bluuebunny/crossref_metadata_2025 huggingface dataset:
Are all mouthguards the same and safe to use? The influence of occlusal supporting mouthguards in decreasing bone distortion and fractures
Authors: Takeda Tomotaka , Ishigami Keiichi , Ogawa Tohru , Nakajima Kazunori , Shibusawa Mami , Shimada Atsushi , Regner Connell Wayne
Published: May 2004

Abstract –  The safety benefits of mouthguards have been demonstrated in many studies, with many authors and sports dentists strongly recommending the wearing of mouthguards. However, wearing a mouthguard with incorrect occlusion might cause a variety of problems. It comes as no surprise that a traumatic blow to the chin, while wearing an insufficient mouthguard lacking anterior contact, can result in severe distortions to the mandibular bone, and bone fractures. The aim of this study was to clarify how ineffective insufficient occlusal supporting mouthguards are and how dangerous they can be to use. Consequently, in this study, occlusal supportive areas were varied and accelerations of head and distortions of the mandible were measured using an artificial skull model and a pendulum impact device. As a result, the distortions of the mandible tended to increase as the supported area decreased. On the contrary, accelerations of the head decreased as the occlusion part decreased. Thus, a lot of impact energy was consumed in the distortion of the mandible; accordingly, it seemed that only a little destructive energy was transferred to the head. From this study, it would seem that wearing a mouthguard, which is insufficient in the occlusion, has the potential of causing a bone fracture of the mandible. Consequently, mouthguards should have proper occlusion.
A dressing history
Authors: Queen Douglas , Orsted Heather , Sanada Hiromi , Sussman Geoff
Published: May 2004

Over the past 30 years as caregivers, clinicians have been exposed to a plethora of new advanced wound dressings. The moist wound care revolution began in the 1970s with the introduction of film and hydrocolloid dressings, and today these are the traditional types of dressings of the advanced dressing categories. Wound‐healing science has progressed significantly over the same period, as a result of intense clinical and scientific research around these product introductions. Today, the clinician understands moist wound healing, occlusion, cost effectiveness, wound bed preparation and MMP activity to name but a few of the many concepts in wound care that have flourished as a result of technology and product advancement. This review article presents a condensed history of dressing development over the past 30 years. However, in addition, such advancement is discussed in respect to its adoption in different parts of the world. The largest single markets of the world are generally the United States of America and Europe; as such, the development of both practice and technology generally begins there. Much has been written about these markets in previous review articles. For the purposes of this review, the development of wound care and the maturing of practice is discussed in respect to Canada, Japan and Australia representing smaller geographical areas where the development has been more recent but nonetheless significant.
Cardiff Wound Impact Schedule: the development of a condition‐specific questionnaire to assess health‐related quality of life in patients with chronic wounds of the lower limb
Authors: Price Patricia , Harding Keith
Published: May 2004

The purpose of this study was to develop and validate a questionnaire to measure the impact of chronic wounds (leg ulcers and diabetic foot ulcers) on patient health‐related quality of life (HRQoL) and identify areas of patient concern. The Cardiff Wound Impact Schedule (CWIS) was created following a three‐stage process. Stage 1 included a focus group (n = 10) and a series of semi‐structured interviews (n = 13) to generate items for the questionnaire. Stage 2 involved piloting the 28‐item questionnaire on 124 patients (64·5% with leg ulceration and 35·5% with diabetic foot ulceration): data from this stage were analysed using factor analysis. The reliability, validity and reproducibility of the resulting scale were assessed in Stage 3, a 3‐month follow‐up study of 135 patients in which patients completed the CWIS and the SF‐36. Factor analysis identified three domains of HRQoL: physical symptoms and daily living, social life and well‐being. There were no significant differences in scores across the wound types. Internal consistency was good (α = 0·77–0·96) as was reproducibility (P < 0·001). The CWIS was able to discriminate between those with healed ulcers and active ulcers (P < 0·01). Construct validity was demonstrated by strong correlations between related items on CWIS and SF‐36 (P < 0·01–P < 0·0001). The data suggest that CWIS has high internal consistency and the ability to discriminate between health states and good reproducibility. CWIS is a valid tool for studying the impact of chronic wounds of the lower leg on HRQoL. CWIS allows clinicians to identify items of patient concern, which can then be used to negotiate options of care most suited to individual patients.
Wound bed preparation and a brief history of TIME
Authors: Schultz Gregory S , Barillo David J , Mozingo David W , Chin Gloria A
Published: May 2004

