bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation and Validation of a PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping
Vibrio parahaemolyticus
: an International Multicenter Collaborative Study
Authors: Kam Kai Man , Luey Cindy K. Y. , Parsons Michele B. , Cooper Kara L. F. , Nair G. B. , Alam M. , Islam M. Atiqul , Cheung Danny T. L. , Chu Y. W. , Ramamurthy T. , Pazhani G. P. , Bhattacharya S. K. , Watanabe H. , Terajima J. , Arakawa E. , Ratchtrachenchai O.-A. , Huttayananont S. , Ribot Efrain M. , Gerner-Smidt Peter , Swaminathan Bala
Published: June 2008

The pandemic spread of
Vibrio parahaemolyticus
is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for
V. parahaemolyticus
that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized
V. parahaemolyticus
pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized
V. parahaemolyticus
isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic
V. parahaemolyticus
strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet
V. parahaemolyticus
protocol was found to be both robust and reproducible among the collaborating laboratories.
Multicenter Evaluation of the New Vitek 2
Neisseria
-
Haemophilus
Identification Card
Authors: Rennie Robert P. , Brosnikoff Cheryl , Shokoples Sandy , Reller L. Barth , Mirrett Stanley , Janda William , Ristow Kathy , Krilcich Ann
Published: June 2008

The new
Neisseria-Haemophilus
identification (NH) card for Vitek 2 was compared with 16S rRNA gene sequencing (16S) as the reference method for accurate identification of
Neisseria
spp.,
Haemophilus
spp., and other fastidious gram-negative bacteria. Testing was performed on the Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains tested 20 times over a minimum of 10 days at all three sites. A challenge set of 30 strains with known identifications and 371 recent fresh and frozen clinical isolates were also tested. Expected positive and negative biochemical reactions were also evaluated for substrate reproducibility. All microorganisms were tested on the NH card, and all clinical and stock isolates were saved for 16S testing. All reproducibility tests yielded expected results within a 95% confidence interval. For challenge microorganisms, there was 98% overall correct identification, including 8% low discrimination, 2% incorrect identification, and 0% unidentified. For clinical strains, there was 96.5% overall correct identification, including 10.2% low discrimination, 2.7% incorrect identification, and 0.8% unidentified. The 2.7% (10/371) of clinical isolates that gave an incorrect identification consisted of 7 isolates correct to genus and 3 strains incorrect to genus. There were an additional 27 strains (primarily
Neisseria
species) for which the 16S identification result was different from the NH card result. These were all unclaimed species by the system. The new NH card met all performance criteria within a 95% confidence interval compared to identification of clinical isolates by 16S.
Correlation of MIC with Outcome for
Candida
Species Tested against Caspofungin, Anidulafungin, and Micafungin: Analysis and Proposal for Interpretive MIC Breakpoints
Authors: Pfaller M. A. , Diekema D. J. , Ostrosky-Zeichner L. , Rex J. H. , Alexander B. D. , Andes D. , Brown S. D. , Chaturvedi V. , Ghannoum M. A. , Knapp C. C. , Sheehan D. J. , Walsh T. J.
Published: June 2008

The CLSI Antifungal Subcommittee followed the M23-A2 “blueprint” to develop interpretive MIC breakpoints for anidulafungin, caspofungin, and micafungin against
Candida
species. MICs of ≤2 μg/ml for all three echinocandins encompass 98.8 to 100% of all clinical isolates of
Candida
spp. without bisecting any species group and represent a concentration that is easily maintained throughout the dosing period. Data from phase III clinical trials demonstrate that the standard dosing regimens for each of these agents may be used to treat infections due to
Candida
spp. for which MICs are as high as 2 μg/ml. An MIC predictive of resistance to these agents cannot be defined based on the data from clinical trials due to the paucity of isolates for which MICs exceed 2 μg/ml. The clinical data set included only three isolates from patients treated with an echinocandin (caspofungin) for which the MICs were >2 μg/ml (two
C. parapsilosis
isolates at 4 μg/ml and one
C. rugosa
isolate at 8 μg/ml). Based on these data, the CLSI subcommittee has decided to recommend a “susceptible only” breakpoint MIC of ≤2 μg/ml due to the lack of echinocandin resistance in the population of
Candida
isolates thus far. Isolates for which MICs exceed 2 μg/ml should be designated “nonsusceptible” (NS). For strains yielding results suggestive of an NS category, the organism identification and antimicrobial-susceptibility test results should be confirmed. Subsequently, the isolates should be submitted to a reference laboratory that will confirm the results by using a CLSI reference dilution method.
European Emergence of Ciprofloxacin-Resistant
Escherichia coli
Clonal Groups O25:H4-ST 131 and O15:K52:H1 Causing Community-Acquired Uncomplicated Cystitis
Authors: Cagnacci Simone , Gualco Laura , Debbia Eugenio , Schito Gian Carlo , Marchese Anna
Published: June 2008

