bluuebunny/crossref_metadata_2025 huggingface dataset:
Depth Perception from Image Defocus in a Jumping Spider
Authors: Nagata Takashi , Koyanagi Mitsumasa , Tsukamoto Hisao , Saeki Shinjiro , Isono Kunio , Shichida Yoshinori , Tokunaga Fumio , Kinoshita Michiyo , Arikawa Kentaro , Terakita Akihisa
Published: January 2012

A Good Judge of Distance

Jumping spiders actively pursue their prey, often jumping relatively long distances in order to catch them. Such feats require accurate depth perception.

Nagata
et al.

(p.
469
; see the Perspective by

Herberstein and Kemp

) show that jumping spiders use a process called image defocus, which allows depth perception to be obtained through the comparison of a nonfocused image to a focused image within the same eye. A single layer within the spider's eye that could not focus green light nevertheless contained a green sensitive pigment. Thus, this layer always receives an unfocused image, while other layers receive images in focus. Confirming this eye arrangement's role in depth perception, spiders unlucky enough to be bathed in green light nearly always jumped short of their target.
Unimpeded Permeation of Water Through Helium-Leak–Tight Graphene-Based Membranes
Authors: Nair R. R. , Wu H. A. , Jayaram P. N. , Grigorieva I. V. , Geim A. K.
Published: January 2012

Porous Membranes

Thin semi-permeable membranes are commonly used as chemical barriers or for filtration purposes. While the size of the pores will influence which molecules are able to pass, other factors—including the surface chemistry of the pore walls, electrostatic interactions, and differences in solubility—can also affect the diffusion rates. There is also a trade-off between the thickness of the membrane regarding strength and permeation rates (see the Perspective by

Paul

).

Karan
et al.

(p.
444
) fabricated membranes from amorphous carbon, which showed excellent strength and could be used for filtrations involving organic solvents.

Nair
et al.

(p.
442
) observed unusual behavior in graphene-based membranes which were able to prevent the diffusion of many small-molecule gases, including helium, but showed almost barrier-free movement of water.
Reversible Reduction of Oxygen to Peroxide Facilitated by Molecular Recognition
Authors: Lopez Nazario , Graham Daniel J. , McGuire Robert , Alliger Glen E. , Shao-Horn Yang , Cummins Christopher C. , Nocera Daniel G.
Published: January 2012

Boxing in Peroxide

Hydrogen peroxide (H
2
O
2
) is a powerful oxidant, and its reactivity is exploited in numerous biological, as well as synthetic, contexts.

Lopez
et al.

(p.
450
) have now managed to capture its dianion (O
2
2-
) in a cryptand—essentially a molecular box assembled from benzamide derivatives—keeping the dianion stable in organic solution for days through a net of well-placed internal hydrogen-bond donors. The encapsulated dianion exhibited clean oxidative reactivity back to O
2
either by chemical or by electrochemical means.
Asymmetric Segregation of Polarized Antigen on B Cell Division Shapes Presentation Capacity
Authors: Thaunat Olivier , Granja Aitor G. , Barral Patricia , Filby Andrew , Montaner Beatriz , Collinson Lucy , Martinez-Martin Nuria , Harwood Naomi E. , Bruckbauer Andreas , Batista Facundo D.
Published: January 2012

Antigen Polarity in B Cell Differentiation

Communication received through cell contact is critical for the differentiation of specialized effector cell populations during the immune response. For example, B lymphocytes acquire antigen that they present to helper T lymphocytes. T lymphocytes, in turn, provide key differentiation signals to B lymphocytes. In order to learn more about this process,

Thaunat
et al.

(p.
475
; see the Perspective by

Dustin and Meyer-Hermann

) used multiphoton microscopy and imaging flow cytometry to visualize the localization of antigen in B lymphocytes during an immune response. Antigen acquired by B lymphocytes exhibited a polarized distribution that was sustained over several rounds of cell division. This produced a population of activated B lymphocytes that contained very low levels of antigen. Daughter cells that received more antigen were better able to stimulate T cells. Because cues received through T lymphocyte interactions are likely to influence B lymphocyte fate decisions, unequal distribution of antigen in dividing B lymphocytes may influence their differentiation.
Heavy Livestock Grazing Promotes Locust Outbreaks by Lowering Plant Nitrogen Content
Authors: Cease Arianne J. , Elser James J. , Ford Colleen F. , Hao Shuguang , Kang Le , Harrison Jon F.
Published: January 2012

Locust Heaven

Locust outbreaks have severe consequences for agriculture, but the conditions that promote an outbreak are unknown.

