The impact of leucine supplementation on body composition and glucose tolerance following energy restriction: an 8-week RCT in adults at risk of the metabolic syndrome.

BACKGROUND: L-Leucine (Leu) supplementation may benefit fat-free mass (FFM) per se and glucose metabolism. OBJECTIVES: To determine whether Leu supplementation during energy restriction blunted the loss of FFM, enhanced the loss of fat mass (FM) and improved glucose tolerance. DESIGN: Thirty-seven adults, aged 20-65 years, with increased waist circumference and at least one other metabolic syndrome (MetS) component, were selected. We employed a two-arm parallel, double blind, randomized control trial (RCT) design. Participants were randomly assigned to an intervention group (leucine - 3 g/d) or placebo (lactose - 2.67 g/d), while following an individualised energy restricted diet for an 8-week period. Detailed body composition (DEXA), oral glucose tolerance test (OGTT), insulin and components of MetS were measured before and after the trial. Analysis of covariance (ANCOVA) assessed the effect of Leu on an intention-to-treat (ITT) principle. Bootstrapping method with 1000 bootstrap samples was used to derive parameter estimates, standard errors, p-values, and 95% confidence intervals for all outcomes. RESULTS: Adjusted for baseline values and other covariates, FFM (p = 0.045) and lean tissue mass (LTM) (p = 0.050) were significantly higher following Leu. These outcomes were modified by a significant treatment x sex interaction that indicated Leu had the greater effect in men. However, on adjustment for body composition changes, there was no difference in insulin sensitivity, oral glucose tolerance, or change in MetS components following Leu. CONCLUSION: Short-term leucine supplementation during energy restriction resulted in a greater preservation of FFM and LTM particularly in men, but did not impact glucose metabolism.

Effect of household cooking on the retention of vitamin D2 and 25-hydroxyvitamin D2 in pulse UV-irradiated, air-dried button mushrooms (Agaricus bisporus).

Vitamin D deficiency has widespread global prevalence. Fresh mushrooms exposed to ultraviolet (UV) radiation generate vitamin D2 which remains after drying. It is not clear if vitamin D2 is retained after rehydration and cooking of dried mushrooms. The aim of this study was to determine the true retention of both vitamin D2 and 25-hydroxyvitamin D2 (25(OH)D2) after cooking UV-irradiated, air-dried, then rehydrated button mushrooms (Agaricus bisporus). Mushrooms were exposed to pulsed UV radiation, then air-dried in a convection oven, followed by rehydration in warm water. Samples were cooked in three different ways: frying (5 min), baking (10 min, 200 °C) and boiling (20 min, 90 °C). Compared to rehydrated, uncooked controls, there was a high retention of D vitamers (≥95%) after cooking. Frying and baking resulted in significantly higher vitamin D2 retention compared to boiling (p < 0.0001). UV-irradiated, dried mushrooms are a valuable source of vitamin D2 after rehydration and cooking.

The Retention of Vitamin D2 and 25-Hydroxyvitamin D2 in Pulse UV-Irradiated Dried Button Mushrooms (Agaricus bisporus) after 12 Months of Storage.

Fresh mushrooms exposed to ultraviolet (UV) radiation prior to drying generate high concentrations of vitamin D2. The aim of this study was to determine the retention of D vitamers in mushrooms that were pulse UV irradiated, then air dried, and stored for up to 12 months. Fresh button mushrooms (A. bisporus) were exposed to pulsed UV radiation (dose 200 mJ/cm2, peak of 17.5 W/cm2), air dried and vacuum sealed before being stored in the dark at room temperature. After storage, samples were freeze dried and quantified for D vitamers using triple quadrupole mass spectrometry. After 3, 6 and 12 months of storage, there was 100% (11.0 ± 0.8 µg/g dry weight (DW), 93% (10.1 ± 0.6 µg/g DW) and 58% (5.5 ± 0.6 µg/g DW) retention of vitamin D2 and 88% (0.14 ± 0.01 µg/g DW), 71% (0.11 ± 0.01 µg/g DW) and 68% (0.1 ± 0.01 µg/g DW) retention of 25-hydroxyvitamin D2 (25(OH)D2), respectively. Compared to the irradiated dried mushrooms that were not stored, the D vitamer concentration was statistically significantly lower (p < 0.05) at 6 and 12 months for 25(OH)D2 and at 12 months for vitamin D2. Sufficient vitamin D2 (99 µg) remained after 12 months storage to provide at least 100% of daily dietary vitamin D requirements in a 20 g serving.

