bluuebunny/crossref_metadata_2025 huggingface dataset:
Imaging Local Electrochemical Current via Surface Plasmon Resonance
Authors: Shan Xiaonan , Patel Urmez , Wang Shaopeng , Iglesias Rodrigo , Tao Nongjian
Published: March 2010

An Electrochemical Landscape

Electrochemical detection is an analytical method that has been used for a wide range of purposes, including trace chemical analysis, glucose and neurotransmitter monitoring, DNA and protein detection, and electrocatalysis. Scanning electrochemical microscopy maps changes in the local electrochemical current along a surface in a serial way, but serial probing can disrupt the process under study.

Shan
et al.

(p.
1363
) show that optical measurements of surface plasmon resonances can be used as a less disruptive way of determining the concentration of electrochemically active species on gold-coated glass slides and their current density. This method can be used for a wide range of applications from analyzing DNA and protein microarrays and enzyme-amplified biosensors to probing the activities of cells.
Analysis of Genetic Inheritance in a Family Quartet by Whole-Genome Sequencing
Authors: Roach Jared C. , Glusman Gustavo , Smit Arian F. A. , Huff Chad D. , Hubley Robert , Shannon Paul T. , Rowen Lee , Pant Krishna P. , Goodman Nathan , Bamshad Michael , Shendure Jay , Drmanac Radoje , Jorde Lynn B. , Hood Leroy , Galas David J.
Published: March 2010

Runs in the Family

The power to detect mutations involved in disease by genome sequencing is enhanced when combined with the ability to discover specific mutations that may have arisen between offspring and parents.

Roach
et al.

(p.
636
, published online 10 March) present the sequence of a family with two offspring affected with two genetic disorders: Miller syndrome and primary ciliary dyskinesia. Sequence analysis of the children and their parents not only showed that the intergenerational mutation rate was lower than anticipated but also revealed recombination sites and the occurrence of rare polymorphisms.
Remote Enzyme Microsurgery
Authors: Bollinger J. Martin , Matthews Megan L.
Published: March 2010

Protein structures reveal a surprising mechanism for construction of a complex enzyme cofactor from standard amino acids.
Bad Theories Can Harm Victims
The Trauma Myth
The Truth About the Sexual Abuse of Children—and Its Aftermath

by Susan A. Clancy

Basic Books, New York, 2010. 252 pp. $25, C$31.95, £14.99. ISBN 9780465016884.
Authors: Loftus Elizabeth F. , Frenda Steven J.
Published: March 2010

Clancy argues that children generally do not experience abuse as traumatic when it occurs but that often the trauma develops later, when "therapeutic culture dictates to victims how they should feel about their experience."
Enforcing Order on Signaling
Authors: Paszek Matthew , Weaver Valerie
Published: March 2010

Mechanical forces constrain the spatial organization of a cell surface receptor, thereby altering its signaling function and cell behavior.
A Civil Conversation About Animals in Research
Authors: Miller Greg
Published: March 2010

Last week, neuroscientist Dario Ringach of the University of California, Los Angeles, spoke with
Science
about a recent panel discussion he co-organized on animal research and the resulting renewed attention he's gotten from animal-rights extremists after having given up primate research in 2006.
Matchmaking Is Part of the Party as ARPA-E Marks Its First Birthday
Authors: Kintisch Eli
Published: March 2010

Last week, 1700 politicians, scientists, industrialists, and investors attended a 3-day summit in a suburb of Washington, D.C., to mark the first anniversary of the Advanced Research Projects Agency–Energy.
On Rarity and Richness
Authors: Pennisi Elizabeth
Published: March 2010

Two researchers have taken a stab at explaining why oceans have far fewer species than terrestrial habitats.
Chlamydia pneumoniae-Induced Memory CD4+T-Cell Activation in Human Peripheral Blood Correlates with Distinct Antibody Response Patterns
Authors: Bunk Sebastian , Schaffert Hanne , Schmid Bianca , Goletz Christoph , Zeller Sabine , Borisova Marina , Kern Florian , Rupp Jan , Hermann Corinna
Published: March 2010

