Induced Apoptosis and Inflammatory Cytokine Expression
Authors: Abend Johanna R. , Uldrick Thomas , Ziegelbauer Joseph M. 
Published: September 2010

ABSTRACT

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, the second most common AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs) during latency. To identify cellular targets of KSHV miRNAs, we have analyzed a previously reported series of microarrays examining changes in cellular gene expression in the presence of KSHV miRNAs. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor (TWEAKR) was among the most consistently and robustly downregulated genes in the presence of KSHV miR-K12-10a (miR-K10a). Results from luciferase assays with reporter plasmids containing the 3′ untranslated region (UTR) of TWEAKR suggest a targeting of TWEAKR by miR-K10a. The mutation of two predicted miR-K10a recognition sites within the 3′ UTR of TWEAKR completely disrupts inhibition by miR-K10a. The expression of TWEAKR was downregulated in cells transfected with miR-K10a as well as during
de novo
KSHV infection. In a KS tumor-derived endothelial cell line, the downregulation of TWEAKR by miR-K10a resulted in reduced levels of TWEAK-induced caspase activation. In addition, cells transfected with miR-K10a showed less induction of apoptosis by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Finally, the downregulation of TWEAKR by miR-K10a in primary human endothelial cells resulted in a decrease in levels of expression of the proinflammatory cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to TWEAK. These results identify and validate an important cellular target of KSHV miRNAs. Furthermore, we demonstrate that a viral miRNA protects cells from apoptosis and suppresses a proinflammatory response, which may have significant implications in the complex context of KS lesions.
Antagonism of Type I Interferon Responses by New World Hantaviruses
Authors: Levine Jessica R. , Prescott Joseph , Brown Kyle S. , Best Sonja M. , Ebihara Hideki , Feldmann Heinz 
Published: September 2010

ABSTRACT
Evasion of interferon (IFN)-mediated antiviral signaling is a common defense strategy for pathogenic RNA viruses. To date, research on IFN antagonism by hantaviruses is limited and has focused on only a subset of the numerous recognized hantavirus species. The host IFN response has two phases, an initiation phase, resulting in the induction of alpha/beta IFN (IFN-α/β), and an amplification phase, whereby IFN-α/β signals through the Jak/STAT pathway, resulting in the establishment of the cellular antiviral state. We examined interactions between these critical host responses and the New World hantaviruses. We observed delayed cellular responses in both Andes virus (ANDV)- and Sin Nombre virus (SNV)-infected A549 and Huh7-TLR3 cells. We found that IFN-β induction is inhibited by coexpression of ANDV nucleocapsid protein (NP) and glycoprotein precursor (GPC) and is robustly inhibited by SNV GPC alone. Downstream amplification by Jak/STAT signaling is also inhibited by SNV GPC and by either NP or GPC of ANDV. Therefore, ANDV- and SNV-encoded proteins have the potential for inhibiting both IFN-β induction and signaling, with SNV exhibiting the more potent antagonism ability. Herein we identify ANDV NP, a previously unrecognized inhibitor of Jak/STAT signaling, and show that IFN antagonism by ANDV relies on expression of both the glycoproteins and NP, whereas the glycoproteins appear to be sufficient for antagonism by SNV. These data suggest that IFN antagonism strategies by hantaviruses are quite variable, even between species with similar disease phenotypes, and may help to better elucidate species-specific pathogenesis.
Potent and Broadly Reactive HIV-2 Neutralizing Antibodies Elicited by a Vaccinia Virus Vector Prime-C2V3C3 Polypeptide Boost Immunization Strategy
Authors: Marcelino José Maria , Borrego Pedro , Rocha Cheila , Barroso Helena , Quintas Alexandre , Novo Carlos , Taveira Nuno 
Published: September 2010

ABSTRACTHuman immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.
Whole-Genome Characterization of Human and Simian Immunodeficiency Virus Intrahost Diversity by Ultradeep Pyrosequencing
Authors: Bimber Benjamin N. , Dudley Dawn M. , Lauck Michael , Becker Ericka A. , Chin Emily N. , Lank Simon M. , Grunenwald Haiying L. , Caruccio Nicholas C. , Maffitt Mark , Wilson Nancy A. , Reed Jason S. , Sosman James M. , Tarosso Leandro F. , Sanabani Sabri , Kallas Esper G. , Hughes Austin L. , O'Connor David H. 
Published: September 2010

ABSTRACTRapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.
Interaction of Hantavirus Nucleocapsid Protein with Ribosomal Protein S19
Authors: Haque Absarul , Mir Mohammad A. 
Published: September 2010

ABSTRACT

Hantaviruses, members of the
Bunyaviridae
family, are emerging category A pathogens that initiate the translation of their capped mRNAs by a novel mechanism mediated by viral nucleocapsid protein (N). N specifically binds to the mRNA 5′ m7G cap and 40S ribosomal subunit, a complex of 18S rRNA and multiple ribosomal proteins. Here, we show that N specifically interacts with the ribosomal protein S19 (RPS19), located at the head region of the 40S subunit. We suggest that this N-RPS19 interaction facilitates ribosome loading on capped mRNAs during N-mediated translation initiation.
Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance
Authors: Fradet-Turcotte Amélie , Moody Cary , Laimins Laimonis A. , Archambault Jacques 
Published: September 2010

ABSTRACT

The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication
in vivo
but dispensable
in vitro
. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.