bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissemination of a single Vibrio cholerae clone in cholera outbreaks during 2005 in Iran
Authors: Pourshafie M. R. , Bakhshi B. , Ranjbar R. , Sedaghat M. , Sadeghifard N. , Zaemi Yazdi J. , Parzadeh M. , Raesi J.
Published: November 2007

In this study, 50 Vibrio cholerae O1 serotype Inaba isolates were collected during several cholera outbreaks throughout Iran during the summer of 2005. The results of antibiotic susceptibility testing showed that 86, 84, 84 and 82 % of the isolates were resistant to streptomycin, chloramphenicol, co-trimoxazole and tetracycline, respectively. The strains were genotyped using randomly amplified polymorphic DNA (RAPD), PFGE and ribotyping techniques. PCR showed that 100, 98 and 98 % carried the ctx, zot and ace genes, respectively. Biochemical fingerprinting of the isolates using the PhenePlate (PhP) system showed a low diversity index level (0.755), suggesting that the strains were highly homogeneous. Among the strains, 100 and 96 % showed an identical ribotype and PFGE patterns, respectively. The two isolates showing different PFGE patterns also exhibited discrete PhP types. RAPD was able to discriminate the isolates into six distinct groups, suggesting some genetic dissimilarity was present among the strains. These ribotyping, PFGE and PhP techniques revealed the clonal dissemination of a single V. cholerae strain throughout Iran in 2005.
Evidence for a clonally different origin of the two cholera epidemics of 2001–2002 and 1980–1987 in South Africa
Authors: Keddy Karen H. , Nadan Sandrama , Govind Chetna , Sturm A. Willem ,   for the Group for Enteric, Respiratory and Meningeal Disease Surveillance in South Africa
Published: November 2007

Vibrio cholerae O1 serotype Ogawa and serotype Inaba isolates from the cholera epidemic that occurred in 2001 and 2002 in South Africa were compared with isolates of V. cholerae O1 serotype Inaba from the epidemic that occurred between 1980 and 1987. PFGE using NotI digestion was used to compare stored isolates received during the 1980s epidemic with those received during the epidemic in 2001/2002. A selected number of these isolates were then sequenced to compare the sequence of the wbeT gene in the V. cholerae O1 Ogawa strains of 2001/2002 with that in the V. cholerae O1 Inaba strains of the 1980s and 2001/2002. Isolates from the recent epidemic were shown to be related, irrespective of serotype, and had comparable banding patterns on PFGE, using NotI. They were distinctly different from those from the previous epidemic. Sequencing of the wbeT gene showed that the gene was highly conserved between the two epidemics. A single deletional mutation of an adenine residue was observed in the V. cholerae serotype Inaba isolates from the 2001/2002 epidemic, resulting in the serotype switch between the V. cholerae O1 strains from the recent epidemic. The distinct differences in PFGE patterns among isolates from the first and second epidemics exclude the possibility that the Inaba strain from the 1980s became dormant in the environment and mutated to serotype Ogawa, causing the 2001/2002 epidemic, despite the apparent consistency in the site of mutation in the Inaba serotypes between the two epidemics.
Epidemiological, biochemical and antimicrobial susceptibility characteristics of Streptococcus pseudoporcinus isolated in Quebec, Canada, from 1997 to 2006
Authors: Gaudreau Christiane , Simoneau Esther , Labrecque Olivia , Laurence Robert A. , Laferrière Céline , Miller Mark , Raynal Lélia , Rallu Fabien
Published: November 2007

From 1997 to 2006, in the province of Quebec, Canada, 15 isolates of Streptococcus
pseudoporcinus from 1 urine and 14 vaginorectal cultures were recovered from the genitourinary tract of pregnant women. All these women originated from the Caribbean or sub-Saharan Africa (P=0.00045 compared with a suitable control group). The S. pseudoporcinus isolates were compared to eight isolates of Streptococcus porcinus identified in Quebec from 1995 to 2006, all from animals, of which five were swine. 16S rRNA gene sequencing was required to differentiate between S. pseudoporcinus and S. porcinus animal isolates.
An iron-regulated outer-membrane protein of Proteus mirabilis is a haem receptor that plays an important role in urinary tract infection and in in vivo growth
Authors: Lima Analía , Zunino Pablo , D'Alessandro Bruno , Piccini Claudia
Published: November 2007

