bluuebunny/crossref_metadata_2025 huggingface dataset:
Marek's Disease Virus-Encoded vIL-8 Gene Is Involved in Early Cytolytic Infection but Dispensable for Establishment of Latency
Authors: Cui Xiaoping , Lee Lucy F. , Reed Willie M. , Kung Hsing-Jien , Reddy Sanjay M.
Published: April 2004

Marek's disease, a lymphoproliferative disease of chickens, is caused by an alphaherpesvirus, Marek's disease virus (MDV). This virus encodes a virokine, vIL-8, with general homology to cellular CXC chemokines such as interleukin-8 (IL-8) and Gro-α. To study the function of vIL-8 gene, we deleted both copies of vIL-8 residing in the terminal repeat long and internal repeat long region of the viral genome and generated a mutant virus with vIL-8 deleted, rMd5/ΔvIL-8. Growth kinetics study showed that vIL-8 gene is dispensable for virus replication in cell culture. In vivo, the vIL-8 gene is involved in early cytolytic infections in lymphoid organs, as evidenced by limited viral antigen expression of rMd5/ΔvIL-8. However, the rMd5/ΔvIL-8 virus is unimpaired in virus replication in the feather follicle epithelium. vIL-8 does not appear to be important for establishment of latency, since rMd5/ΔvIL-8 and the wild-type virus have similar viremia titers at 14 days postinfection, a period when the virus titer comes primarily from reactivated latent genomes. Nevertheless, because of the impaired cytolytic infections, the overall transformation efficiency of the virus with vIL-8 deleted is much lower, as reflected by the reduced number of transformed cells at 5 weeks postinoculation and the presence of fewer gross tumors. Importantly, the revertant virus that restored the expression of vIL-8 gene also restored the wild-type phenotype, indicating the deficient phenotypes are results of vIL-8 deletion. One of the interesting differences between the MDV vIL-8 gene and its cellular counterpart is the presence of a DKR (Asp-Lys-Arg) motif instead of ELR (Glu-Leu-Arg) preceding the invariable CXC motif. To study the significance of this variation, we generated recombinant MDV, rMd5/vIL-8-ELR, carrying the ELR motif. Both in vitro and in vivo studies revealed that the DKR motif is as competent as ELR in pathogenesis of MDV.
Safety and Efficacy of Chimeric Yellow Fever-Dengue Virus Tetravalent Vaccine Formulations in Nonhuman Primates
Authors: Guirakhoo F. , Pugachev K. , Zhang Z. , Myers G. , Levenbook I. , Draper K. , Lang J. , Ocran S. , Mitchell F. , Parsons M. , Brown N. , Brandler S. , Fournier C. , Barrere B. , Rizvi F. , Travassos A. , Nichols R. , Trent D. , Monath T.
Published: April 2004

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.
Scrapie Protein Degradation by Cysteine Proteases in CD11c
+
Dendritic Cells and GT1-1 Neuronal Cells
Authors: Luhr Katarina M. , Nordström Elin K. , Löw Peter , Ljunggren Hans-Gustaf , Taraboulos Albert , Kristensson Krister
Published: April 2004

Dendritic cells (DC) of the CD11c
+
myeloid phenotype have been implicated in the spread of scrapie in the host. Previously, we have shown that CD11c
+
DC can cause a rapid degradation of proteinase K-resistant prion proteins (PrP
Sc
) in vitro, indicating a possible role of these cells in the clearance of PrP
Sc
. To determine the mechanisms of PrP
Sc
degradation, CD11c
+
DC that had been exposed to PrP
Sc
derived from a neuronal cell line (GT1-1) infected with scrapie (ScGT1-1) were treated with a battery of protease inhibitors. Following treatment with the cysteine protease inhibitors (2S,3S)-
trans
-epoxysuccinyl-
l
-leucylamido-3-methylbutane (E-64c), its ethyl ester (E-64d), and leupeptin, the degradation of PrP
Sc
was inhibited, while inhibitors of serine and aspartic and metalloproteases (aprotinin, pepstatin, and phosphoramidon) had no effect. An endogenous degradation of PrP
Sc
in ScGT1-1 cells was revealed by inhibiting the expression of cellular PrP (PrP
C
) by RNA interference, and this degradation could also be inhibited by the cysteine protease inhibitors. Our data show that PrP
Sc
is proteolytically cleaved preferentially by cysteine proteases in both CD11c
+
DC and ScGT1-1 cells and that the degradation of PrP
Sc
by proteases is different from that of PrP
C
. Interference by protease inhibitors with DC-induced processing of PrP
Sc
has the potential to modify prion spread, clearance, and immunization in a host.
Recruitment of Cellular Recombination and Repair Proteins to Sites of Herpes Simplex Virus Type 1 DNA Replication Is Dependent on the Composition of Viral Proteins within Prereplicative Sites and Correlates with the Induction of the DNA Damage Response
Authors: Wilkinson Dianna E. , Weller Sandra K.
Published: April 2004

