bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the PrrB/PrrA Two-Component System in Nitrite Respiration in
Rhodobacter sphaeroides
2.4.3: Evidence for Transcriptional Regulation
Authors: Laratta William P. , Choi Peter S. , Tosques Ivan E. , Shapleigh James P.
Published: July 2002

Rhodobacter sphaeroides
strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification. The regulation of many
Rhodobacter
genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator. Four strains of 2.4.3 carrying mutations within the
prrB
gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite. Studies revealed that the expression of
nirK
, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3. Disruption of
prrA
also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium. Complementation with
prrA
restored the wild-type phenotype. The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a
nirK
-
lacZ
fusion. Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using
nirK
expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant. Inactivation of
prrA
had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of
nirK
. Inactivation of
ccoN
, part of the
cbb
3
-type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both
nirK
expression and Nir activity. This was unexpected, since PrrA-P accumulates in the
ccoN
strain. Together, these results demonstrate that PrrBA plays an essential role in the regulation of
nirK
.
Evidence for Lateral Transfer of the Suilysin Gene Region of
Streptococcus suis
Authors: Takamatsu Daisuke , Osaki Makoto , Sekizaki Tsutomu
Published: July 2002

Suilysin is a cholesterol-binding cytolysin encoded by
sly
in
Streptococcus suis
. DNA sequence determination of the
sly
locus in a strain lacking
sly
revealed the presence of another gene, designated
orf102
, in the place of
sly
. No transposable element or long-repeat sequence was found in the close vicinity. Except for six strains whose corresponding loci have been rearranged, all of the remaining 62 strains examined had either
sly
or
orf102
at the same locus and their flanking regions were conserved. The genetic organizations having either
sly
or
orf102
were found in the strains whose 16S rRNA sequences were identical. These results suggest that
S. suis
acquired
sly
or
orf102
from a foreign source and that these genes subsequently spread among
S. suis
strains by homologous recombination.
Evaluation of the Vitek Card GPS105 and VTK-RO7.01 Software for Detection of Oxacillin Resistance in Clinically Relevant Coagulase-Negative Staphylococci
Authors: Martinez Fernando , Chandler Laura J. , Reisner Barbara S. , Woods Gail L.
Published: July 2002

The performance of Vitek cards GPS105 with software version VTK-R07.01 for detection of oxacillin resistance in coagulase-negative staphylococci (CoNS) was compared to disk diffusion and PCR detection for
mecA
. The sensitivity and specificity of the Vitek GPS105 method were 97.6 and 85.5%, respectively.
Clonal Relationship between U.S. and French Serotype V Group B Streptococcus Isolates
Authors: Le Thomas-Bories Isabelle , Fitoussi Frederic , Mariani-Kurkdjian Patricia , Raymond Josette , Brahimi Naima , Bidet Philippe , Lefranc Valerie , Bingen Edouard
Published: July 2002

We examined the genetic diversity of serotype V group B streptococcus (GBS) isolates in the Paris area and compared them with the predominant American serotype V clone. Pulsed-field gel electrophoresis yielded 11 patterns for 64 French GBS. One pattern was obtained with 60% of the isolates tested and was indistinguishable from that of the predominant American clone.
Dissemination of Clonally Unrelated Erythromycin- and Glycopeptide-Resistant
Enterococcus faecium
Isolates in a Tertiary Greek Hospital
Authors: Maniatis Antonios N. , Pournaras Spyros , Kanellopoulou Maria , Kontos Fanourios , Dimitroulia Evangelia , Papafrangas Evangelos , Tsakris Athanassios
Published: July 2002

Between September 1999 to February 2001, 25 glycopeptide-resistant
Enterococcus faecium
(GRE) isolates were recovered from a Greek hospital. The isolates exhibited 13 distinct chromosomal macrorestriction types by pulsed-field gel electrophoresis, and all were erythromycin and vancomycin resistant, carrying the genes
vanA
and
ermB
. Vancomycin resistance, always linked with erythromycin resistance, was transferable from 17 isolates. The dissemination of erythromycin-resistant GRE strains may, at least in part, reflect the extensive use of macrolides in husbandry in Greece.
Comparative Evaluation of Semiautomated COBAS AMPLICOR Hepatitis B Virus (HBV) MONITOR Test and Manual Microwell Plate-Based AMPLICOR HBV MONITOR Test
Authors: Marin Irena J. , Poljak Mario , Seme Katja , Meglic̆-Volkar Jelka , Matic̆ic̆ Mojca , Les̆nic̆ar Gorazd , Brinovec Vladimir
Published: July 2002

