bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions between the Inner and Outer Capsids of Bluetongue Virus
Authors: Nason Emma L. , Rothagel Rosalba , Mukherjee Sharmila K. , Kar Alak Kanti , Forzan Mario , Prasad B. V. Venkataram , Roy Polly
Published: July 2004

Bluetongue virus is a large and structurally complex virus composed of three concentric capsid layers that surround 10 segments of a double-stranded RNA genome. X-ray crystallographic analysis of the particles without the outer capsid layer has provided atomic structural details of VP3 and VP7, which form the inner two layers. However, limited structural information is available on the other five proteins in the virion—two of which are important for receptor recognition, hemagglutination, and membrane interaction—are in the outer layer, and the others, important for endogenous transcriptase activity are internal. Here we report the electron cryomicroscopy (cryo-EM) reconstruction of the mature particle, which shows that the outer layer has a unique non-T = 13 icosahedral organization consisting of two distinct triskelion and globular motifs interacting extensively with the underlying T = 13 layer. Comparative cryo-EM analysis of the recombinant corelike particles has shown that VP1 (viral polymerase) and VP4 (capping enzyme) together form a flower-shaped structure attached to the underside of VP3, directly beneath the fivefold axis. The structural data have been substantiated by biochemical studies demonstrating the interactions between the individual outer and inner capsid proteins.
Isolation of Capsid Protein Dimers from the Tick-Borne Encephalitis Flavivirus and In Vitro Assembly of Capsid-Like Particles
Authors: Kiermayr Stefan , Kofler Regina M. , Mandl Christian W. , Messner Paul , Heinz Franz X.
Published: July 2004

Flaviviruses have a spherical capsid that is composed of multiple copies of a single capsid protein and, in contrast to the viral envelope, apparently does not have an icosahedral structure. So far, attempts to isolate distinct particulate capsids and soluble forms of the capsid protein from purified virions as well as to assemble capsid-like particles in vitro have been largely unsuccessful. Here we describe the isolation of nucleocapsids from tick-borne encephalitis (TBE) virus and their disintegration into a capsid protein dimer by high-salt treatment. Purified capsid protein dimers could be assembled in vitro into capsid-like particles when combined with in vitro transcribed viral RNA. Particulate structures could also be obtained when single-stranded DNA oligonucleotides were used. These data suggest that the dimeric capsid protein functions as a basic building block in the assembly process of flaviviruses.
Appearance of the Bona Fide Spiral Tubule of Orf Virus Is Dependent on an Intact 10-Kilodalton Viral Protein
Authors: Spehner D. , De Carlo S. , Drillien R. , Weiland F. , Mildner K. , Hanau D. , Rziha H.-J.
Published: July 2004

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the
Parapoxvirus
genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.
Epitope Mapping of the Major Capsid Protein of Type 2 Porcine Circovirus (PCV2) by Using Chimeric PCV1 and PCV2
Authors: Lekcharoensuk Porntippa , Morozov Igor , Paul Prem S. , Thangthumniyom Nattarat , Wajjawalku Worawidh , Meng X. J.
Published: July 2004

Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein.
Structure and Function of the 3′ Terminal Six Nucleotides of the West Nile Virus Genome in Viral Replication
Authors: Tilgner Mark , Shi Pei-Yong
Published: July 2004

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3′-terminal 6 nucleotides (nt) (5′-GGAUCU
OH
-3′) of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3′-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i) The flavivirus-conserved terminal 3′ U is optimal for WN virus replication. Replacement of the wild-type 3′ U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3′ A or G mutants, with only partial reversion. (ii) The flavivirus-conserved 3′ penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3′-terminal CU
OH
, are absolutely essential for viral replication. (iii) The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv) The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v) Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3′ flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.
Calicivirus 3C-Like Proteinase Inhibits Cellular Translation by Cleavage of Poly(A)-Binding Protein
Authors: Kuyumcu-Martinez Muge , Belliot Gaël , Sosnovtsev Stanislav V. , Chang Kyeong-Ok , Green Kim Y. , Lloyd Richard E.
Published: July 2004

Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus
Norovirus
) and feline calicivirus (FCV) (genus
Vesivirus
), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL
pro
) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL
pro
PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C
pro
cleavage, while the FCV r3CL
pro
products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C
pro
cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL
pro
was analyzed in HeLa cell translation extracts, and the presence of r3CL
pro
inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL
pro
, like PV 3C
pro
, mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.
Sequential Roles of Receptor Binding and Low pH in Forming Prehairpin and Hairpin Conformations of a Retroviral Envelope Glycoprotein
Authors: Matsuyama Shutoku , Delos Sue Ellen , White Judith M.
Published: July 2004

A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37°C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.
Characterization of the Murine Alpha Interferon Gene Family
Authors: van Pesch Vincent , Lanaya Hanane , Renauld Jean-Christophe , Michiels Thomas
Published: July 2004

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-α) subtypes. IFN-α, as well as IFN-β, IFN-κ, IFN-ε, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-α subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-α family. A total of 14 IFN-α genes were detected in the mouse genome, in addition to three IFN-α pseudogenes. Four IFN-α genes (IFN-α1, IFN-α7/10, IFN-α8/6, and IFN-α11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-α subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-α4, IFN-α11, IFN-α12, IFN-β, and limitin) showed higher biological activity levels than others, whereas IFN-α7/10 exhibited lower activity. Most murine IFN-α turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-α subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-α subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I•C) transfection or to viral infection were, however, similar.
Progression or Resolution of Coxsackievirus B4-Induced Pancreatitis: a Genomic Analysis
Authors: Ostrowski Stephanie E. , Reilly Andrew A. , Collins Doris N. , Ramsingh Arlene I.
Published: July 2004

Group B coxsackieviruses are associated with chronic inflammatory diseases of the pancreas, heart, and central nervous system. Chronic pancreatitis, which can develop from acute pancreatitis, is considered a premalignant disorder because it is a major risk factor for pancreatic cancer. To explore the genetic events underlying the progression of acute to chronic disease, a comparative analysis of global gene expression during coxsackievirus B4-induced acute and chronic pancreatitis was undertaken. A key feature of acute pancreatitis that resolved was tissue regeneration, which was accompanied by increased expression of genes involved in cell growth, inhibition of apoptosis, and embryogenesis and by increased division of acinar cells. Acute pancreatitis that progressed to chronic pancreatitis was characterized by lack of tissue repair, and the expression map highlighted genes involved in apoptosis, acinoductular metaplasia, remodeling of the extracellular matrix, and fibrosis. Furthermore, immune responses appeared skewed toward development of alternatively activated (M2) macrophages and T helper 2 (Th2) cells during disease that resolved and toward classically activated (M1) macrophages and Th1 cells during disease that progressed. Our hypothesis is that growth and differentiation signals coupled with the M2/Th2 milieu favor acinar cell proliferation, while diminished growth signals and the M1/Th1 milieu favor apoptosis of acinar cells and remodeling/proliferation of the extracellular matrix, resulting in fibrosis.
APOBEC3G Targets Specific Virus Species
Authors: Kobayashi Masayuki , Takaori-Kondo Akifumi , Shindo Keisuke , Abudu Aierken , Fukunaga Keiko , Uchiyama Takashi
Published: July 2004

