What health problems overcrowd hospital emergency departments in Queensland and has this changed over time? A case study of two hospitals in Queensland 2001 - 2009

Aim: to describe what health problems patients attending emergency department with and whether this changed over time. Methods: Electronic data was retrieved from EDIS (Emergency Department Information System) and HBCIS (Hospital Based Clinical Information System) in two hospitals in Queensland in the period 2001-2009. The ICD-10 code of patient's diagnosis was then extrapolated and then group into ICD-10 chapters, such that the health problem can be presented. Results: Among the specific health problems, Chapter XIX 'Injury and poisoning' ranked number one consistently (ranging from 22.1% to 31.2% of the total presentations) in both the urban and remote hospitals in Queensland. The top ten specific presenting health problems in both the urban and remote hospital include Chapter XI 'Digestive system', Chapter XIV 'Genitourinary system', Chapter IX 'Circulatory system', and Chapter XIII 'Musculoskeletal system and connective tissue'. Chapter X 'Respiratory system' made the top ten presenting Chapters in both hospitals, but ranked much higher (number four consistently for the eight years, ranging from 6.8% to 8.3%) in the remote hospital. Chapter XV 'Pregnancy childbirth and puerperium' made to the top ten in the urban hospital only while Chapter XII 'Skin and subcutaneous tissue', Chapter I 'Infectious and parasitic diseases' made the top ten in the remote hospital only. Conclusion: The number one health problem presenting to both the urban and remote hospitals in Queensland is Chapter XIX 'Injury and poisoning', and it did not change in the period 211 - 2009.

Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Age of blood and recipient factors determine the severity of transfusion-related acute lung injury (TRALI)

Introduction Critical care patients frequently receive blood transfusions. Some reports show an association between aged or stored blood and increased morbidity and mortality, including the development of transfusion-related acute lung injury (TRALI). However, the existence of conflicting data endorses the need for research to either reject this association, or to confirm it and elucidate the underlying mechanisms. Methods Twenty-eight sheep were randomised into two groups, receiving saline or lipopolysaccharide (LPS). Sheep were further randomised to also receive transfusion of pooled and heat-inactivated supernatant from fresh (Day 1) or stored (Day 42) non-leucoreduced human packed red blood cells (PRBC) or an infusion of saline. TRALI was defined by hypoxaemia during or within two hours of transfusion and histological evidence of pulmonary oedema. Regression modelling compared physiology between groups, and to a previous study, using stored platelet concentrates (PLT). Samples of the transfused blood products also underwent cytokine array and biochemical analyses, and their neutrophil priming ability was measured in vitro. Results TRALI did not develop in sheep that first received saline-infusion. In contrast, 80% of sheep that first received LPS-infusion developed TRALI following transfusion with "stored PRBC." The decreased mean arterial pressure and cardiac output as well as increased central venous pressure and body temperature were more severe for TRALI induced by "stored PRBC" than by "stored PLT." Storage-related accumulation of several factors was demonstrated in both "stored PRBC" and "stored PLT", and was associated with increased in vitro neutrophil priming. Concentrations of several factors were higher in the "stored PRBC" than in the "stored PLT," however, there was no difference to neutrophil priming in vitro. Conclusions In this in vivo ovine model, both recipient and blood product factors contributed to the development of TRALI. Sick (LPS infused) sheep rather than healthy (saline infused) sheep predominantly developed TRALI when transfused with supernatant from stored but not fresh PRBC. "Stored PRBC" induced a more severe injury than "stored PLT" and had a different storage lesion profile, suggesting that these outcomes may be associated with storage lesion factors unique to each blood product type. Therefore, the transfusion of fresh rather than stored PRBC may minimise the risk of TRALI.