Management of chronic wounds has progressed from merely assessing the status of a wound to understanding the underlying molecular and cellular abnormalities that prevent the wound from healing. The concept of wound bed preparation has simultaneously evolved to provide a systematic approach to removing these barriers to natural healing and enhancing the effects of advanced therapies. This brief review of wound bed preparation traces the development of these concepts and explains how to apply systematic wound management using the TIME acronym – tissue (non viable or deficient), infection/inflammation, moisture (imbalance) and edge (non advancing or undermined).
Rheumatoid arthritis‐associated inflammatory leg ulcers: a new treatment for recalcitrant wounds
Authors: Coelho Sunita , Amarelo Marlena , Ryan Siobhan , Reddy Madhuri , Sibbald R Gary
Published: May 2004

Patients with rheumatoid arthritis may develop leg ulcers of varied aetiologies, including venous disease, infection and inflammation (vasculitis or pyoderma gangrenosum). The leg ulcers in rheumatoid arthritis patients may involve several of these aetiological factors and are often difficult to heal. Both the ulcers and the treatments are often painful, and these ulcers may be present for years. A new oxidised regenerated cellulose and collagen dressing has been developed for slow‐to‐heal wounds and may have a role in the management of superficial inflammation that may persist in many of these ulcers, although clinically it is difficult to distinguish this from critical colonisation or frank infection. Venous disease requires compression therapy. Deep compartment infection should be treated with systemic antimicrobials, and inflammatory processes extending beyond the superficial wound base require disease‐specific systemic anti‐inflammatory agents. Four patients with recalcitrant wounds resistant to best practice were treated successfully with this new dressing combined with a strategy to control bacterial burden.
An experiment in rotational motion with linear and quadratic drag
Authors: Thompson B. G. , Smith P. A.
Published: May 2004

We describe a study of velocity-dependent drag in rotational motion that is suitable for an undergraduate laboratory experiment. Using standard teaching-laboratory equipment to obtain the data, we found that a drag force that is linear in the angular speed describes the data very well; however, the model residuals reveal that quadratic drag is also present. When a combined model is used, the residuals are reduced to the level of measurement uncertainties. An investigation of the effect of airflow on the air bearing used in the experiment confirms that there is a turbine effect on the disk and that the drag is dependent on the airflow suspending the disk. The experiment is simple to perform and analyze, yet reveals interesting physical and analytical complexity with deeper investigation.
Classical solutions for a free particle in a confocal elliptic billiard
Authors: Bandres Miguel A. , Gutiérrez-Vega Julio C.
Published: May 2004

The classical dynamics of a free particle constrained to move in an integrable two-dimensional confocal elliptic billiard is investigated. We derive the characteristic equations for periodic orbits, classify the orbits, present the Poincaré maps, give expressions for the lengths of the trajectories, and do a stability analysis of special orbits. We also explore some interesting geometrical constructions for the billiard which can be extended to the confocal elliptic billiard. The latter provides a well-motivated and relatively straightforward example of Hamilton–Jacobi theory, elliptic integrals, and Jacobi elliptic functions in a way that is seldom discussed in the undergraduate curriculum.
Kinematic subtleties in Einstein’s first derivation of the Lorentz transformations
Authors: Martı́nez Alberto A.
Published: May 2004

We analyze Albert Einstein’s derivation of the Lorentz transformations in his paper, “Zur Elektrodynamik bewegter Körper,” originally published in 1905. The analysis clarifies various misunderstandings in the secondary literature and reveals reasons why Einstein’s work entailed interpretive difficulties.
Clouds in planetary atmospheres: A useful application of the Clausius–Clapeyron equation
Authors: Sánchez-Lavega Agustı́n , Pérez-Hoyos Santiago , Hueso Ricardo
Published: May 2004

