bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of
Mycobacterium bohemicum
Isolated from Human, Veterinary, and Environmental Sources
Authors: Torkko Pirjo , Suomalainen Sini , Iivanainen Eila , Suutari Merja , Paulin Lars , Rudbäck Eeva , Tortoli Enrico , Vincent Véronique , Mattila Rauni , Katila Marja-Leena
Published: July 2002

Chemotaxonomic and genetic properties were determined for 14 mycobacterial isolates identified as members of a newly described species
Mycobacterium bohemicum
. The isolates recovered from clinical, veterinary, and environmental sources were compared for lipid composition, biochemical test results, and sequencing of the 16S ribosomal DNA (rDNA) and the 16S-23S rDNA internal transcribed spacer (ITS) regions. The isolates had a lipid composition that was different from those of other known species. Though the isolates formed a distinct entity, some variations were detected in the features analyzed. Combined results of the phenotypic and genotypic analyses were used to group the isolates into three clusters. The major cluster (cluster A), very homogenous in all respects, comprised the
M. bohemicum
type strain, nine clinical and veterinary isolates, and two of the five environmental isolates. Three other environmental isolates displayed an insertion of 14 nucleotides in the ITS region; they also differed from cluster A in fatty alcohol composition and produced a positive result in the Tween 80 hydrolysis test. Among these three, two isolates were identical (cluster B), but one isolate (cluster C) had a unique high-performance liquid chromatography profile, and its gas liquid chromatography profile lacked 2-octadecanol, which was present in all other isolates analyzed. Thus, sequence variation in the 16S-23S ITS region was associated with interesting variations in lipid composition. Two of the isolates analyzed were regarded as potential inducers of human or veterinary infections. Each of the environmental isolates, all of which were unrelated to the cases presented, was cultured from the water of a different stream. Hence, natural waters are potential reservoirs of
M. bohemicum
.
Differentiation of
Candida dubliniensis
from
Candida albicans
on Staib Agar and Caffeic Acid-Ferric Citrate Agar
Authors: Al Mosaid Asmaa , Sullivan Derek , Salkin Ira F. , Shanley Diarmuid , Coleman David C.
Published: July 2002

The methods currently available for the identification of the pathogenic yeast
Candida dubliniensis
all have disadvantages in that they are time-consuming, expensive, and/or, in some cases, unreliable. In a recent study (P. Staib and J. Morschhäuser, Mycoses 42:521–524; 1999) of 14
C. dubliniensis
and 11
C. albicans
isolates, it was suggested that the ability of
C. dubliniensis
to produce rough colonies and chlamydospores (chlamydoconidia) on Staib agar (SA) provided a simple means of differentiating it from its close relative
C. albicans
. In the present investigation, we examined the colony morphology and chlamydospore production of 130
C. dubliniensis
and 166
C. albicans
isolates on SA and on the related defined medium caffeic acid-ferric citrate agar (CAF). All of the
C. dubliniensis
and
C. albicans
isolates produced chlamydospores on the control medium, i.e., rice-agar-Tween agar. However, while none of the
C. albicans
isolates produced chlamydospores on either SA or CAF, 85.4 and 83.8% of the
C. dubliniensis
isolates produced chlamydospores on SA and CAF, respectively. All of the
C. albicans
isolates grew as smooth, shiny colonies on SA after 48 to 72 h of incubation at 30°C, while 97.7% of the
C. dubliniensis
isolates grew as rough colonies, many (65%) with a hyphal fringe. In contrast, 87.4% of the
C. albicans
and 93.8% of the
C. dubliniensis
isolates yielded rough colonies on CAF. Although the results of this study confirm that SA is a good medium for distinguishing between
C. dubliniensis
and
C. albicans
, we believe that discrimination between these two species is best achieved on the basis of colony morphology rather than chlamydospore production.
Genetic Diversity of
Histoplasma capsulatum
Strains Isolated from Soil, Animals, and Clinical Specimens in Rio de Janeiro State, Brazil, by a PCR-Based Random Amplified Polymorphic DNA Assay
Authors: Muniz Mauro de Medeiros , Pizzini Claudia Vera , Peralta José Mauro , Reiss Errol , Zancopé-Oliveira Rosely M.
Published: July 2002