Chlamydia pneumoniaeis a frequent pathogen of the respiratory tract, and persistent infections with this obligate intracellular bacterium have been associated with different severe sequelae. Although T-cell activation during acuteC. pneumoniaeinfections has been described, little is known about the frequency or the role of theC. pneumoniae-specific memory T cells that reside in the human body after the resolution of the infection. In the present study, theC. pneumoniae-induced T-cell responses in peripheral blood mononuclear cells of 56 healthy volunteers were analyzed and compared to the donor's serum antibody reactivity toward wholeC. pneumoniaeas well as recombinantC. pneumoniaeantigens. Following short-term stimulation withC. pneumoniae, both gamma interferon (IFN-γ)- and interleukin-2 (IL-2)-producing CD4+T-cell responses could be detected in 16 of 56 healthy individuals.C. pneumoniae-activated CD4+T cells expressed CD154, a marker for T-cell receptor-dependent activation, and displayed a phenotype of central memory T cells showing dominant IL-2 production but also IFN-γ production. Interestingly, individuals with both IFN-γ- and IL-2-producing responses showed significantly decreased immunoglobulin G reactivity towardC. pneumoniaeRpoA and DnaK, antigens known to be strongly upregulated during chlamydial persistence, compared to IgG reactivity of seropositive individuals with no T-cell response or CD4+T-cell responses involving the production of a single cytokine (IFN-γ or IL-2). Our results demonstrate that memory CD4+T cells responding toC. pneumoniaestimulation can be detected in the circulation of healthy donors. Furthermore, among seropositive individuals, the presence or the absence of dual IFN-γ- and IL-2-producing T-cell responses was associated with distinct patterns of antibody responses toward persistence-associatedC. pneumoniaeantigens.
Performance of the Enferplex TB Assay with Cattle in Great Britain and Assessment of Its Suitability as a Test To Distinguish Infected and Vaccinated Animals
Authors: Whelan Clare , Whelan Adam O. , Shuralev Eduard , Kwok Hang Fai , Hewinson Glyn , Clarke John , Vordermeier H. Martin
Published: March 2010

Rapid, simple, and accurate antemortem tests for tuberculosis (TB) in cattle need to be developed in order to augment the existing screening methods. In particular, as cattle vaccines are developed, such tests would allow the continuation of test-and-slaughter policies alongside vaccination. Therefore, the development of an assay that
d
istinguishes
i
nfected from
v
accinated
a
nimals (a DIVA test) is an urgent research requirement. In this study, we assessed the performance of a novel multiplex serological test with sera collected from 96 skin-tested animals with bovine tuberculosis, 93 TB-free animals, and 39 cattle vaccinated with
Mycobacterium bovis
BCG. Our results indicate that the test has a relative sensitivity range of 77.0% to 86.5% at corresponding specificity levels of 100.0% to 77.6%. Comparison with the Bovigam gamma interferon antemortem test revealed that this serology test was significantly more sensitive at specificities above 97.9%, while the Bovigam test was, on average, about 10% more sensitive when the test specificity was set below 97%. Importantly, this serological multiplex assay does not react with sera from BCG-vaccinated calves and is therefore suitable as a DIVA test alongside BCG-based vaccine strategies.
Modulation of Gene Expression Related to Toll-Like Receptor Signaling in Dendritic Cells by Poly(γ-Glutamic Acid) Nanoparticles
Authors: Hamasaki Takayuki , Uto Tomofumi , Akagi Takami , Akashi Mitsuru , Baba Masanori
Published: March 2010

Poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) have previously been reported as an efficient antigen delivery system with adjuvant activity. In this study, the gene expression in murine bone marrow-derived dendritic cells (DCs) treated with γ-PGA NPs was examined by oligonucleotide microarray analysis and compared with that in cells treated with other adjuvants. The gene expression of proinflammatory chemokines, cytokines, and costimulatory molecules was upregulated considerably in DCs treated with γ-PGA NPs. The upregulation pattern was similar to that in DCs treated with lipopolysaccharide (LPS) but not to that in DCs treated with unparticulate γ-PGA. The activation of DCs by γ-PGA NPs was confirmed by real-time reverse transcriptase PCR (RT-PCR) analysis of genes related to Toll-like receptor (TLR) signaling. The effect of γ-PGA NPs on DCs was not annihilated by treatment with polymyxin B, an inhibitor of LPS. Furthermore, the immunization of mice with γ-PGA NPs carrying ovalbumin (OVA) as an antigen significantly induced antigen-specific CD8+T cells and antigen-specific production of interleukin-2, tumor necrosis factor alpha, and gamma interferon from the cells. Such activities of γ-PGA NPs were more potent than those obtained with immunization with OVA plus aluminum hydroxide or OVA plus complete Freund's adjuvant. These results suggest that γ-PGA NPs induce a CD8+T-cell response by activating innate immunity in a fashion different from that of LPS. Thus, γ-PGA NPs may be an attractive candidate to be developed further as a vaccine adjuvant.
Updated Postlicensure Surveillance of the Meningococcal C Conjugate Vaccine in England and Wales: Effectiveness, Validation of Serological Correlates of Protection, and Modeling Predictions of the Duration of Herd Immunity
Authors: Campbell Helen , Andrews Nick , Borrow Ray , Trotter Caroline , Miller Elizabeth
Published: March 2010

