bluuebunny/crossref_metadata_2025 huggingface dataset:
The Rate and Molecular Spectrum of Spontaneous Mutations in
Arabidopsis thaliana
Authors: Ossowski Stephan , Schneeberger Korbinian , Lucas-Lledó José Ignacio , Warthmann Norman , Clark Richard M. , Shaw Ruth G. , Weigel Detlef , Lynch Michael
Published: December 2009

Evolution in Action

Rates of evolution in gene and genome sequences have been estimated, but these estimates are subject to error because many of the steps of evolution over the ages are not directly measurable or are hidden under subsequent changes.

Ossowski
et al.

(p.
92
) now provide a more accurate measurement of how often spontaneous mutations arise in a nuclear genome. Mutations arising over 30 generations were compared by sequencing DNA from individual
Arabidopsis thaliana
plants. UV- and deamination-induced mutagenesis appeared to bias the type of mutations found.
Solid Nanoparticles that Catalyze Biofuel Upgrade Reactions at the Water/Oil Interface
Authors: Crossley Steven , Faria Jimmy , Shen Min , Resasco Daniel E.
Published: December 2009

Metal in the Middle

Biphasic reaction mixtures allow the isolation of sensitive products, which can form in one solvent and then shift rapidly into another, protected from side reactions and interfering by-products. However, ensuring that catalysts remain in the proper phase can be challenging, and surfactants added to induce efficient mixing often prove difficult to separate from the product stream.

Crossley
et al.

(p.
68
; see the Perspective by

Cole-Hamilton

) tackle these issues by preparing easily recoverable amphiphilic nanoparticles that simultaneously stabilize aqueous-organic emulsions and catalyze organic reactions. The particles combine hydrophobic nanotubes with hydrophilic oxides, causing them to accumulate at water-hydrocarbon interfaces. Depositing palladium on specific portions of the particles' surfaces thus localizes the metal in one or both phases, facilitating hydrogenation of several compounds of interest in biofuel refining.
Translocation of Single-Stranded DNA Through Single-Walled Carbon Nanotubes
Authors: Liu Haitao , He Jin , Tang Jinyao , Liu Hao , Pang Pei , Cao Di , Krstic Predrag , Joseph Sony , Lindsay Stuart , Nuckolls Colin
Published: December 2009

Carbon Nanotube Bridge for DNA Transport

The nanoporosity of carbon nanotubes has been exploited in the control of molecular transport—for example, in creating membranes.

Liu
et al.

(p.
64
) fabricated devices in which one single-walled carbon nanotube connects two fluid reservoirs. In some of these devices, apparently those in which the nanotube is metallic, the ionic conductivity is anomalously higher than that expected from the bulk resistivity of the electrolyte. This high conductivity was exploited for the transport of single-stranded DNA, which was accompanied by large but transient increases in the ion current.
Masquerade: Camouflage Without Crypsis
Authors: Skelhorn John , Rowland Hannah M. , Speed Michael P. , Ruxton Graeme D.
Published: December 2009

Caterpillars masquerading as twigs are misidentified by chick predators as inanimate objects, rather than remaining undetected.
Serendipitous Astronomy
Authors: Lang Kenneth R.
Published: December 2009

Many of the seminal discoveries in astronomy have been unanticipated.
Brain Activity to Rely On?
Authors: Schwarzkopf D. Sam , Rees Geraint
Published: December 2009

The characteristics of neuronal activity that mark whether consciousness arises include how reproducible neuronal response patterns are to a sensory stimulus.
Molecular Donuts and Donut Holes
Authors: Whitmire Kenton H.
Published: December 2009

A transient template steers the self-assembly of a “giant” metal oxide wheel.
Janus Catalysts Direct Nanoparticle Reactivity
Authors: Cole-Hamilton David J.
Published: December 2009

Metal oxide nanoparticles decorated with carbon nanotubes can be turned into readily recovered catalysts that function at the interface between oil and water phases.
Opportunities for Research and NIH
Authors: Collins Francis S.
Published: December 2009

The promise of fundamental advances in diagnosis, prevention, and treatment of disease has never been greater.
Clarity on Honey Bee Collapse?
Authors: Ratnieks Francis L. W. , Carreck Norman L.
Published: January 2010

