bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnosis of Bacteriuria by Detection of Volatile Organic Compounds in Urine Using an Automated Headspace Analyzer with Multiple Conducting Polymer Sensors
Authors: Aathithan S. , Plant J. C. , Chaudry A. N. , French G. L.
Published: July 2002

The Osmetech Microbial Analyzer (OMA) is an automated headspace analyzer fitted with a novel detector system consisting of an array of polymer sensors, each of which responds to different volatile organic compounds. The system can be used for screening clinical urine specimens for significant bacteriuria by sampling urine headspace and subjecting the output of the multiple-detector response to principal component analysis. The OMA readily distinguished artificially infected urine samples from sterile controls. The OMA was then used to analyze 534 unselected clinical urine specimens, of which 21.5% had significant bacteriuria (containing >105CFU of bacteria/ml). The sensitivity and specificity of the OMA compared with conventional culture were 83.5 and 87.6%, respectively. The OMA is a promising automated system for the rapid routine screening of urine specimens, and further clinical trials are in progress.
Population-Based Survey of Antimicrobial Susceptibility and Serotype Distribution of
Streptococcus pneumoniae
from Meningitis Patients in Salvador, Brazil
Authors: Reis Joice Neves , Cordeiro Soraia Machado , Coppola Steven J. , Salgado Kátia , Carvalho Maria G. S. , Teixeira Lúcia M. , Thompson Terry A. , Facklam Richard R. , Reis Mitermayer G. , Ko Albert I.
Published: July 2002

Penicillin-nonsusceptible strains were isolated from 15% of 303 individuals with pneumococcal meningitis identified during a 4-year surveillance study in Salvador, Brazil. The estimated rate of coverage of the seven-valent conjugate vaccine was 74% among patients <5 years of age and 94% among those infected with nonsusceptible isolates, indicating that the use of conjugate vaccines may be an approach to the control of emerging penicillin resistance in Brazil.
Diagnosis of Respiratory Syncytial Virus Infection: Comparison of Reverse Transcription-PCR to Viral Culture and Serology in Adults with Respiratory Illness
Authors: Falsey Ann R. , Formica Maria A. , Walsh Edward E.
Published: July 2002

Diagnosis of respiratory syncytial virus (RSV) during acute infection in adults is difficult because of the poor sensitivity of viral culture and antigen detection. A recently developed single-tube nested reverse transcription-PCR (RT-PCR) was compared to viral culture and serology by enzyme immunoassay for the diagnosis of RSV in adults with respiratory illness. Nasal swab samples were collected during respiratory illnesses from five groups of subjects: healthy young adults, healthy elderly adults, adults with chronic heart and lung disease, nursing home residents, and adults admitted to the hospital during the winter with cardiopulmonary illnesses. Of 1,112 samples for which all three tests were available, 117 were positive by at least one method and 995 were negative by all methods. One hundred ten were considered true positives because culture and/or serology was positive. Of these, 80 (73%) were PCR positive compared to 43 (39%) that were culture positive. Seven PCR results were considered false positives due to negative culture and serology. The overall RT-PCR sensitivity was 73%, and specificity was 99%. These data indicate that RT-PCR is an excellent method for the diagnosis of acute RSV infection in adults.
Distinguishing Cytomegalovirus (CMV) Infection and Disease with CMV Nucleic Acid Assays
Authors: Caliendo Angela M. , St. George Kirsten , Allega Jessica , Bullotta Arlene C. , Gilbane Lisa , Rinaldo Charles R.
Published: July 2002

