bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythropoietin reduces Schwann cell TNF‐α, Wallerian degeneration and pain‐related behaviors after peripheral nerve injury
Authors: Campana W. Marie , Li Xiaoqing , Shubayev Veronica I. , Angert Mila , Cai Karen , Myers Robert R.
Published: February 2006

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain‐like behaviors. These effects are mediated in large part by pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNF‐α). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF‐α has been implicated in the development of pain‐related behaviors, we measured TNF‐α mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF‐α mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF‐α in Schwann cells, axoplasm and macrophages. In rhEpo‐treated animals, TNF‐α immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF‐α in CCI by blocking expression of TNF‐α in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF‐α expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF‐αin vitro. These results indicated that rhEpo regulates TNF‐α by multiple mechanisms; rhEpo regulates TNF‐α mRNA expression by Schwann cells but also may directly counteract TNF‐α signaling pathways that lead to injury, chronic pain and/or death.
Recruitment of native parasitoids by an exotic leaf miner,Cameraria ohridella: host‐parasitoid synchronization and influence of the environment
Authors: Girardoz Sandrine , Kenis Marc , Quicke Donald L. J.
Published: February 2006

Abstract 

The horse‐chestnut leaf miner,Cameraria ohridella,is a moth of unknown origin that has recently invaded Europe and severely defoliates the European horse‐chestnut, an important ornamental tree.

Several indigenous parasitoids have colonized this new host, but parasitism remains low. One of the hypotheses suggested to explain the low parasitism is that candidate parasitoids emerge too early in spring to attack the first host generation and, thus, need early‐occurring leaf miners as alternate hosts. This hypothesis was tested by observing the synchronization between the phenology of the moth and that of its main parasitoids, and by comparing parasitism rates and parasitoid richness in different environments with various levels of biological diversity.

In spring, the bulk of the parasitoids emerge at least 5 weeks before the occurrence of the first suitable larvae ofC. ohridellawhereas most parasitoid adults reared outdoors die within 5 weeks after emergence.

Parasitism rates and parasitoid richness do not increase with biological diversity, suggesting that most parasitoids attacking the first generation ofC. ohridellado not come from alternate hosts. Parasitism does not increase later in the year in the subsequent generations, when host‐parasitoid synchronization becomes less critical.

We conclude that, although the spring emergence of parasitoids is not synchronized with the phenology ofC. ohridella, the parasitoids attacking the first generation are probably old or late‐emerging adults of the overwintering generation. The lack of synchronization is probably not the only reason for the poor recruitment of native parasitoids byC. ohridella.
Carry over effects of the entomopathogen Bacillus thuringiensis ssp. Kurstaki on Choristoneura fumiferana (Lepidoptera: Tortricidae) progeny under various stressful environmental conditions
Authors: Bauce Éric , Carisey Nathalie , Dupont Alain
Published: February 2006

Abstract 

In the present study, we documented the lethal and sublethal effects of the entomopathogen Btk on spruce budworm and its progeny under various environmental conditions. We hypothesized that aerial spray of Bacillus thuringiensis ssp. kurstaki (Btk) could affect the biological performance of the surviving spruce budworm (Choristoneura fumiferana Clem.) populations and their progenies and that Btk sublethal effects could be widened by other types of stress (i.e. temperature conditions and changes in food suitability from year to year).

The results from a 3‐year field experiment indicated that Btk treatments decreased the fitness of the surviving larvae whatever the prevailing temperature and nutritional conditions.

The detrimental Btk effects on the parental generation carried over to the offspring. The percent of egg hatch and first‐instar survival were negatively affected by Btk whatever other stress spruce budworm parents underwent.

The present study also highlighted the fact that the effects of temperature and nutritional stress suffered by the parents could carry over to the next generation. Balsam fir flowering, which provided larvae with pollen rich in nitrogen, favoured both the parental generation and the fitness of their offspring. Spruce budworm mothers allocated to their progenies large amounts of energy reserves (triglycerides and glycogen) that greatly enhanced the survival of the early stages.

