Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to Fusarium oxysporum f.sp. cubense race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

Rapid Global Expansion of the Fungal Disease Chytridiomycosis into Declining and Healthy Amphibian Populations.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance.

Global banana disease management - getting serious with sustainability and food security

Much research in understanding plant diseases has been undertaken, but there has been insufficient attention given to dealing with coordinated approaches to preventing and managing diseases. A global management approach is essential to the long-term sustainability of banana production. This approach would involve coordinated surveys, capacity building in developing countries, development of disease outbreak contingency plans and coordinated quarantine awareness, including on-line training in impact risk assessment and web-based diagnostic software. Free movement of banana plants and products between some banana-producing countries is causing significant pressure on the ability to manage diseases in banana. The rapid spread of Fusarium oxysporum f. sp. cubense 'tropical race 4' in Asia, bacterial wilts in Africa and Asia and black leaf streak [Mycosphaerella fijiensis] in Brazil and elsewhere are cases in point. The impact of these diseases is devastating, severely cutting family incomes and jeopardising food security around the globe. Agreements urgently need to be reached between governments to halt the movement of banana plants and products between banana-producing countries before it is too late and global food security is irreparably harmed. Black leaf streak, arguably the most serious banana disease, has become extremely difficult to control in commercial plantations in various parts of the world. Sometimes in excess of 50 fungicide sprays have to be applied each year. Disease eradication and effective disease control is not possible because there is no control of disease inoculum in non-commercial plantings in these locations. Additionally, there have been enormous sums of money invested in international banana breeding programmes over many years only to see the value of hybrid products lost too soon. 'Goldfinger' (AAAB, syn. 'FHIA-01'), for example, has recently been observed severely affected by black leaf streak in Samoa. Resistant cultivars alone cannot be relied upon in the fight against this disease. Real progress in control may only come when the local communities are engaged and become actively involved in regional programmes. Global recommendations are long overdue and urgently needed to help ensure the long-term sustainable utilisation of the products of the breeding programmes.

Controlled exposure as a management tool for Glasser's disease.

In this study, nasal swabs taken from multiparous sows at weaning time or from sick pigs displaying symptoms of Glasser's disease from farms in Australia [date not given] were cultured and analysed by polymerase chain reaction (PCR). Within each genotype detected on a farm, representative isolates were serotyped by gel diffusion (GD) testing or indirect haemagglutination (IHA) test. Isolates which did not react in any of the tests were regarded as non-typable and were termed serovar NT. Serovars 1, 5, 12, 13 and 14 were classified as highly pathogenic; serovars 2, 4 and 15 being moderately pathogenic; serovar 8 being slightly pathogenic and serovars 3, 6, 7, 9 and 11 being non-pathogenic. Sows were inoculated with the strain of Haemophilus parasuis (serovars 4, 6 and 9 from Farms 1, 2 and 4, respectively) used for controlled challenge 3 and 5 weeks before farrowing. Before farrowing the sows were divided into control and treatment groups. Five to seven days after birth, the piglets of the treatment group were challenged with a strain from the farm which had were used to vaccinate the sows. The effectiveness of the controlled exposure was evaluated by number of piglets displaying clinical signs possibly related to infection, number of antibiotic treatments and pig mortality. Nasal swabs of sick pigs were taken twice a week to find a correlation to infection. A subsample of pigs was weighed after leaving the weaning sheds. The specificity of a realtime PCR amplifying the infB gene was evaluated with 68 H. parasuis isolates and 36 strains of closely related species. 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were also tested with the realtime PCR, and the results compared with culture and a conventional PCR. The farm experiments showed that none of the controlled challenge pigs showed any signs of illness due to Glasser's disease, although the treatment groups required more antibiotics than the controls. A total of 556 H. parasuis isolates were genotyped, while 150 isolates were serotyped. H. parasuis was detected on 19 of 20 farms, including 2 farms with an extensive history of freedom from Glasser's disease. Isolates belonging to serovars regarded as potentially pathogenic were obtained from healthy pigs at weaning on 8 of the 10 farms with a history of Glasser's disease outbreaks. Sampling 213 sick pigs yielded 115 isolates, 99 of which belonged to serovars that were either potentially pathogenic or of unknown pathogenicity. Only 16 isolates from these sick pigs were of a serovar known to be non-pathogenic. Healthy pigs also had H. parasuis, even on farms free of Glasser's disease. The realtime PCR gave positive results for all 68 H. parasuis isolates and negative results for all 36 non-target bacteria. When used on the clinical material from experimental infections, the realtime PCR produced significantly more positive results than the conventional PCR (165 compared to 86).