The worldwide losses of honey bee colonies continue to puzzle researchers and the beekeeping industry.
Promoting Scientific Standards
Authors: Alberts Bruce
Published: December 2009

The scientific enterprise is built on a foundation of trust. As Kenneth Shine and I emphasized 15 years ago in this journal, if science is to flourish and attain its appropriate role in aiding human progress, “It is incumbent upon all of us in the scientific community to help provide a research environment that, through its adherence to high ethical standards and creative productivity, will attract and retain individuals of outstanding intellect and character to one of society's most important professions.”
*
Obama Backs New Launcher And Bigger NASA Budget
Authors: Lawler Andrew
Published: December 2009

President Barack Obama plans to ask Congress to cancel work on a new rocket and instead fund a heavy-lift launcher to take humans to the moon, asteroids, and the moons of Mars.
Senate Looms as Bigger Hurdle After Copenhagen
Authors: Kintisch Eli
Published: December 2009

As hard as it was to negotiate a deal in Denmark, President Barack Obama faces an even tougher time in Washington trying to convince the U.S. Senate to pass cap-and-trade legislation. And he's up against a ticking clock: the November 2010 elections that will decide whether his party retains control of Congress.
Protests by Staff, Advisers Rattle Australian Synchrotron
Authors: Finkel Elizabeth
Published: December 2009

Australian Synchrotron (AS) Director Robert Lamb and board members have refused to discuss details of his recent firing. But many observers say that a gaping rift developed between the board of directors on one hand and AS staff and the international scientific advisory committee on the other over the slow pace of developing long-term plans.
U.K. Physicists Cry Foul At Major Budget Cuts
Authors: Clery Daniel
Published: December 2009

Administrative changes and the ravages of the economy have left the Science and Technology Facilities Council, the British body responsible for particle physics, astronomy, nuclear physics, and space science, with a gaping hole in its finances. In the 5-year funding plan announced in mid-December, nuclear physics has been pared to the bone, with just £30 million for the next 5 years, amounting to a cut of 29% over that period.
Recession Hits Some Sciences Hard At Florida State University
Authors: Kerr Richard A.
Published: December 2009

At Florida State University—one of the "big four" Florida state schools—a statewide fiscal crisis has turned into a ravaging torrent for a few departments, most of them in the sciences.
In the Afterglow Of the Big Bang
Authors: Bhattacharjee Yudhijit
Published: December 2009

Toiling behind the Iron Curtain under a tough mentor, a Russian astrophysicist uncovered secrets of the universe that have led to discoveries 4 decades later.
2010 Science Budget Not Apocalyptic, as Feared
Authors: Normile Dennis
Published: January 2010

Although some projects will absorb big hits, the first budget of Japan's new administration, approved by the Cabinet at the end of December, calls for relatively minor changes in science priorities.
Europe's Bats Resist Fungal Scourge of North America
Authors: Stokstad Erik
Published: January 2010

The same fungus that has devastated bat colonies in the northeastern United States has been identified for the first time in Europe—in a healthy bat.
Genotypes Coding for Low Serum Levels of Mannose-Binding Lectin Are Underrepresented among Individuals Suffering from Noninfectious Systemic Inflammatory Response Syndrome
Authors: Smithson Alex , Perello Rafael , Aibar Jesus , Espinosa Gerard , Tassies Dolors , Freire Carolina , Castro Pedro , Suarez Belen , Lozano Francisco , Nicolas Josep-Maria
Published: December 2009

Gene polymorphisms, giving rise to low serum levels of mannose-binding lectin (MBL) or MBL-associated protease 2 (MASP2), have been associated with an increased risk of infections. The objective of this study was to assess the outcome of intensive care unit (ICU) patients with systemic inflammatory response syndrome (SIRS) regarding the existence of functionally relevantMBL2andMASP2gene polymorphisms. The study included 243 ICU patients with SIRS admitted to our hospital, as well as 104 healthy control subjects.MBL2andMASP2single nucleotide polymorphisms were genotyped using a sequence-based typing technique. No differences were observed regarding the frequencies of low-MBL genotypes (O/O and XA/O) andMASP2polymorphisms between patients with SIRS and healthy controls. Interestingly, ICU patients with a noninfectious SIRS had a lower frequency for low-MBL genotypes and a higher frequency for high-MBL genotypes (A/A and A/XA) than either ICU patients with an infectious SIRS or healthy controls. The existence of low- or /high-MBL genotypes or aMASP2polymorphism had no impact on the mortality rates of the included patients. The presence of high-MBL-producing genotypes in patients with a noninfectious insult is a risk factor for SIRS and ICU admission.
Proof of Principle for Successful Characterization of Methicillin-Resistant Coagulase-Negative Staphylococci Isolated from Skin by Use of Raman Spectroscopy and Pulsed-Field Gel Electrophoresis
Authors: Willemse-Erix H. F. M. , Jachtenberg J. , Barutçi H. , Puppels G. J. , van Belkum A. , Vos M. C. , Maquelin K.
Published: December 2009

