bluuebunny/crossref_metadata_2025 huggingface dataset:
A Multicistronic DNA Vaccine Induces Significant Protection against Tuberculosis in Mice and Offers Flexibility in the Expressed Antigen Repertoire
Authors: Mir Fayaz-Ahmad , Kaufmann Stefan H. E. , Eddine Ali Nasser
Published: August 2009

Concerns about the safety and efficacy of
Mycobacterium bovis
bacillus Calmette-Guérin (BCG) emphasize the need for alternative tuberculosis (TB) vaccines. DNA vaccines are interesting candidates but are limited by the restricted antigen repertoire that they express. Traditional polycistronic vectors are large and have imbalanced expression. Recent advances in molecular genetics and cellular immunology have paved the way toward the rational design of an efficacious vaccine. We exploited self-cleaving peptide 2A from the foot-and-mouth disease virus, because of its small size and high cleavage activity, to generate an efficient TB DNA vaccine (V-2A). V-2A expresses three mycobacterial antigens, Rv3407, Ag85A, and HspX, in a single open reading frame joined by the 2A sequences, which lead to the segmentation of the long translated polypeptide into individual proteins by posttranslational modification. Our in vitro measurements revealed no differences at the transcriptional or translational level between V-2A and the monocistronic expression of the individual antigens. Mice vaccinated with V-2A developed antigen-specific cellular and humoral responses against all three antigens, imparting protection against
Mycobacterium tuberculosis
aerosol challenge equivalent to that imparted by BCG. These results have important implications for the rational design and development of efficacious recombinant subunit vaccines.
Evidence-Based Approach for Interpretation of Epstein-Barr Virus Serological Patterns
Authors: Klutts J. S. , Ford B. A. , Perez N. R. , Gronowski A. M.
Published: August 2009

Diagnosis of Epstein-Barr virus (EBV) infection is based on clinical symptoms and serological markers, including the following: immunoglobulin G (IgG) and IgM antibodies to the viral capsid antigen (VCA), heterophile antibodies, and IgG antibodies to the EBV early antigen-diffuse (EA-D) and nuclear antigen (EBNA-1). The use of all five markers results in 32 possible serological patterns. As a result, interpretation of EBV serologies remains a challenge. The purpose of this study was to use a large population of patients to develop evidence-based tools for interpreting EBV results. This study utilized 1,846 serum specimens sent to the laboratory for physician-ordered EBV testing. Chart review was performed for more than 800 patients, and diagnoses were assigned based on physician-ordered testing, clinical presentation, and patient history. Testing for all five EBV antibodies was performed separately on all serum samples using the Bio-Rad BioPlex 2200 system. Presumed EBV diagnosis (based on previous publications) was compared to EBV diagnosis based on a medical record review for each serological pattern. Interestingly, of the 32 possible serological patterns, only 12 occurred in ≥10 patients. The remaining 20 patterns were uninterpretable because they occurred with such infrequency. Two easy-to-use tables were created to interpret EBV serological patterns based on whether three (EBV VCA IgG, IgM, and heterophile) or five markers are utilized. The use of these two tables allows for interpretation of >95% of BioPlex serological results. This is the first evidence-based study of its kind for EBV serology.
Campylobacter coli
Prosthetic Hip Infection Associated with Ingestion of Contaminated Oysters
Authors: Sharp Susan E.
Published: August 2009

This report describes the first reported case of a prosthetic hip joint infection due to
Campylobacter coli
. The infection presumably resulted from ingestion of contaminated raw oysters. Issues regarding original smear interpretation, culture isolation, and susceptibility testing are presented.
Highly Sensitive Fetal Goat Tongue Cell Line for Detection and Isolation of Foot-and-Mouth Disease Virus
Authors: Brehm K. E. , Ferris N. P. , Lenk M. , Riebe R. , Haas B.
Published: August 2009

A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.
First Report of Acute Cholecystitis with Sepsis Caused by
Cellulomonas denverensis
Authors: Ohtaki Hirofumi , Ohkusu Kiyofumi , Sawamura Haruki , Ohta Hirotoshi , Inoue Rina , Iwasa Junpei , Ito Hiroyasu , Murakami Nobuo , Ezaki Takayuki , Moriwaki Hisataka , Seishima Mitsuru
Published: August 2009