Zebrafish as a model of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. Its etiology is unknown and no disease-modifying drugs are available. Thus, more information concerning its pathogenesis is needed. Among other genes, mutated PTEN-induced kinase 1 (PINK1) has been linked to early-onset and sporadic PD, but its mode of action is poorly understood. Most animal models of PD are based on the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to MPP+ by monoamine oxidase B (MAO B) and causes cell death of dopaminergic neurons in the substantia nigra in mammals. Zebrafish has been a widely used model organism in developmental biology, but is now emerging as a model for human diseases due to its ideal combination of properties. Zebrafish are inexpensive and easy to maintain, develop rapidly, breed in large quantities producing transparent embryos, and are readily manipulated by various methods, particularly genetic ones. In addition, zebrafish are vertebrate animals and results derived from zebrafish may be more applicable to mammals than results from invertebrate genetic models such as Drosophila melanogaster and Caenorhabditis elegans. However, the similarity cannot be taken for granted. The aim of this study was to establish and test a PD model using larval zebrafish. The developing monoaminergic neuronal systems of larval zebrafish were investigated. We identified and classified 17 catecholaminergic and 9 serotonergic neuron populations in the zebrafish brain. A 3-dimensional atlas was created to facilitate future research. Only one gene encoding MAO was found in the zebrafish genome. Zebrafish MAO showed MAO A-type substrate specificity, but non-A-non-B inhibitor specificity. Distribution of MAO in larval and adult zebrafish brains was both diffuse and distinctly cellular. Inhibition of MAO during larval development led to markedly elevated 5-hydroxytryptamine (serotonin, 5-HT) levels, which decreased the locomotion of the fish. MPTP exposure caused a transient loss of cells in specific aminergic cell populations and decreased locomotion. MPTP-induced changes could be rescued by the MAO B inhibitor deprenyl, suggesting a role for MAO in MPTP toxicity. MPP+ affected only one catecholaminergic cell population; thus, the action of MPP+ was more selective than that of MPTP. The zebrafish PINK1 gene was cloned in zebrafish, and morpholino oligonucleotides were used to suppress its expression in larval zebrafish. The functional domains and expression pattern of zebrafish PINK1 resembled those of other vertebrates, suggesting that zebrafish is a feasible model for studying PINK1. Translation inhibition resulted in cell loss of the same catecholaminergic cell populations as MPTP and MPP+. Inactivation of PINK1 sensitized larval zebrafish to subefficacious doses of MPTP, causing a decrease in locomotion and cell loss in one dopaminergic cell population. Zebrafish appears to be a feasible model for studying PD, since its aminergic systems, mode of action of MPTP, and functions of PINK1 resemble those of mammalians. However, the functions of zebrafish MAO differ from the two forms of MAO found in mammals. Future studies using zebrafish PD models should utilize the advantages specific to zebrafish, such as the ability to execute large-scale genetic or drug screens.

The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Estimation and partitioning of polygenic variation captured by common SNPs for Alzheimer's disease, multiple sclerosis and endometriosis

Common diseases such as endometriosis (ED), Alzheimer's disease (AD) and multiple sclerosis (MS) account for a significant proportion of the health care burden in many countries. Genome-wide association studies (GWASs) for these diseases have identified a number of individual genetic variants contributing to the risk of those diseases. However, the effect size for most variants is small and collectively the known variants explain only a small proportion of the estimated heritability. We used a linear mixed model to fit all single nucleotide polymorphisms (SNPs) simultaneously, and estimated genetic variances on the liability scale using SNPs from GWASs in unrelated individuals for these three diseases. For each of the three diseases, case and control samples were not all genotyped in the same laboratory. We demonstrate that a careful analysis can obtain robust estimates, but also that insufficient quality control (QC) of SNPs can lead to spurious results and that too stringent QC is likely to remove real genetic signals. Our estimates show that common SNPs on commercially available genotyping chips capture significant variation contributing to liability for all three diseases. The estimated proportion of total variation tagged by all SNPs was 0.26 (SE 0.04) for ED, 0.24 (SE 0.03) for AD and 0.30 (SE 0.03) for MS. Further, we partitioned the genetic variance explained into five categories by a minor allele frequency (MAF), by chromosomes and gene annotation. We provide strong evidence that a substantial proportion of variation in liability is explained by common SNPs, and thereby give insights into the genetic architecture of the diseases.

