The tick-borne flaviviruses (TBFV) occur worldwide and the tick-borne encephalitis virus (TBEV) members of the group often cause severe, debilitating neurological disease in humans. Although the primary route of infection is through the bite of an infected tick, alimentary infection through the consumption of TBEV-contaminated dairy products is also well-documented and is responsible for some disease in endemic areas. Experimental infection of goats, cattle, and sheep with TBEV shows that the virus can be excreted in the milk of infected animals. Additionally, the virus remains infectious after exposure to low pH levels, similar to those found in the stomach. To evaluate the survival of virus in milk, we studied the stability of the BSL-2 TBFV, Langat virus, in unpasteurized goat milk over time and after different thermal treatments. Virus was stable in milk maintained under refrigeration conditions; however, there was a marked reduction in virus titer after incubation at room temperature. High temperature, short time pasteurization protocols completely inactivated the virus. Interestingly, simulation of a typical thermal regime utilized for cheese did not completely inactivate the virus in milk. These findings stress the importance of proper milk handling and pasteurization processes in areas endemic for TBEV. Flaviviridae family. TBEV are commonly distributed across Europe and Asia and are further divided, based on geography, into three main subtypes: European, Siberian, and Far Eastern. There are two autochthonous TBFV in North America: Powassan virus and the closely related deer tick virus  group of tick-borne flaviviruses (TBFV) are pathogenic members of the us Ebel, . All of us Ebel, , but eveus Ebel, . The natus Ebel, .via the bite of an infected tick; however, there are well-documented reports of alimentary transmission from infected, domesticated animals in endemic areas. This most often occurs through consumption of unpasteurized “raw” goat milk  than that seen from a tick bite (7–14 days)  cells (ATCC) were maintained in Dulbecco’s minimal essential media  supplemented with 50 μg/ml gentamicin and 10% fetal bovine serum (FBS) (complete DMEM) at 37°C in 5% COvia immunofocus assay, as previously described   was prepared by infection of Vero cell cultures at a multiplicity of infection of 0.005. Virus was titrated 6 focus forming units per milliliter (ffu/ml) in either unpasteurized goat milk or complete DMEM. Five replicate 1 ml aliquots were aliquoted into sterile screw cap tubes (Sarstedt) for each time point, media, and incubation temperature. Samples were incubated at either 4 or 22°C for 0, 8, 24, 48, or 72 h. At each time point, five replicates for each milk and DMEM were placed at −80°C and stored until viral titration was performed (as above). Serial dilutions of each replicate were made in complete DMEM (10−1 to 10−5) and tested along with the undiluted sample.Freshly collected, unpasteurized goat milk was obtained from a local supplier, stored at 4°C, and used the same day. LGTV was adjusted to a final concentration of 106 ffu/ml in unpasteurized goat milk or complete DMEM. Samples of the inoculated media (milk or DMEM) were aliquoted into 0.2 ml PCR tube strips . A DNA engine tetrad thermocycler (Bio-Rad) was used for performing heat pasteurization of the samples. Samples were subjected to “high temperature, short time” pasteurization (HTST) by heating to 72°C for 15 s, immediately cooled to 4°C, and then placed at −80°C until viral titration was performed. To simulate the heating process involved in cheese making, samples were also slowly heated at 0.1°C/min to 30°C, then cooled to 22°C, and incubated for 16 h; samples were then placed at −80°C until viral titration. Viral titration was done by pooling eight 100 μl samples (one tube strip) per replicate.To simulate heat pasteurization conditions . Mean values were computed for the repetitions and two-way ANOVA performed using Sidak multiple comparisons test. Results were not considered to be statistically significant unless a 6 ffu/ml LGTV TP21 in quintuplicate 1 ml aliquots. Aliquots were incubated after inoculation for 0, 8, 24, 48, or 72 h at a temperature of either 4 or 22°C. After all time points had been collected, residual virus titer was determined by immunofocus.Fresh, unpasteurized goat milk was obtained from a local goat breeder and, along with complete DMEM, was added to a final concentration of 1010 decrease over the 72-h period    is one of the most common ways that milk is pasteurized commercially for consumption  and by approximately 0.5 log10 in DMEM samples . Thus, the treatment reduced the quantity of virus, but there was still residual virus in the samples.Some reports describe TBEV infection following consumption of cheese made with milk from infected animals  with the globules upon release into the lumen  under various laboratory conditions. Similar to virus in milk, no significant change was seen in viral titer over the course of a 72-h incubation period under refrigeration conditions. Surprisingly, the virus was significantly more stable at room temperature and during heating to 30°C in complete DMEM supplemented with glucose and FBS than in milk. The reason for this is not immediately obvious.In summary, our study using LGTV, a BSL-2 model for TBEV, strongly supports alimentary transmission as a valid route for TBFV infection in endemic regions. Pasteurization of milk before ingestion or further processing into dairy products was shown to prevent retention of infectious virus, while heating to 30°C fails to eliminate the risk of disease. Further studies to characterize how virus gets into the milk of infected animals might be warranted.DO and NC performed the experiments. DO and MB designed the experiment and analyzed data. DO and MB wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Type 2 diabetes (T2D) and cardiovascular disease (CVD) are leading causes of mortality and two of the most costly diet-related ailments worldwide. Consumption of fiber-rich diets has been repeatedly associated with favorable impacts on these co-epidemics, however, the healthcare cost-related economic value of altered dietary fiber intakes remains poorly understood. In this study, we estimated the annual cost savings accruing to the Canadian healthcare system in association with reductions in T2D and CVD rates, separately, following increased intakes of dietary fiber by adults.Methods: A three-step cost-of-illness analysis was conducted to identify the percentage of individuals expected to consume fiber-rich diets in Canada, estimate increased fiber intakes in relation to T2D and CVD reduction rates, and independently assess the potential annual savings in healthcare costs associated with the reductions in rates of these two epidemics. The economic model employed a sensitivity analysis of four scenarios  to cover a range of assumptions within each step.Results: Non-trivial healthcare and related savings of CAD$35.9-$718.8 million in T2D costs and CAD$64.8 million–$1.3 billion in CVD costs were calculated under a scenario where cereal fiber was used to increase current intakes of dietary fiber to the recommended levels of 38 g per day for men and 25 g per day for women. Each 1 g per day increase in fiber consumption resulted in annual CAD$2.6 to $51.1 million savings for T2D and $4.6 to $92.1 million savings for CVD.Conclusion: Findings of this analysis shed light on the economic value of optimal dietary fiber intakes. Strategies to increase consumers’ general knowledge of the recommended intakes of dietary fiber, as part of healthy diet, and to facilitate stakeholder synergy are warranted to enable better management of healthcare and related costs associated with T2D and CVD in Canada. Globally, the growing prevalence of type 2 diabetes (T2D) and CVD has been accompanied by escalating costs related to healthcare and society’s loss of productivity, putting these two diet-related epidemics among the world’s top public health policy priorities. Approximately 9% of adults around the world were diagnosed with diabetes in Dietary behaviors consistent with guidelines for healthy eating have the potential to produce substantial health and economic benefits. Previous research has demonstrated that, for example, increased consumption of dairy, as well as reduced intakes of calories, sodium, and saturated fat would facilitate considerable health and economic benefits ranging from US$2 to $58 billion annually . From a n = 239,485) showed a 19% lower risk of diabetes  among individuals in the highest quintile of dietary fiber intake  demonstrated that, compared to the lowest quintile of fiber intake, the highest levels of dietary fiber reduced risk of CHD by 29%  Independent analysis of the T2D and CVD reduction rates that would result from consumption of dietary fiber, and (iii) Estimation of the healthcare cost savings associated with reductions in T2D and CVD rates. Additionally, a sensitivity analysis of four scenarios  was created to cover a range of predictions within each of these steps.Utilizing data from the current medical literature and recent healthcare cost estimates from national databases, a three-step variation of a cost-of-illness analysis was conducted to evaluate the healthcare-related economic benefits of fiber consumption: (i) Determination of the Table 1 summarizes the input parameters of the model. This analysis was applied to Canadian adults, which were defined as men and women ≥18 years of age. Demographic populace data was from 2014 and attained from Statistics Canada  and women . For the purpose of this modeling exercise, it is assumed that the relative risk reduction of T2D and CVD per g cereal fiber intake corresponds to a decrease in the population-wide incidence of T2D and CVD of the same magnitude.Based on the meta-analytic data from Individual consumers are the primary beneficiary of any health improvement following increased consumption of dietary fiber. However, in a publicly funded healthcare system, such as the system adopted by Canada, more widespread benefits can also be expected. A reduction in T2D and CVD rates will result in the diversion of fewer resources to the treatment of these highly prevalent health conditions. Given a subsequent reduction in morbidity and mortality that are secondary to improved dietary habits of the population, society will also benefit from increased productivity. In this way, although consumers make deliberate dietary choices, such as increasing fiber intake, without considering the effect on healthcare expenses, the overall impact of their actions on society can be considerable.EBIC 2005–2008 Report .The 8 report  recentlyEBIC 2005–2008 report utilized the friction cost approach, which assumes that sick and deceased workers can be replaced after a certain period of time known as the ‘friction period.’ Cost estimates of this method are normally lower than those derived from the classical human capital method. A reduction in the incidence of T2D is assumed to lead to decreases in both components of the indirect costs.Estimated at CAD$1.3 billion in 2014 dollars, drugs constituted the largest direct costs of T2D, which includes the costs of prescribed and non-prescribed medications purchased in retail stores. Since many different medications are used to treat T2D, it is reasonable that a decrease in the overall incidence of the disease would subsequently lead to a decrease in T2D drug-related costs. Hospitalization costs of T2D, estimated at CAD$527.2 million in 2014 dollars, were calculated on the basis of bed occupation and aggregated by diagnostic category. Generally, hospital costs are largely the fixed costs related to operating and maintaining hospital facilities, as well as the salaries of the medical professionals and support staff. The more variable components of hospitalization include the cost of medications administered to the hospitalized patients, the cost of food and accommodation, and the cost of diagnostic procedures carried out in hospitals. A reduction in the incidence of T2D would be anticipated to lead to fewer hospitalizations resulting from this disorder and, as a consequence, reductions in variable costs. Finally, the physician costs were calculated based on fee-for-service billings submitted to provincial health insurance plans and are allocated on the basis of the primary diagnostic category. For example, if a patient visits a physician’s office for a follow-up treatment after a first diagnosis of T2D, this cost would be attributed to the cost of T2D. The estimated cost of T2D-related visits to physicians was approximately CAD$521.4 million in 2014 dollars. It follows that a reduction in overall T2D levels will result in fewer doctor visits, which will reduce these costs. Total indirect costs for T2D were estimated at CAD$155.4 million in 2014 dollars, including $13.2 million for mortality and $142.2 million for morbidity. In estimating the mortality costs of T2D, The reduction in the cost of T2D was assumed to be linear when a decline in T2D incidence is observed. The exception was for the variable costs related to hospitalization. As described earlier, fixed and variable costs exist in hospitalization. The former is incurred regardless of the prevalence of any disease, whereas the latter is largely affected by the number of admissions. A comprehensive breakdown of hospital care costs in Canada is, to our knowledge, not available. As a result, it was necessary to approximate the portion of hospital costs that are fixed and not affected from reduced incidence of T2D. Previous research has provided a breakdown of fixed and variable costs in American hospitals and found that hospital costs are approximately 84% fixed and 16% variable . Thus, fTable 3.It is reasonable to assume that fewer individuals with T2D will require less medication for treatment. As such, a proportional reduction was assumed for drug costs. Similarly, given that the physician care costs are based on physician billings, which are in turn based on T2D patient visits to doctors’ offices, a reduction in T2D was assumed to lead to a proportional reduction in T2D-related physician costs. Finally, as the number of cases with T2D decreases, costs associated with mortality and morbidity were assumed to follow in a proportional manner. A summary of the relationship between T2D incidence and associated costs is provided in EBIC 2005–2008 , public health, administration, and ambulance services from the 3 report . A reducEBIC 2005–2008, these costs were derived using the friction cost approach. It is logical that a reduction in the incidence of CVD and the corresponding decrease in mortality will reduce this cost. Morbidity, or disability, costs arise when productivity is lost due to illness for a period of time. The estimated economic burden of morbidity resulting from CVD in the EBIC 2005–2008 report was CAD$288.5 million in 2014 dollars. Akin to the mortality component, morbidly costs are expected to decrease with a reduction in the incidence of CVD.In estimating the CVD-related mortality costs of CAD$98.9 million in 2014 dollars, similar to the analysis for T2D by Table 5). Unlike the previous report in EBIC 2005–2008 report does not provide cost estimates for services by “other health providers” for CVD, which necessitated estimations using figures from the NHEX 1973–2013 and EBIC 1998 reports as previously described. The costs for “Other” (5.4%), within the “Other Professionals” category  hospitalization costs, drug costs, and physician costs . Likewise, savings in CVD healthcare and related costs were estimated to range between CAD$12.3 and $81.4 million (very pessimistic), $37.0 and $244.2 million (pessimistic), $123.3 and $813.9 million (optimistic), and $246.7 million and $1.6 billion  with increasing the current fiber intakes of Canadian adults to 20, 25, 30, and 35 g per day, respectively (Table 11).Summarized in Using a cost-of-illness analysis, this study assessed the potential savings in costs of T2D and CVD within the Canadian healthcare system following higher intakes of dietary cereal fiber among adults. The potential economic benefits that resulted from improved intake of cereal fiber were significant. Specifically, if between 5 and 100% of the Canadian adults were to utilize cereal fiber to adopt intakes of dietary fiber that correspond to the IOM’s adequate intake levels , approxiThe rising costs of healthcare are a growing concern, especially in Canada where direct costs of treating disease are borne largely by an increasingly aging public. Any opportunity to reduce these costs should be fully studied. The health benefits of increased dietary fiber intakes are well-recognized. Accumulating evidence suggests protection against a range of major public health concerns, including diabetes , CVD Li, obesityClinical and epidemiological research continues to delineate the benefits of foods and nutrients with respect to general health as well as reduced risk of chronic disease. Such data are useful for developing and adjusting dietary recommendations. Given that the definition of dietary fiber is broad in scope and can include indigestible carbohydrate from a variety of dietary sources , it is rFor T2D and CVD, results from this study complement those from While this cost-of-illness analysis demonstrated the potential for substantial healthcare savings with increased consumption of cereal fiber, it is reasonable that there would be costs associated with the implementation of initiatives that impose such dietary changes for Canadians. These costs would majorly be incurred by two of the aforementioned stakeholders: government(s) and the consumer. Government spending would manifest as programs that promote the consumption foods that impact the Canadian healthcare economic landscape, such as dietary sources of cereal fiber. Initiatives could include increased emphasis for cereal-derived fiber within current programs such as Canada’s Food Guide  as well For the second stakeholder, the consumer, the most significant costs incurred would be those required for imposing dietary change. While this analysis provided cost savings per g increase in cereal fiber consumption, it is ideal if increased consumption of cereal fiber among Canadians surpassed one g per day. That being said, within Canada and other regions, foods with cereal fiber are ubiquitous within the food supply as foods with whole grain and/or added bran. Moreover, dietary recommendations across jurisdictions promote the consumption of whole grains, which are sources of cereal fiber . Whole gIn addition to government and consumers, industry is also a critical stakeholder for facilitating dietary changes among Canadians, including increased consumption of cereal fiber. Given that it is the food industry that manufactures and supplies a significant proportion of the foods that comprise the diets of Canadians, it is reasonable that increased demand for sources of dietary cereal fiber would also incur an upfront cost to industry. These costs would come from new product innovation, renovation of existing products, manufacturing, shipping, listing fees, and marketing. While upfront costs to the food industry are realized, strong demand for nutritious food with cereal fiber will drive continued support for the development of foods that contain whole grains and/or added bran. Governments already impose regulations that outline thresholds regarding the minimum levels of dietary fiber required to make “source of fiber” claims of varying magnitude . MeetingAs discussed previously, to our knowledge, this work is the first to examine the potential savings in costs to the Canadian healthcare system attributed to higher intakes of dietary fiber manifesting in lower rates of T2D and CVD. We utilized the most recent literature and national databases for findings pertaining to identification of the consumer trends and healthcare costs. Developing three sets of analyses enabled the prediction of healthcare cost savings that are mostly communicable to policy makers, dietitians, and other healthcare providers. For example, while IOM target levels of fiber intakes are likely most beneficial for government policy makers and healthcare associations that set guidelines for clinical practice, incremental targets are mainly beneficial to the general public and healthcare practitioners who focus on a stepwise approach for assisting patients with increasing fiber consumption.This study also has limitations. Estimates of risk reduction per g cereal fiber were derived from studies that gave prescribed levels of cereal fiber. Thus, it was assumed that the relationship between cereal fiber dose and risk reduction was linear. However, in some cases a linear dose effect may not exist across all levels of cereal fiber consumed; and, in those instances, thresholds for cereal fiber intake must be met for increased risk reduction. Recently, data from the InterAct Consortium demonstrated that an inverse effect of cereal fiber on T2D risk was linear with a steeper slope at >10 g/day . That beAnother limitation of the present work may relate to the fact that isolating the reported health benefits of dietary cereal fiber intakes from other associated lifestyle and environmental factors is difficult. Fiber intake from certain sources, such as fruits and vegetables, usually associates with better socio-economic status , such asGiven the economic burden of T2D and CVD in Canada and worldwide, the novel findings of this analysis shed light on the economic valuation of optimal dietary fiber intakes and reach beyond the well-established direct health benefits. Strategies to increase the consumers’ general knowledge of the recommended intakes of dietary fiber, and benefits of cereal fiber, as part of healthy diet and overall lifestyle, and to facilitate stakeholder synergy, are highly warranted. Ultimately, such evidence-based strategies are expected to enable better management of healthcare and related costs associated with T2D and CVD.MA and CG designed the economic model, reviewed the literature, and carried out the monetary analyses. MA interpreted the data and drafted the manuscript with assistance from CM. CM, JC, and PJ conceived the study question. All authors contributed to the interpretation of data, critically reviewed the manuscript for important intellectual content, and approved the final version.Funding from Kellogg Canada Inc. directly supported the efforts of Mohammad Abdullah, Collin Gyles, Jared Carlberg, and Peter Jones. The funding organization had no role in defining the study design; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication. At the time of the manuscript’s inception and acceptance for publication, CM was an employee of Kellogg Canada. The authors declare that they have no other competing interests.


Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses. For example, the cell-culture adapted strains of measles virus (MeV) such as Edmonston and Hallé attach to target cells by binding to the complement regulatory protein CD46 e.g. vaccine strains) may alter the antigenicity of the viral haemagglutinin and thus modulate the sensitivity of the virus to neutralising antibodies. Indeed, while phylogenetically all morbilliviruses are closely related, with each viral species forming a single serotype Paramyxoviruses are widespread in nature post mortem, and even then not all primary isolates of virus grow in SLAM-expressing cells in toto. MeV H and F have been expressed successfully on retroviral pseudotypes in trans from transfected plasmids, the amino acid sequences of the H and F are not subject to the antigenic drift that affects stocks of live virus following repeated in vitro passage. In effect, viral pseudotypes “freeze the virus in time”, ensuring that the neutralising response is always measured against virions with identical haemagglutinins, facilitating comparisons between animals, viruses and sampling dates. Moreover, neutralising determinants may be mapped on the viral glycoproteins by site-directed mutagenesis.In order to circumvent the use of cell culture-adapted morbilliviruses in live virus neutralisation assays, systems have been developed to facilitate the isolation and culture of primary strains of virus. By engineering Vero cells to stably express the SLAM molecule from the natural host species of the virus Globally, CDV is the second most common cause of death due to infectious disease in domestic dogs. However, CDV is not solely a pathogen of dogs, it is a widespread pathogen of carnivores The measurement of neutralising antibodies to CDV is routinely performed using live virus microneutralisation tests, a test format that is slow and relies on the visual recording of a cytopathic effect. In contrast, the majority of PPRV antibody tests use enzyme-linked immunosorbent assays (ELISAs), primarily because PPRV microneutralisation tests are labour-intensive and require access to high-level biocontainment facilities. Both CDV and PPRV neutralisation assays are limited to a small number of cell culture-adapted strains of virus. As such, there is currently no means to assess accurately whether there are qualitative differences in the specificity of the neutralising antibodies present in either convalescent sera or sera from vaccinates. Our aim was to develop a flexible system with which we could measure neutralising responses against diverse strains of virus rapidly and with a high sensitivity. Here, we describe the production of VSVΔG pseudotypes bearing CDV H and F glycoproteins and their use in assays for neutralising antibodies. By coupling VSVΔG(CDV) with SLAM expressing target cells, we demonstrate that morbillivirus pseudotypes may be used for the sensitive, rapid and reliable measurement of virus neutralising antibodies.22.1®, Life Technologies Ltd.). Vero (African green monkey) cells HEK293 2.2Sera submitted previously to the Veterinary Diagnostic Services laboratory at the University of Glasgow for post-vaccination CDV antibody titre testing were derived from domestic dogs in the United Kingdom. Additional samples were obtained from dogs and lions in Tanzania and diverse mesocarnivore species in Russia. Ruminant sera were collected from ruminants in Tanzania, and experimentally infected ruminants.2.3luc) has been described luc bearing VSVG was used to infect 293T cells transfected with the VSV-G expression vector pMDG luc (VSVG) pseudotypes were recovered, titrated on 293T cells and used to prepare a working stock of VSVΔGluc (VSV-G) pseudotypes. To prepare CDV H and F expression constructs, viral RNA was prepared from CDV pelleted by centrifugation , used to prepare first strand cDNA , and then used as template in PCR reactions with the following primers: CDV H Sal: 5′-GTCGAC-ACC-ATGCTCCCCTACCAAGACAAGGT-3′, CDV H Not: 5′-GGGCGGCCGC-TTAACGGTTACATGAGAATCTTA-3′: CDV F Sal: 5′-GGGTCGAC-ACC-ATGCACAGGGGAATCCCCAAAAG-3′ and CDV F D633 Not: 5′-GGGCGGCCGC-TTGCTAGCGTCTTTTACAACAGTAAATCAGCA-3′ . Peste des petits ruminants (PPRV) H and F cDNAs were amplified from the Nigeria 75/1 encoding plasmid pCI-PPRV-delL SalI and NotI and cloned into the eukaryotic expression vector VR1012 . Canine SLAM-F1 (dogSLAM) was amplified from total RNA prepared from mitogen-stimulated canine peripheral blood mononuclear cells using PCR  and the primers dogSLAM Bgl: 5′-GCTCAGATCTGAGAGCTTGATGAATTGCCCAG-3′ and dogSLAM Sal: 5′-GCTCGTCGACGCTCTCTGGGAACGTCAC-3′. Amplifications were performed with the following thermocycling conditions: denaturation at 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, annealing at 65 °C for 30 s and extension at 72 °C for 60 s, with a final extension at 72 °C for 2 min. The amplified cDNA was cloned into the pDisplay eukaryotic expression vector  using BglII and SalI. The nucleic acid sequences of all amplified cDNAs were determined externally by Sanger dideoxy chain termination sequencing . All oligonucleotide primers were obtained from Integrated DNA Technologies, Leuven, Belgium.The recombinant vesicular stomatitis virus (VSV) in which the glycoprotein (G) gene has been deleted (VSVΔG) and replaced with firefly luciferase (luc (CDV) and VSVΔGluc (PPRV) pseudotypes, 293T cells were transfected with the H and F expression vectors from CDV and PPRV respectively, followed by super-infection with VSVΔGluc (VSVG) as described To prepare VSVΔG2.4To prepare target cells for the VSV-ΔG(CDV) pseudotypes, 293 cells were transfected with pDisplay-dogSLAM using linear polyethylenimine, MW 25,000  and selected in complete medium supplemented with 800 μg/ml G418. The stably transfected 293-dogSLAM cells were expanded and the surface expression of SLAM confirmed by flow cytometry using rabbit polyclonal anti-HA tag  followed by phycoerythrin (PE)-conjugated goat anti-rabbit IgG (Sigma) on a BD Accuri flow cytometer . SLAM-F1-expressing cells were compared with cells stably-transfected with the empty vector. The analysis gate was set such that <1% of vector-only control cells were deemed positive.2.550 of virus was added to each serum dilution and incubated in the dark at room temperature for 1 h, followed by a further hour at 4 °C. 1 × 104 Vero cells were then added to each well and the plates were incubated at 37 °C for three days prior to microscopic examination for cytopathicity.Neutralising antibodies were determined by live virus neutralisation assay 2.64 293-dogSLAM cells were plated into each well of a 96-well white flat-bottomed plate . Four-fold serum dilutions were prepared in triplicate in complete medium ranging from 1:8 to 1:32768. The diluted serum samples were then added to the 293-dogSLAM cells followed by 2.5 × 103 TCID50 of VSVΔG(CDV) pseudotype. Plates were incubated for 48–72 h at 37 °C, at which time luciferase substrate was added  and the signal analysed on a Microbeta 1450 Jet luminometer (Perkin Elmer). Antibody titres were calculated by interpolating the point at which there was a 90% reduction in luciferase activity  2 × 103luc). As expression of the luciferase reporter gene was very low in Vero-dogSLAM cells, target cells were generated based on 293 cells. Stable transfection and expression of canine SLAM was confirmed by flow cytometry  pseudotypes bearing a green fluorescent protein (GFP) reporter gene were used previously to define determinants of viral cell tropism 7 TCID50 C.2, 2.5 × 103 and 2.5 × 104 TCID50 per well, our objective being to minimise the volume of viral pseudotypes required per assay while ensuring the accuracy of the determination of antibody titre. Irrespective of the amount of input virus, the measured antibody titre  remained consistent, mean titres of 966, 922 and 970 for inputs of 2.5 × 102, 2.5 × 103 and 2.5 × 104 TCID50 per well respectively. However, given the variability observed at the lowest input of virus (2.5 × 102 TCID50), we elected to use an input of 2.5 × 103 TCID50 per well, equivalent to approximately 1 × 107 CPM of luciferase activity.Next, the application of the VSVΔG(CDV) pseudotypes to assays for virus neutralising antibodies was investigated. Three input amounts of VSVΔG(CDV) Onderstepoort strain pseudotypes were compared D, equivar coefficient of 0.76 , suggesting that the pseudotype assay was a reliable substitute for the live virus-based assay. In general, titres obtained with the pseudotype-based assay were ∼10-fold higher than those obtained with the live virus based assay. Of the 44 samples that scored negative by live virus assay (titre <8), 9 (20%) were found to have detectable neutralising antibody activity by pseudotype assay .34 sera that scored weakly positive by the live virus based assay  were found to have titres of <16 by the pseudotype assay. Given the enhanced sensitivity of the pseudotype-based assay, we would predict that any sample yielding a low titre with the live virus microneutralisation test would give a higher titre with the pseudotype-based test. Accordingly, the finding that the 34 sera reported as weakly positive in the live virus microneutralisation test were negative by the pseudotype test may indicate that these samples represented false positives by the live virus microneutralisation assay. If the 34 sera that scored weakly positive by live virus assay are excluded from the comparison between the tests the correlation between the two assays rises to False positives at high serum concentrations are not uncommon with live virus microneutralisation tests and the definition of a reliable cut-off point in the absence of a “gold-standard” diagnostic test is problematic. A significant concern with live virus-based assays is their reliance on visual inspection of a cytopathic effect within the monolayer of cells for endpoint determination. Non-specific toxic effects of the serum sample, especially at high serum concentrations, may alter the morphology of the monolayer, interfering with the interpretation of the assay end-point. The enhanced sensitivity of the pseudotype based assay reduces the susceptibility of the assay to non-specific toxic effects and accordingly may facilitate the detection of virus neutralising antibodies at lower dilutions of serum, circumventing the potential toxic effects of such sera.An alternative consideration is that the sera scoring weakly positive by live virus neutralisation test might be reacting with an antigen present on live virus that is absent from VSVΔG(CDV) pseudotypes. As the Bussell strain of CDV used in this study was propagated in Vero  cells while the VSVΔG(CDV) pseudotypes were generated in 293T (human embryonic kidney) cells, it is conceivable that the weak reactivity detected in the live virus assay reflects an activity against a Vero cell-derived antigen that is absent from the 293T-derived VSVΔG(CDV) pseudotypes. Similarly, as the live virus assay is