Comparative evaluation of the semiautomated COBAS AMPLICOR hepatitis B virus (HBV) MONITOR Test (COBAS-HBV) and manual AMPLICOR HBV MONITOR Test (AMPLICOR-HBV) on 208 serum samples revealed no significant difference in the sensitivities of the two assays. Twenty samples tested HBV DNA negative and 183 samples tested HBV DNA positive by both assays. Three samples tested positive by COBAS-HBV only and two samples tested positive by AMPLICOR-HBV only. HBV DNA concentrations determined by the two assays were significantly related (
n
= 183,
r
= 0.97,
P
< 0.0001), which indicates that COBAS-HBV could replace AMPLICOR-HBV. The major inconvenience of COBAS-HBV is the required performance of appropriate predilutions of high-titer samples in order to extend the narrow dynamic range of the assay.
Identification of a Cluster of Strains Bearing a New Adhesin among Genetically Diverse Enterotoxigenic
Escherichia coli
Isolates of Serogroup O20
Authors: Pichel Mariana , Binsztein Norma , Gutkind Gabriel , Viboud Gloria
Published: July 2002

About one-third of the enterotoxigenic
Escherichia coli
isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties. Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.
A Member of the Delta Subgroup of Proteobacteria from a Pyogenic Liver Abscess Is a Typical Sulfate Reducer of the Genus
Desulfovibrio
Authors: Schoenborn Liesbeth , Abdollahi Hamid , Tee Wee , Dyall-Smith Michael , Janssen Peter H.
Published: July 2002

Strain FH26001/95 (ATCC 700045) was previously isolated from a pyogenic liver abscess from a human. Comparative 16S rRNA gene sequence analysis showed that this strain is related to members of the delta subgroup of the proteobacteria, within a cluster of sulfate-reducing bacteria (
Desulfovibrio
spp.) and non-sulfate-reducing bacteria (
Bilophila wadsworthia
and
Lawsonia
spp.). The phenotype of strain FH26001/95 was found to be typical of members of the genus
Desulfovibrio
. Growth and substrate transformations were possible at oxygen concentrations of 2 to 5% (vol/vol) but not at oxygen concentrations of 21% (vol/vol) in air. Its isolation from an infection in a human suggests that some members of the genus
Desulfovibrio
can be considered opportunistic pathogens.
Molecular Epidemiology of a
Shigella flexneri
Outbreak in a Mountainous Township in Taiwan, Republic of China
Authors: Chiou Chien-Shun , Hsu Wen-Bin , Wei Hsiao-Lun , Chen Jiann-Hwa
Published: July 2002

An outbreak of shigellosis occurred in a township of Nantou Conuty in central Taiwan from August to October in 1996. The infections extended to two neighboring townships and continued to the end of 1996. Forty cases were confirmed during the period, in contrast to only one confirmed case in Nantou County in 1996 before the outbreak. All of these 41 cases in 1996 were identified as infections with
Shigella flexneri
serotype 2a. In order to trace the source of the infections, the 41 isolates recovered were analyzed by plasmid profile and pulsed-field gel electrophoresis (PFGE). There was no correlation between the plasmid profile results and the PFGE results, and the latter were used for subtyping of the 41 isolates. Twenty-two isolates (53%) had the same
Not
I and
Xba
I PFGE patterns, and 4 isolates (10%) had an additional unstable plasmid band in their
Not
I patterns but otherwise had the same
Not
I and
Xba
I patterns as the 22 isolates. These 26 isolates were designated the outbreak strain, and of these, 24 appeared in eight villages in one township and 2 appeared in a neighboring township. Fourteen of the remaining 15 isolates, including the isolate recovered 7 months before the outbreak, had both
Not
I and
Xba
I PFGE patterns closely related to those of the outbreak strain, indicating that
Shigella
infections were endemic in the area. By tracing the first isolation dates of the outbreak strain in individual villages and the neighboring township, it was found that the strain spread along the major arterial road and its branch road as time passed. Our molecular typing results and epidemiological data demonstrated the endemic nature of the outbreak strain as well as a person-to-person mode of transmission for the widespread infections the strain caused.
Effect of Errors in the Sequence of Optical Densities from the Roche AMPLICOR HIV-1 MONITOR Assay on the Validity of Assay Results
Authors: Brambilla Donald J. , Granger Suzanne , Jennings Cheryl , Bremer James W.
Published: July 2002

