bluuebunny/crossref_metadata_2025 huggingface dataset:
Localized gravity/topography admittance and correlation spectra on Mars: Implications for regional and global evolution
Authors: McGovern Patrick J. , Solomon Sean C. , Smith David E. , Zuber Maria T. , Simons Mark , Wieczorek Mark A. , Phillips Roger J. , Neumann Gregory A. , Aharonson Oded , Head James W.
Published: January 2003

From gravity and topography data collected by the Mars Global Surveyor spacecraft we calculate gravity/topography admittances and correlations in the spectral domain and compare them to those predicted from models of lithospheric flexure. On the basis of these comparisons we estimate the thickness of the Martian elastic lithosphere (Te) required to support the observed topographic load since the time of loading. We convert Te to estimates of heat flux and thermal gradient in the lithosphere through a consideration of the response of an elastic/plastic shell. In regions of high topography on Mars (e.g., the Tharsis rise and associated shield volcanoes), the mass‐sheet (small‐amplitude) approximation for the calculation of gravity from topography is inadequate. A correction that accounts for finite‐amplitude topography tends to increase the amplitude of the predicted gravity signal at spacecraft altitudes. Proper implementation of this correction requires the use of radii from the center of mass (collectively known as the planetary “shape”) in lieu of “topography” referenced to a gravitational equipotential. Anomalously dense surface layers or buried excess masses are not required to explain the observed admittances for the Tharsis Montes or Olympus Mons volcanoes when this correction is applied. Derived Te values generally decrease with increasing age of the lithospheric load, in a manner consistent with a rapid decline of mantle heat flux during the Noachian and more modest rates of decline during subsequent epochs.
Indications, Techniques, and Complications of Lumbar Interbody Fusion
Authors: Tay Bobby B.K. , Berven Sigurd , Pascuzzi Robert M. , Roos Karen L. , Engstrom John W.
Published: January 2003

Lumbar interbody arthrodesis is a surgical technique that results in fusion of the anterior column of the spine. The indications for this procedure have evolved over time, and current indications include spinal deformity, segmental instability, and discogenic low back pain. Arthrodesis in the interbody space can be accomplished through anterior or posterior approaches to the spine, and these techniques are discussed. Surgical approaches in performing an interbody arthrodesis can be complicated by nerve and vascular injury. Clinical outcome studies are important in better defining the role of lumbar interbody arthrodesis as a substitute for posterior fusion in the lumbar spine.
Book Reviews
Authors: Batchelor Matthew , Moody Jane , Batchelor Matthew
Published: January 2003

Print‐on Demand: A New Option for Journal Publishers? On‐Demand Production of Books (A Strategic Futures Report for Full Members of PIRA International) by John Birkenshaw and David Butler PIRA International, www.piranet.com. ISBN 1‐85802‐433‐1

A Guide to Medical Publishing and Writing edited by Peter Richardson Mark Allen Publishing, 2002. ix + 145 pp. £10.99

The Book History Reader edited by David Finkelstein and Alistair McCleery Routledge, 2001. ISBN 0‐415‐22657‐0 (hbk), £60.00; 0‐415‐22658‐9 (pbk), £17.99
Human Papillomavirus and Cervical Cancer
Authors: Burd Eileen M.
Published: January 2003

Of the many types of human papillomavirus (HPV), more than 30 infect the genital tract. The association between certain oncogenic (high-risk) strains of HPV and cervical cancer is well established. Although HPV is essential to the transformation of cervical epithelial cells, it is not sufficient, and a variety of cofactors and molecular events influence whether cervical cancer will develop. Early detection and treatment of precancerous lesions can prevent progression to cervical cancer. Identification of precancerous lesions has been primarily by cytologic screening of cervical cells. Cellular abnormalities, however, may be missed or may not be sufficiently distinct, and a portion of patients with borderline or mildly dyskaryotic cytomorphology will have higher-grade disease identified by subsequent colposcopy and biopsy. Sensitive and specific molecular techniques that detect HPV DNA and distinguish high-risk HPV types from low-risk HPV types have been introduced as an adjunct to cytology. Earlier detection of high-risk HPV types may improve triage, treatment, and follow-up in infected patients. Currently, the clearest role for HPV DNA testing is to improve diagnostic accuracy and limit unnecessary colposcopy in patients with borderline or mildly abnormal cytologic test results.
Concepts in Immunology and Diagnosis of Hydatid Disease
Authors: Zhang Wenbao , Li Jun , McManus Donald P.
Published: January 2003

