The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus.

Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2 are not phylogenetically closely related; however, both use the angiotensin-converting enzyme 2 (ACE2) receptor in humans for cell entry. This is not a universal sarbecovirus trait; for example, many known sarbecoviruses related to SARS-CoV-1 have two deletions in the receptor binding domain of the spike protein that render them incapable of using human ACE2. Here, we report three sequences of a novel sarbecovirus from Rwanda and Uganda that are phylogenetically intermediate to SARS-CoV-1 and SARS-CoV-2 and demonstrate via in vitro studies that they are also unable to utilize human ACE2. Furthermore, we show that the observed pattern of ACE2 usage among sarbecoviruses is best explained by recombination not of SARS-CoV-2, but of SARS-CoV-1 and its relatives. We show that the lineage that includes SARS-CoV-2 is most likely the ancestral ACE2-using lineage, and that recombination with at least one virus from this group conferred ACE2 usage to the lineage including SARS-CoV-1 at some time in the past. We argue that alternative scenarios such as convergent evolution are much less parsimonious; we show that biogeography and patterns of host tropism support the plausibility of a recombination scenario, and we propose a competitive release hypothesis to explain how this recombination event could have occurred and why it is evolutionarily advantageous. The findings provide important insights into the natural history of ACE2 usage for both SARS-CoV-1 and SARS-CoV-2 and a greater understanding of the evolutionary mechanisms that shape zoonotic potential of coronaviruses. This study also underscores the need for increased surveillance for sarbecoviruses in southwestern China, where most ACE2-using viruses have been found to date, as well as other regions such as Africa, where these viruses have only recently been discovered.

Patient crying during psychotherapy intake sessions: Relationship to patient characteristics and early treatment process.

The aim of the present study is to further the understanding of who cries at the beginning of psychotherapy and patients' experience of crying in that process. Intake sessions for 53 patients beginning psychotherapy at a university-based clinic were coded for discrete crying segments. Data about patient characteristics were also collected at intake. Results indicate that crying during intake sessions was related to lower global functioning and higher severity of childhood sexual abuse. Furthermore, patients who cried at intake were over four times more likely to also cry at feedback, and those who cried at feedback were almost 12 times more likely to have cried at intake. Finally, crying in the intake session did not appear to be related to patient- or therapist-rated working alliance. Overall, the present study provides valuable information about characteristics of patients who cry at the outset of the therapy process and patients' experience of crying over time in therapy. Findings suggest the need for further research on patient characteristics and aspects of the therapy process that may predict patient crying over the course of treatment, as well as how these early crying experiences may be related to eventual patient outcomes.

The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus.

SARS-CoV-1 and SARS-CoV-2 are not phylogenetically closely related; however, both use the ACE2 receptor in humans for cell entry. This is not a universal sarbecovirus trait; for example, many known sarbecoviruses related to SARS-CoV-1 have two deletions in the receptor binding domain of the spike protein that render them incapable of using human ACE2. Here, we report three sequences of a novel sarbecovirus from Rwanda and Uganda which are phylogenetically intermediate to SARS-CoV-1 and SARS-CoV-2 and demonstrate via in vitro studies that they are also unable to utilize human ACE2. Furthermore, we show that the observed pattern of ACE2 usage among sarbecoviruses is best explained by recombination not of SARS-CoV-2, but of SARS-CoV-1 and its relatives. We show that the lineage that includes SARS-CoV-2 is most likely the ancestral ACE2-using lineage, and that recombination with at least one virus from this group conferred ACE2 usage to the lineage including SARS-CoV-1 at some time in the past. We argue that alternative scenarios such as convergent evolution are much less parsimonious; we show that biogeography and patterns of host tropism support the plausibility of a recombination scenario; and we propose a competitive release hypothesis to explain how this recombination event could have occurred and why it is evolutionarily advantageous. The findings provide important insights into the natural history of ACE2 usage for both SARS-CoV-1 and SARS-CoV-2, and a greater understanding of the evolutionary mechanisms that shape zoonotic potential of coronaviruses. This study also underscores the need for increased surveillance for sarbecoviruses in southwestern China, where most ACE2-using viruses have been found to date, as well as other regions such as Africa, where these viruses have only recently been discovered.