Specifications for the AMPLICOR HIV-1 MONITOR kit indicate that the results are invalid if the optical densities (ODs) from the PCR-amplified sample that are between 0.1 and 2.3 units are out of sequence. However, among 11,904 assays, results were biased only when ODs of 0.2 to 2.0 units were out of sequence, reducing the rate of invalid results from 3.2 to 0.59%.
Antimicrobial Resistance of
Streptococcus pneumoniae
Recovered from Outpatients with Respiratory Tract Infections in Germany from 1998 to 1999: Results of a National Surveillance Study
Authors: Reinert Ralf René , Simic Smiljana , Al-Lahham Adnan , Reinert Susanne , Lemperle Maria , Lütticken Rudolf
Published: July 2002

Clinically significant pneumococcal isolates were prospectively collected from outpatients with respiratory tract infections by 19 different clinical microbiology laboratories in Germany. Resistance rates in a total of 961 isolates were as follows: penicillin, 6.6%; clarithromycin, 10.6%; tetracycline, 13.9%; and levofloxacin, 0.1%. Among 324 isolates from children, pneumococcal serotypes 19F (17.0%), 23F (13.0%), and 6B (11.7%) were the predominant types.
Comparison of Results Generated by Serotyping, Pulsed-Field Restriction Analysis, Ribotyping, and Repetitive-Sequence PCR Used To Characterize Penicillin-Resistant Pneumococci from the United States
Authors: Dunne W. Michael , Kehl Karen Sue , Holland-Staley Carol A. , Brueggemann Angela B. , Pfaller Michael A. , Doern Gary V.
Published: July 2002

One hundred forty-seven isolates of
Streptococcus pneumoniae
with high-level penicillin resistance collected during a national surveillance program in the United States were characterized by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence (BOX element) PCR. The results generated by each method were compared by frequency of association to examine whether relationships existed between the various typing methods and statistically to determine association with the geographic source of the isolate or the age of the patient from whom the isolate was obtained. When the data were examined by pairwise analysis of individual strain classifications produced by each typing method, no statistically significant relationships between strain type, geographic location, or patient age were identified, suggesting that distinct clones of penicillin-resistant
S. pneumoniae
have been widely distributed throughout the United States. However, we did observed shared expression of two or three typing markers at a high frequency (>50%) among clusters of strains, indicating a certain level of concordance between the various typing methods used to classify penicillin-resistant
S
.
pneumoniae
.
Molecular Epidemiology of
Mycobacterium tuberculosis
in Norwa
y
Authors: Dahle Ulf R. , Sandven Per , Heldal Einar , Caugant Dominique A.
Published: July 2002

The incidence of tuberculosis in Norway is one of the lowest in the world, and approximately half of the cases occur in first- and second-generation immigrants. In the present study, the genetic diversity of 92% of all strains of
Mycobacterium tuberculosis
isolated in Norway in 1994 to 1998 was assessed using restriction fragment length polymorphism (RFLP) analysis, with the insertion sequence IS
6110
and the repetitive element DR as probes, to determine the degree of active transmission between patients. The DR probe was used as a secondary molecular marker to support or rule out clustering of strains with fewer than five copies of IS
6110
. After exclusion of 20 cultures representing laboratory contamination, 573 different IS
6110
patterns were found among the 698 strains analyzed. Of these 573 patterns, 542 were observed only once and 31 were shared by 2 to 14 isolates. Among 81 strains (11.5%) carrying fewer than five copies of IS
6110,
56 RFLP patterns were found when the results of both the IS
6110
and DR methods were combined. Among the 698 strains, 570 were considered to be independent cases. A total of 14.5% of the native Norwegians and 19.7% of the foreign patients were part of a cluster. Thus, the degree of recent transmission of tuberculosis in Norway is low and the great majority of the cases are due to reactivation of previous disease. Transmission between immigrants and native Norwegians is uncommon. Two outbreaks, one among native Norwegians and one mainly among immigrants, have been ongoing for several years, indicating that, even in a low-incidence country such as Norway, with a good national program for tuberculosis surveillance, certain transmission chains are difficult to break.
Quantitation of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Cell-Free Cervicovaginal Secretions: Comparison of Reverse Transcription-PCR Amplification (AMPLICOR HIV-1 MONITOR 1.5) with Enhanced-Sensitivity Branched-DNA Assay (Quantiplex 3.0)
Authors: Si-Mohamed Ali , Andreoletti Laurent , Colombet Isabelle , Carreno Marie-Paule , Lopez Gladys , Chatelier Gilles , Kazatchkine Michel D. , Belec Laurent
Published: July 2002