The Clausius–Clapeyron equation is used to do a comparative study of the properties of the clouds that form in planetary atmospheres. Simple static atmospheric models for various planets, the satellite Titan, and the extrasolar planet HD209458b are used together with the saturation vapor pressure curves of the different kinds of molecules to determine the pressure, density, and scale height of the clouds in each body. This application of the Clausius–Clapeyron equation extends our knowledge of terrestrial water clouds to different exotic clouds present in other planets.
Parametric resonance and nonlinear string vibrations
Authors: Rowland David R.
Published: May 2004

Periodic changes in the tension of a taut string parametrically excite transverse motion in the string when the driving frequency is close to twice the natural frequency of any transverse normal mode of the string. The literature on this phenomenon is synthesized and extended to include the effects of damping as well as nonlinearity. It is shown that it is nonlinearity rather than damping that limits the growth of a resonantly excited mode, although damping is needed for steady-state oscillations to occur. The validity of the usual approximation that the string tension depends only on time and not on space is checked by modeling a string as point masses joined by massless linear springs. It is found that although this approximation is likely to be violated in practice, the violation does not have a significant effect on the results. The source of the disagreement in the literature for the speed of longitudinal waves in a stretched string is identified.
An improved calculation of the mass for the resonant spring pendulum
Authors: Christensen Joseph
Published: May 2004

When the appropriate mass is used to oscillate a spring, the vertical oscillations couple to a pendular swing. Previous calculations of various aspects of this resonance assumed a massless spring as a simple pendulum. This paper improves the estimate of the mass necessary to induce this resonance by describing a massive spring as a physical pendulum and obtains an expression for the mass in terms of the spring constant and various lengths associated with the spring. Several approximations will be considered to simplify the complete expression. Comparisons of the predictive power of these expressions are made for various values of the spring constants. An Appendix discusses the assumption of uniform coil density of a hanging massive spring.
The inertia tensor of a magic cube
Authors: Rogers Adam , Loly Peter
Published: May 2004

Magic cubes are shown to have maximally symmetric inertia tensors if they are interpreted as rigid body mass distributions. This symmetry is due to their semi-magic property where each row, column, and pillar has the same mass sum. The moment of inertia depends only on this property and the number of point masses in each row, column, and pillar. Because magic cubes do not possess detailed cubic symmetry, other scenarios that result in maximally symmetric inertia tensors are discussed.
Context-Dependent Effects of L Domains and Ubiquitination on Viral Budding
Authors: Martin-Serrano Juan , Perez-Caballero David , Bieniasz Paul D.
Published: May 2004

Many enveloped viruses encode late assembly domains, or L domains, that facilitate virion egress. PTAP-type L domains act by recruiting the ESCRT-I (endosomal sorting complex required for transport I) component Tsg101, and YPXL/LXXLF-type L domains recruit AIP-1/ALIX, both of which are class E vacuolar protein sorting (VPS) factors, normally required for the generation of vesicles within endosomes. The binding cofactors for PPXY-type L domains have not been unambiguously resolved but may include Nedd4-like ubiquitin ligases. Largely because they act as autonomous binding sites for host factors, L domains are generally transferable and active in a context-independent manner. Ebola virus matrix protein (EbVP40) contains two overlapping L-domain motifs within the sequence IL
PTAPPEY
MEA. Here, we show that both motifs are required for efficient EbVP40 budding. However, upon transplantation into two different retroviral contexts, the relative contributions of the PTAP and PPEY motifs differ markedly. In a murine leukemia virus carrying the EbVP40 sequence, both motifs contributed to overall L domain activity, and budding proceeded in a partly Tsg101-independent manner. Conversely, when transplanted into the context of human immunodeficiency virus type 1 (HIV-1), EbVP40 L-domain activity was entirely due to a PTAP-Tsg101 interaction. In fact, a number of PPXY-type L domains were inactive in the context of HIV-1. Surprisingly, PTAP and YPXL-type L domains that simulated HIV-1 budding reduced the amount of ubiquitin conjugated to Gag, while inactive PPXY-type L domains increased Gag ubiquitination. These observations suggest that active L domains recruit deubiquitinating enzymes as a consequence of class E VPS factor recruitment. Moreover, context-dependent L-domain function may reflect distinct requirements for host functions during the morphogenesis of different viral particles or the underlying presence of additional, as yet undiscovered L domains.
Influenza B Virus Requires BM2 Protein for Replication
Authors: Hatta Masato , Goto Hideo , Kawaoka Yoshihiro
Published: May 2004

