bluuebunny/crossref_metadata_2025_split huggingface dataset:
18S Ribosomal DNA Typing and Tracking of
Acanthamoeba
Species Isolates from Corneal Scrape Specimens, Contact Lenses, Lens Cases, and Home Water Supplies of
Acanthamoeba
Keratitis Patients in Hong Kong
Authors: Booton G. C. , Kelly D. J. , Chu Y.-W. , Seal D. V. , Houang E. , Lam D. S. C. , Byers T. J. , Fuerst P. A. 
Published: July 2002

ABSTRACT

We examined partial 18S ribosomal DNA (
Rns
) sequences of
Acanthamoeba
isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with
Acanthamoeba
. We also looked for evidence of mixed infections. Identification of
Acanthamoeba Rns
genotypes was based on sequences of ∼113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were
Rns
genotype T4 and the remaining one was
Rns
genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two
Acanthamoeba
keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this
Rns
region for tracking
Acanthamoeba
keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of
Rns
genotype T4 strains in
Acanthamoeba
keratitis infections.
Prevalence of Bacterial Vaginosis and Vaginal Flora Changes in Peri- and Postmenopausal Women
Authors: Cauci Sabina , Driussi Silvia , De Santo Davide , Penacchioni Paola , Iannicelli Teresa , Lanzafame Paolo , De Seta Francesco , Quadrifoglio Franco , de Aloysio Domenico , Guaschino Secondo 
Published: July 2002

ABSTRACT

Our aim was to evaluate the prevalence of bacterial vaginosis and decrease in lactobacillus colonization in women 40 years old or older in relation to menopausal status by evaluation of Gram-stained smears. A total of 1,486 smears from Italian Caucasian women aged 40 to 79 years were examined. Women were classified as follows: fertile (regular cycles) (
n
= 328), perimenopausal (irregular cycles) (
n
= 237), and postmenopausal (
n
= 921), including 331 women on estroprogestinic hormone replacement therapy (HRT). The prevalences of bacterial vaginosis (assessed as a Nugent score of ≥7) in fertile (9.8%) and perimenopausal (11.0%) women were not statistically different, whereas the prevalence was significantly lower overall in postmenopausal women (6.0%) (
P
= 0.02). Specifically, 6.3% of postmenopausal women without HRT and 5.4% of postmenopausal women with HRT were positive for bacterial vaginosis. The Nugent score system was not adequate for evaluating the normal and intermediate vaginal flora in women over the age of 40 years. High numbers of peri- and postmenopausal women had no lactobacilli and no bacterial-vaginosis-associated microorganisms. This nonpathological absence of lactobacilli in women with a Nugent score of 4 was scored as 4∗, and this group was considered separately from the intermediate flora group. A score of 4∗ was obtained for 2.1% of fertile women, 11.4% of perimenopausal women, 44.1% of postmenopausal women without HRT, and 6.9% of postmenopausal women with HRT. The physiological reduction in lactobacillus colonization of the vagina in postmenopausal women does not cause an increase in bacterial-vaginosis prevalence. Reversion of lactobacillus flora to premenopausal levels due to HRT does not increase the prevalence of bacterial vaginosis in postmenopausal women.
Transforming Growth Factor Beta 1 Stimulates Expression of the Epstein-Barr Virus BZLF1 Immediate-Early Gene Product ZEBRA by an Indirect Mechanism Which Requires the MAPK Kinase Pathway
Authors: Fahmi Hassan , Cochet Chantal , Hmama Zakariae , Opolon Paule , Joab Irene 
Published: July 2002

ABSTRACTDisruption of Epstein-Barr virus (EBV) latency is mediated by ZEBRA, the protein product of the immediate-early EBV gene, BZLF1. In vitro, phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), induces reactivation of EBV. However, the physiological stimuli responsible for the disruption of viral latency are not well characterized. Transforming growth factor beta 1 (TGF-β1) has also been shown to trigger the reactivation of EBV in Burkitt lymphoma cell lines; however, the effect of TGF-β1 on ZEBRA expression has not been reported. To further understand this phenomenon, we have investigated the effect of TGF-β1 on ZEBRA expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-β1 induces a time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-β1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on ZEBRA expression. A specific inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect TGF-β1-induced ZEBRA expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction. This suggests that TGF-β1 effect on BZLF1 expression requires the MAPK pathway. However, in Raji and B95-8 cells additional routes can be used, as (i) the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete, whereas these inhibitors completely abolished PMA-induced ZEBRA expression, (ii) TGF-β1 induction of ZEBRA expression occurs in PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of TGF-β1 and PMA are additive. Transient transfection of the EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct (Zp-CAT) showed that under conditions where the BZLF1 promoter is activated by PMA treatment, TGF-β1 had no significant effect on the expression of the chloramphenicol acetyltransferase gene. Furthermore, TGF-β1 induction of BZLF1 transcripts is dependent on de novo protein synthesis, which suggests that TGF-β1 induces BZLF1 expression by an indirect mechanism.
Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein
Authors: Glass Pamela J. , White Laura J. , Ball Judith M. , Leparc-Goffart Isabelle , Hardy Michele E. , Estes Mary K. 
Published: July 2002

ABSTRACTNorwalk virus(NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3′ end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3′ end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.
Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells
Authors: Dmitriev Igor , Kashentseva Elena , Rogers Buck E. , Krasnykh Victor , Curiel David T. 
Published: July 2002