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.
Increased Incidence of Squamous Cell Carcinomas in
Mastomys natalensis
Papillomavirus E6 Transgenic Mice during Two-Stage Skin Carcinogenesis
Authors: Helfrich Iris , Chen Min , Schmidt Rainer , Fürstenberger Gerhard , Kopp-Schneider Annette , Trick David , Gröne Hermann-Josef , zur Hausen Harald , Rösl Frank
Published: April 2004

Papillomaviruses cause certain forms of human cancers, most notably carcinomas of the uterine cervix. In contrast to the well-established involvement of papillomavirus infection in the etiology of cervical carcinomas and in carcinomas of a rare hereditary condition, epidermodysplasia verruciformis, a causative role for cutaneous human papillomavirus types in the development of nonmelanoma skin cancer has not been proven. In order to better understand the functions of individual genes of cutaneous papillomavirus types, we generated transgenic mice carrying oncogene E6 of the
Mastomys natalensis
papillomavirus (MnPV), which causes keratoacanthomas of the skin in its natural host. In the present study, we demonstrate that under conditions of experimental two-stage skin carcinogenesis, fast-paced squamous cell carcinomas develop in nearly 100% of MnPV E6 transgenic mice in comparison to 10% in their nontransgenic littermates (log rank test;
P
< 0.0001). Therefore, we conclude that the MnPV E6 transgene favors the malignant progression of chemically induced tumors. Whereas an activating H-
ras
mutation is a consistent feature in benign and malignant tumors in wild-type mice, the majority of papillomas and keratoacanthomas and all squamous cell carcinomas obtained in MnPV E6 transgenic mice contain nonmutated
ras
alleles. These results indicate that the development of squamous cell carcinomas in MnPV E6 transgenic mice does not depend on an activated H-
ras
oncogene.
A Glycine-Rich Bovine Herpesvirus 5 (BHV-5) gE-Specific Epitope within the Ectodomain Is Important for BHV-5 Neurovirulence
Authors: Al-Mubarak A. , Zhou Y. , Chowdhury S. I.
Published: April 2004

The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5.
A Novel Motif in Geminivirus Replication Proteins Interacts with the Plant Retinoblastoma-Related Protein
Authors: Arguello-Astorga Gerardo , Lopez-Ochoa Luisa , Kong Ling-Jie , Orozco Beverly M. , Settlage Sharon B. , Hanley-Bowdoin Linda
Published: April 2004

The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted α-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.
Norwalk Virus N-Terminal Nonstructural Protein Is Associated with Disassembly of the Golgi Complex in Transfected Cells
Authors: Fernandez-Vega Virneliz , Sosnovtsev Stanislav V. , Belliot Gaël , King Adriene D. , Mitra Tanaji , Gorbalenya Alexander , Green Kim Y.
Published: April 2004

Norwalk virus
is the prototype strain for members of the genus
Norovirus
in the family
Caliciviridae
, which are associated with epidemic gastroenteritis in humans. The nonstructural protein encoded in the N-terminal region of the first open reading frame (ORF1) of the Norwalk virus genome is analogous in gene order to proteins 2A and 2B of the picornaviruses; the latter is known for its membrane-associated activities. Confocal microscopy imaging of cells transfected with a vector plasmid that provided expression of the entire Norwalk virus N-terminal protein (amino acids 1 to 398 of the ORF1 polyprotein) showed colocalization of this protein with cellular proteins of the Golgi apparatus. Furthermore, this colocalization was characteristically associated with a visible disassembly of the Golgi complex into discrete aggregates. Deletion of a predicted hydrophobic region (amino acids 360 to 379) in a potential 2B-like (2BL) region (amino acids 301 to 398) near the C terminus of the Norwalk virus N-terminal protein reduced Golgi colocalization and disassembly. Confocal imaging was conducted to examine the expression characteristics of fusion proteins in which the 2BL region from the N-terminal protein of Norwalk virus (a genogroup I norovirus) or MD145 (a genogroup II norovirus) was fused to the C terminus of enhanced green fluorescent protein. Expression of each fusion protein in cells showed evidence for its colocalization with the Golgi apparatus. These data indicate that the N-terminal protein of Norwalk virus interacts with the Golgi apparatus and may play a 2BL role in the induction of intracellular membrane rearrangements associated with positive-strand RNA virus replication in cells.
In Vitro Replication of Hepatitis E Virus (HEV) Genomes and of an HEV Replicon Expressing Green Fluorescent Protein
Authors: Emerson Suzanne U. , Nguyen Hanh , Graff Judith , Stephany David A. , Brockington Alicia , Purcell Robert H.
Published: April 2004

Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome. Fluorescence-activated cell sorter analysis confirmed that the 7mG cap structure was critical for efficient infectivity, although replication could be initiated at a very low level in its absence. HEV virions were also able to infect a limited number of cells of certain lines.
CCAAT/Enhancer Binding Protein α Binds to the Epstein-Barr Virus (EBV) ZTA Protein through Oligomeric Interactions and Contributes to Cooperative Transcriptional Activation of the ZTA Promoter through Direct Binding to the ZII and ZIIIB Motifs during Induction of the EBV Lytic Cycle
Authors: Wu Frederick Y. , Wang Shizhen Emily , Chen Honglin , Wang Ling , Hayward S. Diane , Hayward Gary S.
Published: April 2004

The Epstein-Barr virus (EBV)-encoded ZTA protein interacts strongly with and stabilizes the cellular CCAAT/enhancer binding protein α (C/EBPα), leading to the induction of p21-mediated G
1
cell cycle arrest. Despite the strong interaction between these two basic leucine zipper (bZIP) family proteins, the ZTA and C/EBPα subunits do not heterodimerize, as indicated by an in vitro cross-linking assay with in vitro-cotranslated
35
S-labeled C/EBPα and
35
S-labeled ZTA protein. Instead, they evidently form a higher-order oligomeric complex that competes with C/EBPα binding but not with ZTA binding in electrophoretic mobility shift assays (EMSAs). Glutathione
S
-transferase affinity assays with mutant ZTA proteins revealed that the basic DNA binding domain and the key leucine zipper residues required for homodimerization are all required for the interaction with C/EBPα. ZTA is known to bind to two ZRE sites within the ZTA promoter and to positively autoregulate its own expression in transient cotransfection assays, but there is conflicting evidence about whether it does so in vivo. Examination of the proximal ZTA upstream promoter region by in vitro EMSA analysis revealed two high-affinity C/EBP binding sites (C-2 and C-3), which overlap the ZII and ZIIIB motifs, implicated as playing a key role in lytic cycle induction. A chromatin immunoprecipitation assay confirmed the in vivo binding of both endogenous C/EBPα and ZTA protein to the ZTA promoter after lytic cycle induction but not during the latent state in EBV-infected Akata cells. Reporter assays revealed that cotransfected C/EBPα activated the ZTA promoter even more effectively than cotransfected ZTA. However, synergistic activation of the ZTA promoter was not observed when ZTA and C/EBPα were cotransfected together in either HeLa or DG75 cells. Mutagenesis of either the ZII or the ZIIIB sites in the ZTA promoter strongly reduced C/EBPα transactivation, suggesting that these sites act cooperatively. Furthermore, the introduction of exogenous C/EBPα into EBV-infected HeLa-BX1 cells induced endogenous ZTA mRNA and protein expression, as demonstrated by both reverse transcription-PCR and immunoblotting assays. Finally, double-label immunofluorescence assays suggested that EAD protein expression was activated even better than ZTA expression in latently infected C/EBPα-transfected Akata cells, perhaps because of the presence of a strong B-cell-specific repressed chromatin conformation on the ZTA promoter itself during EBV latency.
Roles of Target Cells and Virus-Specific Cellular Immunity in Primary Simian Immunodeficiency Virus Infection
Authors: Regoes Roland R. , Antia Rustom , Garber David A. , Silvestri Guido , Feinberg Mark B. , Staprans Silvija I.
Published: April 2004