The BM2 protein of influenza B virus functions as an ion channel, which is suggested to be important for virus uncoating in endosomes of virus-infected cells. Because direct support for this function is lacking, whether BM2 plays an essential role in the viral life cycle remains unknown. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutant viruses possessing M segments with the mutated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still expressed BM2. The introduction of multiple stop codons and a one-nucleotide deletion downstream of the stop-start pentanucleotide, in addition to disablement of the BM2 initiation codon, failed to generate viable mutant viruses, but the mutant M segments still expressed proteins that reacted with the BM2 peptide antiserum. To completely abolish BM2 expression, we generated a mutant M gene whose BM2 open reading frame was deleted. Although this mutant was not able to replicate in normal MDCK cells, it did replicate in a cell line that we established which constitutively expresses BM2. Furthermore, a virus possessing the mutant M gene lacking the BM2 open reading frame and a mutant NA gene containing the BM2 open reading frame instead of the NA open reading frame underwent multiple cycles of replication in MDCK cells, with exogenous sialidase used to supplement the deleted viral sialidase activity. These findings demonstrate that the BM2 protein is essential for influenza B virus replication.
Fatal Disseminated Mouse Adenovirus Type 1 Infection in Mice Lacking B Cells or Bruton's Tyrosine Kinase
Authors: Moore Martin L. , McKissic Erin L. , Brown Corrie C. , Wilkinson John E. , Spindler Katherine R.
Published: May 2004

Mouse adenovirus type 1 (MAV-1) infection of B-cell-deficient and Bruton's tyrosine kinase (Btk)-deficient mice resulted in fatal disseminated disease resembling human adenovirus infections in immunocompromised patients. Mice lacking B cells or Btk were highly susceptible to acute MAV-1 infection, in contrast to controls and mice lacking T cells. To our knowledge, this is the first demonstration that mice with an X-linked immunodeficiency phenotype (Btk deficient) are susceptible to virus-induced disease. Mice lacking B cells or Btk on a C57BL/6 background succumbed with encephalomyelitis, hepatitis, and lymphoid necrosis. Mice lacking B cells on a BALB/c background succumbed with enteritis and hepatitis. Survival of acute MAV-1 infection correlated with early T-cell-independent neutralizing antibody and T-cell-independent antiviral immunoglobulin M. Treatment of MAV-1-infected Btk−/−mice 4 to 9 days postinfection with antiserum harvested 6 to 9 days postinfection from MAV-1-infected Btk+/+mice was therapeutic. Our findings implicate a critical role for B-cell function in preventing disseminated MAV-1 infection, particularly production of early T-cell-independent antiviral immunoglobulin M.
Herpes Simplex Virus 1

U
L

31
and

U
L

34
Gene Products Promote the Late Maturation of Viral Replication Compartments to the Nuclear Periphery
Authors: Simpson-Holley Martha , Baines Joel , Roller Richard , Knipe David M.
Published: May 2004

Herpes simplex virus 1 (HSV-1) forms replication compartments (RCs), domains in which viral DNA replication, late-gene transcription, and encapsidation take place, in the host cell nucleus. The formation of these domains leads to compression and marginalization of host cell chromatin, which forms a dense layer surrounding the viral RCs and constitutes a potential barrier to viral nuclear egress or primary envelopment at the inner nuclear membrane. Surrounding the chromatin layer is the nuclear lamina, a further host cell barrier to egress. In this study, we describe an additional phase in RC maturation that involves disruption of the host chromatin and nuclear lamina so that the RC can approach the nuclear envelope. During this phase, the structure of the chromatin layer is altered so that it no longer forms a continuous layer around the RCs but instead is fragmented, forming islands between which RCs extend to reach the nuclear periphery. Coincident with these changes, the nuclear lamina components lamin A/C and lamin-associated protein 2 appear to be redistributed via a mechanism involving the

