bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluating UK research in speech and language therapy
Authors: Lewison Grant , Carding Paul
Published: September 2003

Background: There has been a steady growth in recent years in British higher‐degree training in speech and language therapy. But what is the standing of UK research in the subject and its component areas which should underpin and inform such training? How can such research be evaluated? Aims: The intention was to compare UK publications relevant to speech and language therapy with those of other countries, both quantitatively and qualitatively. We sought then to examine the UK papers in more detail to analyse their sources of funding, their geographical distribution and the ways in which they could appropriately be evaluated. Methods & Procedures: Papers were selectively retrieved from the Science Citation Index and the Social Sciences Citation Index for 1991‐2000 by means of a filter based on journal names and paper title words. They were subsequently checked to remove many false positives. The papers were classified into one of seven subject areas and by their research level (from clinical to basic). Their importance was estimated through their potential impact on other researchers, as determined by the citation score of their journals, by the numbers of citations they actually received and by the subjective esteem in which the various journals were held by UK speech and language researchers. Outcomes & Results: World output of speech and language therapy papers has averaged 1000 papers per year during the 1990s, and has grown by half over the period. UK output has been about 12% of the total, compared with 10% in biomedicine, and is published in high impact journals relative to the norm for the field, which is quite a low rate compared with biomedicine overall. Almost half the UK papers had no funding acknowledgements, with the private‐non‐profit and industrial sectors playing less of a role than in other biomedical areas. Papers in seven subject areas showed substantial differences in their performance on the four criteria selected. Conclusions: The state of British speech and language research appears to be satisfactory, with an above average output in both quantity and quality. However, it is not attracting funding from some types of sponsors and is not being published in general medical journals where it might have a wider influence on general clinical practice. It is also not clear how best such research can be evaluated, although conventional citation counts may be relevant for some subject areas.
Changes in linguapalatal contact patterns during therapy for velar fronting in a 10‐year‐old with Down's syndrome
Authors: Gibbon Fiona E. , McNeill Alison M. , Wood Sara E. , Watson Jocelynne M. M.
Published: September 2003

Background: Articulation errors in the speech of people with Down's syndrome are frequent and often resistant to speech therapy. This preliminary study investigates the use of electropalatography (EPG) to diagnose and treat abnormal articulation patterns associated with velar fronting in a 10‐year‐old girl. Aims: The study measured changes in the accuracy and stability of linguapalatal (tongue‐palate) contact patterns during a 14‐week course of visual feedback therapy using EPG. Therapy aimed to resolve a pattern of velar fronting whereby targets /k, g, - / had alveolar placement [t, d, n]. Methods & Procedures: The participant was a girl (P) with Down's syndrome aged 10;11 years. P had a moderate‐severe speech disorder, which included velar fronting. Her speech was recorded with EPG on three occasions during a 14‐week course of therapy: first, before therapy; second, midway through therapy; and third, after therapy. Three analyses were conducted on the EPG data. The first used an EPG classification scheme that identified accuracy of placement for /t/ and /k/ targets. The second was a centre of gravity measure that detected whether P produced a significant difference between /t/ and /k/ targets. The third was a variability index that quantified the stability of contact patterns. Outcomes & Results: The results of the EPG classification showed that before therapy, /t/ and /k/ targets had identical alveolar placement, reflecting the process of velar fronting. The results after therapy showed that 87% of /k/ targets had accurate velar placement. The centre of gravity measure showed no difference in contact patterns for /t/ and /k/ before therapy, but a statistically significant difference at the second and third recordings. The variability index showed stable contact patterns before therapy for /t/ and /k/ targets, but both became highly unstable midway through therapy, with a return to stability at the third recording. We embed a discussion of P's increased articulation instability during therapy in a recent theoretical framework–dynamic systems–that attempts to account for the emergence of new behavioural forms. Conclusions: These preliminary results suggest that EPG has potential as an effective diagnostic and therapy procedure for articulation errors in people with Down's syndrome. A major issue still to be addressed, however, is the extent to which others will benefit from this approach to intervention.
Article: Bullying risks of 11‐year‐old children with specific language impairment (SLI): Does school placement matter?
Authors: Knox Emma , Conti‐Ramsden Gina
Published: September 2003