Human APOBEC3G (huAPOBEC3G), also known as CEM15, is a broad antiretroviral host factor that deaminates dC to dU in the minus strand DNA of human immunodeficiency virus type 1 (HIV-1), other lentiviruses, and murine leukemia virus (MLV), thereby creating G-to-A hypermutation in the plus strand DNA to inhibit the infectivity of these viruses. In this study, we examined the antiretroviral function of a murine homologue of APOBEC3G (muAPOBEC3G) on several retrovirus systems with different producer cells. MuAPOBEC3G did not suppress the infectivity of murine retroviral vectors produced from human or murine cells, whereas it showed antiviral activity on both wild-type and Δvif virions of HIV-1 in human cells. In contrast, huAPOBEC3G showed broad antiviral activity on HIV-1 and murine retroviral vectors produced from human cells as well as murine cells. These data suggested that muAPOBEC3G does not possess antiretroviral activity on murine retroviruses and has a different target specificity from that of huAPOBEC3G and that huAPOBEC3G works as a broad antiviral factor not only in human cells but also in murine cells. A functional interaction study between human and murine APOBEC3G supported the former hypothesis. Furthermore, studies on the expression of APOBEC3G in producer cells and its incorporation into virions revealed that muAPOBEC3G is incorporated into HIV-1 virions but not into MLV virions. Thus, muAPOBEC3G cannot suppress the infectivity of murine retrovirus because it is not incorporated into virions. We suggest that murine retroviruses can replicate in murine target cells expressing muAPOBEC3G because they are not targets for this enzyme.
Purification of the
Cucumber Necrosis Virus
Replicase from Yeast Cells: Role of Coexpressed Viral RNA in Stimulation of Replicase Activity
Authors: Panaviene Zivile , Panavas Tadas , Serva Saulius , Nagy Peter D.
Published: July 2004

Purified recombinant viral replicases are useful for studying the mechanism of viral RNA replication in vitro. In this work, we obtained a highly active template-dependent replicase complex for
Cucumber necrosis tombusvirus
(CNV), which is a plus-stranded RNA virus, from
Saccharomyces cerevisiae
. The recombinant CNV replicase showed properties similar to those of the plant-derived CNV replicase (P. D. Nagy and J. Pogany, Virology
276:
279-288, 2000), including the ability (i) to initiate cRNA synthesis de novo on both plus- and minus-stranded templates, (ii) to generate replicase products that are shorter than full length by internal initiation, and (iii) to perform primer extension from the 3′ end of the template. We also found that isolation of functional replicase required the coexpression of the CNV p92 RNA-dependent RNA polymerase and the auxiliary p33 protein in yeast. Moreover, coexpression of a viral RNA template with the replicase proteins in yeast increased the activity of the purified CNV replicase by 40-fold, suggesting that the viral RNA might promote the assembly of the replicase complex and/or that the RNA increases the stability of the replicase. In summary, this paper reports the first purified recombinant tombusvirus replicase showing high activity and template dependence, a finding that will greatly facilitate future studies on RNA replication in vitro.
Recovery of Human Metapneumovirus Genetic Lineages A and B from Cloned cDNA
Authors: Herfst Sander , de Graaf Miranda , Schickli Jeanne H. , Tang Roderick S. , Kaur Jasmine , Yang Chin-Fen , Spaete Richard R. , Haller Aurelia A. , van den Hoogen Bernadette G. , Osterhaus Albert D. M. E. , Fouchier Ron A. M.
Published: July 2004

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10
7.2
and 10
5.7
50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.
Dileucine and YXXL Motifs in the Cytoplasmic Tail of the Bovine Leukemia Virus Transmembrane Envelope Protein Affect Protein Expression on the Cell Surface
Authors: Novakovic Sinisa , Sawai Earl T. , Radke Kathryn
Published: July 2004

Several retroviruses downmodulate the cell surface expression of envelope (Env) proteins through peptide sequences located in the cytoplasmic tail of the transmembrane (TM) subunit. We investigated whether cell surface expression of a chimeric protein containing the cytoplasmic domain of the TM protein (CTM) of bovine leukemia virus (BLV) was regulated by two membrane-proximal dileucine motifs or by tyrosine Y487 or Y498 in YXXL motifs. A chimeric protein composed of the extracellular and membrane-spanning portions of human CD8-α plus a wild-type (wt) BLV CTM was detectable on the surface of only 40% of the cells in which it was transiently expressed. Replacement of either dileucine pair with alanines increased the level of surface display of chimeric proteins. Nearly all cells became surface positive when both dileucine motifs were altered simultaneously and when either an N-terminal segment containing both dileucine motifs or a C-terminal segment containing all YXXL motifs was deleted. In contrast, replacement of Y487 or Y498 with alanine or phenylalanine enabled only small increases in surface display compared with wt levels. Chimeric proteins had similar stabilities but were downmodulated from the cell surface at three different rates. Point mutants segregated into each of the three groups of proteins categorized according to these different rates. Interestingly, Y487 mutants were downmodulated less efficiently than Y498 mutants, which behaved like wt. CD8-CTM chimeric proteins were phosphorylated on serine residues, but the native BLV Env protein was not phosphorylated either in transfected cells or in a lymphoid cell line constitutively producing BLV. Thus, both dileucine and YXXL motifs within the BLV CTM contribute to downmodulation of a protein containing this domain. Interactions with other proteins may influence surface exposure of Env protein complexes in virus-infected cells, assisting in viral evasion of adaptive immunity.
Role of CD8+T Cells in Control of West Nile Virus Infection
Authors: Shrestha Bimmi , Diamond Michael S.
Published: July 2004