Cellulomonas denverensis
is a small and thin gram-positive rod-shaped bacterium that was proposed as a new species in 2005. Here we report a female case of acute cholecystitis and sepsis in which
C. denverensis
was determined to be causative.
Serological Immunoassay for Detection of Hepatitis E Virus on the Basis of Genotype 3 Open Reading Frame 2 Recombinant Proteins Produced in
Trichoplusia ni
Larvae
Authors: Jiménez de Oya Nereida , Galindo Inmaculada , Gironés Olivia , Duizer Erwin , Escribano José M. , Saiz Juan-Carlos
Published: August 2009

Hepatitis E virus (HEV) is a major cause of acute hepatitis in humans, and strains of genotypes 1 and 2 are endemic in many regions with suboptimal sanitary conditions. In many industrialized countries, HEV strains of genotype 3 are highly endemic in swine, and an increased number of autochthonous infections with HEV genotype 3 strains have been reported lately. Serological studies of HEV infection are often conducted with commercial assays based on peptides and recombinant proteins of HEV genotype 1 and 2 strains. For some patients with proven HEV genotype 3 infections, these assays failed to detect specific antibodies, and they are not applicable or validated for the detection of anti-HEV antibodies in swine. To elucidate the incidence of hepatitis E in regions where HEV genotype 3 infections can be expected, and to study the seroprevalence of HEV in swine, new tools with broad specificity for all genotypes of HEV are needed. We present the expression and partial characterization of recombinant HEV genotype 3 open reading frame 2 (ORF-2) proteins and their usefulness as diagnostic antigens in detecting anti-HEV antibodies in humans and swine with proven HEV genotype 3 infections. The recombinant antigens were produced at relatively high yields and at low cost upon infection of
Trichoplusia ni
larvae with recombinant baculoviruses expressing recombinant HEV genotype 3 ORF-2 proteins. The enzyme-linked immunosorbent assay based on the recombinant proteins showed good specificity and sensitivity for anti-HEV genotype 3 immunoglobulin G detection in human and swine sera. These recombinant HEV genotype 3 ORF-2 proteins might be added to diagnostic kits containing HEV genotype 1 and 2 antigens in order to develop a broadly sensitive new tool for the diagnosis of hepatitis E.
Molecular Epidemiology of
Brucella
Genotypes in Patients at a Major Hospital in Central Peru
Authors: Nöckler Karsten , Maves Ryan , Cepeda David , Draeger Angelika , Mayer-Scholl Anne , Chacaltana Jesus , Castañeda María , Espinosa Benjamin , Castillo Rosa , Hall Eric , Al Dahouk Sascha , Gilman Robert H. , Cabeza Franco , Smits Henk L.
Published: August 2009

The multiple-locus variable-number repeat analysis of 90 human
Brucella melitensis
isolates from a large urban area in central Peru revealed variations at 4 (Bruce07, Bruce09, Bruce18, and Bruce42) out of 16 loci investigated, of which 1 (Bruce42) also is used for species identification. Ten genotypes were identified, separated by the number of Bruce42 repeats into two groups that may have distinct phenotypic characteristics. Whereas genotypes with five or six Bruce42 repeats were cultured mainly from adult patients, genotypes with three Bruce42 repeats were isolated from children and young adolescents as well as from adults. In addition, the isolates with three Bruce42 repeats were obtained more often from patients with splenomegaly (
P
= 0.02) or hepatomegaly (
P
= 0.006). An annual variation in the diversity of genotypes was observed, possibly reflecting changes in sources of fresh dairy products, supply routes to city shops and markets, and the movement of infected dairy goat herds.
Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-BrucellaAntibodies in Ruminant Serum Samples
Authors: McGiven John A. , Thompson Iain J. , Commander Nicola J. , Stack Judy A.
Published: August 2009

Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucellamonoclonal antibody with a long-lifetime donor fluorophore andBrucellasmooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n= 73) and uninfected (n= 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases.
Variation in Susceptibility of Bloodstream Isolates ofCandida glabratato Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007
Authors: Pfaller M. A. , Messer S. A. , Hollis R. J. , Boyken L. , Tendolkar S. , Kroeger J. , Diekema D. J.
Published: August 2009