Meningococcal serogroup C conjugate (MCC) vaccines were licensed in the United Kingdom more than 10 years ago based on correlates of protection that had previously been established for serogroup C-containing polysaccharide vaccines by using the serum bactericidal antibody (SBA) assay. These correlates of protection were subsequently validated against postlicensure estimates of observed vaccine effectiveness up to 7 to 9 months after the administration of the MCC vaccine. Vaccine effectiveness was, however, shown to fall significantly more than 1 year after the administration of a 3-dose course in infancy. Despite this finding, the marked impact on serogroup C disease has been sustained, with the lowest recorded incidence (0.02 case per 100,000 population) in the 2008-2009 epidemiological year, mainly due to the indirect herd immunity effect of the vaccine in reducing carriage. Updated estimates of vaccine effectiveness through 30 June 2009 confirmed high short-term protection after vaccination in infancy, at 97% (95% confidence interval [CI], 91% to 99%), falling to 68% (95% CI, −63% to 90%) more than a year after vaccination. The observed vaccine effectiveness more than 12 months postvaccination was consistent with measured declining SBA levels, but confidence intervals were imprecise; vaccine effectiveness estimates were consistent with SBA titers of 1:4 or 1:8 as correlates of long-term protection after a primary course in infants. Modeling suggested that protection against carriage persists for at least 3 years and predicted the stabilization of serogroup C disease at low levels (fewer than 50 cases per year) up to 2015-2016.
Comparison of BD Phoenix AP Workflow with Vitek 2
Authors: Junkins Alan D. , Arbefeville Sophie S. , Howard Wanita J. , Richter Sandra S.
Published: March 2010

The BD Phoenix AP instrument reduced the manual setup time for the Phoenix system by 50%. For batches of 14 organisms, the average manual manipulation time per isolate was 89.5 s for BD Phoenix by the use of the AP instrument and 101 s for Vitek 2 (
P
< 0.001).
Clinical Potential of C-Reactive Protein and Procalcitonin Serum Concentrations To Guide Differential Diagnosis and Clinical Management of Pneumococcal and
Legionella
Pneumonia
Authors: Bellmann-Weiler Rosa , Ausserwinkler Mathias , Kurz Katharina , Theurl Igor , Weiss Guenter
Published: March 2010

We retrospectively analyzed the records of 61 hospitalized patients with community-acquired pneumonia (CAP) caused by
S
treptococcus pneumoniae
or
L
egionella pneumophila
. We found that serum procalcitonin and sodium concentrations were significantly lower, and ferritin levels were significantly higher, in patients infected with
L. pneumophila
than in those infected with
S. pneumoniae
. The ratio of C-reactive protein to procalcitonin significantly distinguished between the groups. High procalcitonin levels were associated with an adverse clinical course.
Citrobacterspp. Simultaneously HarboringblaCTX-M,blaTEM,blaSHV,blaampC, and Insertion Sequences IS26andorf513: an Evolutionary Phenomenon of Recent Concern for Antibiotic Resistance
Authors: Shahid M.
Published: March 2010

A collection of 40 confirmed isolates ofCitrobacterspp., obtained from the culture of 5,732 clinical samples during 2006 to 2008, was studied for the presence ofblaCTX-M,blaTEM,blaSHV, andblaampCby monoplex and multiplex PCRs. These isolates were also looked at for the presence of insertion elements IS26andorf513, which play an important role in the resistance gene pool.blaCTX-M,blaTEM,blaSHV, andblaampCwere noticed in 67.5%, 40%, 25%, and 40% of isolates, respectively. Sequencing for the specific CTX-M type revealed the presence of CTX-M-15-type extended-spectrum beta-lactamases (ESBL).sul1-type integrons were detected in 32.5% (13/40) of isolates, and a single plasmid of ca. ∼22 kb was also noticed in all the isolates harboringblagenes. A total of 48.2% (13/27) of isolates harboringblaCTX-Malleles were found to carry IS26elements and 53.4% (7/13) of isolates harboring thesul1-type integrons were found to carryorf513. Alarmingly, a few isolates simultaneously carried IS26andorf513, reflecting the occurrence of complex evolution. All theCitrobacterisolates were noticed to produce an amplicon of 400 bp in an IS26PCR, similar to that of the predominant UK CTX-M-15-producingEscherichia coliclone (clone A) and suggesting a probable genetic relatedness between the two, which could not be proved with British and IndianE. coliisolates in our earlier studies. Here, a first report is being presented describing the occurrence ofblaCTX-M-15in IndianCitrobacterspp. This is also the first report describing the simultaneous occurrence ofblaTEM,blaSHV, andblaampCalong withblaCTX-Min class 1 integrons. The recent findings indicate that the genetic environment ofblaCTX-M-15has changed through the insertion of IS26andorf513, although the consequences of these events remain uncertain. It perhaps also suggests a “turnover” of these mobile elements in the population over time.
Evaluation of an Immunochromatographic Test for Rapid and Reliable Serodiagnosis of Human Tularemia and Detection ofFrancisella tularensis-Specific Antibodies in Sera from Different Mammalian Species
Authors: Splettstoesser W. , Guglielmo-Viret V. , Seibold E. , Thullier P.
Published: March 2010

