bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of a Fragment of Glycoprotein G-2 Produced in the Baculovirus Expression System for Detecting Herpes Simplex Virus Type 2-Specific Antibodies
Authors: Ikoma Minako , Liljeqvist Jan-Åke , Groen Jan , Glazenburg Koen L. , The T. Hauw , Welling-Wester Sytske 
Published: July 2002

ABSTRACT

Fragments of glycoprotein G (gG-2
281-594His
), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1
t26-189His
), and glycoprotein D of HSV-1 (gD-1
1-313
), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1
t26-189His
and gG-2
281-594His
fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1
t26-189His
and the fragment of gG-2
281-594His
were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1
t26-189His
, sera positive for HSV-2 reacted with the gG-2
281-594His
fragment, and sera positive for both types reacted with gG-1
t26-189His
and gG-2
281-594His
in Western blotting. The human sera recognized polypeptides of gG-2
281-594His
with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1
t26-189His
and the recombinant gG-2
281-594His
fragment were used as type-specific antigens for the detection of HSV-1- and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1
1-313
were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2
281-594His
fragment can be obtained in relatively large quantities at low cost.
Improvement of AMPLICOR Human Immunodeficiency Virus Type 1 Viral Load Test (Version 1.5) by Addition of a Coprecipitant during the RNA Isolation Step
Authors: Xu Min , Chan Yung , Fischer Steven H. , Remaley Alan T. 
Published: July 2002

ABSTRACT
The effect of the addition of a coprecipitant during the RNA isolation step on the analytical performance of the COBAS AMPLICOR human immunodeficiency virus type 1 (HIV-1) Monitor (version 1.5; Roche) viral load test was tested. Thirty-six specimens including patient samples, positive control samples, and negative control samples were processed in the presence and absence of the Pellet Paint coprecipitant. Specimens processed without the coprecipitant had lower RNA yields, as evidenced by a lower signal for the quantitation standard (QS). In addition, the results for all samples processed with the coprecipitant were acceptable on the basis of the optical density (OD) reading for the QS, whereas the result for one specimen processed without the coprecipitant was unacceptable on the basis of the OD reading for the QS, which required the assay to be repeated. Furthermore, the use of the coprecipitant improved the overall precision of the assay.
Antibiotic Resistance in
Staphylococcus
Isolates Obtained from Fecal Samples of Healthy Children
Authors: Domínguez Elena , Zarazaga Myriam , Torres Carmen 
Published: July 2002

ABSTRACT

Thirty-nine
Staphylococcus
isolates with different mechanisms of resistance were recovered from the feces of 50 healthy children and tested for their susceptibilities to 17 antibiotics. The percentages of resistance of the staphylococci to some antibiotics were as follows: penicillin, 87%; erythromycin, 64%; tobramycin, 36%; tetracycline, 20.5%; kanamycin, 15%; and gentamicin, 13%. The
mecA
gene was detected in nine coagulase-negative staphylococci.
Multidrug and Broad-Spectrum Cephalosporin Resistance among
Salmonella enterica
Serotype Enteritidis Clinical Isolates in Southern Italy
Authors: Villa Laura , Mammina Caterina , Miriagou Vivi , Tzouvelekis Leonidas S. , Tassios Panayotis T. , Nastasi Antonino , Carattoli Alessandra 
Published: July 2002

ABSTRACT

From 1992 to 1997, only six sporadic isolates of
Salmonella enterica
serotype Enteritidis from patients with cases of gastroenteritis in southern Italy exhibited resistance to broad-spectrum cephalosporins. Five isolates produced SHV-12, and one isolate encoded a class C β-lactamase. The
bla
SHV-12
gene was located in at least two different self-transferable plasmids, one of which also carried a novel class 1 integron.
Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Gag- and Gag Peptide-Specific CD4+T-Cell Clones from an HIV-1-Seronegative Donor following In Vitro Immunization
Authors: Venturini Sara , Mosier Donald E. , Burton Dennis R. , Poignard Pascal 
Published: July 2002

