bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genotyping of
Francisella tularensis
Strains by Pulsed-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism Fingerprinting, and 16S rRNA Gene Sequencing
Authors: García Del Blanco N. , Dobson M. E. , Vela A. I. , De La Puente V. A. , Gutiérrez C. B. , Hadfield T. L. , Kuhnert P. , Frey J. , Domínguez L. , Rodríguez Ferri E. F. 
Published: October 2002

ABSTRACT

We evaluated three molecular methods for identification of
Francisella
strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54
Francisella tularensis
subsp.
holarctica
, 5
F. tularensis
subsp.
tularensis
, 2
F. tularensis
subsp.
novicida
, and 1
F. philomiragia
strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes
Xho
I and
Bam
HI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of
F. tularensis
subsp.
holarctica.
The AFLP analysis technique produced some degree of discrimination among
F. tularensis
subsp.
holarctica
strains (one primary cluster with three major subclusters and minor variations within subclusters) when
Eco
RI-C and
Mse
I-A,
Eco
RI-T and
Mse
I-T,
Eco
RI-A and
Mse
I-C, and
Eco
RI-0 and
Mse
I-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of
F. tularensis
subsp.
tularensis
,
F. tularensis
subsp.
novicida
, and
F. philomiragia
were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all
F. tularensis
subsp.
holarctica
isolates compared in this study. These results suggest that although limited genetic heterogeneity among
F. tularensis
subsp.
holarctica
isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of
F. tularensis
and suitable techniques for the differentiation of individual strains.
Isolation from a Healthy Carrier and Characterization of a
Neisseria meningitidis
Strain That Is Deficient in γ-Glutamyl Aminopeptidase Activity
Authors: Takahashi Hideyuki , Tanaka Hiroshi , Inouye Hiroo , Kuroki Toshiro , Watanabe Yuko , Yamai Shiro , Watanabe Haruo 
Published: October 2002

ABSTRACT

γ-Glutamyl aminopeptidase (GGT) activity is used as a specific marker for the identification of
Neisseria meningitidis
. We isolated from a healthy carrier and characterized an
N. meningitidis
isolate which lacked the activity due to the insertional mutation of the
ggt
gene, suggesting that naturally occurring
N. meningitidis
isolates do not always possess GGT activity.
Real-Time PCR Detection of
Salmonella
in Suspect Foods from a Gastroenteritis Outbreak in Kerr County, Texas
Authors: Daum Luke T. , Barnes William J. , McAvin James C. , Neidert Margaret S. , Cooper Lynn A. , Huff William B. , Gaul Linda , Riggins W. S. , Morris Sandra , Salmen Ann , Lohman Kenton L. 
Published: October 2002

ABSTRACT

In June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5′-nuclease PCR assay was used to screen for
Salmonella
in nine food items from the buffet line. Barbeque chicken B tested positive for
Salmonella
, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing.
Salmonella enterica
serotype Panama was cultured from both food and patient stool samples.
First Reported Isolation of
Cryptococcus neoformans
var.
gattii
from a Patient in Singapore
Authors: Taylor M. B. , Chadwick D. , Barkham T. 
Published: October 2002

ABSTRACT

Cryptococcal meningitis has long been known to afflict immunocompetent patients in Singapore. We report the first identification of an isolate of
Cryptococcus neoformans
var.
gattii
from a Singaporean resident; this variety can cause invasive disease in the immunocompetent. Meningitis in a traveler returning from this area may be cryptococcal.
Modeling Viral Genome Fitness Evolution Associated with Serial Bottleneck Events: Evidence of Stationary States of Fitness
Authors: Lázaro Ester , Escarmís Cristina , Domingo Esteban , Manrubia Susanna C. 
Published: October 2002

ABSTRACT
Evolution of fitness values upon replication of viral populations is strongly influenced by the size of the virus population that participates in the infections. While large population passages often result in fitness gains, repeated plaque-to-plaque transfers result in average fitness losses. Here we develop a numerical model that describes fitness evolution of viral clones subjected to serial bottleneck events. The model predicts a biphasic evolution of fitness values in that a period of exponential decrease is followed by a stationary state in which fitness values display large fluctuations around an average constant value. This biphasic evolution is in agreement with experimental results of serial plaque-to-plaque transfers carried out with foot-and-mouth disease virus (FMDV) in cell culture. The existence of a stationary phase of fitness values has been further documented by serial plaque-to-plaque transfers of FMDV clones that had reached very low relative fitness values. The statistical properties of the stationary state depend on several parameters of the model, such as the probability of advantageous versus deleterious mutations, initial fitness, and the number of replication rounds. In particular, the size of the bottleneck is critical for determining the trend of fitness evolution.
Direct and Quantitative Single-Cell Analysis of Human Immunodeficiency Virus Type 1 Reactivation from Latency
Authors: Kutsch Olaf , Benveniste Etty N. , Shaw George M. , Levy David N. 
Published: October 2002

ABSTRACTThe ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.
Inhibition of Nuclear Import and Alteration of Nuclear Pore Complex Composition by Rhinovirus
Authors: Gustin Kurt E. , Sarnow Peter 
Published: October 2002

ABSTRACTNucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.
Identification and Characterization of the Pseudorabies Virus Tegument Proteins UL46 and UL47: Role for UL47 in Virion Morphogenesis in the Cytoplasm
Authors: Kopp Martina , Klupp Barbara G. , Granzow Harald , Fuchs Walter , Mettenleiter Thomas C. 
Published: October 2002

ABSTRACT

Proteins encoded by the UL46 and UL47 genes of herpes simplex virus type 1 (HSV-1) constitute major components of the viral tegument. However, their functions have so far not been elucidated in detail. By use of monospecific antisera directed against bacterially expressed glutathione-
S
-transferase fusion proteins, the homologous UL46 and UL47 proteins of the alphaherpesvirus pseudorabies virus (PrV) were identified in virus-infected cells and in virions. The PrV UL46 gene product of 693 amino acids (aa) exhibits an apparent molecular mass of 95 kDa, whereas the UL47 product of 750 aa was identified as a 97-kDa protein. Both are present in purified virions, correlating with their role as tegument proteins. Immunofluorescence analysis by confocal laser scan microscopy showed that late in infection the UL46 product is detectable in the cytoplasm, whereas the UL47 product was observed to be diffuse in the cytoplasm and speckled in the nucleus. Virus mutants lacking either the UL46 or the UL47 gene or both were isolated on noncomplementing cells, demonstrating that these genes either singly or in combination are not required for productive viral replication. However, plaque sizes were decreased. Interestingly, in one-step growth analysis, UL47 deletion mutants exhibited an approximately 10-fold decrease in final titers, whereas the UL46 deletion mutant was not affected. This finding correlated with ultrastructural observations which showed unimpaired virion morphogenesis in the absence of the UL46 protein, whereas in the absence of the UL47 protein intracytoplasmic aggregates of partially tegumented capsids were observed. In summary, we identified the PrV UL46 and UL47 proteins and show that the UL47 protein plays an important role in virion assembly in the cytoplasm.
Hantaan Virus Infection Causes an Acute Neurological Disease That Is Fatal in Adult Laboratory Mice
Authors: Wichmann Dominic , Gröne Hermann-Josef , Frese Michael , Pavlovic Jovan , Anheier Bärbel , Haller Otto , Klenk Hans-Dieter , Feldmann Heinz 
Published: October 2002

ABSTRACT

Hantaan virus, the etiological agent of Korean hemorrhagic fever, is transmitted to humans from persistently infected mice (
Apodemus agrarius
), which serve as the primary reservoir. Here we demonstrate that several strains of adult
Mus musculus domesticus
(C57BL/6, BALB/c, AKR/J, and SJL/J) were susceptible to Hantaan virus infection when infected intraperitoneally. First clinical signs were loss of weight, ruffled fur, and reduced activity, which were followed by neurological symptoms, such as paralyses and convulsions. Within 2 days of disease onset, the animals died of acute encephalitis. PCR analysis indicated a systemic infection with viral RNA present in all major organs. Immunohistochemical and in situ hybridization analyses of postmortem material detected viral antigen and RNA in the central nervous system (predominantly brain), liver, and spleen. In the central nervous system, viral antigen and RNA colocalized with perivascular infiltrations, the predominant pathological finding. To investigate the involvement of the interferon system in Hantaan virus pathogenesis, we infected alpha/beta interferon receptor knockout mice. These animals were more susceptible to Hantaan virus infection, indicating an important role of interferon-induced antiviral defense mechanisms in Hantaan virus pathogenesis. The present model may help to overcome shortcomings in the development of therapeutic and prophylactic measurements against hantavirus infections.
Analysis of the Complete Genome Sequence of the Hz-1 Virus Suggests that It Is Related to Members of the
Baculoviridae
Authors: Cheng Chia-Hsiung , Liu Su-Mei , Chow Teh-Yuan , Hsiao Yu-Yun , Wang Dan-Ping , Huang Jiann-Jang , Chen Hong-Hwa 
Published: October 2002

ABSTRACT

We report the complete sequence of a large rod-shaped DNA virus, called the Hz-1 virus. This virus persistently infects the
Heliothis zea
cell lines. The Hz-1 virus has a double-stranded circular DNA genome of 228,089 bp encoding 154 open reading frames (ORFs) and also expresses a persistence-associated transcript 1, PAT1. The G+C content of the Hz-1 virus genome is 41.8%, with a gene density of one gene per 1.47 kb. Sequence analysis revealed that a 9.6-kb region at 43.6 to 47.8 map units harbors five cellular genes encoding proteins with homology to dUTP pyrophosphatase, matrix metalloproteinase, deoxynucleoside kinase, glycine hydroxymethyltransferase, and ribonucleotide reductase large subunit. Other cellular homologs were also detected dispersed in the viral genome. Several baculovirus homologs were detected in the Hz-1 virus genome. These include PxOrf-70, PxOrf-29, AcOrf-81, AcOrf-96, AcOrf-22, VLF-1, RNA polymerase LEF-8 (orf50), and two structural proteins, p74 and p91. The Hz-1 virus p74 homolog shows high structural conservation with a double transmembrane domain at its C terminus. Phylogenetic analysis of the p74 revealed that the Hz-1 virus is evolutionarily distant from the baculoviruses. Another distinctive feature of the Hz-1 virus genome is a gene that is involved in insect development. However, the remainder of the ORFs (81%) encoded proteins that bear no homology to any known proteins. In conclusion, the sequence differences between the Hz-1 virus and the baculoviruses outnumber the similarities and suggest that the Hz-1 virus may form a new family of viruses distantly related to the
Baculoviridae
.
The Antiviral Response to Gamma Interferon
Authors: Costa-Pereira Ana P. , Williams Timothy M. , Strobl Birgit , Watling Diane , Briscoe James , Kerr Ian M. 
Published: October 2002

