bluuebunny/crossref_metadata_2025 huggingface dataset:
Massive Horizontal Gene Transfer in Bdelloid Rotifers
Authors: Gladyshev Eugene A. , Meselson Matthew , Arkhipova Irina R.
Published: May 2008

Horizontal gene transfer in metazoans has been documented in only a few species and is usually associated with endosymbiosis or parasitism. By contrast, in bdelloid rotifers we found many genes that appear to have originated in bacteria, fungi, and plants, concentrated in telomeric regions along with diverse mobile genetic elements. Bdelloid proximal gene-rich regions, however, appeared to lack foreign genes, thereby resembling those of model metazoan organisms. Some of the foreign genes were defective, whereas others were intact and transcribed; some of the latter contained functional spliceosomal introns. One such gene, apparently of bacterial origin, was overexpressed in
Escherichia coli
and yielded an active enzyme. The capture and functional assimilation of exogenous genes may represent an important force in bdelloid evolution.
A Cytosolic Iron Chaperone That Delivers Iron to Ferritin
Authors: Shi Haifeng , Bencze Krisztina Z. , Stemmler Timothy L. , Philpott Caroline C.
Published: May 2008

Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human poly (rC)–binding protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.
Comparison of Three Commercial Immunoassays for Detection of Herpes Simplex Virus Type 2 Antibodies in Commercial Sex Workers in Yunnan Province, China
Authors: Ngo Thoai D. , Laeyendecker Oliver , Morrow Rhoda Ashley , Lai Shenghan , Quinn Thomas C.
Published: May 2008

Five hundred commercial sex workers in China were tested for herpes simplex virus type 2 by three immunoassays and Western blotting. Sensitivities for the Focus, Kalon, and Biokit assays were 86.7%, 82.3%, and 34.9%, respectively, and specificities were 91.8%, 94.2%, and 60.1%, respectively. The Focus assay performed optimally at an index of 1.5 (95.2% sensitivity and 93.4% specificity), and the Kalon assay performed optimally at an index of 1.2 (93.3% sensitivity and 95.2% specificity).
Influence ofMycobacterium bovisBCG Vaccination on Cellular Immune Response of Guinea Pigs Challenged withMycobacterium tuberculosis
Authors: Ordway Diane , Henao-Tamayo Marcela , Shanley Crystal , Smith Erin E. , Palanisamy Gopinath , Wang Baolin , Basaraba Randall J. , Orme Ian M.
Published: May 2008

Mycobacterium bovisbacillus Calmette-Guérin (BCG) currently remains the only licensed vaccine for the prevention of tuberculosis. In this study, we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs and lymph nodes of naïve and vaccinated guinea pigs challenged by low-dose aerosol infection with virulentMycobacterium tuberculosis. As anticipated, vaccinated guinea pigs controlled the growth of the challenge infection more efficiently than controls did. This early phase of bacterial control in immune animals was associated with increased accumulation of CD4 and CD8 T cells, including cells expressing the activation marker CD45, as well as macrophages expressing class II major histocompatibility complex molecules. As the infection continued, the numbers of T cells in the lungs of vaccinated animals waned, whereas the numbers of these cells expressing CD45 increased. Whereas BCG vaccination reduced the influx of heterophils (neutrophils) into the lungs, an early B-cell influx was observed in these vaccinated animals. Overall, vaccine protection was associated with reduced pathology and lung damage in the vaccinated animals. These data provide the first direct evidence that BCG vaccination accelerates the influx of protective T-cell and macrophage populations into the infected lungs, diminishes the accumulation of nonprotective cell populations, and reduces the severity of lung pathology.
Opa Protein Repertoires of Disease-Causing and Carried Meningococci
Authors: Callaghan Martin J. , Buckee Caroline , McCarthy Noel D. , Ibarz Pavón Ana Belén , Jolley Keith A. , Faust Saul , Gray Stephen J. , Kaczmarski Edward B. , Levin Michael , Kroll J. Simon , Maiden Martin C. J. , Pollard Andrew J.
Published: May 2008