Coagulase-negative staphylococci (CNS) are among the most frequently isolated bacterial species in clinical microbiology, and most CNS-related infections are hospital acquired. Distinguishing between these frequently multiple-antibiotic-resistant isolates is important for both treatment and transmission control. In this study we used isolates of methicillin-resistant coagulase-negative staphylococci (MR-CNS) that were selected from a large surveillance study of the direct spread of MR-CNS. This strain collection was used to evaluate (i) Raman spectroscopy as a typing tool for MR-CNS isolates and (ii) diversity between colonies with identical and different morphologies. Reproducibility was high, with 215 of 216 (99.5%) of the replicate samples for 72 isolates ending up in the same cluster. The concordance with pulsed-field gel electrophoresis (PFGE)-based clusters was 94.4%. We also confirm that the skin of patients can be colonized with multiple MR-CNS types at the same time. Morphological differences between colonies from a single patient sample correlated with differences in Raman and PFGE types. Some morphologically indistinguishable colonies revealed different Raman and PFGE types. This indicates that multiple MR-CNS colonies should be examined to obtain a complete insight into the prevalence of different types and to be able to perform an accurate transmission analysis. Here we show that Raman spectroscopy is a reproducible typing system for MR-CNS isolates. It is a tool for screening variability within a collection of isolates. Because of the high throughput, it enables the analysis of multiple colonies per patient, which will enhance the quality of clinical and epidemiological studies.
Results of the Abbott RealTi
m
e HIV-1 Assay for Specimens Yielding “Target Not Detected” Results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test
Authors: Babady N. Esther , Germer Jeffrey J. , Yao Joseph D. C.
Published: December 2009

No significantly discordant results were observed between the Abbott RealTi
m
e HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding “target not detected” results by CTM.
Italian National Survey of Blood Donors: External Quality Assessment (EQA) of Syphilis Testing
Authors: Vulcano Francesca , Milazzo Luisa , Volpi Sabrina , Battista Mara Maria , Barca Alessandra , Hassan Hamisa Jane , Pimpinelli Fulvia , Giampaolo Adele
Published: December 2009

The detection of syphilis among blood donors may reveal high-risk sexual behavior, which can go unreported at the time of donor selection and compromise the safety of the donated blood. In Italy, blood is collected, tested, and distributed by transfusion services (TSs), which also perform outpatient transfusions. Although the TSs must screen for syphilis by law, there are no indications of the specific type of method to be used, generating discrepancies in the results obtained by the different TSs. To determine the proficiency of the TSs in screening for syphilis, we performed an external quality assessment (EQA). The EQA was based on two shipments of serum panels; 133 and 118 of the 326 existing TSs participated in the first and second shipments, respectively. Each panel consisted of both positive and negative serum samples. The results confirmed that the use of a single nontreponemal test (the Venereal Disease Research Laboratory [VDRL] and the rapid plasma reagin [RPR] tests) is the least sensitive means of identifying samples that are positive for syphilis antibodies. We also found that the interpretation of the results of manual techniques, such as the RPR test, the VDRL test, the
Treponema pallidum
hemagglutination (TPHA) assay, and the
T. pallidum
particle agglutination (TPPA) assay, can vary greatly among different TSs and operators. Total Ig enzyme immunoassays (EIAs) are the most sensitive. However, the determination of syphilis on the basis of the results of a single test is not sufficient for an accurate screening; and all blood units should thus be assessed by two distinct treponemal tests, that is, a total Ig EIA and the TPHA or the TPPA assay.
Clonal Distribution of Disease-Associated and Healthy Carrier Isolates ofNeisseria meningitidisbetween 1983 and 2005 in Cuba
Authors: Climent Yanet , Yero Daniel , Martinez Isabel , Martín Alejandro , Jolley Keith A. , Sotolongo Franklin , Maiden Martin C. J. , Urwin Rachel , Pajón Rolando
Published: December 2009