Infection with West Nile virus (WNV) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. Although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. Previously, we demonstrated that components of humoral immunity, particularly immunoglobulin M (IgM) and IgG, have critical roles in preventing dissemination of WNV infection to the central nervous system. In this study, we addressed the function of CD8+T cells in controlling WNV infection. Mice that lacked CD8+T cells or classical class Ia major histocompatibility complex (MHC) antigens had higher central nervous system viral burdens and increased mortality rates after infection with a low-passage-number WNV isolate. In contrast, an absence of CD8+T cells had no effect on the qualitative or quantitative antibody response and did not alter the kinetics or magnitude of viremia. In the subset of CD8+-T-cell-deficient mice that survived initial WNV challenge, infectious virus was recovered from central nervous system compartments for several weeks. Primary or memory CD8+T cells that were generated in vivo efficiently killed target cells that displayed WNV antigens in a class I MHC-restricted manner. Collectively, our experiments suggest that, while specific antibody is responsible for terminating viremia, CD8+T cells have an important function in clearing infection from tissues and preventing viral persistence.
Hepatitis C Virus Targets DC-SIGN and L-SIGN To Escape Lysosomal Degradation
Authors: Ludwig Irene S. , Lekkerkerker Annemarie N. , Depla Erik , Bosman Fons , Musters René J. P. , Depraetere Stany , van Kooyk Yvette , Geijtenbeek Teunis B. H.
Published: July 2004

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to “hide” within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.
Induction of Mucosal and Systemic Neutralizing Antibodies against Human Immunodeficiency Virus Type 1 (HIV-1) by Oral Immunization with Bovine Papillomavirus-HIV-1 gp41 Chimeric Virus-Like Particles
Authors: Zhang Hongtao , Huang Yujun , Fayad Raja , Spear Gregory T. , Qiao Liang
Published: July 2004

Human immunodeficiency virus type 1 (HIV-1) envelope-specific neutralizing antibodies are generated late after initial infection, and the neutralizing antibody response is weak in the infected individuals. Administration of neutralizing antibodies such as 2F5 to HIV-1-infected individuals resulted in reductions in viral loads. Because HIV-1 is transmitted mainly via mucosa and because HIV-specific neutralizing antibodies reduce HIV-1 in infected individuals, a vaccine that can induce both mucosal and systemic HIV-1-specific neutralizing antibodies may be used to prevent and to treat HIV-1 infection. In this study, we made a bovine papillomavirus (BPV) L1-HIV-1 gp41 fusion protein in which ELDKWA of gp41 was inserted into the N terminus of BPV L1 (amino acids 130 to 136). Expression of the fusion protein in insect cells led to the assembly of chimeric virus-like particles (CVLPs). The CVLPs had sizes similar to those of BPV particles and were able to bind to the cell surface and penetrate the cell membrane. Oral immunization of mice with CVLPs induced gp41-specific serum immunoglobulin G (IgG) and intestinal secretory IgA. However, intramuscular immunization with the CVLPs resulted in similar amounts of gp41-specific IgG but low levels of secretory IgA. The antibodies specifically recognized the fixed HIV-1 gp41 on the cell surface. Importantly, the sera and fecal extracts from mice orally immunized with the CVLPs neutralized HIV-1
MN
in vitro. Thus, BPV-HIV-1 gp41 CVLPs may be used to prevent and to treat HIV-1 infection.
Global Identification of Three Major Genotypes of Varicella-Zoster Virus: Longitudinal Clustering and Strategies for Genotyping
Authors: Loparev Vladimir N. , Gonzalez Antonio , Deleon-Carnes Marlene , Tipples Graham , Fickenscher Helmut , Torfason Einar G. , Schmid D. Scott
Published: July 2004