Little is known about the genetic strain diversity and geographical range of
Histoplasma capsulatum
isolated in Rio de Janeiro State, Brazil. We characterized 13 environmental, 7 animal, and 28 clinical
H. capsulatum
isolates by using a PCR-based random amplified polymorphic DNA (RAPD) assay. DNA fingerprinting of these soil, animal, and clinical specimens was performed with four primers (1253, 1281, D-9355, and D-10513) and generated amplicons with considerable polymorphism. Although all of the isolates exhibited more than 80% genetic relatedness, they could be clustered into four to six genotypes for each primer. The RAPD profiles of
H. capsulatum
isolated from Rio de Janeiro State could be distinguished from those of the U.S. strains included in this study (Downs, G222B, G-186B, and FLS1) by showing less than 70% similarity to each primer. The genetic polymorphisms between
H. capsulatum
strains isolated from animals and soil obtained in the same geographic areas were 100% similar, suggesting that an environmental microniche could be acting as a source of infection for animals and the local human population.
Diagnosing Genital Ulcer Disease in a Clinic for Sexually Transmitted Diseases in Amsterdam, The Netherlands
Authors: Bruisten S. M. , Cairo I. , Fennema H. , Pijl A. , Buimer M. , Peerbooms P. G. H. , Van Dyck E. , Meijer A. , Ossewaarde J. M. , van Doornum G. J. J.
Published: July 2002

The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2,
Treponema pallidum
, and
Haemophilus ducreyi
. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with
T. pallidum
was found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous
T. pallidum
and
H. ducreyi
DNA detection was positive for
T. pallidum
in 3.3% of the samples and for
H. ducreyi
in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for
T. pallidum
detection. Specific
H. ducreyi
immunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.
National Epidemiologic Surveys of
Enterobacter aerogenes
in Belgian Hospitals from 1996 to 1998
Authors: De Gheldre Y. , Struelens M. J. , Glupczynski Y. , De Mol P. , Maes N. , Nonhoff C. , Chetoui H. , Sion C. , Ronveaux O. , Vaneechoutte M. ,   le Groupement Pour Le Dépistage, l'Etude et la Prevention des Infections Hospitalières (gdepih-gospiz)
Published: July 2002

Two national surveys were conducted to describe the incidence and prevalence of
Enterobacter aerogenes
in 21 Belgian hospitals in 1996 and 1997 and to characterize the genotypic diversity and the antimicrobial resistance profiles of clinical strains of
E. aerogenes
isolated from hospitalized patients in Belgium in 1997 and 1998. Twenty-nine hospitals collected 10 isolates of
E. aerogenes
, which were typed by arbitrarily primed PCR (AP-PCR) using two primers and pulsed-field gel electrophoresis. MICs of 10 antimicrobial agents were determined by the agar dilution method. Beta-lactamases were detected by the double-disk diffusion test and characterized by isoelectric point. The median incidence of
E. aerogenes
colonization or infection increased from 3.3 per 1,000 admissions in 1996 to 4.2 per 1000 admissions in the first half of 1997 (
P
< 0.01).
E. aerogenes
strains (
n
= 260) clustered in 25 AP-PCR types. Two major types, BE1 and BE2, included 36 and 38% of strains and were found in 21 and 25 hospitals, respectively. The BE1 type was indistinguishable from a previously described epidemic strain in France. Half of the strains produced an extended-spectrum beta-lactamase, either TEM-24 (in 86% of the strains) or TEM-3 (in 14% of the strains). Over 75% of the isolates were resistant to ceftazidime, piperacillin-tazobactam, and ciprofloxacin. Over 90% of the strains were susceptible to cefepime, carbapenems, and aminoglycosides. In conclusion, these data suggest a nationwide dissemination of two epidemic multiresistant
E. aerogenes
strains in Belgian hospitals. TEM-24 beta-lactamase was frequently harbored by one of these epidemic strains, which appeared to be genotypically related to a TEM-24-producing epidemic strain from France, suggesting international dissemination.
Genetic Polymorphism of
Aspergillus fumigatus
in Clinical Samples from Patients with Invasive Aspergillosis: Investigation Using Multiple Typing Methods
Authors: Bertout S. , Renaud F. , Barton R. , Symoens F. , Burnod J. , Piens M.-A. , Lebeau B. , Viviani M.-A. , Chapuis F. , Bastide J.-M. , Grillot R. , Mallié M. ,   THE EUROPEAN RESEARCH GROUP ON BIOTYPE AND GENOTYPE OF ASPERGILLUS
Published: July 2002

