bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the Lipooligosaccharide Biosynthesis Gene
lic2B
as a Putative Virulence Factor in Strains of Nontypeable
Haemophilus influenzae
That Cause Otitis Media
Authors: Pettigrew M. M. , Foxman B. , Marrs C. F. , Gilsdorf J. R.
Published: July 2002

Nontypeable (NT) strains of
Haemophilus influenzae
are an important cause of acute otitis media (OM). The pathogenic process by which NT
H. influenzae
strains cause OM is poorly understood. In order to identify specific virulence factors important for OM pathogenesis, genomic subtraction of the NT
H. influenzae
middle ear isolate G622 against
H. influenzae
strain Rd was conducted and the resulting subtraction products were used to screen a panel of
H. influenzae
isolates. Subtraction identified 36 PCR fragments unique to strain G622, which were used in a preliminary screen of 48 middle ear isolates and 46 nasopharyngeal and throat isolates to identify genes found more frequently among middle ear isolates. These experiments identified a PCR fragment with high homology to the lipooligosaccharide biosynthesis gene
lic2B
(originally identified in an
H. influenzae
type b strain) among 52% of the middle ear isolates and 9% of nasopharyngeal and throat isolates. The
lic2B
gene cloned from NT
H. influenzae
strain G622 was 99% identical at the amino acid level to that of the
H. influenzae
type b strain RM7004. The
lic2B
gene was used to screen a larger panel of
H. influenzae
isolates including the original 48 middle ear isolates, 40 invasive type b isolates, 90 NT
H. influenzae
throat isolates from children attending day care, and 32 NT
H. influenzae
nasopharyngeal clinical isolates. The
lic2B
gene was found 3.7 times more frequently among middle ear isolates than in throat isolates from children attending day care. These data suggest that a specific NT
H. influenzae
gene is associated with OM.
Cloning and Expression of Two Genes Coding for Sodium Pumps in the Salt-Tolerant Yeast
Debaryomyces hansenii
Authors: Almagro Anabel , Prista Catarina , Benito Begoña , Loureiro-Dias Maria C. , Ramos José
Published: July 2002

Two genes encoding Na
+
-ATPases from
Debaryomyces hansenii
were cloned and sequenced. The genes, designated
ENA1
from
D. hansenii
(
DhENA1
) and
DhENA2
, exhibited high homology with the corresponding genes from
Schwanniomyces occidentalis. DhENA1
was expressed in the presence of high Na
+
concentrations, while the expression of
DhENA2
also required high pH. A mutant of
Saccharomyces cerevisiae
lacking the Na
+
efflux systems and sensitive to Na
+
, when transformed with
DhENA1
or
DhENA2
, recovered Na
+
tolerance and also the ability to extrude Na
+
.
Binary Typing of
Staphylococcus aureus
Strains through Reversed Hybridization Using Digoxigenin-Universal Linkage System-Labeled Bacterial Genomic DNA
Authors: van Leeuwen Willem , Libregts Christel , Schalk Marjolijn , Veuskens Jack , Verbrugh Henri , van Belkum Alex
Published: July 2002

A novel binary typing (BT) procedure, based on reversed hybridization of digoxigenin-universal linkage system-labeled bacterial DNA to strip-immobilized probes, is presented. Chromogenic detection of hybrids was performed.
Staphylococcus aureus
isolates (
n
= 20) were analyzed to establish the feasibility of BT. A technically simple and fast procedure has been developed for application in routine microbiology laboratories.
Accuracy of the VITEK 2 System To Detect Glycopeptide Resistance in Enterococci
Authors: van den Braak Nicole , Goessens Wil , van Belkum Alex , Verbrugh Henri A. , Endtz Hubert P.
Published: July 2002

We evaluated the accuracy of the VITEK 2 fully automated system to detect and identify glycopeptide-resistant enterococci (GRE) compared to a reference agar dilution method. The sensitivity of vancomycin susceptibility testing with VITEK 2 for the detection of
vanA
,
vanB
, and
vanC1
strains was 100%. The sensitivity of vancomycin susceptibility testing of
vanC2
strains was 77%. The sensitivity of teicoplanin susceptibility testing of
vanA
strains was 90%. Of 80
vanC
enterococci, 78 (98%) were correctly identified by VITEK 2 as
Enterococcus gallinarum/Enterococcus casseliflavus
. Since the identification and susceptibility data are produced within 3 and 8 h, respectively, VITEK 2 appears a fast and reliable method for detection of GRE in microbiology laboratories.
Rapid Detection of
Mycobacterium tuberculosis
in Contaminated BACTEC 12B Broth Cultures by Testing with Amplified Mycobacterium Tuberculosis Direct Test
Authors: Zheng Xiaotian , Pang Minnie , Engler Howard D. , Tanaka Sherri , Reppun Thomas
Published: July 2002

