bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular BacteriumChlamydophila abortus
Authors: Everson J. S. , Garner S. A. , Fane B. , Liu B.-L. , Lambden P. R. , Clarke I. N. 
Published: July 2002

ABSTRACTA number of bacteriophages belonging to theMicroviridaehave been described infecting chlamydiae. Phylogenetic studies divide theChlamydiaceaeinto two distinct genera,ChlamydiaandChlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered inChlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds bothC. abortuselementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genusChlamydia(C. trachomatis, C. suis, orC. muridarum). Chp2 can infectC. abortus, C. felis, andC. pecorumbut is unable to infect other members of this genus, includingC. caviaeandC. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.
Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of
Escherichia coli
Authors: Nelson David E. , Ghosh Anindya S. , Paulson Avery L. , Young Kevin D. 
Published: July 2002

ABSTRACT

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of
Escherichia coli
are closely related and have similar
dd
-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of
E. coli
have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other
dd
-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the
dd
-carboxypeptidase enzymatic core.
Regulation ofnapGene Expression and Periplasmic Nitrate Reductase Activity in the Phototrophic BacteriumRhodobacter sphaeroidesDSM158
Authors: Gavira Mónica , Roldán M. Dolores , Castillo Francisco , Moreno-Vivián Conrado 
Published: July 2002

ABSTRACTBacterial periplasmic nitrate reductases (Nap) can play different physiological roles and are expressed under different conditions depending on the organism.Rhodobacter sphaeroidesDSM158 has a Nap system, encoded by thenapKEFDABCgene cluster, but nitrite formed is not further reduced because this strain lacks nitrite reductase. Nap activity increases in the presence of nitrate and oxygen but is unaffected by ammonium. Reverse transcription-PCR and Northern blots demonstrated that thenapKEFDABCgenes constitute an operon transcribed as a single 5.5-kb product. Northern blots andnap-lacZfusions revealed thatnapexpression is threefold higher under aerobic conditions but is regulated by neither nitrate nor ammonium, although it is weakly induced by nitrite. On the other hand, nitrate but not nitrite causes a rapid enzyme activation, explaining the higher Nap activity found in nitrate-grown cells. Translationalnap′-′lacZfusions reveal that thenapKandnapDgenes are not efficiently translated, probably due to mRNA secondary structures occluding the translation initiation sites of these genes. Neither butyrate nor caproate increasesnapexpression, although cells growing phototrophically on these reduced substrates show a very high Nap activity in vivo (nitrite accumulation is sevenfold higher than in medium with malate). Phototrophic growth on butyrate or caproate medium is severely reduced in the NapA−mutants. Taken together, these results indicate that nitrate reduction inR. sphaeroidesis mainly regulated at the level of enzyme activity by both nitrate and electron supply and confirm that the Nap system is involved in redox balancing using nitrate as an ancillary oxidant to dissipate excess reductant.
Detection of Sporadic Cases of Hepatitis E Virus (HEV) Infection in China Using Immunoassays Based on Recombinant Open Reading Frame 2 and 3 Polypeptides from HEV Genotype 4
Authors: Wang Youchun , Zhang Huayuan , Li Zhuo , Gu Wenjie , Lan Haiyuan , Hao Wa , Ling Roger , Li Hemin , Harrison Tim J. 
Published: July 2002