Dyslipidaemia, glucose intolerance and cardiovascular disease mortality and morbidity in Europeans and Asians

Dyslipidaemia, a major risk factor of cardiovascular disease (CVD), is prevalent not only in diabetic patients but also in individuals with impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). The aims of this study were: 1) to investigate lipid levels in relation to glucose in European (Study I) and Asian (Study II) populations without a prior history of diabetes; 2) to study the ethnic difference in lipid profiles controlling for glucose levels (Study III); 3) to estimate the relative risk for cardiovascular mortality (Study IV) and morbidity (Study V) associated with dyslipidaemia in individuals with different glucose tolerance status. Data of 15 European cohorts with 19 476 subjects (I and III) and 13 Asian cohorts with 19 763 individuals (II and III) from 21 countries aged 25-89 years, without a prior history of diabetes at enrollment, representing Asian Indian, Chinese, European, Japanese and Mauritian Indian, were compared. The lipid-CVD relationship was studied in 14 European cohorts of 17 763 men and women which provided with follow-up data on vital status, with 871 CVD deaths occurred during the average 10-year follow-up (IV). The impact of dyslipidaemia on incidence of coronary heart disease (CHD) in persons with different glucose categories (V) was further evaluated in 6 European studies, with 9087 individuals free of CHD at baseline and 457 developed CHD during follow-up. Z-scores of each lipid component were used in the data analysis (I, II, IV and V) to reduce the differences in methodology between studies. Analyses of cardiovascular mortality and morbidity were performed using Cox proportional hazards regression analysis adjusting for potential confounding factors. Within each glucose category, fasting plasma glucose (FPG) levels were correlated with increasing levels of triglycerides (TG), total cholesterol (TC), TC to high-density lipoprotein (HDL) ratio and non-HDL cholesterol (non-HDL-C) (p<0.05 in most of the ethnic groups) and inversely associated with HDL-C (p<0.05 in some, but not all, of the populations). The association of lipids with 2-h plasma glucose (2hPG) followed a similar pattern as that for the FPG, except the stronger association of HDL-C with 2hPG. Compared with Central & Northern (C & N) Europeans, multivariable adjusted odd ratios (95% CIs) for having low HDL-C were 4.74 (4.19-5.37), 5.05 (3.88-6.56), 3.07 (2.15-4.40) and 2.37 (1.67-3.35) in Asian Indian men but 0.12 (0.09-0.16), 0.07 (0.04-0.13), 0.11 (0.07-0.20) and 0.16 (0.08-0.32) in Chinese men who had normoglycaemia, prediabetes, undiagnosed and diagnosed diabetes, respectively. Similar results were obtained for women. The prevalence of low HDL-C remained higher in Asian Indians than in others even in individuals with LDL-C < 3 mmol/l. Dyslipidaemia was associated with increased CVD mortality or CHD incidence in individuals with isolated fasting hyperglycaemia or IFG, but not in those with isolated post-load hyperglycaemia or IGT. In conclusion, hyperglycaemia is associated with adverse lipid profiles in Europeans and Asians without a prior history of diabetes. There are distinct patterns of lipid profiles associated with ethnicity regardless of the glucose levels, suggesting that ethnic-specific strategies and guidelines on risk assessment and prevention of CVD are required. Dyslipidaemia predicts CVD in either diabetic or non-diabetic individuals defined based on the fasting glucose criteria, but not on the 2-hour criteria. The findings may imply considering different management strategies in people with fasting or post-load hyperglycaemia.

