bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Single-Tube, Cell Lysis-Based, Genus-Specific PCR Method for Rapid Identification of Mycobacteria: Optimization of Cell Lysis, PCR Primers and Conditions, and Restriction Pattern Analysis
Authors: Khan Izhar U. H. , Yadav Jagjit S.
Published: January 2004

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i) a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii) an improved
Mycobacterium
-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (
hsp
) gene and optimal PCR amplification conditions, and (iii) a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.
Clinical and Environmental Distributions of
Legionella
Strains in France Are Different
Authors: Doleans Anne , Aurell Helena , Reyrolle Monique , Lina Gerard , Freney Jean , Vandenesch Francois , Etienne Jerome , Jarraud Sophie
Published: January 2004

In France, the clinical distribution of
Legionella
species and serogroups does not correspond to their environmental distribution.
Legionella pneumophila
serogroup 1 is more prevalent among clinical isolates (95.4%) than in the environment (28.2%), whereas
L. anisa
is more frequent in the environment (13.8%) than in the clinical setting (0.8%).
Genetic Diversity and Population Structure ofMycobacterium tuberculosisin Casablanca, a Moroccan City with High Incidence of Tuberculosis
Authors: Tazi Loubna , El Baghdadi Jamila , Lesjean Sarah , Locht Camille , Supply Philip , Tibayrenc Michel , Bañuls Anne-Laure
Published: January 2004

Although lower-resource countries have by far the highest burden of tuberculosis, knowledge ofMycobacterium tuberculosispopulation structure and genetic diversity in these regions remains almost nonexistent. In this paper, 150 MoroccanM. tuberculosisisolates circulating in Casablanca were genotyped by random amplified polymorphic DNA analysis using 10 different primers and by mycobacterial interspersed repetitive units-variable number of tandem repeats typing at 12 loci. The population genetic tests revealed a basically clonal structure for this population, without excluding rare genetic exchanges. Genetic analysis also showed a notable genetic polymorphism for the speciesM. tuberculosis, a weak cluster individualization, and an unexpected genetic diversity for a population in such a high-incidence community. Phylogenetic analyses of this Moroccan sample also supported that these isolates are genetically heterogeneous.
Comparison of Diagnostic Sensitivities of Three Assays (Bartels Enzyme Immunoassay [EIA], Biotest EIA, and Binax NOW Immunochromatographic Test) for Detection of
Legionella pneumophila
Serogroup 1 Antigen in Urine
Authors: Guerrero Carmen , Toldos Carmen M. , Yagüe Genoveva , Ramírez Cristobal , Rodríguez Tomás , Segovia Manuel
Published: January 2004

The Bartels enzyme immunoassay (EIA), Biotest EIA, and Binax NOW immunochromatographic test (ICT) urinary antigen kits for the detection of
Legionella pneumophila
serogroup 1 were compared using 178 frozen urine samples. When nonconcentrated urine samples were used, the sensitivity levels of both enzyme EIAs were significantly higher than the sensitivity level of the ICT (Bartels EIA, 71.3%; Biotest EIA, 65.1%; Binax NOW ICT, 37% [
P
< 0.001]). After concentration of the urine samples, no significant differences in sensitivity were found among the three tests.
Identification and Molecular Characterization of Beta-Hemolytic Streptococci Isolated from Harbor Seals (
Phoca vitulina
) and Grey Seals (
Halichoerus grypus
) of the German North and Baltic Seas
Authors: Vossen A. , Abdulmawjood A. , Lämmler C. , Weiß R. , Siebert U.
Published: January 2004

Bacteriological investigations of seals of the German North and Baltic seas resulted in the isolation of bacteria of the genus
Streptococcus
belonging to Lancefield's serological groups C, F, and L. According to biochemical, serological, and 16S ribosomal DNA analysis, the group C and group F streptococci were identified as
Streptococcus phocae
. The group L streptococci could be classified as
Streptococcus dysgalactiae
subsp.
dysgalactiae
.
Transfer of a
Mycobacterium tuberculosis
Genotyping Method, Spoligotyping, from a Reverse Line-Blot Hybridization, Membrane-Based Assay to the Luminex Multianalyte Profiling System
Authors: Cowan Lauren S. , Diem Lois , Brake Mary Catherine , Crawford Jack T.
Published: January 2004

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.
Disseminated Infection with
Nattrassia mangiferae
in an Immunosuppressed Patient
Authors: Willinger Birgit , Kopetzky Gerhard , Harm Ferdinand , Apfalter Petra , Makristathis Athanasios , Berer Andrea , Bankier Alexander , Winkler Stefan
Published: January 2004