There is an ongoing debate on whether acute human immunodeficiency virus infection is controlled by target cell limitation or by virus-specific cellular immunity. To resolve this question, we developed a novel mathematical modeling scheme which allows us to incorporate measurements of virus load, target cells, and virus-specific immunity and applied it to a comprehensive data set generated in an experiment involving rhesus macaques infected with simian immunodeficiency virus. Half of the macaques studied were treated during the primary infection period with reagents which block T-cell costimulation and as a result displayed severely impaired virus-specific immune responses. Our results show that early viral replication in normal infection is controlled to a large extent by virus-specific CD8+T cells and not by target cell limitation.
Characterization of a Highly Evolved Vaccine-Derived Poliovirus Type 3 Isolated from Sewage in Estonia
Authors: Blomqvist Soile , Savolainen Carita , Laine Pia , Hirttiö Päivi , Lamminsalo Elisa , Penttilä Eija , Jöks Silver , Roivainen Merja , Hovi Tapani
Published: April 2004

Two types of vaccine-derived polioviruses have been recently designated to emphasize the different origins of the evolved viruses: circulating vaccine-derived polioviruses (cVDPV) associated with outbreaks of paralytic disease and strains isolated from chronically infected immunodeficient individuals (iVDPV). We describe here a type 3 VDPV (PV3/EST/02/E252; later E252) isolated from sewage collected in Tallinn, Estonia, in October 2002. Due to aberrant properties in subtyping, the virus was subjected to detailed characterization. Partial genomic sequencing suggested that the closest relative was the oral vaccine strain PV3/Sabin, but the two virus strains shared only 86.7% of the 900 nucleotides (nt) coding for the capsid protein VP1. Phylogenetic analysis of the nearly complete genome [nt 19 to poly(A)] revealed multiple nucleotide substitutions throughout the genome and a possible Sabin 3/Sabin 1-recombination junction site in the 2C coding region. A calculation based on the estimated mutation frequency of the P1 region of polioviruses suggested that the E252 virus might have replicated in one or more individuals for approximately 10 years. No persons chronically excreting poliovirus are known in Estonia. Amino acid substitutions were seen in all known antigenic sites, which was consistent with the observed aberrant antigenic properties of the virus demonstrated by both monoclonal antibodies and human sera from vaccinated children. In spite of the apparent transmission potential, no evidence was obtained for circulation of the virus in the Estonian population.
JC Virus Induces Nonapoptotic Cell Death of Human Central Nervous System Progenitor Cell-Derived Astrocytes
Authors: Seth Pankaj , Diaz Frank , Tao-Cheng Jung-Hwa , Major Eugene O.
Published: April 2004

JC virus (JCV), a human neurotropic polyomavirus, demonstrates a selective glial cell tropism that causes cell death through lytic infection. Whether these cells die via apoptosis or necrosis following infection with JCV remains unclear. To investigate the mechanism of virus-induced cell death, we used a human central nervous system progenitor-derived astrocyte cell culture model developed in our laboratory. Using in situ DNA hybridization, immunocytochemistry, electron microscopy, and an RNase protection assay, we observed that astrocytes support a progressive JCV infection, which eventually leads to nonapoptotic cell death. Infected astrocyte cell cultures showed no difference from noninfected cells in mRNA expression of the caspase family genes or in any ultrastructural features associated with apoptosis. Infected cells demonstrated striking necrotic features such as cytoplasmic vacuolization, watery cytoplasm, and dissolution of organelles. Furthermore, staining for caspase-3 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling were not detected in infected astrocyte cultures. Our findings suggest that JCV-induced cell death of these progenitor cell-derived astrocytes does not utilize an apoptosis pathway but exhibits a pattern of cell destruction consistent with necrotic cell death.
Reemerging H5N1 Influenza Viruses in Hong Kong in 2002 Are Highly Pathogenic to Ducks
Authors: Sturm-Ramirez Katharine M. , Ellis Trevor , Bousfield Barry , Bissett Lucy , Dyrting Kitman , Rehg Jerold E. , Poon Leo , Guan Yi , Peiris Malik , Webster Robert G.
Published: April 2004

Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.
Transduction of Terminally Differentiated Neurons by Avian Sarcoma Virus
Authors: Greger James G. , Katz Richard A. , Taganov Konstantin , Rall Glenn F. , Skalka Anna Marie
Published: April 2004

Recent studies have demonstrated that avian sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.
A Human Immunodeficiency Virus Type 1-Infected Individual with Low Viral Load Harbors a Virus Variant That Exhibits an In Vitro RNA Dimerization Defect
Authors: Huthoff Hendrik , Das Atze T. , Vink Monique , Klaver Bep , Zorgdrager Fokla , Cornelissen Marion , Berkhout Ben
Published: April 2004

