Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and  sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia.  Pteropus (Chiroptera), commonly known as flying-foxes, have been identified as the natural host of Hendra virus, a potentially fatal zoonotic paramyxovirus that causes mortality or morbidity in horses and humans in Australia [Pteropus species endemic to mainland Australia, there is accumulating evidence that the Black flying-fox  and the paraphyletic Spectacled flying-fox (P. conspicillatus) [Bats of the genus ustralia , 2. Of tillatus)  are primillatus) –7.Pteropus species have suggested that physiological and ecological factors may constitute risk factors for Hendra virus infection [Previous studies of nfection –11. ‘Phynfection –16.Assessment of glucocorticoid hormones is a recognised and robust methodology for investigating the putative association between physiological stress, infection and disease in wildlife species . GlucocoPteropus species [et al., [et al., [Plasma glucocorticoid measurement has been employed in a number of chiropteran studies –21, and  species , 23. Mor[et al.,  demonstr[et al.,  reportedUnderstanding Hendra virus infection dynamics in flying-foxes and the contributing risk factors is fundamental to elaborating the disease ecology of the virus and thus effective exposure risk management in horses and humans. In this study, we sought to examine temporal trends in urinary cortisol concentration in two disparate wild Australian flying-fox populations, and elucidate the putative relationship between Hendra virus infection and physiological stress.Roost sites were identified using information from the Queensland Department of Environment and Heritage Protection (EHP) and the (then) Queensland Centre for Emerging Infectious Diseases (QCEID). Fieldwork was conducted under the Queensland Department of Agriculture, Fisheries and Forestry Animal Ethics Committee Permit SA 2011/12/375, and Department of Environment, Heritage and Protection Scientific Purposes Permits WISP05810609 and WISP14100614.Urine samples were collected from beneath roosting flying-foxes at two roosts in each of two disparate regions in the eastern Australian state of Queensland between January 2012 and May 2014: in Southeast Queensland (SEQ) (latitude -27°), at roosts in the rural locations of Boonah and Toowoomba; and in Far North Queensland (FNQ) (latitude -17°) at roosts in the rural location of Yungaburra and the urban location of Cairns . As flyiBoth pooled and individual urine samples were collected from the SEQ Boonah roost; pooled samples only were collected from the FNQ Cairns and Yungaburra roosts; and individual samples only were collected from the SEQ Toowoomba roost. Pooled urine collections were undertaken as a shared component of related studies reported elsewhere , 25. A tet al., [Collection of pooled urine samples was performed as per McMichael et al.,  using plCollection of individual animal under-roost urine samples was typically performed using transects of plastic sheeting (measuring 1 m x 40 m) placed parallel to one another approximately 5 m apart, through accessible areas of the roost. The number of transects varied with roost size and accessibility, with 4 to 5 x 40 m transects at Boonah, and 10 x 5 m transects at the smaller Toowoomba roost. Individual animal urine target sample volume was 50–200 μl. The number of samples collected at a single sampling event ranged from 46 to 109. Individual animal urination events were identified by spatial separation of at least one meter between urine splatter patterns. This criterion was based on simulated urine splatter patterns, wherein 10 ml volumes of coloured water were expressed from a syringe from a 5 m height onto plastic sheeting, producing an average splatter size of 0.5 m x 0.5 m. The urine sample was taken from one droplet or 2–3 closely associated droplets from an individual urination event. Samples were not collected when roosting density was so high as to preclude confident identification of an individual urination event. This occurred on a limited number of occasions associated with large influxes of Little red flying-foxes, and only in sections of the roost. Samples were collected in the hour post-dawn, and transported and stored as for pooled samples.The climatic variables of minimum and maximum daily temperature and daily rainfall for the SEQ and FNQ roosts from January 2012 to June 2014 were collated from the Australian Bureau of Meteorology . To avoiPCR-based Hendra virus detection was undertaken on the individual animal under-roost urine samples. Immediately post-collection, 50 μl of each urine sample was added to a corresponding 130 μL of Magmax lysis buffer (AM 8500) to preserve RNA for extraction and Hendra virus PCR analysis. The remainder of the sample was maintained neat for hormone analysis. Samples for PCR analysis underwent total nucleic acid extraction in the BSL Physical Containment Level 3 (PC3) laboratory using the Kingfisher automated extraction system and the MagMax viral RNA Isolation Kit  according to manufacturer’s instructions. RNA extracts (5 μL) were added to 20 μL reaction mix and assayed in duplicate for Hendra virus genome using a quantitative TaqMan RT-PCR targeting the Hendra virus M gene , 25 usinA novel PCR approach using the total nucleic acid extracted for the Hendra virus assay was used to establish the species and sex of the individual animal samples. The cytochrome B gene was targeted for species differentiation, and the SRY gene of the Y chromosome for sex differentiation. Extracted nucleic acid underwent a quantitative TaqMan RT-PCR using the TaqMan Universal Master Mix II, no UNG (product number 4440040). Primers  were supet al., [P, alecto and P. conspicillatus, the two species in which Hendra virus RNA was detected. Briefly, duplicate pooled urine samples were diluted 1:10 in EIA kit buffer and assayed for cortisol according to manufacturer instructions. Absorbances were read at 405 nm with a Thermo Scientific Multiscan Ascent plate reader using Ascent software (Version 2.6). When measurements fell outside the range of 20–80% B/B0, samples were diluted and re-assayed to ensure the measurements fell within the linear range of the standard curve. Cortisol concentration was determined by analysis of absorbance values using MyAssays Analysis Software Solutions Cortisol "Four Parameter Logistic Curve" online data analysis tool . McMichael et al., [Urinary cortisol concentration was measured using Caymen Chemical Company Cortisol Enzyme Immunoassay (EIA), (Product number 500360), employing a synthetic cortisol-specific mouse monoclonal antibody . The assay was validated by the manufacturers for human urine and plasma, and mouse faeces, with minimum detectable cortisol concentration of 0.2 ng/ml. McMichael et al.,  reported et al.,  examinedGeneralised linear mixed models (GLMM)  were useThe pooled urinary cortisol, the individual urinary cortisol and Hendra virus data were subjected to separate unbalanced generalised linear mixed model (GLMM) analyses. The normal or log normal distribution was adopted as appropriate for continuous variables, and the binomial distribution and logit link for binary variables. Cortisol data proved to be positively skewed with heterogeneous variance and were transformed using the natural log (ln) prior to analysis. The residual plots proved to be approximately normal.In the pooled urinary cortisol data analysis, the response variable was urinary cortisol concentration. The fixed effects were year, month, region, total population, species composition, minimum temperature on the day of sampling, and maximum temperature and daily rainfall on the day prior to sampling. Interaction terms were limited to three-way interactions for model parsimony. Sampling day was the additional random effect. Additional linear and nonlinear regression analyses of the effects of the environmental variables of minimum and maximum temperature and rainfall on urinary cortisol measurements were performed.Two analyses were performed on the individual urinary data. Firstly, the response variable was urinary cortisol concentration, and secondly, the response variable was Hendra virus detection. For each analysis, the fixed effects were month, sex, reproductive activity and their (three-way) interactions. Sampling day was the additional random effect.Data were plotted and trends estimated by sampling day, and then were grouped into monthly data for temporal regression analysis. For the cortisol results, the direct back-transformation from the natural log was adopted for presentation, giving geometric means. Annual patterns for the variables were initially investigated with symmetrical Fourier curves, however Gaussian or double-Gaussian curves were subsequently adopted, as these allow a more general shape plus a direct test of the significance of any second annual peak.P. alecto (the Black flying-fox), P. poliocephalus (the Grey-headed flying-fox), P. conspicillatus (the Spectacled flying-fox), and P. scapulatus (the Little red flying-fox). In SEQ, the Boonah roost consisted primarily of P. alecto and P. poliocephalus in varying ratios, but total population of these species never exceeded an estimated 20,000 individuals. Large numbers of P. scapulatus  were periodically present at the roost site. The Toowoomba roost was consistently smaller than the Boonah roost and consisted primarily of P. alecto and P. poliocephalus in varying ratios with total population ranging from 1,000 to 2,000 with an influx of approximately 500 P. scapulatus in March 2013  Fig 2)P. conspiP. alecto and P. poliocephalus was observed in March each year. At the Boonah roost, 95% adult females were palpably pregnant from June onwards in both 2013 and 2014 [P. conspicillatus was observed in February and March each year. Females were palpably pregnant in June , and first pups were observed in September.At the SEQ roosts, peak mating activity of and 2014 , and visTemperatures in SEQ ranged from -4°C to 44.6°C over the study period, with the highest mean monthly temperatures recorded annually in December and January, and the lowest mean monthly temperatures recorded in July and August. Temperatures in FNQ ranged from 9.8°C to 34.9°C over the study period, with the highest mean monthly temperatures recorded annually between December and February, and the lowest mean monthly temperatures in July and August. Daily rainfall in SEQ ranged from 0 mm to 255 mm over the study period, with the highest mean monthly rainfalls recorded annually in December and January, and the lowest mean monthly rainfalls between July and September. Daily rainfall in FNQ ranged from 0 mm to 241 mm over the study period, with the highest mean monthly rainfalls recorded annually between January and March and the lowest mean monthly rainfalls in August.Pooled animal samples were collected from the SEQ Boonah roost and both FNQ roosts (Cairns and Yungaburra) . Signifi2 = 0.83; p < 0.001), with the former increasing exponentially as temperature decreased , where cortisol concentration increased with decreasing maximum temperature. The relationship with both climatic variables was independent of region, total population size or species composition. Further, the relationships between cortisol measurements and environmental variables were consistent, whereby none of the environmental variables showed a significant interaction with region, population size or species composition. While total population size and species composition varied within and between regions over time, neither had a significant effect on cortisol concentration (p = 0.54 and p = 0.51 respectively).There was a strong inverse correlation between urinary cortisol concentration and minimum temperature on the day of sample collection .P. alecto (see below), the analyses were restricted to this species for parsimony. There was no significant difference between mean individual urinary cortisol concentration between collection events (p = 0.16), but there was an evident pattern , however females during birthing season (n = 17) had elevated urinary cortisol when compared with males at the same time of year (n = 7).Individual animal urine samples were collected from Boonah and Toowoomba roosts in SEQ . As all  pattern  correspoP. alecto samples (n = 34/375), with no detections in P. poliocephalus (n = 0/86) or P. scapulatus (n = 0/103) samples. The mean detection prevalence in P. alecto was 9.1%. Statistical analysis demonstrated a significant effect of month (p < 0.001) on RNA detection, with a