ABSTRACTSubstantial evidence argues that human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells play an important role in the control of HIV-1 replication in infected individuals. Moreover, it is increasingly clear that an HIV vaccine should elicit potent cytotoxic lymphocyte and antibody responses that will likely require an efficient CD4+T-cell response. Therefore, understanding and characterizing HIV-specific CD4+T-cell responses is an important aim. Here we describe the generation of HIV-1 Gag- and Gag peptide-specific CD4+T-cell clones from an HIV-1-seronegative donor by in vitro immunization with HIV-1 Gag peptides. The Gag peptides were able to induce a strong CD4+T-cell immune response in peripheral blood mononuclear cells from the HIV-1-seronegative donor. Six Gag peptide-specific CD4+T-cell clones were isolated and their epitopes were mapped. The region of p24 between amino acids 201 and 300 of Gag was defined as the immunodominant region of Gag. A new T helper epitope in the p6 protein of Gag was identified. Two clones were shown to recognize Gag peptides and processed Gag protein, while the other four clones reacted only to Gag peptides under the experimental conditions used. Functional analysis of the clones indicated that both Th1 and Th2 types of CD4+T cells were obtained. One clone showed direct antigen-specific cytotoxic activity. These clones represent a valuable tool for understanding the cellular immune response to HIV-1, and the study provides new insights into the HIV-1-specific CD4+T-cell response and the induction of an anti-Gag and -Gag peptide cellular primary immune response in vitro.
Herpes Simplex Virus Type 1 Origins of DNA Replication Play No Role in the Regulation of Flanking Promoters
Authors: Summers Bretton C. , Leib David A. 
Published: July 2002

ABSTRACT
Herpes simplex virus (HSV) exhibits altered gene regulation in neuronal compared to nonneuronal tissues. It has been hypothesized that initiation of DNA synthesis at the viral origins of replication (oriS and oriL) is a critical step in the upregulation of transcriptional activity of flanking divergent promoters, thereby increasing productive gene expression in neurons. Notably, oriS is flanked by the immediate-early (IE) ICP4 and ICP22/47 promoters, and oriL is flanked by the early (E) UL29 and UL30 promoters. To test this hypothesis further, a series of constructs were generated in which these promoters were placed upstream of luciferase genes. In addition, DNA replication origins were deleted in the context of these promoter constructs. All cassettes were recombined into the viral genome of HSV type 1 strain KOS at a site distal to its native origins. Recombinant reporter expression was monitored in vitro and in vivo to determine the role of viral origins of DNA replication in the regulation of their flanking promoters. Reporter gene expression was unaffected by the presence or absence of oriS or oriL, with the exception of a twofold increase in ICP22/47 promoter activity in the absence of oriS. DNA synthesis inhibitors resulted in a decrease of both IE- and E-promoter activity in primary cells but not continuous cell cultures. Reporter activity was readily assayed in vivo during acute infection and reactivation from latency and was also sensitive to DNA synthesis inhibitors. In all assays, reporter gene expression was unaffected by the presence or absence of either oriS or oriL. These data support the requirement of DNA synthesis for full viral gene expression in vivo but suggest that the origin elements play no role in the regulation of their flanking promoters.
Processing Map and Essential Cleavage Sites of the Nonstructural Polyprotein Encoded by ORF1 of the Feline Calicivirus Genome
Authors: Sosnovtsev Stanislav V. , Garfield Mark , Green Kim Y. 
Published: July 2002

ABSTRACT

Feline calicivirus (FCV) nonstructural proteins are translated as part of a large polyprotein that undergoes autocatalytic processing by the virus-encoded 3C-like proteinase. In this study, we mapped three new cleavage sites (E
46
/A
47
, E
331
/D
332
, and E
685
/N
686
) recognized by the virus proteinase in the N-terminal part of the open reading frame 1 (ORF1) polyprotein to complete the processing map. Taken together with two sites we identified previously (E
960
/A
961
and E
1071
/S
1072
), the FCV ORF1 polyprotein contains five cleavage sites that define the borders of six proteins with calculated molecular masses of 5.6, 32, 38.9, 30.1, 12.7, and 75.7 kDa, which we designated p5.6, p32, p39 (NTPase), p30, p13 (VPg), and p76 (Pro-Pol), respectively. Mutagenesis of the E to A in each of these cleavage sites in an infectious FCV cDNA clone was lethal for the virus, indicating that these cleavages are essential in a productive virus infection. Mutagenesis of two cleavage sites (E
1345
/T
1346
and E
1419
/G
1420
) within the 75.7-kDa Pro-Pol protein previously mapped in bacterial expression studies was not lethal.
Reactivation of Latent Cytomegalovirus Infection in Mouse Brain Cells Detected after Transfer to Brain Slice Cultures
Authors: Tsutsui Yoshihiro , Kawasaki Hideya , Kosugi Isao 
Published: July 2002

