bluuebunny/crossref_metadata_2025 huggingface dataset:
A high frequency amplitude-steered array for real-time volumetric imaging
Authors: Frazier Catherine H. , Hughes W. Jack , O’Brien William D.
Published: December 2002

Real-time three-dimensional acoustic imaging is difficult in water or tissue because of the slow speed of sound in these media. Conventional pulse–echo data collection, which uses at least one transmit pulse per line in the image, does not allow for the real-time update of a volume of data at practical ranges. Recently, a linear amplitude-steered array was presented that allows the collection of a plane of data with a single transmit pulse by spatially separating frequencies in the lateral direction. Later, by using a linear array with frequency separation in the vertical direction and rotating the array in the horizontal direction, volumetric data were collected with a small number of transmit pulses. By expanding the linear array to a two-dimensional array, data can now be collected for volumetric imaging in real time. In this study, the amplitude-steered array at the heart of a real-time volumetric sonar imaging system is described, giving the design of the array and describing how data are collected and processed to form images. An analysis of lateral resolution in the vertical and horizontal directions shows that resolution is improved in the direction of frequency separation over systems that use a broad transmit beam. Images from simulated data are presented.
A numerical optimization approach to acoustic hull array design
Authors: Wettergren Thomas A. , Casey John P. , Streit Roy L.
Published: December 2002

A numerical optimization approach is presented to optimize passive broadband detection performance of hull arrays through the adjustment of array shading weights. The approach is developed for general hull arrays in low signal-to-noise ratio scenarios, and is shown to converge rapidly to optimal solutions that maximize the array’s deflection coefficient. The beamformer is not redesigned in this approach; only the shading weights of the conventional beamformer are adjusted. This approach allows array designers to use the array to minimize the impact of known sources of noise on detection at the beamformer output while maintaining acoustic array gain against an unknown source. The technique is illustrated through numerical examples using hull-borne structural noise as the noise source; however, the design concept can be applied to other design parameters of the array such as element position, material selection, etc.
Direct magnitude estimation and interval scaling of pleasantness and severity in dysphonic and normal speakers
Authors: Eadie Tanya L. , Doyle Philip C.
Published: December 2002

The purpose of this study was to determine the validity of voice pleasantness and overall voice severity ratings of dysphonic and normal speakers using direct magnitude estimation (DME) and equal-appearing interval (EAI) auditory-perceptual scaling procedures. Twelve naive listeners perceptually evaluated voice pleasantness and severity from connected speech samples produced by 24 adult dysphonic speakers and 6 normal adult speakers. A statistical comparison of the two auditory-perceptual scales yielded a linear relationship representative of a metathetic continuum for voice pleasantness. A statistical relationship that is consistent with a prothetic continuum was revealed for ratings of voice severity. These data provide support for the use of either DME or EAI scales when making auditory-perceptual judgments of pleasantness, but only DME scales when judging overall voice severity for dysphonic speakers. These results suggest further psychophysical study of perceptual dimensions of voice and speech must be undertaken in order to avoid the inappropriate and invalid use of EAI scales used in the auditory-perceptual evaluation of the normal and dysphonic voice.
Short sound decay of ancient Chinese music bells
Authors: Pan Jie , Li Xiaodong , Tian Jing , Lin Tanran
Published: December 2002

Ancient Chinese music bells, which have oval-like cross sections, possess the unique feature of a short sound-decay time that is especially suitable for music with fast tempo. Although recorded in the literature nearly 1000 years ago, this feature has never been truly understood. Recent damping measurement of oval pipes with different axial ratios suggested a challenging task for acousticians as the increase of the modal damping with the pipe’s axial ratio is significant only when the pipe response is measured in air but not in vacuum. In this paper, the decrease of sound-decay time of oval pipes with the increased cross-section ovality is investigated. This is the first time that such short sound-decay feature is explained in terms of the rich sound radiation characteristics of the symmetric modes of music bells and the oval pipes. Finite element and boundary element methods are employed to explain how axial ratio significantly affects the normal velocity distribution, radiation efficiency, and directivity of acoustic modes.
High-frequency laser sonar system
Authors: Cray Benjamin A. , Sarma Ashwin , Kirsteins Ivars P.
Published: December 2002