Pandemic (H1N1) Influenza 2009 and Australian emergency departments : implications for policy, practice and pandemic preparedness

Objective: To describe the reported impact of Pandemic (H1N1) 2009 on EDs, so as to inform future pandemic policy, planning and response management. Methods: This study comprised an issue and theme analysis of publicly accessible literature, data from jurisdictional health departments, and data obtained from two electronic surveys of ED directors and ED staff. The issues identified formed the basis of policy analysis and evaluation. Results: Pandemic (H1N1) 2009 had a significant impact on EDs with presentation for patients with ‘influenza-like illness’ up to three times that of the same time in previous years. Staff reported a range of issues, including poor awareness of pandemic plans, patient and family aggression, chaotic information flow to themselves and the public, heightened stress related to increased workloads and lower levels of staffing due to illness, family care duties and redeployment of staff to flu clinics. Staff identified considerable discomfort associated with prolonged times wearing personal protective equipment. Staff believed that the care of non-flu patients was compromised during the pandemic as a result of overwork, distraction from core business and the difficulties associated with accommodating infectious patients in an environment that was not conducive. Conclusions: This paper describes the breadth of the impact of pandemics on ED operations. It identifies a need to address a range of industrial, management and procedural issues. In particular, there is a need for a single authoritative source of information, the re-engineering of EDs to accommodate infectious patients and organizational changes to enable rapid deployment of alternative sources of care.

The effect of Tenofovir on vitamin D metabolism in HIV-infected adults is dependent on sex and ethnicity

Background: Tenofovir has been associated with renal phosphate wasting, reduced bone mineral density, and higher parathyroid hormone levels. The aim of this study was to carry out a detailed comparison of the effects of tenofovir versus non-tenofovir use on calcium, phosphate and, vitamin D, parathyroid hormone (PTH), and bone mineral density. Methods: A cohort study of 56 HIV-1 infected adults at a single centre in the UK on stable antiretroviral regimes comparing biochemical and bone mineral density parameters between patients receiving either tenofovir or another nucleoside reverse transcriptase inhibitor. Principal Findings: In the unadjusted analysis, there was no significant difference between the two groups in PTH levels (tenofovir mean 5.9 pmol/L, 95% confidence intervals 5.0 to 6.8, versus non-tenofovir; 5.9, 4.9 to 6.9; p = 0.98). Patients on tenofovir had significantly reduced urinary calcium excretion (median 3.01 mmol/24 hours) compared to non-tenofovir users (4.56; p,0.0001). Stratification of the analysis by age and ethnicity revealed that non-white men but not women, on tenofovir had higher PTH levels than non-white men not on tenofovir (mean difference 3.1 pmol/L, 95% CI 5.3 to 0.9; p = 0.007). Those patients with optimal 25-hydroxyvitamin D (.75 nmol/L) on tenofovir had higher 1,25-dihydroxyvitamin D [1,25(OH)2D] (median 48 pg/mL versus 31; p = 0.012), fractional excretion of phosphate (median 26.1%, versus 14.6;p = 0.025) and lower serum phosphate (median 0.79 mmol/L versus 1.02; p = 0.040) than those not taking tenofovir. Conclusions: The effects of tenofovir on PTH levels were modified by sex and ethnicity in this cohort. Vitamin D status also modified the effects of tenofovir on serum concentrations of 1,25(OH)2D and phosphate.

Vaccination to protect against infection of the female reproductive tract

Infection of the female genital tract can result in serious morbidities and mortalities from reproductive disability, pelvic inflammatory disease and cancer, to impacts on the fetus, such as infant blindness. While therapeutic agents are available, frequent testing and treatment is required to prevent the occurrence of the severe disease sequelae. Hence, sexually transmitted infections remain a major public health burden with ongoing social and economic barriers to prevention and treatment. Unfortunately, while there are two success stories in the development of vaccines to protect against HPV infection of the female reproductive tract, many serious infectious agents impacting on the female reproductive tract still have no vaccines available. Vaccination to prevent infection of the female reproductive tract is an inherently difficult target, with many impacting factors, such as appropriate vaccination strategies/mechanisms to induce a suitable protective response locally in the genital tract, variation in the local immune responses due to the hormonal cycle, selection of vaccine antigen(s) that confers effective protection against multiple variants of a single pathogen (e.g., the different serovars of Chlamydia trachomatis) and timing of the vaccine administration prior to infection exposure. Despite these difficulties, there are numerous ongoing efforts to develop effective vaccines against these infectious agents and it is likely that this important human health field will see further major developments in the next 5 years.

