bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Copies of the 16S rRNA Gene in
Nocardia nova
Isolates and Implications for Sequence-Based Identification Procedures
Authors: Conville Patricia S. , Witebsky Frank G.
Published: June 2005

Molecular investigation of two
Nocardia
patient isolates showed unusual restriction fragment length polymorphism patterns with restriction endonuclease assays (REA) using an amplified portion of the 16S rRNA gene. Patterns typical of
Nocardia nova
were obtained with REA of an amplified portion of the 65-kDa heat shock protein gene. Subsequent sequence analysis of the 16S rRNA gene regions of these isolates showed the presence of ambiguous bases within an expected restriction endonuclease recognition site which were not able to be resolved on repeat testing. Cloning of amplified regions of the 16S rRNA genes and subsequent sequencing of the resulting clones from the two patient isolates showed three different 16S rRNA gene sequences which corresponded to sequences found in
N. nova
, a molecular variant of
N. nova
, and a previously undescribed sequence. Hybridization studies using a DNA probe corresponding to an 89-bp conserved region of the 16S rRNA gene confirmed the presence of at least two copies of the 16S rRNA gene in the
N. nova
type strain, in a patient isolate identical to the molecular variant of
N. nova
, and in the two other patient isolates. All isolates were found to belong to the species
N. nova
as determined by DNA-DNA hybridization. Because minimal variation has been found in the 16S rRNA gene sequences of different species of
Nocardia
, those laboratories employing molecular methods for identification of these species must be aware of the potential identification complications that may be caused by the presence of differing 16S rRNA genes in the same isolate.
Rapid Detection and Differentiation of Dengue Virus Serotypes by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay
Authors: Parida Manmohan , Horioke Kouhei , Ishida Hiroyuki , Dash Paban Kumar , Saxena Parag , Jana Asha Mukul , Islam Mohammed Alimul , Inoue Shingo , Hosaka Norimitsu , Morita Kouichi
Published: June 2005

The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3′ noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the
Flavivirus
family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.
Enteropathogenic
Escherichia coli
Strains Carrying Genes Encoding the PER-2 and TEM-116 Extended-Spectrum β-Lactamases Isolated from Children with Diarrhea in Uruguay
Authors: Vignoli Rafael , Varela Gustavo , Mota María Inés , Cordeiro Nicolas F. , Power Pablo , Ingold Elizabet , Gadea Pilar , Sirok Alfredo , Schelotto Felipe , Ayala Juan A. , Gutkind Gabriel
Published: June 2005

We studied 13 extended-spectrum β-lactamase (ESBL)-producing enteropathogenic
Escherichia coli
isolates from children suffering acute diarrhea in Uruguay. ESBL characterization in crude extracts showed a single band at pI 5.4. PCR amplification and sequencing data allowed identification of
bla
PER-2
and
bla
TEM-116
. Retrospective analysis suggests that these strains were disseminated in the community, even if unnoticed, prior to their access to the hospital environment more than a decade ago.
Multiplex PCR for Direct Detection of Shiga Toxigenic
Escherichia coli
Strains Producing the Novel Subtilase Cytotoxin
Authors: Paton Adrienne W. , Paton James C.
Published: June 2005

We have recently described a novel AB
5
subtilase cytotoxin produced by certain Shiga toxigenic
Escherichia coli
(STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. In this study we have developed a trivalent PCR assay for the detection of the novel toxin A subunit gene
subA
, as well as
stx
1
and
stx
2
. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively. The assay can be used for determining the toxin genotype of STEC isolates, as well as for direct detection of toxin genes in primary fecal culture extracts.
Gene Mutations Responsible for Overexpression of AmpC β-Lactamase in Some Clinical Isolates ofEnterobacter cloacae
Authors: Kaneko Kenichi , Okamoto Ryoichi , Nakano Ryuichi , Kawakami Sayoko , Inoue Matsuhisa
Published: June 2005