Echinococcosis is a cosmopolitan zoonosis caused by adult or larval stages of cestodes belonging to the genus Echinococcus (family Taeniidae). The two major species of medical and public health importance are Echinococcus granulosus and E. multilocularis, which cause cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. Both CE and AE are both serious diseases, the latter especially so, with a high fatality rate and poor prognosis if managed inappropriately. This review discusses new concepts and approaches in the immunology and diagnosis of CE, but comparative reference has also been made to AE infection and to earlier pivotal studies of both diseases. The review considers immunity to infection in the intermediate and definitive hosts, innate resistance, evasion of the immune system, and vaccination of intermediate and definitive hosts, and it particularly emphasizes procedures for diagnosis of CE and AE, including the value of immunodiagnostic approaches. There is also discussion of the new advances in recombinant and related DNA technologies, especially application of PCR, that are providing powerful tools in the fields of vaccinology and molecular diagnosis of echinococcosis.
Ehrlichia chaffeensis:a Prototypical Emerging Pathogen
Authors: Paddock Christopher D. , Childs James E.
Published: January 2003

Ehrlichia chaffeensis is an obligately intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts. The moderate to severe disease caused by E. chaffeensis in humans, first identified in 1986 and reported for more than 1,000 patients through 2000, represents a prototypical “emerging infection.” Knowledge of the biology and natural history of E. chaffeensis, and of the epidemiology, clinical features, and laboratory diagnosis of the zoonotic disease it causes (commonly referred to as human monocytic ehrlichiosis [HME]) has expanded considerably in the period since its discovery. In this review, we summarize briefly the current understanding of the microbiology, pathogenesis, and clinical manifestations associated with this pathogen but focus primarily on discussing various ecological factors responsible for the recent recognition of this important and potentially life-threatening tick-borne disease. Perhaps the most pivotal element in the emergence of HME has been the staggering increases in white-tailed deer populations in the eastern United States during the 20th century. This animal serves as a keystone host for all life stages of the principal tick vector (Amblyomma americanum) and is perhaps the most important vertebrate reservoir host for E. chaffeensis. The contributions of other components, including expansion of susceptible human populations, growth and broadening geographical distributions of other potential reservoir species and A. americanum, and improvements in confirmatory diagnostic methods, are also explored.
Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research
Authors: Koelle David M. , Corey Lawrence
Published: January 2003

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause prevalent, chronic infections that have serious outcomes in some individuals. Neonatal herpes may occur when the infant traverses the cervix during maternal genital herpes. Genital herpes is a major risk factor for human immunodeficiency virus type 1 transmission. Considerable efforts have been made to design and test vaccines for HSV, focusing on genital infection with HSV-2. Several protein subunit vaccines based on HSV-2 envelope glycoproteins have reached advanced-phase clinical trials. These antigens were chosen because they are the targets of neutralizing-antibody responses and because they elicit cellular immunity. Encouraging results have been reported in studies of treatment of HSV-seronegative women with a vaccine consisting of truncated glycoprotein D of HSV-2 and a novel adjuvant. Because most sexual HSV transmission occurs during asymptomatic shedding, it is important to evaluate the impact of vaccination on HSV-2 infection, clinically apparent genital herpes, and HSV shedding among vaccine recipients who acquire infection. There are several other attractive formats, including subunit vaccines that target cellular immune responses, live attenuated virus strains, and mutant strains that undergo incomplete lytic replication. HSV vaccines have also been evaluated for the immunotherapy of established HSV infection.
Vigorous Innate and Virus-Specific Cytotoxic T-Lymphocyte Responses to Murine Cytomegalovirus in the Submaxillary Salivary Gland
Authors: Cavanaugh Victoria J. , Deng Yuping , Birkenbach Mark P. , Slater Jacquelyn S. , Campbell Ann E.
Published: January 2003