Proteus mirabilis, a common cause of urinary tract infections, expresses iron-regulated outer-membrane proteins (OMPs) in response to iron restriction. It has been suggested that a 64 kDa OMP is involved in haemoprotein uptake and that this might have a role in pathogenesis. In order to confirm this hypothesis, this study generated a P. mirabilis mutant strain (P7) that did not express the 64 kDa OMP, by insertion of the TnphoA transposon. The nucleotide sequence of the interrupted gene revealed that it corresponded to a haemin receptor precursor. Moreover, in vitro growth assays showed that the mutant was unable to grow using haemoglobin and haemin as unique iron sources. The authors also carried out in vivo growth and infectivity assays and demonstrated that P7 was not able to survive in an in vivo model and was less efficient than wild-type strain Pr 6515 in colonizing the urinary tract. These results confirmed that the P. mirabilis 64 kDa iron-regulated OMP is a haem receptor that has an important role for survival and multiplication of these bacteria in the mammalian host and in the development of urinary tract infection.
Use of an HIV-1 reverse-transcriptase enzyme-activity assay to measure HIV-1 viral load as a potential alternative to nucleic acid-based assay for monitoring antiretroviral therapy in resource-limited settings
Authors: Iqbal H. Syed , Balakrishnan P. , Cecelia Anitha J. , Solomon Suniti , Kumarasamy N. , Madhavan Vidya , Murugavel K. G. , Ganesh Aylur K. , Solomon Sunil Suhas , Mayer Kenneth H. , Crowe Suzanne M.
Published: November 2007

An inexpensive and technically less-demanding methodology to quantify HIV-1 viral load would be of great value for resource-limited settings, where the nucleic-acid amplification technique (NAAT) is impractical and/or resource-prohibitive. In this study, an HIV-1 reverse-transcriptase enzyme-activity assay (ExaVir Load assay, version 1) was compared with the gold standard RT-PCR assay, Roche HIV-1 Amplicor Monitor, version 1.5. A total of 121 plasma specimens were used for the evaluation. ExaVir Load had a sensitivity of 97 % and a specificity of 71 % in identifying specimens with <400 copies ml−1 in the Roche RT-PCR assay as being less than the detection limit of the assay (5500 copies ml−1). The mean difference (95 % limits of agreement) between Roche RT-PCR and ExaVir Load was –0.23 (−1.59 to 1.13) log10(copies ml−1) by Bland–Altman analysis. Significant negative correlations were seen between CD4+ T-cell counts and the ExaVir Load assay (r=−0.32, P<0.05), and between CD4+ T-cell counts and the Roche RT-PCR (r=−0.38, P<0.01). The present study with HIV-1 showed a strong correlation between the ExaVir Load assay and the RT-PCR assay. Hence, the ExaVir Load assay could be considered for use in resource-limited settings as an alternative viral-load assay to the standard NAAT-based assay after further evaluation with prospective specimens.
Xenopus microRNA genes are predominantly located within introns and are differentially expressed in adult frog tissues via post-transcriptional regulation
Authors: Tang Guo-Qing , Maxwell E. Stuart
Published: November 2007

The amphibian Xenopus provides a model organism for investigating microRNA expression during vertebrate embryogenesis and development. Searching available Xenopus genome databases using known human pre-miRNAs as query sequences, more than 300 genes encoding 142 Xenopus tropicalis miRNAs were identified. Analysis of Xenopus tropicalis miRNA genes revealed a predominate positioning within introns of protein-coding and nonprotein-coding RNA Pol II-transcribed genes. MiRNA genes were also located in pre-mRNA exons and positioned intergenically between known protein-coding genes. Many miRNA species were found in multiple locations and in more than one genomic context. MiRNA genes were also clustered throughout the genome, indicating the potential for the cotranscription and coordinate expression of miRNAs located in a given cluster. Northern blot analysis confirmed the expression of many identified miRNAs in both X. tropicalis and X. laevis. Comparison of X. tropicalis and X. laevis blots revealed comparable expression profiles, although several miRNAs exhibited species-specific expression in different tissues. More detailed analysis revealed that for some miRNAs, the tissue-specific expression profile of the pri-miRNA precursor was distinctly different from that of the mature miRNA profile. Differential miRNA precursor processing in both the nucleus and cytoplasm was implicated in the observed tissue-specific differences. These observations indicated that post-transcriptional processing plays an important role in regulating miRNA expression in the amphibian Xenopus.
Genomic copy number and expression variation within the C57BL/6J inbred mouse strain
Authors: Watkins-Chow Dawn E. , Pavan William J.
Published: November 2007