The meningococcal Opa proteins play an important role in pathogenesis by mediating invasion of human cells. The aim of this investigation was to determine whether carried and disease-associated meningococci possess different Opa repertoires and whether the diversity of these proteins is associated with clinical severity of disease. Opa repertoires in 227 disease-associated meningococci, isolated in the United Kingdom over a period of 6 years, were compared to the repertoires in 190 asymptomatically carried meningococci isolated in the United Kingdom from a contemporary, nonepidemic period. Multidimensional scaling (MDS) was employed to investigate the association between Opa repertoires and multilocus sequence typing (MLST) genotypes. Associations with clinical severity were also analyzed statistically. High levels of diversity were observed in
opa
alleles, variable regions, and repertoires, and MDS revealed that MLST genotypes were strongly associated with particular Opa repertoires. Individual Opa proteins or repertoires were not associated with clinical severity, though there was a trend toward an association with the
opaD
locus. Meningococcal Opa repertoire is strongly linked to MLST genotype irrespective of epidemiological sampling and therefore correlates with invasiveness. It is not, however, strongly associated with severity of meningococcal disease.
Multiplex Real-Time PCR Assay for Rapid Identification ofMycobacterium tuberculosisComplex Members to the Species Level
Authors: Pinsky Benjamin A. , Banaei Niaz
Published: May 2008

The species identification of members of theMycobacterium tuberculosiscomplex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiateM. tuberculosisfrom the closely relatedM. bovisandM. bovisBCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identifyM. tuberculosis, M. bovis, M. bovisBCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified asM. tuberculosiscomplex and found 57M. tuberculosisisolates and 3M. bovisBCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified asM. bovisby spoligotyping revealed an isolate ofM. tuberculosisthat had been misidentified. We propose that this assay will allow the routine identification ofM. tuberculosiscomplex members in the clinical laboratory.
Rapid, Novel, Specific, High-Throughput Assay for Diagnosis of
Loa loa
Infection
Authors: Burbelo Peter D. , Ramanathan Roshan , Klion Amy D. , Iadarola Michael J. , Nutman Thomas B.
Published: May 2008

The ability to diagnose
Loa loa
infection readily and accurately remains a demanding task. Among the available diagnostic methods, many are impractical for point-of-care field testing. To investigate whether luciferase immunoprecipitation systems (LIPS) can be used for rapid and specific diagnosis of
L. loa
infection, a LIPS assay was developed based on immunoglobulin G (IgG) and IgG4 subclass antibodies to a recombinant
L. loa
SXP-1 (designated LlSXP-1) antigen and tested with sera from healthy controls or patients with proven infection with
L. loa, Mansonella perstans, Onchocerca volvulus, Strongyloides stercoralis
, or
Wuchereria bancrofti
. A LIPS test measuring IgG antibody against LlSXP-1 readily differentiated
L. loa
-infected from uninfected patients and demonstrated markedly improved sensitivity and specificity compared with an LlSXP-1 IgG4-based enzyme-linked immunosorbent assay (67% sensitivity and 99% specificity). No significant immunoreactivity was observed with
S. stercoralis
-infected sera, but a small number of patients infected with
O. volvulus, M. perstans
, or
W. bancrofti
showed positive immunoreactivity. Measuring anti-IgG4-specific antibodies to LlSXP-1 showed a significant correlation (
r
∼ 0.85;
P
< 0.00001) with the anti-IgG results but showed no advantage over measuring the total IgG response alone. In contrast, a rapid LIPS format (called QLIPS) in which the tests are performed in less than 15 minutes under nonequilibrium conditions significantly improved the specificity for cross-reactive
O. volvulus
patient sera (100% sensitivity and 100% specificity). These results suggest that LIPS (and the even more rapid test QLIPS) represents a major advance in the ability to diagnose
L. loa
infection and may have future applications for point-of-care diagnostics.
Capsular Polysaccharide Synthesis Regions in
Klebsiella pneumoniae
Serotype K57 and a New Capsular Serotype
Authors: Pan Yi-Jiun , Fang Han-Chi , Yang Hui-Ching , Lin Tzu-Lung , Hsieh Pei-Fang , Tsai Feng-Chiao , Keynan Yoav , Wang Jin-Town
Published: May 2008

