bluuebunny/crossref_metadata_2025 huggingface dataset:
Developing Postoperative Pain Management: Utilising the Promoting Action on Research Implementation in Health Services (PARIHS) Framework
Authors: Brown Donna , McCormack Brendan
Published: August 2005

Background: The development of acute pain services (APS), education programmes, and protocols assumed an expectation that health care professionals would integrate evidence into everyday practice. However, research evidence into pain management would suggest that this is not the case.

 Approach: Through a review of the literature, the authors aim to (1) explore the factors that have a significant influence on getting evidence into practice (using the PARIHS model as a guide) and (2) examine the relevance of these factors to postoperative pain practices.

 Findings: The need to assist clinicians with developing a greater awareness of the competing and complex influences that surround pain management practices is outlined.

 Recommendations: There is a necessity to adopt a systematic, rigorous, and multidimensional approach to pain management issues, utilising the PARIHS framework as a guide, to improve pain practices.

Worldviews on Evidence‐Based Nursing 2005; 2(3):131‐141
Performing Subcutaneous Injections: A Literature Review
Authors: Annersten Magdalena , Willman Ania
Published: August 2005

Background:Persons using daily subcutaneous injections to administer medicine perform them in different ways and thereby increase the risk of skin complications related to the injection. It is often part of nurses' role to administer medicine and educate the patient in injection technique. Course literature in nursing education, commercial patient education pamphlets, and instructional leaflets do not give consistent advice regarding subcutaneous injection technique.

Aim:The aim of this review was to identify the scientific foundation for the technical performance of subcutaneous injections. The question to be answered was: How should a subcutaneous injection be administered to achieve the right dose in the right place with minimum complications?

Method:The review included a search in three databases, a screening process at abstract level, followed by a quality assessment of included articles. The quality assessment was done independently by two people and followed specific protocols.

Result:A total of 38 articles were assessed for quality and covered information on dose, location, and complications of subcutaneous injection. The assessed studies are heterogeneous in design and describe different aspects of the subcutaneous injection technique. Therefore, the scientific foundation for technical performance is weak. However, several studies indicate that the amount of subcutaneous fat and appropriate needle length are of high importance for the drug to reach the target tissue.

Conclusion:More research regarding effective subcutaneous injection technique needs to be done.

Worldviews on Evidence‐Based Nursing 2005; 2(3):122‐130
Women’s Sexual Concerns: Data Analysis from a Help–Line
Authors: Papaharitou Stamatis , Nakopoulou Evangelia , Kirana Paraskevi , Iraklidou Maria , Athanasiadis Loukas , Hatzichristou Dimitrios
Published: August 2005

Purpose

To report female sexual problems and concerns, as presented by women calling a help–line, and to evaluate women’s help–seeking behavior regarding sexual matters.

Materials and Methods

The study included all telephone calls from women who called for sexual concerns to a help–line dedicated to sexual problems during a 5–year period. During the call, the counselor addresses demographic characteristics of the caller, the sexual problem reported, their sexual function, any previous doctor contacts, coexisting physical and mental health problems, couple’s relationship, and lifestyle factors that may influence sexual function. Data processing employed descriptive statistics and logistic regression analysis in order to detect possible associations between categorical variables.

Results

Of a total of 3,523 calls made by women, 2,287 full forms were analyzed, reflecting a response rate of 64.9%. Most women (46.6%) called for problems encountered by their partners, 45.1% called for their own sexual problems, while 5.9% were calling for their children. Only 34.3% of them had already consulted a doctor. The most frequently reported difficulties were achieving orgasm (25.6%), reduced sexual desire (16.9%), and pain during intercourse (6.1%). Women in the 40–49 age group had the higher odds ratios for the sexual problems reported (reduced sexual desire: odds ratio [OR] 5.0; difficulties achieving orgasm: OR 6.3; pain during intercourse: OR 5.8). Both married and single women had high risk of experiencing low levels of sexual desire (40% and 30%, respectively).