In response to epidemic levels of serogroup B meningococcal disease in Cuba during the 1980s, the VA-MENGOC-BC vaccine was developed and introduced into the National Infant Immunization Program in 1991. Since then the incidence of meningococcal disease in Cuba has returned to the low levels recorded before the epidemic. A total of 420Neisseria meningitidisstrains collected between 1983 and 2005 in Cuba were analyzed by multilocus sequence typing (MLST). The set of strains comprised 167 isolated from disease cases and 253 obtained from healthy carriers. By MLST analysis, 63 sequence types (STs) were identified, and 32 of these were reported to be a new ST. The Cuban isolates were associated with 12 clonal complexes; and the most common were ST-32 (246 isolates), ST-53 (86 isolates), and ST-41/44 (36 isolates). This study also showed that the application of VA-MENGOC-BC, the Cuban serogroup B and C vaccine, reduced the frequency and diversity of hypervirulent clonal complexes ST-32 (vaccine serogroup B type-strain) and ST-41/44 and also affected other lineages. Lineages ST-8 and ST-11 were no longer found during the postvaccination period. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.
Application of Truncated Immunodominant Polypeptide from Hepatitis E Virus (HEV) ORF2 in an Assay To Exclude Nonspecific Binding in Detecting Anti-HEV Immunoglobulin M
Authors: Pan Jin-Shun , Zhang Kui , Zhou Jinyong , Wu Chao , Zhuang Hui , Zhou Yi-Hua
Published: December 2009

The diagnosis of recent hepatitis E virus (HEV) infection depends on serologic testing for anti-HEV IgM; however, false-positive results may occur. In the present study, we cloned the ORF2 fragment of genotype 4 HEV and demonstrated that a subregion covering amino acids 459 to 607 in ORF2 forms the immunodominant B-cell epitopes, as it does in genotype 1 viruses. Truncation of several residues from either the N or C terminus of the polypeptide abolished the reactivity of anti-HEV from naturally infected persons. By the combination of high reactivity of the immunodominant polypeptide and poor reactivity of the truncated polypeptide, we established an indirect enzyme-linked immunosorbent assay (ELISA) to detect anti-HEV IgM. In this assay, all 37 sera that were HEV RNA positive reacted with the immunodominant polypeptide but not with the truncated one, and none of 159 sera from healthy persons reacted with either of the polypeptides. In retesting of 117 sera that originally tested positive for anti-HEV IgM, using a Genelabs kit, only 34 were positive and 83 were negative. Western blot analyses and other experiments strongly indicated that these 83 discordant sera were negative for anti-HEV IgM. Furthermore, among the 117 sera, 5 reacted with both the immunodominant and truncated polypeptides, with comparable optical densities at 450 nm. However, their reactivity was demonstrated to result from nonspecific binding. Together, the data indicate that the poor reactivity of a truncated ORF2 polypeptide can be used to exclude nonspecific binding in the detection of anti-HEV IgM.
Cytospin-Enhanced Immunofluorescence and Impact of Sample Quality on Detection of Novel Swine Origin (H1N1) Influenza Virus
Authors: Landry Marie L. , Ferguson David
Published: December 2009

Nasopharyngeal swabs (
n
= 601) from 278 adult and 323 pediatric patients were tested within 24 h of receipt by cytospin-enhanced direct immunofluorescence antibody testing (DFA) and real-time reverse transcriptase PCR (RT-PCR) using the CDC assay. Cytospin-enhanced DFA detected 230 (84.6%) of 272 swine influenza A PCR-positive results overall but 25 (92.6%) of 27 positive results in patients less than 5 years old and 208 (96.7%) of 215 positive samples with cycle threshold values of <26.
Computational Analysis Identifies Human Adenovirus Type 55 as a Re-Emergent Acute Respiratory Disease Pathogen
Authors: Walsh Michael P. , Seto Jason , Jones Morris S. , Chodosh James , Xu Wenbo , Seto Donald
Published: December 2009