Cease
et al.

(p.
467
) investigated aspects of the locust diet and found that increased nitrogen content of cereal grasses reduced the size and viability of a herbivorous locust species. This locust prefers low N plants, which result from heavy grazing by livestock and erosion.
Ecocide or Utopia on Easter Island?
The Statues That Walked
Unraveling the Mystery of Easter Island

by Terry Hunt and Carl Lipo

Free Press (Simon and Schuster), New York, 2011. 251 pp. $26, C$29.99. ISBN 9781439150313.
Authors: Torrence Robin
Published: January 2012

Drawing on their own primary research, Hunt and Lipo argue that Easter Island's population did not collapse from human exploitation of the environment.
The Pursuit of Love
Understanding Autism
Parents, Doctors, and the History of a Disorder

by Chloe Silverman

Princeton University Press, Princeton, NJ, 2012. 352 pp. $35, £24.95. ISBN 9780691150468.
Authors: Malow Beth Ann
Published: January 2012

Combining historical and ethnographic perspectives, Silverman explores the various ways in which researchers, practitioners, and activists have interpreted and responded to autism.
The Inner Workings of a Dynamic Duo
Authors: Poulsen Hanne , Nissen Poul
Published: January 2012

Structures of two–pore domain potassium channels reveal key differences from the more widely found tetrameric channels.
The Role of Coevolution
Authors: Thompson John N.
Published: January 2012

Coevolution of a virus and a bacterium leads to the emergence of a key adaptive innovation.
Flu Controversy Spurs Research Moratorium
Authors: Malakoff David
Published: January 2012

Amid a growing global controversy over the potential dangers of experiments involving the H5N1 avian influenza virus, a group of leading influenza researchers last week agreed to a 60-day moratorium on some sensitive flu studies.
An Explosive Return of the 'Great Pox'
Authors: Hvistendahl Mara
Published: January 2012

As a result of widespread migration, rising inequality, and evolving sexual mores, China now holds the dubious title of the nation with the largest increase in reported syphilis cases in the penicillin era.
What It'll Take to Go Exascale
Authors: Service Robert F.
Published: January 2012

Scientists hope the next generation of supercomputers will carry out a million trillion operations per second. But first they must change the way the machines are built and run.
Ferreting Out the Hidden Cracks in the Heart of a Continent
Authors: Lubick Naomi
Published: January 2012

Deep beneath the central United States, researchers find signs of buried faults that have triggered earthquakes in the past—and may still be kicking.
Surgical Stress Resistance Induced by Single Amino Acid Deprivation Requires
Gcn2
in Mice
Authors: Peng Wei , Robertson Lauren , Gallinetti Jordan , Mejia Pedro , Vose Sarah , Charlip Allison , Chu Timothy , Mitchell James R.
Published: January 2012

A protein-free diet or one missing certain amino acids can protect against kidney and liver damage due to ischemia in mice.
Triggers for Research Ethics Consultation
Authors: Havard Molly , Cho Mildred K. , Magnus David
Published: January 2012

Research ethics consultation services can help scientists to address ethical and societal issues that go beyond the purview of existing regulatory frameworks.
Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing
Authors: Calvo Sarah E. , Compton Alison G. , Hershman Steven G. , Lim Sze Chern , Lieber Daniel S. , Tucker Elena J. , Laskowski Adrienne , Garone Caterina , Liu Shangtao , Jaffe David B. , Christodoulou John , Fletcher Janice M. , Bruno Damien L. , Goldblatt Jack , DiMauro Salvatore , Thorburn David R. , Mootha Vamsi K.
Published: January 2012

Applying next-generation sequencing to 42 infants with mitochondrial disease highlights both the potential and the challenge of using this technology in clinical diagnosis.
Mechanisms of Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancers
Authors: Katayama Ryohei , Shaw Alice T. , Khan Tahsin M. , Mino-Kenudson Mari , Solomon Benjamin J. , Halmos Balazs , Jessop Nicholas A. , Wain John C. , Yeo Alan Tien , Benes Cyril , Drew Lisa , Saeh Jamal Carlos , Crosby Katherine , Sequist Lecia V. , Iafrate A. John , Engelman Jeffrey A.
Published: January 2012