Contamination of broth cultures of acid-fast bacilli (AFB) by bacterial species other than
Mycobacterium
species frequently occurs. Many of these contaminated cultures require redecontamination and reincubation before the appropriate tests can be performed for identification, significantly affecting the turnaround time for reporting culture results. In this study, the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe) was performed to detect the
Mycobacterium tuberculosis
complex (MTBC) in 125 BACTEC 12B broth cultures with positive growth indices. Among these, 41 grew non-AFB bacteria only, and all 41 were negative by the MTD. The remaining 84 bottles contained contaminated cultures that grew both AFB and other bacteria or yeasts. Repeat decontamination and reincubation of these specimens required a mean time of 13 days (range, 3 to 40 days). The MTD results were positive for 10 samples, 9 of which were MTBC culture positive and 1 of which grew
Myobacterium celatum
, a species known to cross-react in the MTD. All cultures growing other mycobacterial species were negative by the MTD. The results of this study demonstrate that the MTD is both sensitive and specific in detecting MTBC in contaminated broth cultures and that, when used selectively, the MTD can potentially rule in or out a diagnosis of MTBC as much as 12 days earlier than using nonamplified DNA probe testing alone can.
High Prevalence of Granulocytic Ehrlichiae and
Borrelia burgdorferi
Sensu Lato in
Ixodes ricinus
Ticks from Bulgaria
Authors: Christova Iva , Schouls Leo , van de Pol Ingrid , Park Jinho , Panayotov Stefan , Lefterova Viktoria , Kantardjiev Todor , Dumler J. Stephen
Published: July 2002

Bulgarian
Ixodes ricinus
ticks were examined for
Ehrlichia
and
Borrelia
coinfection: 34 and 32% of adult ticks and at least 2 and 10% of nymphs were positive for these infections, respectively. Coinfections and dual or triple
Borrelia
infections were frequent, although
Ehrlichia phagocytophila
heterogeneity was minimal. Multiple tick-borne bacteria coexist in
I. ricinus
ticks in southeastern Europe.
Genome Typing of Adenovirus Type 34 Isolated in Two Cases of Conjunctivitis in Sapporo, Japan
Authors: Saitoh-Inagawa Waka , Tanaka Keico , Uchio Eiichi , Itoh Norihiko , Ohno Shigeaki , Aoki Koki
Published: July 2002

Two cases of conjunctivitis caused by adenovirus type 34 (Ad34) are reported. The isolates were identified as Ad34 by the neutralization test and the PCR-sequence method of the hexon gene but as Ad14 by PCR-restriction fragment length polymorphism analysis. The genome types of these two isolates were identical to that of Ad34a.
Evaluation of the
Mycobacterium bovis
Restriction Fragment Length Polymorphism Probe pUCD, in Combination with the Direct Repeat Probe, for Molecular Typing of
Mycobacterium tuberculosis
Strains in Ireland
Authors: Cameron Henrietta , O'Brien Rory , Murray Anthony , Cryan Bartley , Hone Rosemary , Rogers Mark
Published: July 2002

A mycobacterial restriction fragment length polymorphism probe, pUCD, has recently been described which represents an effective tool for the strain typing of
Mycobacterium bovis
. The present study evaluated this probe, in combination with the direct repeat probe (DR), for the molecular typing of 90 strains of
Mycobacterium tuberculosis
from 87 patients, looking at a group (62 isolates) of nonselected samples to assess pUCD combined with DR as a general tool and a subset of 32 isolates with a common specific IS
6110
strain type in Ireland. Within the group of 62 isolates, pUCD-DR identified 42 strains and was comparable to both IS
6110
(41 strains) and polymorphic guanine-cytosine-rich sequence (PGRS) (37 strains) analysis. pUCD-DR was found to be comparable to IS
6110
and PGRS in identifying four separate clusters of isolates which were confirmed to be clinically related. pUCD-DR divided the common IS
6110
isolates into six distinct types and was comparable to PGRS (seven strain types). The usefulness of this probe as an epidemiological tool is discussed.
Beyond Serotypes and Virulence-Associated Factors: Detection of Genetic Diversity among O153:H45 CFA/I Heat-Stable Enterotoxigenic
Escherichia coli
Strains
Authors: Pacheco A. B. F. , Ferreira L. C. S. , Pichel M. G. , Almeida D. F. , Binsztein N. , Viboud G. I.
Published: July 2002

Characterization of enterotoxigenic
Escherichia coli
(ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like São Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.
Improved Rate of Isolation of
Neisseria meningitidis
by Direct Plating of Pharyngeal Swabs
Authors: Cunningham Richard , Matthews Roger , Lewendon Gill , Harrison Sarah , Stuart James M.
Published: July 2002

Culturing of pharyngeal swabs for
Neisseria meningitidis
is an important clinical and epidemiological tool. Routine methods include direct plating onto solid medium or later plating in the laboratory. A comparison of these methods used with 490 high school students found a significantly higher carriage rate with direct plating (11.8 versus 6.1%;
P
< 0.001).
Reverse Dot Blot Assay (Insertion Site Typing) for Precise Detection of Sites of IS
6110
Insertion in the
Mycobacterium tuberculosis
Genome
Authors: Steinlein Lauren M. , Crawford Jack T.
Published: July 2002