Human cytomegalovirus (CMV) continues to be a significant cause of morbidity and mortality among transplant recipients. Molecular assays have been developed for the detection and quantification of CMV nucleic acid. In evaluating the clinical utility of these assays, correlations with clinical outcome are essential. The Amplicor CMV Monitor and NucliSens CMV pp67 tests were compared to the CMV antigenemia assay for 45 transplant recipients and 1 patient with Wegener's granulomatosis. Twenty-three patients remained antigenemia negative throughout the monitoring period, none of whom developed CMV disease. In this patient group, both the Amplicor and NucliSens assays showed very high specificity; only 1 of the 324 specimens assayed by NucliSens and none of the 303 specimens assayed by Amplicor were positive. Twenty-three patients were antigenemia positive during the monitoring period, 12 of whom developed 13 episodes of symptomatic CMV disease. In this patient group, the NucliSens assay was positive at or before the development of symptoms in 12 of the 13 episodes of CMV disease. All eight patients with symptomatic CMV disease who were tested by the Amplicor assay were positive at or before the development of disease. For the 11 asymptomatic patients, the NucliSens assay was positive less frequently than the antigenemia or Amplicor assays. The NucliSens assay was more likely to be positive at higher antigenemia or viral load levels. Both the NucliSens and Amplicor assays appear to have clinical utility in monitoring patients for CMV disease.
Differentiation of
Aspergillus ustus
Strains by Random Amplification of Polymorphic DNA
Authors: Rath Peter-Michael , Petermeier Katrin , Verweij Paul E. , Ansorg Rainer
Published: July 2002

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of water-soluble proteins and the randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from 21
Aspergillus ustus
isolates, including 11 reference strains and 10 patient and environmental strains from one hospital, were investigated. All isolates showed identical protein patterns. The RAPD assay discriminated between all reference strains. Comparison of hospital isolates showed identical RAPD patterns in some of the patient and environmental isolates. The data indicate that the RAPD technique is useful for fingerprinting
A. ustus
.
The E4-6/7 Protein Functionally Compensates for the Loss of E1A Expression in Adenovirus Infection
Authors: O'Connor Robert J. , Hearing Patrick
Published: July 2002

The E1A gene products are required and sufficient for activation of adenovirus gene expression in cultured cells. The E4-6/7 gene product induces the binding of the cellular transcription factor E2F to the viral E2a promoter region. The induction of E2F binding to the E2a promoter in vitro is directly correlated with transcriptional activation of the E2a promoter in vivo. The E2 region encodes the viral replication proteins, yet adenoviruses lacking E4-6/7 function demonstrate no defective phenotype in infected cells. Here we show that the E4-6/7 protein can functionally compensate for E1A expression in virus infection. In the absence of the E1A gene products, expression of the E4-6/7 protein is sufficient to displace retinoblastoma protein family members from E2Fs, activate expression of early region 2 via induction of E2F DNA binding to the E2a promoter region, and significantly enhance replication of an E1A-defective adenovirus. These results have implications in the regulation of viral gene expression and for the development of recombinant adenovirus vectors.
Identification of the Pseudorabies Virus Promoter Required for Latency-Associated Transcript Gene Expression in the Natural Host
Authors: Jin Ling , Schnitzlein William M. , Scherba Gail
Published: July 2002

Expression of the latency-associated transcript (LAT) gene is a hallmark of alphaherpesvirus latency, and yet its control and function remain an enigma. Resolution of this problem will require verification and subsequent elimination or disabling of elements regulating LAT gene transcription so that the influence of the resultant RNA can be evaluated. Toward this end, we generated a novel pseudorabies virus (PrV) recombinant in which a 282-bp region containing the LAP1 (first latency-active promoter) consensus sequence was replaced by a reporter cassette. Despite this substitution, replication of the recombinant was comparable to that of the parental and rescuant viruses both in cultured mammalian cells and in the natural host, swine. Furthermore, production of the LAT gene-associated 2.0- and 8.0-kb RNAs during an in vitro lytic infection of cultured neuronal cells was unaffected. However, the otherwise constitutively produced and processed 8.4-kb LAT was not detected in porcine trigeminal ganglia latently infected with this novel recombinant, although the viral genome was shown to be present. Therefore, LAP1 is apparently the basal promoter for PrV LAT gene expression during viral latency but is not required for such activity during an in vitro lytic infection of neuronal cells. More importantly, the ability of PrV to persist in a latent state in the absence of LAT suggests that other factors are responsible for this event in the natural host.
Widespread Distribution of a Group I Intron and Its Three Deletion Derivatives in the Lysin Gene of
Streptococcus thermophilus
Bacteriophages
Authors: Foley Sophie , Bruttin Anne , Brüssow Harald
Published: July 2002