Disseminated infection with the coelomycetous fungus
Nattrassia mangiferae
is a very rare disease affecting only the immunocompromised host. We report the first case of a disseminated infection with spondylodiscitis and granular skin lesions due to
N. mangiferae
in a renal transplant patient.
First Report of a Case of Meningitis Caused by
Cryptococcus adeliensis
in a Patient with Acute Myeloid Leukemia
Authors: Rimek Dagmar , Haase Gerhard , Lück Andreas , Casper Jochen , Podbielski Andreas
Published: January 2004

Cryptococcus adeliensis
is a recently described new fungal species which has been isolated from decaying algae in Terre Adelie, Antarctica. We report the first known case of meningitis caused by
C. adeliensis
in a patient with acute myeloid leukemia undergoing allogeneic peripheral blood stem cell transplantation.
Osteosynthesis-Associated Bone Infection Caused by a Nonproteolytic, Nontoxigenic
Clostridium botulinum
-Like Strain
Authors: Carlier Jean-Philippe , K'ouas Guylène , Lozniewski Alain , Sirveaux François , Cailloux Philippe , Mory Francine
Published: January 2004

A nonproteolytic, nontoxigenic
Clostridium botulinum
strain identified by conventional and molecular techniques as type B-, E-, or F-like (BEF-like) was isolated from a human postsurgical wound. All previous reports of such strains have been from environmental sources. Since toxin production is the main taxonomic denominator for
C. botulinum
, a new name is needed for nonproteolytic, nontoxigenic BEF-like clinical isolates.
Disseminated Mucormycosis in a Patient with Acute Myeloblastic Leukemia Misdiagnosed as Infection by
Enterococcus faecium
Authors: Sammassimo S. , Mazzotta S. , Tozzi M. , Gentili S. , Lenoci M. , Santopietro R. , Bucalossi A. , Bocchia M. , Lauria F.
Published: January 2004

Mucormycosis is a rare complication in cancer patients. This report presents the case of a acute myeloblastic leukemia patient who developed an ascending paralysis due to disseminated mucormycosis. The presentation was unusual because the early symptoms were fever and pain, and the disease was misdiagnosed because of a concomitant infection by
Enterococcus faecium
.
Characterization of a Novel Group of Mycobacteria and Proposal of
Mycobacterium sherrisii
sp. nov
Authors: Selvarangan Rangaraj , Wu Whei-Kuo , Nguyen Trang T. , Carlson La Donna C. , Wallis Carolyn K. , Stiglich Susan K. , Chen Yi-Ching , Jost Kenneth C. , Prentice Jennifer L. , Wallace Richard J. , Barrett Sara L. Rassoulian , Cookson Brad T. , Coyle Marie B.
Published: January 2004

We describe here the characterization of five isolates of
Mycobacterium simiae
-like organisms representing a novel group based on whole-cell fatty acid analysis and genotypic evaluation. Two of the five isolates in this study, W55 and W58, were previously considered to belong to
M. simiae
serotype 2. Analysis of cellular fatty acids by gas-liquid chromatography indicated a close clustering of this group, which was well differentiated from the other
M. simiae
-like species. Molecular characterization was performed by nucleic acid sequencing of the small subunit rRNA gene and the gene encoding the 65-kDa heat shock protein and genomic DNA hybridization. Sequence analysis of the entire 16S rRNA gene showed a unique sequence most closely related to those of
M. triplex
and
M. simiae
. The
hsp65
partial gene sequence was identical for the five isolates, with 97% identity to the
M. simiae
type strain. However, qualitative whole genomic DNA hybridization analysis confirmed that this group is genetically distinct from
M. simiae
and
M. triplex
. Antimicrobial susceptibilities for this group resemble those of
M. simiae
and
M. lentiflavum
. We conclude that this group represents a unique
Mycobacterium
species for which we propose the name
Mycobacterium sherrisii
sp. nov.
Practical Bench Comparison of BBL CHROMagar Orientation and Standard Two-Plate Media for Urine Cultures
Authors: D'Souza Holly A. , Campbell Mary , Baron Ellen Jo
Published: January 2004