Evidence of low vitamin D intakes in the Australian population points to a need for data-driven nutrition policy for improving population vitamin D status.

BACKGROUND: Nearly one in four Australian adults is vitamin D deficient (serum 25-hydroxyvitamin D concentrations [25(OH)D] < 50 nmol L-1 ) and current vitamin D intakes in the Australian population are unknown. Internationally, vitamin D intakes are commonly below recommendations, although estimates generally rely on food composition data that do not include 25(OH)D. We aimed to estimate usual vitamin D intakes in the Australian population. METHODS: Nationally representative food consumption data were collected for Australians aged ≥ 2 years (n = 12,153) as part of the cross-sectional 2011-2013 Australian Health Survey (AHS). New analytical vitamin D food composition data for vitamin D3 , 25(OH)D3 , vitamin D2 and 25(OH)D2 were mapped to foods and beverages that were commonly consumed by AHS participants. Usual vitamin D intakes (µg day-1 ) by sex and age group were estimated using the National Cancer Institute method. RESULTS: Assuming a 25(OH)D bioactivity factor of 1, mean daily intakes of vitamin D ranged between 1.84 and 3.25 µg day-1 . Compared to the estimated average requirement of 10 µg day-1 recommended by the Institute of Medicine, more than 95% of people had inadequate vitamin D intakes. We estimated that no participant exceeded the Institute of Medicine's Upper Level of Intake (63-100 µg day-1 , depending on age group). CONCLUSIONS: Usual vitamin D intakes in Australia are low. This evidence, paired with the high prevalence of vitamin D deficiency in Australia, suggests that data-driven nutrition policy is required to safely increase dietary intakes of vitamin D and improve vitamin D status at the population level.

Incidence of RNA viruses infecting taro and tannia in East Africa and molecular characterisation of dasheen mosaic virus isolates.

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) plants growing in 25 districts across Ethiopia, Kenya, Tanzania and Uganda were surveyed for four RNA viruses. Leaf samples from 392 plants were tested for cucumber mosaic virus (CMV), dasheen mosaic virus (DsMV), taro vein chlorosis virus (TaVCV) and Colocasia bobone disease-associated virus (CBDaV) by RT-PCR. No samples tested positive for TaVCV or CBDaV, while CMV was only detected in three tannia samples with mosaic symptoms from Uganda. DsMV was detected in 40 samples, including 36 out of 171 from Ethiopia, one out of 94 from Uganda and three out of 41 from Tanzania, while none of the 86 samples from Kenya tested positive for any of the four viruses. The complete genomes of nine DsMV isolates from East Africa were cloned and sequenced. Phylogenetic analyses based on the amino acid sequence of the DsMV CP-coding region revealed two distinct clades. Isolates from Ethiopia were distributed in both clades, while samples from Uganda and Tanzania belong to different clades. Seven possible recombination events were identified from the analysis carried out on the available 15 full-length DsMV isolates. Nucleotide substitution ratio analysis revealed that all the DsMV genes are under strong negative selection pressure.

Vitamin D Fortification of Milk Would Increase Vitamin D Intakes in the Australian Population, but a More Comprehensive Strategy Is Required.

Low vitamin D status (serum 25-hydroxyvitamin D (25(OH)D) concentration < 50 nmol/L) is prevalent in Australia, ranging between 15% and 32% in the adolescent and adult populations. Vitamin D intakes are also low across the population and were recently estimated at 1.8−3.2 µg/day on average, assuming equal bioactivity of the D vitamers. In combination, these findings strongly suggest that data-driven nutrition policy is needed to increase vitamin D intake and improve status in the Australian population. Food fortification is a potential strategy. We used up-to-date vitamin D food composition data for vitamin D3, 25(OH)D3, vitamin D2, and 25(OH)D2, and nationally representative food and supplement consumption data from the 2011−2013 Australian Health Survey, to model a fortification scenario of 0.8 µg/100 mL vitamin D for fluid dairy milks and alternatives. Under the modelled fortification scenario, the mean vitamin D intake increased by ~2 µg/day from baseline to 4.9 µg/day from food only (7.2 µg/day including supplements). Almost all individual intakes remained substantially below 10 µg/day, which is the Estimated Average Requirement in North America. In conclusion, this modelling showed that fortification of fluid milks/alternatives with vitamin D at the current permitted level would produce a meaningful increase in vitamin D intake, which could be of potential benefit to those with a low vitamin D status. However, this initial step would be insufficient to ensure that most of the population achieves the North American EAR for vitamin D intake. This approach could be included as an effective component of a more comprehensive strategy that includes vitamin D fortification of a range of foods.