SLAM-independent, it is also possible that the live virus assay detects a very weak response against distinct epitopes on the viral glycoproteins that are critical for SLAM-independent infection but are rendered insignificant in the presence of a high affinity SLAM-interaction as in the VSVΔG(CDV) pseudotype assay.r = 0.79, p < 0.0001; r = 0.65, p < 0.0001; r = 0.90, p < 0.0001; It is widely assumed that antibody responses to CDV form a single serotype, however the majority of assays performed to date have utilised a limited number of Vero cell-adapted strains of virus. If we are to assess accurately the neutralising response against field strains of virus, the assays should be performed using biologically relevant, non-cell culture adapted strains of virus. To investigate the feasibility of this approach we compared the neutralisation sensitivity of VSVΔG(CDV) pseudotypes bearing the H and F proteins of the primary A94-11-15 strain with those bearing the cell culture adapted Onderstepoort strain. A94-11-15 is an isolate from an African dog collected during the 1994 CDV outbreak in the Serengeti p < 0.0001). Thus, the specificity of the CDV neutralising response could be confirmed by comparing the responses against CDV with those against PPRV; a higher titre against CDV than PPRV would suggest that the animal was exposed primarily to CDV.In order to define further the viral species-specificity of the pseudotype based test, we compared the neutralisation sensitivity of VSVΔG (CDV) pseudotypes, with pseudotypes prepared using the H and F proteins of the genetically distant morbillivirus, PPRV (peste des petits ruminants). As the degree of antigenic-relatedness varies between morbilliviruses, we would predict that a high titre antibody response against CDV would encompass weaker, cross-neutralising activity against PPRV. Conversely, a high titre anti-PPRV serum would neutralise CDV pseudotypes more weakly. The sensitivities of VSVΔG (CDV) pseudotypes and VSVΔG (PPRV) pseudotypes to neutralisation by sera from animals exposed to, or immunised against, CDV and PPRV was compared. The CDV sera consisted of dog sera from the UK, Tanzania and Russia, and PPRV sera consisted of ruminants from Africa and Asia. The PPRV sera displayed titres of neutralising antibodies that were ∼100-fold higher against VSVΔG (PPRV) than against VSVΔG (CDV) . Converse.g.Neutralising responses against CDV and PPRV were compared in a collection of post-CDV vaccination sera from dogs . Given t4in vitro growth may alter the neutralisation sensitivity of the virus. In regard to the genus Morbillivirus, this phenomenon has been observed with measles virus, where neutralising epitopes were mapped to the receptor binding sites on the haemagglutinin of the virus in vitro systems for measuring neutralising antibodies should utilise SLAM-F1, or nectin-4-expressing cell lines if they are to recapitulate fully the expected neutralisation sensitivity of the virus in vivo. In this study, we generated canine SLAM-expressing 293 cells as an indicator cell line for the neutralisation assay. The “293dogSLAM” cells expressed the VSVΔG-encoded luciferase gene efficiently. Using these target cells, we generated a luciferase-based assay that was approximately 10-fold more sensitive than the conventional live virus based microneutralisation assay and which yielded data in three days with no requirement for operator interpretation of cytopathic effect by visual inspection. As a glow luciferase-based system, the assay is amenable to high-throughput screening using small sample volumes. Uniquely, the modular nature of the VSVΔG pseudotyping system facilitates the preparation of pseudotypes bearing a range of heterologous glycoproteins from diverse, primary strains of CDV. In proof-of-concept, we prepared pseudotypes bearing H and F glycoproteins from both the Vero cell-adapted Onderstepoort and A92-27-14 strains, and the primary field strains A94-11-14 and A94-11-15. Of the CDV strains tested, the two Vero cell-adapted isolates (Onderstepoort and A92-27-4) yielded higher titres during pseudotype production than the other strains tested, perhaps indicating that adaptation to in vitro growth altered the biological properties of these viruses, favouring more efficient pseudotype production. Efficient pseudotyping of morbilliviral glycoproteins on VSV particles requires optimal H and F expression on the plasma membrane of the transfected 293T cells at the site of VSV assembly and budding. Future studies should investigate whether field strains of other morbilliviruses vary in their relative efficiencies of H and F incorporation and whether this impacts upon receptor usage and sensitivity to neutralising antibodies.In this study, we describe the measurement of morbilliviral neutralising antibodies using a viral pseudotype-based assay. The aim of the study was to develop an assay that was rapid, sensitive and which enabled the analysis of neutralising responses against field strains of virus without a requirement for virus adaptation to Vero cell culture. One of the primary concerns with adapting viruses to cell culture is that the cell-culture adapted viruses no longer reflect the neutralisation sensitivity of field strains of the virus. Field strains of virus are generally isolated onto cells expressing the cognate SLAM molecule, while cell culture-adapted strains of virus are able to replicate in SLAM-negative cell lines such as Vero. The nature of the viral receptor utilised by a virus can determine the sensitivity of the virus to neutralising antibodies. Consequently, alterations in receptor usage during adaptation for Classical live virus microneutralisation assays or PRNT assays rely on the visual inspection of the indicator cells for a cytopathic effect or plaque formation. As such, viral entry events that do not trigger a cytopathic effect or plaque formation will not be quantified and neutralisation of such infectivity will not be recorded. In contrast, the pseudotype based neutralisation assay measures neutralisation of viral entry mediated by the viral receptor SLAM-F1. Accordingly, all successful entry events that lead to gene expression are detected by measuring luciferase activity. Thus, while a live virus assay measures the net result of a complex series of events, from entry, through cell-cell spread and cytopathic effect, a pseudotype-based assay focuses solely on antibodies that prevent viral entry. Given that morbilliviruses have evolved two distinct receptor interactions (SLAM-F1 and nectin-4), it may be possible to distinguish antibodies that prevent cell-free virus infection from those that prevent cell-cell spread and dissemination. Cell-free virus infection would be best modelled using SLAM-F1 expressing target cells whereas the spread of virus across epithelial barriers would be modelled better using nectin-4 expressing target cells. The pseudotype-based assay system will facilitate the dissection of the neutralising response and permit the determination of whether the nature of the viral receptor expressed on the target cell is critical to the outcome of the assay.In order to assess the specificity of the system we generated pseudotypes bearing the H and F glycoproteins of PPRV (Nigeria 75/1 strain). Sera raised against CDV, either through vaccination or natural exposure, neutralised CDV ∼100-fold more efficiently than PPRV. In contrast, sera raised against PPRV neutralised PPRV pseudotypes ∼100-fold more efficiently than CDV pseudotypes. These observations reveal two things about the assay system. Firstly, the specificity of the CDV response can be confirmed by comparing against a distantly related viral species. The specificity of a low titre response against CDV can be confirmed by analysing in parallel, the response against PPRV. Secondly, the assay system provides a simple means of measuring cross-neutralisation between distinct strains and species of virus. Indeed, while some UK dog sera appeared to display good neutralisation of PPRV, a pathogen absent from the UK, others were largely CDV-specific. These data suggest that the humoral responses of UK dogs to vaccination against CDV differ in their epitope specificities; some dogs recognising determinants that are broadly conserved between CDV and PPRV while other dogs mount a largely CDV-specific response. Recent studies have noted that a proportion of human sera contain weak cross-neutralising antibodies against CDV Several reports have described the cross-species transmission of morbilliviruses, including CDV, PDV and PPRV


In this study, we characterize for the first time the viruses associated with a red macroalga, Delisea pulchra. Using transmission electron microscopy we identified diverse morphotypes of virus-like particles in D. pulchra ranging from icosahedral to bacilliform to coiled pleomorphic as well as bacteriophages. Virome sequencing revealed the presence of a diverse group of dsRNA viruses affiliated to the genus Totivirus, known to infect plant pathogenic fungi. We further identified a ssRNA virus belonging to the order Picornavirales with a close phylogenetic relationship to a pathogenic virus infecting marine diatoms. The results of this study shed light on a so far neglected part of the seaweed holobiont, and suggest that some of the identified viruses may be possible pathogens for a host that is already known to be significantly impacted by bacterial infections.Marine seaweeds are holobionts comprised of the macroalgal hosts and their associated microbiota. While the composition of the bacterial component of seaweed microbiomes is increasingly studied, almost nothing is known about the presence, diversity and composition of viruses in macroalgae  Marine seaweeds are colonized by diverse microbial communities , which iSeaweeds are the dominant habitat-forming organisms on temperate rocky reefs, comparable in importance to corals in the tropics . In receEctocarpus sp. and Feldmania sp.  and virome sequencing to characterize the virome component of D. pulchra, as a first step in understanding the interaction between the host and the viral component of its microbiome. We focus on RNA viruses because of their dominance as pathogens of macrophytes in terrestrial systems  in Sydney at 3 m depth in January 2014 and stored in separated zip lock bags on ice until viral extraction. Each individual was washed in filter-sterilized and autoclaved seawater to remove loosely attached unspecific viral particles from the surrounding seawater. The algal tissue was then homogenized, generating samples that contained both surface-associated and intracellular viruses. Individual algae were then homogenized separately in 100 ml seaweed extraction buffer  in a tissue homogenizer for 5 min at 4°C. The homogenate was pressed through cheesecloth and further purified by low speed centrifugation at 2,000 × g for 2 min followed by a second centrifugation step at 10,000 × g for 10 min. The supernatant was passed