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates ofCandida glabrataand grouped the isolates by patient age and geographic location within the United States. Susceptibility ofC. glabratato fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance amongC. glabrataBSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency ofC. glabrataincreased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that wereC. glabrata(32%) and the lowest rate of fluconazole resistance (5%).
Target Cell Type-Dependent Modulation of Human Immunodeficiency Virus Type 1 Capsid Disassembly by Cyclophilin A
Authors: Li Yuan , Kar Alak Kanti , Sodroski Joseph
Published: August 2009

The binding of cyclophilin A (CypA) to the human immunodeficiency virus type 1 (HIV-1) capsid protein (CA protein) is required soon after virus entry into natural target cells. In Jurkat T lymphocytes, disrupting CypA-CA interaction either by cyclosporine (Cs) treatment or by alteration (e.g., P90A) of the CA inhibits HIV-1 infection. In HeLa cells, however, treatment with Cs or Cs analogues minimally inhibits the early phase of HIV-1 infection but selects for a Cs-dependent virus with a change (A92E) in CA. To understand these phenomena, we examined the effects of the P90A and A92E changes in the HIV-1 CA protein on the stability of capsid complexes assembled in vitro and on capsid disassembly in the cytosol of virus-exposed target cells. The A92E change impaired CA-CA interactions in vitro and decreased the amount of particulate capsids in the cytosol of HeLa target cells. Reducing the binding of CypA to the A92E mutant capsid, either by Cs treatment or by an additional P90A change in the CA protein, increased the amount of particulate capsids and viral infectivity in HeLa cells. In contrast, reduction of the binding of CypA to HIV-1 capsids in Jurkat T lymphocytes resulted in a decrease in the amount of particulate capsids and infectivity. Thus, depending on the capsid and the target cell, CypA-CA binding either stabilized or destabilized the capsid, indicating that CypA modulates HIV-1 capsid disassembly. In both cell types examined, decreased stability of the capsid was associated with a decrease in the efficiency of HIV-1 infection.
Role for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen 1 in DNA Replication and Metaphase Chromosome Attachment
Authors: Norseen Julie , Johnson F. Brad , Lieberman Paul M.
Published: August 2009

Latent infection by Epstein-Barr virus (EBV) requires both replication and maintenance of the viral genome. EBV nuclear antigen 1 (EBNA1) is a virus-encoded protein that is critical for the replication and maintenance of the genome during latency in proliferating cells. We have previously demonstrated that EBNA1 recruits the cellular origin recognition complex (ORC) through an RNA-dependent interaction with EBNA1 linking region 1 (LR1) and LR2. We now show that LR1 and LR2 bind to G-rich RNA that is predicted to form G-quadruplex structures. Several chemically distinct G-quadruplex-interacting drugs disrupted the interaction between EBNA1 and ORC. The G-quadruplex-interacting compound BRACO-19 inhibited EBNA1-dependent stimulation of viral DNA replication and preferentially blocked proliferation of EBV-positive cells relative to EBV-negative cell lines. BRACO-19 treatment also disrupted the ability of EBNA1 to tether to metaphase chromosomes, suggesting that maintenance function is also mediated through G-quadruplex recognition. These findings suggest that the EBNA1 replication and maintenance function uses a common G-quadruplex binding capacity of LR1 and LR2, which may be targetable by small-molecule inhibitors.
Turnip Mosaic Virus RNA Replication Complex Vesicles Are Mobile, Align with Microfilaments, and Are Each Derived from a Single Viral Genome
Authors: Cotton Sophie , Grangeon Romain , Thivierge Karine , Mathieu Isabelle , Ide Christine , Wei Taiyun , Wang Aiming , Laliberté Jean-François
Published: August 2009

Nicotiana benthamiana
plants were agroinoculated with an infectious cDNA clone of
Turnip mosaic virus
(TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K
2
protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K
2
GFP and the other expressed 6K
2
mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected
N. benthamiana
leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.
Cell Entry of Borna Disease Virus Follows a Clathrin-Mediated Endocytosis Pathway That Requires Rab5 and Microtubules
Authors: Clemente Roberto , de la Torre Juan C.
Published: August 2009