significant elevation in excretion in June (24.9%) (All Hendra virus RNA detections were in  (24.9%) . StatistP. alecto Hendra virus RNA-positive urine samples (n = 34) were slightly but non-significantly higher than those in P. alecto Hendra virus RNA-negative urine samples (n = 341) (p = 0.80).Urinary cortisol concentrations in individual Urine has previously been established as the primary route of excretion of Hendra virus , and as The collection of pooled urine samples presents few challenges logistically and analytically, and the potential bias consequent to an individual Hendra virus infected animal contributing to multiple pools  is mitigated by our systematic approach of collecting each pooled sample from a discrete section of each sheet. However, the under-roost sampling of individual animals can be problematic. Firstly, when roosting density is high, individual urination events are impossible to identify, and sampling opportunities have to be foregone. Secondly, the small volume of urine from the minimal number of drops collected to ensure that a sample is truly from a single individual limits the number of analyses that can be performed, and in some cases, provides only sufficient volume for molecular analysis. The latter might be overcome in the future with the emergence of molecular techniques for measuring physiological parameters, including those indicative of physiological stress, instead of current serological techniques that require a larger sample volume.P. alecto and P. conspicillatus were the primary Hendra virus reservoir species, secondly, that mean Hendra virus RNA urinary excretion prevalence was higher in SEQ than in FNQ, and thirdly, that virus excretion prevalence peaked in June, July, August (southern hemisphere winter) in lower latitude SEQ, while remaining more or less constant in FNQ [The pooled urine Hendra virus data used in our analyses were part of a larger study which showed, firstly, that t in FNQ . The indt in FNQ .P. conspicillatus in SEQ, their predominance in FNQ, and the frequent inaccessibility of P. alecto roosts in FNQ, precluded this. Thus, we used P. conspicillatus as a proxy for P. alecto in FNQ. The two species are phylogenetically paraphyletic [While it would have been preferable to compare physiological responses in the same species in both SEQ and FNQ, the absence of phyletic , are morphyletic , have nophyletic , and minP. conspicillatus, P. alecto and P. poliocephalus [P. alecto and P. poliocephalus, which in mixed roosts likely exacerbates stress through the combined effects of both intra- and interspecific interactions. However, we found no significant direct association between species and urinary cortisol concentration in the pooled urine models.Levels of circulating glucocorticoids can vary with season, sex, age, social status and breeding stage, and valid interpretation of the data requires that these ‘natural’ variations are recognised . Pooled cephalus . The litcephalus , 29, 34.P. alecto and P. poliocephalus. No similar elevations were evident in either FNQ roost. While a peri-parturient cortisol spike is observed in well-studied domestic species [P. alecto and P. conspicillatus, were the elevated cortisol associated with pregnancy, we would expect to see elevated levels in both SEQ and FNQ. Finally, the individual animal urine samples showed that the SEQ winter elevations in urinary cortisol were equally evident in both males and females, discounting an association with pregnancy. Thus, while the observed elevated urinary cortisol in autumn (common to both regions) is plausibly explained by the life-cycle event of mating, the winter elevation in SEQ is not associated with the subsequent event of pregnancy, and is more consistent with an environmental stressor impacting SEQ roosts but not FNQ roosts.Pooled samples from the SEQ Boonah roost also showed elevations in urinary cortisol in June, July and August, corresponding with southern hemisphere winter, and with mid-late pregnancy in  species , a pregn species . FurtherP. alecto populations in SEQ, and given the absence of evidence of an association with any other measured variable, we suggest that low ambient temperature is a biologically plausible stressor. While testing this proposition was beyond the scope of this study, minimum winter temperatures at the SEQ Boonah roost often fell below zero during the study period, with frosts occurring regularly; at the FNQ roosts, minimum temperatures rarely fell below 10°C. Further, veterinary examination of captured individual P. alecto at the SEQ Boonah roost in winter often revealed peripheral vasoconstriction and frost bite damage to ears and feet that was absent in co-roosting P. poliocephalus, a species historically with a more southerly range. Thus, we hypothesize that the recent extension of the range of P. alecto  into su species  have pre species  identifyr in SEQ . To extrr in SEQ  showed aycerides  to facilWe found a small but statistically significant increase in urinary cortisol in pooled samples positive for Hendra virus RNA. In the individual animal urine samples, we found a small and non-significant increase in urinary cortisol measurements in Hendra virus RNA positive samples. This disparity is most plausibly explained by the greater temporal coverage and larger data set of the pooled sampling methodology. Edson et al.  similarlP. alecto in the lower latitude SEQ roosts. Based on our findings and previous studies, we hypothesize an association between low winter temperatures and increased Hendra virus infection and excretion, putatively mediated by the physiological cost of thermoregulation. Further work is required to elaborate and test this hypothesis.In this study, we sought to examine temporal trends in urinary cortisol concentration in disparate flying-fox populations and elucidate the relationship between Hendra virus infection and putative physiological stress. Our findings indicate that urinary cortisol excretion is modulated by both life cycle and ecological factors. We found no significant correlation between increased urinary cortisol due to normal life cycle events and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in While this study has focused on Australian pteropids and Hendra virus, our findings and our approach have direct relevance to studies of the disease ecology of Nipah virus and other emerging Henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics at any wildlife/livestock/human interface.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Few countries have made much progress in implementing transparent and efficient systems for the allocation of mental health care resources. In England there are ongoing efforts by the National Health Service (NHS) to develop mental health ‘payment by results’ (PbR). The system depends on the ability of patient ‘clusters’ derived from the Health of the Nation Outcome Scales (HoNOS) to predict costs. We therefore investigated the associations of individual HoNOS items and the Total HoNOS score at baseline with mental health service costs at one year follow-up.vs ‘regular costs’ in main analyses.An historical cohort study using secondary care patient records from the UK financial year 2012–2013. Included were 1,343 patients with ‘common mental health problems’, represented by ICD-10 disorders between F32-48. Costs were based on patient contacts with community-based and hospital-based mental health services. The costs outcome was transformed into ‘high costs’ After adjustment for covariates, 11 HoNOS items were not associated with costs. The exception was ‘self-injury’ with an odds ratio of 1.41 (95% CI 1.10–2.99). Population attributable fractions (PAFs) for the contribution of HoNOS items to high costs ranged from 0.6%  to 22.4% (self-injury). After adjustment, the Total HoNOS score was not associated with costs . However, the PAF (33.3%) demonstrated that it might account for a modest proportion of the incidence of high costs.Our findings provide limited support for the utility of the self-injury item and Total HoNOS score in predicting costs. However, the absence of associations for the remaining HoNOS items indicates that current PbR clusters have minimal ability to predict costs, so potentially contributing to a misallocation of NHS resources across England. The findings may inform the development of mental health payment systems internationally, especially since the vast majority of countries have not progressed past the early stages of this development. Discrepancies between our findings with those from Australia and New Zealand point to the need for further international investigations. Few countries have made much progress in implementing transparent and efficient systems for the allocation of mental health care resources. In EnglaPerhaps taking into account the lack of evidence for the utility of diagnostically-defined clusters in predicting costs, and the subsequent exclusion of mental health services from the original Medicare system of the United States,–11 NHS pThere is little existing evidence for the utility of the HoNOS in predicting costs for patients with mental disorders. Reports on ultimately unsuccessful attempts to implement casemix classification systems in Australia and New We carried out an historical cohort study with a one-year follow up, using a comprehensive repository of anonymised electronic patient records, to investigate the utility of the HoNOS in predicting mental health service costs. We initially considered including participants with any psychiatric disorder but there was a large discrepancy in missing HoNOS data between patients with less severe disorders attending community-based services (~25%) and patients with more severe disorders who were using hospital-based care (>65%) so we therefore limited our analysis to patients with ‘common mental health problems’, represented by the recording of an ICD-10 disorder related to (unipolar) depression, anxiety, stress, adjustment or somatic problems (F32-F48). Although we use the term ‘common mental health problems’, our sample of secondary mental health care patients differs from those patients with milder and simpler difficulties treated predominately in primary care in the UK. HoNOS items are used for clustering within mental health PbR but it is commonplace for studies to deploy the total HoNOS score in analyses. To provide a comprehensive assessment of predictive utility, we separately investigated associations of individual HoNOS items and the total HoNOS score with costs.The data source was electronic patient records from the South London & Maudsley NHS Foundation Trust (SLaM) Biomedical Research Centre (BRC) Case Register.  SLaM proWe only included patients experiencing ‘common mental health problems’, represented by the recording of an ICD-10 disorder related to (unipolar) depression, anxiety, stress, adjustment or somatic problems (F32-F48). To enhance representativeness of common mental health problems, patients also needed to have a first SLaM contact in two types of community-based services primarily geared towards the treatment of less severe problems: ‘Assessment Brief Therapy’ and ‘Mood, Anxiety and Personality Disorders’. Older adults (aged 65+) were excluded because their mental health service costs are based on a range of services not attended by working age adults . The sample was followed up for one year from the start of their first treatment episode occurring within the UK financial year 2012–2013 (1 April 2012 to 31 March 2013).Sample characteristics were assessed using the following variables: age (at baseline), gender, ethnicity, marital status, area-level deprivation based on the Index of Multiple Deprivation, ICD-10 dThe HoNOS are a set of 12 scales measuring mental health-related problems in the domains of behaviour, symptoms, impairment, and social functioning.  