In addressing an issue rarely explored in research literature, the prevalence and severity of the risk of being bullied at school was measured in 100 children with specific language impairment (SLI). Participants attended a range of different educational placement types and these were compared for bullying risk. Furthermore, the risk encountered by children with SLI was compared with that of normally developing age‐matched peers. Each participant completed a questionnaire and it was found that 36% of participants with SLI considered themselves at risk of being bullied in school compared with only 12% of the normally developing cohort. No statistically significant difference was found between the risk experienced by participants with SLI attending mainstream education and that by participants attending special education placements. Possible explanations for the results are offered and the relevance of the findings in the context of optimizing the educational experience of children with SLI is highlighted.
Evaluation of speech in people with head and neck cancer: A Pilot Study
Authors: McKinstry A. , Perry A.
Published: September 2003

Background: A paucity of information exists on the effects of cancer on speech, with what little literature exists focussing on the effects of treatment. Baseline assessment of speech in head and neck cancer patients before treatment is important to distinguish accurately the effects of treatment from the effects of the cancer itself. Aims: This prospective study had four major foci: (1) to determine if speech intelligibility and motor speech functions of patients with head and neck cancer before treatment differed from the normal population; (2) to determine if impairments in speech intelligibility and motor speech functions were specific to the original site of cancer; (3) to determine which motor speech functions were most related to overall speech intelligibility; and (4) to determine whether there was a relationship between self‐report of speech intelligibility, motor speech functions and the clinical assessment thereof. This study aimed incidentally to determine whether the Frenchay Dysarthria Assessment is a practicable, valid and reliable tool for assessing speech functions in the head and neck cancer population. Methods & Procedures: Twenty participants, between 47 and 76 years of age and all newly diagnosed with a cancer of the head and neck, participated in the pilot study. In examining speech, participants completed a self‐report questionnaire pertaining to speech intelligibility and motor speech functions and were then assessed clinically using subscales of the Frenchay Dysarthria Assessment before the start of cancer treatment. The motor speech functions examined included: respiratory ability and functions of the lip, soft palate, larynx and tongue. Outcomes & Results: Results indicated that participants with head and neck cancer had a greater reduction in both speech intelligibility and in almost all aspects of speech when compared with the normal population. Results further demonstrated that the site of the cancer dictated the type of impairment experienced with respect to speech function. Not all motor speech characteristics correlated with overall speech intelligibility and, lastly, self‐report and clinical assessment were significantly correlated with respect to motor speech characteristics, but they differed slightly in the assessment of speech intelligibility. Conclusions: This pilot study has highlighted the critical importance of collecting baseline measures and reporting functional results according to the cancer site in future research. It has also shown the usefulness of the Frenchay Dysarthria Assessment as a practicable, valid and reliable protocol of motor speech assessment for the head and neck cancer population.
Identification of a calicivirus isolate of unknown origin
Authors: Oehmig Angelika , Büttner Mathias , Weiland Frank , Werz William , Bergemann Klaus , Pfaff Eberhard
Published: September 2003

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.
Mouse respiratory epithelial cells support efficient replication of human rhinovirus
Authors: Tuthill Tobias J. , Papadopoulos Nikolaos G. , Jourdan Patrick , Challinor Lisa J. , Sharp Nigel A. , Plumpton Chris , Shah Ketaki , Barnard Suzanne , Dash Laura , Burnet Jerome , Killington Richard A. , Rowlands David J. , Clarke Neil J. , Blair Edward D. , Johnston Sebastian L.
Published: September 2003