Facets of Object Representation: Process and Outcome Over the Course of Psychodynamic Psychotherapy.

This study explores the effectiveness of psychodynamic psychotherapy in improving facets of object relations (OR) functioning over the course of treatment. The sample consisted of 75 outpatients engaged in short-term dynamic psychotherapy at a university-based psychological services clinic. Facets of OR functioning were assessed at pre- and posttreatment by independent raters using the Social Cognition and Object Relations Scale-Global rating method (SCORS-G; Stein, Hilsenroth, Slavin-Mulford, & Pinsker, 2011 ; Westen, 1995 ) from in-session patient relational narratives. The Comparative Psychotherapy Process Scale (CPPS; Hilsenroth, Blagys, Ackerman, Bonge, & Blais, 2005 ) was used to assess therapist activity and psychotherapy techniques early in treatment. Independent clinical ratings of OR functioning and psychotherapy technique were conducted and all were found to be in the good to excellent range of reliability. Specific facets of OR functioning improved with medium to large effect changes posttreatment. These adaptive changes were significantly related to the incidence of psychodynamic-interpersonal (PI) techniques. Also, this study identified the role specific psychodynamic techniques had in facilitating change in a number of underlying dimensions of OR. Patient self-reported reliable change in symptomatology and reliable change in facets of OR were significantly related as well. This study highlights the utility of incorporating psychological assessment into psychotherapy practice to assess change at the explicit (symptoms) and implicit (OR) level. Limitations of this study, future research directions, and implications for clinical practice are discussed.

Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus.

The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.

Viral discovery as a tool for pandemic preparedness.

Emerging diseases are frequently caused by novel or previously unrecognised zoonotic viral pathogens, which tend to originate in and emerge from wildlife. When human or animal cases are first recognised, molecular or serological diagnostic assays specific to them do not yet exist, causing a delay in the identification of an outbreak's aetiologic agent as well as its source. Preparing for the next virus to emerge is a major public health challenge, impeded by a poor understanding of the diversity of potential candidates that exist in wildlife reservoirs. Characterising the diversity of viruses in key wildlife species will help to reduce the time between detection and response in an outbreak situation, and inform public health strategies that reduce the risk of spillover from animal reservoirs. Pathogen discovery techniques such as consensus polymerase chain reaction (cPCR) and next-generation sequencing (NGS) have been used to identify known and novel viruses in animals and humans, but have not been widely used in surveillance programmes. Metagenomic studies have identified novel viruses, new strains of known viruses, and have characterised host microbiomes. While NGS represents an unbiased approach to viral sequence detection, it is constrained by lower sensitivity than conventional PCR, requires substantial bioinformatics capabilities, and is cost prohibitive and therefore not widely available in the regions of the world that are most vulnerable to zoonotic disease emergence. In contrast, consensus PCR uses standard and widely available technologies, has greater sensitivity than NGS, and has also been used to identify novel viruses in wildlife, livestock and humans, though it is limited to detecting target genetic sequences conserved across known groups of viruses. The use of cPCR, in combination, if possible, with NGS and serology, can offer a powerful approach to rapidly identifying aetiologic agents in an outbreak and characterising the virome of key wildlife known to carry zoonotic viruses. Here, the authors review pathogen discovery techniques currently being used in human and animal surveillance programmes and the challenges of using viral discovery to identify novel zoonotic pathogens.