Multiple mechanisms of crizotinib resistance were identified in lung cancer patients including new secondary ALK mutations and activation of receptor tyrosine kinases.
Teplizumab Induces Human Gut-Tropic Regulatory Cells in Humanized Mice and Patients
Authors: Waldron-Lynch Frank , Henegariu Octavian , Deng Songyan , Preston-Hurlburt Paula , Tooley James , Flavell Richard , Herold Kevan C.
Published: January 2012

Humanized mice can be successfully used as a preclinical testing platform to identify the mechanism of action of immunotherapies in humans.
Bidirectional Dance of Glutamine
Authors: Emadi Ashkan
Published: January 2012

Under low oxygen conditions, reductive metabolism of glutamine is the major source of essential lipid biogenesis in cancer cells.
Just Be “pH”-tient
Authors: Rytting Erik
Published: January 2012

pH-sensitive nanoparticles can keep therapeutic scavengers of reactive oxygen species inactive until they reach the site of kidney injury.
Smoking Out a New Therapy for Lung Disease
Authors: Shum Anthony K.
Published: January 2012

The common antihypertensive drug losartan may treat chronic lung disease.
Evaluation of Coccidioides Antigen Detection in Dogs with Coccidioidomycosis
Authors: Kirsch Emily J. , Greene Russell T. , Prahl Annalisa , Rubin Stanley I. , Sykes Jane E. , Durkin Michelle M. , Wheat Lawrence J.
Published: January 2012

Antigen detection has been reported to be a promising method for rapid diagnosis of coccidioidomycosis in humans.Coccidioidesantigen detection has not been previously reported in dogs with coccidioidomycosis and was evaluated in 60 cases diagnosed based on detection of anti-Coccidioidesantibodies at titers of 1:16 or more in serum. Controls included dogs with presumed histoplasmosis or blastomycosis, other fungal infections, or nonfungal diseases and healthy dogs. Urine and serum specimens were tested using an enzyme immunoassay forCoccidioidesgalactomannan antigen. Antibody testing was performed at commercial veterinary reference laboratories. Antigen was detected in urine or serum of 12 of 60 (20.0%), urine only in 2 of 57 (3.5%), and serum only in 11 of 58 (19.0%) dogs with coccidioidomycosis. Antigen was detected in the urine of 3 of 43 (7.0%) and serum of 1 of 37 (2.7%) dogs with histoplasmosis or blastomycosis but not in 13 dogs with other fungal infections (serum, 9; urine, 13), 41 dogs with nonfungal diseases (urine, 41; serum, 18), or healthy dogs (serum, 21; urine, 21). Detection of antigen was an insensitive method for diagnosis of coccidioidomycosis in dogs in which the diagnosis was based primarily upon detection of antibodies at titers of 1:16 or higher, and the highest sensitivity was in serum.
Human Papillomavirus Antibody Reference Reagents for Use in Postvaccination Surveillance Serology
Authors: Bissett Sara L. , Wilkinson Dianna , Tettmar Kate I. , Jones Nicky , Stanford Elaine , Panicker Gitika , Faust Helena , Borrow Ray , Soldan Kate , Unger Elizabeth R. , Dillner Joakim , Minor Philip , Beddows Simon
Published: January 2012

Suitably controlled serosurveillance surveys are essential for evaluating human papillomavirus (HPV) immunization programs. A panel of plasma samples from 18-year-old females was assembled, the majority of the samples being from recipients of the bivalent HPV vaccine. Antibody specificities were evaluated by three independent laboratories, and 3 pools that displayed no antibodies to any HPV type tested or intermediate or high levels of antibody to HPV16, HPV18, HPV31, and HPV45 were created. These pools will be useful as control reagents for HPV serology.
Evaluation of Processing Methods To Equitably Aliquot Sputa for Mycobacterial Testing
Authors: Hadad David Jamil , Morais Carlos Gustavo Vieira , Vinhas Solange A. , Fennelly Kevin P. , Dietze Reynaldo , Nascimento Cristina Paula , Palaci Moises
Published: January 2012

We compared bacillary loads after splitting sputum specimens by chemical (
N
-acetyl-
l
-cysteine [NALC]) and mechanical homogenization by vortexing with sterile glass beads. NALC and vortexing with glass beads were equally effective at homogenizing sputum specimens, resulting in an equal distribution of tubercle bacilli in the aliquots.
Evidence of Uncultivated Bacteria in the Adult Female Bladder
Authors: Wolfe Alan J. , Toh Evelyn , Shibata Noriko , Rong Ruichen , Kenton Kimberly , FitzGerald MaryPat , Mueller Elizabeth R. , Schreckenberger Paul , Dong Qunfeng , Nelson David E. , Brubaker Linda
Published: January 2012