A total of 148
E. coli
strains displaying reduced susceptibility to ciprofloxacin (MIC ≥ 2 μg/ml) and causing uncomplicated urinary tract infections in eight European countries during 2003 to 2006 were studied. Their phylogenetic groups, biochemical profiles, and antibiotic susceptibilities were determined. Determination of the O:H serotype, pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) PCR, and multilocus sequence typing provided additional discrimination. The majority (82.4%) of the microorganisms (122/148) carried resistance to two or more additional drugs, with the pattern ciprofloxacin-trimethoprim-sufamethoxazole-tetracycline-ampicillin being the most represented (73 strains out of 148; 49.3%). Extended-spectrum beta-lactamase production was detected in 12/148 strains (8.1%), with CTX-M-15 being the most-common enzyme. Six strains out of the whole collection studied (4.0%) contained a
qnrB
-like gene. Overall, 55 different PFGE or RAPD PCR profiles could be distinguished, indicating a substantial heterogeneity. However, about one-third (51/148) of the strains belonged to two clonal groups: O15:K52:H1 (phylogenetic group B2, lactose-nonfermenting variant, ciprofloxacin MIC of 16 μg/ml) and O25:H4 sequence type 131 (ST-131) (phylogenetic group D, ciprofloxacin MIC of ≥32 μg/ml). With the exception of Poland, strains of these two groups were isolated in samples from all participating countries but more frequently in samples from Spain and Italy. In some representative strains of the two main clonal groups, alterations in GyrA and ParC were the basic mechanism of fluoroquinolone resistance. In some members of the O25:H4 ST-131 group, displaying a ciprofloxacin MIC of >32 μg/ml, additional OmpF loss or pump efflux overexpression was found. In the Mediterranean area, strains belonging to these two clonal groups played a major role in determining the high rate of fluoroquinolone-resistant
E. coli
strains observed in the community.
Single-Step PCR Using (GACA)
4
Primer: Utility for Rapid Identification of Dermatophyte Species and Strains
Authors: Shehata Atef S. , Mukherjee Pranab K. , Aboulatta Hassan N. , El Akhras Atef I. , Abbadi Said H. , Ghannoum Mahmoud A.
Published: June 2008

Dermatophytes are fungi that belong to three genera:
Epidermophyton, Microsporum
, and
Trichophyton
. Identification of dermatophyte species is essential for appropriate diagnosis and treatment of dermatophytosis. Routine identification depends on macroscopic and microscopic morphology, which is time-consuming and does not identify dermatophyte strains. In this study, two PCR-based methods were compared for their abilities to identify 21 dermatophyte isolates obtained from Egyptian patients to the species and strain levels. The first method employed a two-step method: PCR amplification, using ITS1 and ITS4 as primers, followed by restriction enzyme digestion using the endonuclease MvaI. The second method employed a one-step approach employing the repetitive oligonucleotide (GACA)
4
as a primer. Dermatophyte strains were also identified using a conventional culture method. Our results showed that the conventional culture method identified four species:
Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum
, and
Trichophyton violaceum
. Moreover, both PCR methods agreed with the diagnosis made using the conventional approach. Furthermore, ITS1/ITS4-based PCR provided no strain differentiation, while (GACA)
4
-based PCR identified different varieties among the
T. mentagrophytes
isolates. Taken together, our results suggest that (GACA)
4
-based PCR has utility as a simple and rapid method for identification of dermatophyte species as well as utility for differentiation of
T. mentagrophytes
variants.
Human Papillomavirus Genotype Specificity of Hybrid Capture 2
Authors: Castle Philip E. , Solomon Diane , Wheeler Cosette M. , Gravitt Patti E. , Wacholder Sholom , Schiffman Mark
Published: June 2008