Conclusions

Women’s sexual concerns are not devoted to their sexual problems, but also their partner’s and children’s problems. Most frequently reported sexual problems are difficulties in reaching orgasm and reduced sexual desire. However, women are reluctant to seek medical advice on their sexual concerns. There is a need for general practitioners and family doctors to become aware of the possibility of a sexual problem and to be trained on how to manage this at a primary care level.
Sexual Experience of Female Partners of Men with Erectile Dysfunction: The Female Experience of Men’s Attitudes to Life Events and Sexuality (FEMALES) Study
Authors: Fisher William A. , Rosen Raymond C. , Eardley Ian , Sand Michael , Goldstein Irwin
Published: August 2005

Introduction

Much research has explored the experience of erectile dysfunction (ED) among men with ED, but far less attention has been paid to the perceptions and sexual experiences of the female partners of men with ED.

Aim

The objective of this study was to characterize the attitudes, beliefs, and sexual experience of female partners of men with erectile difficulties.

Methods

Female partners of men with ED who had participated in the Men’s Attitudes to Life Events and Sexuality (MALES) study were recruited for this research via mail or Internet, after their male partners consented to this contact. Female partners of men with ED (N=293) responded to questionnaire measures assessing their frequency of sexual activity and the nature of their sexual experience, both before and after the development of their partner’s ED, and in relation to their partner’s use of phosphodiesterase type 5 (PDE5) inhibitors.

Results

Women reported engaging in sexual activity significantly less frequently after their partner developed ED in comparison with before (P<0.001). Moreover, significantly fewer women experienced sexual desire, arousal, or orgasm “almost always” or “most times,” and significantly fewer women reported satisfaction with their sexual relationship after their partner developed ED, compared with before (P<0.001). Decreases in female sexual satisfaction and frequency of orgasm were significantly related to the male partner’s self–reported severity of ED (P<0.01). The proportion of women who experienced sexual desire, arousal, and orgasm “almost always” or “most times” was significantly higher in the group whose partners were currently using a PDE5 inhibitor (P<0.05).

Conclusion

Erectile dysfunction has significant adverse effects on the female partner’s sexual experience. Women with partners who were currently using PDE5 inhibitors had a more satisfying sexual experience than those whose partners did not use a PDE5 inhibitor.
Lowland Canterbury landscapes in the making
Authors: Pawson Eric , Holland Peter
Published: August 2005

Abstract:  This article explores environmental imaginaries of colonization in lowland Canterbury. In 1844 Edward Shortland observed that his Māori companions had an exceptionally detailed geographical knowledge of the area and its resources, yet a few years later European settlers were viewing it as an empty stage on which to envision newly‐constructed landscapes. The terrain was contested, but colonial ‘improvement’, through the creation of spaces of modernity, took no cognizance of this. The legacies of that transformation are a simplified, orderly landscape, and fractured but persistent memories of indigenous ecosystems that are now being revived.
Embracing postcolonial geographies: Contributions by Dame Evelyn Stokes to the development of postcolonial geography in New Zealand
Authors: D’Hauteserre Anne‐Marie
Published: August 2005

Abstract: This paper acknowledges Dame Evelyn Stokes’ introduction of postcolonial critical analysis of Māori/Pākehā relationships before postcolonialism was established as a field of theoretical enquiry. The paper reviews research I have done on some of the issues faced by the Mashantucket Pequots in the wider context of the contribution Dame Evelyn Stokes was making to similar debates in New Zealand. She emphasized that Māori research and knowledge provide a different world view and that this world view needs to be taken into account in any geographical study of New Zealand. Only in this way will it be possible to avoid the continued marginalization of Māoris from nation building in their own country.
Using resource management plans to protect organic production in New Zealand
Authors: Wallace Pip
Published: August 2005

Abstract:  The focus of this article is on examining how resource management plans prepared under the Resource Management Act 1991 can be used to control the effects of activities for the purpose of preserving the integrity of an organic farm. The article examines effects that can potentially threaten an organic farm and identifies methods that could be employed in a District or Regional Plan to avoid or mitigate those effects. It concludes that the use of resource management plans represents a substantial opportunity to secure the position of the organic farm in New Zealand.
GIS goes nano: Vegetation studies in Victoria Land, Antarctica
Authors: Brabyn Lars , Green Allan , Beard Catherine , Seppelt Rod
Published: August 2005