Correction of step artefact associated with MRI scanning of long bones

3D models of long bones are being utilised for a number of fields including orthopaedic implant design. Accurate reconstruction of 3D models is of utmost importance to design accurate implants to allow achieving a good alignment between two bone fragments. Thus for this purpose, CT scanners are employed to acquire accurate bone data exposing an individual to a high amount of ionising radiation. Magnetic resonance imaging (MRI) has been shown to be a potential alternative to computed tomography (CT) for scanning of volunteers for 3D reconstruction of long bones, essentially avoiding the high radiation dose from CT. In MRI imaging of long bones, the artefacts due to random movements of the skeletal system create challenges for researchers as they generate inaccuracies in the 3D models generated by using data sets containing such artefacts. One of the defects that have been observed during an initial study is the lateral shift artefact occurring in the reconstructed 3D models. This artefact is believed to result from volunteers moving the leg during two successive scanning stages (the lower limb has to be scanned in at least five stages due to the limited scanning length of the scanner). As this artefact creates inaccuracies in the implants designed using these models, it needs to be corrected before the application of 3D models to implant design. Therefore, this study aimed to correct the lateral shift artefact using 3D modelling techniques. The femora of five ovine hind limbs were scanned with a 3T MRI scanner using a 3D vibe based protocol. The scanning was conducted in two halves, while maintaining a good overlap between them. A lateral shift was generated by moving the limb several millimetres between two scanning stages. The 3D models were reconstructed using a multi threshold segmentation method. The correction of the artefact was achieved by aligning the two halves using the robust iterative closest point (ICP) algorithm, with the help of the overlapping region between the two. The models with the corrected artefact were compared with the reference model generated by CT scanning of the same sample. The results indicate that the correction of the artefact was achieved with an average deviation of 0.32 ± 0.02 mm between the corrected model and the reference model. In comparison, the model obtained from a single MRI scan generated an average error of 0.25 ± 0.02 mm when compared with the reference model. An average deviation of 0.34 ± 0.04 mm was seen when the models generated after the table was moved were compared to the reference models; thus, the movement of the table is also a contributing factor to the motion artefacts.