Les maladies émergentes sont souvent causées par des virus nouveaux ou précédemment inconnus, de portée zoonotique, qui ont généralement leur source dans la faune sauvage, à partir de laquelle s'effectue leur émergence. Lorsque le premier cas d'infection par un virus de ce type est détecté chez l'homme ou chez les animaux, il n'existe encore aucune épreuve moléculaire ou sérologique de détection de l'agent étiologique, ce qui retarde son identification ainsi que l'élucidation de la source du foyer. La préparation aux futures émergences virales est un véritable défi de santé publique et se voit entravée par les lacunes des connaissances sur la diversité des virus potentiellement candidats. La caractérisation des différents virus qui affectent les principales espèces sauvages permettra de réduire le délai entre le moment où un nouveau foyer est détecté et celui où une réponse lui est apportée, et d'élaborer en connaissance de cause des stratégies de santé publique visant à limiter le risque d'un franchissement d'espèce à partir des réservoirs animaux. Les techniques de découverte d'agents pathogènes, par exemple l'amplification en chaîne par polymérase (PCR) pan-générique ou consensus et le séquençage de nouvelle génération (SNG) sont utilisées pour identifier des virus connus ou nouveaux chez l'homme et l'animal, mais ne sont pas d'une utilisation courante dans les programmes de surveillance. Les études métagénomiques permettent d'identifier des virus nouveaux ainsi que les souches nouvelles de virus connus et servent également à caractériser le microbiome de l'hôte. Le SNG constitue une méthode de détection des séquences virales exempte de biais mais sa sensibilité moindre que celle des PCR classiques, les capacités bio-informatiques considérables qu'il requiert et son coût prohibitif sont des contraintes importantes qui en limitent l'utilisation dans les régions du monde les plus vulnérables à l'émergence des maladies zoonotiques. En revanche, la PCR consensus fait appel à des technologies normalisées et largement disponibles, tout en présentant une meilleure sensibilité que le SNG ; elle permet également d'identifier des virus nouveaux présents dans la faune sauvage, chez les animaux domestiques ou chez l'homme, bien qu'elle ne détecte que des séquences génétiques cibles conservées d'un groupe connu de virus à l'autre. Le recours à la PCR consensus, si possible associé aux techniques de SNG et à la sérologie se révèle une stratégie puissante qui permet d'identifier rapidement les agents responsables d'un foyer et de caractériser le virome d'espèces sauvages jouant un rôle majeur en tant que réservoirs de virus zoonotiques. Après avoir passé en revue les techniques de découverte d'agents pathogènes actuellement utilisées dans les programmes de surveillance des maladies animales et humaines, les auteurs font le point sur les enjeux de ces techniques pour l'identification de nouveaux agents pathogènes zoonotiques.

La causa de las enfermedades emergentes reside muchas veces en virus zoonóticos de aparición reciente o hasta entonces no descritos, que en general se originan y surgen en animales salvajes. Cuando se detectan los primeros casos en personas o animales aún no existen pruebas específicas de diagnóstico, ya sea molecular o serológico, y ello retrasa la identificación del agente etiológico del brote y la determinación de su origen. Prepararse para el próximo virus que vaya a aparecer es un gran objetivo de salud pública, lastrado en la práctica por el escaso conocimiento de la gran diversidad de posibles candidatos que moran en los reservorios de la fauna salvaje. La caracterización de los diversos virus que existen en las principales especies de animales salvajes ayudará a reducir el lapso que media entre la detección y la respuesta en caso de brote y a fundamentar a partir de ahí estrategias de salud pública que reduzcan el riesgo de diseminación desde los reservorios animales. Hasta ahora las técnicas de descubrimiento de patógenos, como la reacción en cadena de la polimerasa (PCR) de consenso (PCRc) o la secuenciación de próxima generación, han servido para identificar virus nuevos o ya conocidos en personas y animales, pero no se han aplicado de forma generalizada a los programas de vigilancia. Gracias a estudios de metagenómica se han podido detectar virus recién aparecidos o nuevas cepas de virus ya conocidos y caracterizar los microbiomas de los organismos anfitriones. Aunque la secuenciación de próxima generación constituye un método exento de sesgos para detectar secuencias víricas, adolece de una menor sensibilidad que la PCR convencional, exige una considerable capacidad de gestión informática de datos biológicos y tiene un costo prohibitivo, por lo que no suele aplicarse en las regiones del mundo que están más expuestas a la aparición de enfermedades zoonóticas. La PCR de consenso, en cambio, reposa en técnicas habituales y muy extendidas, ofrece mayor sensibilidad que la secuenciación de próxima generación y también ha sido utilizada para identificar virus nuevos en personas o animales salvajes y domésticos, si bien solo permite detectar las secuencias genéticas «diana¼ conservadas de entre todos los grupos conocidos de virus. El uso de la PCRc, combinado con la secuenciación de próxima generación y técnicas serológicas cuando sea posible, puede ofrecer un potente método para identificar con rapidez los agentes etiológicos de un brote y caracterizar el viroma de los principales animales salvajes de los que se sabe que son portadores de virus zoonóticos. Los autores pasan revista a las técnicas de descubrimiento de patógenos que se utilizan actualmente en los programas de vigilancia sanitaria y zoosanitaria y exponen las dificultades que presenta el uso del descubrimiento de virus para identificar nuevos patógenos zoonóticos.