Community-acquired pyogenic liver abscess caused by
Klebsiella pneumoniae
is an emerging infectious disease. We explored the capsular polysaccharide synthesis (
cps
) regions of three non-K1, non-K2
K. pneumoniae
strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of
wzy
, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two
cps
regions are very useful in detecting K57 and the new
cps
genotype.
Atomic Force Microscopy Investigation of Vaccinia Virus Structure
Authors: Kuznetsov Y. , Gershon P. D. , McPherson A.
Published: May 2008

Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be “soldered” by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.
Deoxynucleoside Triphosphate Incorporation Mechanism of Foamy Virus (FV) Reverse Transcriptase: Implications for Cell Tropism of FV
Authors: Santos-Velazquez Jose , Kim Baek
Published: May 2008

Here, we investigated the pre-steady-state deoxynucleoside triphosphate (dNTP) incorporation kinetics of primate foamy virus (PFV) reverse transcriptase (RT) in comparison with those of HIV-1 and MuLV RTs. PFV RT displayed a drastic reduction in primer extension at low dNTP concentrations where HIV-1 RT remains highly active, indicating a low dNTP binding affinity in the case of PFV RT. Indeed, kinetic analysis showed that, as observed with MuLV RT, PFV RT exhibits ∼10 to 80 times lower dNTP binding affinity than HIV-1 RT. These three RTs, however, show similar catalytic activities. In conclusion, PFV RT displays mechanistic distinctions in comparison to HIV-1 RT and shares close similarity to MuLV RT.
Asparagine 631 Variants of the Chicken Mx Protein Do Not Inhibit Influenza Virus Replication in Primary Chicken Embryo Fibroblasts or In Vitro Surrogate Assays
Authors: Benfield Camilla T. O. , Lyall Jon W. , Kochs Georg , Tiley Laurence S.
Published: May 2008

Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles.
Identification of Residues outside of the Receptor Binding Domain That Influence the Infectivity and Tropism of Porcine Endogenous Retrovirus
Authors: Argaw Takele , Figueroa Mariel , Salomon Daniel R. , Wilson Carolyn A.
Published: May 2008

Identification of determinants of human tropism of porcine endogenous retrovirus (PERV) is critical to understanding the risk of transmission of PERV to recipients of porcine xenotransplantation products. Previously, we showed that a chimeric envelope cDNA encoding the 360 N-terminal residues of the human-tropic PERV envelope class A (PERV-A) SU and the 130 C-terminal residues of the pig-tropic PERV-C SU and all of TM (PERV-A/C) showed a 100-fold decrease in infectivity titer on human cells (M. Gemeniano, O. Mpanju, D. R. Salomon, M. V. Eiden, and C. A. Wilson, Virology 346:108-117, 2006). To identify residues important for human cell infection, we performed site-directed mutagenesis on each of the nine residues, singly or in combination, that distinguish the C-terminal region of PERV-C from PERV-A. Of the nine amino acids, two single-amino-acid substitutions, Q374R and I412V, restored the infectivity of human cells to the chimeric PERV-A/C to a titer equivalent to that of PERV-A. In contrast, PERV-A/C mutant envelope Q439P resulted in undetectable infection of human cells and an approximately 1,000-fold decrease in control pig cells. Mutation of K441R rescued mutants that carried Q439P, suggesting an incompatibility between the proline residue at this position and the presence of KK in the proteolytic cleavage signal. We confirmed this incompatibility with vectors carrying PERV-A envelope mutant R462K that were also rendered noninfectious. Finally, tropism of vectors carrying PERV-C envelope mutants with only four amino acid changes in the C terminus of PERV-C envelope, NHRQ436YNRP plus K441R, was shifted to one similar to that of PERV-A. Our results show an important and previously unrecognized role for infectivity and tropism for residues at the C terminus of SU.
Visualization of the Externalized VP2 N Termini of Infectious Human Parvovirus B19
Authors: Kaufmann Bärbel , Chipman Paul R. , Kostyuchenko Victor A. , Modrow Susanne , Rossmann Michael G.
Published: May 2008