Human rhinoviruses (HRV) are responsible for the majority of virus infections of the upper respiratory tract. Furthermore, HRV infection is associated with acute exacerbation of asthma and other chronic respiratory diseases of the lower respiratory tract. A small animal model of HRV-induced disease is required for the development of new therapies. However, existing mouse models of HRV infection are difficult to work with and until recently mouse cell lines were thought to be generally non-permissive for HRV replicationin vitro. In this report we demonstrate that a virus of the minor receptor group, HRV1B, can infect and replicate in a mouse respiratory epithelial cell line (LA-4) more efficiently than in a mouse fibroblast cell line (L). The major receptor group virus HRV16 requires human intercellular adhesion molecule-1 (ICAM-1) for cell entry and therefore cannot infect LA-4 cells. However, transfection ofin vitro-transcribed HRV16 RNA resulted in the replication of viral RNA and production of infectious virus. Expression of a chimeric ICAM-1 molecule, comprising mouse ICAM-1 with extracellular domains 1 and 2 replaced by the equivalent human domains, rendered the otherwise non-permissive mouse respiratory epithelial cell line susceptible to entry and efficient replication of HRV16. These observations suggest that the development of mouse models of respiratory tract infection by major as well as minor group HRV should be pursued.
Pathogenesis of poliovirus infection in PVRTg mice: poliovirus replicates in peritoneal macrophages
Authors: Buisman A. M. , Sonsma J. A. J. , van Wijk M. G. S. , Vermeulen J. P. , Roholl P. J. , Kimman T. G.
Published: September 2003

The pathogenesis of poliovirus infection, responsible for the induction of a poliovirus-specific mucosal immune response following intraperitoneal (i.p.) inoculation of virus in mice transgenic for the poliovirus receptor (PVRTg mice), was studied. Following inoculation of poliovirus, replication was determined by increase in virus titre (TCID50) and by PCR of poliovirus-specific negative-strand RNA in peritoneal macrophages, mesenteric lymph nodes, Peyer's patches, duodenum, brain, kidney and liver. The presence of poliovirus antigens in several cell types was detected by immunolabelling. It was demonstrated that poliovirus replicated in the peritoneal macrophages of PVRTg mice, since the virus titre in peritoneal cells was increased compared to the titre in the inoculum. Negative-strand RNA was detected in these cells and most of the poliovirus-immunostained cells had the morphology of macrophages and expressed the macrophage-specific markers CD86 and M1/70 on their surface. Furthermore, in peritoneal lavage, poliovirus was also present in CD19+B cells, but not in dendritic or T cells. Moreover, poliovirus was detected in macrophage-like cells in the lamina propria of the intestine, but not in epithelial cells. Replication of poliovirus in mesenteric lymph nodes, Peyer's patches and brain was followed by excretion of virus in the faeces. This suggests that the virus is transported due to migration of macrophages from the peritoneal cavity to mesenteric lymph nodes and the lamina propria of Peyer's patches. It is likely that this route is responsible for the induction of virus-specific IgA in the gut.
Persistence and transmission of natural type I feline coronavirus infection
Authors: Addie D. D. , Schaap I. A. T. , Nicolson L. , Jarrett O.
Published: September 2003

To examine the mode of natural transmission and persistence of feline coronavirus (FCoV), FCoV strains shed by domestic cats were investigated over periods of up to 7 years. An RT-PCR that amplified part of the 3′ end of the viral spike (S) gene was devised to distinguish FCoV types I and II. All but 1 of 28 strains of FCoV from 43 cats were type I. Nucleotide identities of the amplified 320 bp product from 49 type I FCoVs ranged from 79 to 100 %. The consensus partial S sequence of isolates recovered from persistently infected cats at time intervals spanning years was generally conserved. While most cats were infected with a single strain, a few may have been infected by more than one strain. Cats that were transiently infected and ceased shedding could be re-infected with either the same, or a different, strain. In most cases, whether a cat became persistently or transiently infected was independent of the virus strain. However, one strain was unusual in that it infected the majority of cats in the household simultaneously and was still being shed 18 months later. Factors that influence whether FCoV establishes lifelong infection in some cats and not others are determined mainly by the host response to infection.
Detection of equine arteritis virus (EAV)-specific cytotoxic CD8+ T lymphocyte precursors from EAV-infected ponies
Authors: Castillo-Olivares J. , Tearle J. P. , Montesso F. , Westcott D. , Kydd J. H. , Davis-Poynter N. J. , Hannant D.
Published: September 2003

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a51Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain51Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8+/CD4+ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8+T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus–host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.
Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM
Authors: Catteau Adeline , Kalinina Olga , Wagner Marie-Christine , Deubel Vincent , Courageot Marie-Pierre , Desprès Philippe
Published: September 2003