AmpC regulatory genes in 21 ceftazidime-resistant clinical isolates ofEnterobacter cloacae(MICs of ≥16 μg/ml) were characterized. All isolates exhibited AmpC overproduction due to AmpD mutation. Additionally, we found two AmpR mutants among the isolates. This is the first report of chromosomalampRmutation in clinical isolates ofE. cloacae.
Quinolone-Resistant Uropathogenic
Escherichia coli
Strains from Phylogenetic Group B2 Have Fewer Virulence Factors than Their Susceptible Counterparts
Authors: Horcajada Juan P. , Soto Sara , Gajewski Abby , Smithson Alex , Jiménez de Anta M. Teresa , Mensa Josep , Vila Jordi , Johnson James R.
Published: June 2005

The prevalence of 31 virulence factors was analyzed among nalidixic acid-susceptible and -resistant
Escherichia coli
strains from phylogenetic group B2. Hemolysin, cytotoxic necrotizing factor 1, and S and F1C fimbriae genes were less prevalent among nalidixic acid-resistant
E. coli
strains. Quinolone resistance may be associated with a decrease in the presence of some virulence factors.
Occurrence of
hlyA
and
sheA
Genes in Extraintestinal
Escherichia coli
Strains
Authors: Kerényi Monika , Allison Heather E. , Bátai István , Sonnevend Ágnes , Emödy Levente , Plaveczky Nóra , Pál Tibor
Published: June 2005

The association of a hemolytic phenotype with the carriage of the α-hemolysin gene (
hlyA
) and/or the silent hemolysin gene (
sheA
or
clyA
) among 540 extraintestinal clinical isolates of
Escherichia coli
and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal
E. coli
infection, the role of SheA is not completely clarified. Two hemolytic
sheA
+
E. coli
strains that lacked
hlyA
and possessed no other hemolysin genes were identified. No
hlyA
+
sheA
+
strains were identified, suggesting that there is possible incompatibility between
hlyA
and
sheA
in the chromosome of
E. coli
.
Demonstration of a High Level of Parasite Population Homology by Quantification of
Plasmodium falciparum
Alleles in Matched Peripheral, Placental, and Umbilical Cord Blood Samples
Authors: Jafari-Guemouri Sayeh , Ndam Nicaise Tuikue , Bertin Gwladys , Renart Emily , Sow Sokhna , Le Hesran Jean-Yves , Deloron Philippe
Published: June 2005

Plasmodium falciparum msp-1
and
msp-2
genes were quantified by fragment analysis in matched placental, peripheral, and cord blood samples. In the three compartments, the multiplicity of infection values were similar, and parasite populations only partially overlapped, as reported. However, identical alleles represented 80 to 95% of the overall parasite populations of each compartment, demonstrating much more homogenous parasite populations than previously thought.
Cryptococcal Antigen Test Revisited: Significance for Cryptococcal Meningitis Therapy Monitoring in a Tertiary Chinese Hospital
Authors: Lu Hongzhou , Zhou Yingjie , Yin Youkuan , Pan Xiaozhang , Weng Xinhua
Published: June 2005

For a total of 29 non-human immunodeficiency virus 1 cryptococcal meningitis cases, titer changes in the latex agglutination test before and after therapy were reviewed along with clinical manifestations, laboratory findings, and therapy regimens. The cryptococcal antigen titer decreased for every case after therapy and was correlated to fungal clearance as defined by fungus smear and/or culture. However, cryptococcal antigen can remain at low titers for long periods of time after therapy, even when fungus smears and/or cultures become negative.
Extraction of
Mycobacterium tuberculosis
DNA: a Question of Containment
Authors: Somerville Wendy , Thibert Louise , Schwartzman Kevin , Behr Marcel A.
Published: June 2005

DNA fingerprinting of
Mycobacterium tuberculosis
by IS
6110
restriction fragment length polymorphism analysis requires substantial high-quality DNA. We demonstrated that, despite extraction treatments that might be expected to inactivate this organism,
M. tuberculosis
remained viable during this process. These data suggest that the extraction of
M. tuberculosis
DNA should be performed within containment until complete.
Inability To Make a Premortem Diagnosis ofAcanthamoebaSpecies Infection in a Patient with Fatal Granulomatous Amebic Encephalitis
Authors: Bloch Karen C. , Schuster Frederick L.
Published: June 2005