The C57BL/6J strain is one of the most widely used animal models for biomedical research, and individual mice within the strain are often assumed to be genetically identical after more than 70 yr of inbreeding. Using a single nucleotide polymorphism (SNP) genotyping panel, we assessed if copy number variations (CNVs) could be detected within the C57BL/6J strain by comparing relative allele frequencies in first generation (F1) progeny of C57BL/6J mice. Sequencing, quantitative PCR, breeding, and array comparative genomic hybridization (CGH) together confirmed the presence of two CNVs. Both CNVs span genes encoded on chromosome 19, and quantitative RT-PCR demonstrated that they result in altered expression of the insulin-degrading enzyme (Ide) and fibroblast growth factor binding protein 3 (Fgfbp3) genes. Analysis of 39 different C57BL/6J breeders revealed that 64% of mice from the Jackson Laboratory colony were heterozygous for the CNV spanning Ide. Homozygotes with and without the duplication were present in concordance with Hardy-Weinberg equilibrium (13% and 23%, respectively), and analysis of archived samples from the C57BL/6J colony suggests that the duplication has rapidly reached a high frequency in the colony since 1994. The identification of two CNVs in the small portion of the genome screened demonstrates that individual mice of highly inbred strains are not isogenic and suggests other CNVs may be segregating within C57BL/6J as well as other carefully maintained inbred strains. These differences can influence interpretations of physiological, biomedical, and behavioral experiments and can be exploited to model CNVs apparent in the human genome.
Intercountry adoption and nursing care
Authors: Rykkje Linda
Published: November 2007

Aims and objectives:  This paper is based on a Norwegian study that addressed the importance of nurses understanding adoptive families’ particular needs. The study aimed to provide a diverse picture of adoptive families’ challenges and resources. It addressed adoptive families’ experiences with welfare professions, particularly public health nurse services, and explored their need for support before and after adoption. This paper focuses particularly on challenges confronting ‘transracial’ families.

Participants and method:  The qualitative research interview was used as the methodological approach. Nine couples of adoptive parents, four adoptive mothers, three public health nurses and four adult adoptees were interviewed. The analysis process was an ad hoc generation of meaning, and the data were systematically reviewed and categorized through an intuitive analysis style.

Results:  The study indicates that adoptive families manage their ‘most common’ challenges on their own, but the informants also spoke about a need for professional support and assistance in many cases. Recurring themes in the study were challenges linked to ‘attachment difficulties’, ‘external categorization’, ‘grief’ and ‘the meaning of biological heritage and roots’. Findings indicate that there is a substantial demand for increased knowledge of adoptive families’ special needs, not least in the public health nurse services; there is also limited knowledge within other welfare professions. Nurses particularly need to strengthen their understanding on how external categorization affects transracial adoptive families.

Conclusions:  The study recommendations call for improved follow‐up after adoptions. This can be met by establishing a special programme for adoptive families at maternity and child health centres. In addition, public health nurses can support adoptees in their teenage years through individual consultations.
Encountering the older confused patient: professional carers’ experiences
Authors: Stenwall Ewa , Sandberg Jonas , Jönhagen Maria Eriksdotter , Fagerberg Ingegerd
Published: November 2007

The study’s rationale:  Confusion is a common condition among older patients and often a fearful experience. Opinions vary as to how to communicate with and care for confused patients and professional carers often find the patients’ situation almost as distressing as the patients’ themselves do.

Aims and objectives:  The aim of this study was to describe professional carers’ experiences of their encounters with older confused patients.

Methodological design and justification:  Data was collected from interviews with 10 professional carers working on a ward specializing in the care of older confused patients. A descriptive phenomenological research approach was used to gather knowledge of professional carers’ experiences of encounters with older confused patients.

Ethical issues and approval:  A Regional Board of Research Ethics granted ethical permission for the study. The appropriate ethical principles were followed. The participants were contacted personally and received a letter providing information on the study. Written consent was requested before the interview. If needed, the participants were able to get in contact with the staff health service for a follow‐up after the interview. Names or places have been changed in order to ensure confidentiality.

Results:  The encounter with the confused patient is experienced as an encounter with an unfamiliar person, where the patients’ actions and words are unforeseeable and with a lack of immediate trust. The essential meaning is further illuminated by the meaning constituents: the unforeseeable encounter, always being on guard and using oneself as a tool.

Relevance to clinical practice:  This paper focuses on the importance of encouraging professional carers to pay attention to the complexity of the encounter with the confused patient, reflecting upon their own behaviour within these encounters and the importance of knowledge of the patient’s preferred senses and life stories. Caring for confused patients involves a great responsibility where both the professional carers and the patients are vulnerable and exposed.
Host‐use patterns of saproxylic phloeophagous and xylophagous Coleoptera adults and larvae along the decay gradient in standing dead black spruce and aspen
Authors: Saint‐Germain Michel , Drapeau Pierre , M. Buddle Christopher
Published: November 2007