By analysis of a single, variable, and short DNA sequence of 447 bp located within open reading frame 22 (ORF22), we discriminated three major varicella-zoster virus (VZV) genotypes. VZV isolates from all six inhabited continents that showed nearly complete homology to ORF22 of the European reference strain Dumas were assigned to the European (E) genotype. All Japanese isolates, defined as the Japanese (J) genotype, were identical in the respective genomic region and proved the most divergent from the E strains, carrying four distinct variations. The remaining isolates carried a combination of E- and J-specific variations in the target sequence and thus were collectively termed the mosaic (M) genotype. Three hundred twenty-six isolates collected in 27 countries were genotyped. A distinctive longitudinal distribution of VZV genotypes supports this approach. Among 111 isolates collected from European patients, 96.4% were genotype E. Consistent with this observation, approximately 80% of the VZV strains from the United States were also genotype E. Similarly, genotype E viruses were dominant in the Asian part of Russia and in eastern Australia. M genotype viruses were strongly dominant in tropical regions of Africa, Indochina, and Central America, and they were common in western Australia. However, genotype M viruses were also identified as a minority in several countries worldwide. Two major intertypic variations of genotype M strains were identified, suggesting that the M genotype can be further differentiated into subgenotypes. These data highlight the direction for future VZV genotyping efforts. This approach provides the first simple genotyping method for VZV strains in clinical samples.
Inhibition of Infection and Replication of Human Herpesvirus 8 in Microvascular Endothelial Cells by Alpha Interferon and Phosphonoformic Acid
Authors: Krug Laurie T. , Pozharskaya Veronika P. , Yu Yimin , Inoue Naoki , Offermann Margaret K.
Published: July 2004

Infection of endothelial cells with human herpesvirus 8 (HHV-8) is an essential event in the development of Kaposi's sarcoma. When primary microvascular endothelial cells (MECs) were infected with HHV-8 at a low multiplicity of infection, considerable latent replication of HHV-8 occurred, leading to a time-dependent increase in the percentage of virus-infected cells that was accompanied by cellular spindling and growth to a high density with loss of contact inhibition. Only a low percentage of MECs supported lytic replication of HHV-8 and produced infectious virus. Phosphonoformic acid blocked production of infectious virus but did not inhibit the rapid expansion of latently infected MECs. Pretreatment of MECs with alpha interferon (IFN-α) prior to infection effectively reduced HHV-8 viral gene expression, latent replication, and production of infectious virus. High levels of the double-stranded RNA activated protein kinase (PKR) were expressed in HHV-8-infected cells, and incubation with IFN-α increased PKR expression more in virus-infected cells than in uninfected cells. MECs that were immortalized with simian virus 40 large-T antigen differed from nonimmortalized MECs in their response to infection with HHV-8 and demonstrated that cells with elevated levels of expression of antiviral transcripts expressed viral transcripts at reduced levels. These studies demonstrate that MECs respond to HHV-8 with enhanced expression of cellular antiviral genes and that augmentation of innate antiviral defenses with IFN-α is a more effective strategy than inhibition of viral lytic replication to protect MECs from infection with HHV-8 and to restrict proliferation of virus-infected MECs.
Disparate Regions of Envelope Protein Regulate Syncytium Formation versus Spongiform Encephalopathy in Neurological Disease Induced by Murine Leukemia Virus TR
Authors: Murphy Samuel L. , Honczarenko Marek J. , Dugger Natalie V. , Hoffman Paul M. , Gaulton Glen N.
Published: July 2004