A total of 1,023 urine samples sent for routine culture were plated onto sheep blood and MacConkey agars and a BBL CHROMagar Orientation (CO; Becton Dickinson, Cockeysville, Md.) plate, and the results were compared. Of these, 250 urine samples (24%) grew >10,000 CFU of one or two putative pathogens/ml and 773 showed no growth (NG), mixed growth of <10,000 CFU/ml, or three or more strains (mixed). The CO and conventional medium results agreed completely for 595 cultures with NG or <10,000 CFU/ml. An additional 178 urine samples yielded clinically insignificant differences. Both medium sets essentially agreed on quantities and identification for 400 single-pathogen cultures and 9 mixed cultures. With the caveat that CO cannot differentiate
Klebsiella
,
Enterobacter
, and
Serratia
spp., enteric pathogens were identified only by morphology and color on CO. Direct visual differentiation of group B streptococci from lactobacilli is not possible, but lactobacillus cells always exhibited easily recognizable morphology on Gram stain. Of 108 paired organism susceptibility results encompassing 2,268 drug-pathogen combinations, there were 3% errors and only 1% very major errors. Use of CO allowed a >50% reduction in inoculation time and a >20% reduction in work-up time. For our laboratory, with 50% “no growth” and ca. 25% significant results (50%
Escherichia coli
), CO allowed time and workup cost savings for a majority of cultures. A cost analysis (time and supplies for our laboratory) showed that if CO is used alone, the break-even level for CO pricing is $1.78; if CO and blood agar are both used, the break-even pricing for CO is $1.53.
Detection of Human Anti-Flavivirus Antibodies with a West Nile Virus Recombinant Antigen Microsphere Immunoassay
Authors: Wong Susan J. , Demarest Valerie L. , Boyle Rebekah H. , Wang Tian , Ledizet Michel , Kar Kalipada , Kramer Laura D. , Fikrig Erol , Koski Raymond A.
Published: January 2004

We report a new, suspended-microsphere diagnostic test to detect antibodies to West Nile (WN) virus in human serum and cerebrospinal fluid (CSF). The microsphere immunofluorescence assay can be performed in less than 3 h on specimens of ≤30 μl. A recombinant WN virus envelope (E) protein antigen is covalently coupled to fluorescent polystyrene microspheres. After incubation with diluted serum or CSF, antibodies bound to the E protein antigen are detected with fluorescently labeled anti-human immunoglobulin antibody and flow analysis in a dual-laser Luminex 100 instrument. Retrospective testing of 833 sera from New York patients with suspected viral encephalitis demonstrated concordance with results obtained with the traditional enzyme-linked immunosorbent assay for immunoglobulin G (IgG) antibodies to WN virus (kappa = 0.85). One hundred eighty-eight (22.4%) of the samples, which were collected from June to November 2002, tested positive for antibodies to WN virus in the microsphere assay. Specimens depleted of IgG with anti-IgG antibody were reassayed to measure anti-E protein IgM antibodies and to provide an indication of current or recent WN virus infection. The assay also detects antibodies to E proteins from related flaviviruses, including St. Louis encephalitis, Japanese encephalitis, and dengue viruses. The new microsphere immunoassay provides a sensitive and rapid alternative to traditional enzyme-linked immunosorbent assays that detect antibodies to flavivirus E proteins. This assay can aid physicians and public health workers in the management of outbreaks of WN virus and related flaviviruses.
Identification and Susceptibility Testing of
Enterobacteriaceae
and
Pseudomonas aeruginosa
by Direct Inoculation from Positive BACTEC Blood Culture Bottles into Vitek 2
Authors: Bruins Marjan J. , Bloembergen Peter , Ruijs Gijs J. H. M. , Wolfhagen Maurice J. H. M.
Published: January 2004

Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the “gold standard.” We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of
Enterobacteriaceae
and
Pseudomonas aeruginosa
were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture.
Adaptive Resistance to Biocides inSalmonella entericaandEscherichia coliO157 and Cross-Resistance to Antimicrobial Agents
Authors: Braoudaki M. , Hilton A. C.
Published: January 2004

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential ofSalmonella entericaandEscherichia coliO157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for bothS. entericaandE. coliO157. Cross-resistance in adaptively resistantS. entericavaried with the serotype;Salmonella entericaserovar Enteritidis expressed cross-resistance to chloramphenicol, whereasSalmonella entericaserovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistantSalmonella entericaserovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistantSalmonellaserovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms.E. coliO157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.
Sensitivity of Respiratory Virus Culture When Screening with R-Mix Fresh Cells
Authors: Dunn James J. , Woolstenhulme R. Dean , Langer Janine , Carroll Karen C.
Published: January 2004

Use of R-Mix Fresh Cells has been shown to be a rapid and sensitive method for the detection and identification of respiratory viruses. We prospectively evaluated the impact of incorporation of R-Mix shell vials on the sensitivity and time to detection of seven respiratory viruses recovered in a comprehensive culture during the course of an entire respiratory season in a high-volume clinical laboratory. In this study, R-Mix shell vials were used as part of the culture of 3,803 respiratory specimens. A total of 428 respiratory viruses were recovered. Staining of R-Mix vials after overnight incubation allowed initial detection of 274 of 279 influenza viruses, 33 of 38 parainfluenza viruses, 35 of 51 adenoviruses, and 52 of 60 respiratory syncytial viruses (RSVs). The time to reporting of all positive cultures after in-lab specimen receipt was 2.9 days on average and those initially detected in R-Mix cells were reported in 2.3 days on average. A combination of direct fluorescent-antibody (DFA) staining and virus culture was performed on a subset of 711 respiratory specimens. Of 152 viruses identified, 57 were observed only with DFA testing (55 RSV and 2 influenza A viruses) and 31 were recovered only in cell culture. After overnight incubation, R-Mix cells detected 87.1% of respiratory viruses not observed by DFA testing and 96.9% of viruses positive by both methods. The sensitivities of DFA testing and R-Mix cells for identification of influenza viruses were 70.5% and 96.7%, respectively. The R-Mix method detected influenza virus in 18 samples that were negative by DFA testing.
Dynamics of
Streptococcus agalactiae
Colonization in Women during and after Pregnancy and in Their Infants
Authors: Hansen Søren Mose , Uldbjerg Niels , Kilian Mogens , Sørensen Uffe B. Skov
Published: January 2004