We investigated the in vitro RNA dimerization properties of the untranslated leader RNA derived from human immunodeficiency virus type 1 variants circulating in an individual with a low viral load and slow disease progression. The leader sequences of these viruses contain highly unusual polymorphisms within the dimerization initiation site (DIS): an insert that abolishes dimerization and a compensatory substitution. The dimerization of leader RNA from late stages of infection is further improved by additional mutations outside the DIS motif that facilitate a secondary structure switch from a dimerization-incompetent to a dimerization-competent RNA conformation.
Delivery of Short Hairpin RNA Sequences by Using a Replication-Competent Avian Retroviral Vector
Authors: Bromberg-White Jennifer L. , Webb Craig P. , Patacsil Veronique S. , Miranti Cindy K. , Williams Bart O. , Holmen Sheri L.
Published: April 2004

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.
Inability of Simian Virus 40 To Establish Productive Infection of Lymphoblastic Cell Lines
Authors: Shaikh Sophie , Skoczylas Christine , Longnecker Richard , Rundell Kathleen
Published: April 2004

Lymphoblastic cell lines were infected with simian virus 40 (SV40) and then monitored for evidence of a productive infection. No evidence of early gene expression was found 2 days following infection, as determined by assaying viral mRNAs and early antigens. Furthermore, only small amounts of virus could be detected by plaque assay 2 days after infection, and levels slowly declined until they were undetectable after a few weeks in culture. Thus, human lymphocytes are not readily infectible with SV40 and do not provide a simple model for studying interactions of SV40 with a human cell type.
Role of Hydrophobic Residues in the Central Ectodomain of gp41 in Maintaining the Association between Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Subunits gp120 and gp41
Authors: York Joanne , Nunberg Jack H.
Published: April 2004

The interaction between the gp120 and gp41 subunits of the human immunodeficiency virus envelope glycoprotein serves to stabilize the virion form of the complex and to transmit receptor-induced conformational changes in gp120 to trigger the membrane fusion activity of gp41. In this study, we used site-directed mutagenesis to identify amino acid residues in the central ectodomain of gp41 that contribute to the stability of the gp120-gp41 association. We identified alanine mutations at six positions, including four tryptophan residues, which result in mutant envelope glycoprotein complexes that fail to retain gp120 on the cell surface. These envelope glycoproteins readily shed their gp120 and are unable to mediate cell-cell fusion. These findings suggest an important role for the conserved bulky hydrophobic residues in stabilizing the gp120-gp41 complex.
Intraepithelial γδ T Cells May Bridge a Gap between Innate Immunity and Acquired Immunity to Herpes Simplex Virus Type 2
Authors: Nishimura Hitoshi , Yajima Toshiki , Kagimoto Yoshiko , Ohata Mutsuhiro , Watase Taketo , Kishihara Kenji , Goshima Fumi , Nishiyama Yukihiro , Yoshikai Yasunobu
Published: April 2004

Mice depleted of γδ T cells by in vivo administration of anti-TCRγδ monoclonal antibodies showed susceptibility against an intravaginal infection with herpes simplex virus type 2 (HSV-2). The systemic Th1 response was impaired in the γδ T-cell-depleted mice. Mice deficient in the Vδ1 T subset were susceptible to an intravaginal infection with HSV-2. Intraepithelial γδ T cells bearing Vδ1 may help protect against intravaginal infection with HSV-2 through promoting the systemic Th1 response.
Mutagenesis of Tyrosine 24 in the VPg Protein Is Lethal for Feline Calicivirus
Authors: Mitra Tanaji , Sosnovtsev Stanislav V. , Green Kim Y.
Published: April 2004

The genome of feline calicivirus (FCV) is an ∼7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3′ end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5′ end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.
The Herpesvirus Saimiri Open Reading Frame (ORF) 50 (Rta) Protein Contains an AT Hook Required for Binding to the ORF 50 Response Element in Delayed-Early Promoters
Authors: Walters Matthew S. , Hall Kersten T. , Whitehouse Adrian
Published: April 2004