The structures of infectious human parvovirus B19 and empty wild-type particles were determined by cryoelectron microscopy (cryoEM) to 7.5-Å and 11.3-Å resolution, respectively, assuming icosahedral symmetry. Both of these, DNA filled and empty, wild-type particles contain a few copies of the minor capsid protein VP1. Comparison of wild-type B19 with the crystal structure and cryoEM reconstruction of recombinant B19 particles consisting of only the major capsid protein VP2 showed structural differences in the vicinity of the icosahedral fivefold axes. Although the unique N-terminal region of VP1 could not be visualized in the icosahedrally averaged maps, the N terminus of VP2 was shown to be exposed on the viral surface adjacent to the fivefold β-cylinder. The conserved glycine-rich region is positioned between two neighboring, fivefold-symmetrically related VP subunits and not in the fivefold channel as observed for other parvoviruses.
Synthesis of Genomic and Subgenomic RNA in Mosquito Cells Infected with Two Sindbis Virus nsP4 Mutants: Influence of Intracellular Nucleoside Triphosphate Concentrations
Authors: Li Mei-Ling , Kwan Tzu-Yu , Simmonds H. Anne , Stollar Victor
Published: May 2008

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.
Different Mechanisms of Cell Entry by Human-Pathogenic Old World and New World Arenaviruses
Authors: Rojek Jillian M. , Sanchez Ana B. , Nguyen Ngoc Thao , de la Torre Juan-Carlos , Kunz Stefan
Published: May 2008

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.
Induction of Plasma (TRAIL), TNFR-2, Fas Ligand, and Plasma Microparticles after Human Immunodeficiency Virus Type 1 (HIV-1) Transmission: Implications for HIV-1 Vaccine Design
Authors: Gasper-Smith Nancy , Crossman Deanna M. , Whitesides John F. , Mensali Nadia , Ottinger Janet S. , Plonk Steven G. , Moody M. Anthony , Ferrari Guido , Weinhold Kent J. , Miller Sara E. , Reich Charles F. , Qin Li , Self Stephen G. , Shaw George M. , Denny Thomas N. , Jones Laura E. , Pisetsky David S. , Haynes Barton F.
Published: May 2008

The death of CD4
+
CCR5
+
T cells is a hallmark of human immunodeficiency virus (HIV) infection. We studied the plasma levels of cell death mediators and products—tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas ligand, TNF receptor type 2 (TNFR-2), and plasma microparticles—during the earliest stages of infection following HIV type 1 (HIV-1) transmission in plasma samples from U.S. plasma donors. Significant plasma TRAIL level elevations occurred a mean of 7.2 days before the peak of plasma viral load (VL), while TNFR-2, Fas ligand, and microparticle level elevations occurred concurrently with maximum VL. Microparticles had been previously shown to mediate immunosuppressive effects on T cells and macrophages. We found that T-cell apoptotic microparticles also potently suppressed in vitro immunoglobulin G (IgG) and IgA antibody production by memory B cells. Thus, release of TRAIL during the onset of plasma viremia (i.e., the eclipse phase) in HIV-1 transmission may initiate or amplify early HIV-1-induced cell death. The window of opportunity for a HIV-1 vaccine is from the time of HIV-1 transmission until establishment of the latently infected CD4
+
T cells. Release of products of cell death and subsequent immunosuppression following HIV-1 transmission could potentially narrow the window of opportunity during which a vaccine is able to extinguish HIV-1 infection and could place severe constraints on the amount of time available for the immune system to respond to the transmitted virus.
Virological Consequences of Early Events following Cell-Cell Contact between Human Immunodeficiency Virus Type 1-Infected and Uninfected CD4
+
Cells
Authors: Ruggiero Eliana , Bona Roberta , Muratori Claudia , Federico Maurizio
Published: May 2008

Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4
+
cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4
+
cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4
+
lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4
+
cell can occur through different mechanisms, leading to distinguishable virological outcomes.
NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly
Authors: Ma Yinghong , Yates Jeremy , Liang Yuqiong , Lemon Stanley M. , Yi MinKyung
Published: May 2008

A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.
Cryoelectron Microscopy Map of
Atadenovirus
Reveals Cross-Genus Structural Differences from Human Adenovirus
Authors: Pantelic Radosav S. , Lockett Linda J. , Rothnagel Rosalba , Hankamer Ben , Both Gerald W.
Published: May 2008

A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype
Atadenovirus
(OAdV [an ovine adenovirus isolate]) showing information at a 10.6-Å resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a
Mastadenovirus
, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent “knobs” on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.
Human Immunodeficiency Virus Type 1 Infection Increases the In Vivo Capacity of Peripheral Monocytes To Cross the Blood-Brain Barrier into the Brain and the In Vivo Sensitivity of the Blood-Brain Barrier to Disruption by Lipopolysaccharide
Authors: Wang Hongwei , Sun Jinglin , Goldstein Harris
Published: May 2008

Human immunodeficiency virus type 1 (HIV-1), introduced into the brain by HIV-1-infected monocytes which migrate across the blood-brain barrier (BBB), infects resident macrophages and microglia and initiates a process that causes HIV-1-associated neurocognitive disorders. The mechanism by which HIV-1 infection circumvents the BBB-restricted passage of systemic leukocytes into the brain and disrupts the integrity of the BBB is not known. Circulating lipopolysaccharide (LPS), which can compromise the integrity of the BBB, is significantly increased in HIV-1-infected individuals. We hypothesized that HIV-1 infection increases monocyte capacity to migrate across the BBB, which is further facilitated by a compromise of BBB integrity mediated by the increased systemic LPS levels present in HIV-1-infected individuals. To investigate this possibility, we examined the in vivo BBB migration of monocytes derived from our novel mouse model, JR-CSF/EYFP mice, which are transgenic for both a long terminal repeat-regulated full-length infectious HIV-1 provirus and ROSA-26-regulated enhanced yellow fluorescent protein. We demonstrated that JR-CSF/EYFP mouse monocytes displayed an increased capacity to enter the brain by crossing either an intact BBB or a BBB whose integrity was partially compromised by systemic LPS. We also demonstrated that the JR-CSF mouse BBB was more susceptible to disruption by systemic LPS than the control wild-type mouse BBB. These results demonstrated that HIV-1 infection increased the ability of monocytes to enter the brain and increased the sensitivity of the BBB to disruption by systemic LPS, which is elevated in HIV-1-infected individuals. These mice represent a new in vivo system for studying the mechanism by which HIV-1-infected monocytes migrate into the brain.
Reduction in Severity of a Herpes Simplex Virus Type 1 Murine Infection by Treatment with a Ribozyme Targeting the U
L
20 Gene RNA
Authors: Liu Jia , Lewin Alfred S. , Tuli Sonal S. , Ghivizzani Steven C. , Schultz Gregory S. , Bloom David C.
Published: May 2008