Of 62
Streptococcus thermophilus
bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or
S. thermophilus
bacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from
Bacillus
phage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and
Bacillus
phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns from
Lactococcus
,
Lactobacillus
, and
Bacillus
phages.
Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System
Authors: Smith R. L. , Traul D. L. , Schaack J. , Clayton G. H. , Staley K. J. , Wilcox C. L.
Published: July 2002

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of theEscherichia coli lacZgene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.
The Host Range Phenotype Displayed by a Sindbis Virus Glycoprotein Variant Results from Virion Aggregation and Retention on the Surface of Mosquito Cells
Authors: Boehme Karl W. , Popov Vsevolod L. , Heidner Hans W.
Published: July 2002

The Sindbis virus variant NE2G216 is a PE2-containing host range mutant that is growth restricted in cultured mosquito cells (C6/36) due to inefficient release of virions from this cell type. The maturation defect of NE2G216 has been linked to the structures of N-linked oligosaccharides synthesized by arthropod cells. Analysis of C6/36 cells infected with NE2G216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates adhered to the cell surface. The virus aggregation phenotype of NE2G216 was reproduced in vertebrate cells (Pro-5) by the addition of 1-deoxymannojirimycin, an inhibitor of carbohydrate processing which limits the processing of N-linked oligosaccharides to structures that are structurally similar, albeit not identical, to those synthesized in C6/36 cells. We conclude that defective maturation of NE2G216 in mosquito cells is due to virion aggregation and retention on the cell surface and that this phenotype is directly linked to the carbohydrate-processing properties of these cells.
Lentivirus Gene Transfer in Murine Hematopoietic Progenitor Cells Is Compromised by a Delay in Proviral Integration and Results in Transduction Mosaicism and Heterogeneous Gene Expression in Progeny Cells
Authors: Mikkola Hanna , Woods Niels-Bjarne , Sjögren Marketa , Helgadottir Hildur , Hamaguchi Isao , Jacobsen Sten-Eirik , Trono Didier , Karlsson Stefan
Published: July 2002

Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin
−
c-kit
+
Sca1
+
primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% ± 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42.0% ± 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 ± 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 ± 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP
+
lentivirus vector-transduced colonies revealed vector PCR
+
GFP
+
(42%), vector PCR
−
GFP
−
(46%), and vector PCR
+
GFP
−
(13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.
Cytoplasm-to-Nucleus Translocation of a Herpesvirus Tegument Protein during Cell Division
Authors: Elliott Gillian , O'Hare Peter
Published: July 2002

We have previously shown that the herpes simplex virus tegument protein VP22 localizes predominantly to the cytoplasm of expressing cells. We have also shown that VP22 has the unusual property of intercellular spread, which involves the movement of VP22 from the cytoplasm of these expressing cells into the nuclei of nonexpressing cells. Thus, VP22 can localize in two distinct subcellular patterns. By utilizing time-lapse confocal microscopy of live cells expressing a green fluorescent protein-tagged protein, we now report in detail the intracellular trafficking properties of VP22 in expressing cells, as opposed to the intercellular trafficking of VP22 between expressing and nonexpressing cells. Our results show that during interphase VP22 appears to be targeted exclusively to the cytoplasm of the expressing cell. However, at the early stages of mitosis VP22 translocates from the cytoplasm to the nucleus, where it immediately binds to the condensing cellular chromatin and remains bound there through all stages of mitosis and chromatin decondensation into the G
1
stage of the next cycle. Hence, in VP22-expressing cells the subcellular localization of the protein is regulated by the cell cycle such that initially cytoplasmic protein becomes nuclear during cell division, resulting in a gradual increase over time in the number of nuclear VP22-expressing cells. Importantly, we demonstrate that this process is a feature not only of VP22 expressed in isolation but also of VP22 expressed during virus infection. Thus, VP22 utilizes an unusual pathway for nuclear targeting in cells expressing the protein which differs from the nuclear targeting pathway used during intercellular trafficking.
In Vivo Attenuation of Simian Immunodeficiency Virus by Disruption of a Tyrosine-Dependent Sorting Signal in the Envelope Glycoprotein Cytoplasmic Tail
Authors: Fultz Patricia N. , Vance Patricia J. , Endres Michael J. , Tao Binli , Dvorin Jeffrey D. , Davis Ian C. , Lifson Jeffrey D. , Montefiori David C. , Marsh Mark , Malim Michael H. , Hoxie James A.
Published: July 2002