The induction of apoptotic cell death is a prominent cytopathic effect of dengue (DEN) viruses. One of the key questions to be addressed is which viral components induce apoptosis in DEN virus-infected cells. This study investigated whether the small membrane (M) protein was involved in the induction of apoptosis by DEN virus. This was addressed by using a series of enhanced green fluorescent protein-fused DEN proteins. Evidence is provided that intracellular production of the M ectodomains (residues M-1 to M-40) of all four DEN serotypes triggered apoptosis in host cells such as mouse neuroblastoma Neuro 2a and human hepatoma HepG2 cells. The M ectodomains of the wild-type strains of Japanese encephalitis, West Nile and yellow fever viruses also had proapoptotic properties. The export of the M ectodomain from the Golgi apparatus to the plasma membrane appeared to be essential for the initiation of apoptosis. The study found that anti-apoptosis protein Bcl-2 protected HepG2 cells against the death-promoting activity of the DEN M ectodomain. This suggests that the M ectodomain exerts its cytotoxic effects by activating a mitochondrial apoptotic pathway. The cytotoxicity of the DEN M ectodomain reflected the intrinsic proapoptotic properties of the nine carboxy-terminal amino acids (residues M-32 to M-40) designatedApoptoM. Residue M-36 was unique in that it modulated the death-promoting activity of the M ectodomain. Defining theApoptoM-activated signalling pathways leading to apoptosis will provide the basis for studying how the M protein might play a key role in the fate of the flavivirus-infected cells.
In vivo characteristics of human immunodeficiency virus type 1 intersubtype recombination: determination of hot spots and correlation with sequence similarity
Authors: Magiorkinis Gkikas , Paraskevis Dimitrios , Vandamme Anne-Mieke , Magiorkinis Emmanouil , Sypsa Vana , Hatzakis Angelos
Published: September 2003

Recombination plays a pivotal role in the evolutionary process of many different virus species, including retroviruses. Analysis of all human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants revealed that they are more complex than described initially. Recombination frequency is higher within certain genomic regions, such as partial reverse transcriptase (RT), vif/vpr, the first exons of tat/rev, vpu and gp41. A direct correlation was observed between recombination frequency and sequence similarity across the HIV-1 genome, indicating that sufficient sequence similarity is required upstream of the recombination breakpoint. This finding suggests that recombination in vivo may occur preferentially during reverse transcription through the strand displacement-assimilation model rather than the copy-choice model.
In vivo transcription of the Epstein–Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders
Authors: van Beek Josine , Brink Antoinette A. T. P. , Vervoort Marcel B. H. J. , van Zijp Mireille J. M. , Meijer Chris J. L. M. , van den Brule Adriaan J. C. , Middeldorp Jaap M.
Published: September 2003

Thein vivoexpression of the Epstein–Barr virus (EBV)BamHI-A rightward transcripts (BARTs) as well as the putative BART-encoded BARF0 and RK-BARF0 proteins in various EBV-associated malignancies was investigated. RT-PCRs specific for the different splice variants of the BARTs and both a nucleic acid sequence-based amplification assay and an RT-PCR specific for the BARF0 ORF were used. Abundant transcription of BARTs was found in EBV-associated Hodgkin's lymphomas, Burkitt's lymphomas (BL), T-cell non-Hodgkin's lymphomas, post-transplant lymphoproliferative disorders, AIDS-related lymphomas and gastric carcinomas. Using RNAin situhybridization (RISH), BARTs were detected within the neoplastic cells of these malignancies. BARTs encoding RK-BARF0 were not detected. The BARTs detected were shown possibly to encode the RPMS1 and BARF0 proteins, based on their splicing. However, BARTs actually harbouring the BARF0 ORF were detected only in specimens containing a relatively large number of EBV-positive cells. New monoclonal antibodies against the BARF0 protein were generated that efficiently recognized prokaryotic and eukaryotic recombinant BARF0. However, the BARF0 protein was not detected in clinical samples, nor in EBV-positive cell lines, even though these were positive for BARTs by RISH and/or BARF0 RNAin vitroanalysis. Using immunoblot analysis, no antibodies against baculovirus-expressed BARF0 protein were detected in the sera of nasopharyngeal carcinoma patients, BL patients and Hodgkin's disease patients, patients with chronic EBV infection, infectious mononucleosis patients or EBV-positive healthy donors. Thus, BARTs containing the BARF0 ORF are expressedin vivobut the BARF0 protein cannot be detected and may be expressed only marginally. It is concluded that the BARF0 protein is unlikely to play a rolein vivoin EBV-positive malignancies.
Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein
Authors: Chiou Chun-Tang , Hu Chih-Chi Andrew , Chen Pi-Hsin , Liao Ching-Len , Lin Yi-Ling , Wang Jaang-Jiun
Published: September 2003