To better understand the immunological mechanisms that permit prolonged shedding of murine cytomegalovirus (MCMV) from the salivary gland, the phenotypic and functional characteristics of leukocytes infiltrating the submaxillary gland (SMG) were analyzed in infected BALB/c mice. A robust innate immune response, comprised of CD11c+major histocompatibility complex class II+CD11b−CD8α+dendritic cells and γ/δ T-cell receptor-bearing CD3+T cells was prominent through at least 28 days postinfection. Concurrently, a dramatic increase in pan-NK (DX5+) CD3+and CD8+T cells was observed, while CD4+T cells, known to be essential for viral clearance from this tissue, increased slightly. The expression particularly of gamma interferon but also of interleukin-10 and CC chemokines was extraordinarily high in the SMG in response to MCMV infection. The gamma interferon was produced primarily by CD4+and CD8+T lymphocytes and DX5+CD3+T cells. The SMG CD8+T cells were highly cytolytic ex vivo, and a significant proportion of these cells were specific to an immunodominant MCMV peptide. These peptide-specific clones were not exhausted by the presence of high virus titers, which persisted in the SMG despite the strength of the cell-mediated responses. In contrast, MCMV replication was efficiently cleared from the draining cervical and periglandular lymph nodes, a tissue displaying a substantially weaker antiviral response. Our data indicated that vigorous innate and acquired immune responses are elicited, activated, and retained in response to mucosal inflammation from persistent MCMV infection of the submaxillary gland.
The Natural Host Range Shift and Subsequent Evolution of Canine Parvovirus Resulted from Virus-Specific Binding to the Canine Transferrin Receptor
Authors: Hueffer Karsten , Parker John S. L. , Weichert Wendy S. , Geisel Rachel E. , Sgro Jean-Yves , Parrish Colin R.
Published: January 2003

Canine parvovirus (CPV) is a host range variant of a feline virus that acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37°C only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type 2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs the later variant strain of CPV gained the ability to more efficiently use the canine TfR for infection and also showed reduced binding to feline and canine cells compared to CPV type 2. Differences on the top and the side of the threefold spike of the capsid surface controlled specific TfR binding and the efficiency of binding to feline and canine cells, and these differences also determined the cell infection properties of the viruses.
A Structurally Disordered Region at the C Terminus of Capsid Plays Essential Roles in Multimerization and Membrane Binding of the Gag Protein of Human Immunodeficiency Virus Type 1
Authors: Liang Chen , Hu Jing , Whitney James B. , Kleiman Lawrence , Wainberg Mark A.
Published: January 2003

Crystal structures of human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) reveal that the last 11 C-terminal amino acids are disordered. This disordered region contains a glycine-rich sequence 353-GVGGP-357 (numbering refers to the initiation methionine of Gag) that is highly conserved within the Gag proteins of HIV-1, HIV-2, and simian immunodeficiency virus, which suggests the importance of this sequence in virus replication. In the present study, we demonstrate that changing any individual residue within this short region in the context of the full-length HIV-1 genome virtually abolishes production of extracellular virus particles, in either the presence or absence of viral protease activity. This severe defect in virus particle production results from impaired Gag multimerization, as well as from decreased Gag association with the cellular membranes, as demonstrated by the results of gradient sedimentation and membrane flotation centrifugation assays. These findings are further supported by the diffuse distribution pattern of the mutant Gag within the cytoplasm, as opposed to the punctate distribution of the wild-type Gag on the plasma membrane. On the basis of these results, we propose that the disordered feature of amino acid stretch 353-GVGGP-357 in the CA crystal forms may have allowed Gag to adopt multiple conformations and that such structural flexibility is needed by Gag in order to construct geometrically complex particles.
The Core 2 β-1,6-
N
-Acetylglucosaminyltransferase-Mucin Encoded by Bovine Herpesvirus 4 Was Acquired from an Ancestor of the African Buffalo
Authors: Markine-Goriaynoff N. , Georgin J.-P. , Goltz M. , Zimmermann W. , Broll H. , Wamwayi H. M. , Pastoret P.-P. , Sharp P. M. , Vanderplasschen A.
Published: January 2003

The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only viral gene known to date that encodes a homologue of the cellular core 2 β-1,6-
N
-acetylglucosaminyltransferase-mucin type (C2GnT-M). To investigate the origin and evolution of the Bo17 gene, we analyzed its distribution among BoHV-4 strains and determined the sequences of Bo17 from nine representative strains and of the C2GnT-M gene from six species of ruminants expected to encompass the group within which the gene acquisition occurred. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. Phylogenetic analyses indicated that Bo17 was acquired from a recent ancestor of the African buffalo, implying that cattle subsequently acquired BoHV-4 by cross-species transmission. The rate of synonymous nucleotide substitution in Bo17 was estimated at 5 × 10
−8
to 6 × 10
−8
substitutions/site/year, consistent with previous estimates made under the assumption that herpesviruses have cospeciated with their hosts. The Bo17 gene acquisition was dated to around 1.5 million years ago. Bo17 sequences from BoHV-4 strains from African buffalo and from cattle formed two separate clades, estimated to have split about 700,000 years ago. Analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions revealed a burst of amino acid replacements subsequent to the transfer of the cellular gene to the viral genome, followed by a return to a strong constraint on nonsynonymous changes during the divergence of contemporary BoHV-4 strains. The Bo17 gene represents the most recent of the known herpesvirus gene acquisitions and provides the best opportunity for learning more about this important process of viral evolution.
Alphavirus Minus-Strand Synthesis and Persistence in Mouse Embryo Fibroblasts Derived from Mice Lacking RNase L and Protein Kinase R
Authors: Sawicki Dorothea L. , Silverman Robert H. , Williams Bryan R. , Sawicki Stanley G.
Published: January 2003