The genotypes of 52 strains of
Aspergillus fumigatus
isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of
A. fumigatus
should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of
A. fumigatus
and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.
Evaluation of VITEK 2 Rapid Identification and Susceptibility Testing System against Gram-Negative Clinical Isolates
Authors: Ling Thomas K. W. , Tam P. C. , Liu Z. K. , Cheng Augustine F. B.
Published: July 2002

A total of 281 strains of miscellaneous members of the family
Enterobacteriaceae
,
Pseudomonas aeruginosa
, and other gram-negative bacteria were evaluated by use of identification tests with the VITEK 2 system (bioMérieux) and an API identification system (bioMérieux). A total of 237 (95%) strains were correctly identified to the species level. Only six (2.1%) strains were misidentified, and eight (2.8%) strains were not identified. Among 14 strains with discrepant identifications, 8 (57.1%) strains were nonfermenters. The susceptibilities of 228 strains to 11 antibiotics including amikacin, netilmicin, tobramycin, gentamicin, ciprofloxacin, imipenem, meropenem, ceftazidime, cefepime, piperacillin, and piperacillin in combination with tazobactam were tested with the VITEK 2 AST-No. 12 card and by the broth microdilution (MB) method, according to NCCLS guidelines, as a reference. For the 2,508 organism-antibiotic combinations, the rates at which duplicate MICs correlated within ±1 dilution ranged from 84.2 to 95.6%. Only 13 (0.5%) and 10 (0.4%) of the susceptibility tests gave major errors (resistant with the VITEK 2 system but sensitive by the MB method) and very major errors (sensitive with the VITEK 2 system but resistant by the MB method), respectively. Both VITEK 2 ID-GNB (an identification system) and VITEK 2 AST-No. 12 (a susceptibility testing system) card systems gave rapid, reliable, and highly reproducible results.
Novel Method for Detection, Typing, and Quantification of Human Papillomaviruses in Clinical Samples
Authors: Hart Keith W. , Williams O. Martin , Thelwell Nicola , Fiander Alison N. , Brown Tom , Borysiewicz Lesek K. , Gelder Colin M.
Published: July 2002

We report the development of a novel detection and typing methodology for human papillomaviruses (HPV) based on real-time PCR with the self-probing fluorescent primers known as Scorpions. This technique is quick, simple, specific, sensitive, and capable of estimating viral load per cell. We report the results of over 100 typing reactions performed on cell lines, biopsies, and cervical cytobrush samples which, when compared to the current reference HPV detection and typing technique, present a κ value of 0.89. We further report preliminary data suggesting a relationship between viral load per cell and grade of cervical disease.
Antigenic and Genetic Characterization of Influenza C Viruses Which Caused Two Outbreaks in Yamagata City, Japan, in 1996 and 1998
Authors: Matsuzaki Y. , Sugawara K. , Mizuta K. , Tsuchiya E. , Muraki Y. , Hongo S. , Suzuki H. , Nakamura K.
Published: July 2002