Tularemia is a highly contagious infectious zoonosis caused by the bacterial agentFrancisella tularensis. Serology is still considered to be a cornerstone in tularemia diagnosis due to the low sensitivity of bacterial culture and the lack of standardization in PCR methodology for the direct identification of the pathogen. We developed a novel immunochromatographic test (ICT) to efficiently detectF. tularensis-specific antibodies in sera from humans and other mammalian species (nonhuman primate, pig, and rabbit). This new tool requires none or minimal laboratory equipment, and the results are obtained within 15 min. When compared to the method of microagglutination, which was shown to be more specific than the enzyme-linked immunosorbent assay, the ICT had a sensitivity of 98.3% (58 positive sera were tested) and a specificity of 96.5% (58 negative sera were tested) on human sera. On animal sera, the overall sensitivity was 100% (22 positive sera were tested) and specificity was also 100% (70 negative sera were tested). This rapid test preferentially detects IgG antibodies that may occur early in the course of human tularemia, but further evaluation with human sera is important to prove that the ICT can be a valuable field test to support a presumptive diagnosis of tularemia. The ICT can also be a useful tool to monitor successful vaccination with subunit vaccines or live vaccine strains containing lipopolysaccharide (e.g., LVS) and to detect seropositive individuals or animals in outbreak situations or in the context of epidemiologic surveillance programs in areas of endemicity as recently recommended by the World Health Organization.
Development and Preliminary Evaluation of a Rapid Oligochromatographic Assay for Specific Detection of New Human Influenza A H1N1 Virus
Authors: Pérez-Ruiz Mercedes , Navarro-Marí José-María , Bautista-Marín María-Fé , Pedrosa-Corral Irene , Sanbonmatsu-Gámez Sara , Camacho Ana G. , Rojas José , Ruiz-Ortiz Jorge , Rodríguez-Granger Javier , Carrillo José Antonio
Published: March 2010

A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments.
Identification of Atypical
Rhodococcus
-Like Clinical Isolates as
Dietzia
spp. by 16S rRNA Gene Sequencing
Authors: Pilares Lilian , Agüero Jesús , Vázquez-Boland José A. , Martínez-Martínez Luis , Navas Jesús
Published: March 2010

Rhodococcus equi
and
Dietzia
spp. are closely related actinomycetes that show similar phenotypic properties. In humans,
R. equi
is an opportunistic pathogen associated with severe immunodeficiency.
Dietzia
spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as
R. equi
by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true
R. equi
strains, whereas the other eight were identified as members of the genus
Dietzia
, including
Dietzia maris
(four isolates),
Dietzia natronolimnaea
(two isolates), and
Dietzia timorensis
and
Dietzia
sp. (one isolate each). The eight
Dietzia
isolates were highly sensitive to 12 antimicrobial compounds.
Comparison of Inhibitory Mold Agar to Sabouraud Dextrose Agar as a Primary Medium for Isolation of Fungi
Authors: Scognamiglio Theresa , Zinchuk Riva , Gumpeni Pramod , Larone Davise H.
Published: March 2010

Clinical specimens cultured on two selective fungal media, inhibitory mold agar (IMA) and Sabouraud dextrose agar (SDA), were compared with respect to recovery of fungi. Of the 840 fungal isolates recovered, 69.3% grew on both IMA and SDA; 24.9% grew only on IMA; and 5.8% grew only on SDA, showing that IMA is superior (
P
= 0.003).
Reliability of Antimalarial Sensitivity Tests Depends on Drug Mechanisms of Action
Authors: Wein Sharon , Maynadier Marjorie , Tran Van Ba Christophe , Cerdan Rachel , Peyrottes Suzanne , Fraisse Laurent , Vial Henri
Published: March 2010