InternalCosting data were provided through SLaM internal financial records. We chose a one year follow-up period for two reasons: (1) in PbR, the interval for review of allocation to clusters covering less severe and more common disorders is typically one year; (2) the one year period is likely to even out the seasonal effects seen in admissions and mental health problems. For each patient, the mental health service costs outcome was calculated by adding costs of treatment by any community mental health team, whether generic or specialised, and the costs of any hospital treatment, taking as the start point the start of their initial treatment episode within the observation period. Both community mental health team and inpatient costs were calculated based on unit costs calculated at the individual team/ward level using SLaM internal financial data and the total relevant activity over that financial year . On account of a highly skewed distribution, the outcome was transformed into ‘high costs’ (the top 10% of costs) and ‘regular costs’ (remaining 90% of costs). This cut-off was based on research demonstrating that a minority of ‘high cost’ patients (i.e. the top 5%–10% for costs incurred) account for at least a 50% share of costs –33, and All statistical analyses were undertaken using Stata 12. Descriptive statistics were used for sample characteristics. The utility of both individual HoNOS items and the Total HoNOS score (at baseline) in predicting costs (at one year follow up) were determined in separate analyses. HoNOS items were simultaneously entered into all relevant predictive models. Based on previous research showing their associations with mental health service costs,,38 additAs a precursor to the main analyses, t-tests were used to examine differences in both individual item and total HoNOS scores between ‘regular cost’ and ‘high cost’ patients. Associations of baseline HoNOS scores with ‘one-year’ costs were determined using unadjusted and adjusted odds ratios with 95% confidence intervals (CIs), modelled through logistic regression analysis. Population attributable fractions (PAFs)- representing the percentage decrease in the number of ‘high cost’ patients that would theoretically arise if a problem within a given HoNOS domain could be removed from the study population- were calculated and applied to fully-adjusted models. As the ‘aflogit’ command for PAFs is not supported for use with imputed data by Stata 12, PAFs were applied to complete-case analyses.Complete data pertaining to the costs outcome were available but HoNOS scores had sizeable missing data (i.e. 24.8%). We deployed multiple imputation by chained equations (MICE) to impute this missing data. Multiple imputation uses patterns in observed data to impute missing values, repeating this process multiple times to account for uncertainty in the imputed values. MICE facTwo sets of sensitivity analyses were undertaken: (1) complete case analyses using the fully-adjusted logistic regression models (for comparison of results with those derived from multiple imputation); (2) fully-adjusted associations of baseline HoNOS scores with a continuous ‘one year’ costs outcome, analysed using generalised linear models (GLM) with log link functions and gamma distributions (for comparison of results with those involving the dichotomized costs outcome). The use of ‘GLM-log-gammas’ accounted for the skewed distribution of the continuous costs outcome in latter analyses.Initially, we did not separate costs by admitted and non-admitted settings in analysis because such separation of costs is not prioritised in mental health PbR and only 5% of the sample had an inpatient admission in the follow-up period, limiting the potential influence of setting on the findings. However, previous international studies of casemix classification systems for mental health services have separated costs by setting. For additional comparison with these studies, we present the fully-adjusted associations of HoNOS scores with inpatient admission and community health service costs in a supplementary file .n = 1,343). The sample was ethnically diverse with a large proportion living in deprived areas. Most participants remained under the care of CMHTs at the end of their first treatment episode within the observation period.t = 3.72, p < 0.01) and on the Total HoNOS .The distribution of costs by HONOS items is presented in β = 1.17; 95% CI 1.01–1.36). However, a significant cost association was yielded for the Total HoNOS score .Findings pertaining to the associations of baseline HoNOS items and the Total HoNOS score with costs at one year follow up are summarised in turn. After adjustment for covariates, 11 of the 12 HoNOS items were not significantly associated with (‘regular vs high’) mental health service costs for patients with common mental health problems. The exception was the ‘self-injury’ item with an odds ratio of 1.41 (95% CI 1.15–1.72). PAFs for the contribution of HoNOS items to high costs ranged from 0.6%  to 22.4% (self-injury). After adjustment, the Total HoNOS score was not significantly associated with costs in the main analysis, although the association was significant for total costs as a continuous outcome and in supplementary analyses which split costs by setting . AssuminAs high levels of missing HoNOS data led to their exclusion, the findings are not applicable to patients with more severe mental disorders and service needs. This reflects the challenge of collecting comparable mental health service data across diverse settings and clinical populations. But the findings are applicable to the first two mental health clusters from PbR, which cover the most common mental health problems. The findings are less applicable to patients with milder and simpler ‘common mental health problems’ treated predominantly in primary care in the UK. The proportion of missing data for other clinical measures was very high , and thiThe study benefits from its use of an established case register, which provided a large clinical sample from a defined catchment area covering a population of 1.2 million people, which is demographically and socio-economically similar to other deprived areas in London .It is thTwo studies, reporting on the development of casemix classification systems in Australia  and New Comparisons with the mixed findings from previous health service use studies –23 are lComparing the findings with previous research not involving the HoNOS, the association of self-injury with high costs corresponds with a previous report of an association of self-injury frequency with long-term health service costs. In supplThe predominant absence of cost associations for HoNOS items raises concerns about the decision by policy-makers to assign a key role to these items within mental health PbR, largely based on their presumed utility in predicting costs. These concerns are compounded by the lack of robust evidence from other studies for associations of HoNOS scores with costs,–19,21–23https://www.ihpa.gov.au/what-we-do/mental-health-care). Another proposal is for clinicians to judge the most appropriate care pathway option based on the detailed assessment of problems in nine domains of mental health and 12 domains of everyday living, using the MRC Needs For Care Assessment Schedule.[Our findings also highlight the need for assessments of alternative approaches to developing payment systems for mental health services. Monitor—the NHS regulator—has suggested that payments should be closely linked to agreed patient outcome standards to incentivise quality of care. AlthoughSchedule.,50 As asSchedule.,45 furthFuture investigations of the associations of HoNOS scores with costs should address the limitations of the present study. For example, a costs outcome derived from patient contacts with the full range of health services would add generalisability. Examining a broader costs outcome would also raise the issue of whether or not mental health services should be compensated for their involvement in patient care for heavy consumers of primary care services. Greater integration of service sectors, in financing as well as commissioning, planning and delivery, would be required to facilitate this reimbursement approach. Costs information could be collected directly from participants using measures such as the Client Services Receipt Inventory, althoughS1 Table(DOCX)Click here for additional data file.


We report the first whole-genome sequence of mumps virus isolated from a two-year-old girl with bilateral parotitis from a Chikkahallivana village in the Davangere district of Karnataka State, India. The genome of the Davangere mumps isolate was 15,384 bp in length and identical to previously published mumps virus (MuV) genomes from India. BLAST results show 99.1% identity with previously sequenced genotype C viruses isolated from the states of Maharashtra, Tamil Nadu, and Uttar Pradesh. Mumps is mainly a childhood disease characterized by fever and swelling of the parotids, and it also spreads to different organs, i.e., genital organs, pancreas, thyroid, salivary glands, urinary tract, and central nervous system. Mumps-containing vaccines have not been included in India’s Universal Immunization Program. The Indian Academy of Pediatrics suggested inclusion of the first dose of mumps vaccine at nine months and a second dose at 16 to 24 months , hemagglutinin-neuraminidase (HN), nucleoprotein (NP), phosphoprotein (V/P/I), matrix protein (M), large protein (L), and small hydrophobic (SH) proteins (Mumps virus (MuV) is a member of the proteins . A standRecently, seroprevalence studies, vaccine studies, outbreak investigations, virus isolation, and virus genotyping studies were reviewed from India . This reAn outbreak of mumps was investigated during January 2014 in Chikkahallivana village in the Davangere district of Karnataka State, India . During KX953297 (MuVi/Davangere.IND/01.14[C]).The whole-genome sequence obtained from Karnataka State has been deposited in GenBank under the accession number


Betula sp.) from different European stands affected by the “birch leaf-roll disease” (BLRD) a novel virus is identified by means of RNA-Seq virome analysis. The virus represents a new member in the genus Badnavirus, family Caulimoviridae, tentatively named Birch leaf roll-associated virus (BLRaV) and it is the first badnavirus found to infect birch. Complete genome sequences  of three viral isolates of Finnish and German origin have been determined. The virus sequences show a typical badnavirus organization with three major open reading frames (ORFs) and a fourth potential ORF overlapping with the end of ORF3. ORFs 1-2-3 show low level of amino acid identity to the corresponding proteins encoded by other badnaviruses, reaching a maximum of 44% identity (ORF3). Grapevine vein-clearing virus appears as the closest badnavirus when considering the polymerase region. So far, we can exclude evidence for presence of endogenous BLRaV elements in the birch genome, while evidence for the episomal activity of BLRaV is provided. The viral population holds significant haplotype diversity, while co-infection by different BLRaV variants are observed in single hosts. BLRaV presence is associated with the BLRD in both silver (B. pendula) and downy birch (B. pubescens). These results challenge the earlier hypothesis of a causal role of Cherry leaf roll virus in BLRD. Further work is now needed to finally prove that BLRaV is the causal agent for the BLRD.In declining birches ( Betula species were reported throughout Finland, in the subarctic zone of Norway as well as in Sweden, in urban parks and forests as well as exactly at the birch alpine tree line. Similar symptoms had been previously sporadically reported in Great Britain [With more and more results accumulating, it is now clear that virus-associated birch epidemics currently constitute a serious problem for the health of birch forest and urban stands throughout Europe –5. For f Britain , 10 and  Britain –14. The  Britain .Cherry leaf roll virus (CLRV), as the leaf symptoms resembled those observed in CLRV-infected birches from Germany [Betula trees of various species by applying conventional molecular methods like IC-RT-PCR [B. pubescens donor trees from Rovaniemi, Finland to non-symptomatic B. pubescens rootstocks successfully transmitted CLRV [Viral detection efforts in symptomatic birches originating from Finland initially targeted the nepovirus,  Germany , 12, 13.C-RT-PCR  or nesteC-RT-PCR . FurtherC-RT-PCR . Graftinted CLRV . HoweverB. pubescens seedlings are infected by a novel badnavirus. Badnaviruses are double-stranded DNA pararetroviruses and are one of the eight genera of the family Caulimoviridae. They have bacilliform particles and replicate through an RNA intermediate [To investigate the hypothesis that other viruses might be involved in the etiology of this emerging disease, an RNA-Seq metagenomic analysis is performed and the results obtained are reported here. The use of high-throughput sequencing coupled to bioinformatics analysis constitutes a novel approach that has remarkably changed our ability to detect known or novel viruses without the need for any prior knowledge –19. The rmediate . They arrmediate . In the B. pubescens donor tree (Bpub3) with severe leaf symptoms  from Rovaniemi, Finland were grafted on two non-symptomatic B. pubescens rootstocks (grafted seedlings BpubFin407501_3A and BpubFin407507_3I). In parallel, one twig originating from a symptomatic, CLRV-positive B. pendula tree (Bpen 5) from Berlin, Germany was grafted on a non-symptomatic B. pendula rootstock (grafted seedling BpenGer407526_5M). The following growing season, in May 2012, symptoms similar to the ones exhibited by the donor trees could be observed on the grafted birches (B. pubescens seedling (BpubGerNo4) and a CLRV-negative B. pendula seedling (BpenGerM0197542), obtained from the same nursery and not exhibiting any symptoms were used as negative controls. For the graftings and for the controls, two-year-old sprouting birch rootstocks were used .Virus transmission was carried out by grafting in 2011. Two twigs originating from a  birches . Two bir® Spin Plant RNA Mini Kit , followed by removal of remaining DNA with rDNase according to the supplier protocol  and RNA purification using NucleoSpin® RNA Clean-up . Ribosomal RNA depletion was performed using the RiboMinus Plant Kit for RNA-Seq (Invitrogen). One to two micrograms of RiboMinus RNA of each sample were used for cDNA synthesis with the Maxima H Minus double-stranded cDNA synthesis Kit (Thermo Scientific) primed with random hexamers. One to two micrograms purified double-stranded cDNA from each sample were sent to BaseClear (Netherlands) for RNA-Seq analysis with the Illumina HiSeq2500 system and 100 bp-long paired-end sequence reads corresponding to 50–100 Mb data/sample were generated.Pooled samples of three to five leaves randomly selected from the canopy were used for RNA extractions from the symptomatic or from the asymptomatic control seedlings. Total RNAs were isolated using the InviTrapAll NGS data processing and analysis were performed using CLC Genomics Workbench version 7.0.4. Reads were first submitted to quality filtering and trimming. The resulting cleaned reads  were then assembled into contigs, generating between 263 and 1,055 contigs longer than the 300 nucleotide cut-off . Contigs2, 50 mM KCl, 0.2 mM dNTPs, PolyBadna-F1/R1 primers at 1 μM each, and 1 U of Advantage 2 Polymerase Mix (Clontech). After an initial denaturation step of 1 min at 98°C, 40 cycles were applied, each of 95°C for 30 sec, 60°C for 30 sec, and 68°C for 1 min, followed by a final extension step of 10 min at 68°C.A pair of specific, outward-facing primers , PCR products were ligated into the pGEM-T Easy cloning vector using the pGEM-T Easy vector system I (Promega). Transformation of JM109 competent cells was conducted following standard procedures (Promega).When needed, PCR products were first cloned prior to sequencing, in order to identify haplotype-specific rather than consensus sequences. Following purification with the MSBRice tungro bacilliform virus, the type member of the genus Tungrovirus (family Caulimoviridae), was used as outgroup. Bootstrapped Maximum likelihood (ML) and Neighbour-Joining (NJ) phylogenetic trees were constructed with MEGA6 . Robustness of nodes of the phylogenetic tree was assessed from 1,000 bootstrap resampling, and values > 70% were used for internal nodes.Multiple nucleotide or amino acid sequence alignments were performed using CLUSTALW in Bioedit 7.4 , as well as pairwise sequence identity calculations. For the phylogenetic comparisons of complete ORF3 sequences, a representative sequence was used for each badnavirus. RNA-Seq analysis was performed for five birch plants. Three of these plants  were BLRD-affected symptomatic young seedlings that had been graft-inoculated from infected sources of Finnish or German origin . The twode novo assembly process allowed the reconstruction of long contiguous near complete badnaviral genome sequences of 7,916 base pairs (bp) (BpubFin407501_3A), 7,973 bp (BpubFin407507_3I) and 7,846 bp (BpenGer407526_5M). In each case, deep average coverage was obtained for the scaffolds, ranging from 35.1X to 68.7X and giving excellent confidence in the assembled sequences.Ultimately, contigs could be assembled into scaffolds covering near complete badnaviral genomes for plants BpenGer407526_5M and BpubFin407501_3A. In the case of plant BpubFin407507_3I, the single contig obtained already represented a nearly complete genome. Overall, the Betula species.PCR assays were performed using the outward-facing primer pair PolyBadna-F1/R1 to prove the circularity of the genome and to complete the missing region in the assemblies . A 780 bFurther analysis of the contigs from the three BLRD plants revealed in the sample BpubFin407501_3A additional badnavirus contigs that differed from the completely assembled isolate, suggesting the presence of a second, divergent isolate in this plant. These various contigs could be assembled in a scaffold with several large gaps and having only a low 2.5X average coverage. The largest contigs integrated in this scaffold was 2,080 bp-long and the scaffold showed about 8.3%—10.4% divergence from the fully assembled genomes. Due to the low coverage, it was not possible to complete the genome of this variant from the RNA-Seq data.Grapevine vein-clearing virus [Grapevine Roditis leaf discoloration-associated virus , which is within the range of typical badnavirus genomes . The fewng virus . ORF2 lang virus , it coul. ORF3 hGrapevine vein-clearing virus (GVCG), Rubus yellow net virus (RYNV), Gooseberry vein banding-associated virus (GVBaV) and Pagoda yellow mosaic-associated virus (PYMaV), most closely clustered to the latest. The new virus is clearly distantly related phylogenetically to all badnaviruses currently represented in the GenBank  is proposed.Considering only the polymerase region (RT plus RNase H domains) that is typically used for taxonomic discrimination, the divergence level from the known badnavirus genomes is 27.6 to 40.6%, with  ranges; ), the baThe presence of the novel badnavirus was confirmed by means of RT-PCR in 100% of the tested symptomatic trees, corresponding to 22 trees from six different birch stands in Berlin, and three trees located in distant urban green spots of Rovaniemi. The amplified fragment lying within the ORF3 coding region showed significant sequence variability between trees, with an average nucleotide divergence of 9.1% +/- 1% between isolates. Remarkably, in only one case did two neighbouring trees  share a common virus variant. On the other hand, and as expected, the ORF3 variants obtained from the birch trees used for the NGS analysis were proved identical (in the cases of the trees BpubFin407501_3A and BpubFin407507_3I) or nearly identical (in the case of the tree BpenGer407526_5M) to the sequences assembled from the NGS data . In the All BLRaV samples from Finland and Germany used in the phylogenetic analysis cluster into a single subgroup together with the samples originating from the seedlings BpubFin407501_3A, BpubFin407507_3I and BpendGer407526, from which the full-length genome sequences were assembled in the present study. Based on the virus nucleotide variability assessed to date and the corresponding phylogenetic analysis, the existence of a single BLRaV species is suggested. Furthermore, the analysis of the phylogenetic relationships between the variants from Finland and Germany failed to provide evidence for significant clustering of isolates based on their geographical origin, despite the fact that the geographical distribution of the analysed samples within each of the sampling locations (Rovaniemi and Berlin) was very restricted .It is remarkable that mixed infection by several variants was observed in several trees, first as PCR products yielding ambiguous sequences upon direct sequencing. Following cloning of PCR products and sequencing of individual clones, the presence of multiple variants in single plants was confirmed. Multiple variants occurred in Berlin trees MO099, MO199, MO200, MO223, while in Rovaniemi two trees showed contrasting patterns. In tree Bpub19 all five clones sequenced were different, but formed two distinct sub- clusters, while in tree Bpub20 all five clones sequenced revealed the same variant .Badnavirus species for which the name Birch leaf roll-associated virus (BLRaV) is proposed.A novel badnavirus has been identified by means of RNA-Seq while investigating the etiology of a widespread disease in birch forests and urban greens throughout Europe, the “birch leaf-roll disease” (BRLD). Complete genome sequences were assembled from three birch seedlings that had been graft-inoculated with disease sources of different origins, two from Finland and one from Germany. The virus presence was confirmed in numerous trees from both countries, in different birch stands. Sequence and phylogenetic comparison of the full-genome sequences and of the ORF3 sequences of these three virus variants demonstrate that they represent a new Canna yellow mottle virus, Kalanchoë top-spotting virus, Rubus yellow net virus, Fig badnavirus 1, Grapevine Roditis leaf discoloration-associated virus and Gooseberry vein banding-associated virus. The first report of a badnavirus in Betula species not only increases the list of hosts affected by members of this genus but also significantly expands the genus geographic distribution towards its northern limits. Given the wide distribution of Betula species in northern temperate and boreal climates over North America and Eurasia, the spatial distribution of this new virus species could potentially be very broad.To date, badnaviruses are known to be distributed in tropical and temperate regions, with the majority of currently known species infecting tropical and subtropical crops such as banana, black pepper, citrus, cocoa, sugarcane, taro, and yam . Each viSimilar to other pararetroviruses, badnaviruses are often also present as integrated, complete or fragmented and/or re-arranged genomic sequences in some host plant genomes and are then referred to as endogenous pararetroviruses or endogenous viral elements (EVEs) , 30. TheFirst, BLRaV was efficiently transmitted by grafting to non-infected seedlings. An origin of the virus from the rootstocks rather than from the grafted scion is excluded by (i) the absence of infection in the plants grafted with non symptomatic material or in the control rootstocks themselves and (ii) the detection of identical viral variants in the grafted plants and in the original source plants.After analysing by RNA-Seq a sample of symptomatic leaves from the original source tree (Bpub3), a 3,285 nt contig with 99.99% sequence similarity with the corresponding sequence of the full-length variant identified in the grafted seedlings (BpubFin407501_3A and BpubFin407507_3I) could be recovered (data not shown). This demonstrates that the homologies between the isolates infecting the source tree and the grafted ones extends much further than the short, 241 nt region amplified by RT-PCR in the variability analysis. These results further support the hypothesis that the only way that the grafted seedlings in Berlin could share the same BLRaV variant as the original Bpub3 tree in Rovaniemi, is by acquiring this variant through graft transmission, demonstrating the episomal gene activity of BLRaV.Similarly, the multiple co-infections observed in both Finnish and German trees, with up to five different variants detected in a single tree, and the high BLRaV diversity (9.1% average divergence between randomly selected isolates) suggest the existence of a highly diverse and dynamic virus population with large population size (high variant diversity), effective natural transmission pathway(s) and wide geographic distribution . These viral population features, in combination with the described emergence of the “birch leaf-roll disease” contemporaneously in various European regions  – 5, furBetula species cannot therefore be excluded.Even in cases where episomal replication is demonstrated, the presence of endogenous forms of the virus in the host(s) genome cannot be excluded. Some banana streak viruses (BSVs) are found in episomal form having an endogenous counterpart (eBSV), while there are other endogenous banana sequences that have no episomal counterpart . InfectiBetula nana) has been initiated in 2013 [Betula species found to host BLRaV so far (B. pubescens and B. pendula) are genetically different from B. nana, this observation would seem to rule out ancestral integration events prior to the separation between these species.The whole genome sequencing of dwarf birch  and Rovaniemi (Finland).(PDF)Click here for additional data file.