Hybrid Capture 2 (hc2), a clinical test for carcinogenic human papillomavirus (HPV) DNA, has proven to be a sensitive but only modestly specific predictor of cervical precancer and cancer risk. Some of its nonspecificity for clinical end points can be ascribed to cross-reactivity with noncarcinogenic HPV genotypes. However, the reference genotyping tests that have been used for these comparisons are also imperfect. We therefore sought to describe further the HPV genotype specificity of hc2 by comparing the hc2 results to paired results from two related PGMY09/11 L1 primer-based HPV genotyping assays: Linear Array (LA) and its prototype predecessor, the line blot assay (LBA). LA and LBA results were considered separately and combined (detection by either assay or both assays) for 37 individual HPV genotypes and HPV risk group categories (carcinogenic HPV > noncarcinogenic HPV > negative). Baseline specimens from 3,179 of 3,488 (91.5%) women referred to ALTS (a clinical trial to evaluate the management strategies for women with atypical squamous cells of undetermined significance [ASCUS] or low-grade squamous intraepithelial lesions) because of an ASCUS Papanicolaou smear were tested by all three assays. Among single-genotype infections with genotypes targeted by hc2 as detected by either PCR assay, HPV genotype 35 (HPV35) (86.4%), HPV56 (84.2%), and HPV58 (76.9%) were the most likely to test positive by hc2. Among single-genotype infections with genotypes not targeted by hc2 as detected by either assay, HPV82 (80.0%), HPV66 (60.0%) (recently classified as carcinogenic), HPV70 (59.1%), and HPV67 (56.3%) were the most likely to test positive by hc2. Among women who tested negative for carcinogenic HPV by both PCR tests and were positive for noncarcinogenic HPV by either test, 28% of women were hc2 positive. Conversely, 7.8% of all hc2-positive results in this population were due to cross-reactivity of hc2 with untargeted, noncarcinogenic HPV genotypes. In conclusion, hc2 cross-reacts with certain untargeted, noncarcinogenic HPV genotypes that are phylogenetically related to the targeted genotypes, but the degree of cross-reactivity may be less than previously reported.
Purulent Pericarditis with Greenish Pericardial Effusion Caused by
Shewanella algae
Authors: Tan Che-Kim , Lai Chih-Cheng , Kuar Wei-Khie , Hsueh Po-Ren
Published: June 2008

We report the first case of purulent pericarditis with greenish pericardial effusion caused by
Shewanella algae
in a patient with gastric and gallbladder cancer. This case expands the reported spectrum of infection caused by
S. algae
and raises the possibility that
S. algae
is a causative pathogen for purulent pericarditis.
Autofluorescence of Mycobacteria as a Tool for Detection of
Mycobacterium tuberculosis
Authors: Patiño Sol , Alamo Lorenzo , Cimino Mena , Casart Yveth , Bartoli Fulvia , García María J. , Salazar Leiria
Published: June 2008

The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for
Mycobacterium tuberculosis
. Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of
Mycobacterium tuberculosis
was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that <10
6
bacilli ml
−1
of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 μl of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries.
CD4 Binding Affinity Determines Human Immunodeficiency Virus Type 1-Induced Alpha Interferon Production in Plasmacytoid Dendritic Cells
Authors: Haupt Sabrina , Donhauser Norbert , Chaipan Chawaree , Schuster Philipp , Puffer Bridget , Daniels Rod S. , Greenough Thomas C. , Kirchhoff Frank , Schmidt Barbara
Published: June 2008

Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-α) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primaryenvalleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-α induction. Our results illustrate the importance of the gp120-CD4 interaction in determining HIV-1-induced immune stimulation via IFN-α production.
Magnitude and Quality of Vaccine-Elicited T-Cell Responses in the Control of Immunodeficiency Virus Replication in Rhesus Monkeys
Authors: Sun Yue , Santra Sampa , Schmitz Jörn E. , Roederer Mario , Letvin Norman L.
Published: June 2008