Borna disease virus (BDV), the prototypic member of theBornaviridaefamily within the orderMononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.
Venezuelan Equine Encephalitis Virus Disrupts STAT1 Signaling by Distinct Mechanisms Independent of Host Shutoff
Authors: Simmons Jason D. , White Laura J. , Morrison Thomas E. , Montgomery Stephanie A. , Whitmore Alan C. , Johnston Robert E. , Heise Mark T.
Published: August 2009

Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-β and IFN-γ. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-β treatment but did inhibit Jak1 and Jak2 activation in response to IFN-γ, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.
The Matrix Protein of Measles Virus Regulates Viral RNA Synthesis and Assembly by Interacting with the Nucleocapsid Protein
Authors: Iwasaki Masaharu , Takeda Makoto , Shirogane Yuta , Nakatsu Yuichiro , Nakamura Takanori , Yanagi Yusuke
Published: August 2009

The genome of measles virus (MV) is encapsidated by the nucleocapsid (N) protein and associates with RNA-dependent RNA polymerase to form the ribonucleoprotein complex. The matrix (M) protein is believed to play an important role in MV assembly by linking the ribonucleoprotein complex with envelope glycoproteins. Analyses using a yeast two-hybrid system and coimmunoprecipitation in mammalian cells revealed that the M protein interacts with the N protein and that two leucine residues at the carboxyl terminus of the N protein (L523 and L524) are critical for the interaction. In MV minigenome reporter gene assays, the M protein inhibited viral RNA synthesis only when it was able to interact with the N protein. The N protein colocalized with the M protein at the plasma membrane when the proteins were coexpressed in plasmid-transfected or MV-infected cells. In contrast, the N protein formed small dots in the perinuclear area when it was expressed without the M protein, or it was incapable of interacting with the M protein. Furthermore, a recombinant MV possessing a mutant N protein incapable of interacting with the M protein grew much less efficiently than the parental virus. Since the M protein has an intrinsic ability to associate with the plasma membrane, it may retain the ribonucleoprotein complex at the plasma membrane by binding to the N protein, thereby stopping viral RNA synthesis and promoting viral particle production. Consequently, our results indicate that the M protein regulates MV RNA synthesis and assembly via its interaction with the N protein.
Complete Protection from Repeated Vaginal Simian-Human Immunodeficiency Virus Exposures in Macaques by a Topical Gel Containing Tenofovir Alone or with Emtricitabine
Authors: Parikh Urvi M. , Dobard Charles , Sharma Sunita , Cong Mian-er , Jia Hongwei , Martin Amy , Pau Chou-Pong , Hanson Debra L. , Guenthner Patricia , Smith James , Kersh Ellen , Garcia-Lerma J. Gerardo , Novembre Francis J. , Otten Ron , Folks Thomas , Heneine Walid
Published: August 2009

New-generation gels that deliver potent antiretroviral drugs against human immunodeficiency virus type 1 have renewed hopes for topical prophylaxis as a prevention strategy. Previous preclinical research with monkey models suggested that high concentrations and drug combinations are needed for high efficacy. We evaluated two long-acting reverse transcriptase inhibitors, tenofovir (TFV) and emtricitabine (FTC), by using a twice-weekly repeat challenge macaque model and showed that a preexposure vaginal application of gel with 1% TFV alone or in combination with 5% FTC fully protected macaques from a total of 20 exposures to simian-human immunodeficiency virus SF162p3. FTC and TFV were detected in plasma 30 min after vaginal application, suggesting rapid absorption. FTC was detected more frequently than TFV and showed higher levels, reflecting the fivefold-higher concentration of this drug than of TFV. Two of 12 repeatedly exposed but protected macaques showed limited T-cell priming, which did not induce resistance to infection when macaques were rechallenged. Thus, single drugs with durable antiviral activity can provide highly effective topical prophylaxis and overcome the need for noncoital use or for drug combinations which are more complex and costly to formulate and approve.
Passage of Dengue Virus Type 4 Vaccine Candidates in Fetal Rhesus Lung Cells Selects Heparin-Sensitive Variants That Result in Loss of Infectivity and Immunogenicity in Rhesus Macaques
Authors: Añez Germán , Men Ruhe , Eckels Kenneth H. , Lai Ching-Juh
Published: August 2009

Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3′ noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu
327
-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu
327
-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu
327
-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu
327
-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy.
Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p
Authors: Kyratsous Christos A. , Walters Matthew S. , Panagiotidis Christos A. , Silverstein Saul J.
Published: August 2009

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0's RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.
Stimulus Duration and Response Time Independently Influence the Kinetics of Lytic Cycle Reactivation of Epstein-Barr Virus
Authors: Countryman Jill , Gradoville Lyndle , Bhaduri-McIntosh Sumita , Ye Jianjiang , Heston Lee , Himmelfarb Sarah , Shedd Duane , Miller George
Published: August 2009

Epstein-Barr virus (EBV) can be reactivated from latency into the lytic cycle by many stimuli believed to operate by different mechanisms. Cell lines containing EBV differ in their responses to inducing stimuli, yet all stimuli require de novo protein synthesis (44). A crucial step preliminary to identifying these proteins and determining when they are required is to measure the duration of stimulus and response time needed for activation of expression of EBV BRLF1 and BZLF1, the earliest viral indicators of reactivation. Here we show, with four EBV-containing cell lines that respond to different inducing agents, that stimuli that are effective at reactivating EBV can be divided into two main groups. The histone deacetylase inhibitors sodium butyrate and trichostatin A require a relatively long period of exposure, from 2 to 4 h or longer. Phorbol esters, anti-immunoglobulin G (anti-IgG), and, surprisingly, 5-aza-2′-deoxycytidine require short exposures of 15 min or less. The cell/virus background influences the response time. Expression of the EBV BZLF1 and BRLF1 genes can be detected before 2 h in Akata cells treated with anti-IgG, but both long- and short-duration stimuli required 4 or more hr to activate BZLF1 and BRLF1 expression in HH514-16, Raji, or B95-8 cells. Thus, stimulus duration and response time are independent variables. Neither stimulus duration nor response time can be predicted by the number of cells activated into the lytic cycle. These experiments shed new light on the earliest events leading to lytic cycle reactivation of EBV.
Probing the Spatial Organization of Measles Virus Fusion Complexes
Authors: Paal Tanja , Brindley Melinda A. , St. Clair Courtney , Prussia Andrew , Gaus Dominika , Krumm Stefanie A. , Snyder James P. , Plemper Richard K.
Published: August 2009

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.
Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons
Authors: Meng Xiangzhi , Jiang Canhua , Arsenio Janilyn , Dick Kevin , Cao Jingxin , Xiang Yan
Published: August 2009

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L−C7L−) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L−C7L−at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L−C7L−fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.
Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD
Authors: Avitabile Elisa , Forghieri Cristina , Campadelli-Fiume Gabriella
Published: August 2009

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.
Analysis of Human Cytomegalovirus-Encoded MicroRNA Activity during Infection
Authors: Stern-Ginossar Noam , Saleh Niveen , Goldberg Miri D. , Prichard Mark , Wolf Dana G. , Mandelboim Ofer
Published: August 2009

MicroRNAs (miRNAs) are expressed in a wide variety of organisms, ranging from plants to animals, and are key posttranscriptional regulators of gene expression. Virally encoded miRNAs are unique in that they could potentially target both viral and host genes. Indeed, we have previously demonstrated that a human cytomegalovirus (HCMV)-encoded miRNA, miR-UL112, downregulates the expression of a host immune gene, MICB. Remarkably, it was shown that the same miRNA also downregulates immediate-early viral genes and that its ectopic expression resulted in reduced viral replication and viral titers. The targets for most of the viral miRNAs, and hence their functions, are still unknown. Here we demonstrate that miR-UL112 also targets the UL114 gene, and we present evidence that the reduction of UL114 by miR-UL112 reduces its activity as uracil DNA glycosylase but only minimally affects virus growth. In addition, we show that two additional HCMV-encoded miRNAs, miR-US25-1 and miR-US25-2, reduce the viral replication and DNA synthesis not only of HCMV but also of other viruses, suggesting that these two miRNAs target cellular genes that are essential for virus growth. Thus, we suggest that in addition to miR-UL112, two additional HCMV miRNAs control the life cycle of the virus.
A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly
Authors: Hughes Mair , Gretton Sarah , Shelton Holly , Brown David D. , McCormick Christopher J. , Angus Allan G. N. , Patel Arvind H. , Griffin Stephen , Harris Mark
Published: August 2009