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxφ) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (ΔGY). To assess the in vivo replication competence, all viruses contained a stop codon innefthat has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239ΔGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4+T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239ΔGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, thenefstop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxφ signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations innef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.
Polyomavirus Small t Antigen Prevents Retinoic Acid-Induced Retinoblastoma Protein Hypophosphorylation and Redirects Retinoic Acid-Induced G
0
Arrest and Differentiation to Apoptosis
Authors: Yen Andrew , Placanica Lisa , Bloom Stephen , Varvayanis Susi
Published: July 2002

Polyomavirus small t antigen (ST) impedes late features of retinoic acid (RA)-induced HL-60 myeloid differentiation as well as growth arrest, causing apoptosis instead. HL-60 cells were stably transfected with ST. ST slowed the cell cycle, retarding G
2
/M in particular. Treated with RA, the ST transfectants continued to proliferate and underwent apoptosis. ST also impeded the normally RA-induced hypophosphorylation of the retinoblastoma tumor suppressor protein consistent with failure of the cells to arrest growth. The RA-treated transfectants expressed CD11b, an early cell surface differentiation marker, but inducible oxidative metabolism, a later and more mature functional differentiation marker, was largely inhibited. Instead, the cells underwent apoptosis. ST affected significant known components of RA signaling that result in G
0
growth arrest and differentiation in wild-type HL-60. ST increased the basal amount of activated ERK2, which normally increases when wild-type cells are treated with RA. ST caused increased RARα expression, which is normally down regulated in RA-treated wild-type cells. The effects of ST on RA-induced myeloid differentiation did not extend to monocytic differentiation and G
0
arrest induced by 1,25-dihydroxy vitamin D
3
, whose receptor is also a member of the steroid-thyroid hormone superfamily. In this case, ST abolished the usually induced G
0
arrest and retarded, but did not block, differentiation without inducing apoptosis, thus uncoupling growth arrest and differentiation. In sum, the data show that ST disrupted the normal RA-induced program of G
0
arrest and differentiation, causing the cells to abort differentiation and undergo apoptosis.
Theiler's Murine Encephalomyelitis Virus Induces Apoptosis in Gamma Interferon-Activated M1 Differentiated Myelomonocytic Cells through a Mechanism Involving Tumor Necrosis Factor Alpha (TNF-α) and TNF-α-Related Apoptosis-Inducing Ligand
Authors: Jelachich Mary Lou , Lipton Howard L.
Published: July 2002

Infection of susceptible mice with the low-neurovirulence Theiler's murine encephalomyelitis virus strain BeAn results in an inflammatory demyelinating disease similar to multiple sclerosis. While the majority of virus antigen is detected in central nervous system macrophages (Mφs), few infiltrating Mφs are infected. We used the myelomonocytic precursor M1 cell line to study BeAn virus-Mφ interactions in vitro to elucidate mechanisms for restricted virus expression. We have shown that restricted BeAn infection of M1 cells differentiated in vitro (M1-D) results in apoptosis. In this study, BeAn infection of gamma interferon (IFN-γ)-activated M1-D cells also resulted in apoptosis but with no evidence of virus replication or protein expression. RNase protection assays of M1-D cellular RNA revealed up-regulation of Fas and the p55 chain of the tumor necrosis factor alpha (TNF-α) receptor transcripts with IFN-γ activation. BeAn infection of activated cells resulted in increased caspase 8 mRNA transcripts and the appearance of TNF-α-related apoptosis-inducing ligand (TRAIL) 4 h postinfection. Both unactivated and activated M1-D cells expressed TRAIL receptors (R1 and R2), but only activated cells were killed by soluble TRAIL. Activated cells were also susceptible to soluble FasL- and TNF-α-induced apoptosis. The data suggest that IFN-γ-activated M1-D cell death receptors become susceptible to their ligands and that the cells respond to BeAn virus infection by producing the ligands TNF-α and TRAIL to kill the susceptible cells. Unactivated cells are not susceptible to FasL or TRAIL and require virus replication to initiate apoptosis. Therefore, two mechanisms of apoptosis induction can be triggered by BeAn infection: an intrinsic pathway requiring virus replication and an extrinsic pathway signaling through the death receptors.
A Predicted Secondary Structural Domain within the Internal Ribosome Entry Site of Echovirus 12 Mediates a Cell-Type-Specific Block to Viral Replication
Authors: Bradrick Shelton S. , Lieben Elizabeth A. , Carden Bruce M. , Romero José R.
Published: July 2002