While a diversity of immunogens that elicit qualitatively different cellular immune responses are being assessed in clinical human immunodeficiency virus vaccine trials, the consequences of those varied responses for viral control remain poorly understood. In the present study, we evaluated the induction of virus-specific T-cell responses in rhesus monkeys using a series of diverse vaccine vectors. We assessed both the magnitude and the functional profile of the virus-specific CD8
+
T cells by measuring gamma interferon, interleukin-2, and tumor necrosis factor alpha production. We found that the different vectors generated virus-specific T-cell responses of different magnitudes and with different functional profiles. Heterologous prime-boost vaccine regimens induced particularly high-frequency virus-specific T-cell responses with polyfunctional repertoires. Yet, immediately after a pathogenic simian-human immunodeficiency virus (SHIV) challenge, no significant differences were observed between these cohorts of vaccinated monkeys in the magnitudes or the functional profiles of their virus-specific CD8
+
T cells. This finding suggests that the high viral load shapes the functional repertoire of the cellular immune response during primary infection. Nevertheless, in all vaccination regimens, higher frequency and more polyfunctional vaccine-elicited virus-specific CD8
+
T-cell responses were associated with better viral control after SHIV challenge. These observations highlight the contributions of both the quality and the magnitude of vaccine-elicited cellular immune responses in the control of immunodeficiency virus replication.
Vectorial Entry and Release of Hepatitis A Virus in Polarized Human Hepatocytes
Authors: Snooks Michelle J. , Bhat Purnima , Mackenzie Jason , Counihan Natalie A. , Vaughan Nicola , Anderson David A.
Published: June 2008

Hepatitis A virus (HAV) is an enterically transmitted virus that replicates predominantly in hepatocytes within the liver before excretion via bile through feces. Hepatocytes are polarized epithelial cells, and it has been assumed that the virus load in bile results from direct export of HAV via the apical domain of polarized hepatocytes. We have developed a subclone of hepatocyte-derived HepG2 cells (clone N6) that maintains functional characteristics of polarized hepatocytes but displays morphology typical of columnar epithelial cells, rather than the complex morphology that is typical of hepatocytes. N6 cells form microcolonies of polarized cells when grown on glass and confluent monolayers of polarized cells on semipermeable membranes. When N6 microcolonies were exposed to HAV, infection was restricted to peripheral cells of polarized colonies, whereas all cells could be infected in colonies of nonpolarized HepG2 cells (clone C11) or following disruption of tight junctions in N6 colonies with EGTA. This suggests that viral entry occurs predominantly via the basolateral plasma membrane, consistent with uptake of virus from the bloodstream after enteric exposure, as expected. Viral export was also found to be markedly vectorial in N6 but not C11 cells. However, rather than being exported from the apical domain as expected, more than 95% of HAV was exported via the basolateral domain of N6 cells, suggesting that virus is first excreted from infected hepatocytes into the bloodstream rather than to the biliary tree. Enteric excretion of HAV may therefore rely on reuptake and transcytosis of progeny HAV across hepatocytes into the bile. These studies provide the first example of the interactions between viruses and polarized hepatocytes.
Ligand-Independent Exhaustion of Killer Immunoglobulin-Like Receptor-Positive CD8
+
T Cells in Human Immunodeficiency Virus Type 1 Infection
Authors: Alter Galit , Rihn Suzannah , Streeck Hendrik , Teigen Nickolas , Piechocka-Trocha Alicja , Moss Kristin , Cohen Kristen , Meier Angela , Pereyra Florencia , Walker Bruce , Altfeld Marcus
Published: June 2008

Virus-specific CD8
+
T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8
+
T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8
+
T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8
+
T cells, in regulating CD8
+
T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8
+
T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8
+
T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR
+
CD8
+
T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8
+
T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR
+
CD8
+
T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8
+
T-cell response in infected individuals.
Association of Respiratory Syncytial Virus M Protein with Viral Nucleocapsids Is Mediated by the M2-1 Protein
Authors: Li Dongsheng , Jans David A. , Bardin Phillip G. , Meanger Jayesh , Mills John , Ghildyal Reena
Published: June 2008

Cytoplasmic inclusions in respiratory syncytial virus-infected cells comprising viral nucleocapsid proteins (L, N, P, and M2-1) and the viral genome are sites of viral transcription. Although not believed to be necessary for transcription, the matrix (M) protein is also present in these inclusions, and we have previously shown that M inhibits viral transcription. In this study, we have investigated the mechanisms for the association of the M protein with cytoplasmic inclusions. Our data demonstrate for the first time that the M protein associates with cytoplasmic inclusions via an interaction with the M2-1 protein. The M protein colocalizes with M2-1 in the cytoplasm of cells expressing only the M and M2-1 proteins and directly interacts with M2-1 in a cell-free binding assay. Using a cotransfection system, we confirmed that the N and P proteins are sufficient to form cytoplasmic inclusions and that M2-1 localizes to these inclusions; additionally, we show that M associates with cytoplasmic inclusions only in the presence of the M2-1 protein. Using truncated mutants, we show that the N-terminal 110 amino acids of M mediate the interaction with M2-1 and the subsequent association with nucleocapsids. The interaction of M2-1 with M and, in particular, the N-terminal region of M may represent a target for novel antivirals that block the association of M with nucleocapsids, thereby inhibiting virus assembly.
Changes in p19
Arf
Localization Accompany Apoptotic Crisis during Pre-B-Cell Transformation by Abelson Murine Leukemia Virus
Authors: Zimmerman Rebekah Stackpole , Rosenberg Naomi
Published: June 2008