Two commercially available hypersensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation, AMPLICOR HIV-1 Monitor Test 1.5 and Quantiplex HIV RNA 3.0, were compared to detect and quantify HIV-1 RNA in the cell-free fraction of cervicovaginal secretions collected by vaginal washing. Three panel specimens were used: pooled cervicovaginal secretions spiked with HIV-1 subtype A or HIV-1 subtype B and cervicovaginal lavages from HIV-positive and HIV-negative women. Compared to the AMPLICOR HIV-1 Monitor Test 1.5 assay, the Quantiplex HIV-1 3.0 assay yielded higher estimates of HIV-1 RNA concentrations in several tested samples spiked with HIV-1 RNA subtype A, as well as subtype B, particularly samples containing low amounts of HIV-1 RNA. The sensitivity and specificity of the AMPLICOR HIV-1 Monitor Test 1.5 assay were 93 and 100%, respectively; the sensitivity and specificity of the Quantiplex HIV RNA 3.0 assay were 97 and 50%, respectively. In conclusion, in quantifying HIV-1 RNA in cervicovaginal secretions, the Quantiplex HIV RNA 3.0 may lack specificity, and the AMPLICOR HIV-1 Monitor Test 1.5 assay, although highly specific, may lack sensitivity.
Reemergence of Gentamicin-Susceptible Strains of Methicillin-Resistant
Staphylococcus aureus
in France: a Phylogenetic Approach
Authors: Blanc D. S. , Francioli P. , Le Coustumier A. , Gazagne L. , Lecaillon E. , Gueudet P. , Vandenesch F. , Etienne J.
Published: July 2002

The reemergence of gentamicin-susceptible (Gen
s
) methicillin-resistant
Staphylococcus aureus
(MRSA) isolates in France between 1992 and 1996 was investigated using a phylogenetic approach (multiprimer randomly amplified polymorphic DNA typing). Eighty-six percent (65 of 85) of the French strains were grouped into one phylogenetic cluster within which all but one Gen
s
strain were grouped into a subcluster. Thus, the reemergence of Gen
s
MRSA strains in France was likely due to the spread of one specific clone which belonged to a cluster comprising most French gentamicin-resistant (Gen
r
) strains. This suggests that the Gen
s
clone has emerged from a Gen
r
strain of this cluster.
Rapid Diagnosis of
Histoplasma capsulatum
Endocarditis Using the AccuProbe on an Excised Valve
Authors: Chemaly Roy F. , Tomford J. Walton , Hall Gerri S. , Sholtis Mary , Chua Jimmy D. , Procop Gary W.
Published: July 2002

Histoplasma capsulatum
is an infrequent but serious cause of endocarditis. The definitive diagnosis requires culture, which may require a long incubation. We demonstrated the ability of the
Histoplasma capsulatum
AccuProbe to accurately identify this organism when applied directly on an excised valve that contained abundant yeast forms consistent with
H. capsulatum
.
Use of a Reverse Dot Blot Procedure To Identify the Presence of Multiple Serovars inChlamydia trachomatisUrogenital Infection
Authors: Stothard Diane R.
Published: July 2002

Epidemiologic research requires identification ofChlamydia trachomatisserovars and detection of mixed infection. Antibody-based serotyping is unworkable when specimens are urine or vaginal swabs. We developed a reverse dot blot (RDB) to screen for multiple serotypes in these specimens. RDB yielded the predicted results on all artificially mixed samples and on seven of eight clinically mixed samples.
Multicenter Evaluation of a Pathogenic
Mycobacterium
Screening Probe
Authors: Emler Stefan , Feldmann Knut , Giacuzzo Véronique , Hewitt Peter L. , Klapper Paul E. , Lagrange Philippe H. , Wilkins Ed W. , Young Karen K. Y. , Herrmann Jean-Louis
Published: July 2002