The population dynamics of
Streptococcus agalactiae
(group B streptococci [GBS]) colonization of the vagina and anorectal area was investigated in a cohort of 77 Danish women during and after their pregnancy by a new sensitive method. The mean carriage rate among individual observations was 36%, and the cumulative carriage rate over the entire observation period was 54%. Examination of more than 1,500 GBS isolates by pulsed-field gel electrophoresis demonstrated that the GBS population was remarkably homogeneous and stable in each carrier. Virtually all carriers were colonized by a single GBS clone on all occasions spanning up to 2 years. Repeated detection of the same clone even in women who were recorded as intermittent carriers suggests that the actual carrier rate exceeds 50% but that fluctuations in the GBS proportions of the flora occasionally preclude their detection. Newborns and young infants usually carried the same GBS clone as their mothers. However, only twice were identical clones of GBS detected in different women in contrast to the observed clonal relationships of clinical isolates. These observations strongly suggest differences in the properties and epidemiology of virulent GBS clones compared to clones commonly carried by healthy individuals.
RickettsiaSpecies InfectingAmblyomma cooperiTicks from an Area in the State of São Paulo, Brazil, Where Brazilian Spotted Fever Is Endemic
Authors: Labruna Marcelo B. , Whitworth Ted , Horta Maurício C. , Bouyer Donald H. , McBride Jere W. , Pinter Adriano , Popov Vsevolod , Gennari Solange M. , Walker David H.
Published: January 2004

Owing to the potential role of the tickAmblyomma cooperiin the enzootic cycle ofRickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection byRickettsiaspecies inA. cooperiticks collected from an area in Brazil where BSF is endemic. Among a total of 40A. cooperiadult ticks collected in an area of BSF endemicity in the state of São Paulo, PCR analysis detected DNA ofRickettsia belliiin 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individualA. cooperiticks resulted in two isolates ofR. belliiand one isolate genotypically characterized as an SFG rickettsia. The twoR. belliiisolates were established in Vero cell cultures in the laboratory and were confirmed to beR. belliiby molecular analysis of thegltAand 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related toRickettsia parkeri,Rickettsia africae, andRickettsia sibirica.These results do not support the role ofA. cooperiin the ecology ofR. rickettsiiin the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.
Infection with a Proposed New Subspecies ofBabesia canis,Babesia canissubsp.presentii, in Domestic Cats
Authors: Baneth Gad , Kenny Martin J. , Tasker Séverine , Anug Yigal , Shkap Varda , Levy Amos , Shaw Susan E.
Published: January 2004

Parasitemia with a largeBabesiaspecies was identified in two domestic cats from Israel. One cat, also coinfected with feline immunodeficiency virus and “CandidatusMycoplasma haemominutum,” had profound icterus and anemia which resolved after therapy, whereas a second cat was an asymptomatic carrier. Amplification and sequencing of the 18S rRNA gene, followed by phylogenetic analyses, indicated that infection was caused byBabesia canis. However, the sequences of the internal transcribed and 5.8S rRNA regions of the ribosomal operon used for subspeciation ofB. caniswere markedly different from the recognized subspecies ofB. canis, which includeB. canis vogeli,B. canis canis, andB. canis rossi. Based on phylogenetic comparisons of the 18S rRNA gene, 5.8S, and internal transcribed spacer sequences of the isolates from the cats and on the smaller sizes of the merozoite and trophozoite stages of this parasite, which distinguish it from the subspecies ofB. canispresent in dogs, we propose to identify the novel feline genotype ofB. canisdescribed in the present study as a new subspecies,B. canissubsp.presentii.
A Nonproliferating Parvovirus Vaccine Vector Elicits Sustained, Protective Humoral Immunity following a Single Intravenous or Intranasal Inoculation
Authors: Palmer Gene A. , Brogdon Jennifer L. , Constant Stephanie L. , Tattersall Peter
Published: January 2004

