bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multilocus Sequence Typing of
Bordetella pertussis
Based on Surface Protein Genes
Authors: van Loo Inge H. M. , Heuvelman Kees J. , King Audrey J. , Mooi Frits R. 
Published: July 2002

ABSTRACT

Despite more than 50 years of vaccination,
Bordetella pertussis
has remained endemic in The Netherlands, causing epidemic outbreaks every 3 to 5 years. Strain variation may play a role in the persistence of
B. pertussis
and was studied by sequencing 15 genes coding for surface proteins, including genes for all five components of acellular pertussis vaccines: pertussis toxin (Ptx), pertactin (Prn), filamentous hemagglutinin, and fimbriae (Fim2 and Fim3). A low level of allelic variation was observed, confirming a recent evolutionary origin of
B. pertussis
. In modern isolates, polymorphism was observed only in
prn
,
ptxS1
,
ptxS3
, and
tcfA
. Polymorphism in
ptxS1
,
ptxS3
, and
tcfA
was used to categorize isolates in multilocus sequence types (MLSTs). Analysis of Dutch isolates from 1949 to 1999 revealed five MLSTs, which showed a highly dynamic temporal behavior. We observed significant changes in the MLSTs after the introduction of pertussis vaccination in The Netherlands. Epidemic years were found to be associated with the expansion of MLST-4 or MLST-5. MLST-5 showed a remarkable expansion from 10% in 1997 to 80% in 1999. The MLST analysis was extended to a number of widely separated geographic regions: Finland, Italy, Japan, and the United States. MLST-4 and MLST-5 were found to dominate in Italy and the United States. In Finland and Japan, MLST-3 and MLST-2, respectively, were predominant. Thus, although each region showed distinctive MLST frequencies, in three of the five regions MLST-4 and MLST-5 were predominant. These types may represent newly emerged, successful clones. The identification of highly successful clones may shed light on the question of how
B. pertussis
is able to maintain itself in vaccinated populations.
Fatal Endocarditis in a Neonate Caused by the Dematiaceous FungusPhialemonium obovatum: Case Report and Review of the Literature
Authors: Gavin Patrick J. , Sutton Deanna A. , Katz Ben Z. 
Published: July 2002

ABSTRACTPhialemoniumspecies are grouped by most authorities among the dematiaceous fungi. Like several other darkly pigmented fungi, they appear to be an emerging cause of human disease, especially in the immunocompromised host. As numbers of immunocompromised patients increase, the trend of unusual fungi emerging as new pathogens is set to continue. Previous experience suggests that reports of rare fungal infections among selected patients often heralds the emergence of infection in a wider population of patients. We report a case of fatal endocarditis caused byPhialemonium obovatumin a premature neonate. To our knowledge this is the first documented case of native valve endocarditis due to this species, and we review the literature on invasive disease caused by the species. Unfamiliar fungal isolates are frequently misidentified or mistaken for environmental contaminants, and because of their relative rarity, data upon which to base antifungal treatment are limited. Thus, the diagnosis and treatment of unusual fungal pathogens present a significant challenge to clinicians and microbiologists alike. Early referral of such isolates to a specialist reference laboratory is advisable.
Evaluation of Etest Strips for Rapid Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Freixo I. M. , Caldas P. C. S. , Martins F. , Brito R. C. , Ferreira R. M. C. , Fonseca L. S. , Saad M. H. F. 
Published: July 2002

ABSTRACT

In this study, our objective was to evaluate Etest strips containing exponential gradients of isoniazid (INH), rifampin (RIF), and streptomycin (STR) for susceptibility testing of
Mycobacterium tuberculosis. M. tuberculosis
isolates were tested for antimicrobial susceptibilities by the standard proportion method using Löwenstein-Jensen (LJ) medium and by the Etest. The MICs determined by the Etest were obtained at 5, 7, or 10 days. In some strains with Etest-discrepant results, radiometric susceptibility testing (BACTEC) was performed to determine a consensus result.
M. tuberculosis
concordance between the two methods was 97% (86 of 89 isolates) for RIF, 96% for INH (84 of 87 isolates), and 80% (61 of 76 isolates) for STR. Most of the MICs determined by the Etest were easy to interpret and readable within 5 days. Results correlated well with those obtained by the LJ proportion and BACTEC methods for INH and RIF. However, a high proportion of false-sensitive and false-resistant results were observed, most often for STR. We also observed that variations in the inoculum size of
M. tuberculosis
isolates affected the MICs to a substantial degree. These discrepancies, along with the expense of the media, the Etest strips, and the specialized equipment required (CO
2
incubator), make this method less useful in developing countries.
The Cys
3
-His
1
Motif of the Respiratory Syncytial Virus M2-1 Protein Is Essential for Protein Function
Authors: Hardy Richard W. , Wertz Gail W. 
Published: July 2002