The enterovirus 5′ nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5′ (m
7
GpppN) cap-independent manner, and
cis
-acting signals for positive-strand RNA replication. For several enteroviruses, the 5′ NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5′NTR)CBV3, replicates similarly to CBV3 in human and simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37°C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5′NTR)CBV3 was isolated which possessed three transition mutations and had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5′ NTRs of ECV12 and CBV3 exhibit variable intracellular requirements for function and serve as secondary determinants of tissue or species tropism.
Host Switching in
Lyssavirus
History from the Chiroptera to the Carnivora Orders
Authors: Badrane Hassan , Tordo Noël
Published: July 2002

Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora and Chiroptera orders. We studied 36 carnivoran and 17 chiropteran lyssaviruses representing the main genotypes and variants. We compared their genes encoding the surface glycoprotein, which is responsible for receptor recognition and membrane fusion. The glycoprotein is the main protecting antigen and bears virulence determinants. Point mutation is the main force in lyssavirus evolution, as Sawyer's test and phylogenetic analysis showed no evidence of recombination. Tests of neutrality indicated a neutral model of evolution, also supported by globally high ratios of synonymous substitutions (

d
S

) to nonsynonymous substitutions (

d
N

) (>7). Relative-rate tests suggested similar rates of evolution for all lyssavirus lineages. Therefore, the absence of recombination and similar evolutionary rates make phylogeny-based conclusions reliable. Phylogenetic reconstruction strongly supported the hypothesis that host switching occurred in the history of lyssaviruses. Indeed, lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies, very likely following spillovers from bats. Using dated isolates, the average rate of evolution was estimated to be roughly 4.3 × 10
−4

d
S

/site/year. Consequently, the emergence of carnivoran rabies from chiropteran lyssaviruses was determined to have occurred 888 to 1,459 years ago. Glycoprotein segments accumulating more

d
N

than

d
S

were distinctly detected in carnivoran and chiropteran lyssaviruses. They may have contributed to the adaptation of the virus to the two distinct mammal orders. In carnivoran lyssaviruses they overlapped the main antigenic sites, II and III, whereas in chiropteran lyssaviruses they were located in regions of unknown functions.
LFA-1 Expression on Target Cells Promotes Human Immunodeficiency Virus Type 1 Infection and Transmission
Authors: Hioe Catarina E. , Chien Peter C. , Lu ChaFen , Springer Timothy A. , Wang Xiao-Hong , Bandres Juan , Tuen Michael
Published: July 2002

While CD4 and the chemokine receptors are the principal receptors for human immunodeficiency virus (HIV), other cellular proteins, such as LFA-1, are also involved in HIV infection. LFA-1 and its ligands, ICAM-1, ICAM-2, and ICAM-3, can be expressed on the cells infected by HIV, as well as on the HIV virions themselves. To examine the role of LFA-1 expressed on target cells in HIV infection, Jurkat-derived Jβ2.7 T-cell lines that express either wild-type LFA-1, a constitutively active mutant LFA-1, or no LFA-1 were used. The presence of wild-type LFA-1 enhanced the initial processes of HIV infection, as well as the subsequent replication and transmission from cell to cell. In contrast, the constitutively active LFA-1 mutant failed to promote virus replication and spread, even though this mutant could help HIV enter cells and establish the initial infection. This study clearly demonstrates the contribution of LFA-1 in the different stages of HIV infection. Moreover, not only is LFA-1 expression important for initial HIV-cell interaction, subsequent replication, and transmission, but its activity must also be properly regulated.
Positively Charged Termini of the L2 Minor Capsid Protein Are Necessary for Papillomavirus Infection
Authors: Roden Richard B. S. , Day Patricia M. , Bronzo Brian K. , Yutzy William H. , Yang Yanqin , Lowy Douglas R. , Schiller John T.
Published: July 2002