The introduction of nucleic acid amplification assays into the clinical laboratory has reduced the time needed to diagnose diseases caused by members of the
Mycobacterium tuberculosis
complex (MTBC). However, several mycobacterial species other than those of the MTBC are known to cause disease, especially in immunocompromised individuals. A screening assay has been developed for the detection of the major pathogenic mycobacterial species. The assay utilizes pan-genus primers to amplify mycobacterial DNA and a screening probe (KY493) that detects all major pathogenic mycobacteria. A multicenter European study was conducted to assess the performance of the screening probe in the clinical laboratory. The screening probe was evaluated against individual probes specific for
M. tuberculosis
,
M. avium
, and
M. intracellulare
, a genus-specific probe with broader species coverage, and culture. The screening probe had a sensitivity equivalent to that of the species-specific probes; all specimens positive with any of the species-specific probes were also positive with the screening probes. Compared to culture, the sensitivity of the screening probe was 89% (154 of 173) for all culture-positive specimens tested. This value was 89.6% for the genus-specific probe. The screening probe was more specific than the genus-specific probe. Specificity was 93.9% (661 of 704) compared to culture results alone. The comparable specificity value for the genus-specific probe was 84.8%. When clinical data were taken into consideration, the sensitivity of the screening assay was similar to that of culture (81% versus 76.2%) but the positive predictive value of the test was lower (76.2% versus 100% for culture). However, the screening probe was more sensitive than smear and may be a useful tool in the rapid diagnosis of mycobacterial disease.
Characterization of
Borrelia burgdorferi
Isolated from Erythema Migrans Lesions: Interrelationship of Three Molecular Typing Methods
Authors: Iyer Radha , Liveris Dionysios , Adams Atoya , Nowakowski John , McKenna Donna , Bittker Susan , Cooper Denise , Wormser Gary P. , Schwartz Ira
Published: July 2002

Genetic diversity among
Borrelia burgdorferi
isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content (
P
< 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.
Concomitant Infection of Enterotoxigenic
Escherichia coli
in an Outbreak of Cholera Caused by
Vibrio cholerae
O1 and O139 in Ahmedabad, India
Authors: Chakraborty Subhra , Deokule J. S. , Garg Pallavi , Bhattacharya S. K. , Nandy R. K. , Nair G. Balakrish , Yamasaki S. , Takeda Y. , Ramamurthy T.
Published: July 2002

In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by
Vibrio cholerae
O1 Ogawa El Tor,
V. cholerae
O139, and multiple serotypes of enterotoxigenic
Escherichia coli
(ETEC) occurred in January 2000. All of the representative
V. cholerae
O1 and O139 isolates examined harbored the
ctxA
gene (encoding the A subunit of cholera toxin) and the El Tor variant of the
tcpA
gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the
elt
gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of
V. cholerae
O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of
V. cholerae
O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the
V. cholerae
O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the
E. coli
isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which
V. cholerae
and ETEC were concomitantly involved.
Comparison of Two Culture Methods for Detection of Tobramycin-Resistant Gram-Negative Organisms in the Sputum of Patients with Cystic Fibrosis
Authors: Van Dalfsen Jill M. , Stapp Jenny R. , Phelps Charles , Stewart Patricia , Burns Jane L.
Published: July 2002

A culture method utilizing quantitative plating on antibiotic-containing media has been proposed as a technique for the detection of tobramycin-resistant organisms that is more sensitive than standard methods. Typical sputum culture methods quantitate the relative amounts of each distinct morphotype, followed by antibiotic susceptibility testing of a single colony of each morphotype. Sputum specimens from 240 cystic fibrosis patients were homogenized, serially diluted, and processed in parallel by the standard method (MacConkey agar and OF basal medium with agar, polymyxin, bacitracin, and lactose) and by plating on antibiotic-containing media (MacConkey agar with tobramycin added at 25 μg/ml [MAC-25] and 100 μg/ml [MAC-100]). MICs of tobramycin were determined for all
Pseudomonas aeruginosa
isolates by broth microdilution. Growth of
P. aeruginosa
on MAC-25 was considered to be equivalent to a tobramycin MIC of ≥16 μg/ml, and growth on MAC-100 was considered to be equivalent to a tobramycin MIC of ≥128 μg/ml. Analysis of method-specific detection rates showed that tobramycin-containing medium was more sensitive than the standard method for the detection of tobramycin-resistant
P. aeruginosa, Stenotrophomonas maltophilia
, and
Achromobacter xylosoxidans
but was less sensitive for the detection of
Burkholderia cepacia
than the standard method. When MICs for
P. aeruginosa
that grew on tobramycin-containing medium were tested by broth microdilution, the MICs for 28 of 121 strains (23%) growing on MAC-25 and 22 of 56 strains (39%) growing on MAC-100 were MICs <16 and <128 μg/ml, respectively. Addition of a tobramycin-containing MacConkey plate to the routine media for sputum culture may provide additional, clinically relevant microbiologic data.
Ratio of Two Successive Optical Densities from the Roche HIV-1 Monitor Test as a Measure of Accuracy of Estimates of Human Immunodeficiency Virus RNA Concentration
Authors: Jennings Cheryl , Brambilla Donald J. , Bremer James W.
Published: July 2002