A set of measurements recently completed at the Naval Undersea Warfare Center (NUWC) demonstrated that a laser-based sonar system can be used to detect acoustic particle velocity on the surface of a thin acoustically-compliant plate embedded beneath a standard acoustic window. The theoretical acoustic and measured surface particle velocity varied by less than 1 dB (reference m/s) over a wide frequency band (10 kHz to 100 kHz). However, the Polytec Model PSV-100 Scanning Laser Vibrometer System (SLVS) used in the experiments had relatively poor acoustic sensitivity, presumably due to high electronic noise, speckle noise, stand-off distance, and drifting laser focus. The laser’s acoustic sensitivity appears to be inversely proportional to the backscatter signal level. The existing SLVS can sample a grid of 512 by 512 points, with each grid point having a spot size of approximately 10 mm (0.0004 in.). Such fine sampling may be used to create essentially a continuous aperture, eliminating acoustic grating lobes at all frequencies of practical sonar interest.
Maintenance of Serological Memory by Polyclonal Activation of Human Memory B Cells
Authors: Bernasconi Nadia L. , Traggiai Elisabetta , Lanzavecchia Antonio
Published: December 2002

Production of antibodies can last for a lifetime, through mechanisms that remain poorly understood. Here, we show that human memory B lymphocytes proliferate and differentiate into plasma cells in response to polyclonal stimuli, such as bystander T cell help and CpG DNA. Furthermore, plasma cells secreting antibodies to recall antigens are produced in vivo at levels proportional to the frequency of specific memory B cells, even several years after antigenic stimulation. Although antigen boosting leads to a transient increase in specific antibody levels, ongoing polyclonal activation of memory B cells offers a means to maintain serological memory for a human lifetime.
Rainfall Variability, Carbon Cycling, and Plant Species Diversity in a Mesic Grassland
Authors: Knapp Alan K. , Fay Philip A. , Blair John M. , Collins Scott L. , Smith Melinda D. , Carlisle Jonathan D. , Harper Christopher W. , Danner Brett T. , Lett Michelle S. , McCarron James K.
Published: December 2002

Ecosystem responses to increased variability in rainfall, a prediction of general circulation models, were assessed in native grassland by reducing storm frequency and increasing rainfall quantity per storm during a 4-year experiment. More extreme rainfall patterns, without concurrent changes in total rainfall quantity, increased temporal variability in soil moisture and plant species diversity. However, carbon cycling processes such as soil CO
2
flux, CO
2
uptake by the dominant grasses, and aboveground net primary productivity (ANPP) were reduced, and ANPP was more responsive to soil moisture variability than to mean soil water content. Our results show that projected increases in rainfall variability can rapidly alter key carbon cycling processes and plant community composition, independent of changes in total precipitation.
Role of Melanopsin in Circadian Responses to Light
Authors: Ruby Norman F. , Brennan Thomas J. , Xie Xinmin , Cao Vinh , Franken Paul , Heller H. Craig , O'Hara Bruce F.
Published: December 2002

Melanopsin has been proposed as an important photoreceptive molecule for the mammalian circadian system. Its importance in this role was tested in melanopsin knockout mice. These mice entrained to a light/dark cycle, phase-shifted after a light pulse, and increased circadian period when light intensity increased. Induction of the immediate-early gene c-
fos
was observed after a nighttime light pulse in both wild-type and knockout mice. However, the magnitude of these behavioral responses in knockout mice was 40% lower than in wild-type mice. Although melanopsin is not essential for the circadian clock to receive photic input, it contributes significantly to the magnitude of photic responses.
Shape-Controlled Synthesis of Gold and Silver Nanoparticles
Authors: Sun Yugang , Xia Younan
Published: December 2002

Monodisperse samples of silver nanocubes were synthesized in large quantities by reducing silver nitrate with ethylene glycol in the presence of poly(vinyl pyrrolidone) (PVP). These cubes were single crystals and were characterized by a slightly truncated shape bounded by {100}, {110}, and {111} facets. The presence of PVP and its molar ratio (in terms of repeating unit) relative to silver nitrate both played important roles in determining the geometric shape and size of the product. The silver cubes could serve as sacrificial templates to generate single-crystalline nanoboxes of gold: hollow polyhedra bounded by six {100} and eight {111} facets. Controlling the size, shape, and structure of metal nanoparticles is technologically important because of the strong correlation between these parameters and optical, electrical, and catalytic properties.
Evidence for Antibody-Catalyzed Ozone Formation in Bacterial Killing and Inflammation
Authors: Wentworth Paul , McDunn Jonathan E. , Wentworth Anita D. , Takeuchi Cindy , Nieva Jorge , Jones Teresa , Bautista Cristina , Ruedi Julie M. , Gutierrez Abel , Janda Kim D. , Babior Bernard M. , Eschenmoser Albert , Lerner Richard A.
Published: December 2002