We report our studies to probe the possible role of the host response to double-stranded RNA in cessation of alphavirus minus-strand synthesis. Mouse embryo fibroblasts (MEF) from Mx1-deficient mice that also lack either the protein kinase R (PKR) or the latent RNase L or both PKR and RNase L were screened. In RNase L-deficient but not wild-type or PKR-deficient MEF, there was continuous synthesis of minus-strand templates and the formation of new replication complexes producing viral plus strands. Inhibiting translation caused minus-strand synthesis to stop and a loss of transcription activity of the mature replication complexes. This turnover of replication complexes that were stable in cells containing RNase L suggested that RNase L plays some role, albeit possibly indirect, in the formation of stable replication complexes during alphavirus infection. In addition, confluent monolayers of RNase L-deficient murine cells readily established persistent infections and were not killed. This phenotype is contrary to what has been observed for infection in vertebrate cells with a presumably functional RNase L gene and more resembled alphavirus replication in
Aedes
mosquito cells, in which the activity of replication complexes making plus stands was also found to decay with inhibition of translation.
Compartmentalization and Transmission of Multiple Epstein-Barr Virus Strains in Asymptomatic Carriers
Authors: Sitki-Green Diane , Covington Mary , Raab-Traub Nancy
Published: January 2003

Infection with the Epstein-Barr virus (EBV) is often subclinical in the presence of a healthy immune response; thus, asymptomatic infection is largely uncharacterized. This study analyzed the nature of EBV infection in 20 asymptomatic immunocompetent hosts over time through the identification of EBV strain variants in the peripheral blood and oral cavity. A heteroduplex tracking assay specific for the EBV geneLMP1precisely identified the presence of multiple EBV strains in each subject. The strains present in the peripheral blood and oral cavity were often completely discordant, indicating the existence of distinct infections, and the strains present and their relative abundance changed considerably between time points. The possible transmission of strains between the oral cavity and peripheral blood compartments could be tracked within subjects, suggesting that reactivation in the oral cavity and subsequent reinfection of B lymphocytes that reenter the periphery contribute to the maintenance of persistence. In addition, distinct virus strains persisted in the oral cavity over many time points, suggesting an important role for epithelial cells in the maintenance of persistence. Asymptomatic individuals without tonsillar tissue, which is believed to be an important source of virus for the oral cavity, also exhibited multiple strains and a cyclic pattern of transmission between compartments. This study revealed that the majority of patients with infectious mononucleosis were infected with multiple strains of EBV that were also compartmentalized, suggesting that primary infection involves the transmission of multiple strains. Both the primary and carrier states of infection with EBV are more complex than previously thought.
A Mutant Form of the Tax Protein of Bovine Leukemia Virus (BLV), with Enhanced Transactivation Activity, Increases Expression and Propagation of BLV In Vitro but Not In Vivo
Authors: Tajima Shigeru , Takahashi Masahiko , Takeshima Shin-nosuke , Konnai Satoru , Yin Shan Ai , Watarai Shinobu , Tanaka Yoshimasa , Onuma Misao , Okada Kosuke , Aida Yoko
Published: January 2003

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.
Independent Segregation of Human Immunodeficiency Virus Type 1 Gag Protein Complexes and Lipid Rafts
Authors: Ding Lingmei , Derdowski Aaron , Wang Jaang-Jiun , Spearman Paul
Published: January 2003

Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55
Gag
polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55
Gag
mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55
Gag
interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55
Gag
that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55
Gag
forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains.
HLA Class II Polymorphisms and Susceptibility to Recurrent Respiratory Papillomatosis
Authors: Gelder Colin M. , Williams O. Martin , Hart Keith W. , Wall Siôn , Williams Gareth , Ingrams Duncan , Bull Peter , Bunce Mike , Welsh Ken , Marshall Sara E. F. , Borysiewicz Leszek
Published: January 2003