Granulomatous amebic encephalitis (GAE), an infection of immunocompromised hosts, is almost uniformly fatal. A case of GAE in a patient who failed to mount a serologic response toAcanthamoeba polyphagais presented. AlthoughAcanthamoeba polyphagathat is sensitive to multiple antimicrobials grew from brain tissue, an inability to make a premortem diagnosis precluded therapy.
Infection by
Chlamydophilia avium
in an Elderly Couple Working in a Pet Shop
Authors: Saito Takashi , Ohnishi Jitsuko , Mori Yoshihiro , Iinuma Yoshitsugu , Ichiyama Satoshi , Kohi Fumikazu
Published: June 2005

Psittacosis infection is usually reported in adults aged around 30 to 60 years. We report here two cases of psittacosis in an elderly couple (76 and 77 years old) who jointly ran a pet shop. Psittacosis was diagnosed from a history of exposure to birds and from serological testing for
Chlamydophilia avium
.
First Reported Case of Endocarditis Caused by
Candida dubliniensis
Authors: Carr Michael J. , Clarke Susan , O'Connell Finbar , Sullivan Derek J. , Coleman David C. , O'Connell Brian
Published: June 2005

Candida dubliniensis
is an uncommon cause of bloodstream infection. We describe the first reported case of endocarditis caused by
C. dubliniensis
and the use of a rapid and novel real-time PCR assay based on the internal transcribed spacer 2 variable region of the rRNA operon that was used to identify this organism.
Junctional Adhesion Molecule A Serves as a Receptor for Prototype and Field-Isolate Strains of Mammalian Reovirus
Authors: Campbell Jacquelyn A. , Schelling Pierre , Wetzel J. Denise , Johnson Elizabeth M. , Forrest J. Craig , Wilson Greame A. R. , Aurrand-Lions Michel , Imhof Beat A. , Stehle Thilo , Dermody Terence S.
Published: June 2005

Reovirus infections are initiated by the binding of viral attachment protein σ1 to receptors on the surface of host cells. The σ1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 σ1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the σ1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced σ1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of σ1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the σ1 head domain.
Enhanced Breadth of CD4 T-Cell Immunity by DNA Prime and Adenovirus Boost Immunization to Human Immunodeficiency Virus Env and Gag Immunogens
Authors: Wu Lan , Kong Wing-pui , Nabel Gary J.
Published: June 2005

A variety of gene-based vaccination approaches have been used to enhance the immune response to viral pathogens. Among them, the ability to perform heterologous immunization by priming with DNA and boosting with replication-defective adenoviral (ADV) vectors encoding foreign antigens has proven particularly effective in eliciting enhanced cellular and humoral immunity compared to either agent alone. Because adenoviral vector immunization alone can elicit substantial cellular and humoral immune responses in a shorter period of time, we asked whether the immune response induced by the prime-boost immunization was different from adenoviral vaccines with respect to the potency and breadth of T-cell recognition. While DNA/ADV immunization stimulated the CD8 response, it was directed to the same epitopes in Gag and Env immunogens of human immunodeficiency virus as DNA or ADV alone. In contrast, the CD4 response to these immunogens diversified after DNA/ADV immunization compared to each vector alone. These findings suggest that the diversity of the CD4 immune response is increased by DNA/ADV prime-boost vaccination and that these components work synergistically to enhance T-cell epitope recognition.
Investigation by Atomic Force Microscopy of the Structure of Ty3 Retrotransposon Particles
Authors: Kuznetsov Yurii G. , Zhang Min , Menees Thomas M. , McPherson Alexander , Sandmeyer Suzanne
Published: June 2005