Hammerhead ribozymes were designed to target mRNA of several essential herpes simplex virus type 1 (HSV-1) genes. A ribozyme specific for the late gene U
L
20 was packaged in an adenovirus vector (Ad-U
L
20 Rz) and evaluated for its capacity to inhibit the viral replication of several HSV-1 strains, including that of the wild-type HSV-1 (17
syn
+ and KOS) and several acycloguanosine-resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-U
L
20 Rz was also tested for its ability to block an HSV-1 infection, using the mouse footpad model. Mouse footpads were treated with either the Ad-U
L
20 Rz or an adenoviral vector expressing green fluorescent protein (Ad-GFP) and then infected immediately thereafter with 10
4
PFU of HSV-1 strain 17
syn
+. Ad-U
L
20 ribozyme treatment consistently led to a 90% rate of protection for mice from lethal HSV-1 infection, while the survival rate in the control groups was less than 45%. Consistent with this protective effect, treatment with the Ad-U
L
20 Rz reduced the viral DNA load in the feet, the dorsal root ganglia, and the spinal cord relative to that of the Ad-GFP-treated animals. This study suggests that ribozymes targeting essential genes of the late kinetic class may represent a new therapeutic strategy for inhibiting HSV infection.
Induction of Broad CD4+and CD8+T-Cell Responses and Cross- Neutralizing Antibodies against Hepatitis C Virus by Vaccination with Th1-Adjuvanted Polypeptides Followed by Defective Alphaviral Particles Expressing Envelope Glycoproteins gpE1 and gpE2 and Nonstructural Proteins 3, 4, and 5
Authors: Lin Yinling , Kwon Taewoo , Polo John , Zhu Yi-Fei , Coates Stephen , Crawford Kevin , Dong Christine , Wininger Mark , Hall John , Selby Mark , Coit Doris , Medina-Selby Angelica , McCoin Colin , Ng Philip , Drane Debbie , Chien David , Han Jang , Vajdy Michael , Houghton Michael
Published: May 2008

Broad, multispecific CD4+and CD8+T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8+T-cell responses but low CD4+T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4+T helper responses but no CD8+T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4+T helper responses but no CD8+T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4+and CD8+T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.
What to tell and how to tell: a qualitative study of information sharing in research for adults with intellectual disability
Authors: Andre-Barron D , Strydom A , Hassiotis A
Published: May 2008

Objectives:

To explore opinions and attitudes regarding the current information-giving practices in research involving adults with intellectual disabilities.

Design:

Qualitative focus group study with a purposive sample.

Setting:

An intellectual disabilities service within the NHS

Participants:

A sample of 26 individuals including adults with mild intellectual disability, carers, clinicians, care managers and the charitable sector.

Results:

Three main themes were identified: process, format, and content. There was agreement that there is a need for improvement in the process and quality of information giving. With regard to the content of information, there was little discrepancy between the study findings and existing guidance.

Conclusions:

Information should be presented in different formats and prepared with input from appropriate professions. Additionally the roles of peers, carers and other professionals in the process of information giving should be considered.
Scientific responsibility for the dissemination and interpretation of genetic research: lessons from the “warrior gene” controversy
Authors: Wensley D , King M
Published: May 2008

This paper discusses the announcement by a team of researchers that they identified a genetic influence for a range of “antisocial” behaviours in the New Zealand Māori population (dubbed the “warrior gene”). The behaviours included criminality, violence, gambling and alcoholism. The reported link between genetics and behaviour met with much controversy. The scientists were described as hiding behind a veneer of supposedly “objective” western science, using it to perpetuate “racist and oppressive discourses”.

In this paper we examine what went wrong in the dissemination of the research. We chose as our framework the debate around the “internal/external” responsibilities of scientists. Using this discourse we argue that when the researchers ventured to explain their research in terms of social phenomena, they assumed a duty to ensure that their findings were placed “in context”. By “in context”, we argue that evidence of any genetic influence on behavioural characteristics should not be reported in isolation, but instead presented alongside other environmental, cultural and socio-economic influences that may also contribute to the studied behaviour. Rather than imposing a new obligation on scientists, we find this duty to contextualise results is in keeping with the spirit of codes of ethics already in place. Lessons from the “warrior gene” controversy may assist researchers elsewhere to identify potential areas of conflictbeforethey jeopardise research relationships, or disseminate findings in a manner that fuels misleading and/or potentially discriminatory attitudes in society.
National HIV treatment guidelines in Tanzania and Ethiopia: are they legitimate rationing tools?: Table 1
Authors: Johansson K A , Jerene D , Norheim O F
Published: May 2008

Objective:

To provide an ethical analysis of whether the Ethiopian and Tanzanian national HIV/AIDS treatment guidelines can be considered legitimate and fair rationing tools.