Novel human adenoviruses (HAdVs) arise from genome recombination. Analysis of HAdV type 55 from an outbreak in China shows a hexon recombination between HAdV-B11 and HAdV-B14, resulting in a genome that is 97.4% HAdV-B14. Sporadic appearances as a re-emergent pathogen and misidentification as “HAdV-B11a” are due to this partial hexon.
Multilocus Sequence Typing of
Clostridium difficile
Authors: Griffiths David , Fawley Warren , Kachrimanidou Melina , Bowden Rory , Crook Derrick W. , Fung Rowena , Golubchik Tanya , Harding Rosalind M. , Jeffery Katie J. M. , Jolley Keith A. , Kirton Richard , Peto Tim E. , Rees Gareth , Stoesser Nicole , Vaughan Alison , Walker A. Sarah , Young Bernadette C. , Wilcox Mark , Dingle Kate E.
Published: December 2009

A robust high-throughput multilocus sequence typing (MLST) scheme for
Clostridium difficile
was developed and validated using a diverse collection of 50 reference isolates representing 45 different PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (
http://pubmlst.org/cdifficile/
) to facilitate the dissemination and comparison of
C. difficile
MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of
C. difficile
in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.
Evidence of Emergence of New GGII.4 Norovirus Variants from Gastroenteritis Outbreak Survey in France during the 2007-to-2008 and 2008-to-2009 Winter Seasons
Authors: Belliot Gaël , Kamel A. H. , Estienney M. , Ambert-Balay K. , Pothier P.
Published: December 2009

Analysis of 316 outbreaks of gastroenteritis in France from September 2007 through March 2009 showed that genogroup II.4 (GGII.4) noroviruses were predominant and mostly belonged to the 2006b variant. However, the new GGII.4 variants, variant 2008 and the newly discovered Cairo variant from the Middle East, were also detected. The epidemiological survey suggests that these new variants might become the next predominant strains.
Emergence of SCC
mec
Type IV and SCC
mec
Type V Methicillin-Resistant
Staphylococcus aureus
Containing the Panton-Valentine Leukocidin Genes in a Large Academic Teaching Hospital in Central Switzerland: External Invaders or Persisting Circulators?
Authors: Valsesia Giorgia , Rossi Marco , Bertschy Sonja , Pfyffer Gaby E.
Published: December 2009

The hospital epidemiology of methicillin-resistant
Staphylococcus aureus
(MRSA) has changed in the past few years due to the encroachment of community-associated MRSA (CA-MRSA) strains into health care settings. MRSA strains that were isolated during a 2-year period from patients of the Luzerner Kantonsspital were analyzed to elucidate their epidemiology. Moreover, extended surveillance of individuals who were contacts of those patients was carried out for 6 months to identify the routes of spread and to assess the quality of the infection control measures used in our setting. Patient data were collected to distinguish CA-MRSA strains from health care-associated MRSA (HA-MRSA) strains by epidemiological criteria, as defined by the Centers for Disease Control and Prevention (CDC). On the basis of the CDC definition, the majority of the strains were considered to be HA-MRSA. However, 87% of them belonged to staphylococcal cassette chromosome
mec
(SCC
mec
) types IV and V, which are traditionally associated with CA-MRSA. Surprisingly, classical nosocomial SCC
mec
types I and II represented a minority, whereas SCC
mec
type III was completely absent. By PFGE analysis, four predominant clonal lineages and 21 highly variable sporadic genotypes were detected. Twenty-eight percent of the MRSA strains studied carried the genes encoding the Panton-Valentine leukocidin (PVL), of which 21% and 83% were associated with SCC
mec
types IV and V, respectively. Among 289 contact individuals screened for MRSA carriage throughout the extended surveillance, a single secondary patient was discovered. The possibility of nosocomial transmission could be excluded. The high proportions of SCC
mec
type IV and V strains as well as PVL-positive strains suggest strong infiltration of CA-MRSA into our institution. Moreover, the low endemic prevalence of MRSA demonstrates that current infection control measures are sufficient to limit its spreading and the emergence of large epidemic outbreaks.
Sequence-Based Identification of Filamentous Basidiomycetous Fungi from Clinical Specimens: a Cautionary Note
Authors: Romanelli Anna M. , Sutton Deanna A. , Thompson Elizabeth H. , Rinaldi Michael G. , Wickes Brian L.
Published: December 2009