ABSTRACT

Cytomegalovirus (CMV) is the most significant infectious cause of brain disorders in humans involving the developing brain. It is hypothesized that the brain disorders occur after recurrent reactivation of the latent infection in some kinds of cells in the brains. In order to test this hypothesis, we examined the reactivation of latent murine CMV (MCMV) infection in the mouse brain by transfer to brain slice culture. We infected neonatal and young adult mice intracerebrally with recombinant MCMV in which the
lacZ
gene was inserted into a late gene. The brains were removed 6 months after infection and used to prepare brain slices that were then cultured for up to 4 weeks. Reactivation of latent infection in the brains was detected by β-galactosidase (β-Gal) staining to assess β-galactosidase expression. Viral replication was also confirmed by the plaque assay. Reactivation was observed in about 75% of the mice infected during the neonatal period 6 months after infection. Unexpectedly, reactivation was also observed in 75% of mice infected as young adults, although the infection ratio in the brain slices was significantly lower than that in neonatally infected mice. β-Gal-positive cells were observed in marginal regions of the brains or immature neural cells in the ventricular walls. Immunohistochemical staining showed that the β-Gal-positive reactivated cells were neural stem or progenitor cells. These results suggest that brain disorders may occur long after infection by reactivation of latent infection in the immature neural cells in the brain.
The Mannose-Dependent Epitope for Neutralizing Antibody 2G12 on Human Immunodeficiency Virus Type 1 Glycoprotein gp120
Authors: Sanders Rogier W. , Venturi Miro , Schiffner Linnea , Kalyanaraman Roopa , Katinger Hermann , Lloyd Kenneth O. , Kwong Peter D. , Moore John P. 
Published: July 2002

ABSTRACTWe have analyzed the unique epitope for the broadly neutralizing human monoclonal antibody (MAb) 2G12 on the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequence analysis, focusing on the conservation of relevant residues across multiple HIV-1 isolates, refined the epitope that was defined previously by substitutional mutagenesis (A. Trkola, M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger, J. Virol. 70:1100-1108, 1996). In a biochemical study, we digested recombinant gp120 with various glycosidase enzymes of known specificities and showed that the 2G12 epitope is lost when gp120 is treated with mannosidases. Computational analyses were used to position the epitope in the context of the virion-associated envelope glycoprotein complex, to determine the variability of the surrounding surface, and to calculate the surface accessibility of possible glycan- and polypeptide-epitope components. Together, these analyses suggest that the 2G12 epitope is centered on the high-mannose and/or hybrid glycans of residues 295, 332, and 392, with peripheral glycans from 386 and 448 on either flank. The epitope is mannose dependent and composed primarily of carbohydrate, with probably no direct involvement of the gp120 polypeptide surface. It resides on a face orthogonal to the CD4 binding face, on a surface proximal to, but distinct from, that implicated in coreceptor binding. Its conservation amidst an otherwise highly variable gp120 surface suggests a functional role for the 2G12 binding site, perhaps related to the mannose-dependent attachment of HIV-1 to DC-SIGN or related lectins that facilitate virus entry into susceptible target cells.
Modulating the Function of the Measles Virus RNA-Dependent RNA Polymerase by Insertion of Green Fluorescent Protein into the Open Reading Frame
Authors: Duprex W. Paul , Collins Fergal M. , Rima Bert K. 
Published: July 2002

ABSTRACT

Measles virus
(MV) is the type species of the
Morbillivirus
genus and its RNA-dependent RNA polymerase complex is comprised of two viral polypeptides, the large (L) and the phospho- (P) proteins. Sequence alignments of morbillivirus L polymerases have demonstrated the existence of three well-conserved domains (D1, D2, and D3) which are linked by two variable hinges (H1 and H2). Epitope tags (c-Myc) were introduced into H1 and H2 to investigate the tolerance of the variable regions to insertions and to probe the flexibility of the proposed domain structures to spatial reorientation. Insertion into H1 abolished polymerase activity whereas introduction into H2 had no effect. The open reading frame of enhanced green fluorescent protein was also inserted into the H2 region of the MV L gene to extend these observations. This resulted in a recombinant protein that was both functional and autofluorescent, although the overall polymerase activity was reduced by over 40%. Two recombinant viruses which contained the chimeric L genes EdtagL(MMc-mycM) and EdtagL(MMEGFPM) were generated. Tagged L proteins were detectable, by indirect immunofluorescence in the case of EdtagL(MMc-mycM) and by autofluorescence in the case of EdtagL(MMEGFPM). We suggest that D3 enjoys a limited conformational independence from the other domains, indicating that the L polymerases of the
Mononegavirales
may function as multidomain proteins.
Viral Gene Expression Patterns in Human Herpesvirus 6B-Infected T Cells
Authors: Øster Bodil , Höllsberg Per 
Published: July 2002