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.
A reconnaissance survey of late Quaternary sea levels, Bella Bella/Bella Coola region, central British Columbia coast
Authors: Andrews J. T. , Retherford R. M.
Published: August 2009

A preliminary relative sea level curve that covers the last 10 200 years is derived for the area of the islands and outer mainland centered on Bella Bella and Namu, the central coast of British Columbia. The curve shows postglacial emergence of 17 m over this period. The rate of emergence was ~0.6 m/100 year about 9000 BP, and present sea level was attained between 7000 and 8000 BP. Relative sea level continued to fall until the last few hundred to one thousand years BP when a marine transgression led to a rise of sea level and resultant erosion of many coastal Indian middens. Marine limits on the outer islands may reach 120 m asl, whereas in the middle part of the fiord country observed delta surfaces are lower (54–75 m asl). Elevations of raised deltas then attain ~150 m at fiord heads. A readvance of the ice front ≤ 12 210 ± 330 BP (GSC-1351) is suggested by the stratigraphy of one section.
Simulation of limestone diagenesis — a model based on strontium depletion
Authors: Morrow D. W. , Mayers I. R.
Published: August 2009

Limestone diagenesis has been simulated by a computer model based on strontium and zinc depletion in meteoric groundwater. This has enabled estimates to be made of the minimum number of pore volumes of groundwater that must have infiltrated many limestones and the accompanying degree of recrystallization that must have occurred in order to produce the observed reductions in the trace element concentrations. The amount of strontium lost by Barbados and Barbuda aragonite during early diagenesis indicates that the 0.05 value of Katz et al. (1972) for the distribution coefficient of strontium during the aragonite-to-calcite transformation is more accurate than the 0.14 value of Kinsman and Holland (1969). About 3000–6000 pore volumes of meteoric groundwater are required to transform aragonitic limestones on Barbados and Barbuda to low-magnesian calcite with average strontium contents of 1700 and 500 ppm, respectively. Generally, the model suggests that early diagenesis will not decrease the strontium contents of limestones below about 400 ppm.Extrapolation to late diagenesis indicates that during the recrystallization of low-magnesian calcite, tens of thousands to hundreds of thousands of pore volumes of meteoric solution must pass through limestones to reduce their strontium contents to between 100 and 200 ppm. Variation in the degree of solution and reprecipitation of CaCO3 that characterizes the residence periods of pore volumes of solution infiltrating low-magnesian calcite limestones affects the number of bulk recrystallizations, but has little influence on the total volume that passes through these limestones during late diagenesis.Porosity differences and variations in groundwater acidity have more effect on limestone strontium contents during late diagenesis than do the initial mineralogical differences. Facies-controlled porosity variations may have partly produced the range of strontium contents exhibited by ancient limestones.The directional trends of strontium concentrations and the low average strontium content (143 ppm) in the limestones of the Dunedin Formation in British Columbia indicate that it underwent late diagenesis by solutions that, during their passage, had a residence period of many solution–reprecipitation events.Zinc in solution does not undergo a significant increase in concentration during meteoric diagenesis because its distribution coefficient is greater than 1. The observed absence of directional trends of zinc within the Dunedin Formation supports this model prediction.Abnormally high concentrations of strontium are retained in hydrocarbon-bearing limestones because the solution–reprecipitation process is inhibited by the presence of hydrocarbons. This suggests that strontium anomaly surveying would provide a useful adjunct to present hydrocarbon exploration techniques.
The age of metasomatic anhydrite in Mississippian reservoir carbonates, southeastern Saskatchewan
Authors: Kendall Alan C. , Walters Ken L.
Published: August 2009