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H
2
O
2
) from singlet molecular oxygen (
1
O
2
*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H
2
O
2
production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.
Heart Regeneration in Zebrafish
Authors: Poss Kenneth D. , Wilson Lindsay G. , Keating Mark T.
Published: December 2002

Cardiac injury in mammals and amphibians typically leads to scarring, with minimal regeneration of heart muscle. Here, we demonstrate histologically that zebrafish fully regenerate hearts within 2 months of 20% ventricular resection. Regeneration occurs through robust proliferation of cardiomyocytes localized at the leading epicardial edge of the new myocardium. The hearts of zebrafish with mutations in the Mps1 mitotic checkpoint kinase, a critical cell cycle regulator, failed to regenerate and formed scars. Thus, injury-induced cardiomyocyte proliferation in zebrafish can overcome scar formation, allowing cardiac muscle regeneration. These findings indicate that zebrafish will be useful for genetically dissecting the molecular mechanisms of cardiac regeneration.
Short-Lived Nuclides in Hibonite Grains from Murchison: Evidence for Solar System Evolution
Authors: Marhas K. K. , Goswami J. N. , Davis A. M.
Published: December 2002

Records of now-extinct short-lived nuclides in meteorites provide information about the formation and evolution of the solar system. We have found excess
10
B that we attribute to the decay of short-lived
10
Be (half-life 1.5 million years) in hibonite grains from the Murchison meteorite. The grains show no evidence of decay of two other short-lived nuclides—
26
Al (half-life 700,000 years) and
41
Ca (half-life 100,000 years)—that may be present in early solar system solids. One plausible source of the observed
10
Be is energetic particle irradiation of material in the solar nebula. An effective irradiation dose of ∼2 × 10
18
protons per square centimeter with a kinetic energy of ≥10 megaelectronvolts per atomic mass unit can explain our measurements. The presence of
10
Be, coupled with the absence of
41
Ca and
26
Al, may rule out energetic particle irradiation as the primary source of
41
Ca and
26
Al present in some early solar system solids and strengthens the case of a stellar source for
41
Ca and
26
Al.
Everything You Want to Know and Moa
Authors: Steadman David W.
Published: December 2002

The Lost World of the Moa
Prehistoric Life of New Zealand. Trevor H. Worthy and Richard N. Holdaway. Indiana University Press, Bloomington, IN, 2002. 755 pp. $89.95. ISBN 0-253-34034-9.

The authors discuss the composition, ecology, and fate of the Pleistocene vertebrate fauna of New Zealand; their emphasis is on the large flightless birds, but they cover everything from frogs to bats.
The Draft Genome of
Ciona intestinalis
: Insights into Chordate and Vertebrate Origins
Authors: Dehal Paramvir , Satou Yutaka , Campbell Robert K. , Chapman Jarrod , Degnan Bernard , De Tomaso Anthony , Davidson Brad , Di Gregorio Anna , Gelpke Maarten , Goodstein David M. , Harafuji Naoe , Hastings Kenneth E. M. , Ho Isaac , Hotta Kohji , Huang Wayne , Kawashima Takeshi , Lemaire Patrick , Martinez Diego , Meinertzhagen Ian A. , Necula Simona , Nonaka Masaru , Putnam Nik , Rash Sam , Saiga Hidetoshi , Satake Masanobu , Terry Astrid , Yamada Lixy , Wang Hong-Gang , Awazu Satoko , Azumi Kaoru , Boore Jeffrey , Branno Margherita , Chin-bow Stephen , DeSantis Rosaria , Doyle Sharon , Francino Pilar , Keys David N. , Haga Shinobu , Hayashi Hiroko , Hino Kyosuke , Imai Kaoru S. , Inaba Kazuo , Kano Shungo , Kobayashi Kenji , Kobayashi Mari , Lee Byung-In , Makabe Kazuhiro W. , Manohar Chitra , Matassi Giorgio , Medina Monica , Mochizuki Yasuaki , Mount Steve , Morishita Tomomi , Miura Sachiko , Nakayama Akie , Nishizaka Satoko , Nomoto Hisayo , Ohta Fumiko , Oishi Kazuko , Rigoutsos Isidore , Sano Masako , Sasaki Akane , Sasakura Yasunori , Shoguchi Eiichi , Shin-i Tadasu , Spagnuolo Antoinetta , Stainier Didier , Suzuki Miho M. , Tassy Olivier , Takatori Naohito , Tokuoka Miki , Yagi Kasumi , Yoshizaki Fumiko , Wada Shuichi , Zhang Cindy , Hyatt P. Douglas , Larimer Frank , Detter Chris , Doggett Norman , Glavina Tijana , Hawkins Trevor , Richardson Paul , Lucas Susan , Kohara Yuji , Levine Michael , Satoh Nori , Rokhsar Daniel S.
Published: December 2002