Coexpression of bovine papillomavirus L1 with L2 mutants lacking either eight N-terminal or nine C-terminal amino acids that encode positively charged domains resulted in wild-type levels of viral genome encapsidation. Despite wild-type binding to the cell surface, the resulting virions were noninfectious. An L2 mutant encoding a scrambled version of the nine C-terminal residues restored infectivity, in contrast to an L2 mutant encoding a scrambled version of the N-terminal residues.
Cross-Reactivity between Hepatitis C Virus and Influenza A Virus Determinant-Specific Cytotoxic T Cells
Authors: Wedemeyer Heiner , Mizukoshi Eishiro , Davis Anthony R. , Bennink Jack R. , Rehermann Barbara
Published: July 2002

The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8+T cells of HCV-infected patients is the HCVNS3-1073peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10−8M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.
Identification of BV/ODV-C42, an
Autographa californica
Nucleopolyhedrovirus
orf101
-Encoded Structural Protein Detected in Infected-Cell Complexes with ODV-EC27 and p78/83
Authors: Braunagel Sharon C. , Guidry Paula A. , Rosas-Acosta German , Engelking Luke , Summers Max D.
Published: July 2002

orf101
is a late gene of
Autographa californica
nucleopolyhedrovirus (Ac
M
NPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of
orf101
was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (
1629K
) and ODV-EC27 (
orf144
). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.
Partial Activation and Induction of Apoptosis in CD4+and CD8+T Lymphocytes by Conformationally Authentic Noninfectious Human Immunodeficiency Virus Type 1
Authors: Esser Mark T. , Bess Julian W. , Suryanarayana Kalachar , Chertova Elena , Marti Darlene , Carrington Mary , Arthur Larry O. , Lifson Jeffrey D.
Published: July 2002

Increased levels of apoptosis are seen in human immunodeficiency virus (HIV) infection, and this has been proposed as an important mechanism contributing to HIV pathogenesis. However, interpretation of in vitro studies aimed at understanding HIV-related apoptosis has been complicated by the use of high concentrations of recombinant proteins or by direct cytopathic effects of replicating virus. We have developed an inactivation procedure that destroys retroviral infectivity while preserving the structural and functional integrity of the HIV surface proteins. These noninfectious virions interact authentically with target cells, providing a powerful tool to dissect mechanisms of HIV pathogenesis that do or do not require viral replication. Noninfectious CXCR4-tropic HIV-1 virions, but not microvesicles, partially activated freshly isolated CD4+and CD8+peripheral blood mononuclear cell T lymphocytes to express FasL and Fas, but not CD69 or CD25 (interleukin-2 receptor alpha) and eventually die via apoptosis starting 4 to 6 days postexposure. These effects required conformationally intact virions, as heat-denatured virions or equivalent amounts of recombinant gp120 did not induce apoptosis. The maximal apoptotic effect was dependent on major histocompatibility complex (MHC) class II proteins being present on the virion, but was not MHC restricted. The results suggest that the immunopathogenesis of HIV infection may not depend solely on direct cytopathic effects of HIV replication, but that effects due to noninfectious HIV-1 virions may also contribute importantly.
Human Immunodeficiency Virus Type 1 Nef Functions at the Level of Virus Entry by Enhancing Cytoplasmic Delivery of Virions
Authors: Schaeffer Evelyne , Geleziunas Romas , Greene Warner C.
Published: July 2002