Abstract:  Vegetation in Antarctica consists mainly of algae, moss and lichen and is interesting to research because of the isolation and extreme growing conditions. An understanding of this vegetation is important for both the management of tourism in Antarctica, and because it provides a potential barometer of global climate and environmental change. This paper demonstrates two applications of GIS to mapping vegetation in Victoria Land, Antarctica. The first application computes the changes that have occurred to the vegetation within a 120 m by 28 m plot between 1962 and 2004. The second application maps and analyses the growth of a lichen specimen (Buellia frigida) during the same period. These applications demonstrate that GIS is a multiscale technology that can be used to detect detailed change in vegetation growth.
Key Golgi Factors for Structural and Functional Maturation of Bunyamwera Virus
Authors: Novoa Reyes R. , Calderita Gloria , Cabezas Pilar , Elliott Richard M. , Risco Cristina
Published: August 2005

Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a
trans
-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-β-
N
-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the
trans
-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.
High-Level Expression of Single-Chain Fv-Fc Fusion Protein in Serum and Egg White of Genetically Manipulated Chickens by Using a Retroviral Vector
Authors: Kamihira Masamichi , Ono Ken-ichiro , Esaka Kazuhisa , Nishijima Ken-ichi , Kigaku Ryoko , Komatsu Hiroyuki , Yamashita Takashi , Kyogoku Kenji , Iijima Shinji
Published: August 2005

We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.
Human Endogenous Retrovirus Expression Profiles in Samples from Brains of Patients with Schizophrenia and Bipolar Disorders
Authors: Frank Oliver , Giehl Michelle , Zheng Chun , Hehlmann Rüdiger , Leib-Mösch Christine , Seifarth Wolfgang
Published: August 2005

The detection and identification of retroviral transcripts in brain samples, cerebrospinal fluid, and plasma of individuals with recent-onset schizophrenia and schizoaffective disorders suggest that activation or upregulation of distinct human endogenous retroviruses (HERVs) may play a role in the etiopathogenesis of neuropsychiatric diseases. To test this hypothesis, we performed a comprehensive microarray-based analysis of HERV transcriptional activity in human brains. We investigated 50 representative members of 20 HERV families in a total of 215 brain samples derived from individuals with schizophrenia or bipolar disorders and matched controls. A characteristic brain-specific retroviral activity profile was found that consists of members of the class I families HERV-E, HERV-F, and ERV9 and members of HERV-K taxa. In addition to these constitutively expressed HERVs, a number of differentially active HERV elements were identified in all brain samples independent of the disease pattern that may reflect differences in the genetic background of the tested individuals. Only a subgroup of the HML-2 family (HERV-K10) was significantly overrepresented in both bipolar-disorder- and schizophrenia-associated samples compared to healthy brains, suggesting a potential association with disease. Real-time PCR analysis of HERVenvtranscripts with coding capacity potentially involved in neuroinflammatory conditions revealed thatenvexpression of HERV-W, HERV-FRD, and HML-2 remains unaffected regardless of the clinical picture. Our data suggest that HERV transcription in brains is weakly correlated with schizophrenia and related diseases but may be influenced by the individual genetic background, brain-infiltrating immune cells, or medical treatment.
Identification of Domains in
Autographa californica
Multiple Nucleopolyhedrovirus Late Expression Factor 3 Required for Nuclear Transport of P143
Authors: Chen Zhilin , Carstens Eric B.
Published: August 2005