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian,
Ciona intestinalis
. The
Ciona
genome contains ∼16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in
Ciona
, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.
Nota Bene: The Scuttlebutt on Science
Authors: Riddihough Guy
Published: December 2002

Eurekas and Euphorias
. Walter Gratzer. Oxford University Press, New York, 2002. 311 pp. $28, £16.99. ISBN 0-19-280403-0.

Gratzer offers readers a delightful sampling of interesting, revealing, and often humorous incidents from the lives of scientists.
Ubiquitin-Dependent Proteolysis of Cyclin D1 Is Associated with Coxsackievirus-Induced Cell Growth Arrest
Authors: Luo Honglin , Zhang Jingchun , Dastvan Frank , Yanagawa Bobby , Reidy Michael A. , Zhang Huifang M. , Yang Decheng , Wilson Janet E. , McManus Bruce M.
Published: December 2002

Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G
1
phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [
3
H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G
1
progression and G
1
/S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G
1
cyclin-dependent kinase activities, accompanied by decreased levels of G
1
cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3β pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3β independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G
1
/S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.
Evidence for Antibody-Mediated Enhancement of Simian Immunodeficiency Virus (SIV) Gag Antigen Processing and Cross Presentation in SIV-Infected Rhesus Macaques
Authors: Villinger Francois , Mayne Ann E. , Bostik Pavel , Mori Kazuyasu , Jensen Peter E. , Ahmed Rafi , Ansari Aftab A.
Published: December 2002

By using the dominant simian immunodeficiency virus (SIV) Gag Mamu-A01 restricted major histocompatibility complex (MHC) class I epitope p11CM, we demonstrate antibody-mediated enhanced MHC class I cross presentation of SIV Gag. In vitro restimulation of peripheral blood mononuclear cells from SIV-infected rhesus macaques with recombinant full-length SIV Gag p55 plus p55 affinity-purified immunoglobulin G (p55 Gag/p55-IgG) led to the generation of markedly higher frequencies of p11CM specific precursor cytotoxic T lymphocytes (p-CTLs) compared with restimulation with (i) SIV Gag p55 alone or (ii) optimal concentrations of the p11CM peptide alone. These results, along with the finding that CD4 depletion abrogated the enhancement, suggest a prominent role for CD4+T cells. Testing for p-CTLs against other Mamu-A01-restricted SIV Gag epitopes suggested that this mechanism favored recognition of the dominant p11CM peptide, potentially further skewing of the CTL response. The p-CTL enhancing effect was also decreased or abrogated by pepsin digestion of the p55-specific IgG or by the addition of monoclonal antibodies to Fc receptor (FcR) II/III, suggesting that the effect was dependent on FcR-mediated uptake of the immune-complexed antigen. Finally, incubation of antigen-presenting cells with SIV Gag p55 immune complexes in the presence of lactacystin or of bafilomycin indicated that the mechanism of antibody-mediated enhancement of cross presentation required both the proteasomal and the endosomal pathways. These data demonstrate for the first time the cross presentation of antigens via immune complexes in lentiviral infection and indicate a heretofore-unrecognized role for antibodies in modulating the magnitude and potentially also the breadth of MHC class I-restricted antigen processing and presentation and CTL responses.
Epstein-Barr Virus LMP2A Interferes with Global Transcription Factor Regulation When Expressed during B-Lymphocyte Development
Authors: Portis Toni , Longnecker Richard
Published: December 2002