Mast cells and endothelial erosion: Implications for human atherothrombotic disease

More than 40% of all deaths in Finland are caused by atherosclerosis. The complications of atherosclerosis are due to either detachment of the luminal endothelium (erosion) or rupture of the fibrous cap of an atherosclerotic plaque (rupture). As a result, a thrombus is formed at the site of the intimal lesion. Indeed, erosions cause roughly 40% of sudden atherothrombotic deaths and 25% of all atherothrombotic deaths. Erosions are overrepresented in young subjects, diabetics, smokers and women. This dissertation focuses on endothelial erosion. Endothelial erosions were studied in the context of arterial grafting and vascular inflammation. Special attention was given to the role of intimal mast cells and the methodological viewpoints of reliable identification of endothelial erosions. Mast cells are inflammatory cells mostly known for their ability to cause allergic symptoms. In addition to occurring in skin and mucosal surfaces, mast cells are abundant in arterial intima and adventitia. In this study, mast cells were found to associate with endothelial erosions in non-lesional and atherosclerotic human coronary arteries. Thus, mast cells may participate in atherogenesis at the initial phases of the disease process already. We also showed that the mast cell proteases tryptase, chymase, and cathepsin G are all capable of cleaving molecules essential for endothelial cell-to-cell and cell-to-extracellular matrix interactions, such as VE-cadherin and fibronectin. Symptom-causing carotid plaques were found to contain more inflammatory cells, especially mast cells, than non-symptom-causing plaques. Furthermore, the atherogenic serum lipid profile and the degree of carotid stenosis turned out to correlate with the density of carotid plaque mast cells. Apoptotic and proliferating cells were more abundant in non-symptom causing plaques (active renewal of endothelial cells), but erosions were larger in symptom-causing plaques (capacity of endothelial regeneration exceeded). The process of identifying endothelial erosions with immunostainings has been ambiguous, since both endothelial cells and platelets express largely the same antigens. This may have caused inaccurate interpretations of the presence of endothelial erosion. In the last substudy of this thesis we developed a double immunostaining method for simultaneous identification of endothelial cells and platelets. This method enables more reliable identification of endothelial erosions.

Banana leaf disease diagnostics

Provide a leaf disease diagnostic service for samples received from throughout all banana growing areas of Queensland. This extends to both commercial and non-commercial situations.

The epidemiology of severe Streptococcus pyogenes disease in Europe

Diseases caused by the Lancefield group A streptococcus, Streptococcus pyogenes, are amongst the most challenging to clinicians and public health specialists alike. Although severe infections caused by S. pyogenes are relatively uncommon, affecting around 3 per 100,000 of the population per annum in developed countries, the case fatality is high relative to many other infections. Despite a long scientific tradition of studying their occurrence and characteristics, many aspects of their epidemiology remain poorly understood, and potential control measures undefined. Epidemiological studies can play an important role in identifying host, pathogen and environmental factors associated with risk of disease, manifestation of particular syndromes or poor survival. This can be of value in targeting prevention activities, as well directing further basic research, potentially paving the way for the identification of novel therapeutic targets. The formation of a European network, Strep-EURO, provided an opportunity to explore epidemiological patterns across Europe. Funded by the Fifth Framework Programme of the European Commission s Directorate-General for Research (QLK2.CT.2002.01398), the Strep-EURO network was launched in September 2002. Twelve participants across eleven countries took part, led by the University of Lund in Sweden. Cases were defined as patients with S. pyogenes isolated from a normally sterile site, or non-sterile site in combination with clinical signs of streptococcal toxic shock syndrome (STSS). All participating countries undertook prospective enhanced surveillance between 1st January 2003 and 31st December 2004 to identify cases diagnosed during this period. A standardised surveillance dataset was defined, comprising demographic, clinical and risk factor information collected through a questionnaire. Isolates were collected by the national reference laboratories and characterised according to their M protein using conventional serological and emm gene typing. Descriptive statistics and multivariable analyses were undertaken to compare characteristics of cases between countries and identify factors associated with increased risk of death or development of STSS. Crude and age-adjusted rates of infection were calculated for each country where a catchment population could be defined. The project succeeded in establishing the first European surveillance network for severe S. pyogenes infections, with 5522 cases identified over the two years. Analysis of data gathered in the eleven countries yielded important new information on the epidemiology of severe S. pyogenes infections in Europe during the 2000s. Comprehensive epidemiological data on these infections were obtained for the first time from France, Greece and Romania. Incidence estimates identified a general north-south gradient, from high to low. Remarkably similar age-standardised rates were observed among the three Nordic participants, between 2.2 and 2.3 per 100,000 population. Rates in the UK were higher still, 2.9/100,000, elevated by an upsurge in drug injectors. Rates from these northern countries were reasonably close to those observed in the USA and Australia during this period. In contrast, rates of reports in the more central and southern countries (Czech Republic, Romania, Cyprus and Italy) were substantially lower, 0.3 to 1.5 per 100,000 population, a likely reflection of poorer uptake of microbiological diagnostic methods within these countries. Analysis of project data brought some new insights into risk factors for severe S. pyogenes infection, especially the importance of injecting drug users in the UK, with infections in this group fundamentally reshaping the epidemiology of these infections during this period. Several novel findings arose through this work, including the high degree of congruence in seasonal patterns between countries and the seasonal changes in case fatality rates. Elderly patients, those with compromised immune systems, those who developed STSS and those infected with an emm/M78, emm/M5, emm/M3 or emm/M1 were found to be most likely to die as a result of their infection, whereas those diagnosed with cellulitis, septic arthritis, puerperal sepsis or with non-focal infection were associated with low risk of death, as were infections occurring during October. Analysis of augmented data from the UK found use of NSAIDs to be significantly associated with development of STSS, adding further fuel to the debate surrounding the role of NSAIDs in the development of severe disease. As a largely community-acquired infection, occurring sporadically and diffusely throughout the population, opportunities for control of severe infections caused by S. pyogenes remain limited, primarily involving contact chemoprophylaxis where clusters arise. Analysis of UK Strep-EURO data were used to quantify the risk to household contacts of cases, forming the basis of national guidance on the management of infection. Vaccines currently under development could offer a more effective control programme in future. Surveillance of invasive infections caused by S. pyogenes is of considerable public health importance as a means of identifying long and short-term trends in incidence, allowing the need for, or impact of, public health measures to be evaluated. As a dynamic pathogen co-existing among a dynamic population, new opportunities for exploitation of its human host are likely to arise periodically, and as such continued monitoring remains essential.