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B–NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.
Infection of macrophages by a lymphotropic herpesvirus: a new tropism for Marek's disease virus
Authors: Barrow Alexander D. , Burgess Shane C. , Baigent Susan J. , Howes Ken , Nair Venugopal K.
Published: September 2003

Marek's disease virus (MDV) is classified as an oncogenic lymphotropic herpesvirus of chickens. MDV productively and cytolytically infects B,αβT andγδT lymphocytes and latently infects T-helper lymphocytes. The aims of this study were to identify whether MDV infects macrophagesin vivoand, if so, whether quantitative differences in macrophage infection are associated with MDV strain virulence. Chickens were infected with either virulent MDV (HPRS-16) or ‘hypervirulent’ MDV (C12/130). Flow cytometry with monoclonal antibodies recognizing MDV pp38 antigen and leukocyte antigens was used to identify MDV lytically infected cells. Macrophages from HPRS-16- and C12/130-infected chickens were pp38+. It is demonstrated that macrophages are pp38+because they are infected and not because they have phagocytosed MDV antigens, as assessed by confocal microscopy using antibodies recognizing MDV antigens of the three herpesvirus kinetic classes: infected cell protein 4 (ICP4, immediate early), pp38 (early) and glycoprotein B (gB, late). Spleen macrophages from MDV-infected chickens were ICP4+, pp38+and gB+, and ICP4 had nuclear localization denoting infection. Finally, MDV pp38+macrophages had high inherent death rates, confirming cytolytic MDV infection, although production of virus particles has not been detected yet. These results have two fundamental implications for understanding MDV pathogenesis: (i) MDV evolved to perturb innate, in addition to acquired, immunity and (ii) macrophages are excellent candidates for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.
Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen
Authors: Wu Han-Chung , Jung Mei-Ying , Chiu Chien-Yu , Chao Ting-Ting , Lai Szu-Chia , Jan Jia-Tsrong , Shaio Men-Fang
Published: September 2003

In this study, a serotype-specific monoclonal antibody (mAb), D216-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His–Arg/Lys–Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.
Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus
Authors: Suradhat Sanipa , Thanawongnuwech Roongroje
Published: September 2003

Recent studies suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system by upregulating interleukin (IL)-10 gene expression. To determine the effect of PRRSV on porcine cytokine gene expression in vivo, we infected pigs with either the European or North American strain of PRRSV and monitored cytokine gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) using a multiplex PCR assay. Our results showed that both European and North American strains of PRRSV significantly upregulated IL-10 gene expression in PBMC of infected pigs from 5 days post-infection (p.i.). In addition, upregulation of IL-10 and interferon (IFN)-γ gene expression was observed in BALC starting from 9 days p.i. The upregulation of cytokine gene expression in BALC was observed concurrent with an increased percentage of lymphocytes in the BALC population, suggesting a role for peripheral leukocytes in cytokine production in lungs. Our results showed that PRRSV infection resulted in an upregulation of IL-10 gene expression in vivo and that both European and North American strains induced comparable levels of IL-10 gene expression in the infected pigs, despite differences in the clinical signs. Our data support the notion that induction of IL-10 production may be one of the strategies used by PRRSV to modulate the host's immune responses, and this may contribute to the unique clinical picture observed following PRRSV infection.
Association of an 11–12 kDa protease-resistant prion protein fragment with subtypes of dura graft-associated Creutzfeldt–Jakob disease and other prion diseases
Authors: Satoh Katsuya , Muramoto Tamaki , Tanaka Tomoyuki , Kitamoto Noritoshi , Ironside James W. , Nagashima Kazuo , Yamada Masahito , Sato Takeshi , Mohri Shirou , Kitamoto Tetsuyuki
Published: September 2003