The Nef protein of the type 1 human immunodeficiency virus (HIV-1) plays a key although poorly understood role in accelerating the progression of clinical disease in vivo. Nef exerts several biological effects in vitro, including enhancement of virion infectivity, downregulation of CD4 and major histocompatibility complex class I receptor expression, and modulation of various intracellular signaling pathways. The positive effect of Nef on virion infectivity requires its expression in the producer cell, although its effect is manifested in the subsequent target cell of infection. Prior studies suggest that Nef does not alter viral entry into target cells; nevertheless, it enhances proviral DNA synthesis, arguing for an action of Nef at the level of viral uncoating or reverse transcription. However, these early studies discounting an effect of Nef on virion entry may be confounded by the recent finding that HIV enters cells by both fusion and endocytosis. Using epifluorescence microscopy to monitor green fluorescent protein-Vpr-labeled HIV virion entry into HeLa cells, we find that endocytosis forms a very active pathway for virus uptake. Virions entering via the endocytic pathway do not support productive infection of the host cell, presumably reflecting their inability to escape from the endosomes. Conversely, our studies now demonstrate that HIV Nef significantly enhances CD4- and chemokine receptor-dependent entry of HIV virions into the cytoplasmic compartment of target cells. Mutations in Nef either impairing its ability to downregulate CD4 or disrupting its polyproline helix compromise virion entry into the cytoplasm. We conclude that Nef acts at least in part as a regulator of cytosolic viral entry and that this action contributes to its positive effects on viral infectivity.
Characterization of Intracellular Reverse Transcription Complexes of Human Immunodeficiency Virus Type 1
Authors: Fassati Ariberto , Goff Stephen P.
Published: July 2002

To examine the early events of the life cycle of human immunodeficiency virus type 1 (HIV-1), we analyzed the intracellular complexes mediating reverse transcription isolated from acutely infected cells. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that two species of RTCs are formed but only one species is able to synthesize DNA. Most of the capsid, matrix, and reverse transcriptase (RT) proteins dissociate from the complex soon after cell infection, but Vpr remains associated with the RTC. The RTCs isolated 1, 4, and 7 h after infection are competent for reverse transcription in vitro, indicating that a small proportion of RT remains associated with them. HIV RTCs isolated early after infection have a sedimentation velocity of approximately 560S. Later, different species with a sedimentation velocity ranging from 350S to 100S appear. Nuclear-associated RTCs have a sedimentation velocity of 80S. Shortly after initiation of reverse transcription, the viral strong-stop DNA within the RTC is sensitive to nuclease digestion and becomes protected when reverse transcription is almost completed.
Biophysical Characterization and Vector-Specific Antagonist Activity of Domain III of the Tick-Borne Flavivirus Envelope Protein
Authors: Bhardwaj S. , Holbrook M. , Shope R. E. , Barrett A. D. T. , Watowich S. J.
Published: July 2002

The molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain III of the envelope protein has been implicated in these functions. We examined the solution properties and antagonist activity of Langat virus domain III. Our results suggest that domain III adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. Three clusters of phylogenetically conserved residues are identified that may be responsible for the vector-specific antagonist activity of domain III.
Rapid and Irreversible CD4+T-Cell Depletion Induced by the Highly Pathogenic Simian/Human Immunodeficiency Virus SHIVDH12RIs Systemic and Synchronous
Authors: Igarashi Tatsuhiko , Brown Charles R. , Byrum Russell A. , Nishimura Yoshiaki , Endo Yasuyuki , Plishka Ronald J. , Buckler Charles , Buckler-White Alicia , Miller Georgina , Hirsch Vanessa M. , Martin Malcolm A.
Published: July 2002

Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+T-lymphocyte loss.
Hepatic DR5 Induces Apoptosis and Limits Adenovirus Gene Therapy Product Expression in the Liver
Authors: Zhang Huang-Ge , Xie Jinfu , Xu Liang , Yang Pingar , Xu Xin , Sun Sheher , Wang Yongming , Curiel David T. , Hsu Hui-Chen , Mountz John D.
Published: July 2002