An ideal vaccine delivery system would elicit persistent protection following a single administration, preferably by a noninvasive route, and be safe even in the face of immunosuppression, either inherited or acquired, of the recipient. We have exploited the unique life cycle of the autonomous parvoviruses to develop a nonproliferating vaccine platform that appears to both induce priming and continually boost a protective immune response following a single inoculation. A crippled parvovirus vector was constructed, based on a chimera between minute virus of mice (MVM) and LuIII, which expressesBorrelia burgdorferiouter surface protein A (OspA) instead of its coat protein. The vector was packaged into an MVM lymphotropic capsid and inoculated into naive C3H/HeNcr mice. Vaccination with a single vector dose, either intravenously or intranasally, elicited high-titer anti-OspA-specific antibody that provided protection from live spirochete challenge and was sustained over the lifetime of the animal. Both humoral and cell-mediated Th1immunity was induced, as shown by anti-OspA immunoglobulin G2a antibody and preferential gamma interferon production by OspA-specific CD4+T cells.
Downregulation of Major Histocompatibility Complex Class I by Human Ubiquitin Ligases Related to Viral Immune Evasion Proteins
Authors: Bartee Eric , Mansouri Mandana , Hovey Nerenberg Bianca T. , Gouveia Kristine , Früh Klaus
Published: January 2004

Poxviruses and gamma-2 herpesviruses share the K3 family of viral immune evasion proteins that inhibit the surface expression of glycoproteins such as major histocompatibility complex class I (MHC-I), B7.2, ICAM-1, and CD95(Fas). K3 family proteins contain an amino-terminal PHD/LAP or RING-CH domain followed by two transmembrane domains. To examine whether human homologues are functionally related to the viral immunoevasins, we studied seven membrane-associated RING-CH (MARCH) proteins. All MARCH proteins located to subcellular membranes, and several MARCH proteins reduced surface levels of known substrates of the viral K3 family. Two closely related proteins, MARCH-IV and MARCH-IX, reduced surface expression of MHC-I molecules. In the presence of MARCH-IV or MARCH-IX, MHC-I was ubiquitinated and rapidly internalized by endocytosis, whereas MHC-I molecules lacking lysines in their cytoplasmic tail were resistant to downregulation. The amino-terminal regions containing the RING-CH domain of several MARCH proteins examined catalyzed multiubiquitin formation in vitro, suggesting that MARCH proteins are ubiquitin ligases. The functional similarity of the MARCH family and the K3 family suggests that the viral immune evasion proteins were derived from MARCH proteins, a novel family of transmembrane ubiquitin ligases that seems to target glycoproteins for lysosomal destruction via ubiquitination of the cytoplasmic tail.
Template Sequence near the Initiation Nucleotide Can Modulate Brome Mosaic Virus RNA Accumulation in Plant Protoplasts
Authors: Hema M. , Kao C. Cheng
Published: January 2004

Bromoviral templates for plus-strand RNA synthesis are rich in A or U nucleotides in comparison to templates for minus-strand RNA synthesis. Previous studies demonstrated that plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase is more efficient if the template contains an A/U-rich template sequence near the initiation site (K. Sivakumaran and C. C. Kao, J. Virol.
73:
6415-6423, 1999). These observations led us to examine the effects of nucleotide changes near the template's initiation site on the accumulation of BMV RNA3 genomic minus-strand, genomic plus-strand, and subgenomic RNAs in barley protoplasts transfected with wild-type and mutant BMV transcripts. Mutations in the template for minus-strand synthesis had only modest effects on BMV replication in barley protoplasts. Mutants with changes to the +3, +5, and +7 template nucleotides accumulated minus-strand RNA at levels similar to the the wild-type level. However, mutations at positions adjacent to the initiation cytidylate in the templates for genomic and subgenomic plus-strand RNA synthesis significantly decreased RNA accumulation. For example, changes at the third template nucleotide for plus-strand RNA3 synthesis resulted in RNA accumulation at between 18 and 24% of the wild-type level, and mutations in the third template nucleotide for subgenomic RNA4 resulted in accumulations at between 7 and 14% of the wild-type level. The effects of the mutations generally decreased as the mutations occurred further from the initiation nucleotide. These findings demonstrate that there are different requirements of the template sequence near the initiation nucleotide for BMV RNA accumulation in plant cells.
Identification of Host-Specificity Determinants in Betanodaviruses by Using Reassortants between Striped Jack Nervous Necrosis Virus and Sevenband Grouper Nervous Necrosis Virus
Authors: Iwamoto Tokinori , Okinaka Yasushi , Mise Kazuyuki , Mori Koh-Ichiro , Arimoto Misao , Okuno Tetsuro , Nakai Toshihiro
Published: January 2004

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.
Impaired Processing and Presentation of Cytotoxic-T-Lymphocyte (CTL) Epitopes Are Major Escape Mechanisms from CTL Immune Pressure in Human Immunodeficiency Virus Type 1 Infection
Authors: Yokomaku Yoshiyuki , Miura Hideka , Tomiyama Hiroko , Kawana-Tachikawa Ai , Takiguchi Masafumi , Kojima Asato , Nagai Yoshiyuki , Iwamoto Aikichi , Matsuda Zene , Ariyoshi Koya
Published: January 2004

Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the
gag
gene of six field isolates by adopting an
env/nef
-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SL
F
NTVA
V
L and S
V
YNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KY
R
LKH
L
VW,
Q
YRLKHIVW, and
R
Y
R
LKH
L
VW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells. Thus, their escape is likely due to the changes that occur during the processing and presentation of epitopes in the infected cells. Mutations responsible for this mode of escape were located within the epitope regions rather than the flanking regions, and such mutations did not influence the virus replication. The results suggest that the impaired antigen processing and presentation often occur in HIV-1 field isolates and thus are one of the major mechanisms that enable HIV-1 to escape from CTL recognition. We emphasize the importance of testing HIV-1 variants in an endogenous expression system.
An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function
Authors: Jones Thomas R. , Lee Shi-Wu
Published: January 2004

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.
Removal of N-Linked Glycosylation Sites in the V1 Region of Simian Immunodeficiency Virus gp120 Results in Redirection of B-Cell Responses to V3
Authors: Cole Kelly Stefano , Steckbeck Jonathan D. , Rowles Jennifer L. , Desrosiers Ronald C. , Montelaro Ronald C.
Published: January 2004

One mechanism of immune evasion utilized by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope glycoproteins is the presence of a dense carbohydrate shield. Accumulating evidence from in vitro and in vivo experiments suggests that alterations in N-linked glycosylation of SIV gp120 can enhance host humoral immune responses that may be involved in immune control. The present study was designed to determine the ability of glycosylation mutant viruses to redirect antibody responses to shielded envelope epitopes. The influence of glycosylation on the maturation and specificity of antibody responses elicited by glycosylation mutant viruses containing mutations of specific N-linked sites in and near the V1 and V2 regions of SIVmac239 gp120 was determined. Results from these studies demonstrated a remarkably similar maturation of antibody responses to native, fully glycosylated envelope proteins. However, analyses of antibodies to defined envelope domains revealed that mutation of glycosylation sites in V1 resulted in increased antibody recognition to epitopes in V1. In addition, we demonstrated for the first time that mutation of glycosylation sites in V1 resulted in a redirection of antibody responses to the V3 loop. Taken together, these results demonstrate that N-linked glycosylation is a determinant of SIV envelope B-cell immunogenicity in addition to in vitro antigenicity. In addition, our results demonstrate that the absence of N-linked carbohydrates at specific sites can influence the exposure of epitopes quite distant in the linear sequence.
Cross-Genotype Immunity to Hepatitis C Virus
Authors: Lanford Robert E. , Guerra Bernadette , Chavez Deborah , Bigger Catherine , Brasky Kathleen M. , Wang Xiao-Hong , Ray Stuart C. , Thomas David L.
Published: January 2004

Recent studies in humans and chimpanzees suggest that immunity can be induced to diminish the incidence of chronic hepatitis C virus (HCV) infection. However, the immunity that promotes viral recovery is poorly understood, and whether the breadth of this adaptive immunity is sufficient to overcome the substantial intergenotype antigenic diversity represents a final obstacle to demonstrating the feasibility of vaccine development. Here we demonstrate that recovery from a genotype 1 HCV infection protects chimpanzees against infection with representatives of other genotypes that exhibit up to 30% divergence at the amino acid level, including challenges with genotype 4, a mixture of genotypes 2 and 3, and a complex inoculum containing genotypes 1, 2, 3, and 4. In each instance, the level and duration of viremia were markedly reduced in comparison to the primary infection in the same animal. The data indicate that epitopes conserved between genotypes must play an essential role in immunity. The inocula used in the rechallenge studies induced typical primary infection profiles in naïve chimpanzees. Rechallenge infections were associated with rapid increases in the intrahepatic transcripts of interferon-stimulated genes, even in animals exhibiting apparent sterilizing immunity. Protective immunity was often associated with an early increase in gamma interferon transcripts in the liver and increases in intrahepatic transcripts of Mig, a T-cell chemokine that is a gamma interferon response gene. These studies are the first to show that cross-genotype immunity can be induced to HCV, demonstrating the feasibility of developing a vaccine protective against all HCV strains.
The Movement Protein of
Cowpea Mosaic Virus
Binds GTP and Single-Stranded Nucleic Acid In Vitro
Authors: Carvalho C. M. , Pouwels J. , van Lent J. W. M. , Bisseling T. , Goldbach R. W. , Wellink J.
Published: January 2004

The movement protein (MP) of
Cowpea mosaic virus
forms tubules in plasmodesmata to enable the transport of mature virions. Here it is shown that the MP is capable of specifically binding riboguanosine triphosphate and that mutational analysis suggests that GTP binding plays a role in the targeted transport of the MP. Furthermore, the MP is capable of binding both single-stranded RNA and single-stranded DNA in a non-sequence-specific manner, and the GTP- and RNA-binding sites do not overlap.
Ability of Wild-Type and Mutant Lysyl-tRNA Synthetase To Facilitate tRNA
Lys
Incorporation into Human Immunodeficiency Virus Type 1
Authors: Cen Shan , Javanbakht Hassan , Niu Meijuan , Kleiman Lawrence
Published: January 2004

The major human tRNA
Lys
isoacceptors, tRNA1,2Lys and tRNA3Lys, are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where tRNA3Lys acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNA
Lys
, via its interaction with Gag, and it is a strong candidate for being the signal that specifically targets tRNA
Lys
for viral incorporation. Expression of exogenous wild-type LysRS in cells results in an approximately twofold increase in the viral packaging of both LysRS and tRNA
Lys
. Herein, we show that this increase in tRNA
Lys
incorporation into virions is dependent upon the ability of LysRS to bind to tRNA
Lys
but not upon its ability to aminoacylate the tRNA
Lys
. COS7 cells were cotransfected with plasmids coding for both HIV-1 and either wild-type or mutant human LysRS, all of which are incorporated into virions with similar efficiency. However, N-terminally truncated LysRS, which binds poorly to tRNA
Lys
, does not increase tRNA
Lys
packaging into viruses, while C-terminally truncated LysRS, which binds to but does not aminoacylate tRNA
Lys
, still facilitates an increase in tRNA
Lys
packaging into virions.
SINGLE SCALE ANALYSIS OF MANY FERMION SYSTEMS PART 1: INSULATORS
Authors: FELDMAN JOEL , KNÖRRER HORST , TRUBOWITZ EUGENE
Published: January 2004

We construct, using fermionic functional integrals, thermodynamic Green's functions for a weakly coupled fermion gas whose Fermi energy lies in a gap. Estimates on the Green's functions are obtained that are characteristic of the size of the gap. This prepares the way for the analysis of single scale renormalization group maps for a system of fermions at temperature zero without a gap.
SINGLE SCALE ANALYSIS OF MANY FERMION SYSTEMS PART 2: THE FIRST SCALE
Authors: FELDMAN JOEL , KNÖRRER HORST , TRUBOWITZ EUGENE
Published: January 2004

The first renormalization group map arising from the momentum space decomposition of a weakly coupled system of fermions at temperature zero differs from all subsequent maps. Namely, the component of momentum dual to temperature may be arbitrarily large — there is no ultraviolet cutoff. The methods of Part 1 are supplemented to control this special case.
SINGLE SCALE ANALYSIS OF MANY FERMION SYSTEMS PART 3: SECTORIZED NORMS
Authors: FELDMAN JOEL , KNÖRRER HORST , TRUBOWITZ EUGENE
Published: January 2004

The generic renormalization group map associated to a weakly coupled system of fermions at temperature zero is treated by supplementing the methods of Part 1. The interplay between position and momentum space is captured by "sectors". It is shown that the difference between the complete four-legged vertex and its "ladder" part is irrelevant for the sequence of renormalization group maps.
SINGLE SCALE ANALYSIS OF MANY FERMION SYSTEMS PART 4: SECTOR COUNTING
Authors: FELDMAN JOEL , KNÖRRER HORST , TRUBOWITZ EUGENE
Published: January 2004

For a two-dimensional, weakly coupled system of fermions at temperature zero, one principal ingredient used to control the composition of the associated renormalization group maps is the careful counting of the number of quartets of sectors that are consistent with conservation of momentum. A similar counting argument is made to show that particle–particle ladders are irrelevant in the case of an asymmetric Fermi curve.
ON THE INFLUENCE OF ACOUSTIC WAVES ON COHERENT BREMSSTRAHLUNG IN CRYSTALS
Authors: SAHARIAN A. A. , MKRTCHYAN A. R. , PARAZIAN V. V. , GRIGORYAN L. SH.
Published: January 2004

We investigate the coherent bremsstrahlung by relativistic electrons in a single crystal excited by hypersonic vibrations. The formula for the corresponding differential cross-section is derived in the case of a sinusoidal wave. The conditions are specified under which the influence of the hypersound is essential. The case is considered in detail when the electron enters into the crystal at small angles with respect to a crystallographic axis. It is shown that in dependence of the parameters, the presence of hypersonic waves can either enhance or reduce the bremsstrahlung cross-section.
A NOTE ON THE CASIMIR ENERGY OF A MASSIVE SCALAR FIELD IN POSITIVE CURVATURE SPACE
Authors: ELIZALDE E. , TORT A. C.
Published: January 2004