Creutzfeldt–Jakob disease can develop in subjects given a cadaveric dura mater graft (dCJD). This disease has a phenotypic heterogeneity despite the lack of genetic variation. Numerous plaque-type prion protein (PrP) deposits are found in the brain of some but not all subjects; hence, there may be two subtypes of this clinical entity. To validate dCJD subtypes further, we carried out a larger-scale clinicopathological analysis and typing of protease-resistant PrP (PrPSc) in dCJD cases. Cases with plaque-type PrP deposits (p-dCJD) were shown to be distinct from those without PrP plaques (np-dCJD), from several clinicopathological aspects. Analysis of PrPSc revealed that, while the major PrPSc species from both subtypes was of 21 kDa after deglycosylation (type 1 PrPSc), a C-terminal PrP fragment of 11–12 kDa (fPrP11–12) was associated with np-dCJD but not with p-dCJD. The disease type-specific association of fPrP11–12 was also observed in subjects with other prion diseases. An fPrP11–12-like C-terminal PrP fragment was detected in brain lysates from patients associated with fPrP11–12, but not from patients or normal subjects unassociated with fPrP11–12. Results indicated that fPrP was produced by CJD-associated processes in vivo. The present data provide several lines of evidence that support the need for subtyping of dCJD and contribute to the understanding of the processing of disease-specific PrP species. The unique relationship of fPrP11–12 with CJD phenotype supports the view that the phenotypic heterogeneity of CJD is related to the formation of different types of disease-specific PrP and fragments thereof.
Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants
Authors: Dietrich Christof , Maiss Edgar
Published: September 2003

The distribution of potyviruses in mixed infected Nicotiana benthamiana plants was investigated by using green and red fluorescent proteins (GFP, DsRed). Full-length cDNA clones of Plum pox virus (PPV-NAT-AgfpS; PPV-NAT-red), Tobacco vein mottling virus (TVMV-gfp; TVMV-red) and Clover yellow vein virus (ClYVV-GFP) expressing fluorescent proteins, referred to here as labelled viruses, were used to characterize the distribution of different potyviral populations (e.g. TVMV-gfp/PPV-NAT-red), as well as populations of identical, but differently labelled potyviruses (e.g. PPV-NAT-AgfpS/PPV-NAT-red) or in mixed infections of potyviruses with labelled Potato virus X (PVX). Plants infected by any of the PVX/potyvirus combinations exhibited synergistic symptoms and large numbers of cells were doubly infected. In contrast, co-infections of differently labelled potyvirus populations appeared non-synergistic and remained predominantly separate in the infected plants, independent of whether different viruses or identical but differently labelled viruses were co-infecting. Contact of differently labelled virus populations that exhibited spatial separation was restricted to a small number of cells at the border of different fluorescent cell clusters.
A novel, divergent simian T-cell lymphotropic virus type 3 in a wild-caught red-capped mangabey (Cercocebus torquatus torquatus) from Nigeria
Authors: Meertens L. , Shanmugam V. , Gessain A. , Beer B. E. , Tooze Z. , Heneine W. , Switzer W. M.
Published: September 2003

We present here a novel, distinct simian T-cell lymphotropic virus (STLV) found in a red-capped mangabey (Cercocebus torquatus) (CTO-NG409), wild-caught in Nigeria, that showed an HTLV-2-like Western blot (WB) seroreactivity. The complete genome (8920 bp) of CTO-NG409 STLV was related to but different from STLV-3/PHA-PH969 (13·5 %) and STLV-3/PPA-F3 (7·6 %), and STLV-3/CTO604 (11·3 %), found in Eritrean and Senegalese baboons, and red-capped mangabeys from Cameroon, respectively. Phylogenetic analysis of a conservedtax(180 bp) sequence and theenvgene (1482 bp) confirmed the relatedness of STLV-3/CTO-NG409 to the STLV-3 subgroup. Molecular clock analysis ofenvestimated that STLV-3/CTO-NG409 diverged from East and West/Central African STLV-3s about 140 900±12 400 years ago, suggesting an ancient African origin of STLV-3. Since phylogenetic evidence suggests multiple interspecies transmissions of STLV-1 to humans, and given the antiquity and wide distribution of STLV-3 in Africa, a search for STLV-3 in human African populations with HTLV-2-like WB patterns is warranted.
Suppressor activity of potyviral and cucumoviral infections in potyvirus-induced transgene silencing
Authors: Simón-Mateo Carmen , López-Moya Juan José , Guo Hui Shan , González Elena , García Juan Antonio
Published: September 2003