The species-level identification of sterile and/or arthroconidium-forming filamentous fungi presumed to be basidiomycetes based upon morphological or physiological features alone is usually not possible due to the limited amount of hyphal differentiation. Therefore, a reliable molecular approach capable of the unambiguous identification of clinical isolates is needed. One hundred sixty-eight presumptive basidiomycetes were screened by sequence analysis of the internal transcribed spacer (ITS) and D1/D2 ribosomal DNA regions in an effort to obtain a species identification. Through the use of this approach, identification of a basidiomycetous fungus to the species level was obtained for 167/168 of the isolates. However, comparison of the BLAST results for each isolate for both regions revealed that only 28.6% (48/168) of the isolates had the same species identification by use of both the ITS and the D1/D2 regions, regardless of the percent identity. At the less stringent genus-only level, the identities for only 48.8% (82/168) of the isolates agreed for both regions. Investigation of the causes for this low level of agreement revealed that 14% of the species lacked an ITS region deposit and 16% lacked a D1/D2 region deposit. Few GenBank deposits were found to be complete for either region, with only 8% of the isolates having a complete ITS region and 10% having a complete D1/D2 region. This study demonstrates that while sequence-based identification is a powerful tool for many fungi, sequence data derived from filamentous basidiomycetes should be interpreted carefully, particularly in the context of missing or incomplete GenBank data, and, whenever possible, should be evaluated in light of compatible morphological features.
MIC Quality Control Guidelines and Disk Diffusion Test Optimization for CEM-101, a Novel Fluoroketolide
Authors: Jones Ronald N. , Ross James E. , Rhomberg Paul R.
Published: December 2009

The development of diagnostic susceptibility tests for CEM-101, a new fluoroketolide, was addressed by structured studies to determine the optimal disk diffusion test concentration and effects of various testing conditions or supplements and to establish the quality control (QC) ranges for reference broth microdilution tests. The 15-μg CEM-101 disk was selected, and MIC ranges for a total of four QC organisms were proposed, with only three doubling dilutions each that included 95.6 to 99.7% of values reported from the eight-laboratory investigation.
Efficacy of Multivalent Adenovirus-Based Vaccine against Simian Immunodeficiency Virus Challenge
Authors: Casimiro Danilo R. , Cox Kara , Tang Aimin , Sykes Kara J. , Feng Meizhen , Wang Fubao , Bett Andrew , Schleif William A. , Liang Xiaoping , Flynn Jessica , Tobery Timothy W. , Wilson Keith , Finnefrock Adam , Huang Lingyi , Vitelli Salvatore , Lin Jing , Patel Deepa , Davies Mary-Ellen , Heidecker Gwendolyn J. , Freed Daniel C. , Dubey Sheri , O'Connor David H. , Watkins David I. , Zhang Zhi-Qiang , Shiver John W.
Published: December 2009

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts ofMamu-A*01+/B*17−Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, aMamu-A*01+cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ≈ Gag/Pol > Gag ≈ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.
Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu
Authors: Lucas Tiffany M. , Lyddon Terri D. , Cannon Paula M. , Johnson Marc C.
Published: December 2009

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu
RD
, was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.
Activation of Peroxisome Proliferator-Activated Receptor Gamma by Human Cytomegalovirus for
De Novo
Replication Impairs Migration and Invasiveness of Cytotrophoblasts from Early Placentas
Authors: Rauwel Benjamin , Mariamé Bernard , Martin Hélène , Nielsen Ronni , Allart Sophie , Pipy Bernard , Mandrup Susanne , Devignes Marie Dominique , Evain-Brion Danièle , Fournier Thierry , Davrinche Christian
Published: December 2009

Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activity in infected cells. We demonstrate that the PPARγ antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARγ, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARγ in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARγ human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established
in vitro
models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.
Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression
Authors: Sawatsky Bevan , von Messling Veronika
Published: December 2009

Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression.
The Capsid Proteins of Aleutian Mink Disease Virus Activate Caspases and Are Specifically Cleaved during Infection
Authors: Cheng Fang , Chen Aaron Yun , Best Sonja M. , Bloom Marshall E. , Pintel David , Qiu Jianming
Published: December 2009