In the Roche Amplicor HIV-1 Monitor Test, the ratio of the optical density used to estimate the RNA concentration and the optical density of the preceding or the following dilution can be used to identify specimens with inaccurate results which should be retested. We present an algorithm for the identification of such results.
Four Strains of
Escherichia coli
O157:H7 Isolated from Patients during an Outbreak of Disease Associated with Ground Beef: Importance of Evaluating Multiple Colonies from an Outbreak-Associated Product
Authors: Proctor Mary E. , Kurzynski Terry , Koschmann Cindy , Archer John R. , Davis Jeffrey P.
Published: July 2002

This report describes the investigation of a ground-beef-associated outbreak that involved five genetically distinct patient strains of
Escherichia coli
O157:H7. Human and product isolates were evaluated by pulsed-field gel electrophoresis (PFGE) with two endonucleases. The multiple-strain etiology of this outbreak underscores the importance of isolating and evaluating multiple colonies from outbreak-related products and comparing two endonuclease PFGE patterns of all product and human isolates identified during outbreak periods. This investigation emphasizes the importance of interviewing all confirmed and suspected case patients during the outbreak period, regardless of the PFGE pattern of their isolate, to confirm or rule out an epidemiologic link to the outbreak.
Invasive Meningococcal Disease in Scotland, 1994 to 1999, with Emphasis on Group B Meningococcal Disease
Authors: Kyaw Moe H. , Clarke Stuart C. , Christie Peter , Jones Ian G. , Campbell Harry
Published: July 2002

A review was carried out on 774 invasive meningococcal isolates reported to the active meningococcal surveillance system in Scotland from 1994 to 1999. This showed that serogroups B (51.7%) and C (39.2%) caused the majority of disease. The six common PorB proteins (4, 1, 15, 2B, 12, and 21) and PorA proteins (serosubtypes) (P1.4, P1.15, P1.9, P1.14, P1.7, and P1.16) accounted for 50 and 51% of all group B isolates, respectively, during the study period.
Determining the Genomic Locations of Repetitive DNA Sequences with a Whole-Genome Microarray: IS
6110
in
Mycobacterium tuberculosis
Authors: Kivi Mårten , Liu Xuemin , Raychaudhuri Soumya , Altman Russ B. , Small Peter M.
Published: July 2002

The mycobacterial insertion sequence IS
6110
has been exploited extensively as a clonal marker in molecular epidemiologic studies of tuberculosis. In addition, it has been hypothesized that this element is an important driving force behind genotypic variability that may have phenotypic consequences. We present here a novel, DNA microarray-based methodology, designated SiteMapping, that simultaneously maps the locations and orientations of multiple copies of IS
6110
within the genome. To investigate the sensitivity, accuracy, and limitations of the technique, it was applied to eight
Mycobacterium tuberculosis
strains for which complete or partial IS
6110
insertion site information had been determined previously. SiteMapping correctly located 64% (38 of 59) of the IS
6110
copies predicted by restriction fragment length polymorphism analysis. The technique is highly specific; 97% of the predicted insertion sites were true insertions. Eight previously unknown insertions were identified and confirmed by PCR or sequencing. The performance could be improved by modifications in the experimental protocol and in the approach to data analysis. SiteMapping has general applicability and demonstrates an expansion in the applications of microarrays that complements conventional approaches in the study of genome architecture.
Deletion of a Short, Untranslated Region Adjacent to the Polypurine Tract in Moloney Murine Leukemia Virus Leads to Formation of Aberrant 5′ Plus-Strand DNA Ends In Vivo
Authors: Bacharach Eran , Gonsky Jason , Lim David , Goff Stephen P.
Published: July 2002