Transformation by Abelson murine leukemia virus (Ab-MLV) is a multistep process in which growth-stimulatory signals from the v-Abl oncoprotein and growth-suppressive signals from the p19
Arf
-p53 tumor suppressor pathway oppose each other and influence the outcome of infection. The process involves a proliferative phase during which highly viable primary transformants expand, followed by a period of marked apoptosis (called “crisis”) that is dependent on the presence of p19
Arf
and p53; rare cells that survive this phase emerge as fully transformed and malignant. To understand the way in which v-Abl expression affects p19
Arf
expression, we examined changes in expression of
Arf
during all stages of Ab-MLV transformation process. As is consistent with the ability of v-Abl to stimulate Myc, a transcription factor known to induce p19
Arf
,
Myc
and
Arf
are induced soon after infection and p19
Arf
is expressed. At these early time points, the infected cells remain highly viable. The onset of crisis is marked by an increase in p19
Arf
expression and a change in localization of the protein from the nucleoplasm to the nucleolus. These data together suggest that the localization and expression levels of p19
Arf
modulate the effects of the protein during oncogenesis and reveal that the p19
Arf
-mediated response is subject to multiple layers of regulation that influence its function during Ab-MLV-mediated transformation.
Structural Domains within the 3′ Untranslated Region of Turnip Crinkle Virus
Authors: McCormack John C. , Yuan Xuefeng , Yingling Yaroslava G. , Kasprzak Wojciech , Zamora Rodolfo E. , Shapiro Bruce A. , Simon Anne E.
Published: June 2008

The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3′ end of
Turnip crinkle virus
(TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (Ψ
3
) within the 3′-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, Ψ
3
and a second pseudoknot (Ψ
2
) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, Ψ
3
and a pseudoknot that connects H5 and the 3′ end (Ψ
1
). Stepwise replacements of TCV elements with the comparable elements from
Cardamine chlorotic fleck virus
indicated that the complete 142-nt 3′ end, and subsets containing Ψ
3
, H4a, and H4b or Ψ
3
, H4a, H4b, H5, and Ψ
2
, form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, Ψ
3
, and Ψ
2
are capable of simultaneous existence and bears some resemblance to a tRNA. The related
Japanese iris necrotic ring virus
does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3′ untranslated region sequences such as translation and replication.
Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site
Authors: Alymova Irina V. , Taylor Garry , Mishin Vasiliy P. , Watanabe Makiko , Murti K. Gopal , Boyd Kelli , Chand Pooran , Babu Y. Sudhakara , Portner Allen
Published: June 2008

BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-
d
-glycero-
d
-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.
Preservation of FoxP3+Regulatory T Cells in the Peripheral Blood of Human Immunodeficiency Virus Type 1-Infected Elite Suppressors Correlates with Low CD4+T-Cell Activation
Authors: Chase Amanda J. , Yang Hung-Chih , Zhang Hao , Blankson Joel N. , Siliciano Robert F.
Published: June 2008

Elite suppressors (ES) are untreated human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4+T-cell counts and control viremia to levels that are below the limit of detection of current assays. The mechanisms involved in long-term control of viremia have not been fully elucidated. CD4+CD25+regulatory T cells (Tregs) downmodulate chronic inflammation by suppressing the activation and proliferation of effector lymphocytes. We found that while Tregs were functional in ES and patients on highly active antiretroviral therapy (HAART), ES maintained high levels of Tregs in peripheral blood mononuclear cells whereas patients on HAART had evidence of Treg depletion. We also demonstrated that Tregs can serve as reservoirs for HIV-1 in vivo. These data suggest that both direct infection by HIV-1 and tissue redistribution are possible explanations for declining FoxP3+Tregs in progressive HIV-1 infection. Furthermore, the maintenance of Tregs may be one mechanism associated with the nonprogressive nature of HIV-1 infection in ES.
Evidence for a Structural Relationship between BRCT Domains and the Helicase Domains of the Replication Initiators Encoded by thePolyomaviridaeandPapillomaviridaeFamilies of DNA Tumor Viruses
Authors: Kumar Anuradha , Joo Woo S. , Meinke Gretchen , Moine Stephanie , Naumova Elena N. , Bullock Peter A.
Published: June 2008