Epstein-Barr virus (EBV) is associated with the development of malignant lymphomas and lymphoproliferative disorders in immunocompromised individuals. The LMP2A protein of EBV is thought to play a central role in this process by allowing the virus to persist in latently infected B lymphocytes. We have demonstrated that LMP2A, when expressed in B cells of transgenic mice, allows normal B-cell developmental checkpoints to be bypassed. To identify cellular genes targeted by LMP2A that are involved in this process, we have utilized DNA microarrays to compare gene transcription in B cells from wild-type versus LMP2A transgenic mice. In B cells from LMP2A transgenic mice, we observed decreased expression of many genes associated with normal B-cell development as well as reduced levels of the transcription factors that regulate their expression. In particular, expression of the transcription factor E2A was down-regulated in bone marrow and splenic B cells. Furthermore, E2A activity was inhibited in these cells as determined by decreased DNA binding and reduced expression of its target genes, including the transcription factors early B-cell factor and Pax-5. Expression of two E2A inhibitors, Id2 and SCL, was up-regulated in splenic B cells expressing LMP2A, suggesting a possible mechanism for E2A inhibition. These results indicate that LMP2A deregulates transcription factor expression and activity in developing B cells, and this likely allows for a bypass of normal signaling events required for proper B-cell development. The ability of LMP2A to interfere with B-cell transcription factor regulation has important implications regarding its role in EBV latency.
Impaired Binding of Standard Initiation Factors Mediates Poliovirus Translation Attenuation
Authors: Ochs Kerstin , Zeller Amandus , Saleh Lanja , Bassili Gergis , Song Yutong , Sonntag Anja , Niepmann Michael
Published: December 2002

In the oral poliovirus vaccine, three attenuated virus strains generated by Albert Sabin are used. However, insufficient genetic stability of these strains causes major problems in poliovirus eradication. In infected cells, translation of the plus-strand poliovirus RNA genome is directed by the internal ribosome entry site (IRES), a
cis
-acting RNA element that facilitates the cap-independent binding of ribosomes to an internal site of the viral RNA. In each Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence attenuation. Here we report how these decisive mutations in the IRES confer a reduction in poliovirus translation efficiency. These single-nucleotide exchanges impair the interaction of the standard translation initiation factor eIF4G with the IRES domain V. Moreover, binding of eIF4B and the polypyrimidine tract-binding protein and the association of ribosomes with the viral RNA are affected by these mutations. However, the negative effects of the IRES mutations are completely relieved by addition of purified eIF4F. This indicates that eIF4G is the crucial factor that initially binds to the poliovirus IRES and recruits the IRES to the other components of the translational apparatus, while impaired binding of eIF4G plays a key role in attenuation of poliovirus neurovirulence.
A Direct Transposon Insertion Tool for Modification and Functional Analysis of Viral Genomes
Authors: Vilen Heikki , Aalto Juha-Matti , Kassinen Anna , Paulin Lars , Savilahti Harri
Published: December 2002

Advances in DNA transposition technology have recently generated efficient tools for various types of functional genetic analyses. We demonstrate here the power of the bacteriophage Mu-derived in vitro DNA transposition system for modification and functional characterization of a complete bacterial virus genome. The linear double-stranded DNA genome ofEscherichia colibacteriophage PRD1 was studied by insertion mutagenesis with reporter mini-Mu transposons that were integrated in vitro into isolated genomic DNA. After introduction into bacterial cells by electroporation, recombinant transposon-containing virus clones were identified by autoradiography or visual blue-white screening employing α-complementation ofE. coliβ-galactosidase. Additionally, a modified transposon with engineeredNotI sites at both ends was used to introduce novel restriction sites into the phage genome. Analysis of the transposon integration sites in the genomes of viable recombinant phage generated a functional map, collectively indicating genes and genomic regions essential and nonessential for virus propagation. Moreover, promoterless transposons defined the direction of transcription within several insert-tolerant genomic regions. These strategies for the analysis of viral genomes are of a general nature and therefore may be applied to functional genomics studies in all prokaryotic and eukaryotic cell viruses.
Disulfide-Linked Integrase Oligomers Involving C280 Residues Are Formed In Vitro and In Vivo but Are Not Essential for Human Immunodeficiency Virus Replication
Authors: Bischerour Julien , Leh Hervé , Deprez Eric , Brochon Jean-Claude , Mouscadet Jean-François
Published: December 2002