Experiments were performed to determine the function of a 28-nucleotide untranslated sequence lying between the envelope gene and the polypurine tract (PPT) sequence in the Moloney murine leukemia virus (Mo-MuLV) genome. A mutant virus carrying a deletion of this sequence (Mo-MuLVΔ28) replicated more slowly than wild-type (wt) virus and reverted by recombination with endogenous sequences during growth in NIH 3T3 cells. We show that this deletion did not affect the level of viral protein expression or genomic RNA packaging. Mo-MuLVΔ28 served as a helper virus as efficiently as the wt virus; in contrast, a retroviral vector harboring this mutation exhibited reduced transduction efficiency, indicating that the mutation acts not in
trans
but in
cis
. Analysis of acutely infected cells revealed that reduced levels of viral DNA were generated by reverse transcription of the Mo-MuLVΔ28 RNA as compared to the wt RNA. Analysis of DNA circle junctions revealed that plus-strand DNA of Mo-MuLVΔ28 but not wt virus often retained the PPT and additional upstream sequences. These structures suggest that aberrant 5′ ends of plus-strand DNA were generated by a failure to remove the PPT RNA primer and/or by mispriming at sites upstream of the PPT. These data demonstrate that the major role of the sequences immediately upstream of the PPT is specifying efficient and accurate plus-strand DNA synthesis.
Interactions between Hepatitis Delta Virus Proteins
Authors: Moraleda Gloria , Dingle Kate , Biswas Preetha , Chang Jinhong , Zuccola Harmon , Hogle James , Taylor John
Published: July 2002

The 195- and 214-amino-acid (aa) forms of the delta protein (δAg-S and δAg-L, respectively) of hepatitis delta virus (HDV) differ only in the 19-aa C-terminal extension unique to δAg-L. δAg-S is needed for genome replication, while δAg-L is needed for particle assembly. These proteins share a region at aa 12 to 60, which mediates protein-protein interactions essential for HDV replication. H. Zuccola et al. (Structure 6:821–830, 1998) reported a crystal structure for a peptide spanning this region which demonstrates an antiparallel coiled-coil dimer interaction with the potential to form tetramers of dimers. Our studies tested whether predictions based on this structure could be extrapolated to conditions where the peptide was replaced by full-length δAg-S or δAg-L, and when the assays were not in vitro but in vivo. Nine amino acids that are conserved between several isolates of HDV and predicted to be important in multimerization were mutated to alanine on both δAg-S and δAg-L. We found that the predicted hierarchy of importance of these nine mutations correlated to a significant extent with the observed in vivo effects on the ability of these proteins to (i) support in
trans
the replication of the HDV genome when expressed on δAg-S and (ii) act as dominant-negative inhibitors of replication when expressed on δAg-L. We thus infer that these biological activities of δAg depend on ordered protein-protein interactions.
Simian and Human Immunodeficiency Virus Nef Proteins Use Different Surfaces To Downregulate Class I Major Histocompatibility Complex Antigen Expression
Authors: Swigut Tomek , Iafrate A. John , Muench Jan , Kirchhoff Frank , Skowronski Jacek
Published: July 2002

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.
Evidence for the Packaging of Multiple Copies of Tf1 mRNA into Particles and the
trans
Priming of Reverse Transcription
Authors: Haag Amanda Leigh , Lin Jia-Hwei , Levin Henry L.
Published: July 2002

Long terminal repeat (LTR)-containing retrotransposons and retroviruses are close relatives that possess similar mechanisms of reverse transcription. The particles of retroviruses package two copies of viral mRNA that both function as templates for the reverse transcription of the element. We studied the LTR-retrotransposon Tf1 of
Schizosaccharomyces pombe
to test whether multiple copies of transposon mRNA participate in the production of cDNA. Using the unique self-priming property of Tf1, we obtained evidence that multiple copies of Tf1 mRNA were packaged into virus-like particles. By coexpressing two distinct versions of Tf1, we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others. In addition, the first 11 nucleotides of one mRNA were able to prime, in
trans
, the reverse transcription of another mRNA.
Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors
Authors: Suzuki Nobuhiro , Geletka Lynn M. , Nuss Donald L.
Published: July 2002

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus
Cryphonectria parasitica
, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from
Aequorea victoria
, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.
Selective Inhibition of Splicing at the Avian Sarcoma Virus
src
3′ Splice Site by Direct-Repeat Posttranscriptional
cis
Elements
Authors: Guo Wei , Winistorfer Stanley C. , Stoltzfus C. Martin
Published: July 2002