ABSTRACT

We reported previously on the complete sequence of hepatitis E virus (HEV) genotype 4, isolated from patients with sporadic cases of acute HEV infection in China. At least eight HEV genotypes have now been described worldwide, and further isolates await classification. Current immunoassays for the detection of anti-HEV antibodies are based on polypeptides from genotypes 1 and 2 only and may be inadequate for the reliable detection of other genotypes. Because genotypes 1 and 4 predominate in China, we wished to investigate the antigenic reactivities of HEV genotype 4 proteins. Four overlapping regions of open reading frame 2 (ORF2) (FB5, amino acids [aa] 1 to 130; E4, aa 67 to 308; F2-2, aa 288 to 461; E5, aa 414 to 672) and the entire ORF3 product were expressed in
Escherichia coli
as fusion proteins. Enzyme immunoassays based on each of the five purified polypeptides were evaluated with sera from patients with sporadic cases of acute HEV infection. Individual immunoassays derived from HEV genotype 4 detected more cases of acute hepatitis E than a commercial assay. Some serum samples, which were positive for anti-HEV immunoglobulin G only by assays based on HEV genotype 4, were positive for HEV RNA by reverse transcription-PCR. Polypeptide FB5, from the N terminus of ORF2, had the greatest immunoreactivity with sera from patients with acute hepatitis E. These data indicate that the N terminus of ORF2 may provide epitopes which are highly reactive with acute-phase sera and that assays based on genotypes 1 and 2 alone may be inadequate for the detection of HEV infection in China, where sporadic cases of HEV infection are caused predominantly by HEV genotypes 4 and 1.
Outbreak of Subclinical Mastitis in a Flock of Dairy Sheep Associated with
Burkholderia cepacia
Complex Infection
Authors: Berriatua E. , Ziluaga I. , Miguel-Virto C. , Uribarren P. , Juste R. , Laevens S. , Vandamme P. , Govan J. R. W. 
Published: July 2002

ABSTRACT

An outbreak of subclinical mastitis in a flock of 620 milking sheep was investigated. Microbiological and epidemiological analyses identified the causative agent as belonging to the
Burkholderia cepacia
complex (formerly
Pseudomonas cepacia
). Every ewe in the milking flock was individually tested for subclinical mastitis on two separate occasions, 6 weeks apart, by the California (rapid) mastitis test (CMT). The proportion of CMT-positive ewes was 69 of 393 (17.6%) on the first sampling and 27 of 490 (5.5%) on the second sampling. Pure
B. cepacia
cultures identified with the API 20 NE system were grown from 64 of 96 (66.7%) CMT-positive ewes and from 1 of 33 (3.0%) CMT-negative ewes. Statistical analysis confirmed the significant association between a positive CMT result and a positive culture result for
B. cepacia
complex. Additional polyphasic taxonomic analyses of eight isolates showed that seven belonged to
B. cepacia
genomovar III; the remaining isolate was identified as
Burkholderia vietnamiensis
(formerly
B. cepacia
genomovar V). Bacteriological investigation of samples from milking equipment and other environmental sites failed to identify “
B. cepacia
” in any of the samples taken. To our knowledge, this is the first report of an outbreak of natural infection in animals caused by
B. cepacia
complex and the first description of
B. cepacia
complex infection in sheep.
Electrophoretic Karyotype Analysis of Sequential
Candida parapsilosis
Isolates from Patients with Persistent or Recurrent Fungemia
Authors: Shin Jong Hee , Shin Dong Hyeon , Song Jeong Won , Kee Seung Jung , Suh Soon Pal , Ryang Dong Wook 
Published: July 2002

ABSTRACT

We assessed the genetic relatedness of sequential isolates of
Candida parapsilosis
during persistent or recurrent fungemia and the effect of central venous catheter (CVC) removal. Serial isolates of
C. parapsilosis
were obtained from 17 patients with persistent or recurrent fungemia over periods of up to 5 months. Forty-eight
C. parapsilosis
isolates from the blood of 17 patients were analyzed by electrophoretic karyotyping (EK) with pulsed-field gel electrophoresis (PFGE), revealing 25 different karyotypes. The strains sequentially isolated from each of seven patients whose fungemia resolved following CVC removal had the same karyotype. Two patients with fungemia that cleared without CVC removal each had two sequential isolates with different karyotypes. In six (75%) of the eight patients whose fungemia was recurrent even after CVC removal, the karyotypes of the pre- and post-CVC removal isolates were different, implying the emergence of a new strain. Overall, the sequential strains from each patient had identical karyotypes in 53% (9 of 17) of the patients and two different karyotypes in 47% (8 of 17). This study shows that EK with PFGE is useful for investigating persistent or recurrent fungemia due to
C. parapsilosis
and that recurrent fungemia due to
C. parapsilosis
is more likely caused by reinfection with a second strain.
Evaluation of Mycobacteria Growth Indicator Tube for Direct and Indirect Drug Susceptibility Testing of
Mycobacterium tuberculosis
from Respiratory Specimens in a Siberian Prison Hospital
Authors: Goloubeva Vera , Lecocq Maryvonne , Lassowsky Piotr , Matthys Francine , Portaels Françoise , Bastian Ivan 
Published: July 2002