U
L

31
and

U
L

34
gene products. Viruses in which the

U
L

31
or

U
L

34
gene has been deleted are unable to undergo this phase of chromatin reorganization and lamina alterations and instead form RCs which are bounded by an intact host cell chromatin layer and nuclear lamina. We postulate that these defects in chromatin restructuring and lamina reorganization explain the previously documented growth defects of these mutant viruses.
Parvovirus Infection of Cells by Using Variants of the Feline Transferrin Receptor Altering Clathrin-Mediated Endocytosis, Membrane Domain Localization, and Capsid-Binding Domains
Authors: Hueffer Karsten , Palermo Laura M. , Parrish Colin R.
Published: May 2004

The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.
Nipah Virus V and W Proteins Have a Common STAT1-Binding Domain yet Inhibit STAT1 Activation from the Cytoplasmic and Nuclear Compartments, Respectively
Authors: Shaw Megan L. , García-Sastre Adolfo , Palese Peter , Basler Christopher F.
Published: May 2004

In previous reports it was demonstrated that the Nipah virus V and W proteins have interferon (IFN) antagonist activity due to their ability to block signaling from the IFN-α/β receptor (J. J. Rodriguez, J. P. Parisien, and C. M. Horvath, J. Virol.
76:
11476-11483, 2002; M. S. Park et al., J. Virol.
77:
1501-1511, 2003). The V, W, and P proteins are all encoded by the same viral gene and share an identical 407-amino-acid N-terminal region but have distinct C-terminal sequences. We now show that the P protein also has anti-IFN function, confirming that the common N-terminal domain is responsible for the antagonist activity. Truncation of this N-terminal domain revealed that amino acids 50 to 150 retain the ability to block IFN and to bind STAT1, a key component of the IFN signaling pathway. Subcellular localization studies demonstrate that the V and P proteins are predominantly cytoplasmic whereas the W protein is localized to the nucleus. In all cases, STAT1 colocalizes with the corresponding Nipah virus protein. These interactions are sufficient to inhibit STAT1 activation, as demonstrated by the lack of STAT1 phosphorylation on tyrosine 701 in IFN-stimulated cells expressing P, V, or W. Therefore, despite their common STAT1-binding domain, the Nipah virus V and P proteins act by retaining STAT1 in the cytoplasm while the W protein sequesters STAT1 in the nucleus, creating both a cytoplasmic and a nuclear block for STAT1. We also show that the IFN antagonist activity of the P protein is not as strong as that of V or W, perhaps explaining why Nipah virus has evolved to express these two edited products.
pH-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus Is Mediated by the Spike Glycoprotein and Enhanced by Dendritic Cell Transfer through DC-SIGN
Authors: Yang Zhi-Yong , Huang Yue , Ganesh Lakshmanan , Leung Kwanyee , Kong Wing-Pui , Schwartz Owen , Subbarao Kanta , Nabel Gary J.
Published: May 2004

The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.
Murine Coronavirus Replication Induces Cell Cycle Arrest in G0/G1Phase
Authors: Chen Chun-Jen , Makino Shinji
Published: May 2004

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G0/G1phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G0phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G1and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21Cip1, p27Kip1, and p16INK4adid not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G1cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G1cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G0/G1phase.
A Bunyamwera Virus Minireplicon System in Mosquito Cells
Authors: Kohl Alain , Hart Timothy J. , Noonan Carol , Royall Elizabeth , Roberts Lisa O. , Elliott Richard M.
Published: May 2004