During the 3 years from January 1996 to December 1998, a total of 33 strains of influenza C virus were isolated from 10,726 throat swab specimens collected from children with acute respiratory illness who visited two pediatric clinics in Yamagata City, Japan. These 33 strains were isolated in clusters during two different periods, 20 strains in May to August 1996 and the remaining 13 in March to June 1998. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein and phylogenetic analysis of seven RNA segments showed that the 33 influenza C viruses isolated were antigenically and genetically similar and that they were reassortant viruses which had obtained PB2, PB1, HE, M, and NS genes from a C/pig/Beijing/115/81-like virus and P3 and NP genes from a C/Mississippi/80-like virus. These observations suggest strongly that during the survey period of 3 years, two outbreaks of influenza C occurred in Yamagata City, both of which were caused by a reassortant virus having the genome composition described above.
Antibiotic Resistance of
Helicobacter pylori
Strains in Japanese Children
Authors: Kato Seiichi , Fujimura Shigeru , Udagawa Hirokazu , Shimizu Toshiaki , Maisawa Shunichi , Ozawa Kyoko , Iinuma Kazuie
Published: July 2002

The resistance of
Helicobacter pylori
to the recently available antibiotic treatment regimens has been a growing problem. We investigated the prevalence of
H. pylori
resistance to clarithromycin, metronidazole, and amoxicillin among 51
H. pylori
isolates from Japanese children. In addition, the mutations of the corresponding gene were studied by PCR and restriction fragment length polymorphism analysis. Primary resistance to clarithromycin, metronidazole, and amoxicillin was detected in 29, 24, and 0% of strains, respectively. The eradication rates in clarithromycin-susceptible and -resistant strains were 89 and 56%, respectively (
P
< 0.05). The prevalence of strains with acquired resistance to clarithromycin (78%) was higher than that of strains with primary resistance (
P
< 0.01). Among the clarithromycin-resistant strains studied, 92% showed cross-resistance to azithromycin. No acquired resistance to amoxicillin was demonstrated. The A2144G mutation in the 23S rRNA gene was detected in 11 of 12 (92%) clarithromycin-resistant strains tested, whereas the mutation was not detected in any of the 15 susceptible strains. The deletion of the
rdxA
gene was not demonstrated in any of the strains. The results indicate that a high prevalence of clarithromycin-resistant strains is associated with eradication failure. Testing of susceptibility to clarithromycin is recommended.
Production of BRO β-Lactamases and Resistance to Complement in European
Moraxella catarrhalis
Isolates
Authors: Schmitz Franz-Josef , Beeck Andreas , Perdikouli Mirella , Boos Mechthild , Mayer Susanne , Scheuring Sybille , Köhrer Karl , Verhoef Jan , Fluit Ad C.
Published: July 2002

Of the 419
Moraxella catarrhalis
isolates collected during the 1997-1999 European SENTRY surveillance study, 385 (92%) were β-lactamase positive. Twenty-two (5.7%) produced BRO-2 β-lactamase. Twenty-one new mutations were found in the putative promoter region of the
bro
genes. Nineteen percent of all isolates tested were complement sensitive. Resistance to β-lactams is not linked to the phylogenetic lineages associated with susceptibility to complement.
Early Therapy of Vertical Human Immunodeficiency Virus Type 1 (HIV-1) Infection: Control of Viral Replication and Absence of Persistent HIV-1-Specific Immune Responses
Authors: Luzuriaga Katherine , McManus Margaret , Catalina Michelle , Mayack Shane , Sharkey Mark , Stevenson Mario , Sullivan John L.
Published: July 2002

Studies of potent antiretroviral combination regimens were undertaken in young infants to evaluate the potential for long-term suppression of viral replication and to evaluate the immune consequences of such therapies. Early combination antiretroviral therapy led to a loss of plasma viremia, cultivable virus, and labile extrachromosomal replication intermediates. Despite preservation of immune function, persistent human immunodeficiency type 1 (HIV-1)-specific immune responses were not detected in most infants. The absence of detectable, persisting immune responses in most HIV-1-infected infants treated early contrasts with what is typically seen in adults who are treated early. These results are consistent with the notion that early combination antiretroviral therapy of HIV-1-infected infants allows the long-term suppression of viral replication.
Genetics of Mouse Mammary Tumor Virus-Induced Mammary Tumors: Linkage of Tumor Induction to thegagGene
Authors: Hook Lauren M. , Agafonova Yelena , Ross Susan R. , Turner Stephanie J. , Golovkina Tatyana V.
Published: July 2002