Wood‐feeding insects play important functional roles in forest ecosystems, contributing significantly to wood decay processes. However, sampling these species in a direct and quantitative way is difficult because they live most of their lives as larvae deep into the wood; knowledge of species‐specific host‐use patterns along the decay gradient is thus lacking in this group. To cope with these difficulties, we used a novel approach, snag dissection, to investigate occurrence patterns of such Coleoptera adults and larvae. We selected 80 snags of both black spruce and aspen along four classes of decay in five different stands distributed over the tree species’ ranges within the province of Quebec, Canada, and dissected a one‐meter section of each. All adults and larvae of Buprestidae, Cerambycidae and Scolytinae (Coleoptera: Curculionidae) were collected and identified to the lowest taxonomical level possible. Wood density and snag age were also calculated for each sampled snag. In black spruce, host‐use was mostly concentrated at the beginning of the decay gradient. Patterns observed in aspen were opposite, as few insects were found in fresh snags, while most snags in middle to late stages of decay contained insects, often in large numbers, in some reaching densities of over 1000 cerambycid larvae m−3. For both tree species, patterns observed were similar across regions sampled. Differences in host‐use patterns between the coniferous and deciduous host species may be due to differences in secondary chemistry, mechanical defence mechanisms or the stand dynamics typically associated with each tree species.
Behavioural mechanisms that undermine species envelope models: the causes of patchiness in the distribution of great bustardsOtis tardain Spain
Authors: Osborne Patrick E. , Suárez‐Seoane Susana , Carlos Alonso Juan
Published: November 2007

Despite the general success of species envelope models, capturing the fine‐scale detail of patchiness in the distributions of some species is problematic. For great bustards in Spain, apparently suitable habitat patches remain unoccupied and cannot be distinguished from occupied patches in current distribution models. We consider philopatry and conspecific attraction as main behavioural mechanisms which could account for this patchiness, and then look for evidence of their influence on the distribution of great bustards across the whole of Spain. We compared the characteristics of habitat patches classed as suitable by a distribution model according to whether they were actually used or not. Occupied patches were larger than unoccupied patches and over‐used in proportion to their size, suggesting aggregation and a metapopulation structure. Arguing that conspecific attraction may serve to transfer information about site history and environmental predictability (at least over a short time period), we compared the coefficients of variation in time‐series of vegetation and climatic factors at occupied and unoccupied sites. Great bustards chose sites which were more environmentally stable at critical periods in the breeding cycle, “public information” that can only be gained from others rather than through sampling. There is thus evidence that both metapopulation dynamics and conspecific attraction influence the large scale distribution of great bustards in Spain. We discuss how alternative predictor variables and multi‐stage analyses may help us to incorporate behavioural mechanisms into distribution models, but acknowledge that there are limits to the value of species envelope models for animal species making decisions.
Inconsistencies Between Pangean Reconstructions and Basic Climate Controls
Authors: Rowe Clinton M. , Loope David B. , Oglesby Robert J. , Van der Voo Rob , Broadwater Charles E.
Published: November 2007

The supercontinent Pangea dominated our planet from the Permian into the Jurassic. Paleomagnetic reconstructions have been used to estimate the latitudinal position of Pangea during this 100-million-year period. Atmospheric circulation, recorded by eolian sandstones in the southwestern United States, shows a broad sweep of northeasterly winds over their northernmost extent, curving to become northwesterly in the south: This evidence is consistent with paleomagnetic reconstructions of the region straddling the equator in the Early Permian but is at odds with its northward movement to about 20°N by the Early Jurassic. At least one of the following scenarios must be true: The latitude based on paleomagnetism is incorrect; the interpretation of how winds shaped the dunes is mistaken; the basic climate controls in the Jurassic were different from those of today; or the paleogeographic reconstructions available are insufficient to adequately reproduce the wind fields responsible for dune formation.
Evaluation of Two Types of Sponges Used To Collect Cervical Secretions and Assessment of Antibody Extraction Protocols for Recovery of Neutralizing Anti-Human Papillomavirus Type 16 Antibodies
Authors: Kemp Troy J. , Hildesheim Allan , Falk Roni T. , Schiller John T. , Lowy Douglas R. , Rodriguez Ana Cecilia , Pinto Ligia A.
Published: November 2007

Immunogenicity evaluations in human papillomavirus (HPV) vaccine trials have relied on serological samples, yet cervical antibodies are likely to be most relevant for protection against infection. In order to assess functional antibody levels at the cervix, the secreted-alkaline-phosphatase neutralization assay (SEAPNA) was used to measure HPV-neutralizing activity. We assessed the variability of the SEAPNA with serum samples after vaccination with an HPV type 16 (HPV16) L1 virus-like particle vaccine and whether the SEAPNA can be used to monitor neutralizing activity at the cervix. The SEAPNA has an overall coefficient of variation of 29.3%. Recovery from ophthalmic sponges was assessed by spiking V5 (mouse anti-HPV16) antibody onto and extracting it from sterile Merocel and Ultracell sponges and sponges used to collect specimens from participants. V5 recovery from sterile Merocel sponges was complete, yet that from Ultracell sponges was null. The mean V5 recoveries from participant Ultracell and Merocel sponges were 61.2% and 93.5%, respectively, suggesting that Merocel sponges are more appropriate for specimen collection. The SEAPNA can be applied to determine the surrogates of protection and to examine the durability of protection at the cervix.
Cloning, Expression, and Immunological Characterization of the P30 Protein of
Mycoplasma pneumoniae
Authors: Varshney Avanish Kumar , Chaudhry Rama , Kabra Sushil Kumar , Malhotra Pawan
Published: November 2007