Phenuiviridae, genus Phlebovirus) was discovered in sand flies collected in Albania and genetically characterised as a member of the Sandfly fever Naples species complex. To gain knowledge concerning the geographical area where exposure to BALKV exists, entomological surveys were conducted in 2014 and 2015, in Croatia, Bosnia and Herzegovina (BH), Kosovo, Republic of Macedonia and Serbia.Recently, Balkan virus  from the others and that Croatian and BH sequences were grouped . The sand fly infection rate of BALKV was 0.26% in BH and 0.27% in Croatia. Identification of the species content of pools using Phlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.We report here (i) the first direct evidence that the Balkan virus initially described in coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomological results suggesting that Balkan virus is likely transmitted by  Phenuiviridae) are arthropod-borne viruses transmitted by mosquitoes, ticks and sand flies to vertebrate hosts ) from the others, and (ii) that Croatian and BH sequences were grouped  (GenBank: KY662276–KY662287).Phlebotomus neglectus sequences in all five pools; P. neglectus was the unique species in four pools, whereas P. tobbi DNA was present in 1 pool from Croatia  and similar to Fermo virus (0.20%) . Identif species . TogethePhlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.We report here (i) the first direct evidence that Balkan virus initially described in Coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomologic results suggesting that Balkan virus is likely transmitted by


The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus–host interactions will provide new targets for crop improvement. Arabidopsis [The ubiquitin proteasome system (UPS) contributes to all aspects of cell biology and is widely conserved among eukaryotes. The significance of the UPS in plants is reflected by the more than 1400 genes that encode putative E3 ubiquitin (Ub)-ligases in bidopsis ,3,4,5. MArabidopsis, there are approximately 469 E3 Ub-ligase genes encoding RING domain containing proteins, and very few are characterized [There are four subfamilies of E3 Ub-ligases, which include the HECT (Homologous to E3 associated protein C-terminus), RING , U-box and CRL (Cullin–RING ligases) . The moscterized . Thus, tMalformed nascent proteins in the ER are identified and redirected for turnover to prevent their release to subcellular compartments where they can cause toxic damage. In this regard, ERAD essentially functions as a quality control machinery. ERAD is vital to the maintenance of healthy cells across eukaryotes . The corN-glycans that show faulty maturation [ERAD pathways are identified as ERAD-L, ERAD-M and ERAD-C based on their targets and components ,11,12,13turation ,17. EBS7In yeast, the ER bound, HRD1 functions with two E2 Ub-conjugating enzymes identified as Ubc6p and Ubc7p ,19. In mA cytosolic ERAD pathway (ERAD-C) is maintained by another conserved E3 Ub-ligase machinery, named for the founding member in yeast Doa10p C. The yeTombusvirus) and cymbidium ringspot virus . Yeast has been used as a model genetic system to uncover critical host factors that contribute to tombusvirus infection [In recent years, there has been mounting evidence linking protein ubiquitination machinery, the proteasome, or both to the activation and subcellular localization of virus replication or movement protein complexes ,27,28. Tnfection ,30,31, enfection ,33,34. HOne major finding relevant to this discussion is that ubiquitin plays an important role in tombusvirus infection. Rad6/ubc2 and cdc34p are E2 Ub-conjugating enzymes that are components of the viral replication complexes identified in pull down assays ,35. BothRsp5p is an HECT domain E3 Ub-ligase that binds to the tombusvirus p33 and p92 protein and functions along with Uba1p (E1 enzyme) and Ubc1p (E2 enzyme). Rsp5p has three domains known as C1, WW and HECT domains . DeletioRpn11p, which is a central, stabilizing component of the 26S proteasome, is hijacked by TBSV, possibly to assist in suppression of RNA recombination in the spherular environment. Normally, Rpn11p functions to couple de-ubiquitination of substrates with proteasomal degradation, and also contributes to tombusvirus replication ,38,39. RTymoviruses), the UPS judiciously degrades the 66K protein, which is the RNA dependent RNA polymerase (RdRp) component of the viral replicase machinery and is derived from a polyprotein. The outcome is lower levels of accumulating 66K protein relative to other proteins involved in the viral replication complex. Specifically, translation of the TYMV genomic RNA produces two nonstructural proteins named for their molecular weights, the 69K movement protein and the 206K polyprotein. The 206K polyprotein is proteolytically processed to produce a 66K protein and a membrane anchored 140K protein. The 140K is an intermediate polyprotein that can be further processed to the mature 98K and 42K products. The 140K polyprotein intermediate binds to the 66K polymerase and drives the complex to the chloroplast membrane where the viral replicase assembles [Other examples of virus interactions with the UPS are either less well understood or demonstrate that the virus uses the UPS to degrade preferred targets. With respect to turnip yellow mosaic virus  coat protein can become poly-ubiquitinated during infection ,45. The The CRL are the largest category of E3 Ub-ligases, and are multi-subunit enzymes. CRLs are involved in plant hormone regulation, cell cycle regulation, development, and many other dynamic events . Cullin Arabidopsis and plants showed more lateral roots than the non-transgenic controls, had higher levels of auxin, and were more responsive to exogenous applications of 2,4-Dichlorophenoxyacetic acid  [Arabidopsis plants. One F-box protein named the AtFBK protein is a homolog of the sugar beet FBK protein, which binds to the P25 protein, but this interaction does not involve the substrate binding domain. The FBK protein also interacts with SKP1, ascribing this F box protein to the SCF complex. In Arabidopsis, there are two homologues of SKP1 known as ASK1 and ASK2. Competitor studies suggest that P25 interferes with FBK and ASK1 interactions but not FBK and ASK2 interactions. These data suggest that P25 directs FBK to a specific adaptor protein and monitors SCF complex formation. The data argue that P25 targets the F-box protein to downregulate cellular necrosis while promoting rhizomania symptoms [R gene, it is also possible that the P25-F box interaction eliminates the ability of the SCF complex to function in de-repressing R gene function , the P25 proteins are identified as a pathogenicity factor responsible for rhizomania symptoms, also known as “crazy root” symptoms . The cla analog) . Three gsymptoms . If we afunction . Such a Fijivirus) P7-2 protein, which is likely incorporated into cellular SCF complexes. P7-2 has a Mr of 36 kDA and interacts with the ZmSKP1 protein [Poleroviruses interestingly encode the P0 protein that combines the F-box protein interactions to target degradation of the host gene silencing machinery. The polerovirus P0 protein can be incorporated into the SCF complex and identifies substrate Argonaute 1 (AGO1) for degradation. AGO1 is central to the RNA-induced silencing complexes (RISC), which incorporates anti-viral small interfering RNAs (siRNAs) as part of the gene silencing machinery. The polerovirus P0 protein is a silencing suppressor that enables turnover of AGO1 to limit anti-viral defenses ,60. Anot protein . Unlike  protein  . The N-gene belongs to one class of toll-interleukin 1-receptor (TIR) nucleotide-binding site leucine-rich repeat (NB-LRR) genes [With respect to ETI, the role for the SCF complex in the arms race between pathogen and host is exemplified in studies involving TMV and n potato B. The N-R) genes  and Rx bR) genes . During R) genes ,78. In tR) genes . HoweverBeyond virus control through PTI related RLKs or ETI related NLR proteins, geminiviruses, which have single strand circular DNA genomes and replicate in the nucleus, provide an example of how a certain class of viruses can use the SCF-mediated ubiquitin system to elude host defenses. This goes beyond the poleroviruses or fijivirus encoded F-box proteins that can be incorporated into SCF complexes ,71. To bThe UPS is also central to the arms race involving geminiviruses because, in addition to the example of C2 disruption of the SCF complex, the UPS can also play a central role in anti-viral mechanisms C. SpecifThe UPS machinery contributes to adaptive cellular responses to environmental stressors, change in ER conditions, and virus infection. Given the plethora of genes that are categorically ascribed to the UPS, we are still uncovering the various contributions of this system to plant cell biology. However, there are some notable themes emerging that indicate that the UPS is central to plant virus infection. One theme that stands out is the role of ubiquitin machinery supporting tombusvirus and tymovirus replication. With respect to tombusvirus replication, Rad6/ubc2, cdc34p and Rsp5p provide three very interesting examples where separate E2, E3, and proteasome components are incorporated into the viral replication machinery to support interactions with a subset of ESCRT proteins needed to shape a membrane environment along the peroxisome needed to support virus replication. In addition, at least one component of the proteasome is known to be highjacked by the virus, possibly to assist in suppression of RNA recombination in the spherular environment. These data highlight the controlling role that the UPS machinery plays in virus infection making individual component factors useful targets for crop improvement. The application of these discoveries is to identify the best component factors where introducing a mutation into the gene could suppress virus infection and with perhaps limited impact on crop yield. Given the new technologies for transformation, this becomes a reasonable strategy for transferring this knowledge to crop improvement. The tombusvirus example, in fact, highlights the value of in-depth studying of the molecular mechanisms of plant virus infection for identifying valuable targets for crop improvement while understanding how these targets function.With respect to the role of UPS in virus intercellular and systemic movement, studies of TMV and PVX suggest that the UPS machinery is required for rapid turnover of viral proteins to reduce membrane damage and cellular toxicity. The UPS machinery also contributes to antiviral immunity either by monitoring negative repressors of NLR activation, degrading viral effectors, or degrading AGO to suppress gene silencing.With respect to ERAD, the ERAD-C, -L, and -M pathways for protein degradation are well described in mammals and yeast, but far less is known in plants. Data thus far in plants confirm that the HRD1 pathway regulates misfolded proteins in the ER lumen and intramembrane domains, while the SUD1/Doa10/TEB4 pathway regulates malformed proteins in the cytosol. Until now, research into the topic of plant virus related protein ubiquitination and degradation has not uncovered a role for ER resident HRD1 or SUD1. The generally limited information surrounding the nature of the ERAD machinery in plants also limits our understanding of its role in plant virus infection. It is worth noting that most of the research to uncover ERAD machinery in plants has employed abiotic stressors, microbial effectors, and hormone applications. Similar research carried out using plant viruses as stressors are likely to uncover novel aspects of the machinery that could propel our understanding of cellular protein degradation machinery and ERAD substrates in new directions.The SCF complex is the best studied of the E3 Ub-ligases in plants, and is thus far identified in the turnover of AGO1, NLR gene mediated anti-virus innate immunity as well as the direct regulation of plant virus infection. The models presented thus far indicate that ubiquitin is important for intervention during host defense responses to virus infection and can be used to compromise the host gene silencing machinery. A combination of genetic and biochemical approaches will be needed to provide new understanding of the mechanisms that are exploited by viruses to achieve successful biotrophic infections.The ER is central to plant virus replication, translation, maturation, and egress. ER resident chaperones can be hijacked from the ER lumen or cytosolic side of the ER and incorporated into viral replication complexes to promote viral RNA accumulation. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome are vital for the regulation of infection. ER associated ubiquitination as well as cytosolic ubiquitin ligases can be targeted by viruses as a means to direct the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of AGO to suppress gene silencing. Combining the results of unrelated studies, many positive strand RNA plant viruses, as well as the single strand DNA geminiviruses, appear to interact with E3 Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serves to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In some instances, knockout mutations in the UPS can hamper virus infection. In other instances, knockout mutations lead to higher levels of virus accumulation, more aggressive disease, and/or cell death. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus–host interactions will provide new targets for crop improvement.


Rhizoctonia solani represents an important plant pathogenic Basidiomycota species complex and the host of many different mycoviruses, as indicated by frequent detection of dsRNA elements in natural populations of the fungus. To date, eight different mycoviruses have been characterized in Rhizoctonia and some of them have been reported to modulate its virulence. DsRNA extracts of the avirulent R. solani isolate DC17 (AG2-2-IV) displayed a diverse pattern, indicating multiple infections with mycoviruses. Deep sequencing analysis of the dsRNA extract, converted to cDNA, revealed that this isolate harbors at least 17 different mycovirus species. Based on the alignment of the conserved RNA-dependent RNA-polymerase (RdRp) domain, this viral community included putative members of the families Narnaviridae, Endornaviridae, Partitiviridae and Megabirnaviridae as well as of the order Tymovirales. Furthermore, viruses, which could not be assigned to any existing family or order, but showed similarities to so far unassigned species like Sclerotinia sclerotiorum RNA virus L, Rhizoctonia solani dsRNA virus 1, Aspergillus foetidus slow virus 2 or Rhizoctonia fumigata virus 1, were identified. This is the first report of a fungal isolate infected by 17 different viral species and a valuable study case to explore the diversity of mycoviruses infecting R. solani. Rhizoctonia solani Kühn [teleomorph: Thanatephorus cucumeris (Frank) Donk] represents an important species complex of known plant pathogens, which infect a wide variety of plant species including many economically important crops [Rhizoctonia is considered as ubiquitous in soil, but has limited abilities of long distance spread, because it does not produce asexual spores [Rhizoctonia, especially regarding the disease suppression observed in association with monoculture of host plants, the interaction of the fungus with its environment and, in particular, the microflora has been studied by many researchers [Rhizoctonia is significantly affected by suppressive organisms, but the nature of this biocontrol is still a matter of debate [Rhizoctonia can be successfully used for biocontrol purposes, raised also interest in the possible involvement of mycoviruses [nt crops . Due to nt crops . Rhizoctl spores , 3. To uearchers . Today, f debate . The finoviruses , 6.Geminiviridae, which is reported to be transmitted via spray application of purified virions and induces hypovirulence [Cryphonectria parasitica) caused by hypoviruses, raises hope to exploit also other mycoviruses as biocontrol agents [R. solani infecting mycoviruses [Partitiviridae, Endornaviridae, Narnaviridae and a few unclassified mycoviruses [Mycoviruses have been identified in all major fungal taxa and the number of newly described species has expanded enormously during the last years . The incirulence . Neverthirulence , 11 and irulence . The advirulence , to seveirulence . Using mirulence . Successl agents . Neverthoviruses –20. Howeoviruses –28. Deepoviruses , 29. Untoviruses . This leoviruses –32. The oviruses . Consideoviruses , 34. Howoviruses , 35. FurThe fungal strain DC17, originally isolated from sugar beet, was derived from the IRS  and identified as anastomosis group 2-2IV by sequencing of the internal transcribed spacer region . The iso2) containing 0.25 g polyvinylpyrrolidone. Nucleic acids were extracted by addition of 2 mL beta-mercaptoethanol, 0.5 mL SDS (20%), 20 mL phenol/chloroform (1:1) and the suspension was incubated on a shaker for 30 min at RT, followed by centrifugation for 20 min at 5000 xg. The aqueous phase was supplemented with pure ethanol to a final concentration of 16%, mixed with 2 g CF-11 cellulose (Whatman) and incubated for another 20 min at RT. Contaminants were removed by three washing steps with STE16 (STE + 16% EtOH) followed by centrifugation for 2 min at 500 xg. The cellulose was resuspended in 20 mL STE16 and transferred into Econo-Columns (Biorad). After an additional washing step with 20 mL STE16, the dsRNA was eluted by the addition of 10 mL 2xSTE. To remove contaminating genomic DNA and RNA, the eluate was treated with T1-RNase (Roche) (1 U/mL)  followed by the addition of 3.3% 1 M MgCl2 and a treatment with DNase I (Roche) (2 U/mL) for 30 minutes at 37°C each. Thereafter the extract was supplemented with EtOH to a final concentration of 16% and 3.3% 0.5 M EDTA was added. The eluate was mixed with 2 g cellulose and treated as described above. The dsRNA was eluted with 8 mL STE. For precipitation, 80% isopropanol was added. The mixture was incubated at -20°C over night and centrifuged for 30 min at 10 000 xg. After a washing step with 70% EtOH, the dsRNA was resolved in TE buffer and checked by gel electrophoresis for quantity and quality control.The dsRNA was extracted using a modified version of the protocol published by Valverde and colleagues . Myceliu2, 0.15 pmol Primer (GCCGGAGCTCTGCAGAATTC), 12.5 nmol dNTPs, 1 U Dreamtaq (Thermo Scientific) and 5 μL 10x Dreamtaq buffer in a total volume of 50 μL. PCR conditions were as follows: Initial denaturation at 94°C for 1 min, followed by 30 cycles of 20 sec 94°C, 30 seconds 55°C, 2 min 72°C and a final elongation step of 10 min at 72°C. PCR products were analyzed by gel electrophoresis and fragments between 0.5 and 2 kb were excised and extracted from the gel using the NucleoSpin, PCR clean-up, gel extraction kit. Fragments were sheared and adaptors for Illumina sequencing were ligated [The cDNA library was prepared following a modified version of the protocol of Froussard . Four μL ligated .R. solani DC17 virome was established on the Illumina MiSeq system by a pair-end sequencing run (2 × 300 bp) with a distance range of about 500 bp. A de novo assembly was performed using the gsAssembler software (version 2.8.) with default settings. Resulting virus sequences were in silico finished [http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Predicted amino acid sequences were subsequently compared to the NCBI database using BLASTP with standard settings and conserved domains were identified using the conserved domain database at the NCBI website (http://www.ncbi.nlm.nih.gov/cdd).DNA quality was assessed by gel-electrophoresis and the quantity was estimated by a fluorescence-based method using the Quant-iT PicoGreen dsDNA kit (Invitrogen) and the Tecan Infinite 200 Microplate Reader (Tecan Deutschland GmbH). The finished , importefinished  and refiTo complete partial RdRp domains derived from smaller contigs, cDNA was synthesized using a sequence specific primer based on known sequence information targeting the whole RdRp domain . cDNA sySpecific primers were designed according to the results of the Illumina sequencing, flanking the region of the conserved RdRp domains. Primer sequences are listed in Nucleotide sequences, which were confirmed by resequencing with specific primers, were translated into amino acid sequences and aligned to reference genomes from the NCBI database. Conserved motifs were identified by comparisons with well characterized RdRps based on work published by Bruenn 2003, Černý et al. 2014, Koonin et al. 1993, Boehr et al. 2014 and Xu et al. 2003 , 42–44. R. solani isolate DC17 showed a diverse pattern of dsRNA fragments after gel electrophoresis indicating the presence of multiple mycoviruses. At least six dsRNA fragments with a mobility corresponding to the sizes of approximately 0.85 to 12 kb were identified . Based on the assembled virome data, it was possible to identify 17 contigs encoding different viral RdRp domains. For detailed analysis, these RdRp domains were re-sequenced based on whole RNA extracts and partial domains were completed. These data were used for taxonomic classification. The identified RdRps belong to four different protein families (Pfam) corresponding to either the alphavirus-like superfamily, the picornavirus-like super family or the Narnaviridae are the simplest of all RNA viruses consisting only of one small single-stranded RNA of 2.3 to 3.6 kb, which codes for a single RdRp gene [Narnavirus and Mitovirus. The members of the Narnaviridae are only distantly related to other RNA viruses infecting eukaryotes and share higher sequence similarity to bacteriophages of the family Leviviridae [R. solani isolate DC17 is the host of six different mitoviruses. Sequence comparisons with all recognized members of the genus Mitovirus allowed the identification of the four conserved motifs I to IV previously described for mitoviruses by Kitahara and coworkers [Rhizoctonia, the majority of them in R. solani [Rhizoctonia solani mitovirus 16 to 20 (RsMV-16 to 20). The RdRp domain of one mitovirus identified in the analyzed virome shows high sequence similarity