Clinical urine specimens are usually considered to be sterile when they do not yield uropathogens using standard clinical cultivation procedures. Our aim was to test if the adult female bladder might contain bacteria that are not identified by these routine procedures. An additional aim was to identify and recommend the appropriate urine collection method for the study of bacterial communities in the female bladder. Consenting participants who were free of known urinary tract infection provided urine samples by voided, transurethral, and/or suprapubic collection methods. The presence of bacteria in these samples was assessed by bacterial culture, light microscopy, and 16S rRNA gene sequencing. Bacteria that are not or cannot be routinely cultivated (hereinafter called uncultivated bacteria) were common in voided urine, urine collected by transurethral catheter (TUC), and urine collected by suprapubic aspirate (SPA), regardless of whether the subjects had urinary symptoms. Voided urine samples contained mixtures of urinary and genital tract bacteria. Communities identified in parallel urine samples collected by TUC and SPA were similar. Uncultivated bacteria are clearly present in the bladders of some women. It remains unclear if these bacteria are viable and/or if their presence is relevant to idiopathic urinary tract conditions.
Frequency of Decreased Susceptibility and Resistance to Echinocandins among Fluconazole-Resistant Bloodstream Isolates of Candida glabrata
Authors: Pfaller M. A. , Castanheira M. , Lockhart S. R. , Ahlquist A. M. , Messer S. A. , Jones R. N.
Published: January 2012

The echinocandin class of antifungal agents is considered to be the first-line treatment of bloodstream infections (BSI) due to
Candida glabrata
. Recent reports of BSI due to strains of
C. glabrata
resistant to both fluconazole and the echinocandins are of concern and prompted us to review the experience of two large surveillance programs, the SENTRY Antimicrobial Surveillance Program for the years 2006 through 2010 and the Centers for Disease Control and Prevention population-based surveillance conducted in 2008 to 2010. The
in vitro
susceptibilities of 1,669 BSI isolates of
C. glabrata
to fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin were determined by CLSI broth microdilution methods. Fluconazole MICs of ≥64 μg/ml were considered resistant. Strains for which anidulafungin and caspofungin MICs were ≥0.5 μg/ml and for which micafungin MICs were ≥0.25 μg/ml were considered resistant. A total of 162 isolates (9.7%) were resistant to fluconazole, of which 98.8% were nonsusceptible to voriconazole (MIC > 0.5 μg/ml) and 9.3%, 9.3%, and 8.0% were resistant to anidulafungin, caspofungin, and micafungin, respectively. There were 18 fluconazole-resistant isolates that were resistant to one or more of the echinocandins (11.1% of all fluconazole-resistant isolates), all of which contained an acquired mutation in
fks1
or
fks2
. By comparison, there were no echinocandin-resistant strains detected among 110 fluconazole-resistant isolates of
C. glabrata
tested in 2001 to 2004. These data document the broad emergence of coresistance over time to both azoles and echinocandins in clinical isolates of
C. glabrata
.
ϕ
2
GFP10, a High-Intensity Fluorophage, Enables Detection and Rapid Drug Susceptibility Testing of Mycobacterium tuberculosis Directly from Sputum Samples
Authors: Jain Paras , Hartman Travis E. , Eisenberg Nell , O'Donnell Max R. , Kriakov Jordan , Govender Karnishree , Makume Mantha , Thaler David S. , Hatfull Graham F. , Sturm A. Willem , Larsen Michelle H. , Moodley Preshnie , Jacobs William R.
Published: January 2012

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (ϕ
2
GFP10) that for the first time allows direct visualization of
Mycobacterium tuberculosis
in clinical sputum samples. Engineered features distinguishing ϕ
2
GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. ϕ
2
GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of ϕ
2
GFP10 detected
M. tuberculosis
in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with ϕ
2
GFP10. Fluorophage ϕ
2
GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for
M. tuberculosis
infection and for rapid DST.
Detection of Toxigenic Clostridium difficile: Comparison of the Cell Culture Neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile Assays
Authors: Pancholi P. , Kelly C. , Raczkowski M. , Balada-Llasat J. M.
Published: January 2012