Optimisation of Banana streak virus (BSV) diagnostic assays

Bananas are one of the world�fs most important crops, serving as a staple food and an important source of income for millions of people in the subtropics. Pests and diseases are a major constraint to banana production. To prevent the spread of pests and disease, farmers are encouraged to use disease�] and insect�]free planting material obtained by micropropagation. This option, however, does not always exclude viruses and concern remains on the quality of planting material. Therefore, there is a demand for effective and reliable virus indexing procedures for tissue culture (TC) material. Reliable diagnostic tests are currently available for all of the economically important viruses of bananas with the exception of Banana streak viruses (BSV, Caulimoviridae, Badnavirus). Development of a reliable diagnostic test for BSV is complicated by the significant serological and genetic variation reported for BSV isolates, and the presence of endogenous BSV (eBSV). Current PCR�] and serological�]based diagnostic methods for BSV may not detect all species of BSV, and PCR�]based methods may give false positives because of the presence of eBSV. Rolling circle amplification (RCA) has been reported as a technique to detect BSV which can also discriminate between episomal and endogenous BSV sequences. However, the method is too expensive for large scale screening of samples in developing countries, and little information is available regarding its sensitivity. Therefore the development of reliable PCR�]based assays is still considered the most appropriate option for large scale screening of banana plants for BSV. This MSc project aimed to refine and optimise the protocols for BSV detection, with a particular focus on developing reliable PCR�]based diagnostics Initially, the appropriateness and reliability of PCR and RCA as diagnostic tests for BSV detection were assessed by testing 45 field samples of banana collected from nine districts in the Eastern region of Uganda in February 2010. This research was also aimed at investigating the diversity of BSV in eastern Uganda, identifying the BSV species present and characterising any new BSV species. Out of the 45 samples tested, 38 and 40 samples were considered positive by PCR and RCA, respectively. Six different species of BSV, namely Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), were detected by PCR and confirmed by RCA and sequencing. No new species were detected, but this was the first report of BSMYV in Uganda. Although RCA was demonstrated to be suitable for broad�]range detection of BSV, it proved time�]consuming and laborious for identification in field samples. Due to the disadvantages associated with RCA, attempts were made to develop a reliable PCR�]based assay for the specific detection of episomal BSOLV, Banana streak GF virus (BSGFV), BSMYV and BSIMV. For BSOLV and BSGFV, the integrated sequences exist in rearranged, repeated and partially inverted portions at their site of integration. Therefore, for these two viruses, primers sets were designed by mapping previously published sequences of their endogenous counterparts onto published sequences of the episomal genomes. For BSOLV, two primer sets were designed while, for BSGFV, a single primer set was designed. The episomalspecificity of these primer sets was assessed by testing 106 plant samples collected during surveys in Kenya and Uganda, and 33 leaf samples from a wide range of banana cultivars maintained in TC at the Maroochy Research Station of the Department of Employment, Economic Development and Innovation (DEEDI), Queensland. All of these samples had previously been tested for episomal BSV by RCA and for both BSOLV and BSGFV by PCR using published primer sets. The outcome from these analyses was that the newly designed primer sets for BSOLV and BSGFV were able to distinguish between episomal BSV and eBSV in most cultivars with some B�]genome component. In some samples, however, amplification was observed using the putative episomal�]specific primer sets where episomal BSV was not identified using RCA. This may reflect a difference in the sensitivity of PCR compared to RCA, or possibly the presence of an eBSV sequence of different conformation. Since the sequences of the respective eBSV for BSMYV and BSIMV in the M. balbisiana genome are not available, a series of random primer combinations were tested in an attempt to find potential episomal�]specific primer sets for BSMYV and BSIMV. Of an initial 20 primer combinations screened for BSMYV detection on a small number of control samples, 11 primers sets appeared to be episomal�]specific. However, subsequent testing of two of these primer combinations on a larger number of control samples resulted in some inconsistent results which will require further investigation. Testing of the 25 primer combinations for episomal�]specific detection of BSIMV on a number of control samples showed that none were able to discriminate between episomal and endogenous BSIMV. The final component of this research project was the development of an infectious clone of a BSV endemic in Australia, namely BSMYV. This was considered important to enable the generation of large amounts of diseased plant material needed for further research. A terminally redundant fragment (.1.3 �~ BSMYV genome) was cloned and transformed into Agrobacterium tumefaciens strain AGL1, and used to inoculate 12 healthy banana plants of the cultivars Cavendish (Williams) by three different methods. At 12 weeks post�]inoculation, (i) four of the five banana plants inoculated by corm injection showed characteristic BSV symptoms while the remaining plant was wilting/dying, (ii) three of the five banana plants inoculated by needle�]pricking of the stem showed BSV symptoms, one plant was symptomless while the remaining had died and (iii) both banana plants inoculated by leaf infiltration were symptomless. When banana leaf samples were tested for BSMYV by PCR and RCA, BSMYV was confirmed in all banana plants showing symptoms including those were wilting and/or dying. The results from this research have provided several avenues for further research. By completely sequencing all variants of eBSOLV and eBSGFV and fully sequencing the eBSIMV and eBSMYV regions, episomal BSV�]specific primer sets for all eBSVs could potentially be designed that could avoid all integrants of that particular BSV species. Furthermore, the development of an infectious BSV clone will enable large numbers of BSVinfected plants to be generated for the further testing of the sensitivity of RCA compared to other more established assays such as PCR. The development of infectious clones also opens the possibility for virus induced gene silencing studies in banana.

Using the collaborative evidence based practice model : a systematic review and uptake of chlorhexidine-impregnated sponge dressings on central venous access devices in a tertiary cancer centre

Background: Greater research utilisation in cancer nursing practice is needed, in order to provide well-informed and effective nursing care to people affected by cancer. This paper aims to report on the implementation of evidence-based practice in a tertiary cancer centre. Methods: Using a case report design, this paper reports on the use of the Collaborative Model for Evidence Based Practice (CMEBP) in an Australian tertiary cancer centre. The clinical case is the uptake of routine application of chlorhexidine-impregnated sponge dressings for preventing centrally inserted catheter-related bloodstream infections. In this case report, a number of processes that resulted in a service-wide practice change are described. Results: This model was considered a feasible method for successful research utilisation. In this case report, chlorhexidine-impregnated sponge dressings were proposed and implemented in the tertiary cancer centre with an aim of reducing the incidence of centrally inserted catheter-related bloodstream infections and potentially improving patient health outcomes. Conclusion: The CMEBP is feasible and effective for implementing clinical evidence into cancer nursing practice. Cancer nurses and health administrators need to ensure a supportive infrastructure and environment for clinical inquiry and research utilisation exists, in order to enable successful implementation of evidence-based practice in their cancer centres.