Infectivity of an Infectious Clone of Banana Streak CA Virus in A-Genome Bananas (Musa acuminata ssp.).

We have characterized the complete genome sequence of an Australian isolate of banana streak CA virus (BSCAV). A greater-than-full-length, cloned copy of the virus genome was assembled and agroinoculated into five tissue-cultured plants of nine different Musa acuminata banana accessions. BSCAV was highly infectious in all nine accessions. All five inoculated plants from eight accessions developed symptoms by 28 weeks post-inoculation, while all five plants of M. acuminata AA subsp. zebrina remained symptomless. Symptoms were mild in six accessions but were severe in Khae Phrae (M. acuminata subsp. siamea) and the East African Highland banana accession Igisahira Gisanzwe. This is the first full-length BSCAV genome sequence reported from Australia and the first report of the infectivity of an infectious clone of banana streak virus.

Vitamin D Food Fortification and Biofortification Increases Serum 25-Hydroxyvitamin D Concentrations in Adults and Children: An Updated and Extended Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Low vitamin D status is a global public health issue that vitamin D food fortification and biofortification may help to alleviate. OBJECTIVES: We investigated the effect of vitamin D food fortification and biofortification on circulating 25-hydroxyvitamin D (25(OH)D) concentrations. We expanded the scope of earlier reviews to include adults and children, to evaluate effects by vitamin D vitamer, and investigate linear and nonlinear dose-response relations. METHODS: We conducted a systematic review and meta-analysis. We searched CINAHL, MEDLINE, PubMed, Embase, the Cochrane Library, and gray and unpublished literature sites for randomized controlled trials, including people of all ages, with the criteria: absence of illness affecting vitamin D absorption, duration ≥4 wk, equivalent placebo food control, dose quantification, dose ≥5 µg/d, baseline and endpoint or absolute change in 25(OH)D concentrations reported, random allocation, and participant blinding. Quality was assessed using the Jadad Scale. RESULTS: Data from 34 publications (2398 adults: 1345 intervention, 1053 controls; 1532 children: 970 intervention, 562 controls) were included. Random-effects meta-analysis of all studies combined (mean dose 16.2 µg/d) indicated a pooled treatment effect of 21.2 nmol/L (95% CI: 16.2, 26.2), with a greater effect for studies using cholecalciferol than ergocalciferol. Heterogeneity was high (I2  > 75%). Metaregression analyses for all studies combined suggested positive effect differences for baseline circulating 25(OH)D concentrations <50 nmol/L, dose ≥10 µg/d and a negative effect difference when the intervention arm included a calcium dose ≥500 mg/d greater than the control arm. Dose-response rates were found to be nonlinear (Wald test for nonlinearity P < 0.001). For all studies combined, a threshold occurred at ∼26 nmol/L for a dose of ∼21 µg/d. CONCLUSIONS: These results support use of vitamin D food fortification to improve circulating 25(OH)D circulations in populations. This work was registered with PROSPERO as CRD42020145497.

Vitamin D composition of Australian foods.

Australia needs accurate vitamin D food composition data to support public health initiatives. Previously, limitations in analytical methodology have precluded development of a comprehensive database. We used liquid chromatography with triple quadrupole mass spectrometry (LC-QQQ) to analyse 149 composite samples representing 98 foods (primary samples n = 896) in duplicate for vitamin D3, 25-hydroxyvitamin D3 (25(OH)D3), vitamin D2, 25(OH)D2. The greatest concentrations of vitamin D3 were found in canned salmon and a malted chocolate drink powder (fortified); chicken eggs and chicken leg meat contained the most 25(OH)D3. Margarine (fortified) and chocolate contained the greatest concentrations of vitamin D2, with smaller amounts found in various meat products. 25(OH)D2 was detected in various foods, including meats, and was quantitated in lamb liver. These data advance knowledge of dietary vitamin D in Australia and highlight the importance of analysis of these four forms of vitamin D to accurately represent the vitamin D content of food.

Effectiveness of a Singaporean Community-Based Physical Activity and Nutrition Intervention: A Cluster Randomized Controlled Trial.