A major limitation of adenovirus (Ad) gene therapy product expression in the liver is subsequent elimination of the hepatocytes expressing the gene therapy product. This elimination is caused by both necrosis and apoptosis related to the innate and cell-mediated immune response to the Ad. Apoptosis of hepatocytes can be induced by the innate immune response by signaling through death domain receptors on hepatocytes including the tumor necrosis factor alpha (TNF-α) receptor (TNFR), Fas, and death domain receptors DR4 and DR5. We have previously shown that blocking signaling through TNFR enhances and prolongs gene therapy product expression in the liver. In the present study, we constructed an Ad that produces a soluble DR5-Fc (AdsDR5), which is capable of neutralizing TNF-related apoptosis-inducing ligand (TRAIL). AdsDR5 prevents TRAIL-mediated apoptosis of CD3-activated T cells and decreases hepatocyte apoptosis after AdCMVLacZ administration and enhances the level and duration oflacZtransgene expression in the liver. In addition to blocking TRAIL and directly inhibiting apoptosis, AdsDR5 decreases production of gamma interferon (IFN-γ) and TNF-α and decreases NK cell activation, all of which limit Ad-mediated transgene expression in the liver. These results indicate that (i) AdsDR5 produces a DR5-Fc capable of neutralizing TRAIL, (ii) AdsDR5 can reduce activation of NK cells and reduce induction of IFN-γ and TNF-α after Ad administration, and (iii) administration of AdsDR5 can enhance Ad gene therapy in the liver.
Identification of NF-κB-Dependent Gene Networks in Respiratory Syncytial Virus-Infected Cells
Authors: Tian Bing , Zhang Yuhong , Luxon Bruce A. , Garofalo Roberto P. , Casola Antonella , Sinha Mala , Brasier Allan R.
Published: July 2002

Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. In epithelial cells, RSV replication activates nuclear translocation of the inducible transcription factor nuclear factor κB (NF-κB) through proteolysis of its cytoplasmic inhibitor, IκB. In spite of a putative role in mediating virus-inducible gene expression, the spectrum of NF-κB-dependent genes induced by RSV infection has not yet been determined. To address this, we developed a tightly regulated cell system expressing a nondegradable, epitope-tagged IκBα isoform (Flag-IκBα Mut) whose expression could be controlled by exogenous addition of nontoxic concentrations of doxycycline. Flag-IκBα Mut expression potently inhibited IκBα proteolysis, NF-κB binding, and NF-κB-dependent gene transcription in cells stimulated with the prototypical NF-κB-activating cytokine tumor necrosis factor alpha (TNF-α) and in response to RSV infection. High-density oligonucleotide microarrays were then used to profile constitutive and RSV-induced gene expression in the absence or presence of Flag-IκBα Mut. Comparison of these profiles revealed 380 genes whose expression was significantly changed by the dominant-negative NF-κB. Of these, 236 genes were constitutive (not RSV regulated), and surprisingly, only 144 genes were RSV regulated, representing numerically ∼10% of the total population of RSV-inducible genes at this time point. Hierarchical clustering of the 144 RSV- and Flag-IκBα Mut-regulated genes identified two discrete gene clusters. The first group had high constitutive expression, and its expression levels fell in response to RSV infection. In this group, constitutive mRNA expression was increased by Flag-IκBα Mut expression, and the RSV-induced decrease in expression was partly inhibited. In the second group, constitutive expression was very low (or undetectable) and, after RSV infection, expression levels strongly increased. In this group, NF-κB was required for RSV-inducible expression because Flag-IκBα Mut expression blocked their induction by RSV. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (complement components and growth factor regulators). These data suggest that NF-κB action induces global cellular responses after viral infection.
Purification and Characterization of Oligomeric Envelope Glycoprotein from a Primary R5 Subtype B Human Immunodeficiency Virus
Authors: Srivastava Indresh K. , Stamatatos Leonidas , Legg Harold , Kan Elaine , Fong Anne , Coates Stephen R. , Leung Louisa , Wininger Mark , Donnelly John J. , Ulmer Jeffrey B. , Barnett Susan W.
Published: July 2002

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1US4. We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (Rh) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smallerRhof the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.