Method:

Qualitative study and ethical analysis involving guideline documents and interviews with nine key members involved in the development of the guidelines. The analysis followed an editing organising style. The theoretical framework was a guideline-specific framework based on theories of just resource allocation in healthcare and conditions that ensure fair processes in guideline development. According to this framework, legitimate rationing requires reasons for patient selection to be explicit, public and relevant, and decisions must be open to question and revision.

Results:

The only explicit rationing criteria that both guidelines recommended were clinical antiretroviral treatment indications. Explicit non-clinical rationing criteria were expressed in a separate Ethiopian implementation guideline. Neither of the guideline development processes fully satisfies minimal requirements of procedural fairness. There is a lack of transparency. The reasons for decisions are rarely given and are not publicly available. This reduces the opportunity for public questioning, debate and revisions. The guidelines were based on expert opinion and consensus. Recommendations from the WHO were copied without much discussion, disagreement or adjustment.

Conclusions:

The two national HIV treatment guidelines discussed are de facto mechanisms for rationing but were developed using methods that do not fully satisfy the requirements of fair processes.
Croatian physicians’ and nurses’ experience with ethical issues in clinical practice
Authors: Sorta-Bilajac I , Baždarić K , Brozović B , Agich G J
Published: May 2008

Aim:

To assess ethical issues in everyday clinical practice among physicians and nurses of the University Hospital Rijeka, Rijeka, Croatia.

Subjects and methods:

We surveyed the entire population of internal medicine, oncology and intensive care specialists and associated nurses employed at the University Hospital Rijeka, Rijeka, Croatia (n = 532). An anonymous questionnaire was used to explore the type and frequency of ethical dilemmas, rank of their difficulty, access to and use of ethics support services, training in ethics and confidence about knowledge in ethics. Physicians (n = 113, 55% of them female) ranged in age from 27 to 61 years, and nurses (n = 251, 95% female), from 20 to 52.

Results:

The most frequent ethical dilemmas concerned uncertain or impaired decision-making capacity (66% of physicians, 47% of nurses, p = 0.008), limitation of treatment at the end of life (60% of physicians, 31% of nurses, p<0.001) and disagreements among family members (47% of physicians, 31% of nurses, p = 0.025). The most difficult dilemmas concerned euthanasia and physician-assisted suicide (49% of physicians, 52% of nurses) and limitation of treatment at the end of life (14% of physicians, 18% of nurses). Only a minority reported ever using any kind of ethics support services (12% of physicians, 3% of nurses, p = 0.001) or being very confident about knowledge in ethics (5% of physicians, 6% of nurses).

Conclusions:

Similar ethical difficulties are present in the clinical practice of both physicians and nurses, with important differences in access and use of ethics support services. A need for systematic ethics educational activities was identified. Inclusion of individual ethics consultants in Croatian healthcare ethics support services is strongly advised.
Nurses’ attitudes towards artificial food or fluid administration in patients with dementia and in terminally ill patients: a review of the literature
Authors: Bryon E , de Casterlé B Dierckx , Gastmans C
Published: May 2008

Objective:

Although nurses have an important role in the care process surrounding artificial food or fluid administration in patients with dementia or in terminally ill patients, little is known about their attitudes towards this issue. The purpose of this study was to thoroughly examine nurses’ attitudes by means of a literature review.

Method:

An extensive systematic search of the electronic databases PubMed, Cinahl, PsycINFO, The Cochrane Library, FRANCIS, Philosopher’s Index and Social Sciences Citation Index was conducted to identify pertinent articles published from January 1990 to January 2007.