We have developed an amplification-based reverse dot blot assay for the detection of specific sites of insertion of the
Mycobacterium tuberculosis
insertion sequence IS
6110
. In this assay, a set of biotin-labeled amplicons representing the various copies of IS
6110
and their flanking sequences is generated by linker-mediated PCR. The amplicons are then hybridized to immobilized oligonucleotide probes that are specific for known IS
6110
insertion sites. The method was evaluated using an array of oligonucleotide probes corresponding to IS
6110
insertion sites from
M. tuberculosis
strains CDC1551, Erdman, and H37Rv, and multidrug-resistant strain W. A set of 72 DNA samples from 60
M. tuberculosis
clinical isolates was analyzed for the presence or absence of these insertion sites, and the assay was found to be highly reproducible. This method of identifying insertion sites has been named “insite” and can be used for the genotyping of
M. tuberculosis
complex strains based on IS
6110
insertion site profiles.
Biotyping of
Penicillium marneffei
Reveals Concentration-Dependent Growth Inhibition by Galactose
Authors: Wong Samson S. Y. , Ho Timothy Y. C. , Ngan Antonio H. Y. , Woo Patrick C. Y. , Que Tak-Lun , Yuen Kwok-Yung
Published: July 2002

Thirty-two isolates of the dimorphic fungus
Penicillium marneffei
were studied for their biochemical properties. All isolates possessed the enzyme urease and were inhibited by 500 mg of cycloheximide per liter. No strain fermented glucose, and thus no strain fermented any of the other five sugars tested. All assimilated glucose, maltose, and cellobiose; only one of the isolates did not assimilate salicin. Totals of 65.6, 84.4, and 71.9% of the isolates assimilated trehalose, xylose, and nitrate, respectively. Twelve strains possessed the enzyme β-galactosidase. Overall, 17 different biotypes were recognized, but no association was found between the human immunodeficiency virus status of the patients and the biotype. A novel finding of concentration-dependent growth inhibition of
P. marneffei
by galactose is described. Inhibition of growth occurred at a low concentration of galactose (0.015 to 0.25%) when galactose was the sole carbon source in the medium. Morphological changes of the fungal cells were observed in the presence of galactose.
Monitoring the Emergence of Hepatitis B Virus Polymerase Gene Variants during Lamivudine Therapy Using the LightCycler
Authors: Whalley Simon A. , Brown Dave , Teo Chong Gee , Dusheiko Geoffrey M. , Saunders Nicholas A.
Published: July 2002

Treatment of chronic hepatitis B virus (HBV) infection with lamivudine is associated with the appearance in the circulation of HBV variants with mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the polymerase gene. Fluorometric real-time PCR with the LightCycler assay was used for the detection of resistant variants. Differences in the hybridization melting curve kinetics of probes bound to the sequences encoding the wild-type or the mutant YMDD motifs (YIDD or YVDD in which the methionine residue is altered to an isoleucine or a valine, respectively) distinguished the single-base changes responsible for the resistance phenotype. The LightCycler probe hybridization assay was applied to 40 serum specimens from 19 patients, and the results were correlated with the nucleotide sequences determined for the corresponding PCR products. All three variants could be identified in the specimens. PCR clones obtained from four patients early in the course and prior to lamivudine therapy were investigated for the appearance of YIDD and YVDD variants with the LightCycler assay. In one patient, a transient appearance of the YIDD variant was observed 6 weeks into therapy. Subsequently, after 11 months of lamivudine therapy, the YVDD variant emerged in that patient.
Controlled Comparison of Original Vented Aerobic FAN Medium with New Nonvented BacT/ALERT FA Medium for Culturing Blood
Authors: Mirrett Stanley , Everts Richard J. , Reller L. Barth
Published: July 2002

To evaluate the performance of BacT/ALERT FA (FA) medium, a new aerobic BacT/ALERT FAN (FAN) medium (Organon Teknika Corporation, Durham, N.C.) that does not require the added cost and inconvenience of a venting unit, we inoculated blood specimens from adult patients with suspected sepsis into an original FAN aerobic culture bottle and an FA bottle. Of 7,745 blood culture sets containing both bottles, 5,256 (68%) met the criteria for adequacy of filling. A total of 466 isolates judged to represent the causes of true infections were recovered from 276 patients; 271 isolates were recovered from both bottles, 82 were recovered from the FAN bottle only, and 113 were recovered from the FA bottle only (
P
< 0.05). More
Burkholderia cepacia
isolates (
P
< 0.01),
Candida albicans
isolates (
P
< 0.001),
Cryptococcus neoformans
isolates (
P
< 0.01), yeasts overall (
P
< 0.001), and total microorganisms (
P
< 0.05) were recovered from FA bottles. Of cultures found to be positive within the first 72 h of incubation, the mean times to detection were almost identical for FAN (20.4 h) and FA (20.7 h) bottles. Of 263 isolates that caused monomicrobic episodes of bloodstream infections, 180 were detected in both bottles, 32 were detected in FAN bottles only, and 51 were detected in FA bottles only (
P
< 0.05). Of 186 isolates considered to be contaminants, 63 were detected in both media, 64 were detected in FAN bottles only, and 59 were detected in FA bottles only (
P
was not significant). The number of false-positive results were comparable: 69 (1.3%) in FAN bottles and 56 (1.1%) in FA bottles. However, there were 14 isolates with false-negative results (6 yeasts, 6 nonfermenters, and 1 isolate each of
Propionibacterium acnes
and coagulase-negative staphylococci) in FAN bottles, whereas there were none in FA bottles. On the basis of these results, we conclude that the new nonvented FA bottle is superior to the original vented FAN medium for the recovery of
B. cepacia
and yeasts, especially
C. albicans
and
C. neoformans
, and is comparable to FAN medium for other microorganisms.
Recognition of Two Groups of Methicillin-Resistant
Staphylococcus aureus
Strains Based on Epidemiology, Antimicrobial Susceptibility, Hypervariable-Region Type, and Ribotype in Finland
Authors: Salmenlinna Saara , Vuopio-Varkila Jaana
Published: July 2002