Egg hatch and the survival of first‐instar larval progeny were drastically affected when their parents had reduced larval growth as a result of exposure to cool temperatures that had desynchronized insect and bud phenology.

Budworm mothers submitted to negative impacts of previous defoliation allocated low amounts of energy reserves to their progeny. This lack of energy associated with unfavourable temperature conditions (i.e. high temperatures in late summer and in early fall and an extended cool period in spring) drastically reduced survival of diapausing second‐instar larvae.

These results highlight the importance of considering the various sources of stress when attempting to evaluate the impact of a control agent on an insect pest population and its progenies.
Validation of Three‐Dimensional Cardiac Image Integration: Use of Integrated CT Image into Electroanatomic Mapping System to Perform Catheter Ablation of Atrial Fibrillation
Authors: KISTLER PETER M. , EARLEY MARK J. , HARRIS STUART , ABRAMS DOMINIC , ELLIS STEPHEN , SPORTON SIMON C. , SCHILLING RICHARD J.
Published: February 2006

Introduction: Accurate visualization of the complex left atrial (LA) anatomy and the location of an ablation catheter within the chamber is important in the success and safety of ablation for atrial fibrillation (AF). We describe the integration of CT into an electroanatomic mapping (EAM) system and its validation in patients undergoing catheter ablation for AF.

Methods and Results: Thirty patients (59.2 ± 8 years, 25 M) with paroxysmal (12) and persistent (18) AF underwent ablation using CT image integration into an electroanatomic mapping system. CT registration using the pulmonary veins as markers (landmark) was achieved with an error of 6.4 ± 2.8 mm with repeat registration required in two patients. Registration of the CT by best fit to a electroanatomic geometry (surface) was achieved with an error of 2.3 ± 0.4 mm. There was no significant difference in the regional LA registration error at superior (1.7 ± 0.7 mm), inferior (2.2 ± 1.4 mm), septal (1.7 ± 0.8 mm), and lateral (1.7 ± 0.7 mm, P = 0.13) sites. Cardiac rhythm at the time of CT did not have a significant effect on total or regional surface registration accuracy (mean total 2.5 ± 0.3 in AF patients vs 2.3 ± 0.5 in SR patients, P = 0.22). The integrated CT was used to guide the encirclement of the pulmonary veins (PV) in pairs with electrical isolation achieved by maintaining ablation along the ablation line in 58 of 60 PV pairs. Postprocedural PV angiography did not demonstrate significant stenosis.

Conclusion: CT image integration into an EAM system was successfully performed in patients undergoing catheter ablation for AF. With a greater appreciation of the complex and variable nature of the PV and LA anatomy this new technology may improve the efficacy and safety of the procedure.
WORKPLACE SAFETY IN ATLANTA'S CONSTRUCTION INDUSTRY: INSTITUTIONAL FAILURE IN TEMPORARY STAFFING ARRANGEMENTS
Authors: Mehta Chirag , Theodore Nik
Published: February 2006

This article examines workplace safety conditions in Atlanta's construction industry in light of the growing presence of temporary staffing agencies in the industry. Using data collected from surveys of building contractors, temporary staffing agencies, and temp workers, the article explores the ways in which the use of agency‐supplied temps by construction contractors subverts workplace health and safety regulations, thereby exposing construction workers to heightened risk of injury.
Wind‐aerated lagoons for sustainable treatment of wastewaters from small communities
Authors: Horan N. J. , Salih A. , Walkinshaw T.
Published: February 2006