Clostridium difficile
is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect
C. difficile
toxins or the genes that encode them in fecal samples. We evaluated the Xpert
C. difficile
and Xpert
C. difficile
/Epi (Cepheid, CA) that detect the toxin B gene (
tcdB
) and
tcdB
,
cdt
, and a deletion in
tcdC
associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene
C. difficile
(Meridian Bioscience, Inc.) that detects the toxin A gene (
tcdA
) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert
C. difficile
, Xpert
C. difficile
/Epi, and Illumigene
C. difficile
assays. Of the 200 prospective stools tested, 10.5% (
n
= 23) were determined to be positive by CCNA, 17.5% (
n
= 35) were determined to be positive by Illumigene
C. difficile
, and 21.5% (
n
= 43) were determined to be positive by Xpert
C. difficile
and Xpert
C. difficile
/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (
n
= 47) were determined to be positive by Illumigene
C. difficile
and 100% (
n
= 50) were determined to be positive by Xpert
C. difficile
and Xpert
C. difficile
/Epi. Of the 11 discrepant results (i.e., negative by Illumigene
C. difficile
but positive by Xpert
C. difficile
and Xpert
C. difficile
/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert
C. difficile
/Epi as the 027/NAP1/BI strain. The Xpert
C. difficile
and Xpert
C. difficile
/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic
C. difficile
in stool specimens.
Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples
Authors: Wessels Els , Schelfaut Jacqueline J. G. , Bernards Alexandra T. , Claas Eric C. J.
Published: January 2012

The identification and detection of mitis group streptococci, which contain
Streptococcus pneumoniae
, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of
S. pneumoniae
and
Streptococcus pseudopneumoniae
and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23
S. pneumoniae
, 2
S. pseudopneumoniae
, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of
S. pneumoniae
showed concordant results with optochin susceptibility testing (CO
2
atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O
2
atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the
tuf
and
rpoB
genes resulted in several false-positive, false-negative, or inconclusive results. The
S. pseudopneumoniae
isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of
S. pneumoniae
and
S. pseudopneumoniae
isolates. Using this method,
S. pneumoniae
and
S. pseudopneumoniae
DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.
Species Specificity of Protein Kinase R Antagonism by Cytomegalovirus TRS1 Genes
Authors: Child Stephanie J. , Brennan Greg , Braggin Jacquelyn E. , Geballe Adam P.
Published: January 2012

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed by many different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted to changes in PKR. As a result of this “arms race,” modern TRS1 alleles in CMVs may function differently in cells derived from alternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replication of a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not have these activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfills these functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNA and, in the cell types in which they can rescue VVΔE3L replication, they also bind to PKR and prevent phosphorylation of the α-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophosphorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations in this critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanism of PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of the factors that contributes to species specificity of cytomegalovirus replication.
Canine Distemper Virus Epithelial Cell Infection Is Required for Clinical Disease but Not for Immunosuppression
Authors: Sawatsky Bevan , Wong Xiao-Xiang , Hinkelmann Sarah , Cattaneo Roberto , von Messling Veronika
Published: January 2012

To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.
Cell Entry-Associated Conformational Changes in Reovirus Particles Are Controlled by Host Protease Activity
Authors: Madren Jillann A. , Sarkar Payel , Danthi Pranav
Published: January 2012

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s.
In vitro
incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the μ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.
Roles of the Coding and Noncoding Regions of Rift Valley Fever Virus RNA Genome Segments in Viral RNA Packaging
Authors: Murakami Shin , Terasaki Kaori , Narayanan Krishna , Makino Shinji
Published: January 2012

We characterized the RNA elements involved in the packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5′-terminal 25 nucleotides of each RNA segment were equally competent for RNA packaging and carried an RNA packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA packaging.
Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells
Authors: Chahal Jasdave S. , Flint S. J.
Published: January 2012