Recurrent respiratory papillomatosis (RRP) is characterised by multiple laryngeal papillomas. Left untreated, the lesions enlarge, spread, and endanger the airway. Medical treatments are unsatisfactory, and repeated surgery remains the mainstay of therapy. RRP is caused by human papillomavirus (HPV) infection. However, since oral HPV infection is common and RRP is rare, other host and/or viral factors may contribute to pathogenesis. In an attempt to identify such factors, we have investigated 60 patients. The patients were HLA class I, II, and tumor necrosis factor TNF typed by sequence-specific primer PCR, and the results compared to those for 554 healthy controls by using Fisher's exact test. Peripheral blood mononuclear cell proliferative responses of 25 controls and 10 patients to HPV-11 L1 virus-like particles (VLP) were compared. Short-term VLP-specific T-cell lines were established, and recognition of L1 was analyzed. Finally, the L1 open reading frames of HPV isolates from four patients were sequenced. Susceptibility to RRP was associated with HLA DRB1*0301 (33 of 60 patients versus 136 of 554 controls,
P
< 0.0001). The three most severely affected patients were homozygous for this allele. A range of T-cell proliferative responses to HPV-11 VLP were observed in DRB1*0301-positive healthy donors which were comparable to those in DRB1*0301-negative controls. Individuals with juvenile-onset RRP also mounted a range of VLP responses, and their magnitude was negatively correlated with the clinical staging score (
P
= 0.012 by the Spearman rank correlation). DRB1*0301-positive patients who responded to L1 recognized the same epitope as did matched controls and produced similar cytokines. Sequencing of clinical isolates excluded the possibility that nonresponsiveness was the result of mutation(s) in L1.
Molecular and Functional Analysis of an Interferon Gene from the Zebrafish,
Danio rerio
Authors: Altmann Stephen M. , Mellon Mark T. , Distel Daniel L. , Kim Carol H.
Published: January 2003

The interferon (IFN) family consisting of alpha IFN (IFN-α), IFN-β, IFN-ω, IFN-δ, IFN-κ, and IFN-τ is a large group of cytokines involved in the innate immune response against various microorganisms. Genes for IFN have been cloned from a variety of mammalian and avian species; however, IFN genes from lower-order vertebrates have not been forthcoming. Here, we report the cloning and characterization of the IFN gene from the zebrafish,
Danio rerio
. Zebrafish IFN (zfIFN) is 185 amino acids in length, with the first 22 amino acids representing a putative signal peptide. Treatment with the known IFN inducer polyinosinic acid-polycytidylic acid (poly[I]-poly[C]) resulted in an increase in zfIFN mRNA transcripts. zfIFN was also able to activate the IFN-inducible Mx promoter when cotransfected with a plasmid containing the zebrafish Mx promoter upstream of a luciferase reporter gene. To demonstrate antiviral activity, zebrafish cells were transfected with zfIFN and challenged with a fish rhabdovirus. A 36% reduction in plaque number was seen in zfIFN-transfected cells, compared to cells transfected with a control vector. Phylogenetic analysis has shown zfIFN to be approximately equally divergent from avian and mammalian IFN, consistent with its origin from an IFN present in the most recent common ancestor of these divergent lineages. A putative IFN from puffer,
Fugu rubripes
, was also found when zfIFN was used to search the fugu genome database, demonstrating that zfIFN can be used to find additional fish IFN genes. These results demonstrate that zebrafish can be used as an effective model for studying innate immunity and immune response to infectious disease.
Multiple Amino Acid Residues within the Rabbit Prion Protein Inhibit Formation of Its Abnormal Isoform
Authors: Vorberg Ina , Groschup Martin H. , Pfaff Eberhard , Priola Suzette A.
Published: January 2003

Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.
Hepatitis C Virus RNA Synthesis in a Cell-Free System Isolated from Replicon-Containing Hepatoma Cells
Authors: Hardy Richard W. , Marcotrigiano Joseph , Blight Keril J. , Majors John E. , Rice Charles M.
Published: January 2003

A number of hepatitis C virus (HCV) proteins, including NS5B, the RNA-dependent RNA polymerase, were detected in membrane fractions from Huh7 cells containing autonomously replicating HCV RNA replicons. These membrane fractions were used in a cell-free system for the analysis of HCV RNA replication. Initial characterization revealed a reaction in which the production of replicon RNA increased over time at temperatures ranging from 25 to 40°C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn
2+
and Mg
2+
cations could be used in the reaction; however, concentrations of Mn
2+
greater than 1 mM were inhibitory. Compounds shown to inhibit recombinant NS3 and NS5B activity in vitro were found to inhibit RNA synthesis in the cell-free system. This system should be useful for biochemical analysis of HCV RNA synthesis by a multisubunit membrane-associated replicase and for evaluating potential antiviral agents identified in biochemical or cell-based screens.
A Subpopulation of Tegument Protein vhs Localizes to Detergent-Insoluble Lipid Rafts in Herpes Simplex Virus-Infected Cells
Authors: Lee Grace E. , Church Geoffrey A. , Wilson Duncan W.
Published: January 2003