ABSTRACT

The M2 gene of respiratory syncytial (RS) virus has two open reading frames (ORFs). ORF1 encodes a 22-kDa protein termed M2-1. The M2-1 protein contains a Cys
3
-His
1
motif (C-X
7
-C-X
5
-C-X
3
-H) near the amino terminus. This motif is conserved in all human, bovine, and ovine strains of RS virus. A similar motif found in the mammalian transcription factor Nup475 has been shown to bind zinc. The M2-1 protein of human RS virus functions as a transcription factor which increases polymerase processivity, and it enhances readthrough of intergenic junctions during RS virus transcription, thereby acting as a transcription antiterminator. The M2-1 protein also interacts with the nucleocapsid protein. We examined the effects of mutations of cysteine and histidine residues predicted to coordinate zinc in the Cys
3
-His
1
motif on transcription antitermination and N protein binding. We found that mutating the predicted zinc-coordinating residues, the cysteine residues at amino acid positions 7 and 15 and the histidine residue at position 25, prevented M2-1 from enhancing transcriptional readthrough. In contrast, mutations of amino acids within this motif not predicted to coordinate zinc had no effect. Mutations of the predicted zinc-coordinating residues in the Cys
3
-His
1
motif also prevented M2-1 from interacting with the nucleocapsid protein. One mutation of a noncoordinating residue in the motif which did not affect readthrough during transcription, E10G, prevented interaction with the nucleocapsid protein. This suggests that M2-1 does not require interaction with the nucleocapsid protein in order to function during transcription. Analysis of the M2-1 protein in reducing sodium dodecyl sulfate-polyacrylamide gels revealed two major forms distinguished by their mobilities. The slower migrating form was shown to be phosphorylated, whereas the faster migrating form was not. Mutations in the Cys
3
-His
1
motif caused a change in distribution of the M2-1 protein from the slower to the faster migrating form. The data presented here show that the Cys
3
-His
1
motif of M2-1 is essential for maintaining the functional integrity of the protein.
Structural, Functional, and Genetic Comparisons of Epstein-Barr Virus Nuclear Antigen 3A, 3B, and 3C Homologues Encoded by the Rhesus Lymphocryptovirus
Authors: Jiang Hua , Cho Young-gyu , Wang Fred 
Published: July 2002

ABSTRACT

EBNA-3A, -3B, and -3C are three latent infection nuclear proteins important for
Epstein-Barr virus
(EBV)-induced B-cell immortalization and the immune response to EBV infection. All three are hypothesized to function as transcriptional transactivators, but little is known about their precise mechanism of action or their role in EBV pathogenesis. We have cloned and studied the three EBNA-3 homologues from a closely related lymphocryptovirus (LCV) which naturally infects rhesus monkeys. The rhesus LCV EBNA-3A, -3B, and -3C homologues have 37, 40, and 36% amino acid identity with the EBV genes, respectively. Function, as measured by in vitro assays, also appears to be conserved with the EBV genes, since the rhesus LCV EBNA-3s can interact with the transcription factor RBP-Jκ and the rhesus LCV EBNA-3C encodes a Q/P-rich domain with transcriptional activation properties. In order to better understand the relationship between these EBV and rhesus LCV latent infection genes, we asked if the rhesus LCV EBNA-3 locus could be recombined into the EBV genome and if it could substitute for the EBV EBNA-3s when assayed for human B-cell immortalization. Recombination between the EBV genome and rhesus LCV DNA was reasonably efficient. However, these studies suggest that the rhesus LCV EBNA-3 locus was not completely interchangeable with the EBV EBNA-3 locus for B-cell immortalization and that at least one determinant of the species restriction for LCV-induced B-cell immortalization maps to the EBNA-3 locus. The overall conservation of EBNA-3 structure and function between EBV and rhesus LCV indicates that rhesus LCV infection of rhesus monkeys can provide an important animal model for studying the role of the EBNA-3 genes in LCV pathogenesis.
Coreceptor-Dependent Inhibition of the Cell Fusion Activity of Simian Immunodeficiency Virus Env Proteins
Authors: Yang Chinglai , Yang Qingyuan , Compans Richard W. 
Published: July 2002