Previous studies have indicated that the adenovirus type 5 E1B 55-kDa protein facilitates viral DNA synthesis in normal human foreskin fibroblasts (HFFs) but not in primary epithelial cells. To investigate this apparent difference further, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected by the mutant AdEasyE1Δ2347, which carries the Hr6 frameshift mutation that prevents production of the E1B 55-kDa protein, in an E1-containing derivative of AdEasy. Impaired viral DNA synthesis was observed in normal HFFs but not in normal human bronchial epithelial cells infected by this mutant. However, acceleration of progression through the early phase, which is significantly slower in HFFs than in epithelial cells, eliminated the dependence of efficient viral DNA synthesis in HFFs on the E1B 55-kDa protein. These observations suggest that timely synthesis of the E1B 55-kDa protein protects normal cells against a host defense that inhibits adenoviral genome replication. One such defense is mediated by the Mre11-Rad50-Nbs1 complex. Nevertheless, examination of the localization of Mre11 and viral proteins by immunofluorescence suggested that this complex is inactivated similarly in AdEasyE1Δ2347 mutant-infected and AdEasyE1-infected HFFs.
The Multibasic Cleavage Site in H5N1 Virus Is Critical for Systemic Spread along the Olfactory and Hematogenous Routes in Ferrets
Authors: Schrauwen Eefje J. A. , Herfst Sander , Leijten Lonneke M. , van Run Peter , Bestebroer Theo M. , Linster Martin , Bodewes Rogier , Kreijtz Joost H. C. M. , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E. , Fouchier Ron A. M. , Kuiken Thijs , van Riel Debby
Published: January 2012

The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space—including along the olfactory route—and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.
Draft Genome Sequence of the Coccolithovirus Emiliania huxleyi Virus 202
Authors: Nissimov Jozef I. , Worthy Charlotte A. , Rooks Paul , Napier Johnathan A. , Kimmance Susan A. , Henn Matthew R. , Ogata Hiroyuki , Allen Michael J.
Published: January 2012

Emiliania huxleyi
virus 202 (EhV-202) is a member of the
Coccolithoviridae
, a group of viruses that infect the marine coccolithophorid
Emiliania huxleyi
. EhV-202 has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 407 kbp, consisting of 485 coding sequences (CDSs). Here we describe the genomic features of EhV-202, together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.
Structural Basis for Broad Detection of Genogroup II Noroviruses by a Monoclonal Antibody That Binds to a Site Occluded in the Viral Particle
Authors: Hansman Grant S. , Taylor David W. , McLellan Jason S. , Smith Thomas J. , Georgiev Ivelin , Tame Jeremy R. H. , Park Sam-Yong , Yamazaki Makoto , Gondaira Fumio , Miki Motohiro , Katayama Kazuhiko , Murata Kazuyoshi , Kwong Peter D.
Published: January 2012

Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.
Recombinant Dengue Type 2 Viruses with Altered E Protein Domain III Epitopes Are Efficiently Neutralized by Human Immune Sera
Authors: Wahala Wahala M. P. B. , Huang Claire , Butrapet Siritorn , White Laura J. , de Silva Aravinda M.
Published: January 2012

Humans develop polyclonal, serotype-specific neutralizing antibody responses after dengue virus (DENV) infection. Many mouse antibodies that neutralize DENV bind to the lateral ridge or A strand epitopes on domain III of the viral envelope (EDIII) protein. It has been assumed that these epitopes are also the main target of human neutralizing antibodies. Using recombinant dengue serotype 2 viruses with altered EDIII epitopes, we demonstrate that EDIII epitopes are not the main target of human neutralizing antibody.
Distinct Transformation Tropism Exhibited by Human T Lymphotropic Virus Type 1 (HTLV-1) and HTLV-2 Is the Result of Postinfection T Cell Clonal Expansion
Authors: Kannian Priya , Yin Han , Doueiri Rami , Lairmore Michael D. , Fernandez Soledad , Green Patrick L.
Published: January 2012

Human T lymphotropic virus type 1 (HTLV-1) and HTLV-2 are related but pathogenically distinct viruses. HTLV-1 mainly causes adult T cell leukemia, while HTLV-2 is not associated with leukemia.
In vitro
, HTLV-1 and HTLV-2 predominantly transform CD4
+
and CD8
+
T cells, respectively: the genetic determinant maps to the viral envelope. Herein, we investigate whether this transformation tropism occurs during initial infection or subsequently during the cellular transformation process. Since most individuals are chronically infected at the time of detection, we utilized an established rabbit model to longitudinally measure the early HTLV-1 and HTLV-2 infection and replication kinetics in purified CD4
+
and CD8
+
T cells. HTLV-1 and HTLV-2 were detected in both CD4
+
and CD8
+
T cells within 1 week postinoculation. In HTLV-1-infected rabbit CD4
+
T cells, proviral burden and
tax
/
rex
mRNA expression peaked early, and expression levels were directly proportional to each other. The late expression of the antisense transcript (
Hbz
or
Aph-2
) correlated directly with a late proviral burden peak in HTLV-1- or HTLV-2-infected rabbit CD8
+
T cells, respectively. This study provides the first
in vivo
evidence that these viruses do not exhibit cellular preference during initial infection. We further evaluated the transformation tropism of HTLV-1 and HTLV-2 over a 9-week period using
in vitro
cell growth/immortalization assays. At the early weeks, both HTLV-1 and HTLV-2 showed proportionate growth of CD4
+
and CD8
+
T cells. However, beyond week 5, the predominance of one particular T cell type emerged, supporting the conclusion that transformation tropism is a postinfection event due to selective clonal expansion over time.
Hepatitis C Virus Infection of Human T Lymphocytes Is Mediated by CD5
Authors: Sarhan Mohammed A. , Pham Tram N. Q. , Chen Annie Y. , Michalak Tomasz I.
Published: January 2012