Sindbis virus and Pogosta disease in Finland

Sindbis virus (SINV) (genus Alphavirus, family Togaviridae) is an enveloped virus with a genome of single-stranded, positive-polarity RNA of 11.7 kilobases. SINV is widespread in Eurasia, Africa, and Australia, but clinical infection only occurs in a few geographically restricted areas, mainly in Northern Europe. In Europe, antibodies to SINV were detected from patients with fever, rash, and arthritis for the first time in the early 1980s in Finland. It became evident that the causative agent of this syndrome, named Pogosta disease, was closely related to SINV. The disease is also found in Sweden (Ockelbo disease) and in Russia (Karelian fever). Since 1974, for unknown reason, the disease has occurred as large outbreaks every seven years in Finland. This study is to a large degree based on the material collected during the 2002 Pogosta disease outbreak in Finland. We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years.

The Distribution of Henipaviruses in Southeast Asia and Australasia: Is Wallace's Line a Barrier to Nipah Virus?

Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia's closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace's Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace's Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.

Identification of a new Polerovirus (family Luteoviridae) associated with cotton bunchy top disease in Australia

Cotton bunchy top (CBT) disease has caused significant yield losses in Australia and is now managed by control of its vector, the cotton aphid (Aphis gossypii). Its mode of transmission and similarities in symptoms to cotton Blue Disease suggested it may also be caused by a luteovirus or related virus. Degenerate primers to conserved regions of the genomes of the family Luteoviridae were used to amplify viral cDNAs from CBT-affected cotton leaf tissue that were not present in healthy plants. Partial genome sequence of a new virus (Cotton bunchy top virus, CBTV) was obtained spanning part of the RNA-dependent-RNA-polymerase (RdRP), all of the coat protein and part of the aphid-transmission protein. CBTV sequences could be detected in viruliferous aphids able to transmit CBT, but not aphids from non-symptomatic plants, indicating that it is associated with the disease and may be the causal agent. All CBTV open-reading frames had their closest similarity to viruses of the genus Polerovirus. The partial RdRP had 90 % amino acid identity to the RdRP of Cotton leafroll dwarf virus (CLRDV) that causes cotton blue disease, while other parts of the genome were more similar to other poleroviruses. The sequence similarity and genome organization of CBTV suggest that it should be considered a new member of the genus Polerovirus. This partial genome sequence of CBTV opens up the possibility for developing diagnostic tests for detection of the virus in cotton plants, aphids and weeds as well as alternative strategies for engineering CBT resistance in cotton plants through biotechnology. © 2012 Australasian Plant Pathology Society Inc.