Studies of DNA tumor viruses have provided important insights into fundamental cellular processes and oncogenic transformation. They have revealed, for example, that upon expression of virally encoded proteins, cellular pathways involved in DNA repair and cell cycle control are disrupted. Herein, evidence is presented that BRCT-related regions are present in the helicase domains of the viral initiators encoded by thePolyomaviridaeandPapillomaviridaeviral families. Of interest, BRCT domains in cellular proteins recruit factors involved in diverse pathways, including DNA repair and the regulation of cell cycle progression. Therefore, the viral BRCT-related regions may compete with host BRCT domains for particular cellular ligands, a process that would help to explain the pleiotropic effects associated with infections with many DNA tumor viruses.
Human Papillomavirus Type 16 E7 Oncoprotein Associates with E2F6
Authors: McLaughlin-Drubin Margaret E. , Huh Kyung-Won , Münger Karl
Published: June 2008

The papillomavirus life cycle is intimately coupled to the differentiation state of the infected epithelium. Since papillomaviruses lack most of the rate-limiting enzymes required for genome synthesis, they need to uncouple keratinocyte differentiation from cell cycle arrest and maintain or reestablish a replication-competent state within terminally differentiated keratinocytes. The human papillomavirus (HPV) E7 protein appears to be a major determinant for this activity and induces aberrant S-phase entry through the inactivation of the retinoblastoma tumor suppressor and related pocket proteins. In addition, E7 can abrogate p21 and p27. Together, this leads to the activation of E2F1 to E2F5, enhanced expression of E2F-responsive genes, and increased cdk2 activity. E2F6 is a pRB-independent, noncanonical member of the E2F transcription factor family that acts as a transcriptional repressor. E2F6 expression is activated in S phase through an E2F-dependent mechanism and thus may provide a negative-feedback mechanism that slows down S-phase progression and/or exit in response to the activation of the other E2F transcription factors. Here, we show that low- and high-risk HPV E7 proteins, as well as simian virus 40 T antigen and adenovirus E1A, can associate with and inactivate the transcriptional repression activity of E2F6, thereby subverting a critical cellular defense mechanism. This may result in the extended S-phase competence of HPV-infected cells. E2F6 is a component of polycomb group complexes, which bind to silenced chromatin and are critical for the maintenance of cell fate. We show that E7-expressing cells show decreased staining for E2F6/polycomb complexes and that this is at least in part dependent on the association with E2F6.
Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity
Authors: Farquhar Michelle J. , Harris Helen J. , Diskar Mandy , Jones Sarah , Mee Christopher J. , Nielsen Søren U. , Brimacombe Claire L. , Molina Sonia , Toms Geoffrey L. , Maurel Patrick , Howl John , Herberg Friedrich W. , van IJzendoorn Sven C. D. , Balfe Peter , McKeating Jane A.
Published: June 2008

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.
Influenza Virus Protecting RNA: an Effective Prophylactic and Therapeutic Antiviral
Authors: Dimmock Nigel J. , Rainsford Edward W. , Scott Paul D. , Marriott Anthony C.
Published: June 2008

Another influenza pandemic is inevitable, and new measures to combat this and seasonal influenza are urgently needed. Here we describe a new concept in antivirals based on a defined, naturally occurring defective influenza virus RNA that has the potential to protect against any influenza A virus in any animal host. This “protecting RNA” (244 RNA) is incorporated into virions which, although noninfectious, deliver the RNA to those cells of the respiratory tract that are naturally targeted by infectious influenza virus. A 120-ng intranasal dose of this 244 protecting virus completely protected mice against a simultaneous challenge of 10 50% lethal doses with influenza A/WSN (H1N1) virus. The 244 virus also protected mice against strong challenge doses of all other subtypes tested (i.e., H2N2, H3N2, and H3N8). This prophylactic activity was maintained in the animal for at least 1 week prior to challenge. The 244 virus was 10- to 100-fold more active than previously characterized defective influenza A viruses, and the protecting activity was confirmed to reside in the 244 RNA molecule by recovering a protecting virus entirely from cloned cDNA. There was a clear therapeutic benefit when the 244 virus was administered 24 to 48 h after a lethal challenge, an effect which has not been previously observed with any defective virus. Protecting virus reduced, but did not abolish, replication of challenge virus in mouse lungs during both prophylactic and therapeutic treatments. Protecting virus is a novel antiviral, having the potential to combat human influenza virus infections, particularly when the infecting strain is not known or is resistant to antiviral drugs.
STAT2 Is a Primary Target for Measles Virus V Protein-Mediated Alpha/Beta Interferon Signaling Inhibition
Authors: Ramachandran Aparna , Parisien Jean-Patrick , Horvath Curt M.
Published: June 2008