Findings:

Nurses’ arguments for or against could be categorised as ethical-legal, clinical or social-professional. The most important arguments explicitly for artificial food and fluid administration in patients with dementia or in terminally ill patients were sanctity of life, considering artificial food and fluid administration as basic nursing care, and giving reliable nutrition, hydration or medication. An explicit counter-argument was the high cost of treatment. Arguments used by opponents and proponents were quality of life and dignified death. The arguments were not strikingly different for the two patient populations. It turned out that the nurses’ ethical arguments remarkably reflected the current ethical debate. But some of their clinical presuppositions contradicted current clinical evidence.

Conclusion:

The interaction between clinical facts and ethical reflections makes the findings of this review extremely relevant for clinical ethics. A large need exists to clearly communicate to nurses the latest clinical evidence and the main results of ongoing ethical debates.
Systematic reviews of empirical bioethics
Authors: Strech D , Synofzik M , Marckmann G
Published: May 2008

Background:

Publications and discussions of survey research in empirical bioethics have steadily increased over the past two decades. However, findings often differ among studies with similar research questions. As a consequence, ethical reasoning that considers only parts of the existing literature and does not apply systematic reviews tends to be biased. To date, we lack a systematic review (SR) methodology that takes into account the specific conceptual and practical challenges of empirical bioethics.

Methods:

The steps of systematically reviewing empirical findings in bioethics are presented and critically discussed. In particular, (a) the limitations of traditional SR methodologies in the field of empirical bioethics are critically discussed, and (b) conceptual and practical recommendations for SRs in empirical bioethics that are (c) based on the authors’ review experiences in healthcare ethics are presented.

Results:

A 7-step approach for SRs of empirical bioethics is proposed: (1) careful definition of review question; (2) selection of relevant databases; (3) application of ancillary search strategies; (4) development of search algorithms; (5) relevance assessment of the retrieved references; (6) quality assessment of included studies; and (7) data analysis and presentation. Conceptual and practical challenges arise because of various peculiarities in reviewing empirical bioethics literature and can lead to biased results if they are not taken into account.

Conclusions:

If suitably adapted to the peculiarities of the field, SRs of empirical bioethics provide transparent information for ethical reasoning and decision-making that is less biased than single studies.
How do elderly spouse care givers of people with Alzheimer disease experience the disclosure of dementia diagnosis and subsequent care?
Authors: Laakkonen M-L , Raivio M M , Eloniemi-Sulkava U , Saarenheimo M , Pietilä M , Tilvis R S , Pitkälä K H
Published: May 2008

Objectives:

To examine the experiences of spousal care givers of Alzheimer patients to disclosure of dementia diagnosis and subsequent care.

Methods:

A random sample of 1943 spousal care givers of people receiving medication for Alzheimer disease (AD) was sent a cross-sectional postal survey about their opinions on the disclosure of dementia and follow-up care. A smaller qualitative study (n = 63) included open-ended questions concerning their experiences of the same topics.

Results:

The response rate for the survey was 77%. Of the respondents, 1214 of 1434 acknowledged themselves as their spouse’s care giver. The mean age of the care givers was 78.2 years, and that of demented spouses, 80.5 years. Of the care givers, 63% were women. The couples had long-lasting marriages (mean 52 years). Of the care givers, 93% reported that dementia had been disclosed openly to their spouse; 97% also preferred that physicians openly inform the patients of the dementia diagnosis, although 55% of their spouses with AD had developed depressive symptoms after the disclosure. Of the care givers, 71% felt they had received sufficient information about dementia. However, only 50% estimated that their spouses’ follow-up care had been well organised. The responses in the qualitative study indicated that many care givers felt grief and anxiety. They also expressed feelings of loneliness and uncertainty about how to deal with follow-up care for dementia.

Conclusions:

Elderly spousal care givers were quite satisfied with the information given them about dementia. However, the support with regard to the follow-up care of care-giving families failed to meet their needs adequately.