This study examined the effectiveness of a 6-month intervention to improve the health behaviors and outcomes among women aged 50 years and older. A sample of 580 (intervention n = 295; control n = 285) women was recruited from 26 recreational centers. Only the intervention group participated in the Singapore Physical Activity (PA) and Nutrition Study (SPANS), received health resources (calendar, recipe, and booklets) and motivational support from program ambassadors. The intervention group showed significant improvements in moderate-intensity PA, vigorous-intensity PA, and total PA (P < .001), increased intake frequency of fruit and vegetables (P = .049), a reduction in salt and sugary beverage intake (P ≤ .042), and reductions in systolic blood pressure (BP; -3.68 mm Hg), diastolic BP (-3.54 mm Hg), and percentage body fat (-2.13%; P ≤ .020) when compared with the control group. The SPANS appeared to be efficacious in improving PA and dietary behaviors, reducing BP and percentage body fat among Singaporean women.

Process evaluation of the 'Singapore Physical Activity and Nutrition Study'.

INTRODUCTION: The Singapore Physical Activity and Nutrition Study (SPANS) aimed to improve the physical activity (PA) and nutrition behaviours of Singaporean women aged 50 years and over. The SPANS program consisted of PA classes, nutrition workshops, telephone dietary counselling, health booklets, a health calendar and program ambassadors. This study aimed to assess and understand the implementation of the program strategies and gain insight into process evaluation components to inform future programs. METHODS: The evaluation was guided by a process evaluation framework and collected data via questionnaires (n = 209), program ambassador documentation and exit interviews with program completers (n = 13) and non-completers (n = 12). RESULTS: In total, 295 participants completed the program (response rate = 84 %). Participants reported high levels of satisfaction with the overall program (99.5 %) and program activities (96.7 %), and also rated program ambassadors highly. Participation rates were highest for telephone dietary counselling sessions. The main reason for not attending program activities was having a 'busy schedule' (n = 158). Participants cited a need for improved recreational centre facilities and increased flexibility around program delivery. CONCLUSIONS: The process evaluation showed that the program strategies were implemented as planned and were deemed suitable for supporting behaviour change among Singaporean women aged 50 years and over. The program reached and involved the majority of participants throughout the six months. The combination of practical educational resources and supportive program ambassadors were key strategies that facilitated positive PA and dietary behaviours. However, there needs to be some flexibility in the delivery of programs. The findings of this research may inform other programs in the region.

Long-term sustainability of a physical activity and nutrition intervention for rural adults with or at risk of metabolic syndrome.

OBJECTIVE: To determine longer-term (18-month) sustainability of a six-month physical activity and nutrition intervention for 50-69-year-olds with or at risk of metabolic syndrome residing in a rural Australian community. METHODS: Participants (n=151) were followed-up at 12 and 18 months post-intervention. Changes in nutrition behaviours (fat and fibre barometer); physical activity behaviours (IPAQ); anthropometry (waist-hip ratio, weight, BMI), blood pressure, blood parameters (triglycerides, glucose, LDL-, HDL-, non-HDL, total-cholesterol) were analysed using t-tests and repeated measures ANOVA. RESULTS: Across three time points (6, 12 and 18 months) marginal decrease was observed for waist circumference (p=0.001), a modest increase was observed for diastolic blood pressure (p=0.010) and other outcome measures remained stable. CONCLUSION: Maintenance and ongoing improvement of health behaviours in the longer-term is challenging. Future studies must look for ways to embed interventions into communities so they are sustainable and investigate new approaches to reduce the risk of chronic disease. Implications for public health: Metabolic syndrome is a major health issue in Australia and worldwide. Early identification and management are required to prevent the progression to chronic disease. This 18-month follow-up showed that outcomes measures remained relatively stable; however, there is a need to investigate opportunities for embedded community interventions to support long-term health behaviour change.

Efficacy of vitamin D food fortification and biofortification in children and adults: a systematic review protocol.

