The symptom and genetic diversity of cassava brown streak viruses infecting cassava in East Africa

The genetic and symptom diversity of six virus isolates causing cassava brown streak disease (CBSD) in the endemic (Kenya, Mozambique, and Tanzania) and the recently affected epidemic areas (Uganda) of eastern Africa was studied. Five cassava varieties; Albert, Colombian, Ebwanateraka, TMS60444 (all susceptible) and Kiroba (tolerant) were graft inoculated with each isolate. Based on a number of parameters including the severity of leaf and root symptoms, and the extent of virus transmission by grafting, the viruses were classified as either severe or relatively mild. These results were further confirmed by the mechanical inoculation of 13 herbaceous hosts in which the virulent isolates caused plant death in Nicotiana clevelandii and N. benthamiana whereas the milder isolates did not. Phylogenetic analysis of complete coat protein gene sequences of these isolates together with sequences obtained from 14 other field-collected samples from Kenya and Zanzibar, and reference sequences grouped them into two distinct clusters, representing the two species of cassava brown streak viruses. Put together, these results did not suggest the association of a hypervirulent form of the virus with the current CBSD epidemic in Uganda. Identification of the severe and milder isolates, however, has further implications for disease management and quarantine requirements

The role of the whitefly, Bemisia tabaci (Gennadius), and farmer practices in the spread of cassava brown streak ipomoviruses

Cassava brown streak disease (CBSD) is arguably the most dangerous current threat to cassava, which is Africa's most important food security crop. CBSD is caused by two RNA viruses: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). The roles of the whitefly Bemisia tabaci (Gennadius) and farmer practices in the spread of CBSD were investigated in a set of field and laboratory experiments. The virus was acquired and transmitted by B. tabaci within a short time (5–10 min each for virus acquisition and inoculation), and was retained for up to 48 hr. Highest virus transmission (60%) was achieved using 20–25 suspected viruliferous whiteflies per plant that were given acquisition and inoculation periods of 24 and 48 hr, respectively. Experiments mimicking the agronomic practices of cassava leaf picking or the use of contaminated tools for making cassava stem cuttings did not show the transmission of CBSV or UCBSV. Screenhouse and field experiments in Tanzania showed that the spread of CBSD next to spreader rows was high, and that the rate of spread decreased with increasing distance from the source of inoculum. The disease spread in the field up to a maximum of 17 m in a cropping season. These results collectively confirm that CBSV and UCBSV are transmitted by B. tabaci semipersistently, but for only short distances in the field. This implies that spread over longer distances is due to movements of infected stem cuttings used for planting material. These findings have important implications for developing appropriate management strategies for CBSD

Molecular diagnostics, genetic diversity and generating infectious clones for cassava brown streak viruses

Cassava brown streak disease (CBSD) threatens cassava production in eastern and southern African countries. Diagnostic protocols currently available for the causal agents of CBSD, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV), were unreliable but were urgently needed. In this study, sampling procedures and diagnostic protocols were developed for accurate and reliable detection of both CBSV and CBSUV. The cetyltrimethylammonium bromide (CTAB) method of RNA extraction was optimized for sample preparation from infected cassava plants and compared with the commercial kit RNeasy (Qiagen) for sensitivity and reproducibility. Results showed that both protocols were reliable but CTAB was more cost-effective and ideal for resource-poor laboratories. Mixed infections of cassava mosaic begomoviruses (CMBs) that cause cassava mosaic disease (CMD), CBSV and CBSUV have become more common with the recent spread of CBSD at mid-altitudes. A multiplex PCR for the simultaneous detection of viruses that cause both diseases, the first of its kind for cassava, was therefore developed to detect CBSV and CBSUV along with the three commonly occurring CMBs (African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), and East African cassava mosaic virus-Uganda (EACMVUG) in eastern Africa. Similarly, a duplex PCR was developed for the simultaneous detection of CBSV and CBSUV, both viruses being detected in field-collected samples from Tanzania and Kenya. The genetic diversity of more than 40 CBSD isolates from Kenya, Tanzania, Uganda, and Mozambique was further examined by sequencing the coat protein (CP) gene and partial HAM1 gene sequences. The phylogenetic tree clustered the CBSD isolates into two groups reflecting the two virus species causing CBSD. In this study, various strategies were carried out for generating infectious clones of CBSV; gateway cloning, in vivo and in vitro transcription methods, and amplification of the viral genome in three fragments. Although 3 overlapping CBSV fragments were successfully cloned, the presence of an unexpected mutation at one of the cloning sites unfortunately did not allow reassembling of the fragments to construct the full-length cDNA

Towards a standardised approach for evaluating guidelines and guidance documents on palliative sedation: Study protocol