The murine leukemia virus (MLV) TR1.3 provides an excellent model to study the wide range of retrovirus-induced central nervous system (CNS) pathology and disease. TR1.3 rapidly induces thrombotic events in brain microvessels and causes cell-specific syncytium formation of brain capillary endothelial cells (BCEC). A single amino acid substitution, W102G, in the MLV envelope protein (Env) regulates the pathogenic effects. The role of Env in determining this disease phenotype compared to the induction of spongiform encephalomyelitis with a longer latency, as seen in several other MLV and in human retroviruses, was determined by studying in vitro-attenuated TR1.3. Virus cloned from this selection, termed TRM, induced progressive neurological disease characterized by ataxia and paralysis and the appearance of spongiform neurodegeneration throughout the brain stem and spinal cord. This disease was associated with virus replication in both BCEC and highly ramified glial cells. TRM did not induce syncytium formation, either in vivo or in vitro. Sequence and mutational analyses demonstrated that TRM contained a reversion of Env G102W but that neurological disease mapped to the single amino acid substitution Env S159P. The results demonstrate that single nucleotide changes within disparate regions of Env control dramatically different CNS disease patterns.
The Virion Host Shutoff Protein of Herpes Simplex Virus Type 1 Has RNA Degradation Activity in Primary Neurons
Authors: Strand Stephanie S. , Vanheyningen Tambryn K. , Leib David A.
Published: July 2004

The virion host shutoff protein (Vhs) of herpes simplex virus type 1 induces destabilization of mRNA following infection. Our study of primary neurons from CD-1 mice demonstrates that
vhs
is functional in neurons but that more Vhs is required to mediate RNA degradation in neurons than in other susceptible cells.
Epstein-Barr Virus Latent Membrane Protein 1 (LMP-1) Half-Life in Epithelial Cells Is Down-Regulated by Lytic LMP-1
Authors: Pandya Jyotsna , Walling Dennis M.
Published: July 2004

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in
trans
down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the
cis
ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.
Role of intravenous urogram in investigation of urinary tract infection: an observational study
Authors: Padmakumar B , Carty H M , Hughes D A , Judd B A
Published: July 2004

Objectives: To examine the value of an intravenous urogram (IVU) in patients with abnormal differential 99mTc dimercaptosuccinic acid (DMSA) uptake without scarring or ultrasound abnormality.

Study design: Forty patients (age 0–19 years) were identified over a two year period in whom the differential renal uptake was >10%, who had smooth renal outlines, and had no evidence of scarring. All patients had an ultrasound examination. Two had marked urological abnormalities on ultrasound and eight had a duplex system in the kidney with greater DMSA uptake. In 18 patients where no explanation was apparent for the discrepant DMSA uptake, an IVU was performed.

Results: Eight patients had a normal IVU. In the remaining 10 patients, six had duplex systems without scarring and four had appearances of scarring in the kidney with reduced DMSA uptake.

Conclusions: In this small selected group an IVU will identify a significant number of patients with normal kidneys, unrecognised simple duplex systems, or scarring where the DMSA scan has been inconclusive. This will help in planning long term follow up.
Non-clinical factors influencing the selection of patients with acute coronary syndromes for angiography
Authors: Quaas A , Curzen N , Garratt C
Published: July 2004

Objective: To analyse clinical and non-clinical factors determining the selection for coronary angiography in patients with acute coronary syndromes (ACS).

Design: Single centre, prospective cohort study.

Participants: Eighty consecutive patients admitted with a diagnosis of ACS during the period 21 May 2001 to 4 July 2001.

Setting: Coronary care unit of a tertiary referral centre, the Manchester Royal Infirmary.

Data collection: Information concerning baseline patient characteristics, clinical presentation, and the selection for angiography was collected from the patient notes.

Data analysis: Windows SPSS version 9.0 using cross tabulations with χ2 estimation and binomial logistic regression analysis.

Main outcome measure: Selection for angiography in ACS.