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease. Although infection of hepatocytes is mainly responsible for manifestations of hepatitis C, the virus also invades the immune system by a yet-to-be-identified mechanism. Using human T cell lines and primary T lymphocytes as targets and patient-derived HCV as inocula, we aimed to identify how HCV gains entry into these cells. HCV replication was determined by detection of the HCV RNA replicative (negative) strand and viral proteins, while specific antibodies, knocking down gene expression and making otherwise-resistant cells prone to HCV, were employed to identify a receptor molecule determining T lymphocyte permissiveness to HCV infection. The results revealed that T cell susceptibility to HCV requires CD5, a lymphocyte-specific glycoprotein belonging to the scavenger receptor cysteine-rich family. Blocking of T cell CD5 with antibody or silencing with specific short hairpin RNA (shRNA) decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the opposite effect. Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated infection of these otherwise HCV-resistant cells. In contrast to T cells, hepatocytes do not express CD5. The data revealed that CD5 is a molecule important for HCV entry into human T lymphocytes. This finding provides direct insight into the mechanism of HCV lymphotropism and defines a target for potential interventions against HCV propagating in this extrahepatic compartment.
Depletion of Intracellular Zinc Inhibits the Ubiquitin Ligase Activity of Viral Regulatory Protein ICP0 and Restricts Herpes Simplex Virus 1 Replication in Cell Culture
Authors: Grant Kyle , Grant Louise , Tong Lily , Boutell Chris
Published: January 2012

The viral ubiquitin ligase ICP0 stimulates the onset of HSV-1 lytic infection and productive reactivation of viral genomes from latency. In order to mediate these processes, it requires its C3HC4 RING finger domain, a tertiary structural fold that is coordinated by the binding of two zinc (Zn
2+
) atoms. Here we formally demonstrate that Zn
2+
binding and intracellular Zn
2+
levels are critical for ICP0's biochemical activity and that depletion of intracellular Zn
2+
severely attenuates HSV-1 replication.
Identification and Characterization of Equine Herpesvirus Type 1 pUL56 and Its Role in Virus-Induced Downregulation of Major Histocompatibility Complex Class I
Authors: Ma Guanggang , Feineis Silke , Osterrieder Nikolaus , Van de Walle Gerlinde R.
Published: January 2012

Major histocompatibility complex class I (MHC-I) molecules play an important role in host immunity to infection by presenting antigenic peptides to cytotoxic T lymphocytes (CTLs), which recognize and destroy virus-infected cells. Members of the
Herpesviridae
have developed multiple mechanisms to avoid CTL recognition by virtue of downregulation of MHC-I on the cell surface. We report here on an immunomodulatory protein involved in this process, pUL56, which is encoded by
ORF1
of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus. We show that EHV-1 pUL56 is a phosphorylated early protein which is expressed as different forms and predominantly localizes to Golgi membranes. In addition, the transmembrane (TM) domain of the type II membrane protein was shown to be indispensable for correct subcellular localization and a proper function. pUL56 by itself is not functional with respect to interference with MHC-I and likely needs another unidentified viral protein(s) to perform this action. Surprisingly, pUL49.5, an inhibitor of the transporter associated with antigen processing (TAP) and encoded by EHV-1 and related viruses, appeared not to be required for pUL56-induced early MHC-I downmodulation in infected cells. In conclusion, our data identify a new immunomodulatory protein, pUL56, involved in MHC-I downregulation which is unable to perform its function outside the context of viral infection.
Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide
Authors: Lehoux Michaël , Fradet-Turcotte Amélie , Lussier-Price Mathieu , Omichinski James G. , Archambault Jacques
Published: January 2012