ABSTRACT

The manual Mycobacteria Growth Indicator Tube (MGIT) method was evaluated for performing direct and indirect drug susceptibility testing (DST) of
Mycobacterium tuberculosis
for isoniazid and rifampin on 101 strongly smear-positive sputum specimens in a Siberian prison hospital. Using the indirect method of proportion (MOP) as the “gold standard,” the accuracies of isoniazid and rifampin susceptibility testing by the direct MGIT system were 97.0 and 94.1%, respectively. The accuracy of the indirect MGIT system was 98.0% for both drugs. The turnaround times from specimen processing to reporting of the DST results ranged between 4 and 23 (mean, 9.2) days by the direct MGIT method, 9 and 30 (mean, 15.3) days by the indirect MGIT method, and 26 and 101 (mean, 59.6) days by the indirect MOP. MGIT appears to be a reliable, rapid, and convenient method for performing direct and indirect DSTs in low-resource settings, but further studies are required to refine the direct DST protocol. Cost is the only factor prohibiting widespread implementation of MGIT.
Discrimination between
Candida albicans
and Other Pathogenic Species of the Genus
Candida
by Their Differential Sensitivities to Toxins of a Panel of Killer Yeasts
Authors: Buzzini P. , Martini A. 
Published: July 2002

ABSTRACT

The differential sensitivities to toxins produced by a short panel of four killer yeasts allowed discrimination between 91 strains of the yeast
Candida albicans
and 223 non-
C. albicans Candida
strains. One hundred percent of
C. albicans
isolates exhibited negative results to the toxin panel, while 100% of non-
C. albicans
cultures gave well-defined and reproducible positive results to at least one of the four killer toxins. Among
C. albicans
strains only 96 and 87% gave germ tube (GT)- and chlamydospore-positive results, respectively. In addition a few GT-false-positive strains were detected among non-
C. albicans
isolates. Susceptibility to the toxin panel is apparently expressed more consistently than either GT or chlamydospore production and may constitute a promising basis for a new simple and easy-to-use procedure for routine discrimination between the species
C. albicans
and other species of the genus
Candida
.
Development and Evaluation of a DNA Enzyme Immunoassay Method for
env
Genotyping of Subtypes A through G of Human Immunodeficiency Virus Type 1 Group M, with Discrimination of the Circulating Recombinant Forms CRF01_AE and CRF02_AG
Authors: Plantier Jean-Christophe , Vergne Laurence , Damond Florence , MBoup Souleymane , MPoudi-NGole Eitel , Buzelay Laurence , Farfara Isabelle , Brand Denys , Peeters Martine , Brun-Vézinet Françoise , Delaporte Eric , Barin Francis 
Published: July 2002