Results: Cross tabulations with χ2 analysis and logistic regression analysis identified significant non-clinical factors predicting the use of angiography. Although clinical factors such as recurrent ischaemia (odds ratio 5.11) influenced the decision to undergo coronary angiography, non-clinical factors such as young age (odds ratio 6.88 for <65 years old), gender (odds ratio 3.81 for males), admission on a weekday (odds ratio 0.2488 for admission on the weekend), and consultant in charge (odds ratio 0.111 for consultant “2”) independently predicted the use of angiography in ACS.

Conclusion: The selection of patients for angiography in ACS is not based purely on clinical criteria. Awareness of the apparent sources of bias among clinical decision makers may improve management of these patients.
Scabies: more than just an irritation
Authors: McCarthy J S , Kemp D J , Walton S F , Currie B J
Published: July 2004

Human scabies, caused by skin infestation with the arthropod mite, Sarcoptes scabiei, typically results in a papular, intensely pruritic eruption involving the interdigital spaces, and flexure creases. Recent research has led to a reassessment of the morbidity attributable to this parasite in endemic communities, particularly resulting from secondary skin sepsis and postinfective complications including glomerulonephritis. This has led to studies of the benefits of community based control programmes, and to concerns regarding the emergence of drug resistance when such strategies are employed. The renewed research interest into the biology of this infection has resulted in the application of molecular tools. This has established that canine and human scabies populations are genetically distinct, a finding with major implications for the formulation of public health control policies. Further research is needed to increase understanding of drug resistance, and to identify new drug targets and potential vaccine candidates.
Subclinical hyperthyroidism: to treat or not to treat?
Authors: Hoogendoorn E H , den Heijer M , van Dijk A P J , Hermus A R
Published: July 2004

Subclinical hyperthyroidism may be defined as the presence of free thyroxine and tri-iodothyronine levels within the reference range and a reduced serum thyroid stimulating hormone (TSH) level. In this review the prevalence of low TSH in the population and health consequences of subclinical hyperthyroidism, for example, effects on heart and bone mass, are discussed. Guidelines for treatment are given, based on expert opinion.
Management of haematemesis and melaena
Authors: Palmer K
Published: July 2004

Acute upper gastrointestinal bleeding is a common medical emergency which carries hospital mortality in excess of 10%. The most important causes are peptic ulcer and varices. Varices are treated by endoscopic band ligation or injection sclerotherapy and management of the underlying liver disease. Ulcers with major stigmata are treated by injection with dilute adrenaline, thrombin, or fibrin glue; application of heat using the heater probe, multipolar electrocoagulation, or Argon plasma coagulation; or endoclips. Intravenous omeprazole reduces the risk of re-bleeding in ulcer patients undergoing endoscopic therapy. Repeat endoscopic therapy or operative surgery are required if bleeding recurs.
Caring for “incurables”: the 150th anniversary of the Royal Hospital for Neuro-Disability, Putney
Authors: Cook G C
Published: July 2004

The Royal Hospital for Incurables (RHI), now known as the Royal Hospital for Neuro-Disability and situated on West Hill, Putney, was founded by Andrew Reed DD exactly 150 years ago. The RHI was thus the pioneer in modern times of long stay institutions for the sick and dying. It became one of the great Victorian charities, and remained independent of the National Health Service, which was introduced in 1948. Originally the long stay patients suffered from a multiplicity of diseases; in recent years chronic neurological disease has dominated the scenario. This institution has also become a major centre for genetic and trauma-associated neurological damage, and rehabilitation.
Imbibition response of green foxtail, canola, wild mustard, and wild oat seeds to different osmotic potentials
Authors: Boyd Nathan S , Van Acker R C
Published: July 2004