of 92% to Rhizoctonia solani mitovirus 9 (KP900918.1). Since only a partial sequence of this virus has been deposited in the NCBI database, no comparison of the complete RdRp protein was possible, to check whether both viruses fulfill the species demarcation criterion of less than 90% amino acid sequence similarity over the complete RdRp protein [Rhizoctonia solani mitovirus 9 DC17.Viruses of the family dRp gene . The famiviridae . The anaoworkers . Multipl. solani , 50. Ana9 KP90091.1. SinceEndornaviridae are found in algae, plants and fungi [Soybean-associated endornavirus 1, which is the species in the NCBI database showing the highest level of similarity [Endornaviridae were identified by sequence alignments with all recognized members of this family (Rhizoctonia solani endornavirus 3 (RsEV-3).Viruses belonging to the family nd fungi . They pond fungi . The polnd fungi . The CDDmilarity . Seven cs family . This inEndornaviridae. So far, three mycoviruses belonging to the family of the Endornaviridae were described to infect the genus Rhizoctonia, namely Rhizoctonia solani endornavirus—RS002 (RsEV-1), Rhizoctonia solani endornavirus 2 (RsEV-2) and Rhizoctonia cerealis endornavirus 1 (RcEV-1) [Endornaviridae can also be high, like it is the case for the Bell pepper endornavirus and Phaseoulus vulgaris endornavirus, which share a sequence homology of 74% within their RdRp domains.This finding was confirmed by a phylogenetic analysis of the conserved RdRp domain within the alphavirus-like superfamily which placed this novel virus within the family (RcEV-1) ,50, 53. Tymovirales comprises four viral families. Viruses belonging to the Alphaflexiviridae, Betaflexiviridae and Gammaflexiviridae feature a filamentous morphology, whereas members of the Tymoviridae are icosahedral. So far, three mycoviruses belonging to the order Tymovirales have been recognized by the ICTV; all of them represent own genera [Botrytis virus X (Botrexvirus) and Sclerotinia sclerotiorum debilitation-associated RNA virus (Sclerodarnavirus) belong to the Alphaflexiviridae, Botrytis virus F represents the only member of the family Gammaflexiviridae [R. solani DC17 revealed the presence of two domains assigned to pfam00978, which were distantly related to each other and to several members of the order Tymovirales. Sequence comparisons with the type species of all genera within the order allowed the identification of the seven conserved motifs A to G  and Rhizoctonia solani flexivirus 2 (RSFV-2).The order n genera . Whereasiviridae –56. The s A to G . A phyloovirales . They foR. solani DC17 virome also revealed the presence of three domains, representing the pfam00978 group that were closely rated to each other, but showed no clear affiliation to any of the recognized families of the alphavirus-like superfamily. Instead, they display similarities to the so far unassigned mycovirus Sclerotinia sclerotiorum RNA virus L (SsRV-L), as indicated by BLAST analysis. SsRV-L is a positive-strand RNA virus of 6,043 nts, which carries one large ORF of 1965 aa featuring conserved domains for a methyltransferase, helicase and RdRp [R. solani DC17 form a separate clade together with SsRV-L. This group shows a distant relationship to the families of the Hepeviridae and Alphatetraviridae  [Rhizoctonia solani RNA virus 1 (RsRV-1), Rhizoctonia solani RNA virus 2 (RsRV-2), Rhizoctonia solani RNA virus 3 (RsRV-3).The search for conserved RdRp domains in the and RdRp . Multiplaviridae . Two of aviridae , indicatRosellinia necatrix partitivirus 7. An alignment with several recognized members of the family Partitiviridae allowed the identification of the conserved domains A to F. It is reported that motif G is not well conserved within this viral family [Alphapartitivirus , is a new member of the genus Alphapartitivirus.The virome analysis revealed the presence of one contig of 1958 nt, which codes for a putative protein of 633 aa. The protein contains a conserved RdRp domain showing the highest sequence similarity of 56% to the conserved RdRp domain of l family . Neverthitivirus . A seconR. solani DC17 virome showed the presence of a domain belonging to the cd01699 group. Different members of the picornavirus-like superfamily, like the Claciviridae or the Partitiviridae, share this group of domain. A multiple alignment with different members of the picornavirus-like superfamily allowed the identification of the conserved motifs A to G (Aspergillus foetidus slow virus 2 (AfsV-2). The virus present in R. solani DC17 was named Rhizoctonia solani mycovirus 1 (RsMV-1). The phylogenetic analysis grouped RsMV-1 with AfsV-2, Penicillium aurantiogriseum foetidus-like virus (PaflV) and Rosellinia necatrix mycovirus 2 (RnMV-2) into one clade, which is distantly related to the Calciviridae  is an unclassified bi-segmented dsRNA virus, which was isolated from R. solani AG-1 IA [Partitiviridae and both share a RdRp of pfam00680 group. Nevertheless, a phylogenetic analysis indicates that it is only distantly related to the family Partitiviridae [R. solani DC17 showed that one of the RdRps is very similar to the one of RsRV-1 carrying the same conserved motifs A to G . Both viruses are closely related, which was also confirmed by the phylogenetic analysis of the RdRp domain. Both viruses cluster together and form a distinct clade together with some so far unclassified viruses like Heterobasidion RNA virus 6 or Curvularia thermal tolerance virus  is the only recognized member of the family Megabirnaviridae. It possesses a bi-segmented double-stranded RNA genome and the segments are encapsidated separately into isometric particles. The first segment carries two ORFs, which code for an RdRp of the pfam02123 protein family and a coat protein. The second segment encodes two proteins of unknown function [R. solani DC17, two conserved RdRp domains of the pfam02123 group were detected. One showed significant similarity of 68% to the RdRp of RnMBV-1, whereas the second RdRp domain displayed lower similarity of only 29%. Sequence comparisons with different members of the picornavirus-like superfamily allowed the identification of the conserved motifs A to G in both novel RdRps  [Phlebiopsis gigantean large virus-1 (PgV-1) and Lentinula edodes mycovirus (HKB and HKA) [R. solani DC17 contains two mycoviruses, which are related to the family Megabirnaviridae. The first virus seems to be a member of the genus Megabirnavirus and was named Rhizoctonia solani megabirnavirus 1 (RsMBV-1). The second virus is closely related to a different group of so far unclassified viruses and was named Rhizoctonia solani mycovirus 3 (RsMV-3). To elucidate whether RsMV-3 belongs to a new viral family or a new proposed genus within the Megabirnaviridae [Currently, function , 61. In el RdRps . A phyloaviridae . However (RfV-1) . RfV-1 iand HKA) . The anaaviridae , furtherNarnaviridae , as well as to the Partitiviridae , it is not sure if all mycoviruses are delectable by this method, as interference with RNA silencing seems to be an important trait in mycoviruses [Our results demonstrate that a deep sequencing approach is the method of choice for the analysis of diverse viromes and that the characterization of the conserved RdRp domain is the best way to get a valid overview about viral diversity in such complex RNA virus viromes. However, our study also showed the two main pitfalls of virus characterization in virome samples. In general, virome analysis faces the problem, that no marker gene for targeted amplification like the ribosomal internal transcribed spacer (ITS) is present in viruses. Instead, other methods for the enrichment of viral sequences are used, which all bring along certain disadvantages. The most popular method is the purification of virus particles, which seems natural, as the viruses used to be defined as capsid-coding organisms . Howeveroviruses . The seqoviruses . Especiaoviruses . To overoviruses , 66. FolThe second problem is the limited number of characterized mycoviral sequences, which can be used as reference for species assignment of the generated contigs. Apart from conserved regions, sequence similarities are too low to draw conclusions about the phylogeny of the virus . The RdRR. solani DC17 and allowed an identification of novel viruses which might be of special interest in the fields of biocontrol, virus biology and taxonomy. For example, the analysis revealed the presence of the novel mitovirus RsMV-20, which is closely related to Thanatephorus cucumeris mitovirus (TcMV). An infection of Rhizoctonia with TcMV resulted in decreased virulence of the host [Rhizoctonia (AG-3), infected with TcMV, demonstrated its potential use as biocontrol agent [Pleosporales megabirnavirus 1, which was detected in the virome of marine fungi, were associated with hypovirulence of their hosts [Rosellinia necatrix mycovirus 1, which belongs to a group of novel viruses distantly related to the Totiviridae [Victoriviruses (Totiviridae) [R. solani DC17, RsMV-1 would have to use the CP of another virus, like Rhizoctonia solani partitivirus-1 or Rhizoctonia solani megabirnavirus-1. Either way, a finding like this would be interesting in regard to virus interactions and evolution.This approach enabled us to get a first and valid impression of the viral diversity of the species inhabiting the host , 68. Stuir hosts , 69–71. ir hosts , 58. Theiviridae . In caseStill, for a full-value assignment into viral families and a biological characterization of the novel viruses, more criteria than the phylogeny of the RdRp have to be analyzed, especially when the viruses belong to novel viral groups. The next steps need to be the determination of full-length sequences and the isolation of single virus species to study their biological effects by e.g. purification and transfection of viral particles, the construction of infectious full-length cDNA-clones, or the elimination of certain virus by mycelial fragmentation, hyphal tipping or protoplastation –75.R solani to explore the diversity of mycoviruses in this fungus. By the characterization of the conserved RdRp domains, we identified 17 different mycoviruses of which 16 domains represent novel species not characterized before. To our knowledge no comparable number of virus species belonging to at least eight different families, has been previously reported to infect a single fungal isolate. The research conducted here is a step forward in exploring the mycoviruses of R. solani. The enormous diversity identified in this study raises questions about the interactions of viruses as well as the dynamics of viral communities in fungi. More detailed analysis of the hypovirulent isolate DC17 and its inhabiting viruses might help to get insights into this still unexplored field of mycovirus research. Work, like the one presented here, will help to further populate public databases with viral sequences, which in turn will help to solve difficulties in virus identification and classification in the future [This is the first study characterizing a complex virome from a hypovirulent isolate of e future .S1 Table(DOCX)Click here for additional data file.