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses.
Bunyamwera virus
(BUN; genus
Orthobunyavirus
, family
Bunyaviridae
) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An
Aedes albopictus
C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing
Renilla
luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.
Longitudinal Analysis of CD8+T Cells Specific for Structural and Nonstructural Hepatitis B Virus Proteins in Patients with Chronic Hepatitis B: Implications for Immunotherapy
Authors: Webster George J. M. , Reignat Stephanie , Brown David , Ogg Graham S. , Jones Louise , Seneviratne Suranjith L. , Williams Roger , Dusheiko Geoffrey , Bertoletti Antonio
Published: May 2004

The cytotoxic T-cell response in chronic hepatitis B virus (HBV) infection has been described as weak and mono- or oligospecific in comparison to the more robust virus-specific T-cell response present in resolved infection. However, chronic hepatitis B is a heterogeneous disease with markedly variable levels of virus replication and liver disease activity. Here we analyzed (both directly ex vivo and after in vitro stimulation) the HBV-specific CD8 T-cell responses against structural and nonstructural HBV proteins longitudinally in patients with different patterns of chronic infections. We found that the profiles of virus-specific CD8+-T-cell responses during chronic infections are highly heterogeneous and influenced more by the level of HBV replication than by the activity of liver disease. An HBV DNA load of <107copies/ml appears to be the threshold below which circulating multispecific HBV-specific CD8+T cells are consistently detected. Furthermore, CD8+T cells with different specificities are differentially regulated during chronic infections. HBV core-specific CD8+T cells are associated with viral control, while CD8+T cells specific for envelope and polymerase epitopes can occasionally be found in the setting of high levels (>107copies) of HBV replication. These findings have implications for the design of immunotherapy for chronic HBV infections.
Styrylquinolines, Integrase Inhibitors Acting Prior to Integration: a New Mechanism of Action for Anti-Integrase Agents
Authors: Bonnenfant Sabine , Thomas Claire Marie , Vita Claudio , Subra Frédéric , Deprez Eric , Zouhiri Fatima , Desmaële Didier , d'Angelo Jean , Mouscadet Jean François , Leh Hervé
Published: May 2004

We have previously shown that styrylquinolines (SQLs) are integrase inhibitors in vitro. They compete with the long terminal repeat substrate for integrase. Here, we describe the cellular mode of action of these molecules. We show that SQLs do not interfere with virus entry. In fact, concentrations of up to 20 times the 50% inhibitory concentration did not inhibit cell-to-cell fusion or affect the interaction between GP120 and CD4 in vitro. Moreover, the pseudotype of the retrovirus envelope did not affect drug activity. Quantitative reverse transcription PCR experiments showed that SQLs do not inhibit the entry of the genomic RNA. In contrast, the treatment of human immunodeficiency virus type 1-infected cells with SQLs reduced the amount of the late cDNA, suggesting for the first time that integrase targeting molecules may affect the accumulation of DNA during reverse transcription. The cellular target of SQLs was confirmed by the appearance of mutations in the integrase gene when viruses were grown in the presence of increasing concentrations of SQLs. Finally, these mutations led to SQL-resistant viruses when introduced into the wild-type sequence. In contrast, SQLs were fully active against reverse transcriptase inhibitor- and diketo acid-resistant viruses, positioning SQLs as a second group of anti-integrase compounds.
Disruption of the Actin Cytoskeleton Can Complement the Ability of Nef To Enhance Human Immunodeficiency Virus Type 1 Infectivity
Authors: Campbell Edward M. , Nunez Rafael , Hope Thomas J.
Published: May 2004

The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.
Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-α(1-3) Galactosyl Epitope
Authors: Magre Saema , Takeuchi Yasuhiro , Langford Gillian , Richards Andrew , Patience Clive , Weiss Robin
Published: May 2004

Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-α(1-3) galactosyl sugar structure (α-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing α-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out α-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which α-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed α-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation.
Microarray Analysis Reveals Characteristic Changes of Host Cell Gene Expression in Response to Attenuated Modified Vaccinia Virus Ankara Infection of Human HeLa Cells
Authors: Guerra Susana , López-Fernández Luis A. , Conde Raquel , Pascual-Montano Alberto , Harshman Keith , Esteban Mariano
Published: May 2004