Ty3, a member of the
Metaviridiae
family of long-terminal-repeat retrotransposons found in
Saccharomyces cerevisiae
, encodes homologs of retroviral Gag and Gag-Pol proteins, which, together with genomic RNA, assemble into virus-like particles (VLPs) that undergo processing and reverse transcription. The Ty3 structural proteins, capsid and nucleocapsid, contain major homology and nucleocapsid motifs similar to retrovirus capsid and nucleocapsid proteins, but Ty3 lacks a matrix-like structural domain amino terminal to capsid. Mass spectrometry analysis of Ty3 Gag3 processing products defined an acetylated Ser residue as the amino terminus of Gag3/p34, p27, and CA/p24 species and supported a model where p34 and p27 occur in phosphorylated forms. Using atomic force microscopy, VLPs were imaged from cells producing wild-type and protease and reverse transcriptase mutant Ty3. Wild-type VLPs were found to have a broad range of diameters, but the majority, if not all of the particles, exhibited arrangements of capsomeres on their surfaces which were consistent with icosahedral symmetry. Wild-type particles were in the range of 25 to 52 nm in diameter, with particles in the 42- to 52-nm diameter range consistent with T=7 symmetry. Both classes of mutant VLPs fell into a narrower range of 44 to 53 nm in diameter and appeared to be consistent with T=7 icosahedral symmetry. The smaller particles in the wild-type population likely correspond to VLPs that have progressed to reverse transcription or later stages, which do not occur in the protease and reverse transcriptase mutants. Ty3 VLPs did not undergo major external rearrangements during proteolytic maturation.
Effects of a Temperature Sensitivity Mutation in the J1R Protein Component of a Complex Required for Vaccinia Virus Assembly
Authors: Chiu Wen-Ling , Szajner Patricia , Moss Bernard , Chang Wen
Published: June 2005

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol.
76:
9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus C
ts
45. The mutant C
ts
45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39°C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with C
ts
45 at 39°C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.
Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication
Authors: Casais Rosa , Davies Marc , Cavanagh David , Britton Paul
Published: June 2005

The avian coronavirus
Infectious bronchitis virus
(IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins.
Effect of CD8
+
Lymphocyte Depletion on Virus Containment after Simian Immunodeficiency Virus SIVmac251 Challenge of Live Attenuated SIVmac239Δ3-Vaccinated Rhesus Macaques
Authors: Schmitz Jörn E. , Johnson R. Paul , McClure Harold M. , Manson Kelledy H. , Wyand Michael S. , Kuroda Marcelo J. , Lifton Michelle A. , Khunkhun Rajinder S. , McEvers Kimberly J. , Gillis Jacqueline , Piatak Michael , Lifson Jeffrey D. , Grosschupff Gudrun , Racz Paul , Tenner-Racz Klara , Rieber E. Peter , Kuus-Reichel Kristine , Gelman Rebecca S. , Letvin Norman L. , Montefiori David C. , Ruprecht Ruth M. , Desrosiers Ronald C. , Reimann Keith A.
Published: June 2005

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8
+
lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Δ3 of CD8
+
lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8
+
lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8
+
cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8
+
T-cell responses and lower neutralizing antibody titers than those in
Mamu-A*01
−
animals. Consistent with these findings, the depletion of CD8
+
lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in
Mamu-A*01
+
than in
Mamu-A*01
−
animals. The partial control of postchallenge viremia after CD8
+
lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.
Limited Sequence Evolution within Persistently Targeted CD8 Epitopes in Chronic Human Immunodeficiency Virus Type 1 Infection
Authors: Koibuchi Tomohiko , Allen Todd M. , Lichterfeld Mathias , Mui Stanley K. , O'Sullivan Kristin M. , Trocha Alicja , Kalams Spyros A. , Johnson R. Paul , Walker Bruce D.
Published: June 2005

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-γ) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-γ responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.
New DNA Viruses Identified in Patients with Acute Viral Infection Syndrome
Authors: Jones Morris S. , Kapoor Amit , Lukashov Vladimir V. , Simmonds Peter , Hecht Frederick , Delwart Eric
Published: June 2005

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.
Paracrine Inhibition of Prion Propagation by Anti-PrP Single-Chain Fv Miniantibodies
Authors: Donofrio Gaetano , Heppner Frank L. , Polymenidou Magdalini , Musahl Christine , Aguzzi Adriano
Published: June 2005

Prion diseases are characterized by the deposition of PrP
Sc
, an abnormal form of the cellular prion protein PrP
C
. A growing body of evidence suggests that antibodies to PrP
C
can antagonize deposition of PrP
Sc
. However, host tolerance hampers the induction of immune responses to PrP
C
, and cross-linking of PrP
C
by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-
myc
and His
6
tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP
Sc
, even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.
Envelope Protein Glycosylation Status Influences Mouse Neuroinvasion Phenotype of Genetic Lineage 1 West Nile Virus Strains
Authors: Beasley David W. C. , Whiteman Melissa C. , Zhang Shuliu , Huang Claire Y.-H. , Schneider Bradley S. , Smith Darci R. , Gromowski Gregory D. , Higgs Stephen , Kinney Richard M. , Barrett Alan D. T.
Published: June 2005