In vitro
antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used
in vitro
antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.
Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus
Authors: Nordgren Johan , Bucardo Filemón , Svensson Lennart , Lindgren Per-Eric
Published: March 2010

We have developed a light-upon-extension (LUX) real-time PCR assay for detection, quantification, and genogrouping of group A rotavirus (RV), the most common cause of acute gastroenteritis in children. The LUX system uses a fluorophore attached to one primer and having a self-quenching hairpin structure, making it cost-effective and specific. We designed genogroup-specific primers having different fluorophores, making it possible to differentiate between the two main genogroups of human group A RVs. The assay was applied on clinical stool specimens from Sweden and Central America (
n
= 196) and compared to immunological and conventional PCR assays. The genogrouping ability was further validated against a subset of clinical specimens, which had been genogrouped using monoclonal antibodies. Our real-time PCR assay detected and quantified all positive specimens (
n
= 145) and exhibited higher sensitivity than immunological assays and conventional PCR. The assay exhibited a wide dynamic range, detecting from 5 to >10
7
genes per PCR, resulting in a theoretical lower detection limit of <10,000 viruses per gram of stool. No cross-reaction was observed with specimens containing norovirus, sapovirus, astrovirus, or adenovirus. In total, 22 (15%) of the positive clinical specimens were identified as genogroup I, 122 (84%) were identified as genogroup II, and 1 specimen was found to contain a mix of both genogroups. All genogroup I-positive specimens were associated with capsid glycoprotein 2 (G2). No significant difference in viral load was found between genogroups or geographic region. The detection and quantification, combined with the genogrouping ability, make this assay a valuable tool both for diagnostics and for molecular epidemiological investigations.
Comparison of the Vitek 2 Antifungal Susceptibility System with the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Broth Microdilution Reference Methods and with the Sensititre YeastOne and Etest Techniques forIn VitroDetection of Antifungal Resistance in Yeast Isolates
Authors: Cuenca-Estrella Manuel , Gomez-Lopez Alicia , Alastruey-Izquierdo Ana , Bernal-Martinez Leticia , Cuesta Isabel , Buitrago Maria J. , Rodriguez-Tudela Juan L.
Published: March 2010

The commercial technique Vitek 2 system for antifungal susceptibility testing of yeast species was evaluated. A collection of 154 clinical yeast isolates, including amphotericin B- and azole-resistant organisms, was tested. Results were compared with those obtained by the reference procedures of both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Two other commercial techniques approved for clinical use, the Etest and the Sensititre YeastOne, were included in the comparative exercise as well. The average essential agreement (EA) between the Vitek 2 system and the reference procedures was >95%, comparable with the average EAs observed between the reference procedures and the Sensititre YeastOne and Etest. The EA values were >97% forCandidaspp. and stood at 92% forCryptococcus neoformans. Intraclass correlation coefficients (ICC) between the commercial techniques and the reference procedures were statistically significant (P< 0.01). Percentages of very major errors were 2.6% between Vitek 2 and the EUCAST technique and 1.6% between Vitek 2 and the CLSI technique. The Vitek 2 MIC results were available after 14 to 18 h of incubation for allCandidaspp. (average time to reading, 15.5 h). The Vitek 2 system was shown to be a reliable technique to determine antifungal susceptibility testing of yeast species and a more rapid and easier alternative for clinical laboratories than the procedures developed by either the CLSI or EUCAST.
The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering
Authors: He Lilin , Piper Amanda , Meilleur Flora , Myles Dean A. A. , Hernandez Raquel , Brown Dennis T. , Heller William T.
Published: March 2010

The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.
Human Papillomaviruses Modulate Expression of MicroRNA 203 upon Epithelial Differentiation to Control Levels of p63 Proteins
Authors: Melar-New Marta , Laimins Laimonis A.
Published: March 2010