Virion host shutoff (vhs) is a 58-kDa protein encoded by the UL41 gene of herpes simplex virus (HSV). vhs resides within the tegument of HSV, enters the cell cytoplasm at infection, and destabilizes host cell and viral mRNA. Late in infection, vhs must be assembled into the tegument of progeny virions, a poorly understood process. Using an anti-vhs antiserum and Western blotting of total cell or cytoplasmic extracts, we found that vhs is largely insoluble in HSV-infected cells, even in the presence of high levels of salt and the detergent Triton X-100. Furthermore, a subpopulation of vhs appears to be associated with detergent-insoluble lipid rafts and this raft population is enriched in a cytoplasmic fraction which contains assembling and mature HSV particles. Our data raise the possibility that HSV tegument polypeptides associate with membrane rafts, in common with the matrix proteins of a number of other viruses.
Enhancement of Antibodies to the Human Immunodeficiency Virus Type 1 Envelope by Using the Molecular Adjuvant C3d
Authors: Green Thomas D. , Montefiori David C. , Ross Ted M.
Published: January 2003

DNA vaccines expressing the envelope (Env) protein of the human immunodeficiency virus have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, the murine and human homologues of the complement component, C3d, were used in a DNA vaccine to enhance the titers of antibody to Env. Initially, plasmids expressing a secreted form of Env (sgp120) fused to one, two, or three copies of the murine homologue of C3d (mC3d) were constructed. Mice were inoculated with four vaccinations of DNA or two DNA vaccinations, followed by two boosts of affinity-purified gp120 protein. Analyses of titers demonstrated that multiple copies of mC3d coupled to sgp120 induced long-lasting, high-titer anti-Env antibody. Priming mice with sgp120-mC3d-DNA, followed by inoculation of purified gp120 protein, elicited the strongest antibody titers; however, the avidity maturation of the antibody was accelerated in the mice inoculated with sgp120-mC3d3-DNA. In addition, DNAs expressing sgp120 fused to three copies of the human homologue of C3d (hC3d3) efficiently enhanced the anti-Env antibody in rabbits. Lastly, antisera from both mice and rabbits vaccinated with DNA expressing sgp120-C3d3elicited higher titers of neutralizing antibody than did nonfused forms of Env. These results indicate that C3d, conjugated to sgp120, enhances the antibody responses to Env compared to non-C3d fused forms of Env, and this approach may be one way to overcome the poor ability of DNA vaccines to generate antibodies to Env.
Genome-Based Identification of Cancer Genes by Proviral Tagging in Mouse Retrovirus-Induced T-Cell Lymphomas
Authors: Kim Rachel , Trubetskoy Alla , Suzuki Takeshi , Jenkins Nancy A. , Copeland Neal G. , Lenz Jack
Published: January 2003

The identification of tumor-inducing genes is a driving force for elucidating the molecular mechanisms underlying cancer. Many retroviruses induce tumors by insertion of viral DNA adjacent to cellular oncogenes, resulting in altered expression and/or structure of the encoded proteins. The availability of the mouse genome sequence now allows analysis of retroviral common integration sites in murine tumors to be used as a genetic screen for identification of large numbers of candidate cancer genes. By positioning the sequences of inverse PCR-amplified, virus-host junction fragments within the mouse genome, 19 target genes were identified in T-cell lymphomas induced by the retrovirus SL3-3. The candidate cancer genes included transcription factors (
Fos
,
Gfi1
,
Lef1
,
Myb
,
Myc
,
Runx3
, and
Sox3
), all three D cyclins, Ras signaling pathway components (
Rras2
/
TC21
and
Rasgrp1
), and
Cmkbr7
/
CCR7
. The most frequent target was
Rras2
. Insertions as far as 57 kb away from the transcribed portion were associated with substantially increased transcription of
Rras2
, and no coding sequence mutations, including those typically involved in Ras activation, were detected. These studies demonstrate the power of genome-based analysis of retroviral insertion sites for cancer gene discovery, identify several new genes worth examining for a role in human cancer, and implicate the pathways in which those genes act in lymphomagenesis. They also provide strong genetic evidence that overexpression of unmutated
Rras2
contributes to tumorigenesis, thus suggesting that it may also do so if it is inappropriately expressed in human tumors.
Antibody-Mediated Targeting of an Adenovirus Vector Modified To Contain a Synthetic Immunoglobulin G-Binding Domain in the Capsid
Authors: Volpers Christoph , Thirion Christian , Biermann Volker , Hussmann Stefanie , Kewes Helmut , Dunant Patrick , von der Mark Helga , Herrmann Andreas , Kochanek Stefan , Lochmüller Hanns
Published: January 2003