Mycoplasma pneumoniae
, a self-replicating cell wall-deficient prokaryote, has a differentiated terminal organelle that is essential for cytadherence and gliding motility. P30, an important protein associated with the terminal organelle, is required for the cytadherence and virulence of
M. pneumoniae
. P30 is a transmembrane protein with an intracytoplasmic N terminus and an exposed C terminus. In the present study, we amplified and sequenced the full-length
p30
gene of
Mycoplasma pneumoniae
directly from 18 Indian asthmatic patients. Sequence diversity was observed in the
p30
genes from 16 clinical samples when the sequences were compared with the sequence of strain M-129. We also successfully expressed a fragment of the
p30
gene (P30B) that includes the complete C-terminal proline-rich amino acid sequences in different
Escherichia coli
expression systems. The maltose binding protein (MBP)-P30B fusion protein was recognized by
M. pneumoniae
-infected patient sera in immunoblots, and the protein was immunogenic in mice. We further analyzed the reactivity of the MBP-P30B fusion protein with patient sera in an enzyme-linked immunosorbent assay (ELISA) and compared it with the reactivity obtained with a commercial kit (the Serion ELISA Classic kit). The sensitivity and the specificity of the in-house ELISA were 78.57% and 89.47%, respectively. This study suggests that the P30 protein can be used as an antigen along with other adhesin proteins for the immunodiagnosis of
M. pneumoniae
infection.
Serotype-Specific and Age-Dependent Generation of Pneumococcal Polysaccharide-Specific Memory B-Cell and Antibody Responses to Immunization with a Pneumococcal Conjugate Vaccine
Authors: Clutterbuck Elizabeth A. , Oh Sarah , Hamaluba Mainga , Westcar Sharon , Beverley Peter C. L. , Pollard Andrew J.
Published: November 2007

Glycoconjugate vaccines have dramatically reduced the incidence of encapsulated bacterial diseases in toddlers under 2 years of age, but vaccine-induced antibody levels in this age group wane rapidly. We immunized adults and 12-month-old toddlers with heptavalent pneumococcal conjugate vaccine to determine differences in B-cell and antibody responses. The adults and 12-month-old toddlers received a pneumococcal conjugate vaccine. The toddlers received a second dose at 14 months of age. The frequencies of diphtheria toxoid and serotype 4, 14, and 23F polysaccharide-specific plasma cells and memory B cells were determined by enzyme-linked immunospot assay. The toddlers had no preexisting polysaccharide-specific memory B cells or serum immunoglobulin G (IgG) antibody but had good diphtheria toxoid-specific memory responses. The frequencies of plasma cells and memory B cells increased by day 7 (P< 0.0001) in the adults and the toddlers following a single dose of conjugate, but the polysaccharide responses were significantly lower in the toddlers than in the adults (P= 0.009 to <0.001). IgM dominated the toddler antibody responses, and class switching to the IgG was serotype dependent. A second dose of vaccine enhanced the antibody and memory B-cell responses in the toddlers but not the ex vivo plasma cell responses. Two doses of pneumococcal conjugate vaccine are required in toddlers to generate memory B-cell frequencies and antibody class switching for each pneumococcal polysaccharide equivalent to that seen in adults.
Effects of Lipopolysaccharide andMannheimia haemolyticaLeukotoxin on Bovine Lung Microvascular Endothelial Cells and Alveolar Epithelial Cells
Authors: McClenahan David , Hellenbrand Katrina , Atapattu Dhammika , Aulik Nicole , Carlton David , Kapur Arvinder , Czuprynski Charles
Published: November 2007