Epidemiological evidence suggests that some methicillin-resistant
Staphylococcus aureus
(MRSA) strains are more prone to dissemination than others. We studied 72 MRSA strains, collected through nationwide MRSA surveillance in 1992 through 1999 and known to be either (i) sporadic, (ii) local outbreak strains spread within one hospital, or (iii) epidemic strains spread among hospitals, by antimicrobial susceptibility testing, hybridization of the
mec
hypervariable region (HVR), and ribotyping. Our results show that two main groups can be identified among these strains. The first group includes mainly nonepidemic, nonmultiresistant MRSA strains showing a specific
mec
HVR hybridization pattern, A, in combination with a variety of ribotypes. The other group includes multiresistant strains with
mec
HVR hybridization pattern B or C in association with closely related ribotype a or b. Sixty-four percent (9 of 14) of Finnish epidemic MRSA strains belong to the latter group. These findings support the existence of differences in epidemic potential among MRSA strains.
Lack of Usefulness of Carbon Utilization Tests for Identification of
Mycobacterium mucogenicum
Authors: Conville Patricia S. , Witebsky Frank G.
Published: July 2002

Carbon utilization tests have proven to be useful for the identification of some species of rapidly growing mycobacteria and have been described as one of the few tests useful for the differentiation of
Mycobacterium mucogenicum
from other rapid growers. We have found the carbon utilization tests to be unreliable for the identification of patient isolates of this species. In this study, using 28 isolates of rapidly growing mycobacteria, we examined several variables which might have an effect on results of citrate, inositol, and mannitol utilization: inoculum concentration, incubation temperature, and medium manufacturer. None of these variables affected results obtained for most species of rapid growers or for ATCC strains of
M. mucogenicum
. Results for patient isolates of
M. mucogenicum
were found to be inconsistent regardless of the methodology employed and resulted in an ambiguous identification of these isolates or an incorrect identification as
Mycobacterium chelonae
. Molecular or cell wall analysis may be the best technique to employ for accurate identification of
M. mucogenicum
.
Detection of Precytopathic Effect of Enteroviruses in Clinical Specimens by Centrifugation-Enhanced Antigen Detection
Authors: Lipson Steven M. , David Kathryn , Shaikh Fatima , Qian Lian
Published: July 2002

Rapid enterovirus detection is important for decisions about antibiotic administration and length of hospital stay. The efficacy of rapid antigen detection-cell culture amplification (Ag-CCA) was evaluated with monoclonal antibodies (MAbs) 5-D8/1 (DAKO) and Pan-Enterovirus clone 2E11 (Chemicon) with 10 poliovirus, echovirus, and coxsackievirus type A and B stock isolates and College of American Pathologists check samples. By using Ag-CCA technology, MAb 2E11 was more sensitive than 5-D8/1 at detecting a greater number of stock isolates at or past tube (cytopathic effect [CPE]) culture (TC) end points. The efficacy of Ag-CCA in the clinical setting was subsequently confirmed with 273 consecutively freshly collected nasopharyngeal aspirate or swab specimens, rectal swab, and cerebrospinal fluid specimens during the 1999 enterovirus season. All specimens were tested by Ag-CCA in parallel with rhesus monkey kidney (RhMk), MRC-5, and A549 conventional TCs. Approximately 60% of field specimens were additionally tested with Hep-2 and HNK conventional TCs. Sixty-two percent of the clinical specimens tested were Ag-CCA positive after 48 h. Among 51 isolates, the mean time to CPE or culture confirmation was 5.5 days (range, 2 to 18 days). After 48 h, Ag-CCA achieved sensitivity, specificity, and positive and negative predictive values of 62, 100, 100, and 93%, respectively. During the same period, TC-CPE displayed test parameters of 12, 100, 100, and 85%, respectively. After 5 days, the sensitivity and specificity of Ag-CCA increased to 92 and 98%, respectively. Within the same period, isolation attained sensitivity and specificity of 52 and 100%, respectively. Although Ag-CCA displayed slightly reduced sensitivity and reduced specificity compared with conventional cell culture after 14 days, the markedly superior 48-h enterovirus Ag-CCA detection rate supports incorporation of this assay into the routine clinical setting.
Detection of
rpoB
Mutations in
Mycobacterium tuberculosis
by Biprobe Analysis
Authors: Edwards K. J. , Metherell L. A. , Yates M. , Saunders N. A.
Published: July 2002