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.
Sequence Analysis of Porcine Endogenous Retrovirus Long Terminal Repeats and Identification of Transcriptional Regulatory Regions
Authors: Wilson Carolyn A. , Laeeq Sabahat , Ritzhaupt Armin , Colon-Moran Winston , Yoshimura Fayth K.
Published: December 2002

Porcine cells express endogenous retroviruses, some of which are infectious for human cells. To better understand the replication of these porcine endogenous retroviruses (PERVs) in cells of different types and animal species, we have performed studies of the long terminal repeat (LTR) region of known gammaretroviral isolates of PERV. Nucleotide sequence determination of the LTRs of PERV-NIH, PERV-C, PERV-A, and PERV-B revealed that the PERV-A and PERV-B LTRs are identical, whereas the PERV-NIH and PERV-C LTRs have significant sequence differences in the U3 region between each other and with the LTRs of PERV-A and PERV-B. Sequence analysis revealed a similar organization of basal promoter elements compared with other gammaretroviruses, including the presence of enhancer-like repeat elements. The sequences of the PERV-NIH and PERV-C repeat element are similar to that of the PERV-A and PERV-B element with some differences in the organization of these repeats. The sequence of the PERV enhancer-like repeat elements differs significantly from those of other known gammaretroviral enhancers. The transcriptional activities of the PERV-A, PERV-B, and PERV-C LTRs relative to each other were similar in different cell types of different animal species as determined by transient expression assays. On the other hand, the PERV-NIH LTR was considerably weaker in these cell types. The transcriptional activity of all PERV LTRs was considerably lower in porcine ST-IOWA cells than in cell lines from other species. Deletion mutant analysis of the LTR of a PERV-NIH isolate identified regions that transactivate or repress transcription depending on the cell type.
Extent of Measles Virus Spread and Immune Suppression Differentiates between Wild-Type and Vaccine Strains in the Cotton Rat Model (
Sigmodon hispidus
)
Authors: Pfeuffer Joanna , Püschel Karen , Meulen Volker ter , Schneider-Schaulies Jürgen , Niewiesk Stefan
Published: December 2002

Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model (
Sigmodon hispidus
), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 → Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model.
Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells
Authors: De Silva Frank S. , Moss Bernard
Published: December 2002

Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.
Immunization of Newborn Rhesus Macaques with Simian Immunodeficiency Virus (SIV) Vaccines Prolongs Survival after Oral Challenge with Virulent SIVmac251
Authors: Van Rompay Koen K. A. , Greenier Jennifer L. , Cole Kelly Stefano , Earl Patricia , Moss Bernard , Steckbeck Jonathan D. , Pahar Bapi , Rourke Tracy , Montelaro Ronald C. , Canfield Don R. , Tarara Ross P. , Miller Christopher , McChesney Michael B. , Marthas Marta L.
Published: December 2002

There is an urgent need for active immunization strategies that, if administered shortly after birth, could protect infants in developing countries from acquiring human immunodeficiency virus (HIV) infection through breast-feeding. Better knowledge of the immunogenic properties of vaccine candidates in infants and of the effect of maternal antibodies on vaccine efficacy will aid in the development of such a neonatal HIV vaccine. Simian immunodeficiency virus (SIV) infection of infant macaques is a useful animal model of pediatric HIV infection with which to address these questions. Groups of infant macaques were immunized at birth and 3 weeks of age with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol, and Env (MVA-SIVgpe) or live-attenuated SIVmac1A11. One MVA-SIVgpe-immunized group had maternally derived anti-SIV antibodies prior to immunization. Animals were challenged orally at 4 weeks of age with a genetically heterogeneous stock of virulent SIVmac251. Although all animals became infected, the immunized animals mounted better antiviral antibody responses, controlled virus levels more effectively, and had a longer disease-free survival than the unvaccinated infected monkeys. Maternal antibodies did not significantly reduce the efficacy of the MVA-SIVgpe vaccine. In conclusion, although the tested vaccines delayed the onset of AIDS, further studies are warranted to determine whether a vaccine that elicits stronger early immune responses at the time of virus exposure may be able to prevent viral infection or AIDS in infants.
Functional Expression of Chemokine Receptor CCR5 on CD4+T Cells during Virus-Induced Central Nervous System Disease
Authors: Glass William G. , Lane Thomas E.
Published: December 2002

Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). CD4+T cells are important in amplifying demyelination by attracting macrophages into the central nervous system (CNS) following viral infection; however, the mechanisms governing the entry of these cells into the CNS are poorly understood. The role of chemokine receptor CCR5 in trafficking of virus-specific CD4+T cells into the CNS of MHV-infected mice was investigated. CD4+T cells from immunized CCR5+/+and CCR5−/−mice were expanded in the presence of the immunodominant epitope present in the MHV transmembrane (M) protein encompassing amino acids 133 to 147 (M133-147). Adoptive transfer of CCR5+/+-derived CD4+T cells to MHV-infected RAG1−/−mice resulted in CD4+-T-cell entry into the CNS and clearance of virus from the brain. These mice also displayed robust demyelination correlating with macrophage accumulation within the CNS. Conversely, CD4+T cells from CCR5−/−mice displayed an impaired ability to traffic into the CNS of MHV-infected RAG1−/−recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. Analysis of chemokine receptor mRNA expression by M133-147-expanded CCR5−/−-derived CD4+T cells revealed reduced expression of CCR1, CCR2, and CXCR3, indicating that CCR5 signaling is important in increased expression of these receptors, which aid in trafficking of CD4+T cells into the CNS. Collectively these results demonstrate that CCR5 signaling is important to migration of CD4+T cells to the CNS following MHV infection.
Role of Microglial Cells in Selective Replication of Simian Immunodeficiency Virus Genotypes in the Brain
Authors: Babas Tahar , Muñoz Daniel , Mankowski Joseph L. , Tarwater Patrick M. , Clements Janice E. , Zink M. Christine
Published: December 2002

An accelerated, consistent macaque simian immunodeficiency virus (SIV) model in which over 90% of pigtailed macaques (
Macaca nemestrina
) coinoculated with SIV/17E-Fr and SIV/DeltaB670 developed encephalitis was used to determine whether central nervous system (CNS) lesions are associated with the replication of specific genotypes in the brain and, more specifically, in the microglia. Ten of 11 inoculated macaques had severe (
n
= 3), moderate (
n
= 5), or mild (
n
= 2) encephalitis at 3 months postinoculation. To compare actively replicating viral genotypes in the CNS and in microglia with those in the periphery, the V1 region of the SIV envelope gene was amplified and sequenced from RNA extracted from basal ganglia, from microglial cells isolated from the brain, and from peripheral blood mononuclear cells (PBMC) isolated from blood at the time of death. To distinguish between actively replicating with latent viral genotypes in the CNS, viral genotypes in RNA and DNA from basal ganglia were compared. Two macrophage-tropic, neurovirulent viruses, SIV/17E-Fr and SIV/DeltaB670 Cl-2, predominated in the brain RNA of macaques with encephalitis, comprising 95% of the genotypes detected. The same two viral genotypes were present at the same frequencies in microglial cell RNA, suggesting that microglia are pivotal in the selective replication of neurovirulent viruses. There was a significantly greater number of viral genotypes in DNA than there were in RNA in the brain (
P
= 0.004), including those of both the macrophage- and lymphocyte-tropic viral strains. Furthermore, significantly fewer viral genotypes were detected in brain RNA than in PBMC RNA at the time of death (
P
= 0.004) and the viral strain that predominated in the brain frequently was different from that which predominated in the PBMC of the same animal. These data suggest that many viral genotypes enter the brain, but only a limited subset of macrophage-tropic, neurovirulent viruses replicate terminally in the brains of macaques with encephalitis. They further suggest that the selection of macrophage-tropic, neurovirulent viruses occurs not at the level of the blood-brain barrier but at a stage after virus entry and that microglial cells may play an important role in that selection process.
Novel Member of the
CD209
(
DC-SIGN
) Gene Family in Primates
Authors: Bashirova Arman A. , Wu Li , Cheng Jie , Martin Thomas D. , Martin Maureen P. , Benveniste Raoul E. , Lifson Jeffrey D. , KewalRamani Vineet N. , Hughes Austin , Carrington Mary
Published: December 2002