through a 0.45 μm filter and viral particles were precipitated by the addition of 10% (w/v) PEG 8,000 overnight. Viral particles were pelleted by centrifugation at 7,600 × g for 30 min and re-suspended in VR buffer  overnight. Cellular debris was removed by low speed centrifugation at 7,600 × g for 10 min. The viral containing supernatant was then further purified by density gradient ultracentrifugation. For this the viral suspension was loaded onto a 10–40% Optiprep gradient and centrifuged in a Beckman SW 41 ti swinging bucket rotor at 28,000 rpm for 2 h. After ultracentrifugation one distinct viral band was observed in both samples. These viral bands were removed separately by syringe, diluted 1:3 in VR buffer and pelleted by centrifugation at 28,000 rpm for 2 h. Viruses were re-suspended in 200 μl VR buffer without sodium sulphite. The presence of viral particles was checked by epifluorescence microscopy according to the protocol developed by Two individuals (termed here A and B) of g at room temperature for 5 min. The chloroform:isoamyl alcohol extraction of the supernatant was repeated three times to remove contaminating polysaccharides and proteins. Finally, supernatant was transferred into a new tube and the nucleic acids were precipitated by the addition of 0.7 volume of isopropanol. After an incubation step at -20°C for 2 h nucleic acids were pelleted by centrifugation at 14,000 × g at 4°C for 20 min. The pellet was washed with 75% (v/v) ethanol, air dried and dissolved in 30 μl molecular-grade water. DNA was digested with the TURBOTM DNAse (Life Technology) according to the manufacture’s protocol. RNA was purified by column purification using the Quick-RNATM MiniPrep kit (Zymo Research). The TruSeq stranded-RNA preparation kit (Illumina) was used for reverse transcription and cDNA library preparation. Sequencing was conducted on a MiSeq platform (Illumina) at the Ramaciotti Centre for Genomics .Two-hundred microlitres of purified and re-suspended viral particles per sample prepared as above were used for RNA extraction. Viral RNA was extracted in SDS-extraction buffer  in the presence of 1% (w/v) polyvinylpyrrolidone , 1% 2-mercaptoethanol and proteinase K (0.5 mg/ml) at 37°C for 30 min, followed by a second incubation step at 56°C for 15 min. Twice the amount of RNA-extraction buffer ; 1% 2-mercaptoethanol; pH 7.5) was added and samples were incubated for another 15 min at 56°C. An equal volume of chloroform:isoamyl alcohol (24:1) was then added to the warm solution, mixed thoroughly and centrifuged at 12,000 × e-value <10-4 were classified as contaminations and removed by Deconseq 0.4.3 . All partial or complete viral sequences have been submitted to the GenBank database under the accession no. LIKW00000000  and LIKX000000000 .Sequence reads were trimmed and adaptors removed using Trimmomatic V0.32  and asseeq 0.4.3  from theeq 0.4.3  with an Totivirus: KT455444-KT455461 and Picornaviridae KT455462-KT455464.Reference sequences of the RNA-dependent RNA polymerase (RdRp) from different viruses were obtained from NCBI. Multiple alignment of amino acid sequences of the RdRp were constructed using ClustalW implemented in BioEdit 7.2.5 . PhylogeFigure 1), including icosahedral , bacilliform to coiled pleomorphic (B and C) VLPs as well as bacteriophages (F). We observed two size classes of icosahedral viral particles one with a diameter of 40 nm  and one with a diameter of 30 nm . The pleomorphic forms observed under TEM , while virus-like, may also have been membrane vesicles originating from the homogenized algal tissue.Transmission electron microscopy (TEM) of negative-stained virus-like particles (VLPs) revealed in both samples a range of morphotypes (Table 1). Sequence analysis revealed that both individuals contained single- and double-stranded RNA viruses. ssRNA viruses accounted for 18 and 10% of the viral community compared to 82 and 90% dsRNA viruses for individual A and B, respectively .After removal of contaminating sequences, 267,372 reads of individual A could be assembled into 509 contigs. Based on blastx results 102 could be assigned to viruses. Individual B with 294,226 cleaned reads resulted in 369 contigs, for which 41 viral hits were found . The almost full-length genome sequence of this D. pulchra RNA virus with a length of 9581 bp has been submitted to GenBank under the accession number KT455464. Similar to the Agla RNA virus the D. pulchra RNA virus encodes two major polyproteins; one replication associated and one structural polyprotein . Protein domain analysis via SMART . Within conserved domains of the polyprotein similarity reaches values of 50% for the RdRp and 46% for the structural domain Dcistrvp4. This virus could be detected in both individuals and accounted for 7–14% of the viral composition. Analysis based on the amino acid sequence of the RdRp showed that this D. pulchra RNA virus distinctly clusters with environmental sequences (GenBank accession numbers AY285750-AY285768) generated from geographically and seasonally diverse samples of marine virioplankton communities .Most of the ssRNA viruses identified in this study showed highest sequence similarity (based on blastx results) to viruses known to infect unicellular Heterokontophyta, such as munities  and withPartitiviridae and Totiviridae . The relative abundance of Partitiviridae was comparable in both individuals and showed only a low diversity, essentially being dominated by a vius with similarity to Penicillium stoloniferum virus. In contrast, for the Totiviridae we detected more taxonomic diversity at lower levels. Comparison between the two different individuals showed that some viruses with similarity to Xanthophyllomyces dendrorhous virus, Scheffersomyces segobiensis virus, and Saccharomyces cerevisiae virus had relatively consistent abundances, while other viruses with similarity to Grapevine associated mycovirus (0.6–10%), Black raspberry virus (2.4–14%), and Tuber aestivum virus (5–10%) were more variable between both individuals. Phylogenetic analysis based on the RdRp clearly shows that D. pulchra’s associated Totiviridae form multiple, distinct clusters within the genus Totivirus .In addition, we discovered a diverse group of genetically distinct dsRNA viruses. These dsRNA viruses are taxonomically affiliated with the families D. pulchra viromes with other published marine RNA viromes of the virioplankton community of the Strait of Georgia, Canada  RNA virus, which infects marine diatoms and induces cell lysis  infect the bloom forming alga H. akashiwo . The observed sequence similarities to viruses known to infect Heterokontophyta or diatoms therefore does not strictly speak to these organisms being the host of the viruses we have isolated, e.g., as epiphytes on D. pulchra. While we cannot exclude that some of the isolated viruses might be derived from surface-associated epiphytes, we took great care to select individuals that were visually free from fouling organisms and cleaned the algal surface to remove loosely attached contaminations from planktonic organisms. We thus believe that the RNA virus, found here indeed directly interacts with D. pulchra, which is further supported by the many taxonomic groups found consistently from both individuals. However, due to the method used in this study to isolate viruses we do not know how the viruses may be localized within the seaweed holobiont. They might be surface-associated or originate from inside the algal tissue.We know even less about the viromes of macroalga with the lack of sequence data for macroalgal viruses in public databases significantly constraining the analysis of our data. Most of the viral sequence data in public databases are from pathogens or domesticated plants. The only viruses known from multicellular marine alga are the us virus  and Feldp. virus , both ofDelisea pulchra is arguably one of the best known seaweed holobionts, but to date the focus on the microbiome of this alga has been limited to the bacterial  component. This microbiome is complex (D. pulchra by bacteria ( complex  and when complex . Given tbacteria , and envbacteria . Viral ibacteria . FunctioD. pulchra (Moreover, in addition to the eukaryotic viruses identified by RNA analysis, we also observed bacteriophages by TEM (these were not detected in our sequencing, but RNA containing bacteriophages are less common than those containing DNA . Such ba pulchra  and know pulchra , lectins pulchra , bromoph pulchra , or poly pulchra  have shoD. pulchra. This finding argues that the interactions between the host and its associated viruses should be considered for future studies of marine holobionts. Viruses could play a key role in modulating both positive and negative interactions for the macroalgal host and be an important factor in ensuring the stability of the seaweed holobiont.This study shows that a large diversity of eukaryotic viruses exists in the marine seaweed Conceived and designed the experiments: TL, TT, and PS performed the experiments: TL analyzed the data: TL Contributed reagents/materials/analysis tools:TT and PS wrote the paper: TL, TT, and PS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses , carlaviruses  and allexiviruses , for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic , a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic ( Allium sativum L.) is an example of a live plant propagule for which international trade has grown hugely, by over 70%, in the past decade The international trade in fresh culinary produce has been influenced by a softening of trade barriers through the General Agreement on Tariffs and Trade (GATT) Allium vineale L. , today listed as an environmental weed in parts of Australia, first appeared in a catalogue of plants grown in the Melbourne Botanic Gardens in 1853, where it may have been introduced as a culinary plant Humans have an ancient association with garlic. It originated in the region around the Tian Shan Mountains of Central Asia, where it was first cultivated over 7000 years ago Potyvirus, Carlavirus, Allexivirus and PotexvirusBecause most garlic cultivars do not produce viable seed they are propagated vegetatively; thus, garlic is susceptible to accumulation of a complex of viruses, notably members of the genera Quarantine control of plant pathogens is particularly important for isolated food-producing countries like Australia. Assessment of imported produce for the presence of pathogens can be unreliable when using methods based on visual inspection for the pathogen(s) or symptoms of infection, DNA hybridization (e.g. PCR-based assays) or antibody affinity (e.g. ELISA). Such procedures may not effectively identify pathogens when symptoms are mild, when infected products are rare and/or dispersed amongst large volumes of healthy ones (e.g. grain), when produce is multiply infected (e.g. vegetatively propagated vegetables and flowers), and when unexpected or undescribed pathogens are present. We decided to