ABSTRACTHerpesvirus gene expression is divided into immediate-early (IE) or α genes, early (E) or β genes, and late (L) or γ genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.
Adding Genes to the RNA Genome of Vesicular Stomatitis Virus: Positional Effects on Stability of Expression
Authors: Wertz Gail W. , Moudy Robin , Ball L. Andrew 
Published: July 2002

ABSTRACT
Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.
Subcellular Localization of the Bovine Leukemia Virus R3 and G4 Accessory Proteins
Authors: Lefèbvre Laurent , Ciminale Vincenzo , Vanderplasschen Alain , D'Agostino Donna , Burny Arsène , Willems Luc , Kettmann Richard 
Published: July 2002

ABSTRACT

Bovine leukemia virus
(BLV) is a complex retrovirus that belongs to the
Deltaretrovirus
genus, which also includes
Human T-cell leukemia virus type 1
(HTLV-1). Both viruses contain an X region coding for at least four proteins: Tax and Rex, which are involved in transcriptional and posttranscriptional regulation, respectively, and the accessory proteins R3 and G4 (for BLV) and p12
I
, p13
II
, and p30
II
(for HTLV-1). The present study was aimed at characterizing the subcellular localization of BLV R3 and G4. The results of immunofluorescence experiments on transfected HeLa Tat cells demonstrated that R3 is located in the nucleus and in cellular membranes, as previously reported for HTLV-1 p12
I
. In contrast, G4, like p13
II
, is localized both in the nucleus and in mitochondria. In addition, we have shown that G4 harbors a mitochondrial targeting signal consisting of a hydrophobic region and an amphipathic α-helix. Thus, despite a lack of significant primary sequence homology, R3 and p12
I
and G4 and p13
II
exhibit similar targeting properties, suggesting possible overlap in their functional properties.
Handedness of the Herpes Simplex Virus Capsid and Procapsid
Authors: Cheng Naiqian , Trus Benes L. , Belnap David M. , Newcomb William W. , Brown Jay C. , Steven Alasdair C. 
Published: July 2002

ABSTRACT
The capsid of herpes simplex virus has an icosahedral surface lattice with a nonskew triangulation number, T=16. Nevertheless, the proteins arrayed on this lattice necessarily have an intrinsic handedness. We have determined the handedness of both the herpes simplex virus type 1 capsid and its precursor procapsid by a cryoelectron microscopic tilting method.
1917 Avian Influenza Virus Sequences Suggest that the 1918 Pandemic Virus Did Not Acquire Its Hemagglutinin Directly from Birds
Authors: Fanning Thomas G. , Slemons Richard D. , Reid Ann H. , Janczewski Thomas A. , Dean James , Taubenberger Jeffery K. 
Published: July 2002

ABSTRACT
Wild waterfowl captured between 1915 and 1919 were tested for influenza A virus RNA. One bird, captured in 1917, was infected with a virus of the same hemagglutinin (HA) subtype as that of the 1918 pandemic virus. The 1917 HA is more closely related to that of modern avian viruses than it is to that of the pandemic virus, suggesting (i) that there was little drift in avian sequences over the past 85 years and (ii) that the 1918 pandemic virus did not acquire its HA directly from a bird.
Effects of Varying Sequence Similarity on the Frequency of Repeat Deletion during Reverse Transcription of a Human Immunodeficiency Virus Type 1 Vector
Authors: An Wenfeng , Telesnitsky Alice 
Published: July 2002

ABSTRACT
Genetic recombination contributes to human immunodeficiency virus type 1 (HIV-1) diversity, with homologous recombination being more frequent than nonhomologous recombination. In this study, HIV-1-based vectors were used to assay the effects of various extents of sequence divergence on the frequency of the recombination-related property of repeat deletion. Sequence variation, similar in degree to that which differentiates natural HIV-1 isolates, was introduced by synonymous substitutions into a gene segment. Repeated copies of this segment were then introduced into assay vectors. With the use of a phenotypic screen, the deletion frequency of identical repeats was compared to the frequencies of repeats that differed in sequence by various extents. During HIV-1 reverse transcription, the deletion frequency observed with repeats that differed by 5% was 65% of that observed with identical repeats. The deletion frequency decreased to 26% for repeats that differed by 9%, and when repeats differed by 18%, the deletion frequency was about 5% of the identical repeat value. Deletion frequencies fell to less than 0.3% of identical repeat values when genetic distances of 27% or more were examined. These data argue that genetic variation is not as inhibitory to HIV-1 repeat deletion as it is to the corresponding cellular process and suggest that, for sequences that differ by about 25% or more, HIV-1 recombination directed by sequence homology may be no more frequent than that which is homology independent.