Plants have been explored for many years as inexpensive and versatile platforms for the generation of vaccines and other biopharmaceuticals. Plant viruses have also been engineered to either express subunit vaccines or act as epitope presentation systems. Both icosahedral and helical, filamentous-shaped plant viruses have been used for these purposes. More recently, plant viruses have been utilized as nanoparticles to transport drugs and active molecules into cancer cells. The following review describes the use of both icosahedral and helical plant viruses in a variety of new functions against cancer. The review illustrates the breadth of variation among different plant virus nanoparticles and how this impacts the immune response. Plants as bioreactors for modern pharmaceutical products have been under investigation for many years, and these are only now beginning to hit the marketplace. From microbicides for HIV treatment, vaccines for pandemic influenza, to a therapeutic agent for Gaucher’s disease, plant-made pharmaceuticals are being readily employed for a variety of purposes in medicine today ,2,3. PlaPlant viruses have also undergone research and development for their potential use as expression vectors for pharmaceutical production ,5. PlantOver the past few years, plant viruses have found a novel role as nanoparticles, to not only deliver medical materials such as drugs, but also sources of material for electronics and optics, as examples ,12,13. IPlant viruses are frequently characterized by their icosahedral or helical morphologies; these differences offer a range of attributes which make them suitable for a variety of purposes in medicine as well as in engineering ,12,13. BPlant viruses which possess an icosahedral morphology and have been engineered as nanoparticles include Cowpea mosaic virus (CPMV), Brome Mosaic virus (BMV), Red clover necrotic mosaic virus (RCMNV), Hibiscus chlorotic ringspot virus (HCRSV), Johnson grass chlorotic stripe mosaic virus (JgCSMV), and Physalis Mottle Virus (PhMV) . The higOf all the icosahedral plant viruses that have been developed as nanoparticles, Cowpea mosaic virus (CPMV) has been studied the most extensively. CPMV has been found to bind specifically to vimentin, which is expressed on the surface of many types of cells, including HeLa cells. Upon binding, CPMV can be internalized by endocytosis ,39. UptaA mouse model has been used to demonstrate that CPMV nanoparticles can act as effective immunotherapies for lung metastatic melanoma models and other cancers by inducing an innate immune cell-mediated anti-tumor response . CPMV naMadden et al. (2017) examined the doxorubicin containing Red clover necrotic mosaic virus (RCNMV) as a drug delivery vehicle. The effectiveness RCNMV nanoparticles (PVNs) were compared with PEGylated liposomal doxorubicin (PLD) and small molecule doxorubicin using mouse ovarian cancer and melanoma models . The autAnother example is Hibiscus chlorotic ringspot virus (HCRSV), a positive-sense, RNA icosahedral virus 30 nm in diameter and of the family Tombusviridae. The coat protein of HCRSV is 38 kDa and can self-assemble into empty virus-like particles. The authors Ren et al. (2007) first demonstrated the ability of HCRSV to transport drug molecules polystyrenesulfonic acid (PSA) and polyacrylic acid (PAA) . The autAlemzadeh et al. (2017) used Johnson grass chlorotic stripe mosaic virus (JgCSMV), a member of the Tombusvirus family, to produce empty virus-like particles . JgCSMV E. coli to make p = 3 structured virus-like particles that resemble the wild type virus. Masarapu et al. (2017) found that the N-terminus of the CP can be modified without deleterious effect to virus assembly [Physalis mottle virus is a 30 nm icosahedral member of the tymovirus family, with a positive sense RNA genome. The 21 kDal CP can self-assemble in assembly . VLPs deHelical, or filamentous virus nanoparticles are characterized by having a high aspect ratio, which enables them to be utilized for a whole variety of purposes and can include the tombusviruses such as Tobacco mosaic virus (TMV), the potexviruses such as Potato virus X (PVX) and Papaya mosaic virus (PapMV), and potyviruses such as Zuchinni yellow mosaic virus (ZYMV) ,13 Tabl. Their rOne powerful use for plant virus nanoparticles has been found in the field of personalized medicine. Non-Hodgkin’s Lymphoma (NHL) is a common hematologic malignancy with approximately one quarter of all cases presenting as follicular B-cell lymphoma. Conventional treatment for NHL include chemotherapy, radiation, passive antibody therapy and more recently, active immunotherapy . MalignaTMV has also been engineered as a drug carrying nanoparticle to combat non-Hodgkin’s lymphoma. Kernan et al. (2017) conjugated the antimitotic drug valine-citrulline monomethyl auristatin E (vcMMAE) targeting non-Hodgkin’s lymphoma, to TMV nanoparticles . The autJobsri et al. (2015) used Potato virus X nanoparticles containing on their surface idiotypic tumour antigens to elicit an immune response against murine B cell lymphoma . The autPlant-derived vaccines have also been explored as a potential immunotherapy against solid tumors. For example, the TMV MagnICON system has been used to elicit a strong response against breast cancer, by expressing a fragment of Her2 . PVX nanThe externally exposed side chains of the capsid protein of Potato virus X includes a lysine residue which can be functionalized to conjugate drugs or imaging reagents . These vCarignan et al. (2017), demonstrated that Papaya mosaic virus (PapMV) nanoparticles containing single-stranded RNA could function as a TLR7 agonist and could be employed as an immune modulator in cancer immunotherapy . The autTMV can be further modified to be tumor-specific by functionalizing a tumor-homing peptide, such as cRGD, to its surface; this enables the virus to become internalized into tumor cells . TMV nanTMV has also been used as a hollow nanotube to encapsulate with photosensitizers based on porphyrin dynamic sensors. The nanotubes were engineered to target melanoma cells and deliver their cargo. Photodynamic therapy (PDT) based on reactive oxygen species created during exposure to activation by specific light wavelengths, was used to efficiently kill the melanoma cells . Besides vaccine and epitope carrying and drug delivery systems, other uses for plant virus nanoparticles have included an assortment of biosensors, optoelectronic devices, catalysts and imaging systems ,12. Plant virus nanoparticles are broad in application. For example, Pitek et al. (2017) demonstrated that Tobacco mosaic virus can home in to thrombi and deliver streptokinase via PEG linkers to mouse models of thrombotic cardiovascular disease . The autThe flexuous rod-shaped virions of potyvirus Turnip mosaic virus (TuMV) has also been examined for peptide display. Recently, González-Gamboa et al. (2017) found that a peptide derived from the human thrombin receptor (TR) inhibited infectivity of recombinant TuMV. The authors produced virus-like particles using both wild type as well as recombinant capsid protein . These VMore engineering strategies are being made to further enhance the utilization of TMV as a nanoparticle in medicine. Fischer et al. (2017) expressed the monomeric non-oxygen-dependent fluorescent protein iLOV to the C-terminus of the coat protein of TMV using an N-terminal Foot-and-mouth disease virus (FMDV) 2A ribosome skipping sequence . FluorocThe cysteine side chains of Potato virus X (Cys 121) have recently been identified as reactive groups for modification in addition to lysine . LysinesOver the past two decades, plant viruses are well on their way to becoming established as expression systems for vaccines and other pharmaceutical proteins. More recently, plant viruses have found a place in the fight against cancer, by acting as ‘smart’ nanoparticles that can home in on tumor cells and deliver drugs as cargo, as well as in many cases elicits a highly localized and powerful immune response . Plant vTheir inherent ability to penetrate solid tumors provides plant viruses with an added edge in our current arsenal of nanocarriers for cancer therapy ,33. Drug