The direct-repeat elements (dr1) of avian sarcoma virus (ASV) and leukosis virus have the properties of constitutive transport elements (CTEs), which facilitate cytoplasmic accumulation of unspliced RNA. It is thought that these elements represent binding sites for cellular factors. Previous studies have indicated that in the context of the avian sarcoma virus genome, precise deletion of both ASV dr1 elements results in a very low level of virus replication. This is characterized by a decreased cytoplasmic accumulation of unspliced RNA and a selective increase in spliced
src
mRNA. Deletion of either the upstream or downstream dr1 results in a delayed-replication phenotype. To determine if the same regions of the dr1 mediate inhibition of
src
splicing and unspliced RNA transport, point mutations in the upstream and downstream elements were studied. In the context of viral genomes with single dr1 elements, the effects of the mutations on virus replication and increases in
src
splicing closely paralleled the effects of the mutations on CTE activity. For mutants strongly affecting CTE activity and splicing, unspliced RNA but not spliced RNA turned over in the nucleus more rapidly than wild-type RNA. In the context of wild-type virus containing two dr1 elements, mutations of either element that strongly affect CTE activity caused a marked delay of virus replication and a selective increase in
src
splicing. However, the turnover of the mutant unspliced RNA as well as the spliced mRNA species did not differ significantly from that of the wild type. These results suggest the dr1 elements in ASV act to selectively inhibit
src
splicing and that both elements contribute to the fitness of the wild-type virus. However, a single dr1 element is sufficient to stabilize unspliced RNA.
Nuclear Import of Moloney Murine Leukemia Virus DNA Mediated by Adenovirus Preterminal Protein Is Not Sufficient for Efficient Retroviral Transduction in Nondividing Cells
Authors: Lieber André , Kay Mark A. , Li Zong-Yi
Published: July 2002

Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G
1
/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration.
Roles of the AX
4
GKS and Arginine-Rich Motifs of Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding Activity
Authors: Chang Shin C. , Cheng Ju-Chien , Kou Yi-Hen , Kao Chuan-Hong , Chiu Chiung-Hui , Wu Hung-Yi , Chang Ming-Fu
Published: July 2002

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in
Escherichia coli
, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX
4
GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX
4
GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the γ-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.
Latent Membrane Protein 1 of Epstein-Barr Virus Inhibits as Well as Stimulates Gene Expression
Authors: Sandberg Mark L. , Kaykas Ajamete , Sugden Bill
Published: July 2002

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) functionally resembles a constitutively active, CD40-like receptor and contributes to the maintenance of proliferation of EBV-infected primary human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it binds TRAF, TRADD, and JAK molecules to activate NF-κB-, AP-1-, and STAT-dependent pathways as does CD40. Yet LMP-1 appears to lack a ligand to regulate its signaling. We have found that LMP-1, when expressed at physiologic levels, inhibits gene expression detectably. Higher levels of LMP-1 expression eventually inhibit both the steady-state level of RNA produced from a
Bam
HI C promoter reporter and general cellular protein synthesis. These findings indicate that LMP-1 can limit its signaling and that this control is manifest at two levels. The domain of LMP-1 that binds TRAF, TRADD, and JAK/STAT molecules is not required for this regulation. A derivative of LMP-1 that contains only its amino-terminal and membrane-spanning domains is sufficient to inhibit reporter activity when the reporter genes are expressed from the
Bam
HI C and LMP-1 promoters. This same derivative of LMP-1 in parallel assays is sufficient to inhibit wild-type LMP-1's stimulation of NF-κB-dependent gene expression. We suggest that LMP-1 encodes stimulatory and inhibitory activities; the latter could limit signaling in the apparent absence of ligand-dependent down-regulation.
Effective Induction of Simian Immunodeficiency Virus-Specific Systemic and Mucosal Immune Responses in Primates by Vaccination with Proviral DNA Producing Intact but Noninfectious Virions
Authors: Wang Shainn-Wei , Kozlowski Pamela A. , Schmelz Gretchen , Manson Kelledy , Wyand Michael S. , Glickman Rhona , Montefiori David , Lifson Jeffrey D. , Johnson R. Paul , Neutra Marian R. , Aldovini Anna
Published: July 2002

We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.