OBJECTIVE: The objective of this review is to assess the effect of vitamin D food fortification and biofortification on serum 25-hydroxyvitamin D (25(OH)D) concentrations. INTRODUCTION: Vitamin D deficiency is a global issue with considerable implications for public health. Many people do not receive sufficient sun exposure for adequate endogenous vitamin D synthesis, nor sufficient dietary vitamin D to meet their requirements. Increasing sun exposure may increase the risk of skin cancer, and vitamin D supplementation is not favored as a population-wide solution. Previous systematic reviews have shown a dose-dependent effect of vitamin D-fortified foods on circulating 25(OH)D concentrations. However, previous reviews did not investigate the efficacy of vitamin D fortification and biofortification in children, and several new fortification and biofortification trials in adults have been published since these earlier systematic reviews were conducted. INCLUSION CRITERIA: This review will consider randomized controlled trials that investigate the effect of vitamin D food fortification and biofortification on serum 25(OH)D concentrations in children and adults. METHODS: CINAHL, MEDLINE, Embase, Cochrane Central Register of Controlled Trials, and PubMed will be searched for relevant studies published in English. Following a screening of titles and abstracts by a single author, relevant full-text articles will be independently reviewed by two authors. Two authors will independently assess methodological quality using the JBI critical appraisal checklist for randomized controlled trials. Data will be independently extracted by two authors. A meta-analysis will be conducted, and heterogeneity will be statistically tested. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42020145497.

A randomised controlled crossover trial investigating the short-term effects of different types of vegetables on vascular and metabolic function in middle-aged and older adults with mildly elevated blood pressure: the VEgetableS for vaScular hEaLth (VESSEL) study protocol.

BACKGROUND: A diet rich in fruits and vegetables is recommended for cardiovascular health. However, the majority of Australians do not consume the recommended number of vegetable servings each day. Furthermore, intakes of vegetables considered to have the greatest cardiovascular benefit are often very low. Results from prospective observational studies indicate that a higher consumption of cruciferous vegetables (e.g. broccoli, cabbage, cauliflower) is associated with lower cardiovascular disease risk. This may be due to the presence of specific nutrients and bioactive compounds found almost exclusively, or at relatively high levels, in cruciferous vegetables. Therefore, the aim of this randomised controlled crossover trial is to determine whether regular consumption of cruciferous vegetables results in short-term improvement in measures related to cardiovascular disease risk, including ambulatory blood pressure, arterial stiffness, glycaemic control, and circulating biomarkers of oxidative stress and inflammation. METHODS: Twenty-five participants (50-75 years) with mildly elevated blood pressure (systolic blood pressure 120-160 mmHg) will complete two 2-week intervention periods in random order, separated by a 2-week washout period. During the intervention period, participants will consume 4 servings (~ 300 g) of cruciferous vegetables per day as a soup (~ 500-600 mL/day). The 'control' soup will consist of other commonly consumed vegetables (potato, sweet potato, carrot, pumpkin). Both soups will be approximately matched for energy, protein, fat, and carbohydrate content. All measurements will be performed at the beginning and end of each intervention period. DISCUSSION: The findings of this study will provide evidence regarding the potential cardiometabolic health benefits of cruciferous vegetables, which may contribute to the revision of dietary and clinical guidelines. TRIAL REGISTRATION: The trial was registered with the Australian New Zealand Clinical Trial Registry on 19th September 2019 (ACTRN12619001294145).

Recreational Centres' Facilities and Activities to Support Healthy Ageing in Singapore.

OBJECTIVE: This study examined the physical and social environment (facilities and activities) of Singapore's Recreational Centres (RCs) and female patrons' (>50 years) perception of the RC facilities and activities. MATERIALS AND METHODS: A total of 100 RCs were audited, and 22 face-to-face interviews were undertaken. RESULTS: Physical activity classes were the main activity offered (mean = eight classes per month), with walking (29.8%) and aerobics sessions (17.5%) being the most frequent. Nutrition classes and social activities were offered less often (mean = one class per month). The activities were well received by patrons, offering opportunities to interact while participating in physical activity and nutrition classes. However, the need for staff training, consideration of patron's abilities and the desire to engage in alternative activities were expressed. CONCLUSION: Overall, RCs' facilities and activities were well liked by the patrons but opportunities for improvements were identified. Regular reviews of facilities and activities through consultation with the RC patrons and managers are needed to ensure that the facilities and activities remain relevant and practical to the patrons. This will help to support active lifestyles and healthy eating practices among older adults residing within the community.

High-Intensity Single-Leg Cycling Improves Cardiovascular Disease Risk Factor Profile.