Biological treatment processes that utilise wind‐aerated lagoons have proved popular for small communities because of their negligible sludge production. Scottish Water has recently constructed one such system, known as the Aero‐Fac (North Dakota, USA), and this paper reports on an intensive 12‐month study designed to both monitor the lagoon performance and establish the key design parameters. Over the monitoring period the final effluent complied with both Scottish Environmental Protection Agency discharge consents and Scottish Water performance guarantees for biochemical oxygen demand and total suspended solids, which averaged 9 and 28 mg/L, respectively. The lagoons showed an accelerated growth of algae during the summer months, but this did not adversely affect the final effluent suspended solids. The lagoons also achieved a faecal coliform removal of around 3.4 log and an average effluent ammonia of 7.6 mg/L. However, the ammonia removal was seasonal with a better performance in the summer months, which probably reflects take‐up by the growing algal population over this period. No sludge was wasted from the lagoon over the monitoring period yet it accumulated only sparingly in the lagoons, mainly around the inlet of the primary lagoon. The estimated per capita sludge accumulation rate was 0.0047 m3/person/year. The total capital costs of the scheme were £1.65 million, and of this the lagoon cost was £840 000. The capital costs of £420 per capita make this more expensive than other, similar options, but plant operating costs are significantly reduced.
The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating
Authors: Cui Liying , Aleksandrov Luba , Hou Yue‐Xian , Gentzsch Martina , Chen Jey‐Hsin , Riordan John R. , Aleksandrov Andrei A.
Published: February 2006

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide‐binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild‐type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl‐methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild‐type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function.
Rate dependency of β‐adrenergic modulation of repolarizing currents in the guinea‐pig ventricle
Authors: Rocchetti M. , Freli V. , Perego V. , Altomare C. , Mostacciuolo G. , Zaza A.
Published: February 2006

β‐Adrenergic stimulation modulates ventricular currents and sinus cycle length (CL). We investigated how changes in CL affect the current induced by isoprenaline (Iso) during the action potential (AP) of guinea‐pig ventricular myocytes. Action‐potential clamp was applied at CLs of 250 and 1000 ms to measure: (1) the net current induced by 0.1 μm Iso (IIso); (2) the L‐type Ca2+ current ICaL and slow delayed rectifier current IKs components of IIso (IIsoCa and IIsoK), identified as the Iso‐induced current sensitive to nifedipine and HMR1556, respectively; and (3) IIso persisting after inhibition of both ICa and IKs (IisoR). The pause dependency of IKs and its modulation were evaluated in voltage‐clamp experiments. The rate dependency of the duration of the action potential at 90% repolarization (APD90) and its modulation by isoprenaline were tested in current‐clamp experiments. At a CL of 250 ms IIso was inward during initial repolarization and reversed at 59% of APD90. At a CL of 1000 ms IIso became mostly inward in all cells. Switching to shorter CL did not change IIsoCa and IIsoK amplitudes, but moved their peak amplitudes to earlier repolarization; IIsoR was independent of CL. Acceleration of IIsoK at shorter CL was based on faster pause dependency of IKs activation rate. The ‘restitution’ of activation rates was modulated by isoprenaline. The APD90–CL relation was rotated anticlockwise by isoprenaline and crossed the control curve at a CL of 150 ms (400 beats min−1). We conclude that: (1) isoprenaline induced markedly different current profiles according to pacing rate, involving CL‐dependent ICa and IKs modulation; (2) the effect of isoprenaline on APD90 was CL dependent, and negligible during tachycardia; and (3) during sympathetic activation, repolarization stability may involve matched modulation of sinus rate and repolarizing currents.
Zebrafish MiR-430 Promotes Deadenylation and Clearance of Maternal mRNAs
Authors: Giraldez Antonio J. , Mishima Yuichiro , Rihel Jason , Grocock Russell J. , Van Dongen Stijn , Inoue Kunio , Enright Anton J. , Schier Alexander F.
Published: February 2006

MicroRNAs (miRNAs) comprise 1 to 3% of all vertebrate genes, but their in vivo functions and mechanisms of action remain largely unknown. Zebrafish miR-430 is expressed at the onset of zygotic transcription and regulates morphogenesis during early development. By using a microarray approach and in vivo target validation, we find that miR-430 directly regulates several hundred target messenger RNA molecules (mRNAs). Most targets are maternally expressed mRNAs that accumulate in the absence of miR-430. We also show that miR-430 accelerates the deadenylation of target mRNAs. These results suggest that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early embryogenesis.
Selective Stimulation of T Cell Subsets with Antibody-Cytokine Immune Complexes
Authors: Boyman Onur , Kovar Marek , Rubinstein Mark P. , Surh Charles D. , Sprent Jonathan
Published: February 2006