Adenovirus vectors have been targeted to different cell types by genetic modification of the capsid or by using recombinant or chemically engineered adaptor molecules. However, both genetic capsid modifications and bridging adaptors have to be specifically tailored for each particular targeting situation. Here, we present an efficient and versatile strategy allowing the direct use of monoclonal antibodies against cell surface antigens for targeting of adenovirus vectors. A synthetic 33-amino-acid immunoglobulin G (IgG)-binding domain (Z33) derived from staphylococcal protein A was inserted into the adenovirus fiber protein. The fiber retained the ability to assemble into trimers, bound IgG with high affinity (

K
d

= 2.4 nM), and was incorporated into vector particles. The transduction efficiency of the Z33-modified adenovirus vector in epidermal growth factor receptor (EGFR)-expressing cells was strongly and dose-dependently enhanced by combination with an EGFR-specific monoclonal antibody. The antibody-mediated increase in cellular transduction was abolished in the presence of competing protein A. In targeting experiments with differentiated primary human muscle cells, up to a 77-fold increase in reporter gene transfer was achieved by preincubation of the vector with monoclonal antibodies directed against neuronal cell adhesion molecule or integrin α
7
, respectively. The IgG-binding adenovirus vector holds promise for directed gene transfer to a wide variety of cell types by simply changing the target-specific antibody.
Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection
Authors: Sung Vicky M.-H. , Shimodaira Shigetaka , Doughty Alison L. , Picchio Gaston R. , Can Huong , Yen T. S. Benedict , Lindsay Karen L. , Levine Alexandra M. , Lai Michael M. C.
Published: January 2003

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.
Nuclear Localization of the Duck Hepatitis B Virus Capsid Protein: Detection and Functional Implications of Distinct Subnuclear Bodies in a Compartment Associated with RNA Synthesis and Maturation
Authors: Mabit Hélène , Knaust Andreas , Breiner Klaus M. , Schaller Heinz
Published: January 2003

We have reinvestigated the subcellular distribution of the duck hepatitis B virus (DHBV) core protein in infected duck hepatocytes and in transfected cells. By using indirect immunofluorescence, the protein was found to be localized not only in the cytoplasm, as described previously, but also within the cell nucleus, being concentrated in distinct, brightly staining nuclear core bodies (NCBs). In colocalization studies using confocal microscopy, the NCBs were found exclusively in the periphery of nuclear subdomains characterized as splicing factor compartments and distal to other subnuclear domains. Also relevant for their functional significance is that the NCBs formed during the establishment of virus infection, i.e., at very low overall concentrations of newly synthesized core protein, and persisted throughout all stages of infection. Moreover, a subset of NCBs colocalized with foci of pregenomic DHBV RNA present at concentrations detectable by fluorescence in situ hybridization. Taken together, these findings indicate that a minor fraction of the DHBV core protein molecules escapes the major cytoplasmic assembly pathway to accumulate in specific subnuclear domains, and they furthermore suggest that these NCBs serve a role in the synthesis and/or maturation of the DHBV RNA pregenome.
Effect of Humoral Immune Responses on Controlling Viremia during Primary Infection of Rhesus Monkeys with Simian Immunodeficiency Virus
Authors: Schmitz Jörn E. , Kuroda Marcelo J. , Santra Sampa , Simon Meredith A. , Lifton Michelle A. , Lin Wenyu , Khunkhun Rajinder , Piatak Michael , Lifson Jeffrey D. , Grosschupff Gudrun , Gelman Rebecca S. , Racz Paul , Tenner-Racz Klara , Mansfield Keith A. , Letvin Norman L. , Montefiori David C. , Reimann Keith A.
Published: January 2003

Cellular immune responses mediated by CD8+lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.
In Vivo Induction of Apoptosis Correlating with Reduced Infectivity during Baculovirus Infection
Authors: Clarke Thomas E. , Clem Rollie J.
Published: January 2003