The herpesvirus saimiri open reading frame (ORF) 50 encodes two proteins, which activate transcription directly, following interactions with delayed-early (DE) promoters containing a specific motif. In this report, we demonstrate that ORF 50 contains a DNA binding domain that has homology to an AT hook DNA binding motif. Deletion analysis of this domain reduces ORF 50-mediated transactivation of the DE ORF 6 and ORF 57 promoters by 100 and 90%, respectively. Furthermore, gel retardation experiments demonstrated that the AT hook motif is required for binding the ORF 50 response element in the promoters of DE genes. Single site-directed mutagenesis of the AT hook revealed that mutation of the glycine residue at position 408 to an alanine reduces ORF 50 transactivation of the ORF 57 promoter by 40%. Moreover, the mutation of multiple basic residues in conjunction with the glycine residue within the core element of the AT hook abolishes ORF 50-mediated transactivation. In addition, p50GFPΔAT-hook is capable of functioning as a
trans-
dominant mutant, leading to a reduction in virus production of approximately 50% compared to that for wild-type ORF 50.
Ror
γ (
Rorc
) Is a Common Integration Site in Type B Leukemogenic Virus-Induced T-Cell Lymphomas
Authors: Broussard Dana R. , Lozano Mary M. , Dudley Jaquelin P.
Published: April 2004

The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the
Ror
γ locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more
Ror
γ isoforms in >77% of the tumors tested may complement apoptotic effects of c-
myc
overexpression.
UNAVOIDABLE CONFLICT BETWEEN MASSIVE GRAVITY MODELS AND MASSIVE TOPOLOGICAL TERMS
Authors: ACCIOLY ANTONIO , DIAS MARCO
Published: April 2004

Massive gravity models in (2+1) dimensions, such as those obtained by adding to Einstein's gravity the usual Fierz–Pauli, or the more complicated Ricci scalar squared (R2), terms, are tree level unitary. Interesting enough these seemingly harmless systems have their unitarity spoiled when they are augmented by a Chern–Simons term. Furthermore, if the massive topological term is added to [Formula: see text] gravity, or to [Formula: see text] gravity (higher-derivative gravity), which are nonunitary at the tree level, the resulting models remain nonunitary. Therefore, unlike the common belief, as well as the claims in the literature, the coexistence between three-dimensional massive gravity models and massive topological terms is conflicting.
PRE-INFLATION IN THE PRESENCE OF CONFORMAL COUPLING
Authors: KUIROUKIDIS APOSTOLOS , PAPADOPOULOS DEMETRIOS B.
Published: April 2004

We consider a massless scalar field, conformally coupled to the Ricci scalar curvature, in the pre-inflation era of a closed FLRW Universe. The scalar field potential can be of the form of the Coleman–Weinberg one-loop potential, which is flat at the origin and drives the inflationary evolution. For positive values of the conformal parameter ξ, less than the critical value ξ c =(1/6), the model admits exact solutions with nonzero minimum scale factor and zero initial Hubble parameter. Thus these solutions can be matched smoothly to the so-called Pre-Big-Bang models. At the end of this pre-inflation era one can match inflationary solutions by specifying the form of the potential and the whole solution is of the class C(1).
OPEN-CLOSED DUALITY: LESSONS FROM MATRIX MODEL
Authors: SEN ASHOKE
Published: April 2004

Recent investigations involving the decay of unstable D-branes in string theory suggest that the tree level open string theory which describes the dynamics of the D-brane already knows about the closed string states produced in the decay of the brane. We propose a specific conjecture involving quantum open string field theory to explain this classical result, and show that the recent results in two-dimensional string theory are in exact accordance with this conjecture.
THE OBSERVATION OF GRAVITATIONAL WAVES FROM SPACE USING LISA
Authors: SUMNER TIMOTHY J. , SHAUL DIANA N. A.
Published: April 2004

The observation of gravitational waves using space-borne instrumentation allows the detection of astrophysical sources in a much longer wavelength part of the spectrum than ground-based instruments can reach. This opens up the prospect of a completely different view of the Universe than can be achieved in any other way. This article summarises the scientific goals of the LISA mission in its search for sources within the 10-4 to 10-1 Hz window and describes the current status of the overall project.
THE DIELECTRIC FUNCTION EXCITED BY ρNN TENSOR COUPLING IN NUCLEAR MATTER
Authors: LIU HUI , LI JIA-RONG
Published: April 2004

The dielectric function of nuclear matter excited by ρNN tensor coupling has been studied in the framework of finite temperature field theory. The induced current mechanism has been introduced to explain the three extrema on the dielectric function curve, of which one is in the space-like region and the other two are in the time-like region. It points out that the tensor coupling contributes much more large amplitude than the vector coupling and plays a more important role on the time-like region compared with its effect on the space-like region.
A NEW SYMMETRIC EXPRESSION OF WEYL ORDERING
Authors: FUJII KAZUYUKI , SUZUKI TATSUO
Published: April 2004