Autographa californica multiple nucleopolyhedrovirus
(AcMNPV) late expression factor 3 (LEF-3) is an essential protein for DNA replication in transient assays. P143, a large DNA-binding protein with DNA-unwinding activity, is also essential for viral DNA replication in vivo. Both LEF-3 and P143 are found in the nucleus of AcMNPV-infected cells, but only LEF-3 localizes to the nucleus when expressed in transfected cells on its own from a plasmid expression vector. P143 requires LEF-3 as a transporter to enter the nucleus. To investigate the possibility that LEF-3 carries a nuclear localization signal domain, we constructed a series of LEF-3 deletion mutants and examined the intracellular localization of the products in plasmid-transfected cells. We discovered that the N-terminal 56 amino acid residues of LEF-3 were sufficient for nuclear localization and that this domain, when fused with either the green fluorescent protein reporter gene or P143, was able to direct these proteins to the nucleus. Transient DNA replication assays demonstrated that fusing the LEF-3 nuclear localization signal domain to P143 did not alter the function of P143 in supporting DNA replication but was not sufficient to substitute for whole LEF-3. These data show that although one role for LEF-3 during virus infection is to transport P143 to the nucleus, LEF-3 performs other essential replication functions once inside the nucleus.
Mutations of Epstein-Barr Virus gH That Are Differentially Able To Support Fusion with B Cells or Epithelial Cells
Authors: Wu Liguo , Borza Corina M. , Hutt-Fletcher Lindsey M.
Published: August 2005

The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.
Structural Determinants of Tissue Tropism and In Vivo Pathogenicity for the Parvovirus Minute Virus of Mice
Authors: Kontou Maria , Govindasamy Lakshmanan , Nam Hyun-Joo , Bryant Nathan , Llamas-Saiz Antonio L. , Foces-Foces Concepción , Hernando Eva , Rubio Mari-Paz , McKenna Robert , Almendral José M. , Agbandje-McKenna Mavis
Published: August 2005

Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp
b
) and native wild-type (wt) empty capsids (MVMp
e
), determined and refined to 3.25 and 3.75 Å resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 Å resolution in this study. A comparison of the MVMp
b
and MVMp
e
capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the “shoulder” of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.
The Cotton Rat (
Sigmodon hispidus
) Is a Permissive Small Animal Model of Human Metapneumovirus Infection, Pathogenesis, and Protective Immunity
Authors: Williams John V. , Tollefson Sharon J. , Johnson Joyce E. , Crowe James E.
Published: August 2005

Human metapneumovirus (hMPV) is a newly described paramyxovirus that is an important cause of acute respiratory tract disease. We undertook to develop a small animal model of hMPV infection, pathogenesis, and protection. Hamsters, guinea pigs, cotton rats, and nine inbred strains of mice were inoculated intranasally with hMPV. The animals were sacrificed, and nasal and lung tissue virus yields were determined by plaque titration. None of the animals exhibited respiratory symptoms. The quantity of virus present in the nasal tissue ranged from 4.6 × 10
2
PFU/gram tissue (C3H mice) to greater than 10
5
PFU/gram (hamster). The amount of virus in the lungs was considerably less than in nasal tissue in each species tested, ranging from undetectable (<5 PFU/g; guinea pigs) to 1.8 × 10
5
PFU/gram (cotton rat). The peak virus titer in cotton rat lungs occurred on day 4 postinfection. hMPV-infected cotton rat lungs examined on day 4 postinfection exhibited histopathological changes consisting of peribronchial inflammatory infiltrates. Immunohistochemical staining detected virus only at the luminal surfaces of respiratory epithelial cells throughout the respiratory tract. hMPV-infected cotton rats mounted virus-neutralizing antibody responses and were partially protected against virus shedding and lung pathology on subsequent rechallenge with hMPV. Viral antigen was undetectable in the lungs on challenge of previously infected animals. This study demonstrates that the cotton rat is a permissive small animal model of hMPV infection that exhibits lung histopathology associated with infection and that primary infection protected animals against subsequent infection. This model will allow further in vivo studies of hMPV pathogenesis and evaluation of vaccine candidates.
Hepatitis A Virus Suppresses RIG-I-Mediated IRF-3 Activation To Block Induction of Beta Interferon
Authors: Fensterl Volker , Grotheer Dajana , Berk Iris , Schlemminger Stefanie , Vallbracht Angelika , Dotzauer Andreas
Published: August 2005

Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of double-stranded RNA (dsRNA)-induced beta interferon (IFN-β) gene expression. In this report, we show that this is due to an interaction of HAV with the intracellular dsRNA-induced retinoic acid-inducible gene I (RIG-I)-mediated signaling pathway upstream of the kinases responsible for interferon regulatory factor 3 (IRF-3) phosphorylation (TBK1 and IKKε). In consequence, IRF-3 is not activated for nuclear translocation and gene induction. In addition, we found that HAV reduces TRIF (TIR domain-containing adaptor inducing IFN-β)-mediated IRF-3 activation, which is part of the Toll-like receptor 3 signaling pathway. As IRF-3 is necessary for IFN-β transcription, inhibition of this factor results in efficient suppression of IFN-β synthesis. This ability of HAV seems to be of considerable importance for HAV replication, as HAV is not resistant to IFN-β, and it may allow the virus to establish infection and preserve the sites of virus production in later stages of the infection.
Human Immunodeficiency Virus Type 1 Vpr Induces the Degradation of the UNG and SMUG Uracil-DNA Glycosylases
Authors: Schröfelbauer Bärbel , Yu Qin , Zeitlin Samantha G. , Landau Nathaniel R.
Published: August 2005

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr has previously been shown to bind to the cellular uracil DNA glycosylase UNG. We show here that the binding of Vpr to UNG and to the related enzyme SMUG induces their proteasomal degradation. UNG and SMUG were found to be encapsidated in Δ
vpr
HIV-1 virions but were significantly less abundant in
vpr

+

virions. Δ
vpr
virions contained readily detectable uracil-DNA glycosylase enzymatic activity, while the activity was reduced to undetectable levels in
vpr

+

virions. Consistent with proteasomal degradation, complexes that contained Vpr and the E3 ubiquitin ligase components Cul1 and Cul4 were detected in cell lysates. We hypothesized that the interaction of Vpr might be a means for the virus to reduce the frequency of abasic sites in viral reverse transcripts at uracil residues caused by APOBEC3-catalyzed deamination of cytosine residues. Although APOBEC3 is largely neutralized by the Vif accessory protein, residual enzyme could remain in virions that would generate uracils. In support of this, Δ
vif vpr

+

HIV-1 produced in the presence of limited amounts of APOBEC3G was significantly more infectious than Δ
vif
Δ
vpr
virus. In Addition,
vpr
+
HIV-1 replicated more efficiently than
vpr
−
virus in cells that expressed limited amounts of APOBEC3G. The findings highlight the importance of cytidine deamination in the virus replication cycle and present a novel function for Vpr.
The Product of the Vaccinia Virus L5R Gene Is a Fourth Membrane Protein Encoded by All Poxviruses That Is Required for Cell Entry and Cell-Cell Fusion
Authors: Townsley Alan C. , Senkevich Tatiana G. , Moss Bernard
Published: August 2005

The L5R gene of vaccinia virus is conserved among all sequenced members of the
Poxviridae
but has no predicted function or recognized nonpoxvirus homolog. Here we provide the initial characterization of the L5 protein. L5 is expressed following DNA replication with kinetics typical of a viral late protein, contains a single intramolecular disulfide bond formed by the virus-encoded cytoplasmic redox pathway, and is incorporated into intracellular mature virus particles, where it is exposed on the membrane surface. To determine whether L5 is essential for virus replication, we constructed a mutant that synthesizes L5 only in the presence of an inducer. The mutant exhibited a conditional-lethal phenotype, as cell-to-cell virus spread and formation of infectious progeny were dependent on the inducer. Nevertheless, all stages of replication occurred in the absence of inducer and intracellular and extracellular progeny virions appeared morphologically normal. Noninfectious virions lacking L5 could bind to cells, but the cores did not enter the cytoplasm. In addition, virions lacking L5 were unable to mediate low-pH-triggered cell-cell fusion from within or without. The phenotype of the L5R conditional lethal mutant is identical to that of recently described mutants in which expression of the A21, A28, and H2 genes is repressed. Thus, L5 is the fourth component of the poxvirus cell entry/fusion apparatus that is required for entry of both the intracellular and extracellular infectious forms of vaccinia virus.
Multiple Gene Segments Control the Temperature Sensitivity and Attenuation Phenotypes of
ca
B/Ann Arbor/1/66
Authors: Hoffmann Erich , Mahmood Kutubuddin , Chen Zhongying , Yang Chin-Fen , Spaete Joshua , Greenberg Harry B. , Herlocher M. Louise , Jin Hong , Kemble George
Published: August 2005