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation ofIntprotooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression ofIntgenes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenousMtv1provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to thegagregion of the retrovirus.
Identification of a Second Transforming Function in Bovine Papillomavirus Type 1 E6 and the Role of E6 Interactions with Paxillin, E6BP, and E6AP
Authors: Das Kingshuk , Bohl Joanna , Vande Pol Scott B.
Published: July 2002

Papillomavirus E6 oncoproteins transform mammalian cells through interaction with cellular proteins. Bovine papillomavirus type 1 E6 (BE6) interacts with three previously described cellular targets: the E6AP E3 ubiquitin ligase, the calcium-binding protein E6BP (also known as ERC-55), and paxillin, which is a focal adhesion adapter protein. BE6 interacts strongly with each of these proteins in vitro, binding to similar peptide sequences found in E6AP, E6BP, and paxillin. To determine which BE6 interactions are necessary for transformation by BE6, we used a novel selection strategy for temperature-sensitive BE6 mutants in yeast that could discriminate in their interaction between E6AP, E6BP, and paxillin. All BE6 mutants that retained transforming ability retained association with paxillin, while some mutants that were transformation positive failed to interact with E6AP or E6BP. This study demonstrates that oncogene mutants that are temperature sensitive for transformation can be selected in yeast and that the induction of anchorage-independent cell proliferation by BE6 does not require strong association of BE6 with either E6AP or E6BP. Of particular interest is the identification of a BE6 mutant that interacts strongly with the acidic charged leucine motifs of E6AP, E6BP, and paxillin but is devoid of transformation activity, thereby genetically identifying a second essential transformation function in BE6 that is independent of interaction with acidic charged leucine motifs.
Infectious Epstein-Barr Virus Lacking Major Glycoprotein BLLF1 (gp350/220) Demonstrates the Existence of Additional Viral Ligands
Authors: Janz Annette , Oezel Muhsin , Kurzeder Christian , Mautner Josef , Pich Dagmar , Kost Manuela , Hammerschmidt Wolfgang , Delecluse Henri-Jacques
Published: July 2002

The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.
Respiratory Syncytial Virus Infection and G and/or SH Protein Expression Contribute to Substance P, Which Mediates Inflammation and Enhanced Pulmonary Disease in BALB/c Mice
Authors: Tripp Ralph A. , Moore Deborah , Winter Jorn , Anderson Larry J.
Published: July 2002

A distinct clinical presentation of respiratory syncytial virus (RSV) infection of humans is bronchiolitis, which has clinical features similar to those of asthma. Substance P (SP), a tachykinin neuropeptide, has been associated with neurogenic inflammation and asthma; therefore, we chose to examine SP-induced inflammation with RSV infection. In this study, we examined the production of pulmonary SP associated with RSV infection of BALB/c mice and the effect of anti-SP F(ab)2antibodies on the pulmonary inflammatory response. The peak production of pulmonary SP occurred between days 3 and 5 following primary RSV infection and day 1 after secondary infection. Treatment of RSV-infected mice with anti-SP F(ab)2antibodies suggested that SP may alter the natural killer cell response to primary and secondary infection. In mice challenged after formalin-inactivated RSV vaccination, SP appears to markedly enhance pulmonary eosinophilia as well as increase polymorphonuclear cell trafficking to the lung. Based on studies with a strain of RSV that lacks the G and SH genes, the SP response to RSV infection appears to be associated with G and/or SH protein expression. These data suggest that SP may be an important contributor to the inflammatory response to RSV infection and that anti-SP F(ab)2antibodies might be used to ameliorate RSV-associated disease.
Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease Activity on Partially Single-Stranded Substrates
Authors: Davis Michael D. , Wu Jianwen , Owens Roland A.
Published: July 2002