Evidence of unique genotypes of Beak and feather disease virus in Southern Africa

Psittacine beak and feather disease (PBFD), caused by Beak and feather disease virus (BFDV), is the most significant infectious disease in psittacines. PBFD is thought to have originated in Australia but is now found worldwide; in Africa, it threatens the survival of the indigenous endangered Cape parrot and the vulnerable black-cheeked lovebird. We investigated the genetic diversity of putative BFDVs from southern Africa. Feathers and heparinized blood samples were collected from 27 birds representing 9 psittacine species, all showing clinical signs of PBFD. DNA extracted from these samples was used for PCR amplification of the putative BFDV coat protein (CP) gene. The nucleotide sequences of the CP genes of 19 unique BFDV isolates were determined and compared with the 24 previously described sequences of BFDV isolates from Australasia and America. Phylogenetic analysis revealed eight BFDV lineages, with the southern African isolates representing at least three distinctly unique genotypes; 10 complete genome sequences were determined, representing at least one of every distinct lineage. The nucleotide diversity of the southern African isolates was calculated to be 6.4% and is comparable to that found in Australia and New Zealand. BFDVs in southern Africa have, however, diverged substantially from viruses found in other parts of the world, as the average distance between the southern African isolates and BFDV isolates from Australia ranged from 8.3 to 10.8%. In addition to point mutations, recombination was found to contribute substantially to the level of genetic variation among BFDVs, with evidence of recombination in all but one of the genomes analyzed.

A three-nucleotide mutation altering the Maize streak virus Rep pRBR-interaction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR-Rep binding

Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA-pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA-pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover. © 2005 SGM.

Rapid host adaptation by extensive recombination

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature. © 2009 SGM.

Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan

Most mastreviruses (family Geminiviridae) infect monocotyledonous hosts and are transmitted by leafhopper vectors. Only two mastrevirus species, Tobacco yellow dwarf virus from Australia and Bean yellow dwarf virus (BeYDV) from South Africa, have been identified whose members infect dicotyledonous plants. We have identified two distinct mastreviruses in chickpea stunt disease (CSD)-affected chickpea originating from Pakistan. The first is an isolate of BeYDV, previously only known to occur in South Africa. The second is a member of a new species with the BeYDV isolates as its closest relatives. A PCR-based diagnostic test was developed to differentiate these two virus species. Our results show that BeYDV plays no role in the etiology of CSD in Pakistan, while the second virus occurs widely in chickpea across Pakistan. A genomic clone of the new virus was infectious to chickpea (Cicer arietinum L.) and induced symptoms typical of CSD. We propose the use of the name Chickpea chlorotic dwarf Pakistan virus for the new species. The significance of these findings with respect to our understanding of the evolution, origin and geographic spread of dicot-infecting mastreviruses is discussed. © 2008 Springer-Verlag.

Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants

Maize streak disease is a severe agricultural problem in Africa and the development of maize genotypes resistant to the causal agent, Maize streak virus (MSV), is a priority. A transgenic approach to engineering MSV-resistant maize was developed and tested in this study. A pathogen-derived resistance strategy was adopted by using targeted deletions and nucleotide-substitution mutants of the multifunctional MSV replication-associated protein gene (rep). Various rep gene constructs were tested for their efficacy in limiting replication of wild-type MSV by co-bombardment of maize suspension cells together with an infectious genomic clone of MSV and assaying replicative forms of DNA by quantitative PCR. Digitaria sanguinalis, an MSV-sensitive grass species used as a model monocot, was then transformed with constructs that had inhibited virus replication in the transient-expression system. Challenge experiments using leafhopper-transmitted MSV indicated significant MSV resistance - from highly resistant to immune - in regenerated transgenic D. sanguinalis lines. Whereas regenerated lines containing a mutated full-length rep gene displayed developmental and growth defects, those containing a truncated rep gene both were fertile and displayed no growth defects, making the truncated gene a suitable candidate for the development of transgenic MSV-resistant maize. © 2007 SGM.