The process known as ‘recovery’ by which virus-infected plants become resistant to the infection is an interesting phenomenon where both RNA silencing and virus resistance fully converge. In a previous study, we showed that transgenic Nicotiana benthamiana NIbV3 plants, transformed with a mutated NIb coding sequence from Plum pox virus (PPV), showed a delayed, very specific, resistance phenotype, which was induced by the initial infection. This recovery was the consequence of the activation of an RNA silencing mechanism in the PPV-infected plant, which took place even though PPV encodes a silencing suppressor (HCPro). Making use of plants regenerated from the recovered tissue, which maintained the transgene silencing/virus resistance phenotype, we have demonstrated that both Cucumber mosaic virus (CMV) and Tobacco vein mottling virus (TVMV), expressing the silencing suppressor 2b and HCPro, respectively, were able to reactivate transgene expression. Surprisingly, only the silencing suppression caused by CMV, but not that originating from TVMV, was able to revert the recovered NIbV3 plants to a PPV-susceptible phenotype.
Identification of equine herpesvirus-1 antigens recognized by cytotoxic T lymphocytes
Authors: Soboll Gisela , Whalley J. Millar , Koen Mathew T. , Allen George P. , Fraser Darrilyn G. , Macklin Michael D. , Swain William F. , Lunn D. Paul
Published: September 2003

Equine herpesvirus-1 (EHV-1) causes serious disease in horses throughout the world, despite the frequent use of vaccines. CTLs are thought to be critical for protection from primary and reactivating latent EHV-1 infections. However, the antigen-specificity of EHV-1-specific CTLs is unknown. The aim of this study was to identify EHV-1 genes that encode proteins containing CTL epitopes and to determine their MHC I (or ELA-A in the horse) restriction. Equine dendritic cells, transfected with a series of EHV-1 genes, were used to stimulate autologous CTL precursor populations derived from previously infected horses. Cytotoxicity was subsequently measured against EHV-1-infected PWM lymphoblast targets. Dendritic cells were infected with EHV-1 (positive control) or transfected with plasmids encoding the gB, gC, gD, gE, gH, gI, gL, immediate-early (IE) or early protein of EHV-1 using the PowderJect XR-1 research device. Dendritic cells transfected with the IE gene induced CTL responses in four of six ponies. All four of these ponies shared a common ELA-A3.1 haplotype. Dendritic cells transfected with gC, gD, gI and gL glycoproteins induced CTLs in individual ponies. The cytotoxic activity was ELA-A-restricted, as heterologous targets from ELA-A mismatched ponies were not killed and an MHC I blocking antibody reduced EHV-1-specific killing. This is the first identification of an EHV-1 protein containing ELA-A-restricted CTL epitopes. This assay can now be used to study CTL specificity for EHV-1 proteins in horses with a broad range of ELA-A haplotypes, with the goal of developing a multi-epitope EHV-1 vaccine.
Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses
Authors: Chare Elizabeth R. , Gould Ernest A. , Holmes Edward C.
Published: September 2003

Recombination is increasingly seen as an important means of shaping genetic diversity in RNA viruses. However, observed recombination frequencies vary widely among those viruses studied to date, with only sporadic occurrences reported in RNA viruses with negative-sense genomes. To determine the extent of homologous recombination in negative-sense RNA viruses, phylogenetic analyses of 79 gene sequence alignments from 35 negative-sense RNA viruses (a total of 2154 sequences) were carried out. Powerful evidence was found for recombination, in the form of incongruent phylogenetic trees between different gene regions, in only five sequences from Hantaan virus, Mumps virus and Newcastle disease virus. This is the first report of recombination in these viruses. More tentative evidence for recombination, where conflicting phylogenetic trees were observed (but were without strong bootstrap support) and/or where putative recombinant regions were very short, was found in three alignments from La Crosse virus and Puumala virus. Finally, patterns of sequence variation compatible with the action of recombination, but not definitive evidence for this process, were observed in a further ten viruses: Canine distemper virus, Crimean-Congo haemorrhagic fever virus, Influenza A
virus, Influenza B
virus, Influenza C
virus, Lassa virus, Pirital virus, Rabies virus, Rift Valley Fever virus and Vesicular stomatitis virus. The possibility of recombination in these viruses should be investigated further. Overall, this study reveals that rates of homologous recombination in negative-sense RNA viruses are very much lower than those of mutation, with many viruses seemingly clonal on current data. Consequently, recombination rate is unlikely to be a trait that is set by natural selection to create advantageous or purge deleterious mutations.
Herpes simplex virus type 1 glycoprotein B sorting in hippocampal neurons
Authors: Potel Corinne , Kaelin Karin , Danglot Lydia , Triller Antoine , Vannier Christian , Rozenberg Flore
Published: September 2003