Two
CD209
family genes identified in humans,
CD209
(
DC-SIGN
) and
CD209L
(
DC-SIGNR
/
L-SIGN
), encode C-type lectins that serve as adhesion receptors for ICAM-2 and ICAM-3 and participate in the transmission of human and simian immunodeficiency viruses (HIV and SIV, respectively) to target cells in vitro. Here we characterize the
CD209
gene family in nonhuman primates and show that recent evolutionary alterations have occurred in this family across primate species. All of the primate species tested, specifically, Old World monkeys (OWM) and apes, have orthologues of human
CD209
. In contrast,
CD209L
is missing in OWM but present in apes. A third family member, that we have named
CD209L2
, was cloned from rhesus monkey cDNA and subsequently identified in OWM and apes but not in humans. Rhesus
CD209L2
mRNA was prominently expressed in the liver and axillary lymph nodes, although preliminary data suggest that levels of expression may vary among individuals. Despite a high level of sequence similarity to both human and rhesus CD209, rhesus CD209L2 was substantially less effective at binding ICAM-3 and poorly transmitted HIV type 1 and SIV to target cells relative to CD209. Our data suggest that the
CD209
gene family has undergone recent evolutionary processes involving duplications and deletions, the latter of which may be tolerated because of potentially redundant functional activities of the molecules encoded by these genes.
Identification of a Lytic-Cycle Epstein-Barr Virus Gene Product That Can Regulate PKR Activation
Authors: Poppers Jeremy , Mulvey Matthew , Perez Cesar , Khoo David , Mohr Ian
Published: December 2002

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of viral gene expression. Like many transactivators encoded by herpesviruses, SM transports predominantly unspliced viral mRNA cargo from the nucleus to the cytosol, where it is subsequently translated. This activity likely involves a region of the protein that has homology to the herpes simplex virus type 1 (HSV-1) ICP27 gene product, the first member of this class of regulators to be discovered. However, SM also contains a repetitive segment rich in arginine and proline residues that is dispensable for its effects on RNA transport and splicing. This portion of SM, comprised of RXP triplet repeats, shows homology to the carboxyl-terminal domain of Us11, a double-stranded RNA (dsRNA) binding protein encoded by HSV-1 that inhibits activation of the cellular PKR kinase. To evaluate the intrinsic ability of SM to regulate PKR, we expressed and purified several SM protein derivatives and examined their activity in a variety of biochemical assays. The full-length SM protein bound dsRNA, associated physically with PKR, and prevented PKR activation. Removal of the 37-residue RXP domain significantly compromised all of these activities. Furthermore, the SM RXP domain was itself sufficient to inhibit PKR activation and interact with the kinase. Relative to its Us11 counterpart, the SM RXP segment bound dsRNA with reduced affinity and responded differently to single-stranded competitor polynucleotides. Thus, SM represents the first EBV gene product expressed during the lytic cycle that can prevent PKR activation. In addition, the RXP repeat segment appears to be a conserved herpesvirus motif capable of associating with dsRNA and modulating activation of the PKR kinase, a molecule important for the control of translation and the cellular antiviral response.
Characterization of a Siberian Virus Isolated from a Patient with Progressive Chronic Tick-Borne Encephalitis
Authors: Gritsun T. S. , Frolova T. V. , Zhankov A. I. , Armesto M. , Turner S. L. , Frolova M. P. , Pogodina V. V. , Lashkevich V. A. , Gould E. A.
Published: December 2002

A strain ofTick-borne encephalitis virusdesignated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype ofTick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T277→V and E279→G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.
The Genome Length of Human Parainfluenza Virus Type 2 Follows the Rule of Six, and Recombinant Viruses Recovered from Non-Polyhexameric-Length Antigenomic cDNAs Contain a Biased Distribution of Correcting Mutations
Authors: Skiadopoulos Mario H. , Vogel Leatrice , Riggs Jeffrey M. , Surman Sonja R. , Collins Peter L. , Murphy Brian R.
Published: December 2002

Members of the
Paramyxovirinae
subfamily of the
Paramyxoviridae
family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the “rule of six.” To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.