Bovine respiratory disease resulting from infection withMannheimia haemolyticacommonly results in extensive vascular leakage into the alveoli.M. haemolyticaproduces two substances, lipopolysaccharide (LPS) and leukotoxin (LKT), that are known to be important in inducing some of the pathological changes. In the present study, we examined bovine pulmonary epithelial (BPE) cell and bovine lung microvascular endothelial cell monolayer permeability, as measured by trans-well endothelial and epithelial cell electrical resistance (TEER), after incubation with LPS, LKT, or LPS-activated neutrophils. Endothelial cell monolayers exposed to LPS exhibited significant decreases in TEER that corresponded with increased levels of proinflammatory cytokines, apoptosis, and morphological changes. In contrast, BPE cells exposed to LPS increased the levels of production of inflammatory cytokines but displayed no changes in TEER, apoptosis, or visible morphological changes. Both cell types appeared to express relatively equal levels of the LPS ligand Toll-like receptor 4. However, TEER in BPE cell monolayers was decreased when the cells were incubated with LPS-activated neutrophils. Although the incubation of BPE cells with LKT decreased TEER, this was not reduced by the incubation of LKT with a neutralizing antibody and was reversed when LKT was preincubated with the LPS-neutralizing compound polymyxin B. Because BPE cells did not express the LKT receptor CD11a/CD18, we infer that contaminating LPS was responsible for the decreased TEER. In conclusion, LPS triggered changes in endothelial cells that would be consistent with vascular leakage, but neither LPS nor LKT caused similar changes in epithelial cells, unless neutrophils were also present.
Development of a Fluorescent-Microsphere Immunoassay for Detection of Antibodies Specific to Equine Arteritis Virus and Comparison with the Virus Neutralization Test
Authors: Go Yun Young , Wong Susan J. , Branscum Adam J. , Demarest Valerie L. , Shuck Kathleen M. , Vickers Mary L. , Zhang Jianqiang , McCollum William H. , Timoney Peter J. , Balasuriya Udeni B. R.
Published: November 2007

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5
1-255
], M
1-162
, and N
1-110
), as well as partial sequences of these structural proteins (GP5
1-116
, GP5
75-112
, GP5
55-98
, M
88-162
, and N
1-69
) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in
Escherichia coli
were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5
55-98
protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5
55-98
MIA and VNT outcomes correlated significantly (
r
= 0.84;
P
< 0.0001). Although the GP5
55-98
MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.
Sensitive and Specific Enzyme-Linked Immunosorbent Assay Using Chemiluminescence for Detection of Severe Acute Respiratory Syndrome Viral Infection
Authors: Fujimoto Kotaro , Chan Kwok-Hung , Takeda Kazuhiko , Lo Kam-Fai , Leung Raymond H. K. , Okamoto Takashi
Published: November 2007

Here we report the development of a more-sensitive immunoassay for severe acute respiratory syndrome (SARS) based on an enzyme-linked immunosorbent assay using chemiluminescence (CLEIA) to detect the viral nucleocapsid (N) antigen in nasopharyngeal aspirate (NPA) from patients infected with SARS coronavirus (CoV). The CLEIA was established with an optical combination of monoclonal antibodies (MAbs) against SARS CoV N protein prepared from mice immunized with recombinant N protein without cultivating the virus. The capture and detecting MAbs of the CLEIA reacted to the carboxyl-terminal and amino-terminal peptides of the N protein, respectively. The CLEIA was capable of detecting recombinant N protein at 1.56 pg/ml and viral N protein in SARS CoV cell culture lysates at 0.087 of 50% tissue culture infective doses/ml. The CLEIA showed no cross-reactivities to recombinant N proteins of common human CoV (229E, OC43, and NL63) or lysates of cells infected with 229E and OC43. In addition, an evaluation with 18 SARS-positive NPA samples, all confirmed SARS positive by quantitative PCR and antibodies to SARS CoV, revealed that all (18/18) were found positive by the CLEIA; thus, the sensitivity of detection was 100%. When we tested 20 SARS-negative NPA samples, the CLEIA was shown to have high specificity (100%). The sensitivity of our novel SARS CLEIA was significantly higher than the previous EIA and comparable to the other methods using reverse transcription-PCR.
Testing Strategy To Identify Cases of Acute Hepatitis C Virus (HCV) Infection and To Project HCV Incidence Rates
Authors: Page-Shafer Kimberly , Pappalardo Brandee L. , Tobler Leslie H. , Phelps Bruce H. , Edlin Brian R. , Moss Andrew R. , Wright Teresa L. , Wright David J. , O'Brien Thomas R. , Caglioti Sally , Busch Michael P.
Published: November 2007

Surveillance for hepatitis C virus (HCV) is limited by the challenge of differentiating between acute and chronic infections. In this study, we evaluate a cross-sectional testing strategy that identifies individuals with acute HCV infection and we estimate HCV incidence. Anti-HCV-negative persons from four populations with various risks, i.e., blood donors, Veterans Administration (VA) patients, young injection drug users (IDU), and older IDU, were screened for HCV RNA by minipool or individual sample nucleic acid testing (NAT). The number of detected viremic seronegative infections was combined with the duration of the preseroconversion NAT-positive window period (derived from analysis of frequent serial samples from plasma donors followed from NAT detection to seroconversion) to estimate annual HCV incidence rates. Projected incidence rates were compared to observed incidence rates. Projected HCV incidence rates per 100 person-years were 0.0042 (95% confidence interval [95% CI], 0.0025 to 0.007) for blood donors, 0.86 (95% CI, 0.02 to 0.71) for VA patients, 39.8 (95% CI, 25.9 to 53.7) for young IDU, and 53.7 (95% CI, 23.4 to 108.8) for older IDU. Projected rates were most similar to observed incidence rates for young IDU (33.4; 95% CI, 28.0 to 39.9). This study demonstrates the value of applying a cross-sectional screening strategy to detect acute HCV infections and to estimate HCV incidence.
Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major
Brucella
Clades
Authors: Foster Jeffrey T. , Okinaka Richard T. , Svensson Rita , Shaw Kathryn , De Barun K. , Robison Richard A. , Probert William S. , Kenefic Leo J. , Brown William D. , Keim Paul
Published: November 2007