The introduction ofWest Nile virus(WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.
Flavivirus Activates Phosphatidylinositol 3-Kinase Signaling To Block Caspase-Dependent Apoptotic Cell Death at the Early Stage of Virus Infection
Authors: Lee Chyan-Jang , Liao Ching-Len , Lin Yi-Ling
Published: June 2005

Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an antiapoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.
Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Glycoproteins
Authors: Goffard Anne , Callens Nathalie , Bartosch Birke , Wychowski Czeslaw , Cosset François-Loïc , Montpellier Claire , Dubuisson Jean
Published: June 2005

Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.
Virus Isolates during Acute and Chronic Human Immunodeficiency Virus Type 1 Infection Show Distinct Patterns of Sensitivity to Entry Inhibitors
Authors: Rusert Peter , Kuster Herbert , Joos Beda , Misselwitz Benjamin , Gujer Cornelia , Leemann Christine , Fischer Marek , Stiegler Gabriela , Katinger Hermann , Olson William C. , Weber Rainer , Aceto Leonardo , Günthard Huldrych F. , Trkola Alexandra
Published: June 2005

We studied the effect of entry inhibitors on 58 virus isolates derived during acute and chronic infection to validate these inhibitors in vitro and to probe whether viruses at early and chronic disease stages exhibit general differences in the interaction with entry receptors. We included members of all types of inhibitors currently identified: (i) agents that block gp120 binding to CD4 (CD4-IgG
2
and monoclonal antibody [MAb] IgG
1
b12), (ii) compounds that block the interaction with CCR5 (the chemokine RANTES/CCL5, the small-molecule inhibitor AD101, and the anti-CCR5 antibody PRO 140), (iii) the fusion inhibitor enfuvirtide (T-20), and (iv) neutralizing antibodies directed against gp120 (MAb 2G12) and gp41 (MAbs 2F5 and 4E10). No differences between viruses from acute and chronic infections in the susceptibility to inhibitors targeting the CD4 binding site, CCR5, or fusion or to MAb 2G12 were apparent, rendering treatment with entry inhibitors feasible across disease stages. The notable exceptions were antibodies 2F5 and 4E10, which were more potent in inhibiting viruses from acute infection (
P
= 0.0088 and 0.0005, respectively), although epitopes of these MAbs were equally well preserved in both groups. Activities of these MAbs correlated significantly with each other, suggesting that common features of the viral envelope modulate their potencies.
Analysis of Foot-and-Mouth Disease Virus Internalization Events in Cultured Cells
Authors: O'Donnell Vivian , LaRocco Michael , Duque Hernando , Baxt Barry
Published: June 2005

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the αVsubgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the βG-βH loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the αVβ3and αVβ6integrins, virus adsorbed to the cells at 4°C appears to colocalize almost exclusively with the αVβ6integrin. Upon shifting the infected cells to 37°C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.
Early Events in Integrin αvβ6-Mediated Cell Entry of Foot-and-Mouth Disease Virus
Authors: Berryman Stephen , Clark Stuart , Monaghan Paul , Jackson Terry
Published: June 2005

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin αvβ6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of αvβ6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with αvβ6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37°C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin αvβ6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.
Histidine at Residue 99 and the Transmembrane Region of the Precursor Membrane prM Protein Are Important for the prM-E Heterodimeric Complex Formation of Japanese Encephalitis Virus
Authors: Lin Ying-Ju , Wu Suh-Chin
Published: June 2005