A biprobe assay utilizing LightCycler technology was developed to detect rifampin resistance-associated gene mutations in the
Mycobacterium tuberculosis rpoB
gene. Three biprobes detected all mutations present in the 46 rifampin-resistant isolates. Wild-type sequences were correctly identified in each case. The method was reproducible, accurate, and easy to use.
Immune Reactivity of Human Sera to the Glycoprotein B of Human Herpesvirus 7
Authors: Franti Michael , Aubin Jean-Thierry , De Saint-Maur Guillemette , Kosuge Haruhiko , Yamanishi Koichi , Gautheret-Dejean Agnes , Garbarg-Chenon Antoine , Huraux Jean-Marie , Agut Henri
Published: July 2002

The glycoprotein B (gB) is highly conserved among distinct human herpesvirus 7 (HHV-7) strains. Similarly to other herpesvirus glycoproteins, gB has been assumed to induce a specific human immune response. However, it did not appear as an immunodominant protein in conventional immunoblot assays. Recombinant gB, obtained from either
Escherichia coli
or baculovirus expression systems, did react specifically with HHV-7-seropositive sera, and the main corresponding epitopes were located in its N-terminal part. A 24-amino-acid peptide, corresponding to a predicted hydrophilicity peak and presenting no extensive homology with other betaherpesvirus glycoproteins, was selected in this region at positions 129 to 152 of the gB sequence. When tested by enzyme-linked immunosorbent assay (ELISA), this peptide specifically reacted with HHV-7-seropositive sera. This reactivity was significantly inhibited by the preincubation of sera with the peptide itself, lysates of gB-expressing cells, or lysates of HHV-7-infected cells. The reactivity was not significantly modified when sera were preincubated with lysates of either human cytomegalovirus (HCMV)- or HHV-6-infected cells. In cross-sectional studies including both children and adults, 49 out of 61 serum samples (80%) were found to be positive by HHV-7 ELISA, independent of their reactivity to HCMV. A longitudinal serological study of 17 children during the first 4 years of life showed that the level of ELISA-detected antibodies significantly decreased within a few weeks after birth and then increased in the following months, likely reflecting, respectively, the loss of maternal antibodies and the occurrence of seroconversion. These results demonstrate that gB peptide ELISA might be a useful tool for the serological study of HHV-7 infection.
Staphylococcus
Heterogeneously Resistant to Vancomycin in China and Antimicrobial Activities of Imipenem and Vancomycin in Combination against It
Authors: Benquan Wu , Yingchun Tang , Kouxing Zhang , Tiantuo Zhang , Jiaxing Zhu , Shuqing Tan
Published: July 2002

Of 115 methicillin-resistant
Staphylococcus
strains collected from sputum specimens, 34 strains reduced susceptibility to vancomycin, 9 of which emerged as heterogeneous vancomycin-resistant strains (hetero-VRS), with various degrees of vancomycin resistance at a frequency of 10
−6
or higher. Seventy-six percent (19 of 25) of non-hetero-VRS and 100% (9 of 9) of hetero-VRS were susceptible to synergistic treatment with vancomycin and imipenem. Clinical clearance between 9 hetero-VRS and 25 non-hetero-VRS had an obvious statistical significance (
P
= 0.001). The hetero-VRS may play an important role in vancomycin therapy failure.
Detection of Heat-Stable Antigens of
Campylobacter jejuni
and
C. coli
by Direct Agglutination and Passive Hemagglutination
Authors: Oza A. N. , Thwaites R. T. , Wareing D. R. A. , Bolton F. J. , Frost J. A.
Published: July 2002

The two serotyping schemes for the detection of heat-stable antigens of
Campylobacter jejuni
and
Campylobacter coli
use the same strains for antiserum production but differ in the detection systems used for identifying agglutination. The Penner method uses passive hemagglutination (PHA) while the Laboratory of Enteric Pathogens method uses the same antisera but in a whole-bacterial-cell direct agglutination (DA) protocol.
C. jejuni
produces a polysaccharide capsule, which is antigenic, and is the main component detected by the PHA method. The DA method will detect both capsule antigens and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) surface antigens. Comparison of both methods by using a selection of isolates from human infection has shown a range of variation in agglutination specificity, reflecting the differences in antigens detected by the two methods. While 27.4% of the 416
C. jejuni
isolates reacted with the antisera raised against the same type strains by either method, the majority showed a range of more complex relationships. None of the 37
C. coli
isolates reacted with the same antiserum by both methods. Together the two schemes gave a total of 102 distinct combined serogroups for
C. jejuni
and 16 for
C. coli
. Thus, while some clonally related isolates share the same capsule and LOS or LPS antigens, other strains appear to have a common capsule antigen but differ in their LPS or LOS structures or vice versa.
Development and Application of a New Scheme for Typing
Campylobacter jejuni
and
Campylobacter coli
by PCR-Based Restriction Fragment Length Polymorphism Analysis
Authors: Shi Feng , Chen Yuen Yuen , Wassenaar Trudy M. , Woods Walter H. , Coloe Peter J. , Fry Benjamin N.
Published: July 2002