The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin
in vivo
. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target.
Tissue Exit: a Novel Control Point in the Accumulation of Antigen-Specific CD8 T Cells in the Influenza A Virus-Infected Lung
Authors: Jennrich Silke , Lee Michael H. , Lynn Rachel C. , Dewberry Kristofer , Debes Gudrun F.
Published: January 2012

Memory/effector T cells efficiently migrate into extralymphoid tissues and sites of infection, providing immunosurveillance and a first line of defense against invading pathogens. Even though it is a potential means to regulate the size, quality, and duration of a tissue infiltrate, T cell egress from infected tissues is poorly understood. Using a mouse model of influenza A virus infection, we found that CD8 effector T cells egressed from the infected lung in a CCR7-dependent manner. In contrast, following antigen recognition, effector CD8 T cell egress decreased and CCR7 function was reducedin vivoandin vitro, indicating that the exit of CD8 T cells from infected tissues is tightly regulated. Our data suggest that the regulation of T cell egress is a mechanism to retain antigen-specific effectors at the site of infection to promote viral clearance, while decreasing the numbers of bystander T cells and preventing overt inflammation.
Differential Gag-Specific Polyfunctional T Cell Maturation Patterns in HIV-1 Elite Controllers
Authors: Ferrando-Martínez Sara , Casazza Joseph P. , Leal Manuel , Machmach Kawthar , Muñoz-Fernández Ma Ángeles , Viciana Pompeyo , Koup Richard A. , Ruiz-Mateos Ezequiel
Published: January 2012

A small fraction of HIV-infected individuals (<1%), referred to as elite controllers (EC), are able to maintain undetectable viral loads indefinitely without treatment. The role of the maturational phenotype of T cells in the control of HIV infection in these individuals is not well described. We compared the maturational and functional phenotypes of Gag-specific CD4 and CD8 T cells from EC, who maintain undetectable viral loads without treatment; relative controllers (RC), who maintain viral loads of <1,000 copies/ml without treatment; and noncontrollers (NC), who fail to control viral replication. EC maintained higher frequencies of HIV-specific CD4 T cells, less mature polyfunctional Gag-specific CD4 T cells (CD27
+
CD57
−
CD45RO
+
), and Gag-specific polyfunctional CD4 T cells than those observed in NC. In EC, the frequency of polyfunctional Gag-specific CD8 T cells was higher than that observed in RC and NC. RC had a similar functional phenotype to that observed in NC, despite consistently lower viral loads. Finally, we found a direct correlation between the frequency of Gag-specific CD27
+
CD57
−
CD45RO
+
CD4
+
T cells and the frequency of mature HIV-specific CD8 T cells. Altogether, our data suggest that immature Gag-specific interleukin-2 (IL-2)-producing CD4
+
T cells may play an important role in spontaneous control of HIV viremia by effectively supporting HIV-specific CD8 T lymphocytes. This difference appears to differentiate EC from RC.
Partitioning the Genetic Diversity of a Virus Family: Approach and Evaluation through a Case Study of Picornaviruses
Authors: Lauber Chris , Gorbalenya Alexander E.
Published: January 2012

The recent advent of genome sequences as the only source available to classify many newly discovered viruses challenges the development of virus taxonomy by expert virologists who traditionally rely on extensive virus characterization. In this proof-of-principle study, we address this issue by presenting a computational approach (DEmARC) to classify viruses of a family into groups at hierarchical levels using a sole criterion—intervirus genetic divergence. To quantify genetic divergence, we used pairwise evolutionary distances (PEDs) estimated by maximum likelihood inference on a multiple alignment of family-wide conserved proteins. PEDs were calculated for all virus pairs, and the resulting distribution was modeled via a mixture of probability density functions. The model enables the quantitative inference of regions of distance discontinuity in the family-wide PED distribution, which define the levels of hierarchy. For each level, a limit on genetic divergence, below which two viruses join the same group, was objectively selected among a set of candidates by minimizing violations of intragroup PEDs to the limit. In a case study, we applied the procedure to hundreds of genome sequences of picornaviruses and extensively evaluated it by modulating four key parameters. It was found that the genetics-based classification largely tolerates variations in virus sampling and multiple alignment construction but is affected by the choice of protein and the measure of genetic divergence. In an accompanying paper (C. Lauber and A. E. Gorbalenya, J. Virol. 86:3905–3915, 2012), we analyze the substantial insight gained with the genetics-based classification approach by comparing it with the expert-based picornavirus taxonomy.