Human papillomaviruses (HPV) link their life cycles to epithelial differentiation and induce productive replication of viral DNA in suprabasal cells. Viral-DNA amplification requires cells to remain active in the cell cycle upon differentiation. This is in contrast to normal cells, which lose proliferative capability upon differentiation. One factor that negatively regulates proliferative capability upon differentiation is microRNA 203 (miR-203), which is expressed primarily in suprabasal epithelial cells. Although HPVs do not encode their own microRNAs (miRNAs), they modulate expression of cellular miRNAs to regulate the activities of cellular proteins. We show that the HPV E7 protein downregulates miR-203 expression upon differentiation, which may occur through the mitogen-activated protein (MAP) kinase/protein kinase C (PKC) pathway. One target of miR-203 is the p63 family of transcription factors, and we demonstrate that HPV-positive cells maintain significantly higher levels of these factors upon differentiation than do normal keratinocytes. Several downstream targets of p63, CARM-1, p21, and Bax, were also increased in E7-expressing cells, and their levels were inversely correlated with amounts of miR-203. Introduction of expression vectors for miR-203 into keratinocytes that stably maintain HPV episomes resulted in short-term elevation of HPV genome copy numbers, but these were rapidly lost upon subsequent passage. When HPV-positive cells expressing high levels of miR-203 were induced to differentiate in methylcellulose, impaired genome amplification was observed. We conclude that high levels of miR-203 are inhibitory to HPV amplification and that HPV proteins act to suppress expression of this microRNA to allow productive replication in differentiating cells.
Visualizing Viral Dissemination in the Mouse Nervous System, Using a Green Fluorescent Protein-Expressing Borna Disease Virus Vector
Authors: Ackermann Andreas , Guelzow Timo , Staeheli Peter , Schneider Urs , Heimrich Bernd
Published: March 2010

Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.
Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel
Bombyx mori
Nucleopolyhedrovirus-Based Vector with a Modified
fp25K
Gene
Authors: Nakanishi Tadashi , Goto Chie , Kobayashi Michihiro , Kang WonKyung , Suzuki Takehiro , Dohmae Naoshi , Matsumoto Shogo , Shimada Toru , Katsuma Susumu
Published: March 2010

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant
Bombyx mori
NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional
fp25K
but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of
Autographa californica
multiple NPV (AcMNPV)
fp25K
into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV
fp25K
and examined its potential in BmN cells and
B. mori
larvae. Our results showed that the introduction of AcMNPV
fp25K
significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.
Influenza Virus M2 Ion Channel Protein Is Necessary for Filamentous Virion Formation
Authors: Rossman Jeremy S. , Jing Xianghong , Leser George P. , Balannik Victoria , Pinto Lawrence H. , Lamb Robert A.
Published: March 2010

Influenza A virus buds from cells as spherical (∼100-nm diameter) and filamentous (∼100 nm × 2 to 20 μm) virions. Previous work has determined that the matrix protein (M1) confers the ability of the virus to form filaments; however, additional work has suggested that the influenza virus M2 integral membrane protein also plays a role in viral filament formation. In examining the role of the M2 protein in filament formation, we observed that the cytoplasmic tail of M2 contains several sites that are essential for filament formation. Additionally, whereas M2 is a nonraft protein, expression of other viral proteins in the context of influenza virus infection leads to the colocalization of M2 with sites of virus budding and lipid raft domains. We found that an amphipathic helix located within the M2 cytoplasmic tail is able to bind cholesterol, and we speculate that M2 cholesterol binding is essential for both filament formation and the stability of existing viral filaments.
Anti-gp41 Antibodies Cloned from HIV-Infected Patients with Broadly Neutralizing Serologic Activity
Authors: Pietzsch John , Scheid Johannes F. , Mouquet Hugo , Seaman Michael S. , Broder Christopher C. , Nussenzweig Michel C.
Published: March 2010

Most HIV-infected individuals develop antibodies to the gp120 and gp41 components of the viral spike; however, only a fraction of these individuals mount a broadly neutralizing serum response against HIV. We have cloned anti-HIV antibodies from the memory B-cell compartment of six individuals with variable viral loads and high titers of broadly neutralizing antibodies. Here, we report on the features of the anti-gp41 response in these patients. Competition experiments with previously characterized antibodies targeting defined epitopes on the gp41 ectodomain showed antibodies directed against the “immunodominant region” (cluster I), the carboxy-terminal heptad repeat (cluster II), and the membrane-proximal external region (cluster IV). On the other hand, antibodies directed against the amino-terminal part of the molecule, including the fusion peptide, polar region, and the N-terminal heptad repeat, were not detected. When all patients' data were combined, unique B-cell clones targeting cluster I, II, and IV accounted for 32%, 49%, and 53% of all anti-gp41-reactive B cells, respectively; therefore, no single region was truly immunodominant. Finally, although we found no new neutralizing epitopes or HIV-1-neutralizing activity by any of the gp41 antibodies at concentrations of up to 50 μg/ml, high concentrations of 7 out of 15 anti-cluster I antibodies neutralized tier 2 viruses.
A Human Herpesvirus MicroRNA Inhibits p21 Expression and Attenuates p21-Mediated Cell Cycle Arrest
Authors: Gottwein Eva , Cullen Bryan R.
Published: March 2010

The oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) expresses 12 viral microRNAs (miRNAs) in latently infected cells. Here, we report that cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest, are direct targets for KSHV miR-K1. Ectopically expressed KSHV miR-K1 specifically inhibited the expression of endogenous p21 in KSHV-negative cells and strongly attenuated the cell cycle arrest that normally occurs upon p53 activation, yet miR-K1 did not prevent the induction of other p53-responsive genes. Stable knockdown of miR-K1 in latently KSHV-infected human primary effusion lymphoma (PEL) B cells revealed a derepression of p21 expression and enhanced cell cycle arrest following activation of p53. Our data demonstrate that miR-K1 represses the expression of p21, a protein with known tumor suppressor functions, and suggest that this KSHV miRNA is likely to contribute to the oncogenic potential of this opportunistic viral pathogen.
Glycoprotein D of Bovine Herpesvirus 5 (BoHV-5) Confers an Extended Host Range to BoHV-1 but Does Not Contribute to Invasion of the Brain
Authors: Gabev Evgeni , Tobler Kurt , Abril Carlos , Hilbe Monika , Senn Claudia , Franchini Marco , Campadelli-Fiume Gabriella , Fraefel Cornel , Ackermann Mathias
Published: March 2010

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their
in vitro
and
in vivo
host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins.
In vivo
, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.
Sequence Variability in Clinical and Laboratory Isolates of Herpes Simplex Virus 1 Reveals New Mutations
Authors: Szpara Moriah L. , Parsons Lance , Enquist L. W.
Published: March 2010

Herpes simplex virus 1 (HSV-1) is a well-adapted human pathogen that can invade the peripheral nervous system and persist there as a lifelong latent infection. Despite their ubiquity, only one natural isolate of HSV-1 (strain 17) has been sequenced. Using Illumina high-throughput sequencing of viral DNA, we obtained the genome sequences of both a laboratory strain (F) and a low-passage clinical isolate (H129). These data demonstrated the extent of interstrain variation across the entire genome of HSV-1 in both coding and noncoding regions. We found many amino acid differences distributed across the proteome of the new strain F sequence and the previously known strain 17, demonstrating the spectrum of variability among wild-type HSV-1 proteins. The clinical isolate, strain H129, displays a unique anterograde spread phenotype for which the causal mutations were completely unknown. We have defined the sequence differences in H129 and propose a number of potentially causal genes, including the neurovirulence protein ICP34.5 (RL1). Further studies will be required to demonstrate which change(s) is sufficient to recapitulate the spread defect of strain H129. Unexpectedly, these data also revealed a frameshift mutation in the UL13 kinase in our strain F isolate, demonstrating how deep genome sequencing can reveal the full complement of background mutations in any given strain, particularly those passaged or plaque purified in a laboratory setting. These data increase our knowledge of sequence variation in large DNA viruses and demonstrate the potential of deep sequencing to yield insight into DNA genome evolution and the variation among different pathogen isolates.
Both E Protein Glycans Adversely Affect Dengue Virus Infectivity but Are Beneficial for Virion Release
Authors: Lee Eva , Leang Sook Kwan , Davidson Andrew , Lobigs Mario
Published: March 2010

The E protein of most flaviviruses is modified by Asn-linked glycosylation at residue 153/154 and in the case of the four dengue virus (DENV) serotypes by a second glycan at residue 67. However, the absence of E protein glycosylation among numerous natural isolates of different flaviviruses suggests that the glycan,per se, is not critically important in the virus life cycle. Consistent with this notion, we show that ablation of both glycans from the DENV-2 E protein reduces but does not prevent growth of the variant in mammalian and mosquito cells. We found a pronounced and opposing effect of glycan ablation on two stages of the virus growth cycle: infectivity and release. Loss of either of the two DENV E protein glycans markedly enhanced infectivity of variants for mosquito cells at the expense of efficient virion release. The variants also displayed reduced release in mammalian cells, which was more prominent for viruses lacking the Asn 67-linked glycan than for those lacking the Asn 153-linked glycan, without a marked change in infectivity. Mutations, which compensated for the defect in virus morphogenesis associated with ablation of the Asn 67-linked glycan in mammalian cells but interestingly not in mosquito cells, were identified at the glycosylation acceptor motif and a second site in E protein domain II. The dueling influences of infectivity and release on virus growth affected by the glycans may explain the plasticity in E protein glycosylation among the flaviviruses.
Different Potential of C-Type Lectin-Mediated Entry between Marburg Virus Strains
Authors: Matsuno Keita , Kishida Noriko , Usami Katsuaki , Igarashi Manabu , Yoshida Reiko , Nakayama Eri , Shimojima Masayuki , Feldmann Heinz , Irimura Tatsuro , Kawaoka Yoshihiro , Takada Ayato
Published: March 2010