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (
trs
) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the
trs
via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722–2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.
The Use of Chimeric Venezuelan Equine Encephalitis Viruses as an Approach for the Molecular Identification of Natural Virulence Determinants
Authors: Powers Ann M. , Brault Aaron C. , Kinney Richard M. , Weaver Scott C.
Published: July 2002

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.
Reconstitution of a Functional Duck Hepatitis B Virus Replication Initiation Complex from Separate Reverse Transcriptase Domains Expressed in
Escherichia coli
Authors: Beck Jürgen , Nassal Michael
Published: July 2002

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (ɛ) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by ɛ and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in
Escherichia coli
, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could
trans
complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of ɛ-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
Coxsackievirus A9 VP1 Mutants with Enhanced or Hindered A Particle Formation and Decreased Infectivity
Authors: Airaksinen Antero , Roivainen Merja , Hovi Tapani
Published: July 2002

We have studied coxsackievirus A9 (CAV9) mutants that each have a single amino acid substitution in the conserved 29-PALTAVETGHT-39 motif of VP1 and a reduced capacity to produce infectious progeny virus. After uncoating, all steps in the infection cycle occurred according to the same kinetics as and similar efficiency to the wild-type virus. However, the particle/infectious unit ratio in the progeny was significantly increased. The differences were apparently due to altered stability of the capsid: there were mutant viruses with enhanced or hindered uncoating, and both of these characteristics were found to reduce fitness under standard passaging conditions. At 32°C the instable mutants had an advantage, while the wild-type and the most stable mutant grew poorly. When comparing the newly published CAV9 structure and the other enterovirus structures, we found that the PALTAVETGHT motif is always in exactly the same position, in a cavity formed by the 3 other capsid proteins, with the C terminus of VP4 between this motif and the RNA. In the 7 enterovirus structures determined to date, the most conserved residues of the studied motif have identical contacts to neighboring residues of VP2, VP3, and VP4. We conclude that (i) the mutations affect the uncoating step necessary for infection, resulting in an untimely or hindered externalization of the VP1 N terminus together with the VP4, and (ii) the reason for the studied motif being evolutionarily conserved is its role in maintaining an optimal balance between the protective stability and the functional flexibility of the capsid.
Host RNA Polymerase Requirements for Transcription of the Human Hepatitis Delta Virus Genome
Authors: Moraleda Gloria , Taylor John
Published: July 2002

Replication of the genome of hepatitis delta virus (HDV) requires RNA-directed RNA synthesis using a host polymerase(s). This manuscript reviews the relevant published evidence. It also provides two new studies, both of which made use of transiently transfected Huh7 cells undergoing HDV RNA-directed RNA synthesis. For the first study, RNA transcription inhibitors were added to the transfected cells for periods of 1 to 2 days, after which assays of the effects on the accumulation of processed unit-length genomic HDV RNA were performed. For the second study, nuclei were isolated at 6 days after transfection, and then in vitro runoff transcription was used to assay the effects of RNA transcription inhibitors. Overall, the data support the interpretation that HDV transcription does not require host polymerase I or III (pol I or III) but at least primarily involves an enzyme resembling pol II.
Cytopathic Effects of Non-Syncytium-Inducing and Syncytium-Inducing Human Immunodeficiency Virus Type 1 Variants on Different CD4+-T-Cell Subsets Are Determined Only by Coreceptor Expression
Authors: Kwa David , Vingerhoed Jose , Boeser-Nunnink Brigitte , Broersen Silvia , Schuitemaker Hanneke
Published: July 2002

In peripheral blood mononuclear cells, syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) infected and depleted all CD4+T cells, including naive T cells. Non-SI HIV-1 infected and depleted only the CCR5-expressing T-cell subset. This may explain the accelerated CD4 cell loss after SI conversion in vivo.
Sequences Adjacent to
oriP
Improve the Persistence of Epstein-Barr Virus-Based Episomes in B Cells
Authors: White Robert E. , Wade-Martins Richard , James Michael R.
Published: July 2002

Epstein-Barr virus (EBV)
oriP
and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans
oriP
and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing
oriP
alone.
RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5 Includes Affinity for Double-Stranded RNA
Authors: Vende Patrice , Taraporewala Zenobia F. , Patton John T.
Published: July 2002