Cold-adapted (
ca
) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccine's replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses,
ca
B/Ann Arbor/1/66 is temperature sensitive (
ts
) at 37°C and attenuated (
att
) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of
ca
B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of
ca
B/Ann Arbor/1/66, and these changes reverted both the
ts
and the
att
phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of
ts
, whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the
att
phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the
ts
and the
att
phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.
Characterization of an Antisense Transcript Spanning the UL81-82 Locus of Human Cytomegalovirus
Authors: Bego Mariana , Maciejewski J. , Khaiboullina S. , Pari G. , St. Jeor S.
Published: August 2005

In this study we present the characterization of a novel transcript,
UL81-82ast
, UL81-82 antisense transcript, and its protein product. The transcript was initially found in a cDNA library of monocytes from a seropositive donor. mRNA was obtained from monocytes isolated from a healthy donor with a high antibody titer against human cytomegalovirus (HCMV). The mRNAs were cloned into a lambda phage-derived vector to create the cDNA library. Using PCR,
UL81-82ast
was amplified from the library. The library was tested for the presence of numerous HCMV genes. Neither structural genes nor immediate-early genes were found.
UL81-82ast
was detected in five bone marrow samples from healthy antibody-positive donors. This same transcript was also found in in vitro-infected human fibroblasts early after infection but disappears at the same time that UL82 transcription begins. Not only was the transcript amplified using reverse transcription-PCR and sequenced but its protein product (UL82as protein) was detected by both Western blot and immunofluorescence. Phylogenetic studies using UL82as protein were conducted, showing a high degree of conservation in clinical isolates, laboratory strains of HCMV, and even in chimpanzee CMV. The transcript could be involved in the posttranscriptional regulation of the UL82 gene, affecting its mRNA stability or translation. Since the UL82 product, pp71, functions as an immediate-early transactivator, its posttranscriptional control could have some effect over latency reactivation and lytic replication.
The Expression Strategy of Goose Parvovirus Exhibits Features of both the
Dependovirus
and
Parvovirus
Genera
Authors: Qiu Jianming , Cheng Fang , Yoto Yuko , Zádori Zoltán , Pintel David
Published: August 2005

The RNA transcription profile of the goose parvovirus (GPV) was determined, and it is a surprising hybrid of features of the
Parvovirus
and
Dependovirus
genera of the
Parvovirinae
subfamily of the
Parvoviridae
. Similar to the
Dependovirus
adeno-associated virus type 5, RNAs transcribed from the GPV upstream P9 promoter, which encode the viral nonstructural proteins, were polyadenylated at a high efficiency at a polyadenylation site [(pA)p] located within an intron in the center of the genome. Efficient usage of (pA)p required a downstream element that overlaps with the polypyrimidine tract of the A2 3′ splice site of the central intron. An upstream element required for efficient use of (pA)p was also identified. RNAs transcribed from the P42 promoter, presumed to encode the viral capsid proteins, primarily extended through (pA)p and were polyadenylated at a site, (pA)d, located at the right end of the genome and ultimately spliced at a high efficiency. No promoter analogous to the
Dependovirus
P19 promoter was detected; however, similar to minute virus of mice and other members of the
Parvovirus
genus, a significant portion of pre-mRNAs generated from the P9 promoter were additionally spliced within the putative GPV Rep1 coding region and likely encode an additional, smaller, nonstructural protein. Also similar to members of the
Parvovirus
genus, detectable activity of the GPV P42 promoter was highly dependent on transactivation by the GPV Rep1 protein in a manner dependent on binding to a
cis
-element located in the P42 promoter.
Alpha/Beta Interferon Differentially Modulates the Clearance of Cytoplasmic Encapsidated Replication Intermediates and Nuclear Covalently Closed Circular Hepatitis B Virus (HBV) DNA from the Livers of Hepatocyte Nuclear Factor 1α-Null HBV Transgenic Mice
Authors: Anderson Aimee L. , Banks Krista E. , Pontoglio Marco , Yaniv Moshe , McLachlan Alan
Published: August 2005