We re-evaluate the zero point Casimir energy for the case of a massive scalar field in R1×S3 space, allowing also for deviations from the standard conformal value ξ=1/6, by means of zero temperature zeta function techniques. We show that for the problem at hand this approach is equivalent to the high temperature regularization of the vacuum energy, as conjectured in a previous publication. The analytic continuation can be performed in two ways, which are seen to be equivalent.
PROPAGATOR OF THE INTERACTING RARITA–SCHWINGER FIELD
Authors: KALOSHIN A. E. , LOMOV V. P.
Published: January 2004

We obtain in analytical form the dressed propagator of the massive Rarita–Schwinger field taking into account all spin components and discuss shortly its properties.
COSMOLOGY WITH INTERACTING DARK ENERGY
Authors: MBONYE MANASSE R.
Published: January 2004

The early cosmic inflation, when taken along with the recent observations that the universe is currently dominated by a low density vacuum energy, leads to at least two potential problems which modern cosmology must address. First, there is the old cosmological constant problem, with a new twist: the coincidence problem. Secondly, cosmology still lacks a model to predict the observed current cosmic acceleration and to determine whether or not there is a future exit out of this state (as previously in the inflationary case). This constitutes (what is called here) a dynamical problem. Here a framework is proposed to address these two problems, based on treating the cosmic background vacuum (dark) energy as both dynamical and interacting. The universe behaves as a vacuum-driven cosmic engine which, in search of equilibrium, always back-reacts to vacuum-induced accelerations by increasing its inertia (internal energy) through vacuum energy dissipation. The process couples cosmic vacuum (dark) energy to matter to produce future-directed increasingly comparable amplitudes in these fields by setting up oscillations in the decaying vacuum energy density and corresponding sympathetic ones in the matter fields. By putting bounds on the relative magnitudes of these coupled oscillations the model offers a natural and conceptually simple channel to discuss the coincidence problem, while also suggesting a way to deal with the dynamical problem. A result with important observational implications is an equation of state w(t) which specifically predicts a variable, quasi-periodic, acceleration for the current universe. This result can be directly tested by future observational techniques such as SNAP.
ARE MASSIVE ELEMENTARY PARTICLES BLACK HOLES?
Authors: WARD B. F. L.
Published: January 2004

We use exact results in a new approach to quantum gravity to study the effect of quantum loop corrections on the behavior of the metric of spacetime near the Schwarzschild radius of a massive point particle in the standard model. We show that the classical conclusion that such a system is a black hole is obviated. Phenomenological implications are discussed.
ANOMALY MEDIATED SUPERSYMMETRY BREAKING AND ITS TEST IN LINEAR COLLIDERS
Authors: ROY SOUROV
Published: January 2004

Signatures of anomaly mediated supersymmetry breaking in linear colliders are briefly reviewed after presenting an outline of the theoretical framework. A unique and distinct feature of a large class of models of this type is a winolike chargino which is very closely degenerate in mass with the lightest neutralino. The very slow decay of this chargino results in a heavily ionizing charged track and one soft charged pion with a characteristic momentum distribution, leading to unique signals in linear colliders which are essentially free of background. The determination of chargino and slepton masses from such events is a distinctly interesting possibility.
HAMILTON–JACOBI QUANTIZATION OF TWO-DIMENSIONAL GRAVITY WITH TORSION
Authors: MUSLIH SAMI I.
Published: January 2004

Two-dimensional gravity with torsion is investigated using the Hamilton–Jacobi formalism. The equations of motion and the action integral are obtained as total differential equations in many variables. The integrabilty conditions, lead us to obtain the path integral quantization as an integration over the canonical phase-space coordinates.
IS THE QUARK-MIXING MATRIX MODULI SYMMETRIC?
Authors: CHATURVEDI S. , GUPTA VIRENDRA
Published: January 2004

If the unitary quark-mixing matrix, V, is moduli symmetric, it will depend on three real parameters. This means that there is a relation between the four parameters needed to parametrize a general V. It is shown that there exists a very simple relation involving |V11|2, |V33|2, [Formula: see text] and [Formula: see text]. This relation is compared with the present experimental data. It is concluded that a moduli symmetric V is not ruled out.
PHASE DIAGRAMS OF ONE-DIMENSIONAL MODELS UNDER STRONG RANDOM EXTERNAL FIELD
Authors: KERIMOV AZER
Published: January 2004

We consider one-dimensional models of classical statistical physics and prove that at each fixed value of the temperature for all realizations of additional sufficiently strong random external field the limiting Gibbs state is unique.
RESPONSE OF RANDOM FIELD ISING MODEL DRIVEN BY AN EXTERNAL FIELD
Authors: ROY RATNADEEP , RAY PURUSATTAM
Published: January 2004

We study the dynamics of spin flipping at first order transitions in zero temperature two-dimensional random-field Ising model driven by an external field. We find a critical value of the disorder strength at which a discontinuous sharp jump in magnetization first occurs. We discuss growth morphology of the flipped-spin domains at and away from criticality.