Discovery of a Novel Hepatovirus (Phopivirus of Seals) Related to Human Hepatitis A Virus.

UNLABELLED: Describing the viral diversity of wildlife can provide interesting and useful insights into the natural history of established human pathogens. In this study, we describe a previously unknown picornavirus in harbor seals (tentatively named phopivirus) that is related to human hepatitis A virus (HAV). We show that phopivirus shares several genetic and phenotypic characteristics with HAV, including phylogenetic relatedness across the genome, a specific and seemingly quiescent tropism for hepatocytes, structural conservation in a key functional region of the type III internal ribosomal entry site (IRES), and a codon usage bias consistent with that of HAV. IMPORTANCE: Hepatitis A virus (HAV) is an important viral hepatitis in humans because of the substantial number of cases each year in regions with low socioeconomic status. The origin of HAV is unknown, and no nonprimate HAV-like viruses have been described. Here, we describe the discovery of an HAV-like virus in seals. This finding suggests that the diversity and evolutionary history of these viruses might be far greater than previously thought and may provide insight into the origin and pathogenicity of HAV.

Usability testing of the Internet program: "Teens Taking Charge: Managing My Transplant Online".

Adolescents with SOT demonstrate high rates of medication non-adherence and higher rates of graft loss compared to all other age groups. Self-management interventions encompass information-based material designed to achieve disease-related learning and changes in the participant's knowledge and skill acquisition, while providing social support. These interventions have had some success in chronic disease populations by reducing symptoms and promoting self-efficacy and empowerment. Using findings from a needs assessment, an Internet-based self-management program, Teens Taking Charge: Managing My Transplant Online, for youth with SOT was developed. This program contains information on transplant, self-management and transition skills, and opportunities for peer support. The purpose of this study was to determine the usability and acceptability of the initial three modules (Medication and Vaccines; Diet after Transplant; and Living with a Transplant Organ) of the online program from the perspectives of youth with SOT. Participants were recruited from SOT clinics at a large pediatric tertiary care center in Canada. Three iterative cycles (seven patients per iteration) of usability testing took place to refine the Web site prototype. Study procedures involved participants finding items from a standardized list of features and talking aloud about issues they encountered, followed by a semi-structured interview to generate feedback about what they liked and disliked about the program. All 21 patients (mean age = 14.9 yr) found the Web site content to be trustworthy, they liked the picture content, and they found the videos of peer experiences to be particularly helpful. Participants had some difficulties finding information within submodules and suggested a more simplistic design with easier navigation. This web-based intervention is appealing to teenagers and may foster improved self-management with their SOT. Nine additional teen and two parent modules are being developed, and the completed Web site will undergo usability testing. In the future, a randomized control trial will determine the feasibility and effectiveness of this online self-management program on adherence, self-efficacy, and transition skills.

Patient Crying in Psychotherapy: Who Cries and Why?

AIM: The aim of the present study is to further the understanding of who cries in therapy and the relation of technique with crying behaviour in therapy. METHOD: Psychological assessment feedback sessions, prior to the initiation of formal therapy for 52 patients beginning psychotherapy at a university-based clinic were coded for discrete crying segments. Data about patient characteristics and the process of the session were collected at the time of the session. Therapist's interventions were recorded verbatim and independently rated. RESULTS: The number of times a patient cried during their session correlated negatively with global assessment of functioning scores and positively with measures of borderline personality disorder pathology as well as a measure of severity of childhood sexual abuse. Patients' crying behaviour demonstrated significant negative correlations with the overall experience of the session (bad/good), smoothness and positivity. Group differences between criers and non-criers reflected these trends as well. No significant correlations or group differences were found with regard to patient-rated or therapist-rated alliance as it relates to crying behaviour. Analysis indicates that therapist intervention prior to patient crying most often encouraged the exploration and expression of difficult affect, new perspectives on key issues or the patient's fantasies and wishes. DISCUSSION: Our study addresses a significant gap in the clinical literature on crying. Crying behaviour seems to be related to certain clinical variables and has a negative impact on patient experience of the session in which they cry, although the alliance remained unaffected. LIMITATIONS: Small sample, outpatients with mild/moderate psychopathology and graduate trainees provided therapy. KEY PRACTITIONER MESSAGE: Patients with greater problems in emotional dysregulation, borderline personality disorder symptoms and greater severity of childhood sexual abuse are more likely to display greater affective intensity during the beginning of treatment. Results suggest that the alliance may remain strong despite patients experiencing a session in which they cried as difficult. Therapeutic interventions that focus on affect, new understanding of old patterns and patient fantasies with outpatient clinical populations appeared to be associated with crying in session.