see whether the latest high-throughput sequencing technologies could be used to test normal commercially available imported garlic bulbs, and perhaps overcome some of those problems. We chose to test garlic bulbs because they are live propagules and potentially can be germinated and grown to maturity, most that are sold for commercial or home use in Australia are imported, and partial and/or complete genome sequences of several viruses infecting garlic are known. As many fruit and vegetable stores in Australia declare the country of origin of the fresh produce they sell, we were able to obtain and test garlic grown in the USA, Mexico, Argentina, Spain and China, and also from Australian producers and home gardeners.A. vineale is a pest in Australian wheat fields because vegetative bulbils are produced on the tops of tall stems, and these resemble wheat grains in size. Bulbils contaminating the harvested wheat taint it with a garlic odour, thereby lowering its value. Milk and meat both become ordourous when animals eat the plant.From 2009-12 garlic bulbs imported from China, Mexico, Argentina, the USA, and Spain were collected from retail fruit and vegetable stores in Perth, Western Australia. A locally-grown plant was collected with permission from a home gardener in Torbay, Western Australia who had maintained the same garlic germline for a number of years . Another was purchased from a commercial organic vegetable grower in Pemberton, Western Australia, who kept propagules from the previous crop to grow the subsequent crop . A garlic plant was purchased from a hardware store in Perth, Western Australia, that supplied potted plants to the home garden market. Bulbs were placed in bark/sand potting mix in a vector-free greenhouse. Plants were grown for at least two (vegetative) generations to confirm that viruses were maintained.A. vineale was obtained from a wild plant growing on a road verge near the town of Augusta in southwest Australia. It was transferred to a greenhouse and maintained in the same manner as described for A. sativum.A bulb of De novo sequence assembly applications within Geneious Pro v7 (www.geneious.com) and CLC Genomics Workbench v7 (www.clcbio.com) software packages; resulting contigs greater than 1000 nt were analysed using Blastn and Blastx to interrogate the GenBank database (NCBI); contigs with one or more hits to a known virus within the top 100 hits were identified; reads with no hits or very low hit scores and high e-values to known sequences were identified; raw reads were mapped back to contigs representing putative viral sequences to calculate sequence coverage. After this initial analysis, putative virus-derived contigs with mean sequence coverage of less than 10-fold were discarded. Contigs with no matches to known sequences were analysed for the presence of open-reading frames (ORF) and deduced amino acid sequences of predicted large ORFs were analysed for the presence of conserved domains using the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/cdd/). Presence of viruses was confirmed in original host plants by RT-PCR assays using species-specific primers designed from the original sequences, followed by Sanger sequencing of amplicons. Evolutionary histories of putative virus-derived sequences were inferred using the appropriate model with Maximum Likelihood as implemented within Mega v6 (www.megasoftware.net). Aligned sequences were checked for incongruent relationships resulting from recombination using the programs implemented in Recombination Detection Program v3 (RDP3) (http://web.cbio.uct.ac.za/~darren/rdp.html).Total RNA was extracted from 100 mg of leaf tissue using RNeasy Plant Miniprep columns (Qiagen) following the manufacturer's instructions, or RNA was extracted from 100 mg of leaf tissue using a modified cellulose-based method for enriching for double-stranded RNA A. sativum) plants tested and one isolate of one species was identified from one plant of wild garlic . Viruses belonged to four genera: Carlavirus (family Betaflexiviridae), Potyvirus (family Potyviridae), Allexivirus and Potexvirus (family Alphaflexiviridae) (Mean sequence coverage of most virus genomes found was greater than 60-fold. The exceptions were SLV-SG3 (11-fold), GarVX-Torbay1 (10-fold), and GarVX-USG1 (17-fold). Isolates of one to eight virus species were identified from each of the 10 garlic (viridae) . Raw seqIsolates of two carlaviruses were identified: garlic common latent virus (GCLV) and shallot latent virus (SLV). Both carlaviruses were recently described for the first time in Australia in garlic Isolates of two potyviruses were identified: leek yellow stripe virus (LYSV) and onion yellow dwarf virus (OYDV). Nine isolates of LYSV were identified. Three isolates were from locally grown plants and the other six were from plants imported from China, Mexico, Argentina and Spain. The amino acid sequences of partial polyproteins spanning the 6K2, VPg, NIa-Pro, NIb, and CP region were used to compare LYSV isolates. LYSV isolates from Spain and China were almost identical, sharing 98% identity. The others shared 87–97% sequence identity with one another and with other isolates already known from Asia, the Americas, and Europe. Sequences of seven new isolates of OYDV from bulbs from Australia, Argentina, China, and Spain were determined. They shared >90% identity with one another and with other isolates whose sequences are known   were identified. Deduced phylogenies of the allexivirus sequences described are shown in These are not a well-studied viruses and few complete genome sequences are available from them. All but one plant tested was infected with at least one allexivirus, the exception being the plant from China . SeventeGarVB isolate Bate1  was very similar (95–96% aa identity) to the only other two isolates described from Korea and Japan.GarVC isolates Bate1  and SW3.3A (Mexico) were almost identical to one another (99%), but another isolate from Mexico and two others described previously from Japan were more distant, sharing only 88%–90% sequence identity.The first complete genome sequences of two GarVD isolates were determined from plants imported from Argentina. The three new GarVD isolates were genetically close to two isolates described from Japan and Korea (94–100% aa identity). The two GarVD genomes shared 85% nt identity with one another and 70% nt identity with the genome of the nearest relative, GarVE (GenBank accession NC_004012). The GarVD replicases shared 92% aa identity and their CPs shared 96% aa identity. The new GarVD CP sequences shared 94–99% aa identity with 43 other complete GarVD CP sequences available on GenBank. Between species, the closest matching proteins were homologous ones from GarVE. The two new GarVD isolates and the one available GarVE isolate from China shared 82% aa (73% nt) identity over their replicases, and the CP sequences 76% aa (72–73% nt) identity with those of GarVE. Comparison of deduced amino acid sequences of five partial GarVE CP sequences from China (AJ551498-AJ551502) with 43 homologous sequences from GarVD from Brazil, Japan, Korea, China, UK and Poland revealed that all sequences shared 88% identity or greater.Five new GarVX isolates were detected from plants derived from Australia, the USA, Mexico (two isolates), and Spain. Unusually, a plant from Australia and the one from the USA harbored only one virus isolate, in both cases GarVX. This is indicative that they were probably recently derived from virus-free plants, but subsequently became infected with GarVX via a vector. GarVX isolates from Mexico (isolate SW3.1) and Spain were very close (96–100% aa identity) to previously identified isolates, whereas another isolate from the same plant from Mexico (SW3.4A) was the most genetically distant GarVX isolate identified so far (89–91% aa identity with other described isolates).A. vineale tested harbored a single isolate of Asparagus virus 3 (AV3), and its genome was fully sequenced. The genome was 6,398 nt in length. This is the first report of AV3 occurring in Australia and from A. vineale. Type isolate AV3-Japan was described from Japan in 1980 from asparagus  Allium chinense G.Don) when the complete genome sequence was determined (GenBank accession NC_003400), and the name scallion virus X (ScaVX) proposed Asparagus virus 3 was accepted by the ICTV when the genome sequence of the type isolate (AV3-Japan) became available (GenBank accession code NC010416) The plant of For viruses, encounters with new vectors and host species are major drivers of evolutionary change, but other environmental, ecological, and biological pressures may also play a part i. Population bottlenecks occurred when the Spanish introduced garlic to Cuba in 1505, and subsequently to the continental Americas Allium diversity Allium hosts, perhaps via new vectors. In this scenario, selection pressures associated with encountering new hosts/vectors drove evolution of some allexiviruses. After differentiation, the viruses reinfected garlic.ii. The Americas hold centers of iii. The currently recognized allexivirus species evolved in American alliaceous and/or non-alliaceous species, and subsequently infected garlic when they came into contact with it. The isolates with the greatest branch lengths, i.e. GarVX-SW3.4A (Mexico), GarVA-SW3.1A (Mexico), GarVA-WA6 (Argentina), are basal to other known isolates , therefoAllium species that exist naturally in the regions where garlic has been grown for centuries in Central and South America, and in ancestral garlic populations and other indigenous Allium species of the Tian Shan region of China and Kyrgyzstan will provide evidence for one or more of these scenarios. Preferably, such projects should be done using generic methods  to overcome the inherent biases associated with antibody and RT-PCR based methods.A search for allexiviruses infecting indigenous The garlic-borne carlaviruses and potyviruses described from the Americas were not notably different from those described from elsewhere.A. vineale plants introduced deliberately to Australia in the 1800s A. vineale bulbuls contaminating imported wheat grain. AV3 is a pathogen of concern because it has a wider potential host range than the other viruses detected, not being restricted to the genus Allium.It is not known how AV3 got to Australia or how long it has been there. Conceivably, it infected the original Allium-infecting viruses potentially threaten another estimated 750 wild and cultivated allium species that exist around the world A. cepa (onion), A. oschaninii , A. schoenoprasum (chive), A. porrum (leek), and those grown as ornamentals, e.g. A. giganteum and A. jesdianum.Most of the new virus isolates from imported garlic represent species previously reported from Australia. The exceptions were GarVX, which was detected in both locally grown and imported culinary garlic, and AV3 from wild garlic. The presence of so many virus species, including unusual genotypes, should be of concern to garlic growers and possibly to those responsible for preserving indigenous plant communities. Solanum tuberosum) and sweet potato (Ipomoea batatas), both of which suffer similar accumulations of viruses Although 42 virus isolates are described here, they are derived from only 11 plants. This is not only a clear illustration of the capacity of garlic to accumulate viruses, but it also highlights the limitations of the current study, which provides only a snapshot of the movement of viruses facilitated by a vast international trade network. Indeed, movement of people, animals and plants has always provided a vehicle for viruses to expand their geographical and host ranges. The direct risk of virus expansion is real and recognized, but when trade enables viruses from disparate origins to meet and undergo intra- and inter-species recombination, potentially even more damaging viruses can emerge The new sequencing technologies are already revolutionizing plant virus diagnosis, enabling detection of most, if not all, viruses, known and unknown, infecting the plants tested e.g. . A signi