Interleukin-2 (IL-2), which is a growth factor for T lymphocytes, can also sometimes be inhibitory. Thus, the proliferation of CD8
+
T cells in vivo is increased after the injection of a monoclonal antibody that is specific for IL-2 (IL-2 mAb), perhaps reflecting the removal of IL-2–dependent CD4
+
T regulatory cells (T regs). Instead, we show here that IL-2 mAb augments the proliferation of CD8
+
cells in mice simply by increasing the biological activity of preexisting IL-2 through the formation of immune complexes. When coupled with recombinant IL-2, some IL-2/IL-2 mAb complexes cause massive (>100-fold) expansion of CD8
+
cells in vivo, whereas others selectively stimulate CD4
+
T regs. Thus, different cytokine-antibody complexes can be used to selectively boost or inhibit the immune response.
Comparison of Virus Transcription during Lytic Infection of the Oka Parental and Vaccine Strains of Varicella-Zoster Virus
Authors: Cohrs Randall J. , Gilden Donald H. , Gomi Yasuyuki , Yamanishi Koichi , Cohen Jeffrey I.
Published: February 2006

The
attenuated Oka vaccine (V-Oka) strain of varicella-zoster virus (VZV)
effectively reduces disease produced by primary infection and virus
reactivation. V-Oka was developed by propagation of the Oka parental
(P-Oka) strain of VZV in guinea pig and human embryo fibroblasts.
Complete DNA sequencing of both viruses has revealed 63 sites that
differ between P-Oka and V-Oka, 37 of which are located within 21
unique open reading frames (ORFs). Of the ORFs that differ, ORF 62
contains the greatest number (10) of mutated sites. ORF 62 encodes IE
62, the major immediate-early transactivator of virus genes, and is
essential for lytic virus replication. To determine whether a
disproportionate number of mutations in ORF 62 might account for virus
attenuation, we compared the global pattern of V-Oka gene expression to
that of P-Oka. Transcription of ORFs 62, 65, 66, and 67 was suppressed,
whereas ORF 41 was elevated in V-Oka-infected cells compared to
P-Oka-infected cells (
P
< 0.01; z test). Suppression
of ORF 62, 65, and 66 transcription was confirmed by quantitative dot
blot and Western blot analyses. Transient-transfection assays to
determine whether mutations within V-Oka-derived IE 62 affected its
ability to transactivate VZV gene promoters revealed similar IE 62
transactivation of VZV gene 20, 21, 28, 29, 65, and 66 promoters in
both P-Oka and V-Oka. Together, our results indicate that mutations in
V-Oka IE 62 alone are unlikely to account for vaccine virus
attenuation.
Stability and Function of JC Virus Large T Antigen and T′ Proteins Are Altered by Mutation of Their Phosphorylated Threonine 125 Residues
Authors: Tyagarajan Shiva K. , Frisque Richard J.
Published: February 2006

JC virus (JCV), a human polyomavirus, exhibits oncogenic activity in rodents and primates. The large tumor antigens (TAgs) of the polyomaviruses play key roles in viral replication and oncogenic transformation. Analyses of JCV TAg phosphorylation mutants indicated that the amino-terminal phosphorylation site at threonine 125 (T125) is critical to TAg replication function. This site is also conserved in the TAg splice variants T′
135
, T′
136
, and T′
165
. By constructing stable cell lines expressing JCV T125A and T125D mutants, we show that mutation of this phosphorylation site to alanine generates an unstable TAg; however, the stability of the three T′ proteins is unaffected. JCV T125A mutant proteins bind the retinoblastoma protein (RB) family members p107 and p130 with slightly reduced efficiencies and fail to induce the release of transcriptionally active E2F from RB-E2F complexes. On the other hand, cell lines expressing JCV T125D mutant proteins produce stable TAg and T′ proteins which bind p107 and p130 more efficiently than do the wild-type proteins. In addition, T125D mutant proteins efficiently induce the release of E2F from RB-E2F complexes. T125D mutant cell lines, unlike the T125A mutant lines, continue to grow under conditions of low serum concentration and anchorage independence. Finally, both T125A and T125D mutant viruses are replication defective. Phosphorylation of the T125 site is likely mediated by a cyclin-cyclin-dependent kinase, suggesting that JCV TAg and T′ protein functions that mediate viral replication and oncogenic transformation events are regulated in a cell cycle-dependent manner.
Human Immunodeficiency Virus (HIV) Vaccine Trials: a Novel Assay for Differential Diagnosis of HIV Infections in the Face of Vaccine-Generated Antibodies
Authors: Khurana Surender , Needham James , Mathieson Bonnie , Rodriguez-Chavez Isaac R. , Catanzaro Andrew T. , Bailer Robert T. , Kim Jerome , Polonis Vicky , Cooper David A. , Guerin Jan , Peterson Michael L. , Gurwith Marc , Nguyen Nga , Graham Barney S. , Golding Hana
Published: February 2006