The ability of seeds to imbibe water depends on the difference in water potentials between the seed and the surrounding medium, as well as seed diffusivity. The proportional moisture content and imbibition rate of Brassica napus L. 'Liberty' (canola), Setaria viridis (L.) Beauv. (green foxtail), Brassica kaber (DC.) L.C. Wheeler (wild mustard), and Avena fatua L. (wild oat) were examined over time in water and osmotic solutions of 0.5 and 1 MPa. Average imbibition rate of wild oat was significantly higher than that of all other species studied, while the average imbibition rate of green foxtail was significantly lower than that of all other species studied. Differences between imbibition rates may have been caused by differences in seed size or seed diffusivity. Wild mustard and canola had the highest proportional moisture content at 25 h and were the only two species to achieve greater than 80% germination within this time frame. Germination was reduced in osmotic solutions of 0.5 and 1 MPa compared with water for all species. Species differ in their ability to imbibe water as well as their ability to germinate at lower seed moisture contents.Key words: germination, weed, soil moisture, uptake, seed size.
The nuclear small subunit ribosomal genes of Sphaeronaemella helvellae, Sphaeronaemella fimicola, Gabarnaudia betae, and Cornuvesica falcata: phylogenetic implications
Authors: Hausner Georg , Reid James
Published: July 2004

Sequences were obtained from the nuclear small subunit ribosomal RNA genes for representatives of four ophiostomatoid genera (Ceratocystis, Gondwanamyces, Cornuvesica, and Sphaeronaemella) to resolve their phylogenetic position within the Ascomycota. Phylogenetic analysis suggests that these genera are monophyletic and share common ancestry with members of the Microascales. Based on sequence data, strains representing the mitotic species Gabarnaudia betae (Delacr.) Samson & W. Gams were shown clearly to be derived from Sphaeronaemella species. Sequences were also obtained from strains representing the syntype of Sphaeronaemella fragariae, the exholotype of Sphaeronaemella humicola, and the extype of Gabarnaudia tholispora. The results suggest that putative extype cultures for S. humicola and G. tholispora no longer represent the original material deposited. Our data also support the exclusion of S. fragariae from Sphaeronaemella. Key words: Cornuvesica, Gabarnaudia, Gondwanamyces, Sphaeronaemalla fragariae, ophiostomatoid fungi, ribosomal DNA.
Induced apogamy in Tmesipteris (Psilotaceae)
Authors: Whittier Dean P
Published: July 2004

Gametophytes of Tmesipteris lanceolata Dang., which are mycorrhizal in nature, were grown in axenic culture. If cultured in the light on a nutrient medium containing minerals and 0.5% glucose, they did not become photosynthetic; however, about 15% of them produced apogamous sporophytes with stems and microphylls. The gametophytesporophyte junction had a direct connection between the gametophyte and sporophyte tissues and lacked a foot, which is typical for apogamy. Gametangia were limited to the gametophyte portions of these gametophytesporophyte growths, and the vascular tissue was present only in the sporophyte regions. The apogamous aerial stems had the normal anatomy for a sporophyte, with vascular tissue, epidermal cells, stomata, and chlorenchyma. The origin of the apogamous sporophytes was different from the origin in fern gametophytes. The Tmesipteris sporophytes arose terminally from the gametophyte apices. It appears that the apical meristem of the gametophyte is converted to a shoot apical meristem to form the apogamous aerial shoot.Key words: Tmesipteris, Psilotaceae, apogamy, sporophyte, gametophyte.
Modern and Holocene stomate records of tree-line variations in northwestern Quebec
Authors: Leitner Ruth , Gajewski K
Published: July 2004

Conifer stomates in lake sediments from the forest to tundra transition in northwestern Quebec were analysed to reconstruct tree-line history during the past 6000 years. The pollen from these sites had already been analysed. Stomate concentrations in modern sediments generally conform to the vegetation distribution, although stomates were lacking in some forested sites. Results from nine cores on a transect from boreal forest to tundra show broad-scale correspondence between Picea stomate and pollen concentrations. At a smaller scale, however, discrepancies were found, such as the absence of stomates from a forest tundra site. A broad-scale correspondence between pollen and stomate concentrations of Larix was also found, although the low numbers of Larix stomates make interpretation difficult. Neither stomate absence nor presence can unambiguously be used to indicate the absence or presence of trees in the area immediately surrounding the lake.Key words: tree line, palynology, stomate, vegetation history, Picea, Larix.