West Nile virus in the British Virgin Islands.

West Nile virus (WNV) first emerged in the US in 1999 and has since spread across the Americas. Here, we report the continued expansion of WNV to the British Virgin Islands following its emergence in a flock of free-roaming flamingos. Histologic review of a single chick revealed lesions consistent with WNV infection, subsequently confirmed with PCR, immunohistochemistry and in situ hybridization. Full genome analysis revealed 99% sequence homology to strains circulating in the US over the past decade. This study highlights the need for rapid necropsy of wild bird carcasses to fully understand the impact of WNV on wild populations.

Relationship between patient pre-treatment object relations functioning and psychodynamic techniques early in treatment.

UNLABELLED: This study explored the relationship between patient pre-treatment object relations (OR) functioning and psychodynamic techniques employed during two early sessions (third and ninth). The sample consisted of 76 outpatients engaged in short-term psychodynamic psychotherapy. Results showed that lower levels of patient pre-treatment OR functioning, particularly in the identity and affective domains, were related to a greater use of psychodynamic-interpersonal techniques in sessions. Patients who had more adaptive management of aggressive impulses were related to a greater use of cognitive-behavioural techniques by therapists. In addition, exploratory analyses between OR functioning and specific psychodynamic-interpersonal and cognitive-behavioural techniques showed that lower OR functioning in terms of affect, self-esteem, identity coherence, social causality, emotional investment in relationships as well as Global OR were significantly related to therapist focus on avoidance of important topics and affective changes during the session. Implications for clinical practice and Q1 future research are discussed. KEY PRACTITIONER MESSAGE: Consider more frequent use of psychodynamic techniques early in treatment with patients expressing more pathological object representations, particularly when these deficits are in the affective and identity domains. Lower patient object relations functioning may necessitate an in session focus on issues that are avoided or uncomfortable early in treatment. Lower patient object relations functioning may necessitate the need to address and explore labile affective expressions in session as they occur early in treatment. When patients are able to more adaptively express or manage aggressive impulses early within psychodynamic psychotherapy consider the integration of problem solving, goal oriented, future focused (i.e., CB) techniques.

Coronaviruses in bats from Mexico.

Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (ß)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were ß-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A ß-CoV with 96.5 % amino acid identity to the ß-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

Emergence of fatal avian influenza in New England harbor seals.

UNLABELLED: From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission. IMPORTANCE: The emergence of new strains of influenza virus is always of great public concern, especially when the infection of a new mammalian host has the potential to result in a widespread outbreak of disease. Here we report the emergence of an avian influenza virus (H3N8) in New England harbor seals which caused an outbreak of pneumonia and contributed to a U.S. federally recognized unusual mortality event (UME). This outbreak is particularly significant, not only because of the disease it caused in seals but also because the virus has naturally acquired mutations that are known to increase transmissibility and virulence in mammals. Monitoring the spillover and adaptation of avian viruses in mammalian species is critically important if we are to understand the factors that lead to both epizootic and zoonotic emergence.

RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication.

The evolution of two homologues of the core protein VP6 of epizootic haemorrhagic disease virus (EHDV), which correspond to the geographical origin of the virus.