All current human immunodeficiency virus (HIV) vaccine candidates contain multiple viral components and elicit antibodies that react positively in licensed HIV diagnostic tests, which contain similar viral products. Thus, vaccine trial participants could be falsely diagnosed as infected with HIV. Additionally, uninfected, seropositive vaccinees may encounter long-term social and economic harms. Moreover, this also interferes with early detection of true HIV infections during preventive HIV vaccine trials. An HIV-seropositive test result among uninfected vaccine trial participants is a major public health concern for volunteers who want to participate in future HIV vaccine trials. Based on the increased number of HIV vaccines being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Using a whole-HIV-genome phage display library, we identified conserved sequences in Env-gp41 and Gag-p6 which are recognized soon after infection, do not contain protective epitopes, and are not part of most current HIV vaccines. We established a new HIV serodetection assay based on these peptides. To date, this assay, termed HIV-SELECTEST, demonstrates >99% specificity and sensitivity. Importantly, in testing of plasma samples from multiple HIV vaccine trials, uninfected trial participants scored negative, while all intercurrent infections were detected within 1 to 3 months of HIV infection. The new HIV-SELECTEST is a simple but robust diagnostic tool for easy implementation in HIV vaccine trials and blood banks worldwide.
Retroviral Restriction Factors Fv1 and TRIM5α Act Independently and Can Compete for Incoming Virus before Reverse Transcription
Authors: Passerini Luca D. , Keckesova Zuzana , Towers Greg J.
Published: February 2006

The restriction factors Fv1 and TRIM5α provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5α-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5α for virus before reverse transcription. In human cells expressing Fv1
b
, N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5α by Fv1. Our findings suggest that, like TRIM5α, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5α when expressed in human cells.
Mouse-Adapted Scrapie Infection of SN56 Cells: Greater Efficiency with Microsome-Associated versus Purified PrP-res
Authors: Baron Gerald S. , Magalhães Ana C. , Prado Marco A. M. , Caughey Byron
Published: February 2006

The process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.
Hepatitis B Virus Reverse Transcriptase and ε RNA Sequences Required for Specific Interaction In Vitro
Authors: Hu Jianming , Boyer Morgan
Published: February 2006

Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ε) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific ε binding, we have defined the protein and RNA sequences required for specific HBV RT-ε interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for ε binding. With respect to the ε RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of ε, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-ε interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-ε interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some ε sequences by the RT may be required specifically for viral DNA synthesis.
Comparison of the Replication of Influenza A Viruses in Chinese Ring-Necked Pheasants and Chukar Partridges
Authors: Humberd Jennifer , Guan Yi , Webster Robert G.
Published: February 2006