Members of the genus
Brucella
are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven
Brucella
species. The genomes of the various
Brucella
species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main
Brucella
species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined
Brucella
clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major
Brucella
groups that will be valuable for clinical and forensic applications.
Rapid and Reliable Diagnostic Algorithm for Detection of
Clostridium difficile
Authors: Fenner Lukas , Widmer Andreas F. , Goy Gisela , Rudin Sonja , Frei Reno
Published: November 2007

We evaluated a two-step algorithm for detection of
Clostridium difficile
in 1,468 stool specimens. First, specimens were screened by an immunoassay for
C. difficile
glutamate dehydrogenase antigen (C.DIFF CHEK-60). Second, screen-positive specimens underwent toxin testing by a rapid toxin A/B assay (TOX A/B QUIK CHEK); toxin-negative specimens were subjected to stool culture. This algorithm allowed final results for 92% of specimens with a turnaround time of 4 h.
Multiplex PCR for Rapid Detection of
Staphylococcus aureus
Isolates Suspected to Represent Community-Acquired Strains
Authors: Strommenger B. , Braulke C. , Pasemann B. , Schmidt C. , Witte W.
Published: November 2007

The continuous spread of community-acquired methicillin-resistant
Staphylococcus aureus
(caMRSA) and the introduction of these highly virulent isolates into hospitals represent increasing threats. The timely recognition of caMRSA strains is crucial for infection control purposes. Thus, we developed a PCR-based assay for the easy and rapid determination of those caMRSA clones that currently are the most prevalent in Germany and Central Europe. This assay was able to correctly identify the majority of the isolates as caMRSA of sequence type 80 (ST80), clonal complex 1 (USA400), and ST8 (USA300). In combination with
spa
typing-BURP (based upon repeat pattern) analysis and resistance typing, it provides a means for the extensive characterization of suspicious isolates. Thus, this assay represents a reliable tool for monitoring the emergence and spread of different caMRSA clones. The resulting information, in combination with careful interpretation of the epidemiological records, might help to prevent the further spread of those highly virulent caMRSA clones.
In Vitro Susceptibility of Invasive Isolates of
Candida
spp. to Anidulafungin, Caspofungin, and Micafungin: Six Years of Global Surveillance
Authors: Pfaller M. A. , Boyken L. , Hollis R. J. , Kroeger J. , Messer S. A. , Tendolkar S. , Diekema D. J.
Published: November 2007

The echinocandins are being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to the echinocandins among invasive
Candida
sp. isolates is indicated. We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 5,346 invasive (bloodstream or sterile-site) isolates of
Candida
spp. collected from over 90 medical centers worldwide from 1 January 2001 to 31 December 2006. We performed susceptibility testing according to the CLSI M27-A2 method and used RPMI 1640 broth, 24-h incubation, and a prominent inhibition endpoint for determination of the MICs. Of 5,346 invasive
Candida
sp. isolates, species distribution was 54%
C. albicans
, 14%
C. parapsilosis
, 14%
C. glabrata
, 12%
C. tropicalis
, 3%
C. krusei
, 1%
C. guilliermondii
, and 2% other
Candida
spp. Overall, all three echinocandins were very active against
Candida
: anidulafungin (MIC
50
, 0.06 μg/ml; MIC
90
, 2 μg/ml), caspofungin (MIC
50
, 0.03 μg/ml; MIC
90
, 0.25 μg/ml), micafungin (MIC
50
, 0.015 μg/ml; MIC
90
, 1 μg/ml). More than 99% of isolates were inhibited by ≤2 μg/ml of all three agents. Results by species (expressed as the percentages of isolates inhibited by ≤2 μg/ml of anidulafungin, caspofungin, and micafungin, respectively) were as follows: for
C. albicans
, 99.6%, 100%, and 100%; for
C. parapsilosis
, 92.5%, 99.9%, and 100%; for
C. glabrata
, 99.9%, 99.9%, and 100%; for
C. tropicalis
, 100%, 99.8%, and 100%; for
C. krusei
, 100%, 100%, and 100%; and for
C. guilliermondii
, 90.2%, 95.1%, and 100%. There was no significant change in the activities of the three echinocandins over the 6-year study period and no difference in activity by geographic region. All three echinocandins have excellent in vitro activities against invasive strains of
Candida
isolated from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of
Candida
spp.
Comparison of BD GeneOhm Methicillin-Resistant
Staphylococcus aureus
(MRSA) PCR versus the CHROMagar MRSA Assay for Screening Patients for the Presence of MRSA Strains
Authors: Boyce John M. , Havill Nancy L.
Published: November 2007