Spodoptera frugiperda
caterpillars were infected with a mutant of
Autographa californica M
nucleopolyhedrovirus lacking the antiapoptotic
p35
gene. Viral infectivity, replication, and spread were substantially reduced compared to that of a control revertant virus. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling confirmed that apoptosis occurred in mutant-infected caterpillars, thus directly correlating reduced infectivity and in vivo induction of apoptosis.
Amplification of a Complete Simian Immunodeficiency Virus Genome from Fecal RNA of a Wild Chimpanzee
Authors: Santiago Mario L. , Bibollet-Ruche Frederic , Bailes Elizabeth , Kamenya Shadrack , Muller Martin N. , Lukasik Magdalena , Pusey Anne E. , Collins D. Anthony , Wrangham Richard W. , Goodall Jane , Shaw George M. , Sharp Paul M. , Hahn Beatrice H.
Published: January 2003

Current knowledge of the genetic diversity of simian immunodeficiency virus (SIVcpz) infection of wild chimpanzees (
Pan troglodytes
) is incomplete since few isolates, mostly from captive apes from Cameroon and Gabon, have been characterized; yet this information is critical for understanding the origins of human immunodeficiency virus type 1 (HIV-1) and the circumstances leading to the HIV-1 pandemic. Here, we report the first full-length SIVcpz sequence (TAN1) from a wild chimpanzee (
Pan troglodytes schweinfurthii
) from Gombe National Park (Tanzania), which was obtained noninvasively by amplification of virion RNA from fecal samples collected under field conditions. Using reverse transcription-PCR and a combination of generic and strain-specific primers, we amplified 13 subgenomic fragments which together spanned the entire TAN1 genome (9,326 bp). Distance and phylogenetic tree analyses identified TAN1 unambiguously as a member of the HIV-1/SIVcpz group of viruses but also revealed an extraordinary degree of divergence from all previously characterized SIVcpz and HIV-1 strains. In Gag, Pol, and Env proteins, TAN1 differed from west-central African SIVcpz and HIV-1 strains on average by 36, 30, and 51% of amino acid sequences, respectively, approaching distance values typically found for SIVs from different primate species. The closest relative was SIVcpzANT, also from a
P. t. schweinfurthii
ape, which differed by 30, 25, and 44%, respectively, in these same protein sequences but clustered with TAN1 in all major coding regions in a statistically highly significant manner. These data indicate that east African chimpanzees, like those from west-central Africa, are naturally infected by SIVcpz but that their viruses comprise a second, divergent SIVcpz lineage which appears to have evolved in relative isolation for an extended period of time. Our data also demonstrate that noninvasive molecular epidemiological studies of SIVcpz in wild chimpanzees are feasible and that such an approach may prove essential for unraveling the evolutionary history of SIVcpz/HIV-1 as well as that of other pathogens naturally infecting wild primate populations.
CD154 Blockade Results in Transient Reduction in Theiler's Murine Encephalomyelitis Virus-Induced Demyelinating Disease
Authors: Howard Laurence M. , Neville Katherine L. , Haynes Lia M. , Dal Canto Mauro C. , Miller Stephen D.
Published: January 2003

Transient CD154 blockade at the onset of Theiler's murine encephalomyelitis virus-induced demyelinating disease ameliorated disease progression for 80 days, reduced immune cell infiltration, and transiently increased viral loads in the central nervous system. Peripheral antiviral and autoimmune T-cell responses were normal, and disease severity returned to control levels by day 120.
Protein Phosphatase 2A Enhances Activation of Human Immunodeficiency Virus Type 1 by Phorbol Myristate Acetate
Authors: Faulkner Neil E. , Lane Brian R. , Bock Paul J. , Markovitz David M.
Published: January 2003

The viral replication rate in patients infected with human immunodeficiency virus type 1 (HIV-1) is controlled in part by regulation of the transcription of viral genes. The rate of transcription is determined by a complex interplay between cellular and viral proteins and the promoter elements found in the long terminal repeats. Protein phosphatase 2A (PP2A) is a phosphoprotein that plays important roles in the regulation of signal transduction and cell growth. In this report, we demonstrate that overexpression of the catalytic subunit of protein phosphatase 2A (PP2Ac) increases the basal activity of the HIV-1 promoter and, especially, enhances the promoter's response to the protein kinase C (PKC) activator 12-
O
-tetradecanoyl phorbol-13-acetate (PMA). Additionally, ectopic PP2Ac enhances activation of HIV-1 provirus by PMA. Okadaic acid, a potent inhibitor of PP2A, markedly reduces both HIV-1 enhancer and proviral activation. Fostriecin, a PP2A inhibitor which has been used as an antineoplastic agent in clinical trials, is also able to inhibit PMA-stimulated HIV-1 proviral activation. These observations demonstrate a role for the important cellular phosphatase PP2A in HIV-1 transcription and replication and also suggest that PKC can potentiate the activity of PP2A. PP2A is a potential target for therapeutic intervention in patients infected with HIV-1.