Aleutian mink disease virus (AMDV) is currently the only known member of the genus
Amdovirus
in the family
Parvoviridae
. It is the etiological agent of Aleutian disease of mink. We have previously shown that a small protein with a molecular mass of approximately 26 kDa was present during AMDV infection and following transfection of capsid expression constructs (J. Qiu, F. Cheng, L. R. Burger, and D. Pintel, J. Virol. 80:654-662, 2006). In this study, we report that the capsid proteins were specifically cleaved at aspartic acid residue 420 (D420) during virus infection, resulting in the previously observed cleavage product. Mutation of a single amino acid residue at D420 abolished the specific cleavage. Expression of the capsid proteins alone in Crandell feline kidney (CrFK) cells reproduced the cleavage of the capsid proteins in virus infection. More importantly, capsid protein expression alone induced active caspases, of which caspase-10 was the most active. Active caspases, in turn, cleaved capsid proteins
in vivo
. Our results also showed that active caspase-7 specifically cleaved capsid proteins at D420
in vitro
. These results suggest that viral capsid proteins alone induce caspase activation, resulting in cleavage of capsid proteins. We also provide evidence that AMDV mutants resistant to caspase-mediated capsid cleavage increased virus production approximately 3- to 5-fold in CrFK cells compared to that produced from the parent virus AMDV-G at 37°C but not at 31.8°C. Collectively, our results indicate that caspase activity plays multiple roles in AMDV infection and that cleavage of the capsid proteins might have a role in regulating persistent infection of AMDV.
Rapid Dissociation of HIV-1 from Cultured Cells Severely Limits Infectivity Assays, Causes the Inactivation Ascribed to Entry Inhibitors, and Masks the Inherently High Level of Infectivity of Virions
Authors: Platt Emily J. , Kozak Susan L. , Durnin James P. , Hope Thomas J. , Kabat David
Published: December 2009

By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20- to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.
Evidence for Multiple Recent Host Species Shifts among the Ranaviruses (FamilyIridoviridae)
Authors: Jancovich James K. , Bremont Michel , Touchman Jeffrey W. , Jacobs Bertram L.
Published: December 2009

Members of the genusRanavirus(familyIridoviridae) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.
Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family
Authors: Chandramouli Sumana , Joseph Jeremiah S. , Daudenarde Sophie , Gatchalian Jovylyn , Cornillez-Ty Cromwell , Kuhn Peter
Published: December 2009

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-Å crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.
Adenovirus Type 5 E1A and E6 Proteins of Low-Risk Cutaneous Beta-Human Papillomaviruses Suppress Cell Transformation through Interaction with FOXK1/K2 Transcription Factors
Authors: Komorek Jessica , Kuppuswamy Mohan , Subramanian T. , Vijayalingam S. , Lomonosova Elena , Zhao Ling-jun , Mymryk Joe S. , Schmitt Kimberly , Chinnadurai G.
Published: January 2010

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.
Evolution of the HIV-1
env
Gene in the Rag2
−/−
γ
C
−/−
Humanized Mouse Model
Authors: Ince William L. , Zhang Liguo , Jiang Qi , Arrildt Kathryn , Su Lishan , Swanstrom Ronald
Published: December 2009

Rag2
−/−
γ
C
−/−
mice transplanted with human hematopoietic stem cells (DKO-hu-HSC mice) mimic aspects of human infection with human immunodeficiency virus type 1 (HIV-1), including sustained viral replication and CD4
+
T-cell decline. However, the extent of HIV-1 evolution during long-term infection in these humanized mice, a key feature of the natural infection, has not been assessed fully. In this study, we examined the types of genotypic and phenotypic changes in the viral
env
gene that occur in the viral populations of DKO-hu-HSC mice infected with the CCR5-tropic isolate HIV-1
JRCSF
for up to 44 weeks. The mean rate of divergence of viral populations in mice was similar to that observed in a cohort of humans during a similar period of infection. Many amino acid substitutions were common across mice, including losses of N-linked glycosylation sites and substitutions in the CD4 binding site and in CD4-induced epitopes, indicating common selective pressures between mice. In addition,
env
variants evolved sensitivity to antibodies directed at V3, suggesting a more open conformation for Env. This phenotypic change was associated with increased CD4 binding efficiency and was attributed to specific amino acid substitutions. In one mouse,
env
variants emerged that exhibited a CXCR4-tropic phenotype. These sequences were compartmentalized in the mesenteric lymph node. In summary, viral populations in these mice exhibited dynamic behavior that included sequence evolution, compartmentalization, and the appearance of distinct phenotypic changes. Thus, humanized mice offer a useful model for studying evolutionary processes of HIV-1 in a complex host environment.
Naturally Occurring Human Monoclonal Antibodies Neutralize both 1918 and 2009 Pandemic Influenza A (H1N1) Viruses
Authors: Krause Jens C. , Tumpey Terrence M. , Huffman Chelsey J. , McGraw Patricia A. , Pearce Melissa B. , Tsibane Tshidi , Hai Rong , Basler Christopher F. , Crowe James E.
Published: December 2009