A molecular typing approach for
Campylobacter jejuni
and
Campylobacter coli
was developed with restriction fragment length polymorphism analysis of a 9.6-kb PCR-amplified portion of the lipopolysaccharide gene cluster. Sixty-one Penner serotype reference strains were analyzed with this new genotyping scheme, and 32 genogroups were found. Eleven additional genogroups were obtained from 87 clinical
C
.
jejuni
strains tested. This molecular typing method shows a correlation with the Penner heat-stable serotyping method, a phenotypic typing method based on lipopolysaccharide structures that is often used as a “gold standard” for subtyping
Campylobacter
spp. This strong correlation suggests that the data obtained can be directly compared with epidemiological data collected in the past by classical serotyping of
C. jejuni
and
C. coli.
In contrast to the high percentage of nontypeability by phenotyping, this molecular typing method results in 100% typeability and provides a superior alternative to serotyping.
Utilization of Nonviral Sequences for Minus-Strand DNA Transfer and Gene Reconstitution during Retroviral Replication
Authors: Cheslock Sara Rasmussen , Anderson Jeffrey A. , Hwang Carey K. , Pathak Vinay K. , Hu Wei-Shau
Published: July 2002

Minus-strand DNA transfer, an essential step in retroviral reverse transcription, is mediated by the two repeat (R) regions in the viral genome. It is unclear whether R simply serves as a homologous sequence to mediate the strand transfer or contains specific sequences to promote strand transfer. To test the hypothesis that the molecular mechanism by which R mediates strand transfer is based on homology rather than specific sequences, we examined whether nonviral sequences can be used to facilitate minus-strand DNA transfer. The green fluorescent protein (GFP) gene was divided into GF and FP fragments, containing the 5′ and 3′ portions of GFP, respectively, with an overlapping F fragment (85 bp). FP and GF were inserted into the 5′ and 3′ long terminal repeats, respectively, of a murine leukemia virus-based vector. Utilization of the F fragment to mediate minus-strand DNA transfer should reconstitute GFP during reverse transcription. Flow cytometry analyses demonstrated that GFP was expressed in 73 to 92% of the infected cells, depending on the structure of the viral construct. This indicated that GFP was reconstituted at a high frequency; molecular characterization further confirmed the accurate reconstitution of GFP. These data indicated that nonviral sequences could be used to efficiently mediate minus-strand DNA transfer. Therefore, placement and homology, not specific sequence context, are the important elements in R for minus-strand DNA transfer. In addition, these experiments demonstrate that minus-strand DNA transfer can be used to efficiently reconstitute genes for gene therapy applications.
Efficient Site-Specific Nonribozyme Opening of Hepatitis Delta Virus Genomic RNA in Infected Livers
Authors: Chang Jinhong , Moraleda Gloria , Gudima Severin , Taylor John
Published: July 2002

Examination of the 1,679-nucleotide (nt) unit-length hepatitis delta virus (HDV) RNAs in the livers of two HDV-infected woodchucks showed that 96% of the antigenomic RNA but only 50% of the genomic RNA was circular. We subsequently found that at least half of the linear unit-length genomic RNA was open at a unique location. Using a modified form of RNA ligation-mediated amplification of cDNA ends, we showed that the 5′ end was located at nt 1212. Like the previously described ribozyme cleavage site at nt 686, the new site produced a 5′-OH. Nevertheless, we showed that this novel site was not produced by activity of the HDV genomic ribozyme. We speculate that the 5′ end at nt 1212 reflects a preferred site of posttranscriptional endonucleolytic cleavage of genomic RNA.
Variation in the Nucleotide Sequence of Cottontail Rabbit Papillomavirus a and b Subtypes Affects Wart Regression and Malignant Transformation and Level of Viral Replication in Domestic Rabbits
Authors: Salmon Jérôme , Nonnenmacher Mathieu , Cazé Sandrine , Flamant Patricia , Croissant Odile , Orth Gérard , Breitburd Françoise
Published: July 2002

We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits.
Identification of a Domain in Human Immunodeficiency Virus Type 1 Rev That Is Required for Functional Activity and Modulates Association with Subnuclear Compartments Containing Splicing Factor SC35
Authors: D'Agostino Donna M. , Ferro Tiziana , Zotti Lorenza , Meggio Flavio , Pinna Lorenzo A. , Chieco-Bianchi Luigi , Ciminale Vincenzo
Published: July 2002

The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the ΔLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.
Hepadnavirus Envelope Topology: Insertion of a Loop Region in the Membrane and Role of S in L Protein Translocation
Authors: Grgacic E. V. L. , Kuhn C. , Schaller H.
Published: July 2002