Epizootic haemorrhagic disease virus is a 10-segmented, double-stranded RNA virus. When these ten segments of dsRNA are run on 1% agarose, eastern (Australia, Japan) and western (North America, Africa, Middle-East) strains of the virus can be separated phenotypically based on the migration of genome segments 7-9. In western strains, segments 7-9 are roughly the same size and co-migrate as a single RNA band. In eastern strains, segment 9 is smaller, so while segments 7 and 8 co-migrate, the segment 9 RNA runs faster than its western homologue. Translation experiments demonstrated that these two segment 9 homologues are both functional and produce proteins (VP6) of different sizes-something that has not been reported in any other orbivirus species to date. Sequence analysis suggests that eastern and western versions of segment 9 (VP6) have likely evolved as a response to adaptive selection in different geographical regions via gene duplication and subsequent mutation. These significant findings are considered unusual given the conserved nature of VP6 and its presumed role as the viral helicase. It is not currently known what the biological relevance of each homologue is to the virus.

Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV).

Three unique non-structural (NS) proteins are produced by Epizootic haemorrhagic disease virus (EHDV) during infection of a host cell; NS1, NS2 and NS3. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. Unlike the core, or outer-coat proteins, there are no characteristic genetic or phylogenetic traits common to all of the EHDV NS proteins; indicating that each is evolving under different selection pressures. These differences are discussed. Evidence of genetic recombination in genome segment 8 (coding for NS2) is also presented, together with evidence of gene duplication and mutation, suggesting the EHDV genome may have evolved using mechanisms such as these.

Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus (EHDV).

The core proteins of epizootic haemorrhagic disease virus (EHDV) have important roles to perform in maintaining the structure and function of the virus. A complete genetic and phylogenetic analysis was therefore performed on these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The inner core proteins (VP1, VP3, VP4 and VP6) were characterised by high levels of sequence conservation, and the ability to topotype isolates very strongly into eastern or western groups. This is particularly evident in genome segment 9 (VP6) which exists as two different sized homologues. VP7 did not topotype, but rather exhibited a more random, radial phylogeny suggestive of genetic drift. With the exception of VP6, all of the core proteins also showed high numbers of synonymous mutations in the third base position, suggesting they have been evolving for a long period of time. Interestingly, VP6 did not show this, and possible reasons for this are discussed.

Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup.

The outer-coat proteins, VP2 and VP5, of epizootic haemorrhagic disease virus (EHDV) are important for host cell binding during the initiation of infection. They are also known to determine virus serotype. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each and the correlation of genetic sequence data with serotype. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The results show that VP2 is highly variable, is under great pressure to adapt and can be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP2 but still shows some correlation with serotype. Seven serotypes of EHDV have been defined in this study, although the results do show that some serotypes are extremely closely related--and highlight the benefit of using both molecular and serologic analyses. Analysis of the terminal hexanucleotides showed that the 5' terminus is under greater purifying selection than the 3'. Evidence is also presented that both segments 2 and 6 (coding for VP2 and VP5 respectively) have grown via gene duplication and subsequent mutation.

Perceptions of transitional care needs and experiences in pediatric heart transplant recipients.

Survival following pediatric heart transplantation (HTx) continues to improve. The transition from pediatric to adult care is becoming a pivotal stage in the ongoing medical management of this population. Published data support enhanced outcomes for adolescent patients with increased attention to transitional care. The purpose of this study was to explore the 'transition experience' of adolescent HTx recipients and families. All teens (12-18 years) and parents at a single-center HTx program were invited to participate in semistructured interviews. Qualitative, phenomenological methodology was used to build theoretical knowledge and guided the data collection and analysis. The study population included 14 patients (7 males) with a mean age of 15.7 +/- 1.8 years (11.7-17.8 years) and at a mean of 4.1 +/- 3.3 years post-HTx (0.3-9.2 years) at the time of study participation. Major themes identified included: (i) adolescent disinterest and apathy regarding transition to adult care versus parental anxiety about their child's eventual departure from the pediatric transplant center, (ii) perceived differences in pediatric versus adult care and (iii) identification of strategies described as helpful in facilitating the transition. Understanding the experiences and perceptions of adolescent HTx recipients and their parents is crucial to planning effective transitional care and necessary for evidenced-based practice.

Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains.

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 degrees S and 5 degrees N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).