The potential use of the modified vaccinia virus Ankara (MVA) strain as a live recombinant vector to deliver antigens and elicit protective immune responses against infectious diseases demands a comprehensive understanding of the effect of MVA infection on human host gene expression. We used microarrays containing more than 15,000 human cDNAs to identify gene expression changes in human HeLa cell cultures at 2, 6, and 16 h postinfection. Clustering of the 410 differentially regulated genes identified 11 discrete gene clusters with altered expression patterns after MVA infection. Clusters 1 and 2 (accounting for 16.59% [68 of 410] of the genes) contained 68 transcripts showing a robust induction pattern that was maintained during the course of infection. Changes in cellular gene transcription detected by microarrays after MVA infection were confirmed for selected genes by Northern blot analysis and by real-time reverse transcription-PCR. Upregulated transcripts in clusters 1 and 2 included 20 genes implicated in immune responses, including interleukin 1A (IL-1A), IL-6, IL-7, IL-8, and IL-15 genes. MVA infection also stimulated the expression of NF-κB and components of the NF-κB signal transduction pathway, including p50 and TRAF-interacting protein. A marked increase in the expression of histone family members was also induced during MVA infection. Expression of the Wiskott-Aldrich syndrome family members WAS, WASF1, and the small GTP-binding protein RAC-1, which are involved in actin cytoskeleton reorganization, was enhanced after MVA infection. This study demonstrates that MVA infection triggered the induction of groups of genes, some of which may be involved in host resistance and immune modulation during virus infection.
Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to
l
-Chicoric Acid
Authors: Lee Deborah J. , Robinson W. E.
Published: May 2004

The diketo acids are potent inhibitors of human immunodeficiency virus (HIV) integrase (IN). Mutations in IN, T66I, S153Y, and M154I, as well as T66I-S153Y and T66I-M154I double mutations, confer resistance to diketo acids (D. J. Hazuda et al., Science
287:
646-650, 2000). The effects of these IN mutations on viral replication, enzymatic activity, and susceptibility to other HIV inhibitors are reported herein. By immunofluorescence assay and real-time PCR, all mutant viruses demonstrated a modest delay in viral spread compared to that of reference HIV. These viruses also showed a statistically significant defect in integration without defects in reverse transcription. Recombinant IN containing S153Y, T66I, and M154I-T66I mutations had an approximately twofold decrease in both disintegration and 3′-end-processing-strand transfer activities in vitro. In contrast, IN containing M154I demonstrated a greater than twofold increase in specific activity in both reactions. All mutant HIVs were resistant to
l
-chicoric acid, a dicaffeoyltartaric acid IN inhibitor, both in tissue culture and in biochemical assays, yet remained susceptible to the reverse transcriptase inhibitors zidovudine and nevirapine. Thus, IN mutations conferring resistance to the diketo acids can yield integration defects, attenuated catalysis in vitro, and cross-resistance to
l
-chicoric acid.
Integrase-Specific Enhancement and Suppression of Retroviral DNA Integration by Compacted Chromatin Structure In Vitro
Authors: Taganov Konstantin D. , Cuesta Isabel , Daniel René , Cirillo Lisa Ann , Katz Richard A. , Zaret Kenneth S. , Skalka Anna Marie
Published: May 2004

Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.
The NV Gene of Snakehead Rhabdovirus (SHRV) Is Not Required for Pathogenesis, and a Heterologous Glycoprotein Can Be Incorporated into the SHRV Envelope
Authors: Alonso Marta , Kim Carol H. , Johnson Marc C. , Pressley Meagan , Leong Jo-Ann
Published: May 2004