We investigated the replication and transmission of avian influenza A viruses in two species thought to be intermediate hosts in the spread of influenza A viruses in live poultry markets: Chinese ring-necked pheasants and chukar partridges. All 15 hemagglutinin subtypes replicated in pheasants, and most subtypes transmitted to naïve contact pheasants, primarily via the fecal-oral route. Many viruses were shed from the gastrointestinal tract of experimentally inoculated pheasants for 14 days or longer. Virus was isolated from the cloacal swabs of one contact pheasant for an unprecedented 45 days. Chukar partridges were less susceptible to infection with avian influenza viruses. The viruses that replicated in chukar partridges were isolated for 7 days after experimental inoculation, predominantly from the respiratory tract. We detected high neutralizing antibody titers with correspondingly low levels of serum hemagglutination inhibition antibody titers in pheasants and chukar partridges when chicken red blood cells were used in serological analyses. When horse erythrocytes were used, antibody titers were comparable to those obtained by using the neutralization assay. More importantly, the results suggested that pheasants can serve as a reservoir of influenza virus. Because of their continuous asymptomatic infection and longer stay in the markets, pheasants are ideal “carriers” of influenza A viruses. Their continued presence in live markets contributes to the perpetuation and genetic interaction of influenza viruses there. On the basis of our findings, it does not make good sense to ban quail but not pheasants from the live markets.
Proteomics Analysis of the Tombusvirus Replicase: Hsp70 Molecular Chaperone Is Associated with the Replicase and Enhances Viral RNA Replication
Authors: Serva Saulius , Nagy Peter D.
Published: February 2006

Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown ∼35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (
ssa1 ssa2
) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned
ssa1 ssa2
mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.
RNA Secondary Structure in the Coding Region of Dengue Virus Type 2 Directs Translation Start Codon Selection and Is Required for Viral Replication
Authors: Clyde Karen , Harris Eva
Published: February 2006

Dengue virus
is a positive-strand RNA virus and a member of the genus
Flavivirus
, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region
cis
element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.
Bovine Coronavirus 5′-Proximal Genomic Acceptor Hotspot for Discontinuous Transcription Is 65 Nucleotides Wide
Authors: Wu Hung-Yi , Ozdarendeli Aykut , Brian David A.
Published: February 2006

Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5′-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of bovine coronavirus) near the 3′ end of the 5′-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable bovine coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (∼2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5′-proximal “hotspot.” Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5′-proximal multifunctional structure.
Gradual Elimination of Retroviruses in YBR/Ei Mice
Authors: MacDearmid Cameron C. , Case Laure K. , Starling Christa L. , Golovkina Tatyana V.
Published: February 2006

Mouse mammary tumor virus (MMTV), a well-characterized retrovirus that causes mammary tumors in susceptible mice, is commonly used to investigate virus-host interactions. We have shown that YBR/Ei mice demonstrate a novel, dominant mechanism of resistance to MMTV infection and MMTV-induced mammary tumors. MMTV can both establish infection in YBR/Ei mice and be transmitted by YBR/Ei mice as an infectious virus. However, virus production is severely attenuated, resulting in gradual clearance of infection in successive generations. Our transfer experiments showed that T cells generated in MMTV-infected resistant mice were required to restrict MMTV replication in susceptible mice. These results emphasize the importance of inducing T-cell responses for effective protection against retroviral infections.
Heptad Repeat 2 in Herpes Simplex Virus 1 gH Interacts with Heptad Repeat 1 and Is Critical for Virus Entry and Fusion
Authors: Gianni Tatiana , Piccoli Angela , Bertucci Carlo , Campadelli-Fiume Gabriella
Published: February 2006

Herpes simplex virus 1 (HSV-1) entry into cells and cell-cell fusion mediated by HSV-1 glycoproteins require four glycoproteins, gD, gB, gH, gL. Of these, gH is the only one that so far exhibits structural-functional features typical of viral fusion glycoproteins, i.e., a candidate fusion peptide and, downstream of it, a heptad repeat (HR) segment able to form a coiled coil, named HR-1. Here, we show that gH carries a functional HR-2 capable of physical interaction with HR-1. Specifically, mutational analysis of gH aimed at increasing or decreasing the ability of HR-2 to form a coiled coil resulted in an increase or decrease of fusion activity, respectively. HSV infection was modified accordingly. A mimetic peptide with the HR-2 sequence inhibited HSV-1 infection in a specific and dose-dependent manner. Circular dichroism spectroscopy showed that both HR-2 and HR-1 mimetic peptides adopt mainly random conformation in aqueous solution, while a decrease in peptide environmental polarity determines a conformational change, with a significant increase of the α-helical conformation content, in particular, for the HR-1 peptide. Furthermore, HR-1 and HR-2 mimetic peptides formed a stable complex, as revealed in nondenaturing electrophoresis and by circular dichroism. The mixture of HR-1 and HR-2 peptides reversed the inhibition of HSV infection exerted by the single peptides. Complex formation between HR-1 and HR-2 was independent of the presence of adjacent gH sequences and of additional glycoproteins involved in entry and fusion. Altogether, HR-2 adds to the features typical of class 1 fusion glycoproteins exhibited by HSV-1 gH.
Recognition of Vaccinia Virus-Infected Cells by Human Natural Killer Cells Depends on Natural Cytotoxicity Receptors
Authors: Chisholm Susan E. , Reyburn Hugh T.
Published: February 2006

Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.
Transcriptional Origin of Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs
Authors: Cai Xuezhong , Cullen Bryan R.
Published: February 2006

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.
Latency-Associated Nuclear Antigen (LANA) of Kaposi's Sarcoma-Associated Herpesvirus Interacts with Origin Recognition Complexes at the LANA Binding Sequence within the Terminal Repeats
Authors: Verma Subhash C. , Choudhuri Tathagata , Kaul Rajeev , Robertson Erle S.
Published: February 2006

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the
cis-
acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G
1
/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.
Inhibition of the ATM/p53 Signal Transduction Pathway by Kaposi's Sarcoma-Associated Herpesvirus Interferon Regulatory Factor 1
Authors: Shin Young C. , Nakamura Hiroyuki , Liang Xiaozhen , Feng Pinghui , Chang Heesoon , Kowalik Timothy F. , Jung Jae U.
Published: February 2006

Infected cells recognize viral replication as a DNA damage stress and elicit the ataxia telangiectasia-mutated (ATM)/p53-mediated DNA damage response signal transduction pathway as part of the host surveillance mechanisms, which ultimately induces the irreversible cell cycle arrest and apoptosis. Viruses have evolved a variety of mechanisms to counteract this host intracellular innate immunity. Kaposi's sarcoma-associated herpesvirus (KSHV) viral interferon regulatory factor 1 (vIRF1) interacts with the cellular p53 tumor suppressor through its central DNA binding domain, and this interaction inhibits transcriptional activation of p53. Here, we further demonstrate that KSHV vIRF1 downregulates the total p53 protein level by facilitating its proteasome-mediated degradation. Detailed biochemical study showed that vIRF1 interacted with cellular ATM kinase through its carboxyl-terminal transactivation domain and that this interaction blocked the activation of ATM kinase activity induced by DNA damage stress. As a consequence, vIRF1 expression greatly reduced the level of serine 15 phosphorylation of p53, resulting in an increase of p53 ubiquitination and thereby a decrease of its protein stability. These results indicate that KSHV vIRF1 comprehensively compromises an ATM/p53-mediated DNA damage response checkpoint by targeting both upstream ATM kinase and downstream p53 tumor suppressor, which might circumvent host growth surveillance and facilitate viral replication in infected cells.
A Single Intranasal Inoculation with a Paramyxovirus-Vectored Vaccine Protects Guinea Pigs against a Lethal-Dose Ebola Virus Challenge
Authors: Bukreyev Alexander , Yang Lijuan , Zaki Sherif R. , Shieh Wun-Ju , Rollin Pierre E. , Murphy Brian R. , Collins Peter L. , Sanchez Anthony
Published: February 2006