The glycoproteins (GPs) of filoviruses are responsible for virus entry into cells. It is known that GP interacts with cellular C-type lectins for virus attachment to cells. Since primary target cells of filoviruses express C-type lectins, C-type lectin-mediated entry is thought to be a possible determinant of virus tropism and pathogenesis. We compared the efficiency of C-type lectin-mediated entry between Marburg virus strains Angola and Musoke by using a vesicular stomatitis virus (VSV) pseudotype system. VSV pseudotyped with Angola GP (VSV-Angola) infected K562 cells expressing the C-type lectin, human macrophage galactose-type C-type lectin (hMGL), or dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) more efficiently than VSV pseudotyped with Musoke GP (VSV-Musoke). Unexpectedly, the binding affinity of the C-type lectins to the carbohydrates on GPs did not correlate with the different efficiency of C-type lectin-mediated entry. Site-directed mutagenesis identified the amino acid at position 547, which switched the efficiency of C-type lectin-mediated entry. In a three-dimensional model of GP, this amino acid was in close proximity to the putative site of cathepsin processing. Interestingly, the cathepsin inhibitors reduced the infectivity of VSV-Angola less efficiently than that of VSV-Musoke in C-type lectin-expressing K562 cells, whereas only a limited difference was found in control cells. The amino acid at position 547 was critical for the different effects of the inhibitors on the virus infectivities. These results suggest that the efficiency of C-type lectin-mediated entry of filoviruses is controlled not only by binding affinity between C-type lectins and GP but also by mechanisms underlying endosomal entry, such as proteolytic processing by the cathepsins.
Translation Control by Protein Kinase R Restricts Minute Virus of Mice Infection: Role in Parvovirus Oncolysis
Authors: Ventoso Iván , Berlanga Juan J. , Almendral José M.
Published: March 2010

The relevance of translational control in the gene expression and oncotropism of the autonomous parvoviruses was investigated with MVMp, the prototype strain of minute virus of mice (MVM), infecting normal and transformed rodent and human cells of different tissue origins. Mouse embryo fibroblasts (MEFs) and NIH 3T3 fibroblasts were resistant to MVMp infection, but 3T3 fibroblasts derived from double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) knockout mice (PKR
o/o
) behaved in a manner that was highly permissive to productive MVMp replication. NIH 3T3 resistance correlated with significant phosphorylation of eukaryotic translation initiation factor 2 (eIF2) occurring at early time points after infection. Permissive PKR
o/o
cells were converted to MVMp-restrictive cells after reintroduction of the PKR gene by transfection. Conversely, regulated expression of the vaccinia virus E3 protein, a PKR inhibitor, in MEFs prevented eIF2α phosphorylation and increased MVMp protein synthesis.
I
n vitro
-synthesized genome-length R1 mRNA of MVMp was a potent activator of PKR. Virus-resistant primary MEFs and NIH 3T3 cells responded to MVMp infection with significant increases in eIF2α phosphorylation. In contrast, virus-permissive mouse (PKR
o/o
, BHK21, and A9) and human transformed (NB324K fibroblast, U373 glioma, and HepG2 hepatoma) cells consistently showed no significant increase in the level of eIF2α phosphorylation following MVMp infection. The synthesis of the viral NS1 protein was inversely correlated with the steady-state PKR levels. Our results show that the PKR-mediated antiviral response is an important mechanism for control of productive MVMp infection, and its impairment in human transformed cells allowed efficient MVMp gene expression. PKR translational control may therefore contribute to the oncolysis of MVMp and other autonomous parvoviruses.
Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1
Authors: Niskanen Einari A. , Ihalainen Teemu O. , Kalliolinna Olli , Häkkinen Milla M. , Vihinen-Ranta Maija
Published: March 2010

The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here we have identified key ATP-binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from the conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of infectious viruses. When provided in
trans
, all except the R508A mutation reduced infectivity in a dominant-negative manner, possibly by hindering genome replication. These results suggest that the conserved R510 residue, but not R508, is the arginine finger sensory element of CPV NS1. Moreover, fluorescence recovery after photobleaching (FRAP), complemented by computer simulations, was used to assess the binding properties of mutated fluorescent fusion proteins. These experiments identified ATP-dependent and -independent binding modes for NS1 in living cells. Only the K406M mutant had a single binding site, which was concluded to indicate ATP-independent binding. Furthermore, our data suggest that DNA binding of NS1 is dependent on its ability to both bind and hydrolyze ATP.