Background: Sedation in palliative care has received growing attention in recent years; and so have guidelines, position statements, and related literature that provide recommendations for its practice. Yet little is known collectively about the content, scope and methodological quality of these materials. According to research, there are large variations in palliative sedation practice, depending on the definition and methodology used. However, a standardised approach to comparing and contrasting related documents, across countries, associations and governmental bodies is lacking. This paper reports on a protocol designed to enable thorough and systematic comparison of guidelines and guidance documents on palliative sedation. Methods and design. A multidisciplinary and international group of palliative care researchers, identified themes and clinical issues on palliative sedation based on expert consultations and evidence drawn from the EAPC (European Association of Palliative Care) framework for palliative sedation and AGREE II (Appraisal Guideline Research and Evaluation) instrument for guideline assessment. The most relevant themes were selected and built into a comprehensive checklist. This was tested on people working closely with practitioners and patients, for user-friendliness and comprehensibility, and modified where necessary. Next, a systematic search was conducted for guidelines in English, Dutch, Flemish, or Italian. The search was performed in multiple databases (PubMed, CancerLit, CNAHL, Cochrane Library, NHS Evidence and Google Scholar), and via other Internet resources. Hereafter, the final version of the checklist will be used to extract data from selected literature, and the same will be compiled, entered into SPSS, cleaned and analysed systematically for publication. Discussion. We have together developed a comprehensive checklist in a scientifically rigorous manner to allow standardised and systematic comparison. The protocol is applicable to all guidelines on palliative sedation, and the approach will contribute to rigorous and systematic comparison of international guidelines on any challenging topic such as this. Results from the study will provide valuable insights into common core elements and differences between the selected guidelines, and the extent to which recommendations are derived from, or match those in the EAPC framework. The outcomes of the study will be disseminated via peer-reviewed journals and directly to appropriate audiences

Host and virus effects on reversion in cassava affected by cassava brown streak disease

The phenomenon of virus-infected plants naturally recovering health is known as ‘reversion’, and is a type of resistance mechanism exploited in some crop plants for disease control. Various parameters were investigated that affect reversion from cassava brown streak disease (CBSD) in three cassava varieties (Albert, Kaleso and Kiroba) that differ in levels of resistance to the disease. Cassava plants were inoculated by grafting with two virus species (Ugandan cassava brown streak virus, UCBSV and Cassava brown streak virus, CBSV) that cause CBSD, and the plants grown from them were subsequently assessed for reversion. The rate of reversion depended on the cassava variety, virus species, and the length and position of the stem cuttings used. A significantly high proportion of progenies were virus-free (reverted) for the resistant variety Kaleso (64·1% for UCBSV and 54·9% of CBSV), compared to the tolerant variety Kiroba (56·7 and 45·5%) and the susceptible control Albert (38·9 and 35·1%). The highest number of virus-free plants was generated from short 10 cm long cuttings (e.g. 60·1% for Kaleso for CBSV) compared to 20 cm long stem cuttings (e.g. 21·4% for Albert). Cuttings taken from upper stems of diseased plants produced most virus-free progenies compared to middle and lower parts. More than 50% virus-free plants were obtained in the resistant and tolerant varieties. This is a highly valuable finding and could be exploited for developing strategies to control the current CBSD epidemic in eastern and central Africa

Molecular identification and sequence analysis of bipartite Begomovirus infecting Horsegram legume in India

The molecular diversity of Begomovirus infecting Horsegram yellow Mosaic viruses (HgYMV1 and HgYMV2), from two main horsegram growing farms near Bangalore, Karnataka State, South India was investigated. The viral DNA was amplified from horsegram plants exhibiting mild and severe symptoms by polymerase chain reaction, and complete genome of the HgYMV were identified by their sequence analysis. Isolates of HgYMV1 and HgYMV2 were found to be associated with severe symptom phenotype from HgYMV. HgYMV was most closely related to Mungbean yellow mosaic indian viruse (MYMIV) and Mungbean yellow mosaic virus (MYMV) at 81.8 to 84.8 % nucleotide identity, based on DNA-A and DNA-B component sequences. HgYMV was distantly related to Dolicos yellow mosaic virus from Asia (DoYMV-Ban and DoYMV-DB) and partially to Cowpea golden mosaic virus from Nigeria (CPGMV-[NG]) at 64 and 62 % DNA nucleotide identity. Analysis of the DNA-B sequence of HgYMV revealed a DNA-B component identical to those of Bean golden yellow mosaic virus BGYMV isolates described. Furthermore, the DNA-B component for extant BGYMV isolates and strains were also the closest relatives for the HgYMV1 DNA-B components at 48.7 % nucleotide identity. Therefore, HgYMV could be considered to be a new species of the genus Begomovirus (family Geminiviridae).Keywords: Begomovirus, Horsegram, yellow Mosaic viruses, DNA sequencin

Testing the infectivity of a Begomovirus by particle bombardment method using a gene gun