Replacement anhydrite porphyroblasts are abundant in Mississippian carbonates from south-eastern Saskatchewan and have caused deterioration in oil field reservoir quality. Previously they were interpreted to have formed at the time overlying Mississippian bedded anhydrites were deposited. Porphyroblasts are here reported to be absent from a late diagenetic altered zone, composed of dolomitized and anhydrite-plugged beds, that occurs immediately beneath the Mississippian unconformity. This absence from strata that normally contain them indicates that anhydrite porphyroblast growth occurred after the formation of the sub-unconformity altered zone. This zone is believed to have been rendered impermeable, or otherwise made unsuitable for anhydrite replacement, before introduction of fluids that elsewhere precipitated the anhydrite porophyroblasts. The altered zone is dated as post-Watrous redbeds (post-Triassic); consequently the growth of replacement anhydrite post-dates this period. The occurrence of included bitumen in many porphyroblasts indicates that some replacement occurred after or during the introduction of oil into the Mississippian reservoirs (Late Cretaceous?).An early diagenetic origin for pre-replacement-anhydrite joints, stylolites and dolomite need not be invoked now that replacement anhydrite has been shown to be a late diagenetic feature.
The occurrence of the Ordovician brachiopod Foliomena at Percé, Quebec
Authors: Sheehan Peter M. , Lespérance Pierre J.
Published: August 2009

A new species of brachiopod, Foliomena joliensis, has been found in the White Head Formation of Percé, Quebec. The genus has previously been reported only in Europe where it occurs in deep-water communities. As in Europe, the assemblage containing Foliomena is dominated by vagile animals, and the assemblage has only rare, very small sessile animals. Some associated brachiopods are described and illustrated.
Framboids in Champlain Sea sediments
Authors: Haynes J. , Quigley R. M.
Published: August 2009

This is the first reported occurrence of irregular particles consisting predominantly of framboidal pyrite, quartz and lesser feldspar in Champlain Sea clays in the Arnprior and Hawkesbury areas of Ontario.
Precambrian paleosols in Canada
Authors: Gall Quentin
Published: August 2009

Although at least 38 Precambrian paleosols have been reported in Canada, few have received the attention afforded most other geological phenomena. The most intensively studied of these are widely distributed Early Proterozoic paleosols associated with the sub-Huronian and Matonabbee (new name) unconformities. Criteria that have been most useful in identifying Precambrian paleosols in Canada include macrostructures (ascending protolith disruption, saprolith, and corestones), micromorphology (sepic texture and peds), mineralogical changes (dissolution, transformation), geochemical signatures (ascending depletion of some elements, characteristic weathering indices), and distinctive colour contrast. Diagenetic mechanical and geochemical overprinting of the paleosols, evident in many Canadian examples, includes paleosol compaction, veins cutting pedogenic features, and widespread alkali and alkaline earth element metasomatism. Metamorphism and deformation also tend to mask pedogenic features, as attested by the scarcity of documented occurrences of medium- to high-grade metamorphosed Precambrian paleosols in Canada. The recognition of other Precambrian paleosols in metamorphosed terranes could be aided by systematic detailed studies of rocks beneath unconformities to assess geochemical gradients in metamorphic minerals rich in Si, Al and Fe. Geochronological constraints on Precambrian paleosol development might reinforce previously proposed stratigraphic correlations, suggest new correlations, and serve to delimit times and areas of relative tectonic stability and subaerial exposure.
Sedimentology of the Archean Doré metasediments, Arliss Lake area, southern Michipicoten greenstone belt, Superior Province
Authors: Rice R. J. , Donaldson J. A.
Published: August 2009

Doré metasediments in the Arliss Lake area of the Michipicoten greenstone belt predominantly represent submarine-fan sedimentation in mid- to outer-fan settings; subaerial deposition was rare, occurring in a sandy braided stream environment and as mud-poor debris flows in a probable fan-delta setting. The Arliss Lake subbasin was likely small, and of an irregular configuration which restricted down-fan sediment distribution, promoting aggradational lobe development; it existed on a convergent plate margin. Felsic pyroclastic volcanism was periodically synchronous with epiclastic sedimentation but only rarely dominated. Paleogeographic development, addressed by comparing juxtaposed thrust-fault-bounded lithological packages of similar facing, suggests that the subbasin experienced several episodes of instability, which caused dramatic changes in relative sea level.