Schizophrenia spectrum disorders result in enormous individual suffering and financial burden on patients and on society. In Germany, there are about 1,000,000 individuals suffering from schizophrenia (SZ) or schizoaffective disorder (SAD), a combination of psychotic and affective symptoms. Given the heterogeneous nature of these syndromes, one may assume that there is a difference in treatment costs among patients with paranoid SZ and SAD. However, the current the national system of cost accounting in psychiatry and psychosomatics in Germany assesses all schizophrenia spectrum disorders within one category.The study comprised a retrospective audit of data from 118 patients diagnosed with paranoid SZ (F20.0) and 71 patients with SAD (F25). We used the mean total costs as well as partial cost, i.e., mean costs for medication products, mean personal costs and mean infrastructure costs from each patient for the statistical analysis. We tested for differences in the four variables between SZ and SAD patients using ANCOVA and confirmed the results with bootstrapping.p = .02). Mean total costs were significantly higher for SAD patients (p = .023). Further, we found a significant difference in mean personnel costs (p = .02) between patients with SZ and SAD. However, we found no significant differences in mean pharmaceutical costs (p = .12) but a marginal difference of mean infrastructure costs (p = .05) between SZ and SAD. We found neither a common decrease of costs over time nor a differential decrease in SZ and SAD.SAD patients had a longer duration of stay than patients with SZ (We found evidence for a difference of case related costs of inpatient treatments for paranoid SZ and SAD. The differences in mean total costs seem to be primarily related to the mean personnel costs in patients with paranoid SZ and SAD rather than mean pharmaceutical costs, possibly due to higher personnel effort and infrastructure. Schizophrenia (SZ) is a neurodevelopmental disorder characterized by various combinations of delusions, hallucinations, disorganized thoughts and behavior, cognitive impairment and movement disorders ,2. The pIn 1952, schizoaffective disorder (SAD) as an independent nosological entity was introduced with the first publication of Diagnostic and Statistical Manual of Mental disorders (DSM) . HoweverBecause of missing diagnostic biomarkers, schizophrenia spectrum disorders still pose a diagnostic challenge. In daily clinical practice, especially when deciding admission to inpatient treatment it is not uncommon to reserve the diagnosis of SZ for severe cases presenting with flamboyant psychotic symptoms such as bizarre delusions or first-rank symptoms, reflecting uncertainty in distinguishing SZ from SAD. However, patients with SAD might exhibit more severe depressive and negative symptoms, lower age of onset , less coEven in modern psychiatry, the diagnosis of a mental disorder is still based solely on clinical observations and the patient’s narrative. Because of the very heterogeneous presentation of the two disorders, their diagnosis is still a challenge. Correspondingly, their treatment is demanding and a variety of medications can be used to treat both disorders. On the other hand, it is still important to keep the longitudinal overlap of psychotic and affective symptoms in mind and distinguish between these two disorders in order to establish the appropriate combination and dosage of antipsychotic and mood stabilizing medication in SAD. It can be hypothesized that this issue and the remaining diagnostic uncertainty in SAD and the demanded dual treatment strategy is most likely associated with additional clinical effort and resources compared to SZ.While it is of general interest for psychiatry to test this hypothesis of different resource allocation to SZ and SAD treatments, it is of special importance when economic prerequisites are changing. In Germany, the current lump sum payment scheme for psychiatric services is based on daily treatment costs depending on general categories of human resources allocation in a hospital ward, but independent of patient’s diagnosis. Following the reimbursement in the somatic medicine based on diagnostic related groups (DRGs), in 2009, a new national system of cost accounting in psychiatry and psychosomatics has been introduced. The system is the new basis of daily cost data collection but is still not implemented as the basis of reimbursement. The implementation of this new system as a controlling instrument in the clinical daily routine is currently a very controversial issue.The main topic is the validity of this diagnosis-related classification within a group of schizophrenia spectrum disorders (ICD 10 F2x.x) which does not map single diagnostic entities but rather groups of diagnoses. However, the variety of clinical subtypes within this major diagnostic domain including schizophrenia, schizotypal and delusional disorders challenges approaches to use such major categories to determine the demand of financial resources in of cost accounting. The distinction of SZ and SAD represents a prototype of dilemma in this discussion, as both disorders are summarized in the same category. However, no studies have investigated the treatment costs between SZ and SAD patients in Germany yet. Along these lines, the first aim of the presented analysis was to explore and compare the duration of treatment, daily treatment costs and overall costs associated with SZ and SAD at the Department of General Psychiatry in Heidelberg. A second issue is the assumption of a decrement in treatment costs presumed by the cost accounting system.We believe that a comprehensive comparison of the healthcare costs among SZ and SAD patients might provide more insight into the distribution of costs between diagnostic groups and improve the distribution of available treatment resources.st January 2013 and 31st December 2013. The Department of General Psychiatry has 127 inpatient beds as well as an outpatient department in a predominantly urban area in Heidelberg and a fully integrated health service providing treatment for patients with all kinds of mental disorders. The retrospective data were examined for daily in-patient costs, duration of treatment and overall residential treatment costs. 189 patients were identified with admission and discharge between 1st January 2013 and 31st December 2013. The inclusion criteria were: (1) confirmed diagnoses of paranoid schizophrenia or SAD and (2) age between 16 and 80 years. The exclusion criteria included incomplete medical histories, diagnosis of comorbid dementia, and serious acute or chronic neurological or internal disorders which would require additional medical treatment. This resulted in 118 patients diagnosed with paranoid schizophrenia (F20.0) and 70 patients with SAD (F25.*) being included in the analysis. One of the main goals of this study was to ensure that the case costs were calculated in a comparable way to previous health-care studies in psychiatry. Therefore, the procedure chosen is according to the categories of the “Institute for the Hospital Remuneration System” (“Institut für das Entgeltsystem im Krankenhaus”—InEK) as described in the Costs Accounting Manual, Version 1.0 , but a statistically significant difference in age . According to ANCOVA, SAD patients had a longer duration of stay than patients with SZ (p = .027) (p = .023)]  = 2.39; p = .12)] and a marginal difference of mean infrastructure costs  between paranoid SZ and SAD. The costs for medication products, corresponding to item 4a of the German hospital cost accounting algorithm (see above), represented an average of 1.56% of the total costs in patients with paranoid schizophrenia and 1.52% in patients with SAD.Demographic characteristics and costs of both clinical groups are summarized in  = .027) . Mean to= .023)] . FurtherThe paired samples t-test with bootstrapping with 10,000 samples revealed statistically significant differences between the two groups in the mean total costs (p = 0.035), and mean personnel costs (p = 0.04). We found a marginally significant difference in the mean infrastructure costs (p = 0.053) and mean pharmaceutical costs (p = 0.067), between the two groups. According to the bootstrapping procedure, the differences were located within the 95% confidence intervals, for mean total costs , mean medication costs is , personnel costs  and infrastructure/hotel costs is . According to the paired-sample t-test , the variable group was significant.In the analysis of group x time effects on costs we found no significant effects within the interval of 8 weeks . In AddiTo our knowledge this is the first study to compare inpatient health care costs between paranoid SZ and SAD patients in inpatient treatment. The three main findings challenge the current aggregation of both disorders in a single category of resource allocation algorithms: First, SAD patients had a longer duration of stay than patients with SZ. Second, mean total costs were significantly higher for individuals suffering from SAD than paranoid SZ patients. Third, we found we found statistically significant difference in mean personnel costs and a marginal difference in infrastructure costs. In addition there was no evidence for a general or group specific decrement of costs over time.The extended inpatient period in the SAD group is not surprising for two reasons: First, there is a profound empirical evidence for a lower diagnostic stability of SAD when compared to SZ –23. AccoContradictory to our assumption and the idea of greater symptom severity in SAD, we found no statistically significant difference in pharmaceutical costs between patients with SZ and SAD. But, we found a statistically significant difference in mean personnel costs between both diagnostic groups. On the one hand, we had still expected a difference in allocated pharmaceutical treatment resources given as SAD patients might show more severe affective and negative symptoms. However, SAD patients also have higher levels of cognitive and social functioning when compared to SZ . These pAnother notable issue is the result of repeated measures ANOVA in both patient groups. We found no main effect of time in this analysis indicating that there are no significant increments or decrements in the mean total costs, mean costs for medication products, personnel and infrastructure costs. Based upon these results, we disagree with a previously published recommendation of the “InEK” in Germany to reimburse the hospital costs daily with step-wise decreasing remuneration . This moFinally, we examined the mean total costs between SAD and paranoid SZ. Our results are in line with previous cost-of-illness studies for SZ in Germany . HoweverThis is the first statistically significant evidence for distinguishable costs among paranoid SZ and SAD patients. The findings of this study provide insights into the economic impact of different nosological entities. Our results may provide important clues for further development of cost accounting systems for reimbursement in Germany, which has subsumed both paranoid SZ and SAD within a single diagnoses related category. In the future, we strongly advocate retrospective audit of data from larger patient samples ). Future


SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40–50 °C).In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal  For example, some leukemia can be associated with the so-called Philadelphia chromosome (Ph′ chromosome), which is a result of the reciprocal translocation between human chromosomes 9 and 22 , 2. The  process . Our preDiscrimination of completely complementary and partially complementary DNA duplexes on the sensor surface can be achieved by employing various factors influencing hybridization efficiency, including target length and concentration, temperature, composition of hybridization buffer, posthybridization stringency wash, and a combination of the factors , 6. The Unlike hybridization suppressors, stringency control based on the ionic strength of the hybridization buffer, which strongly influences hybridization of completely complementary and partially complementary DNA duplexes and does not lead to excessive strong refractive index change, appears quite attractive . For SPRbcr-abl hybrid mRNA from those related to the normal bcr gene using two stringency control strategies based on the decrease in ionic strength of the hybridization buffer and the temperature elevation by SPR spectrometry biosensor.The aim of this research was to detect and discriminate the oligonucleotides related to the 2PO4, 6-mercapto-1-hexanol was obtained from Fluka ; all other reagents were of analytical grade. All solutions were made with deionized MilliQ water.Urea, KHTable 2SO4 and 30 % H2O2; WARNING: piranha solution reacts violently with organic compounds and must be handled with extreme care) at room temperature for 2 min, then rinsed thoroughly with distilled and deionized water, and dried in the air. The cleaned plate is mounted on the spectrometer prism using immersion liquid. The flow rate  is controlled by the peristaltic pump “Ismatec.” For immobilization of the thiolated probe, 120 μl 1 μM mod-Ph in 0.5 M KH2PO4 (pH 3.8) is injected into the measuring flow cell and exposed for 1 h. After that, the sites on the gold surface, free from immobilized mod-Ph, were blocked by 1 mM aqueous solution of 6-mercapto-1-hexanol [To investigate the processes of oligonucleotide immobilization and hybridization, we used the two-channel SPR spectrometer “Plasmon SPR-6.” This computer-controlled optoelectronic device in the Kretschmann optical configuration was developed at the V.E. Lashkarev Institute of Semiconductor Physics of the National Academy of Sciences of Ukraine. A 45-nm-thick gold layer on a glass plate serves as a sensor surface. Specific recognition layer on the sensor surface (bioselective element) is prepared as follows. Prior to modification, the gold surface of the glass plate is cleaned with freshly prepared piranha solution  to obtain a stable sensor signal. At the next step, 120 μl target solution of various concentrations in the running buffer solution is injected into the measuring flow cell and exposed for 10 min. To distinguish an actual sensor response caused by interactions between the target and the immobilized probe from that caused by occasional fluctuations of medium refractive index, it is necessary to wash the flow cell before and after each sample by the same buffer solution and only then to determine a value of the SPR response. Regeneration of the bioselective element after hybridization experiments was achieved by denaturation of the surface duplexes by rinsing with 8 M urea.bcr gene and 12 nucleotides of the abl gene of the site of their junction in the hybrid mRNA as a target oligonucleotide P1 for the sensor development. Therefore, completely complementary oligonucleotide mod-Ph is used as a probe for immobilization on the sensor surface.We have chosen a sequence consisting of 12 nucleotides of the 2PO4, pH 3.8) have led to an efficient immobilization of the probe on the gold surface of the SPR biosensor. The changes in the resonance angle at the mod-Ph immobilization were converted into surface density using a factor of 120 mdeg per 100 ng cm−2 [−2 (7.6 × 1012 molecules cm−2). The theoretical coverage of a two-dimensional close-packed full monolayer of single-stranded DNA (ssDNA) is 1 × 1014 molecules cm−2, assuming that the molecules are perfect cylinders 1.1 nm in diameter and are oriented perpendicularly to the plane of the surface [The chosen conditions , the reduced level of hybridization can be observed for both targets, but for the less complementary oligonucleotides, it was somewhat much stronger Fig. b. The rabcr-abl and to the normal bcr genes.Figure Tm) values  of the solution-phase hybridized duplexes of the probe mod-Ph and the targets P1 and Bcrex14  creates less favorable conditions for hybridization, and the corresponding values of This approach using SPR spectrometer can be realized as follows: (1) a level of sensor response corresponding to hybridization of the immobilized oligonucleotide probes with target oligonucleotides in the linear range SPR biosensor at the initial (room) temperature should be determined and (2) SPR biosensor measuring cell should be washed by the buffer solution at a specified elevated temperature for a certain period of time (in this work—5 min). As a result of this procedure, certain duplexes will be melted and the buffer solution flowing through the measuring cell will wash out the separated target oligonucleotides. After this, washout and subsequent cooling of the measuring cell to the initial temperature, sensor response, which will reflect the amount of the remaining duplexes should be determined.Successful application of the thermodiscrimination is not possible without the immobilization stability of the surface-bound thiolated probes at the elevated temperature. However, the wide application of the regeneration of the single-stranded thiolated probe layer after hybridization experiments by rinsing with hot water (> 60 °C) proves the stability of the probe layer , 17, 18.For implementation of the thermodiscrimination, thermoregulated measuring flow cell for the SPR spectrometer “Plasmon 6” and corresponding software were created. It is able to ensure rapid heating of the buffer solution that is pumping through the measuring cell to the desired temperature and control it to within 0.1 °C. In addition, it can clearly trace cooling of the buffer solution to determine the return to the initial temperature. This device allowed to carry out thermodiscrimination of completely complementary oligonucleotide P1 and partially complementary oligonucleotide Bcrex14, an example of which is shown in Fig. In this example, the difference between working and reference channels during oligonucleotide hybridization before heating, during heating, and after cooling to the original level of temperature was monitored. The success of the discrimination was provided by significant difference between sensor responses, caused by processes of hybridization/dehybridization of these two pairs of oligonucleotides. After heating to 40 °C, the duplexes formed by partially complementary oligonucleotide Bcrex14 were melted and almost completely washed out from the measuring cell . In the case of complementary oligonucleotide P1, the opposite situation was observed: at the same procedure of hybridization, heating temperature and cooling the difference of sensor responses of working and reference channels was near the same as that, which was observed at the initial hybridization.The ratio of these sensor responses is the analytical indicator that shows the remaining portion of the duplexes, formed by various oligonucleotides. And, its dependence on temperature for different pairs of oligonucleotides shows that temperature range, in which thermodiscrimination of completely and partially complementary oligonucleotides could be carried out.As shown in Fig. Tm. To obtain these values, a sigmoid fitting function was used and the corresponding values have been taken from the curves from Fig. Tm determined for heterogeneous system (hybridization on the sensor surface) are a few degrees lower than the values obtained using the web server DINAMelt for the solution-phase hybridization  is of importance because of technical simplicity of its realization, but it produced a relatively moderate level of discrimination and therefore requires a careful accounting of the impact of possible high concentrations of partially complementary DNA.For implementation of the thermodiscrimination, thermoregulated measuring flow cell for the SPR spectrometer Plasmon 6 and corresponding software were created. Using this thermoregulated measuring flow cell allowed the absolutely effective thermodiscrimination of the completely complementary oligonucleotide P1 and the partially complementary oligonucleotide Bcrex14.Tm of the duplexes formed by the studied oligonucleotides. As it was expected, the experimental values of Tm determined for the heterogeneous system (hybridization on the sensor surface) are a few degrees lower than the values obtained using the web server DINAMelt for the solution-phase hybridization.Besides that, this approach gives the opportunity to calculate the experimental values of bcr-abl gene related to some types of leukemia.It is necessary to note that this method of thermodiscrimination is very flexible because the developed device can control the temperature to within 0.1 °C and a time of hot wash can be selected almost without restriction. In general, we believe the obtained results  are very important for the future development of the SPR biosensor for clinical detection of the