ABSTRACTHuman adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.
Reovirus-Induced Apoptosis Is Mediated by TRAIL
Authors: Clarke Penny , Meintzer Suzanne M. , Gibson Spencer , Widmann Christian , Garrington Timothy P. , Johnson Gary L. , Tyler Kenneth L. 
Published: July 2002

ABSTRACTMembers of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.
Bovine Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Cooperatively Antagonize Alpha/Beta Interferon-Induced Antiviral Response
Authors: Schlender Jörg , Bossert Birgit , Buchholz Ursula , Conzelmann Karl-Klaus 
Published: July 2002

ABSTRACT
The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-α) receptor. Treatment of cells with recombinant universal IFN-α A/D or IFN-β revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-α/β mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.
Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120
Authors: Derdeyn Cynthia A. , Decker Julie M. , Sfakianos Jeffrey N. , Wu Xiaoyun , O'Brien William A. , Ratner Lee , Kappes John C. , Shaw George M. , Hunter Eric 
Published: July 2002

ABSTRACT

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a
trans
-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC
50
) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log
10
higher than the mean IC
50
for CXCR4 (X4) isolates (
P
= 0.0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC
50
for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log
10
higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC
50
for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log
10
lower than the IC
50
for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.
Relationship between Retroviral DNA Integration and Gene Expression
Authors: Weidhaas Joanne Barnes , Angelichio Elizabeth Lloyd , Fenner Sabine , Coffin John M. 
Published: July 2002

ABSTRACT
Although retroviruses can integrate their DNA into a large number of sites in the host genome, factors controlling the specificity of integration remain controversial and poorly understood. To assess the effects of transcriptional activity on integration in vivo, we created quail cell clones containing a construct with a minigene cassette, whose expression is controlled by the papilloma virus E2 protein. From these clones we derived transcriptionally active subclones expressing the wild-type E2 protein and transcriptionally silent subclones expressing a mutant E2 protein that binds its target DNA but is unable to activate transcription. By infecting both clones and subclones with avian leukosis virus and using a PCR-based assay to determine viral DNA integration patterns, we were able to assess the effects of both protein binding and transcriptional activity on retroviral DNA integration. Contrary to the hypothesis that transcriptional activity enhances integration, we found an overall decrease in integration into our gene cassette in subclones expressing the wild-type E2 protein. We also found a decrease in integration into our gene cassette in subclones expressing the mutant E2 protein, but only into the protein binding region. Based on these findings, we propose that transcriptionally active DNA is not a preferred target for retroviral integration and that transcriptional activity may in fact be correlated with a decrease in integration.
Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid: Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection
Authors: Wobus Christiane E. , Hügle-Dörr Barbara , Girod Anne , Petersen Gabriele , Hallek Michael , Kleinschmidt Jürgen A. 
Published: July 2002

ABSTRACT
The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341–1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.
pRB-Dependent, J Domain-Independent Function of Simian Virus 40 Large T Antigen in Override of p53 Growth Suppression
Authors: Gjoerup Ole , Chao Herta , DeCaprio James A. , Roberts Thomas M. 
Published: July 2002

ABSTRACT
Simian virus 40 (SV40) large T antigen (LT) can immortalize and transform many cell types. These activities are attributed in large part to the binding and functional inactivation by LT of two major tumor suppressors: p53 and the retinoblastoma protein, pRB. Most effects of LT on pRB have been shown to additionally require an intact J domain, which mediates binding to Hsc70. We show here that the J domain is not required for p53 override in full-length LT. Although LT binds p53, it was shown previously that overcoming a p53-induced cell cycle arrest requires binding to pRB family members (R. S. Quartin et al., J. Virol. 68:1334–1341). We demonstrate that an LT mutant defective for pRB family member binding (K1) can be complemented for efficient override of p53 arrest by a construct encoding the first 135 amino acids of LT with a J domain-inactivating mutation, H42Q. Hence, complementation does not require the J domain, and pRB binding by LT is important for more than dissociating pRB-E2F complexes, which is J dependent. In accordance with this notion, LT alleviates pRB small-pocket-mediated transcriptional repression independently of the J domain. The LT K1 mutant can also be complemented for p53 override by small t antigen (st) in a manner independent of its J domain. Our observations underscore the importance of multiple SV40 functions, two in LT and one in st, that act cooperatively to counteract p53 growth suppression.
The Leader Polypeptide of Theiler's Murine Encephalomyelitis Virus Is Required for the Assembly of Virions in Mouse L Cells
Authors: Badshah Cyrus , Calenoff Miriam A. , Rundell Kathleen 
Published: July 2002

ABSTRACT

Deletion of the entire leader polypeptide of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) results in the production of an attenuated virus that grows in baby hamster kidney (BHK) cells but cannot grow at all in mouse L-929 cells. This study examined the reasons for the failure of
dl
-L, the GDVII variant that lacks the leader polypeptide, to grow in mouse cells. At low multiplicities of infection, it was difficult to detect any viral proteins in mouse cells. However, levels of positive- and negative-strand RNA molecules were only moderately reduced in these infections. Viral RNA showed no major defect in translatability, as the mutant viral RNA was nearly as efficient as that of the wild-type (WT) virus in directing protein synthesis in vitro in assays using extracts prepared from mouse L cells. Viral protein synthesis was detected in
dl
-L-infected mouse cells as multiplicities of infection were increased and approached the levels observed in WT infections. Despite this, there was a total lack of virus production in high-multiplicity infections, and this was found to correlate with the failure of viral proteins and early virion precursors to assemble into virions in mouse cells. Thus, the inability of
dl
-L to grow in mouse cells reflects complex effects on various stages of the virus infection but is primarily a defect in virus assembly.