ABSTRACTThe cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors.
Synthesis of Virus-Specific High-Mobility DNA after Temperature Upshift of SC-1 Cells Chronically Infected with Moloney Murine Leukemia Virus Mutant
ts
1
Authors: Szurek Paul F. , Brooks Benjamin Rix 
Published: July 2002

ABSTRACT

Premature termination products of reverse transcription that consist physically of viral minus-sense single-stranded DNA that is shorter than one long terminal repeat and partial DNA duplexes are dramatically increased in the central nervous system (CNS) of FVB/N mice that are infected by
ts
1, a temperature-sensitive mutant of Moloney murine leukemia virus. Due to their migration in agarose gels, these incomplete physical forms of DNA have been designated high-mobility (HM) DNA. In non-CNS tissues, the level of HM DNA is either low or not detectable. In order to determine the conditions that are necessary for the synthesis of HM DNA in vivo, we have characterized the physical forms of HM DNA that were synthesized in vitro in chronically infected SC-1 cells after temperature upshift. At the permissive temperature of 34°C, the chronically infected SC-1 cells did not synthesize HM DNA. After temperature upshift of the cultured cells from 34 to 37°C, the chronically infected SC-1 cells developed extremely high levels of HM DNA. Following temperature downshift of the cultured cells from 37 to 34°C, a decrease in the level of HM DNA and an increase in the level of unintegrated linear proviral DNA occurred simultaneously. These results suggested that the accumulation of HM DNA both in vitro and in vivo may be the result of superinfection.
Conserved and Exposed Epitopes on Intact, Native, Primary Human Immunodeficiency Virus Type 1 Virions of Group M
Authors: Nyambi Phillipe N. , Mbah Henry A. , Burda Sherri , Williams Constance , Gorny Miroslaw K. , Nádas Arthur , Zolla-Pazner Susan 
Published: July 2002

ABSTRACTWe have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates.
Extensive Homologous Recombination among Widely Divergent TT Viruses
Authors: Worobey Michael 
Published: July 2002

ABSTRACT
Analyses of a collection of full-length TT virus genomes showed nearly half of them to be recombinant. The results were highly significant and revealed homologous recombination both within and among genotypes, often involving extremely divergent lineages. Recombination breakpoints were significantly more common in the noncoding region of the TT virus genome than in the coding region.
Effect of the Cytoplasmic Domain of the Simian Immunodeficiency Virus Envelope Protein on Incorporation of Heterologous Envelope Proteins and Sensitivity to Neutralization
Authors: Vzorov A. N. , Compans R. W. 
Published: July 2002