For the creation operator a†and the annihilation operator a of a harmonic oscillator, we consider Weyl ordering expression of (a†a)nand obtain a new symmetric expression of Weyl ordering w.r.t. a†a≡N and aa†=N+1 where N is the number operator. Moreover, we interpret intertwining formulas of various orderings in view of the difference theory. Then we find that the noncommutative parameter corresponds to the increment of the difference operator w.r.t. variable N. Therefore, quantum (noncommutative) calculations of harmonic oscillators are done by classical (commutative) ones of the number operator by using the difference theory. As a by-product, nontrivial relations including the Stirling number of the first kind are also obtained.
INTEGRABILITY AND ACTION FUNCTION IN MULTI-HAMILTONIAN SYSTEMS
Authors: MUSLIH S. I.
Published: April 2004

Multi-Hamiltonian systems are investigated by using the Hamilton–Jacobi method. Integration of a set of total differential equations which includes the equations of motion and the action integral function is discussed. It is shown that this set is integrable if and only if the total variations of the Hamiltonians vanish. Two examples are studied.
STABILITY ISSUES FOR DARK ENERGY
Authors: FRAMPTON PAUL H.
Published: April 2004

Cosmological data reveal a dark energy with a negative equation of state w=P/ρ<0. We discuss its stability under nucleation from Λ>0 to Λ=0 in an assumed first-order phase transition. From arguments about stimulated nucleation it is shown that the critical nucleation radius must be at least of galactic size. The spontaneous nucleation rate is infinitesimal thus explaining the dark energy's extreme stability. Dark energy detection at subgalactic scales will never be possible.
MATERIAL PROPERTIES OF THE SUPRASPINATUS TENDON AT ITS INSERTION — A MEASUREMENT WITH THE SCANNING ACOUSTIC MICROSCOPY
Authors: Sano Hirotaka , Saijo Yoshifumi , Kokubun Shoichi
Published: April 2004

The acoustic properties of the supraspinatus tendon insertions of three adult Japanese white rabbits (male, 3.2–3.5 kg) were measured by scanning acoustic microscopy (SAM). After cutting parallel to the supraspinatus tendon fibers, specimens were fixed with 10% neutralized formalin, embedded in paraffin and sectioned at 5 μm thickness. The sound speed and the attenuation constant along the articular surface of the tendon from the insertion site to the musculotendinous junction were measured by SAM. The two-dimensional distribution of the acoustic properties was displayed with color-coded scales and was compared to the histologic structure. Toluidine-blue staining and immunohistochemical staining against type II collagen were performed to determine the presence of fibrocartilage besides a standard Elastica–Masson staining to assess the overall histologic structure.

The distribution of the sound speed and the attenuation constant showed an almost identical pattern. Both parameters were lower in the non-mineralized fibrocartilage than those in the tendon proper. The area indicating low sound speed corresponded well to the area of metachromasia and type II collagen. The non-mineralized fibrocartilage indicated softer material properties than the tendon proper, which could be interpreted as an adaptation for the various biomechanical stresses at the insertion site.
STAGING OF KÜMMELL'S DISEASE
Authors: Li Kung-Chia , Wong Tak-Uee , Kung Fu-Chi , Li Allen , Hsieh Ching-Hsiang
Published: April 2004

Kümmell's disease is defined as delayed post-traumatic vertebral collapse, common in osteoporotic people with/without a trauma history. Its clinical course is progressive, and treatment modalities have to be tailored to the patient. In the literature, no staging system has been reported. Based on clinical symptomatology, radiographic and MRI imaging, a staging system for Kümmell's disease was developed. This study involved 129 Kümmell's disease patients with 348 compression fractures involving 136 vertebrae. The mean age was 72.5 ± 6.6 years, with a female to male ratio of 103 to 26. Osteoporosis is a common risk factor. Minor trauma can be traced in some of the patients. A three-stage system was proposed. Stage I: Intact or minor compression on plain X-ray films with signs of osteonecrosis on the MRI. Stage II: Body collapse with dynamic mobility, but with intact posterior body wall. Stage III: Collapse of posterior body wall with resultant cord compression. There were 19, 90 and 33 vertebrae respectively classified in Stages I, II and III. Kümmell's disease is most common threat to the thoracolumbar junction. Treatments based on clinical symptomatology and radiographic findings were well correlated with the staging system. The present staging system therefore seems appropriate to describe the natural course of Kümmell's disease and to guide management policy.
THE ASSESSMENT OF THE KINEMATICS OF THE CERVICAL SPINE USING OPEN (INTERVENTIONAL) MRI
Authors: Emohare O. , McGregor A. H. , Wragg P. , Gedroyc W. M. W.
Published: April 2004