A unique feature of the large hepadnavirus envelope protein (L) is its mixed transmembrane topology, resulting from partial posttranslational translocation of the pre-S domain. Using protease protection analysis, we demonstrate for duck hepatitis B virus an essential role for the small envelope protein (S) in this process, providing the first experimental evidence for an S translocation channel. Further analysis revealed that the presumed cytoplasmic loop between TM1 and TM2 in the C-terminal S domain is membrane embedded and protrudes to the particle surface. These data suggest that some L molecules form a highly folded, potentially spring-loaded topology with five membrane-spanning regions and a membrane-traversing pre-S chain.
Degradation of Tobacco Mosaic Virus Movement Protein by the 26S Proteasome
Authors: Reichel Christoph , Beachy Roger N.
Published: July 2002

Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 and
clasto
-lactacystin-β-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.
Patterns of Genomic Sequence Diversity among Their Simian Immunodeficiency Viruses Suggest that L'Hoest Monkeys (
Cercopithecus lhoesti
) Are a Natural Lentivirus Reservoir
Authors: Beer Brigitte E. , Bailes Elizabeth , Dapolito George , Campbell Barbara J. , Goeken Robert M. , Axthelm Michael K. , Markham Philip D. , Bernard Jacky , Zagury Daniel , Franchini Genoveffa , Sharp Paul M. , Hirsch Vanessa M.
Published: July 2002

Recently, we described a novel simian immunodeficiency virus (SIVlhoest) from a wild-caught L'Hoest monkey (
Cercopithecus lhoesti
) from a North American zoo. To investigate whether L'Hoest monkeys are the natural host for these viruses, we have screened blood samples from 14 wild animals from the Democratic Republic of Congo. Eight (57%) were found to be seropositive for SIV. Nearly full-length genome sequences were obtained for SIV isolates from three of these monkeys and compared to the original isolate and to other SIVs. The four samples of SIVlhoest formed a distinct cluster in phylogenetic trees. Two of these isolates differed on average at only about 5% of nucleotides, suggesting that they were epidemiologically linked; otherwise, the SIVlhoest isolates differed on average by 18%. Both the level of diversity and the pattern of its variation along the genome were very similar to those seen among isolates of SIVagm from vervet monkeys, pointing to similarities in the nature of, and constraints on, SIV evolution in these two species. Discordant phylogenetic relationships among the SIVlhoest isolates for different genomic regions indicated that mosaic viruses have been generated by recombination, implying that individual monkeys have been coinfected by more than one strain of SIV. Taken together, these observations provide strong evidence that L'Hoest monkeys constitute a natural reservoir for SIV.
Viral and Cellular Factors That Target the Promyelocytic Leukemia Oncogenic Domains Strongly Activate a Glucocorticoid-Responsive Promoter
Authors: Wienzek Sandra , Dobbelstein Matthias
Published: July 2002

Promyelocytic leukemia (PML) oncogenic domains (PODs) accumulate the transcriptional cofactor named CREB binding protein (CBP) and have been suggested to function as centers of transcription. Transcriptional activation by nuclear hormones, such as glucocorticoids, is augmented by the key constituent of PODs, the PML protein, and decreased by the POD-associated Tax protein of human T-cell leukemia virus type 1 (HTLV-1). This led to the hypothesis that intact PODs might play a positive role in the activation of these promoters. We report here that transiently expressed E4orf3 protein of adenovirus type 5, immediate-early protein 1 of human cytomegalovirus, and the PML-retinoic acid receptor fusion protein from leukemia cells each redistribute CBP within the nucleus. However, unlike the Tax protein of HTLV-1, these factors did not inhibit a glucocorticoid-inducible promoter but strongly enhanced its activity. Thus, at least glucocorticoid-induced transcription does not depend on POD integrity.
Thymic Lesions in Cats Infected with a Pathogenic Molecular Clone or an
ORF-A/2
-Deficient Molecular Clone of Feline Immunodeficiency Virus
Authors: Norway Robert M. , Crawford P. Cynthia , Johnson Calvin M. , Mergia Ayalew
Published: July 2002

Previous studies using feline immunodeficiency virus (FIV) molecular clones lacking the putative transactivator gene (
ORF-A/2
) failed to address the issue of thymus pathogenesis or investigate the levels of viral replication in separate lymphoid compartments (Y. Inoshima, et al., J. Virol. 70:8518–8526, 1996; E. E. Sparger, et al., Virology 205:546–553, 1994). Using a highly pathogenic molecular clone of FIV, JSY3, and an
ORF-A/2
-deficient mutant, JSY3ΔORF-A/2, we compared viral replication and the extent of thymic dysfunction as measured by the formation of lymphoid follicles and alteration of the thymocyte subsets. Viral replication was reduced in JSY3ΔORF-A/2-infected cats as measured by lymphocyte coculture, immunohistochemistry, and quantitative PCR. Cell-associated viral load measured by lymphocyte coculture varied in a tissue-dependent manner with replication highest in lymphocytes isolated from the thymus, lower in those from the peripheral blood, and lowest in those from lymph node. Thymic proviral load and the number of viral p24 Gag-positive cells within the thymus detected by immunohistochemistry were also reduced. In addition, the onset of a reduced peripheral blood CD4/CD8 ratio was delayed in JSY3ΔORF-A/2-infected cats. The formation and extent of thymic lymphoid follicular hyperplasia were similar in JSY3 and JSY3ΔORF-A/2-infected cats as measured by anticytokeratin immunohistochemistry and flow cytometry for percent pan T-negative, immunoglobulin G-positive cells within the thymus. In contrast, comparison of thymocyte subpopulations demonstrated a reduced expansion of single-positive CD4
−
CD8
+
thymocytes in JSY3ΔORF-A/2-infected cats. Level of viral replication, therefore, may not correlate with the formation of thymic lymphoid follicles but may correlate with the expansion of the single-positive CD4
−
CD8
+
thymocyte subpopulation.
Cleavage in and around the DR1 Element of the
a
Sequence of Herpes Simplex Virus Type 1 Relevant to the Excision of DNA Fragments with Length Corresponding to One and Two Units of the
a
Sequence
Authors: Umene Kenichi
Published: July 2002