The 2009 pandemic influenza A (H1N1) virus exhibits hemagglutinin protein sequence homology with the 1918 pandemic influenza virus. We found that human monoclonal antibodies recognized the Sa antigenic site on the head domains of both 1918 and 2009 hemagglutinins, a site that is hypervariable due to immune selection. These antibodies exhibited high potency against the 2009 virusin vitro, and one exerted a marked therapeutic effectin vivo.
Two Kinetic Patterns of Epitope-Specific CD8 T-Cell Responses following Murine Gammaherpesvirus 68 Infection
Authors: Freeman Michael L. , Lanzer Kathleen G. , Cookenham Tres , Peters Bjoern , Sidney John , Wu Ting-Ting , Sun Ren , Woodland David L. , Sette Alessandro , Blackman Marcia A.
Published: January 2010

Murine gammaherpesvirus 68 (γHV68) provides an important experimental model for understanding mechanisms of immune control of the latent human gammaherpesviruses. Antiviral CD8 T cells play a key role throughout three separate phases of the infection: clearance of lytic virus, control of the latency amplification stage, and prevention of reactivation of latently infected cells. Previous analyses have shown that T-cell responses to two well-characterized epitopes derived from ORF6 and ORF61 progress with distinct kinetics. ORF6487-specific cells predominate early in infection and then decline rapidly, whereas ORF61524-specific cells continue to expand through early latency, due to sustained epitope expression. However, the paucity of identified epitopes to this virus has limited our understanding of the overall complexities of CD8 T-cell immune control throughout infection. Here we screened 1,383 predicted H-2b-restricted peptides and identified 33 responses, of which 21 have not previously been reported. Kinetic analysis revealed a spectrum of T-cell responses based on the rapidity of their decline after the peak acute response that generally corresponded to the expression patterns of the two previously characterized epitopes. The slowly declining responses that were maintained during latency amplification proliferated more rapidly and underwent maturation of functional avidity over time. Furthermore, the kinetics of decline was accelerated following infection with a latency-null mutant virus. Overall, the data show that γHV68 infection elicits a highly heterogeneous CD8 T-cell response that segregates into two distinctive kinetic patterns controlled by differential epitope expression during the lytic and latency amplification stages of infection.
N-Linked Glycan at Residue 523 of Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Masks a Second Receptor-Binding Site
Authors: Mishin Vasiliy P. , Watanabe Makiko , Taylor Garry , DeVincenzo John , Bose Michael , Portner Allen , Alymova Irina V.
Published: January 2010

The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second receptor-binding site identified in Newcastle disease virus (NDV) HN (I. A. Alymova, G. Taylor, V. P. Mishin, M. Watanabe, K. G. Murti, K. Boyd, P. Chand, Y. S. Babu, and A. Portner, J. Virol.
82:
8400-8410, 2008). Sequence analysis and superposition of the NDV and hPIV-3 HN dimer structures revealed that, similar to what was seen in hPIV-1, the N-linked glycan at residue 523 on hPIV-3 HN may cover a second receptor-binding site. Removal of this N-linked glycosylation site by an Asn-to-Asp substitution at residue 523 (N523D) changed the spectrum of the mutant virus's receptor specificity, delayed its elution from both turkey and chicken red blood cells, reduced mutant sensitivity (by about half) to the selective HN inhibitor BCX 2855 in hemagglutination inhibition tests, and slowed its growth in LLC-MK
2
cells. The neuraminidase activity of the mutant and its sensitivity to BCX 2855 in neuraminidase inhibition assays did not change, indicating that the mutation did not affect the virus's catalytic-binding site and that all observed effects were caused by the exposure of the purported second receptor-binding site. Our data are consistent with the idea that, similar to the case for hPIV-1, the N-linked glycan shields a second receptor-binding site on hPIV-3 HN.