The scope of serial studies into the kinematics of the cervical vertebrae, have been limited by the methods available for imaging. Plain radiography has been one of the main methods by which data has been collected. The widespread use of this method has, however, been restricted by factors including exposure to ionizing radiation and magnification errors. With the advent of Interventional Magnetic Resonance (iMR) scanners, however, the authors sought to determine the utility of an iMR scanner in obtaining functional images of the cervical vertebrae, from which repeatable measurements of vertebral kinematics can be collected.

20 healthy volunteers (mean age 25.4 ± 3.7 years) were recruited. They had their cervical vertebrae scanned in three positions: neutral, flexed and extended. Sagittal images were obtained for each subject from between the level of the C2 vertebra to the T1 vertebra and measurements of inter-segmental motion were made, using established clinical methodology.

Clear images of the cervical vertebrae were obtained in all positions from which measures of motion were made. Greatest angular motions were found to occur in the mid-cervical level, C4/C5. Non-osseous structures, including the intervertebral discs and nerve roots were well visualized.

Our results suggest that iMR scanning may have an important role in the imaging of cervical spine and its associated structures. It offers a modality that allows the determination of both normal and abnormal kinematics on a large scale.
MECHANICAL STRESS MAY CAUSE THE TEAR OF THE LABRUM IN ACETABULAR DYSPLASIA
Authors: Sano Hirotaka , Yamada Norikazu , Maeda Shingo
Published: April 2004

In the current study, using the arthrogram, we developed two-dimensional finite element (FE) models of the human hip joint. To clarify the relationship between the stress distribution and the degree of acetabular dysplasia, three FE models were established and analyzed. The models varied only in the degree of the bony covering of the femoral head; i.e. the center-edge (CE) angle=20, 10, 0 degrees. An edge load (x=0 N, y=600 N) was then applied on the distal border of the femur to simulate the bearing of the body weight. In the CE=20 degree model, no definite stress concentration was seen at the site of the labrum. On the other hand, the stress concentration was seen from the attachment of the labrum to the superior aspect of the acetabulum in the CE=0 degree model. The site of stress concentration clearly corresponded to the lesions where the acetabular rim pathologies were seen in the clinical practice. Moreover, we found that the Von Mises stress increases dramatically with decreasing the CE angle at the attachment of the labrum. In the dysplastic hip, the mechanical stress increases significantly at the supero-lateral aspect of the acetabulum, which eventually leads to the tearing or detachment of the labrum.
MUSCLES WITHIN MUSCLES: ANATOMICAL AND FUNCTIONAL SEGMENTATION OF SELECTED SHOULDER JOINT MUSCULATURE
Authors: Wickham J. B. , Brown J. M. M. , McAndrew D. J.
Published: April 2004

The objectives of this study were two-fold. Firstly we wished to investigate the segmental anatomy, geometry and gross innervation patterns of selected shoulder joint musculature (pectoralis major, latissimus dorsi and deltoid) to establish if these muscles could be anatomically subdivided according to predetermined criteria. We then wished to use this information to help determine if a segments activation pattern was reliant on an efficient line of action and a large moment arm for the intended movement. Surface electromyography was used for this purpose whilst manipulating the independent variable of contraction intensity (% MVC) during two shoulder joint flexion tasks.

For the anatomical portion of the study the dissection of ten cadaveric shoulders revealed the deltoid to consist of seven segments whilst the pectoralis major and the latissimus dorsi were both ascribed six segments according to predetermined anatomical/functional criteria. Primary nerve branching was evident in each muscle but no apparent relationship existed between the designated anatomical segments and the primary nerve branches. Differences were evident in the geometry of each segment in regard to moment arms and orientations of each segments line of action.

The functional portion of this study, which utilized miniature bipolar surface electrodes during the performance of a static shoulder joint flexion task at 75% and 25% MVC, showed significant differences (p<0.05) in the activation patterns of active segments between the two tasks. Specifically, a "drop out" of segments with smaller flexion moment arms and more diverging lines of action in comparison to the movement plane was evident for all subjects in the 25% MVC task in comparison to the 75% MVC task.