The
a
sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage) to yield unit-length linear HSV-1 DNA prior to or during packaging of HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and 0.5 kb (longer band), the lengths of which correspond to one and two units of the
a
sequence, was identified using acrylamide gel electrophoresis of HSV-1 DNA preparations extracted by the method of Hirt. Twelve DNA fragments from each band were molecularly cloned, and nucleotide sequences were determined. Both termini of eight (67%) DNA clones from the shorter band corresponded to the specific cleavage site on DR1. Five (41%) DNA clones from the longer band had a terminus corresponding to the specific cleavage site on DR1 on one side, but not on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA clones from the longer band were in and around DR1. Thus, cleavage events of DR1 can be classified into three categories: (i) authentic cleavage; (ii) site-specific cleavage on the third DR1 distal from the S component (secondary site-specific cleavage), which is related to the generation of the shorter DNA band in combination with authentic cleavage; and (iii) less-specific cleavage events in and around other DR1 elements which relate to the generation of the longer DNA band.
Evidence for Retroviral Intramolecular Recombinations
Authors: Zhang Jiayou , Ma Yan
Published: July 2002

As a consequence of being diploid, retroviruses have a high recombination rate. Naturally occurring retroviruses contain two repeat sequences (R regions) flanking either end of their RNA genomes, and recombination between these two R regions occurs at a high rate. We deduced that recombination may occur between two sequences within the same RNA molecule (intramolecular) as well as between sequences present within two separate RNA molecules (intermolecular). Intramolecular recombination would usually result in a deletion within the progeny provirus. In this report, we demonstrate that intramolecular recombination between two identical sequences occurred within a chimeric RNA vector. In addition, high rates of recombination between two identical sequences within the same RNA molecule resulted mostly from intramolecular recombination.
Role for p53 in Gene Induction by Double-Stranded RNA
Authors: Hummer B. T. , Li X.-L. , Hassel B. A.
Published: July 2002

Cross talk between p53 and interferon-regulated pathways is implicated in the induction of gene expression by biologic and genotoxic stresses. We demonstrate that the interferon-stimulated gene ISG15 is induced by p53 and that p53 is required for optimal gene induction by double-stranded RNA (dsRNA), but not interferon. Interestingly, virus induces ISG15 in the absence of p53, suggesting that virus and dsRNA employ distinct signaling pathways.
The 2.2-Kilobase Latency-Associated Transcript of Herpes Simplex Virus Type 2 Does Not Modulate Viral Replication, Reactivation, or Establishment of Latency in Transgenic Mice
Authors: Wang Kening , Pesnicak Lesley , Guancial Elizabeth , Krause Philip R. , Straus Stephen E.
Published: July 2002

To better understand the mechanisms responsible for the observed effects of deletions in the promoter region of the latency-associated transcript (LAT) gene in impairing herpes simplex virus (HSV) reactivation, we generated mice transgenic for a 5.5-kb HSV type 2 (HSV-2) genomic fragment spanning the major LAT, along with the LAT promoter and flanking regions, in the C57BL/6 background. The mice expressed abundant 2.2-kb major LATs in trigeminal ganglia (TG) and other tissues. The effects of the transgene on HSV-2 infection, latency, and reactivation were assessed. When infected with wild-type (WT) HSV-2 or its LAT promoter deletion (LAT
−
) mutant, primary lung fibroblast lines established from normal C57BL/6 and transgenic mice supported virus growth equally well. The replication of these viruses in the mouse eye and their spread to TG and brains were similar. The quantities of latent viral DNA in TG of transgenic and normal mice, as determined by real-time PCR, were comparable. UV light-induced reactivation of the LAT
−
mutant from transgenic mice (0 to 7%) was no more frequent than that from normal mice (0 to 14%), while WT virus was reactivated from 13 to 54% of normal mice and 22 to 54% of transgenic mice. The cumulative data indicate that, when expressed transgenically, the HSV-2 major LAT cannot influence HSV-2 infection or latency and cannot complement the defect in reactivation of the LAT
−
mutant. These results imply that the phenotype of reduced reactivation associated with the LAT
−
mutant is related to a function encoded in the LAT promoter but not to the major LAT itself.