To determine whether intranasal inoculation with a paramyxovirus-vectored vaccine can induce protective immunity against Ebola virus (EV), recombinant human parainfluenza virus type 3 (HPIV3) was modified to express either the EV structural glycoprotein (GP) by itself (HPIV3/EboGP) or together with the EV nucleoprotein (NP) (HPIV3/EboGP-NP). Expression of EV GP by these recombinant viruses resulted in its efficient incorporation into virus particles and increased cytopathic effect in Vero cells. HPIV3/EboGP was 100-fold more efficiently neutralized by antibodies to EV than by antibodies to HPIV3. Guinea pigs infected with a single intranasal inoculation of 10
5.3
PFU of HPIV3/EboGP or HPIV3/EboGP-NP showed no apparent signs of disease yet developed a strong humoral response specific to the EV proteins. When these animals were challenged with an intraperitoneal injection of 10
3
PFU of EV, there were no outward signs of disease, no viremia or detectable EV antigen in the blood, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died or developed severe EV disease following challenge. The highly effective immunity achieved with a single vaccine dose suggests that intranasal immunization with live vectored vaccines based on recombinant respiratory viruses may be an advantageous approach to inducing protective responses against severe systemic infections, such as those caused by hemorrhagic fever agents.
Intrahepatic Hepatitis C Virus Replication Correlates with Chronic Hepatitis C Disease Severity In Vivo
Authors: Pal Sampa , Shuhart Margaret C. , Thomassen Lisa , Emerson Scott S. , Su Tao , Feuerborn Nathan , Kae John , Gretch David R.
Published: February 2006

The role of viral factors in the pathogenesis of chronic hepatitis C is unknown. The objective of the present study was to characterize markers of hepatitis C virus (HCV) infection and replication in liver biopsy specimens obtained from 65 genotype 1-infected subjects, including 31 who were coinfected with human immunodeficiency virus (HIV), and to analyze associations between intrahepatic viral markers and hepatitis C disease severity. The percentages of liver cells harboring HCV genomes (%G) and replicative-intermediate RNAs (%RI) were evaluated using strand-specific in situ hybridization, while HCV core and NS3 antigens were assessed by immunocytochemistry. HIV-positive and HIV-negative subjects had similar mean grades and stages of liver disease and had similar indices of HCV infection and replication in liver, even though coinfected subjects had significantly shorter mean disease duration (P= 0.0003). Multivariate analysis showed that %G was not associated with grade or stage of liver disease (P= 0.5 and 0.4, respectively), while %RI was strongly associated with liver inflammation (P< 0.001), liver fibrosis (P< 0.001), and serum alanine aminotransferase levels (P= 0.01). NS3 antigen (but not core) was more frequently detected in HCV RI-positive versus RI-negative specimens (P= 0.028). These findings demonstrate a link between HCV proliferation and hepatitis C disease severity and suggest similar pathogenic mechanisms in HIV-positive and HIV-negative individuals.
YRKL Sequence of Influenza Virus M1 Functions as the L Domain Motif and Interacts with VPS28 and Cdc42
Authors: Hui Eric Ka-Wai , Barman Subrata , Tang Dominic Ho-Ping , France Bryan , Nayak Debi P.
Published: February 2006

Earlier studies have shown that the C-terminal half of helix 6 (H6) of the influenza A virus matrix protein (M1) containing the YRKL sequence is involved in virus budding (E. K.-W. Hui, S. Barman, T. Y. Yang, and D. P. Nayak, J. Virol.
77:
7078-7092, 2003). In this report, we show that the YRKL sequence is the L domain motif of influenza virus. Like other L domains, YRKL can be inserted at different locations on the mutant M1 protein and can restore virus budding in a position-independent manner. Although YRKL is a part of the nuclear localization signal (NLS), the function of YRKL was independent of the NLS activity and the NLS function of M1 was not required for influenza virus replication. Some mutations in YRKL and the adjacent region caused a reduction in the virus titer by blocking virus release, and some affected virus morphology, producing elongated particles. Coimmunoprecipitation and Western blotting analyses showed that VPS28, a component of the ESCRT-I complex, and Cdc42, a member of the Rho family GTP-binding proteins, interacted with the M1 protein via the YRKL motif. In addition, depletion of VPS28 and Cdc42 by small interfering RNA resulted in reduction of influenza virus production. Moreover, overexpression of dominant-negative Cdc42 inhibited influenza virus replication, whereas a constitutively active Cdc42 mutant enhanced virus production in infected cells. These results indicated that VPS28, a component of ESCRT-I, and Cdc42, a small G protein, are associated with the M1 protein and involved in the influenza virus life cycle.