Treatment with alpha interferon is a standard therapy for patients with chronic hepatitis B virus (HBV) infections. This treatment can reduce virus load and ameliorate disease symptoms. However, in the majority of cases, alpha interferon therapy fails to resolve the chronic HBV infection. The reason alpha interferon therapy is inefficient at resolving chronic HBV infections is assumed to be because it fails to eliminate covalently closed circular (CCC) HBV DNA from the nuclei of infected hepatocytes. In an attempt to address this issue, the stability of HBV CCC DNA in response to alpha/beta interferon induction was examined in HNF1α-null HBV transgenic mice. Alpha/beta interferon induction by polyinosinic-polycytidylic acid [poly(I-C)] treatment efficiently eliminated encapsidated cytoplasmic HBV replication intermediates while only modestly reducing nuclear HBV CCC DNA. These observations indicate that nuclear HBV CCC DNA is more stable than cytoplasmic replication intermediates in response to alpha/beta interferon induction. Consequently it appears that for therapies to resolve chronic HBV infection efficiently, they will have to target the elimination of the most stable HBV replication intermediate, nuclear HBV CCC DNA.
The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element
Authors: François Achille , Guilbaud Mickaël , Awedikian Rafi , Chadeuf Gilliane , Moullier Philippe , Salvetti Anna
Published: August 2005

The p5 promoter region of adeno-associated virus type 2 (AAV-2) is a multifunctional element involved in
rep
gene expression, Rep-dependent replication, and site-specific integration. We initially characterized a 350-bp p5 region by its ability to behave like a
cis
-acting replication element in the presence of Rep proteins and adenoviral factors. The objective of this study was to define the minimal elements within the p5 region required for Rep-dependent replication. Assays performed in transfected cells (in vivo) indicated that the minimal p5 element was composed by a 55-bp sequence (nucleotides 250 to 304 of wild-type AAV-2) containing the TATA box, the Rep binding site, the terminal resolution site present at the transcription initiation site (trs
+1
), and a downstream 17-bp region that could potentially form a hairpin structure localizing the trs
+1
at the top of the loop. Interestingly, the TATA box was absolutely required for in vivo but dispensable for in vitro, i.e., cell-free, replication. We also demonstrated that Rep binding and nicking at the trs
+1
was enhanced in the presence of the cellular TATA binding protein, and that overexpression of this cellular factor increased in vivo replication of the minimal p5 element. Together, these studies identified the minimal replication origin present within the AAV-2 p5 promoter region and demonstrated for the first time the involvement of the TATA box, in
cis
, and of the TATA binding protein, in
trans
, for Rep-dependent replication of this viral element.
Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response
Authors: Marques Joao T. , Rebouillat Dominique , Ramana Chilakamarti V. , Murakami Junko , Hill Jason E. , Gudkov Andrei , Silverman Robert H. , Stark George R. , Williams Bryan R. G.
Published: August 2005

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G
1
arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G
1
arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.
Impact of 2-Bromo-5,6-Dichloro-1-β-
d
-Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation
Authors: McVoy Michael A. , Nixon Daniel E.
Published: August 2005

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-β-
d
-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.
Epstein-Barr Virus Can Establish Infection in the Absence of a Classical Memory B-Cell Population
Authors: Conacher Margaret , Callard Robin , McAulay Karen , Chapel Helen , Webster David , Kumararatne Dinakantha , Chandra Anita , Spickett Gavin , Hopwood Paul A. , Crawford Dorothy H.
Published: August 2005