ABSTRACT

The tools currently available for genetic subtyping of human immunodeficiency virus type 1 are laborious or can be used only for the analysis of a limited number of samples and/or subtypes. We developed and evaluated a molecular biology-based method using subtype-specific oligonucleotide probes for
env
genotyping of subtypes A through G, CRF01_AE, and CRF02_AG. DNA enzyme immunoassay (DEIA) genotyping is based on nested PCR amplification of the 5′ end of the
env
gene (proviral DNA), followed by subtype-specific hybridization and immunoenzymatic detection on microplates. DEIA genotyping was validated with a large number of samples (
n
= 128) collected in Europe (France;
n
= 47), West-Central Africa (Cameroon;
n
= 36), and West Africa (Senegal;
n
= 45). Three different formats, depending on the distribution of subtypes in the three countries, were developed. The results were compared with those obtained by sequencing of the V3-V5 region and phylogenetic analysis or an
env
heteroduplex mobility assay. Additional sequencing and phylogenetic analyses of the DEIA region (the first codon of the env coding sequence to the middle of conserved region C1 of gp120) were performed to investigate the reasons for discrepancies. Intense and highly specific reactions between the oligonucleotide probes and the corresponding samples were observed. Overall, correct identification was achieved for 107 of 128 samples (83.6%). One sample was not amplified, 10 (8%) were nontypeable (NT), and 10 (8%) were misidentified. Six of the 10 discordant samples were further investigated by phylogenetic analysis, which indicated that these samples corresponded to recombinants involving the env 5′ end and the V3 and V5 regions of the two parental clades. Sequencing of NT samples showed numerous differences between sample and probe sequences, resulting in a lack of hybridization, and revealed the limitations of the selected probes in terms of specificity and sensitivity. We demonstrated the feasibility of DEIA genotyping: six subtypes plus the two most prevalent circulating recombinant forms were discriminated by using the 5′ end of the
env
gene. This method can be adapted to the local situation by including only probes that correspond to the prevalent strains.
In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation
Authors: Nateri Abdolrahman S. , Hughes Pamela J. , Stanway Glyn 
Published: July 2002

ABSTRACT

Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus,
Parechovirus
, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5′ untranslated region (5′UTR) shows similarities to that of cardio/aphthoviruses in some of its structural domains (A to L). The 5′UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5′UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.
Pathogenic Simian/Human Immunodeficiency Virus SHIV
KU
Inoculated into Immunized Macaques Caused Infection, but Virus Burdens Progressively Declined with Time
Authors: Silverstein Peter S. , Mackay Glenn A. , Mukherjee Sampa , Li Zhuang , Piatak Michael , Lifson Jeffrey D. , Narayan Opendra , Kumar Anil 
Published: July 2002

ABSTRACT

Using the simian immunodeficiency virus/human immunodeficiency virus (SHIV)-macaque model of AIDS, we had shown in a previous report that a live, nonpathogenic strain of SHIV, further attenuated by deletion of the
vpu
gene and inoculated orally into adult macaques, had effectively prevented AIDS following vaginal inoculation with pathogenic SHIV
KU
. Examination of lymph nodes from the animals at 18 weeks postchallenge had shown that all six animals were persistently infected with challenge virus. We report here on a 2-year follow-up study on the nature of the persistent infections in these animals. DNA of the vaccine virus was present in the lymph nodes at all time points tested, as far as 135 weeks postchallenge. In contrast, the DNA of SHIV
KU
became undetectable in one animal by week 55 and in three others by week 63. These four macaques have remained negative for SHIV
KU
DNA as far as the last time point examined at week 135. Quantification of the total viral DNA concentration in lymph nodes during the observation period showed a steady decline. All animals developed neutralizing antibody and cytotoxic-T-lymphocyte responses to SHIV
KU
that persisted throughout the observation period. Vaccine-like viruses were isolated from two animals, and a SHIV
KU
-like virus was isolated from one of the two macaques that remained positive for SHIV
KU
DNA. There was no evidence of recombination between the vaccine and the challenge viruses. Thus, immunization with the live vaccine not only prevented disease but also contributed to the steady decline in the virus burdens in the animals.
Localization of a Binding Site for Herpes Simplex Virus Glycoprotein D on Herpesvirus Entry Mediator C by Using Antireceptor Monoclonal Antibodies
Authors: Krummenacher Claude , Baribaud Isabelle , Ponce de Leon Manuel , Whitbeck J. Charles , Lou Huan , Cohen Gary H. , Eisenberg Roselyn J. 
Published: July 2002

ABSTRACT
The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.