ABSTRACT
A role for alpha/beta interferon (IFN-α/β) in the IFN-γ antiviral response has long been suggested. Accordingly, possible roles for autocrine or double-stranded-RNA (dsRNA)-induced IFN-α/β in the IFN-γ response were investigated. Use was made of wild-type and a variety of mutant human fibrosarcoma cell lines, including mutant U5A cells, which lack a functional IFN-α/β receptor and hence an IFN-α/β response. IFN-γ did not induce detectable levels of IFN-α/β in any of the cell lines, nor was the IFN-γ response per se dependent on autocrine IFN-α/β. On the other hand, a number of responses to dsRNA [poly(I) · poly(C)] and encephalomyocarditis virus were greatly enhanced by IFN-γ pretreatment (priming) of wild-type cells or of mutant cells lacking an IFN-α/β response; these include the primary induction of dsRNA-inducible mRNAs, including IFN-β mRNA, and, to a lesser extent, the dsRNA-mediated activation of the p38 mitogen-activated protein (MAP) kinase(s). IFN-γ priming of mRNA induction by dsRNA is dependent on JAK1 and shows biphasic kinetics, with an initial rapid (<30-min) response being followed by a more substantial effect on overnight incubation. The IFN-γ-primed dsRNA responses appear to be subject to modulation through the p38, phosphatidylinositol 3-kinase, and ERK1/ERK2 MAP kinase pathways. It can be concluded that despite efficient priming of IFN-β production, the IFN-α/β pathways play no significant role in the primary IFN-γ antiviral response in these cell-virus systems. The observed IFN-γ priming of dsRNA responses, on the other hand, will likely play a significant role in combating virus infection in vivo.
C-Terminal gp40 Peptide Analogs Inhibit Feline Immunodeficiency Virus: Cell Fusion and Virus Spread
Authors: Medinas R. J. , Lambert D. M. , Tompkins W. A. 
Published: October 2002

ABSTRACTThe envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1), gp160, is synthesized as a protein precursor that when proteolytically cleaved yields two subunits, gp120 and gp41. gp120 is the surface glycoprotein on HIV-1 responsible for binding to CD4, and gp41 is the transmembrane glycoprotein involved in the membrane fusion process. gp41 is divided into the N-terminal fusion peptide, the heptad repeat 1 (HR1) and HR2 regions, and the C-terminal transmembrane region, which are collectively responsible for virus fusion and entry into the cell. Synthetic peptides derived from the HR2 and HR1 regions of HIV-1LAIhave been shown to prevent virus-cell fusion and infection in vitro. In phase II clinical trials in HIV patients, data revealed that T20 has antiviral efficacy and is well tolerated. Similar results were obtained in vitro with HIV-2 and simian immunodeficiency virus, supporting the conservation of the gp41 ectodomain among lentiviruses. Feline immunodeficiency virus (FIV) infection in the cat has been used as a model to develop potential antivirals for HIV. To determine if synthetic gp40 analogs capable of inhibiting FIV infection could be identified, 15 overlapping 35-amino-acid peptides derived from the C-terminal HR2 domain of FIV gp40 were synthesized. These peptides were tested for efficacy against FIV in a syncytium-forming assay with FIV-infected CrFK cells and HeLa cells expressing the FIV receptor CXCR4. Several peptides exhibited activity at the nanogram level. Antiviral activity was confirmed by suppression of reverse transcriptase in a FIV feline CD4+-T-cell (FCD4-E) acute-infection assay. These data demonstrate that synthetic peptides derived from the HR2 domain of the FIV gp41 protein are effective inhibitors of FIV infection.
Intravirion Processing of the Human Immunodeficiency Virus Type 1 Vif Protein by the Viral Protease May Be Correlated with Vif Function
Authors: Khan Mohammad A. , Akari Hirofumi , Kao Sandra , Aberham Claudia , Davis David , Buckler-White Alicia , Strebel Klaus 
Published: October 2002

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral Pr and was sensitive to Pr inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala
150
. Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function, suggesting that intravirion processing of Vif is important for the production of infectious viruses.
Blockade of Human Immunodeficiency Virus Type 1 Expression by Caveolin-1
Authors: Llano Manuel , Kelly Tara , Vanegas Maria , Peretz Mary , Peterson Timothy E. , Simari Robert D. , Poeschla Eric M. 
Published: October 2002

ABSTRACT
Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.