Measles virus, a member of the
Morbillivirus
family, infects millions of people each year despite the availability of effective vaccines. The V protein of measles virus is an important virulence factor that can interfere with host innate immunity by inactivating alpha/beta interferon (IFN-α/β) and IFN-γ signaling through protein interactions with signal transducer and activator of transcription proteins STAT1 and STAT2. Here we demonstrate that although STAT1 interference results from protein interactions within a V protein N-terminal region encompassed by amino acids 110 to 130, detection of STAT1 interaction and IFN-γ signaling inhibition requires the presence of cellular STAT2. Cell-specific variability in STAT1 interference was observed to correlate with V protein expression level. A more direct target for measles virus V protein-mediated IFN-α/β evasion is STAT2. Results indicate that the widely conserved C-terminal zinc finger domain of measles virus V protein is both necessary and sufficient to bind STAT2 and disrupt IFN-α/β signal transduction. Mutagenesis and molecular modeling define a contact surface for STAT2 association that includes aspartic acid residue 248 as critical for STAT2 interference and IFN antiviral immune suppression. These findings clearly define the molecular determinants for measles virus IFN evasion and validate specific targets as candidates for therapeutic intervention.
Epstein-Barr Virus Latent Membrane Protein 2A Preferentially Signals through the Src Family Kinase Lyn
Authors: Rovedo Mark , Longnecker Richard
Published: June 2008

Latent membrane protein 2A (LMP2A) is a viral protein expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor signal transduction and provides survival and developmental signals to B cells in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, we crossed LMP2A transgenic mice (TgE) with Lyn−/−, Fyn−/−, or Blk−/−mice. TgE Lyn−/−mice had a larger immunoglobulin M (IgM)-positive B-cell population than TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. Both TgE Fyn−/−and TgE Blk−/−mice have an IgM-negative population of splenic B cells, similar to the TgE mice. LMP2A was also transiently transfected into the human EBV-negative B-cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates, whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. From these results, we conclude that LMP2A preferentially associates with and signals through Lyn compared to its association with other SFKs. This preferential association is due in part to the SH2 domain of Lyn associating with LMP2A.
Targeting the Apoptotic Pathway with BCL-2 Inhibitors Sensitizes Primary Chronic Lymphocytic Leukemia Cells to Vesicular Stomatitis Virus-Induced Oncolysis
Authors: Tumilasci Vanessa Fonseca , Olière Stephanie , Nguyên Thi Lien-Ahn , Shamy April , Bell John , Hiscott John
Published: June 2008

Chronic lymphocytic leukemia (CLL) is characterized by clonal accumulation of CD5
+
CD19
+
B lymphocytes that are arrested in the G
0
/G
1
phase of the cell cycle and fail to undergo apoptosis because of overexpression of the antiapoptotic B-cell CLL/lymphoma 2 (BCL-2) protein. Oncolytic viruses, such as vesicular stomatitis virus (VSV), have emerged as potential anticancer agents that selectively target and kill malignant cells via the intrinsic mitochondrial pathway. Although primary CLL cells are largely resistant to VSV oncolysis, we postulated that targeting the apoptotic pathway via inhibition of BCL-2 may sensitize CLL cells to VSV oncolysis. In the present study, we examined the capacity of EM20-25—a small-molecule antagonist of the BCL-2 protein—to overcome CLL resistance to VSV oncolysis. We demonstrate a synergistic effect of the two agents in primary ex vivo CLL cells (combination index of 0.5;
P
< 0.0001). In a direct comparison of peripheral blood mononuclear cells from healthy volunteers with primary CLL, the two agents combined showed a therapeutic index of 19-fold; furthermore, the combination of VSV and EM20-25 increased apoptotic cell death in Karpas-422 and Granta-519 B-lymphoma cell lines (
P
< 0.005) via the intrinsic mitochondrial pathway. Mechanistically, EM20-25 blocked the ability of the BCL-2 protein to dimerize with proapoptotic BAX protein, thus sensitizing CLL to VSV oncolytic stress. Together, these data indicate that the use of BCL-2 inhibitors may improve VSV oncolysis in treatment-resistant hematological malignancies, such as CLL, with characterized defects in the apoptotic response.
Experimental Optic Neuritis Induced by a Demyelinating Strain of Mouse Hepatitis Virus
Authors: Shindler Kenneth S. , Kenyon Lawrence C. , Dutt Mahasweta , Hingley Susan T. , Sarma Jayasri Das
Published: June 2008