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persists in the body for life after primary infection. The primary site of EBV persistence is the memory B lymphocyte, but whether the virus initially infects naïve or memory B cells is still disputed. We have analyzed EBV infection in nine cases of X-linked hyper-immunoglobulin M (hyper-IgM) syndrome who, due to a mutation in CD40 ligand gene, do not have a classical, class-switched memory B-cell population (IgD
−
CD27
+
). We found evidence of EBV infection in 67% of cases, which is similar to the infection rate found in the general United Kingdom population (60 to 70% for the relevant age range). We detected EBV DNA in peripheral blood B cells and showed in one case that the infection was restricted to the small population of nonclassical, germinal center-independent memory B cells (IgD
+
CD27
+
). Detection of EBV small RNAs, latent membrane protein 2, and EBV nuclear antigen 3C expression in peripheral blood suggests full latent viral gene expression in this population. Analysis of EBV DNA in serial samples showed variability over time, suggesting cycles of infection and loss. Our results demonstrate that short-term EBV persistence can occur in the absence of a germinal center reaction and a classical memory B-cell population.
Ubiquitylation of Cdk9 by Skp2 Facilitates Optimal Tat Transactivation
Authors: Barboric Matjaz , Zhang Fan , Besenicar Mojca , Plemenitas Ana , Peterlin B. Matija
Published: August 2005

By recruiting the positive transcriptional elongation factor b (P-TEFb) to paused RNA polymerase II, the transactivator Tat stimulates transcriptional elongation of the human immunodeficiency virus type 1 (HIV-1) genome. We found that cyclin-dependent kinase 9 (Cdk9), the catalytic subunit of P-TEFb, is ubiquitylated in vivo. This ubiquitylation depended on the Skp1/Cul1/F-box protein E3 ubiquitin ligase Skp2. Likewise, Tat required Skp2 since its transactivation of the HIV-1 long terminal repeat decreased in primary mouse embryonic fibroblasts, which lacked Skp2. The ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and transactivation response element. Thus, our findings underscore the requirement of ubiquitylation for the coactivator function in regulating HIV-1 transcriptional elongation.
Ternary Complex Formation of Human Immunodeficiency Virus Type 1 Env, CD4, and Chemokine Receptor Captured as an Intermediate of Membrane Fusion
Authors: Mkrtchyan Samvel R. , Markosyan Ruben M. , Eadon Michael T. , Moore John P. , Melikyan Gregory B. , Cohen Fredric S.
Published: August 2005

Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37°C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23°C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37°C from the 23°C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23°C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23°C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23°C was prolonged, more cells quickly fused upon the raising of the temperature to 37°C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form.
Cross-Reactivity of T Lymphocytes Recognizing a Human Cytotoxic T-Lymphocyte Epitope within BK and JC Virus VP1 Polypeptides
Authors: Krymskaya Ludmila , Sharma Madeva C. , Martinez Joy , Haq Wahajul , Huang Eric C. , Limaye Ajit P. , Diamond Don J. , Lacey Simon F.
Published: August 2005

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol.
168:
499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain
127:
1970-1978, 2004; I. J. Koralnik et al., J. Immunol.
168:
499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol.
71:
115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.
Glycosylation, Disulfide Bond Formation, and the Presence of a WSXWS-Like Motif in the Orf Virus GIF Protein Are Critical for Maintaining the Integrity of Binding to Ovine Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2
Authors: McInnes C. J. , Deane D. , Haig D. , Percival A. , Thomson J. , Wood A. R.
Published: August 2005

Orf virus (ORFV), the type species of the familyParapoxviridae, encodes a protein (GIF) that binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). There is no obvious sequence homology between the ORFV protein and any known mammalian GM-CSF- or IL-2-binding proteins. We demonstrate here that many of the biochemical properties of mammalian GM-CSF receptors that are required for efficient binding of GM-CSF are also critical to the GIF protein for binding to ovine GM-CSF (ovGM-CSF). Site-directed mutagenesis of the GIF protein demonstrated, first, the importance of disulfide bonds, and second, that a sequence motif (WDPWV), related to the WSXWS motif of the type 1 cytokine receptor superfamily, was necessary for biological activity. Finally, glycosylation of the GIF protein was also critical for binding to GM-CSF.