ABSTRACT
In addition to the viral envelope (Env) proteins, host cell-derived proteins have been reported to be present in human immunodeficiency virus and simian immunodeficiency virus (SIV) envelopes, and it has been postulated that they may play a role in infection. We investigated whether the incorporation of host cell proteins is affected by the structure and level of incorporation of viral Env proteins. To compare the cellular components incorporated into SIV particles and how this is influenced by the structure of the cytoplasmic domain, we compared SIV virions with full-length and truncated Env proteins. The levels of HLA-I and HLA-II molecules were found to be significantly (15- to 25-fold) higher in virions with full-length Env than in those with a truncated Env. Virions with a truncated Env were also found to be less susceptible to neutralization by specific antibodies against HLA-I or HLA-II proteins. We also compared the level of incorporation into SIV virions of a coexpressed heterologous viral glycoprotein, the influenza virus hemagglutinin (HA) protein. We found that SIV infection of cells expressing influenza virus HA resulted in the production of phenotypically mixed SIV virions containing influenza virus HA as well as SIV envelope proteins. The HA proteins were more effectively incorporated into virions with full-length Env than in virions with truncated Env. The phenotypically mixed particles with full-length Env, containing higher levels of HA, were sensitive to neutralization with anti-HA antibody, whereas virions with truncated Env proteins and containing lower levels of HA were more resistant to neutralization by anti-HA antibody. In contrast, SIV virions with truncated Env proteins were found to be highly sensitive to neutralization by antisera to SIV, whereas virions with full-length Env proteins were relatively resistant to neutralization. These results indicate that the cytoplasmic domain of SIV Env affects the incorporation of cellular as well as heterologous viral membrane proteins into the SIV envelope and may be an important determinant of the sensitivity of the virus to neutralizing antibodies.
African Swine Fever Virus Protein A238L Interacts with the Cellular Phosphatase Calcineurin via a Binding Domain Similar to That of NFAT
Authors: Miskin James E. , Abrams Charles C. , Dixon Linda K. 
Published: July 2002

ABSTRACT
The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-κB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.
Identification of Upregulated Genes in Scrapie-Infected Brain Tissue
Authors: Riemer Constanze , Queck Ingo , Simon Dietrich , Kurth Reinhard , Baier Michael 
Published: July 2002

ABSTRACTThe pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2′,5′-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and β2-microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2′,5′-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.
Ras-GAP Binding and Phosphorylation by Herpes Simplex Virus Type 2 RR1 PK (ICP10) and Activation of the Ras/MEK/MAPK Mitogenic Pathway Are Required for Timely Onset of Virus Growth
Authors: Smith C. C. , Nelson J. , Aurelian L. , Gober M. , Goswami B. B. 
Published: July 2002

ABSTRACT
We used a herpes simplex virus type 2 (HSV-2) mutant with a deletion in the RR1 (ICP10) PK domain (ICP10ΔPK) and an MEK inhibitor (PD98059) to examine the role of ICP10 PK in virus growth. In HSV-2-infected cells, ICP10 PK binds and phosphorylates the GTPase activating protein Ras-GAP. In vitro binding and peptide competition assays indicated that Ras-GAP N-SH2 and PH domains, respectively, bind ICP10 at phosphothreonines 117 and 141 and a WD40-like motif at positions 160 to 173. Binding and phosphorylation did not occur in cells infected with ICP10ΔPK. GTPase activity was significantly lower in HSV-2- than in ICP10ΔPK-infected cells. Conversely, the levels of activated Ras and mitogen-activated protein kinase (MAPK), and the expression and stabilization of the transcription factor c-Fos were significantly increased in cells infected with HSV-2 or a revertant virus [HSV-2(R)] but not with ICP10ΔPK. PD98059 inhibited MAPK activation and induction-stabilization of c-Fos. Expression from the ICP10 promoter was increased in cells infected with HSV-2 but not with ICP10ΔPK, and increased expression was ablated by PD98059. ICP10 DNA formed a complex with nuclear extracts from HSV-2-infected cells which was supershifted by c-Fos antibody and was not seen with extracts from ICP10ΔPK-infected cells. Complex formation was abrogated by PD98059. Onset of HSV-2 replication was significantly delayed by PD98059 (14 h versus 2 h in untreated cells), a delay similar to that seen for ICP10ΔPK. The data indicate that Ras-GAP phosphorylation by ICP10 PK is involved in the activation of the Ras/MEK/MAPK mitogenic pathway and c-Fos induction and stabilization. This results in increased ICP10 expression and the timely onset of HSV-2 growth.
A Functional NSP4 Enterotoxin Peptide Secreted from Rotavirus-Infected Cells
Authors: Zhang Mingdong , Zeng Carl Q.-Y. , Morris Andrew P. , Estes Mary K. 
Published: July 2002

ABSTRACT
Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis–Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112–175). The secretion of NSP4 aa112–175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112–175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112–175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112–175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112–175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.