We compared the BD GeneOhm methicillin-resistant
Staphylococcus aureus
(MRSA) real-time PCR assay with the CHROMagar MRSA assay for the detection of MRSA in 286 nasal surveillance specimens. Compared with the CHROMagar MRSA assay, PCR had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 98.6%, 95.8%, and 100%, respectively. The mean PCR turnaround time was 14.5 h.
Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding
Authors: Zhang Baoshan , Sun Chengqun , Jin Sha , Cascio Michael , Montelaro Ronald C.
Published: November 2007

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.
A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice
Authors: Jiao Peirong , Tian Guobin , Li Yanbing , Deng Guohua , Jiang Yongping , Liu Chang , Liu Weilong , Bu Zhigao , Kawaoka Yoshihiro , Chen Hualan
Published: November 2007

In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.
Rotavirus Infection Induces the Phosphorylation of eIF2α but Prevents the Formation of Stress Granules
Authors: Montero Hilda , Rojas Margarito , Arias Carlos F. , López Susana
Published: November 2007

Early during the infection process, rotavirus causes the shutoff of cell protein synthesis, with the nonstructural viral protein NSP3 playing a vital role in the phenomenon. In this work, we have found that the translation initiation factor 2α (eIF2α) in infected cells becomes phosphorylated early after virus infection and remains in this state throughout the virus replication cycle, leading to a further inhibition of cell protein synthesis. Under these restrictive conditions, however, the viral proteins and some cellular proteins are efficiently translated. The phosphorylation of eIF2α was shown to depend on the synthesis of three viral proteins, VP2, NSP2, and NSP5, since in cells in which the expression of any of these three proteins was knocked down by RNA interference, the translation factor was not phosphorylated. The modification of this factor is, however, not needed for the replication of the virus, since mutant cells that produce a nonphosphorylatable eIF2α sustained virus replication as efficiently as wild-type cells. In uninfected cells, the phosphorylation of eIF2α induces the formation of stress granules, aggregates of stalled translation complexes that prevent the translation of mRNAs. In rotavirus-infected cells, even though eIF2α is phosphorylated these granules are not formed, suggesting that the virus prevents the assembly of these structures to allow the translation of its mRNAs. Under these conditions, some of the cellular proteins that form part of these structures were found to change their intracellular localization, with some of them having dramatic changes, like the poly(A) binding protein, which relocates from the cytoplasm to the nucleus in infected cells, a relocation that depends on the viral protein NSP3.
Induction of Particle Polymorphism by
Cucumber Necrosis Virus
Coat Protein Mutants In Vivo
Authors: Kakani Kishore , Reade Ron , Katpally Umesh , Smith Thomas , Rochon D'Ann
Published: November 2007

The
Cucumber necrosis virus
(CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles.
Identification of Two Virus Integration Sites in the Brown Alga
Feldmannia
Chromosome
Authors: Meints Russel H. , Ivey Richard G. , Lee Amy M. , Choi Tae-Jin
Published: November 2007

Two similar, large double-stranded DNA viruses,
Feldmannia
species virus 158 (FsV-158) and FsV-178, replicate only in the unilocular reproductive cells (sporangia) of a brown filamentous alga in the genus
Feldmannia
. Virus particles are not present in vegetative cells but they are produced in the sporangia formed on vegetative filaments that have been transferred newly into culture. Thus, we proposed that these viruses exist in the vegetative cells in a latent form (R. G. Ivey, E. C. Henry, A. M. Lee, L. Klepper, S. K. Krueger, and R. H. Meints, Virology 220:267-273, 1996). In this article we present evidence that the two FsV genomes are integrated into the host genome during vegetative growth. The FsV genome integration sites were identified by cloning the regions where the FsV genome is linked to the host DNA. FsV-158 and FsV-178 are integrated into two distinct locations in the algal genome. In contrast, the integration sites in the two viral genomes are identical. Notably, the integration sites in the host and viruses contain GC and CG dinucleotide sequences, respectively, from which the GC sequences are recovered at both host-virus junctions. The splice sites in the two FsV genomes are predicted to form a stem-loop structure with the CG dinucleotide in the loop portion.