The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in
trans
in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of carboxyl-terminally truncated prM mutant baculoviruses was constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex by approximately 40% for the TM1 (at residues 130 to 147 [prM130-147]) truncation and 20% for TM2 (at prM153-167) truncation. Alanine-scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM binding element for stable prM-E heterodimeric complex formation. The single amino acid mutation at the His99 residue of prM abolishing the prM-E interaction was not due to reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse-chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus coexpression of prM and E proteins but not with the prM H99A mutant. Sequence alignment analysis was further conducted with different groups of flaviviruses to show that the prM H99 residues are generally conserved. Our findings are the first report to characterize the minimum binding elements of the prM protein that are involved in prM-E interactions of flaviviruses. This information, concerning a molecular framework for the prM protein, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.
Perforin and Fas Cytolytic Pathways Coordinately Shape the Selection and Diversity of CD8+-T-Cell Escape Variants of Influenza Virus
Authors: Price Graeme E. , Huang Lei , Ou Rong , Zhang Menghua , Moskophidis Demetrius
Published: June 2005

Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.
Real-Time Detection of Virus Particles and Viral Protein Expression with Two-Color Nanoparticle Probes
Authors: Agrawal Amit , Tripp Ralph A. , Anderson Larry J. , Nie Shuming
Published: June 2005

Respiratory syncytial virus (RSV) mediates serious lower respiratory tract illness in infants and young children and is a significant pathogen of the elderly and immune compromised. Rapid and sensitive RSV diagnosis is important to infection control and efforts to develop antiviral drugs. Current RSV detection methods are limited by sensitivity and/or time required for detection. In this study, we show that antibody-conjugated nanoparticles rapidly and sensitively detect RSV and estimate relative levels of surface protein expression. A major development is use of dual-color quantum dots or fluorescence energy transfer nanobeads that can be simultaneously excited with a single light source.
Detection of Human Immunodeficiency Virus Type 1 Nef and CD4 Physical Interaction in Living Human Cells by Using Bioluminescence Resonance Energy Transfer
Authors: Cluet David , Bertsch Christophe , Beyer Christian , Gloeckler Liliane , Erhardt Mathieu , Gut Jean-Pierre , Galzi Jean-Luc , Aubertin Anne-Marie
Published: June 2005

CD4 down-regulation by human immunodeficiency virus type 1 (HIV-1) Nef protein is a key function for virus virulence. This activity may be mediated by a direct Nef-CD4 interaction. We investigated the formation, in situ, of such a complex between proteins using bioluminescence resonance energy transfer technology and coimmunoprecipitations. Our data clearly demonstrate that Nef and CD4 interact in intact human cells. Moreover, our results clearly indicate that the dileucine motif of the CD4 cytoplasmic domain, critical for the Nef-induced CD4 down-regulation, is not implicated in the Nef/CD4 complex formation in the cellular context.
Whole-Genome Transcription Profiling of Rhesus Monkey Rhadinovirus
Authors: Dittmer Dirk P. , Gonzalez Carlos M. , Vahrson Wolfgang , DeWire Scott M. , Hines-Boykin Rebecca , Damania Blossom
Published: June 2005

Rhesus monkey rhadinovirus (RRV) and Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) belong to the gamma-2 grouping of herpesviruses. RRV and KSHV share a high degree of sequence similarity, and their genomes are organized in a similar fashion. RRV serves as an excellent animal model system to study the gamma herpesvirus life cycle both in vitro and in vivo. We have developed a high-sensitivity, high-throughput, high-specificity real-time quantitative reverse transcriptase-based PCR assay for RRV and have used this assay to profile transcription from the whole RRV genome during de novo productive infection of rhesus fibroblasts. Using this assay, we demonstrate that the genome-wide transcription profile for RRV closely parallels the genome-wide transcription profile for KSHV.
Epstein-Barr Virus (EBV) Latent Membrane Protein 2A Regulates B-Cell Receptor-Induced Apoptosis and EBV Reactivation through Tyrosine Phosphorylation
Authors: Fukuda Makoto , Longnecker Richard
Published: June 2005

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection of B cells. Within the immune system, apoptosis is a central mechanism in normal lymphocyte homeostasis both during early lymphocyte development and in response to antigenic stimuli. In this study, we found that latent membrane protein 2A (LMP2A) inhibited B-cell receptor (BCR)-induced apoptosis in Burkitt's lymphoma cell lines. Genistein, a specific inhibitor of tyrosine-specific protein kinases, blocked BCR-induced apoptosis and EBV reactivation in the cells. These findings indicate that LMP2A blocks BCR-induced cell apoptosis and EBV reactivation through the inhibition of activation of tyrosine kinases by BCR cross-linking.