Snakehead rhabdovirus
(SHRV) affects warm-water fish in Southeast Asia and belongs to the genus
Novirhabdovirus
by virtue of its “nonvirion” (NV) gene. To examine the function of the NV gene, we used a recently developed reverse genetic system to produce a viable recombinant SHRV carrying an NV gene deletion. The recombinant virus was produced at the same rate and same final concentrations as the wild-type virus in cultured fish cells in spite of the NV gene deletion. The role of the NV protein in fish pathogenesis was also investigated. Zebra fish (
Danio rerio
) were infected with the NV deletion mutant or with a recombinant virus containing a copy of the SHRV genome, and similar mortality rates as well as final mortalities were recorded, suggesting no apparent role for the NV protein in fish pathogenesis. Interestingly, the unsuccessful rescue of fully viable recombinants with genomes containing deletions in the G/NV gene junction suggested a role for the gene junction in virus transcription and replication. Finally, we demonstrated that the SHRV glycoprotein can be replaced by the glycoprotein of infectious hematopoietic necrosis virus (IHNV) or by a hybrid protein composed of SHRV and IHNV sequences.
Suppression of Proinflammatory Cytokine Expression by Herpes Simplex Virus Type 1
Authors: Mogensen Trine H. , Melchjorsen Jesper , Malmgaard Lene , Casola Antonella , Paludan Søren R.
Published: May 2004

Viral immune evasion strategies are important for establishment and maintenance of infections. Many viruses are in possession of mechanisms to counteract the antiviral response raised by the infected host. Here we show that a herpes simplex virus type 1 (HSV-1) mutant lacking functional viral protein 16 (VP16)—a tegument protein promoting viral gene expression—induced significantly higher levels of proinflammatory cytokines than wild-type HSV-1. This was observed in several cell lines and primary murine macrophages, as well as in peritoneal cells harvested from mice infected in vivo. The enhanced ability to stimulate cytokine expression in the absence of VP16 was not mediated directly by VP16 but was dependent on the viral immediate-early genes for infected cell protein 4 (ICP4) and ICP27, which are expressed in a VP16-dependent manner during primary HSV infection. The virus appeared to target cellular factors other than interferon-induced double-stranded RNA-activated protein kinase R (PKR), since the virus mutants remained stronger inducers of cytokines in cells stably expressing a dominant-negative mutant form of PKR. Finally, mRNA stability assay revealed a significantly longer half-life for interleukin-6 mRNA after infection with the VP16 mutant than after infection with the wild-type virus. Thus, HSV is able to suppress expression of proinflammatory cytokines by decreasing the stability of mRNAs, thereby potentially impeding the antiviral host response to infection.
Processing of a Pestivirus Protein by a Cellular Protease Specific for Light Chain 3 of Microtubule-Associated Proteins
Authors: Fricke Jens , Voss Christiane , Thumm Michael , Meyers Gregor
Published: May 2004

The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with theSaccharomyces cerevisiaeenzyme. The search for a system for functional testing of these putative LC3-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeastAUT2deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.
Intracellular Targeting Signals Contribute to Localization of Coronavirus Spike Proteins near the Virus Assembly Site
Authors: Lontok Erik , Corse Emily , Machamer Carolyn E.
Published: May 2004

Coronavirus budding at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) requires accumulation of the viral envelope proteins at this point in the secretory pathway. Here we demonstrate that the spike (S) protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a canonical dilysine endoplasmic reticulum retrieval signal (-KKXX-COOH) in its cytoplasmic tail. This signal can retain a chimeric reporter protein in the ERGIC and when mutated allows transport of the full-length S protein as well as the chimera to the plasma membrane. Interestingly, the IBV S protein also contains a tyrosine-based endocytosis signal in its cytoplasmic tail, suggesting that any S protein that escapes the ERGIC will be rapidly endocytosed when it reaches the plasma membrane. We also identified a novel dibasic motif (-KXHXX-COOH) in the cytoplasmic tails of S proteins from group 1 coronaviruses and from the newly identified coronavirus implicated in severe acute respiratory syndrome. This dibasic motif also retained a reporter protein in the ERGIC, similar to the dilysine motif in IBV S. The cytoplasmic tails of S proteins from group 2 coronaviruses lack an intracellular localization signal. The inherent differences in S-protein trafficking could point to interesting variations in pathogenesis of coronaviruses, since increased levels of surface S protein could promote syncytium formation and direct cell-to-cell spread of the infection.