INTRODUCTION: Regular exercise can reduce the risk of developing cardiovascular disease through risk factor modification, with high-intensity exercise and more recently small muscle mass training providing alternatives to moderate-intensity exercise. METHODS: This study randomly assigned 53 healthy middle-age adults (age, 62 ± 6 yr) to complete 24 sessions (8 wk; 3 d·wk) of exercise training, using either high-intensity double-leg cycling (n = 17; HITDL), high-intensity single-leg cycling (n = 18; HITSL), or moderate-intensity double-leg cycling (n = 18; MCTDL). Biomarkers of cardiovascular risk (total cholesterol, triglycerides, HDL-c, LDL-c, apo-B48, and glucose), anthropometry measures (body mass, body mass index, waist circumference, and waist-to-hip ratio), resting blood pressure, and aerobic capacity were assessed pre- and postintervention. RESULTS: Total work completed was greater (P < 0.01) in MCTDL (5938 ± 1462 kJ) compared with the HITDL (3462 ± 1063 kJ) and HITSL (4423 ± 1875 kJ). Pre- to posttraining differences were observed for waist-to-hip ratio (0.84 ± 0.09 vs 0.83 ± 0.09; P < 0.01), resting systolic blood pressure (129 ± 11 vs 124 ± 12 mm Hg; P < 0.01), total cholesterol (5.87 ± 1.17 vs 5.55 ± 0.98 mmol·L; P < 0.01), and LDL-c (3.70 ± 1.04 vs 3.44 ± 0.84 mmol·L; P < 0.01), with no differences between conditions. In addition, aerobic capacity increased after training (22.3 ± 6.4 vs 24.9 ± 7.6 mL·kg·min; P < 0.01), with no differences between conditions. CONCLUSION: These findings suggest that all three modes of exercise can be prescribed to achieve cardiovascular risk reduction in an aging population.

Effect of PolyGlycopleX (PGX) Consumption on Blood Lipid Profiles in Healthy, Low CVD Risk Overweight Adults.

Raised blood lipid levels are associated with a risk of a cardiovascular disease (CVD). Moderate reductions in several CVD factors such as total, low-density lipoprotein (LDL) cholesterol and non-high-density lipoprotein (non-HDL) cholesterol concentrations may be more effective in reducing overall risk than a major reduction in just one. A blind, randomised controlled trial was conducted with 120 healthy overweight (BMI 25⁻30) adults aged 25⁻70 years who were non-smokers, not diabetic and of low risk of cardiovascular disease, as assessed by the Framingham risk equation. Participants consumed 4.5 g PolyGlycopleX (PGX) as softgel capsules (PGXS) or 5 g PGX granules (PGXG) or 5 g rice flour (RF) with meals three times a day for 12 weeks. Total, LDL and non-HDL cholesterol were all significantly reduced (-6%, -5% and -3.5%, respectively) post the PGX granule treatment; however, PGX in softgel capsule form did not affect blood lipid profiles. Daily consumption of PGX granules in overweight low CVD risk adults produced lipid changes indicating a CVD preventative benefit.

Molecular characterisation of a putative new polerovirus infecting pumpkin (Cucurbita pepo) in Kenya.

Next-generation sequencing of RNA extracted from a pumpkin plant with mosaic symptoms in Kenya identified the presence of a polerovirus sequence closely related to pepo aphid-borne yellows virus (PABYV). The near-complete polerovirus sequence comprised 5,810 nucleotides and contained seven putative open reading frames (ORFs) with a genome organisation typical of poleroviruses. BLASTp analysis of the translated sequences of ORFs 0, 1 and 2 revealed that their amino acid sequences differed by more than 10% from the corresponding protein sequences of other poleroviruses. These results suggest that this virus is a putative novel member of the genus Polerovirus, which has been provisionally named "pumpkin polerovirus" (PuPV).

Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses.

The genus Badnavirus is characterized by members that are genetically and serologically heterogeneous which presents challenges for their detection and characterization. The presence of integrated badnavirus-like sequences in some host species further complicates detection using PCR-based protocols. To address these challenges, we have assessed and optimized various RCA protocols including random-primed RCA (RP-RCA), primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-RCA) and specific-primed RCA (SP-RCA). Using Dioscorea bacilliform AL virus (DBALV) as an example, we demonstrate that viral DNA amplified using the optimized D-RCA and SP-RCA protocols showed an 85-fold increase in badnavirus NGS reads compared with RP-RCA. The optimized RCA techniques described here were used to detect a range of badnaviruses infecting banana, sugar cane, taro and yam demonstrating the utility of RCA for detection of diverse badnaviruses infecting a variety of host plant species.