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.
P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing
Authors: Pfeffer S. , Dunoyer P. , Heim F. , Richards K. E. , Jonard G. , Ziegler-Graff V.
Published: July 2002

Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves ofNicotiana benthamianacontaining a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5′-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.
Visna Virus-Induced Activation of MAPK Is Required for Virus Replication and Correlates with Virus-Induced Neuropathology
Authors: Barber Sheila A. , Bruett Linda , Douglass Brian R. , Herbst David S. , Zink M. Christine , Clements Janice E.
Published: July 2002

It is well accepted that viruses require access to specific intracellular environments in order to proliferate or, minimally, to secure future proliferative potential as latent reservoirs. Hence, identification of essential virus-cell interactions should both refine current models of virus replication and proffer alternative targets for therapeutic intervention. In the present study, we examined the activation states of mitogen-activated protein kinases (MAPKs), ERK-1/2, in primary cells susceptible to visna virus and report that virus infection induces and sustains activation of the ERK/MAPK pathway. Treatment of infected cells with PD98059, a specific inhibitor of the ERK/MAPK pathway, abolishes visna virus replication, as evidenced by extremely low levels of Gag protein expression and reverse transcriptase activity in culture supernatants. In addition, although visna virus-induced activation of MAPK is detectable within 15 min, early events of viral replication (i.e., reverse transcription, integration, and transcription) are largely unaffected by PD98059. Interestingly, further examination demonstrated that treatment with PD98059 results in decreased cytoplasmic expression ofgagandenv, but notrev, mRNA, highly suggestive of an ERK/MAPK-dependent defect in Rev function. In vivo analysis of ERK-1/2 activation in brains derived from visna virus-infected sheep demonstrates a strong correlation between ERK/MAPK activation and virus-associated encephalitis. Moreover, double-labeling experiments revealed that activation of MAPK occurs not only in cells classically infected by visna virus (i.e., macrophages and microglia), but also in astrocytes, cells not considered to be major targets of visna virus replication, suggesting that activation of the ERK/MAPK pathway may contribute to the virus-induced processes leading to neurodegenerative pathology.
Murine Retroviral Pseudotype Virus Containing Hepatitis B Virus Large and Small Surface Antigens Confers Specific Tropism for Primary Human Hepatocytes: a Potential Liver-Specific Targeting System
Authors: Sung Vicky M.-H. , Lai Michael M. C.
Published: July 2002

We have developed a system for producing murine leukemia virus (MLV) pseudotyped with human hepatitis B virus (HBV) large (L) and small (S) surface antigens (HBsAg) for targeting primary human hepatocytes. Using the MLV(HBV) pseudotype virus containing a β-galactosidase reporter gene, we demonstrated that this pseudotype virus exhibits strict tropism for primary human hepatocytes, similar to the natural target cell specificity of HBV. It does not infect any of the established tissue culture cell lines, including human hepatoma cell lines (HepG2 and Huh-7), or rat primary hepatocytes. The infectivity of MLV(HBV) for human hepatocytes was inhibited by anti-HBs antibody. The L form of HBsAg was both necessary and sufficient for virus infectivity, but the presence of both L and S forms enhanced the surface expression of HBsAg and thus increased virus production. The middle form of HBsAg was not necessary. This pseudotype virus bypasses the requirement for the liver-specific transcription factors for HBV replication, enabling direct study of HBV tissue tropism conferred by the viral envelope proteins. This virus also offers a potential liver-specific targeting system for gene therapy.
Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains
Authors: Eichwald Catherine , Vascotto Fulvia , Fabbretti Elsa , Burrone Oscar R.
Published: July 2002

Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione
S-
transferase-NSP5 mutants were produced in
Escherichia coli
and used to determine phosphoacceptor sites. These were mapped to four serines (Ser
153
, Ser
155
, Ser
163
, and Ser
165
) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.
Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases
Authors: Macnaughton Thomas B. , Shi Stephanie T. , Modahl Lucy E. , Lai Michael M. C.
Published: July 2002

Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [
32
P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 μg/ml) of α-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by α-amanitin at concentrations as low as 2.5 μg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.
Feedback Controlled Chemical Reactor with Feed of Two Reaction Components with Spectrophotometric Indication
Authors: Skopal František , Dušek Václav
Published: July 2002

Theoretical relationships and simplifying conditions have been derived for the feed of two reaction components into a nonisochoric reactor with ideal stirring. The feed of reaction components is controlled by the negative feedback at a constant absorbance of the reaction mixture. The theoretical relationships have been verified using model 2. order oxidation-reduction reactions of Ce(IV)/V(IV) and Fe(III)/V(III) in 1 M sulfuric acid at 20 °C.
Preparation of D-Arabinose in a Laboratory Fluidized Bed Electrode Cell
Authors: Jiřičný Vladimír , Staněk Vladimír
Published: July 2002

Results of experimental work emloying an electrochemical reactor with the fluidized bed electrode to prepare D-arabinose by direct degradative oxidation of sodium D-gluconate without chemical oxidizing agents are presented. The observed yield of D-arabinose exceeded 70% by mass related to the theoretically obtainable amount with the selectivity ranging between 96 and 98.1%. The undesirable product was mainly D-erythrose produced by consecutive reaction from D-arabinose. Typical specific electric power consumption ranged between 7.5 and 14 kWh per kg of D-arabinose.
Some Analogs of 4-(2',4'-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic Acid: Synthesis and Antiinflammatory Activity
Authors: Kuchař Miroslav , Vosátka Václav , Poppová Marie , Knězová Eva , Panajotovová Vladimíra , Tomková Hana , Taimr Jan
Published: July 2002

Analogs of 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (I, flobufen), containing a double bond (II, IV, V, VII, VIII) or a methyl group in position 3 (VI) were prepared. Their antiinflammatory activity was evaluated and compared with that of flobufen. None of the mentioned analogs reached the activity of the standard. Isomerization of the unsaturated derivatives is connected with a shift of the double bond, Z-E transformation or lactonization. Reaction conditions and spectra of the compounds prepared are described.
Siting and Reactivity of the Co Ions in Ferrierite in Selective Catalytic Reduction of NO with CH4
Authors: Kaucký Dalibor , Dědeček Jiří , Vondrová Alena , Sobalík Zdeněk , Wichterlová Blanka
Published: July 2002

Catalytic activity of Co ions in CoNaK- and CoH-ferrierites in selective catalytic reduction of NO with methane depending on Co loading was investigated. The previously determined distribution of the exchanged Co ions in Co-ferrierites at three different cationic sites, differring in coordination of the Co ions to framework oxygens, has been utilized to analyze catalytic activity of the Co ions. The type of coordination of the Co ions dramatically affects their reactivity in the selective catalytic reduction of NO with methane. The Co ions coordinated to four framework oxygens of the wall of the main channel exhibit the highest activity, while the Co ions placed in a "boat shape", the most packed site of ferrierite show the lowest activity.
4,6-Dicarba-8-thia-arachno-nonaborane(10) Revisited. Theoretical Refinement of Its Structure
Authors: Hnyk Drahomír , Hofmann Matthias , Schleyer Paul von Ragué
Published: July 2002

The structure of the nine-vertex arachno-thiadicarbaborane C2SB6H10 has been established employing the ab initio/IGLO/NMR method. Theoretical IGLO 11B chemical shifts support C and S atom placements at the 4, 6 and 8 positions, respectively, and unambiguously rule out the 4,6,5-C2SB6H10 structural alternative, suggested earlier on the basis of IR and Raman spectroscopy. Important structural features of the 4,6,8-C2SB6H10 geometry include a small B(7)-S(8)-B(9) angle (MP2(fc)/6-31G*: 97.9°) and long S-B bonds (MP2(fc)/6-31G*: 1.905 and 1.924 Å) compared to B-B bonds spanning the 1.71-1.85 Å range.