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that spreads in the nervous system in functionally connected neurons. Determining how HSV-1 components are sorted in neurons is critical to elucidate the mechanisms of virus neuroinvasion. By using recombinant viruses expressing glycoprotein B (gB) tagged with green fluorescent protein (GFP), the subcellular localization of this envelope protein was visualized in infected hippocampal neurons in culture. Results obtained using a fully infectious recombinant virus containing GFP inserted into the ectodomain of gB support the view that capsids and gB are transported separately in neuron processes. Moreover, they show that during infection gB is sorted to the dendritic tree and the axons of polarized hippocampal neurons. However, GFP insertion into the cytoplasmic tail of gB impaired the maturation of the resulting fusion protein and caused its retention in the endoplasmic reticulum. The defective protein did not gain access to axons of infected neurons. These results suggest that the cytoplasmic tail of gB plays a role in maturation and transport and subsequently in axonal sorting in differentiated hippocampal neurons.
Recombination analysis of human-tropic porcine endogenous retroviruses
Authors: Klymiuk Nikolai , Müller Mathias , Brem Gottfried , Aigner Bernhard
Published: September 2003

Prevention of cross-species infection of porcine endogenous retroviruses (PERV) is crucial for xenotransplantation. The potential risk of infection is caused by replication-competent PERV as well as by hybrid viruses derived from recombination events of distinct PERV genomes. Recently, human-tropic, replication-competent PERV genomes obtaining hybrid sequences have been observed. Here, complete polymorphism pattern analysis was performed on the full-length PERV γ1 clones and on the complete envelope (env) gene sequences published to date. Several recombined full-length clones and a high number of different recombination patterns in the env gene were identified. In addition, recombinations with retroviral genomes not yet known were found. Thus, the potential risk of infection also exists for recombination products, including defective PERV loci.
Isolation of Kaeng Khoi virus from dead Chaerephon plicata bats in Cambodia
Authors: Osborne J. C. , Rupprecht C. E. , Olson J. G. , Ksiazek T. G. , Rollin P. E. , Niezgoda M. , Goldsmith C. S. , An U. S. , Nichol S. T.
Published: September 2003

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2·6–3·2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.
Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA
Authors: El-Hage Nazira , Luo Guangxiang
Published: September 2003

Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5′-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.
The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg–HCPro and VPg–VPg interactions
Authors: Yambao Ma. Leonora M. , Masuta Chikara , Nakahara Kenji , Uyeda Ichiro
Published: September 2003

Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro–VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro–VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro–VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg–VPg interaction and the central 19 amino acids were needed for the HCPro–VPg interaction.
Molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi
Authors: Gilad Oren , Yun Susan , Adkison Mark A. , Way Keith , Willits Neil H. , Bercovier Herve , Hedrick Ronald P.
Published: September 2003

Koi herpesvirus (KHV) has been associated with devastating losses of common carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) in North America, Europe, Israel and Asia. A comparison of virion polypeptides and genomic restriction fragments of seven geographically diverse isolates of KHV indicated that with one exception they represented a homogeneous group. A principal environmental factor influencing the onset and severity of disease is water temperature. Optimal growth of KHV in a koi fin cell line occurred at temperatures from 15–25 °C. There was no growth or minimal growth at 4, 10, 30 or 37 °C. Experimental infections of koi with KHV at a water temperature of 23 °C resulted in a cumulative mortality of 95·2 %. Disease progressed rapidly but with lower mortality (89·4–95·2 %) at 28 °C. Mortality (85·0 %) also occurred at 18 °C but not at 13 °C. Shifting virus-exposed fish from 13–23 °C resulted in the rapid onset of mortality.