Optic neuritis (ON), an inflammatory demyelinating optic nerve disease, occurs in multiple sclerosis (MS). Pathological mechanisms and potential treatments for ON have been studied via experimental autoimmune MS models. However, evidence suggests that virus-induced inflammation is a likely etiology triggering MS and ON; experimental virus-induced ON models are therefore required. We demonstrate that MHV-A59, a mouse hepatitis virus (MHV) strain that causes brain and spinal cord inflammation and demyelination, induces ON by promoting mixed inflammatory cell infiltration. In contrast, MHV-2, a nondemyelinating MHV strain, does not induce ON. Results reveal a reproducible virus-induced ON model important for the evaluation of novel therapies.
Persistent Equine Arteritis Virus Infection in HeLa Cells
Authors: Zhang Jianqiang , Timoney Peter J. , MacLachlan N. James , McCollum William H. , Balasuriya Udeni B. R.
Published: June 2008

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53→Cys and Val55→Ala), GP2 (Leu15→Ser, Trp31→Arg, Val87→Leu, and Ala112→Thr), GP3 (Ser115→Gly and Leu135→Pro), and GP4 (Tyr4→His and Ile109→Phe) proteins or with a single point mutation in the GP5 protein (Pro98→Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.
Translation Termination Reinitiation between Open Reading Frame 1 (ORF1) and ORF2 Enables Capsid Expression in a Bovine Norovirus without the Need for Production of Viral Subgenomic RNA
Authors: McCormick Christopher J. , Salim Omar , Lambden Paul R. , Clarke Ian N.
Published: June 2008

A generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.
Ras Modifies Proliferation and Invasiveness of Cells Expressing Human Papillomavirus Oncoproteins
Authors: Yoshida Satoshi , Kajitani Naoko , Satsuka Ayano , Nakamura Hiroyasu , Sakai Hiroyuki
Published: June 2008

Infection by human papillomavirus (HPV) is a major risk factor for human cervical carcinoma. However, the HPV infection alone is not sufficient for cancer formation. Cervical carcinogenesis is considered a multistep process accompanied by genetic alterations of the cell. Ras is activated in approximately 20% of human cancers, and it is related to the metastatic conversion of tumor cells. We investigated how Ras activation was involved in the malignant conversion of HPV-infected lesions. The active form of H-ras was introduced into human primary keratinocytes expressing the HPV type 18 (HPV18) oncoproteins E6 and/or E7. We analyzed the keratinocytes’ growth potentials and found that the activation of the Ras pathway induced senescence-like growth arrest. Senescence could be eliminated by high-risk E7 expression, suggesting that the pRb pathway was important for Ras-induced senescence. Then we analyzed the effect of Ras activation on epidermis development by using an organotypic “raft” culture and found that the E7 and H-ras coexpressions conferred invasive potential on the epidermis. This invasiveness resulted from the upregulation of MT1-MMP and MMP9 by H-ras and E7, respectively, in which the activation of the MEK/extracellular signal-regulated kinase pathway was involved. These results indicated that the activation of Ras or the related signal pathways promoted the malignant conversion of HPV-infected cells.
Temporary Depletion of CD11c
+
Dendritic Cells Delays Lymphoinvasion after Intraperitonal Scrapie Infection
Authors: Cordier-Dirikoc Sevda , Chabry Joëlle
Published: June 2008

The involvement of immune cells in prion capture and transport to lymphoid tissues still remains unclear. To investigate the role of dendritic cells (DC), we used DTR
+/+
mice, a transgenic model designed to trigger short-term ablation of DC. Transient depletion of DC around the time of intraperitoneal infection delayed prion replication in the spleen, as followed by PrPsc amount, a specific hallmark of prion diseases. Consequently, neuroinvasion and incubation time of prion disease were delayed. In contrast, no differences were observed after oral infection. These results suggest that DC act as vectors for prions from the peripheral entry site to the spleen.