This study was design to identify the causal agent of Horsegram yellow mosaic disease and to investigate the pathogenicity of Horsegram yellow mosaic viruses (HgYMV) infective clones. The samples were obtained using standard method from the two main horsegram growing areas of Bangalore, Karnataka State of India. The viral DNA from horsegram plants exhibiting severe symptoms was amplified by PCR. An isolate of HgYMV1 and HgYMV2 were associated with severe symptom phenotype from HgYMV. Full-length clones of DNA-A and DNA-B genomic components were constructed and attempts were made to introduce homologous (HgYMV1/HgYMV2) combinations of DNA-A and DNA-B genomic components into Nicotiana benthamiana plants. Inoculation of linearized constructs containing full-length clones or partial head-to-tail dimers of DNA-A and DNAB genomic components resulted in the introduction of DNA-A genomic components into the host plant. However, these combinations of genomic DNA component were not detected in the inoculated plants bombarded using the gene gun. Thus, this study was unable to confirm the pathogenicity of HgYMV infective clones using N. benthamiana as model plant.Keywords: Horsegram, Begomovirus, yellow mosaic viruses, Particle bombardment, Gene gu

Transitions at the end of life for older adults - patient, carer and professional perspectives

BackgroundThe end of life may be a time of high service utilisation for older adults. Transitions between care settings occur frequently, but may produce little improvement in symptom control or quality of life for patients. Ensuring that patients experience co-ordinated care, and moves occur because of individual needs rather than system imperatives, is crucial to patients’ well-being and to containing health-care costs.ObjectiveThe aim of this study was to understand the experiences, influences and consequences of transitions between settings for older adults at the end of life. Three conditions were the focus of study, chosen to represent differing disease trajectories.SettingEngland.ParticipantsThirty patients aged over 75 years, in their last year of life, diagnosed with heart failure, lung cancer and stroke; 118 caregivers of decedents aged 66–98 years, who had died with heart failure, lung cancer, stroke, chronic obstructive pulmonary disease or selected other cancers; and 43 providers and commissioners of services in primary care, hospital, hospice, social care and ambulance services.Design and methodsThis was a mixed-methods study, composed of four parts: (1) in-depth interviews with older adults; (2) qualitative interviews and structured questionnaire with bereaved carers of older adult decedents; (3) telephone interviews with care commissioners and providers using case scenarios derived from the interviews with carers; and (4) analysis of linked Hospital Episode Statistics (HES) and mortality data relating to hospital admissions for heart failure and lung cancer in England 2001–10.ResultsTransitions between care settings in the last year of life were a common component of end-of-life care across all the data sets that made up this study, and many moves were made shortly before death. Patients’ and carers’ experiences of transitions were of a disjointed system in which organisational processes were prioritised over individual needs. In many cases, the family carer was the co-ordinator and provider of care at home, excluded from participation in institutional care but lacking the information and support to extend their role with confidence. The general practitioner (GP) was a valued, central figure in end-of-life care across settings, though other disciplines were critical of GPs’ expertise and adherence to guidelines. Out-of-hours services and care homes were identified by many as contributors to unnecessary transitions. Good relationships and communication between professionals in different settings and sectors was recognised by families as one of the most important influences on transitions but this was rarely acknowledged by staff.ConclusionsDevelopment of a shared understanding of professional and carer roles in end-of-life transitions may be one of the most effective ways of improving patients’ experiences. Patients and carers manage many aspects of end-of-life care for themselves. Identifying ways to extend their skills and strengthen their voices, particularly in hospital settings, would be welcomed and may reduce unnecessary end-of-life transitions. Why the experiences of carers appear to have changed little, despite the implementation of a range of relevant policies, is an important question that has not been answered. Recommendations for future research include the relationship between policy interventions and the experiences of end-of-life carers; identification of ways to harmonise understanding of the carers’ role and strengthen their voice, particularly in hospital settings; identification of ways to reduce the influence of interprofessional tensions in end-of-life care; and development of interventions to enhance patients’ experiences across transitions.FundingThe National Institute for Health Research Health Services and Delivery Research programme

What is case management in palliative care? An expert panel study

Contains fulltext : 110207.pdf (publisher's version ) (Open Access)ABSTRACT: BACKGROUND: Case management is a heterogeneous concept of care that consists of assessment, planning, implementing, coordinating, monitoring, and evaluating the options and services required to meet the client's health and service needs. This paper describes the result of an expert panel procedure to gain insight into the aims and characteristics of case management in palliative care in the Netherlands. METHODS: A modified version of the RAND(R)/University of California at Los Angeles (UCLA) appropriateness method was used to formulate and rate a list of aims and characteristics of case management in palliative care. A total of 76 health care professionals, researchers and policy makers were invited to join the expert panel, of which 61% participated in at least one round. RESULTS: Nine out of ten aims of case management were met with agreement. The most important areas of disagreement with regard to characteristics of case management were hands-on nursing care by the case manager, target group of case management, performance of other tasks besides case management and accessibility of the case manager. CONCLUSIONS: Although aims are agreed upon, case management in palliative care shows a high level of variability in implementation choices. Case management should aim at maintaining continuity of care to ensure that patients and those close to them experience care as personalised, coherent and consistent

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only requires a single incubation temperature (optimum 37 °C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories