Safety strategies and harm reduction for methamphetamine users in the era of fentanyl contamination: A qualitative analysis.

INTRODUCTION: In the United States, methamphetamine use is increasing and the context of its use has changed, with reports of illicitly manufactured fentanyl being mixed with methamphetamine (either deliberately or inadvertently). We explore risk-mitigating actions taken by people who use drugs to protect their health when using methamphetamine in that context. METHODS: We conducted qualitative interviews with 48 adults (18+) who used methamphetamine in the past three months at two sites in Nevada, USA and two sites in New Mexico, USA. Interviews were recorded, transcribed, and analyzed using thematic analysis. RESULTS: Respondents described two rationales for employing harm reduction strategies. First, to prevent harm from methamphetamine containing illicit fentanyl, and second, to maintain their general wellbeing while using methamphetamine. Regarding methamphetamine containing illicit fentanyl, our findings highlight how respondents employ primary strategies like buying from trusted sources and secondary strategies such as spotting and selective use of harm reduction tools (i.e., fentanyl test strips) to reduce risks. To maintain their general wellbeing, participants reduced their use of methamphetamine as reasonably as possible, and used other substances like marijuana and alcohol alongside methamphetamine to counter the unwanted side effects of methamphetamine (i.e., hallucinations and paranoia). Use of these harm reduction strategies varied within situational and social contexts, and respondents usually developed these strategies based on their lived experiences. CONCLUSION: Our findings uniquely demonstrate that people who use methamphetamine prioritize community driven, trust-based strategies within their social networks to mitigate risks in a fentanyl-contaminated drug environment. Additionally, our results indicate that harm reduction behaviors are influenced by multilevel risk environments, which include social, physical, economic, and political factors. Overall, these results highlight the potential for targeted interventions at the network level, which are responsive to complexities and shifts in drug market dynamics- such as illicit fentanyl in methamphetamine.

The effects of including sprouted barley with alfalfa hay in the diet on ruminal health and performance of cow-calf pairs.

The world population is growing exponentially, increasing demand to produce high-quality protein for human consumption. Changes in weather patterns, drought, and decreased land resources due to urbanization have increased the strain on the agriculture sector to meet world demands. An alternative method to combat these issues and continue to produce high-quality livestock feed would be through a controlled environment vertical farming system. Commonly, cereal grains, such as barley, are used in these systems to produce livestock feed. However, there is little information on the viability of feeding sprouted grains to beef cattle. Two diets of either feeder-quality alfalfa hay (n = 10 pairs; ALF) or the same alfalfa hay and sprouted barley (SB; 12.6% dry matter [DM]; n = 10 pairs) were fed for 90 d to Angus pairs with a steer calf during mid to late lactation. On days 0 and 90, body weight (BW), milk, rumen fluid, and body condition score were collected from cows and hip height and BW were recorded for calves. On day 10, BW was recorded for cows and calves and rumen fluid was collected from cows. Rumen fluid was also collected from cows on day 45. On day 55, BW was collected for both cows and calves and milk from cows. Intake was recorded throughout the trial via bunks with Vytelle technology. The PROC MIXED procedure of SAS was used to analyze all data with the day as a repeated measure to determine the main effect of diet. Individual volatile fatty acids (VFA) were measured as a percent of total VFA. No differences (P ≥ 0.16) were observed in calf BW, hip height, milk protein, fat, lactose, calf DM intake (DMI), or cow DMI. Cows fed SB tended (P = 0.08) to have a decreased somatic cell count compared to ALF. Percent butyrate was impacted by diet × day (P = 0.02), but no difference (P > 0.09) at any time points were detected. Additionally, a diet × day effect (P = 0.001) on rumen pH demonstrated that both groups stayed consistent until day 45 and then SB pH decreased the last 45 d. There was a day effect for total VFA (P = 0.0009), acetate:propionate (Ac:Pr; P < 0.0001), acetate (P < 0.0001), and propionate (P < 0.0001) demonstrating that total VFA, acetate, and Ac:Pr all increased throughout the trial, while propionate decreased. These results indicate that SB can be a potential alternative feed at this stage of production as it does not negatively impact health or production, but does affect the rumen pH and proportion of some VFA.

Climate variability and uncertainty associated with weather patterns can greatly impact feed security for cattle producers. Flooding, drought, and temperature extremes can reduce a farmer's ability to produce a consistent crop, resulting in feed prices that can fluctuate greatly. Vertical farming systems that sprout cereal grains in a controlled environment, using precision irrigation, may alleviate the effects of external factors such as climate and resulting feed prices. The objective of this study was to determine if sprouted barley (SB) could be used as an effective alternative feed source for cow-calf pairs. Two diets were fed to 20 cow-calf pairs, a control diet consisting of 100% feeder-quality alfalfa hay, or an experimental diet comprised of feeder-quality alfalfa hay and a 12.6% dry matter inclusion of SB for 90 d. Body weight, feed intake, and feeding behavior were analyzed in the cows and calves. Ruminal health was also assessed in cows by analyzing the ruminal fluid for pH and volatile fatty acid composition. When health and performance metrics were analyzed, no differences were found between the two diets that were administered to the cattle.

A qualitative study of interest in and preferences for potential medications to treat methamphetamine use disorder.

INTRODUCTION: We examined acceptability of and preferences for potential medications for treating methamphetamine use disorder (MUD) among people who use methamphetamine and examined how benefits and drawbacks of methamphetamine use affect perceived acceptability and preferences. METHODS: We conducted qualitative interviews as part of a larger study in 2019-2020. The interview assessed patterns of substance use (including methamphetamine), benefits and drawbacks of methamphetamine use, and interest in a medication to treat MUD. Analysis used an inductive thematic approach, guided by three primary questions: (1) would participants be interested in taking a potential medication for MUD?; (2) what effects would they would like from such a medication?; and (3) what would their ideal treatment route and schedule be (e.g. daily pill, monthly injection)?. RESULTS: We interviewed 20 people reporting methamphetamine use in the past 3 months (10 from Reno, Nevada, USA and 10 from Rio Arriba County, New Mexico, USA). Seven used exclusively methamphetamine, while thirteen used other substances in addition to methamphetamine. Most were enthusiastic about a potential medication to treat MUD. Of those who were not interested (n = 5), all indicated no current concerns about their methamphetamine use. Perceived functional benefits of methamphetamine use (i.e., energy, counteracting opioid sedation, and improved social and emotional wellbeing) informed preferences for a replacement-type medication that would confer the same benefits while mitigating drawbacks (e.g., psychosis, hallucinations, withdrawal). Opinions on preferred dosing varied, with some preferring longer acting medications for convenience, while others preferred daily dosing that would align with existing routines. CONCLUSION: Participants were excited about a potential for a medication to treat MUD. Their preferences were informed by the functional role of methamphetamine in their lives and a desire to maintain the stimulant effects while mitigating harms of illicit methamphetamine. Treatment outcomes that emphasize functioning and wellbeing, rather than abstinence, should be explored.

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.

Patterns of and Rationale for the Co-use of Methamphetamine and Opioids: Findings From Qualitative Interviews in New Mexico and Nevada.

Introduction: Methamphetamine use and methamphetamine-involved deaths have increased dramatically since 2015, and opioid-related deaths now frequently involve methamphetamine. Nevada and New Mexico are states with elevated rates of opioid and methamphetamine use. In this paper, we report results from a qualitative analysis that examined patterns of methamphetamine and opioid co-use over participants' lifespan, factors that influence those patterns, and implications for health outcomes among users. Methods: Project AMPED was a multisite, mixed-methods study of methamphetamine use in Northern New Mexico and Northern Nevada. Between December 2019 and May 2020, qualitative interview participants were asked to describe their patterns of and reasons for co-administration of opioids and methamphetamine. Results: We interviewed 21 people who reported using methamphetamine in the past 3 months. Four primary patterns of methamphetamine and opioid co-use were identified: [1] using both methamphetamine and heroin, either simultaneously or sequentially (n = 12), [2] using methamphetamine along with methadone (n = 4), [3] using prescription opioids and methamphetamine (n = 1), and [4] using only methamphetamine (n = 4). Among those who used methamphetamine and heroin simultaneously or sequentially, motivations drew from a desire to enhance the effect of one drug or another, to feel the "up and down" of the "perfect ratio" of a goofball, or to mitigate unwanted effects of one or the other. Among those who used methamphetamine and methadone, motivations focused on alleviating the sedative effects of methadone. Conclusion: To address the emergent trend of increasing methamphetamine-related deaths, researchers, health care professionals, and community health workers must acknowledge the decision-making processes behind co-use of opioids and methamphetamine, including the perceived benefits and harms of co-use. There is an urgent need to address underlying issues associated with drug use-related harms, and to design interventions and models of treatment that holistically address participants' concerns.

"It's called overamping": experiences of overdose among people who use methamphetamine.

BACKGROUND: The USA is experiencing increases in methamphetamine use and methamphetamine-related or attributed deaths. In the current study, we explore qualitative narratives of methamphetamine overdose and strategies used by people who use drugs to reduce the undesirable effects associated with methamphetamine use. METHODS: We conducted 21 qualitative interviews with people over the age of 18 who reported using methamphetamine in the previous 3 months in Nevada and New Mexico. Interviews were recorded, transcribed, and analyzed using qualitative thematic analysis. RESULTS: Respondents described a constellation of psychological and physical symptoms that they characterized as "overamping," experienced on a continuum from less to more severe. Reports of acute, fatal methamphetamine overdose were rare. Few reported seeking medical attention for undesirable effects (usually related to psychological effects). General self-care strategies such as sleeping and staying hydrated were discussed. CONCLUSIONS: When asked directly, our respondents claimed that acute, fatal methamphetamine overdose is rare or even impossible. However, they described a number of undesirable symptoms associated with overconsumption of methamphetamine and had few clinical or harm reduction strategies at their disposal. Addressing this current wave of drug-related deaths will require attention to the multiple factors that structure experiences of methamphetamine "overdose," and a collaborative effort with PWUDs to devise effective harm reduction and treatment strategies.

The mobile emergency recovery intervention trial (MERIT): Protocol for a 3-year mixed methods observational study of mobile recovery outreach teams in Nevada's emergency departments.

BACKGROUND: The Substance Abuse and Mental Health Administration awarded State Targeted Response grants to support states' efforts to address the opioid epidemic. In Nevada, one component of this grant was mobile recovery outreach teams (MROTs) that utilized peer recovery support specialists to provide care for qualifying patients in emergency departments (EDs). The Mobile Emergency Recovery Intervention Trial (MERIT) is a mixed methods study to assess the feasibility/acceptability and effectiveness of the MROT intervention. This protocol mainly describes the R33 research activities and outcomes. The full protocol can be found protocols.io. METHODS: Data will be derived from state-level data sets containing de-identified emergency department visits, substance use disorder treatment records, and mortality files; in-person mixed methods interviews; participant observation; and self-report process evaluation forms. Primary outcomes include Medication Assisted Treatment (MAT) initiation and non-fatal overdose; secondary outcomes include MAT retention and fatal overdose. Quantitative hypotheses will be tested using generalized linear mixed effects models, Bayesian hierarchical models, and marginal Cox models. Qualitative interview data will be analyzed using an inductive thematic analysis procedure. DISCUSSION: It is impossible to conduct a randomized controlled trial of the effectiveness of the MROTs, given the ethical and logistical considerations of this intervention.This study's innovative design employs a mixed methods formative phase to examine feasibility and acceptability, and a quasi-experimental outcomes evaluation phase employing advanced statistical methods to mitigate bias and suggest causal inference regarding the effectiveness of the MROTs.Innovative interventions have been deployed in many states; evidence regarding their effectiveness is lacking, but critical to informing an effective public health response to the opioid epidemic.

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.

Factors Associated With Calling 911 for an Overdose: An Ethnographic Decision Tree Modeling Approach.

Objectives. To identify factors that influence when people who use drugs (PWUDs) call 911 for an overdose. Methods. We conducted 45 qualitative interviews and 180 surveys with PWUDs who had recently witnessed overdoses in Southern California from 2017 to 2019. We used conditional inference tree and random forest models to generate and validate a model to predict whether 911 would be called. Results. Our model had good in- (83%) and out-of-sample (84%) predictive accuracy. Three aspects of the social and policy environment influenced calling 911 for an overdose: the effectiveness of response strategies employed, the behavior of other bystanders, and whether the responder believes it is their responsibility to call. Conclusions. Even in the presence of policies that provide some protections, PWUDs are faced with difficult decisions about calling 911 and must weigh their own safety against that of an overdose victim. Potential interventions include strengthening training and safety planning for PWUDs, bolstering protections for PWUDs when they call 911, and separating law enforcement response from emergency medical response to overdoses.

Correlates of Medical Mistrust Among Minority Women at Risk for HIV and Their Networks.

BACKGROUND: Medical mistrust is a barrier to engaging in HIV prevention and treatment, including testing and adherence to antiretroviral therapy. Research often focuses on how race and experiences of discrimination relate to medical mistrust, overlooking the role that other characteristics may play (e.g., history of physical abuse, diagnosis of mental illness). Furthermore, studies are often restricted to samples of men who have sex with men and findings may not generalize to other at-risk groups. AIMS: The current study explores a range of demographic, cognitive, behavioral, and social network correlates of medical mistrust. METHOD: This study employed an egocentric network design among a racially diverse sample of at-risk women and women in their social networks (n = 165). RESULTS: Results from multivariable linear regressions stratified by race (Black vs. others) indicate that medical mistrust is associated with both individual-level and network-level characteristics. Across both groups, age and experiences of racial discrimination were associated with higher medical mistrust. Having a regular sex partner and having a higher proportion of network members who are family was significantly associated with medical mistrust among non-Black women. DISCUSSION: Individual-level and network-level variables were significantly associated with medical mistrust. Therefore, interventions that attempt to mitigate medical mistrust as a barrier to HIV prevention and treatment should consider how mistrust may be related to characteristics of individuals and broader contexts. CONCLUSION: Health interventions may benefit from conceiving of medical mistrust as a complex, rational response to cumulative discriminatory life experiences and a reflection of the networks within which individuals are embedded.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

"It's Gonna be a Lifeline": Findings From Focus Group Research to Investigate What People Who Use Opioids Want From Peer-Based Postoverdose Interventions in the Emergency Department.

STUDY OBJECTIVE: Postoverdose interventions that deploy peer recovery support specialists to emergency departments (EDs) are a promising response to opioid overdoses among patients presenting in EDs. The objective of this study was to elicit patients' perspectives regarding the feasibility and acceptability of such an intervention and to ensure that their perspectives are represented in intervention design, implementation, and evaluation. METHOD: In 2019 the study investigators conducted focus groups with people who use opioids to elicit perspectives about a postoverdose intervention delivered in the ED by using a semistructured interview guide that asked about feasibility, acceptability, perceived benefits, and concerns. Focus groups were digitally recorded, transcribed, and analyzed for emerging themes. RESULTS: Nine focus groups with 30 people who use opioids were conducted. Key findings that could improve feasibility and acceptability of the intervention include the following: the importance of balancing the urgency of seeing patients quickly with a need to accommodate the experience of precipitated withdrawal symptoms; the need to address privacy concerns; and the need to address concerns related to cost, insurance coverage, and sustainability. Perceived benefits of the intervention included the ability of the peer recovery support specialist to provide advocacy and support, serve as a model of hope and encouragement for behavior change, and fill key service gaps. CONCLUSION: Postoverdose interventions in the ED provide the opportunity to integrate harm reduction-based interventions into traditional biomedical care facilities. These interventions can fill gaps in services and provide additional care and comfort for people who use opioids, but design, implementation, and evaluation should be informed by a patient-centered care perspective.

Localization of Tobacco Yellow Dwarf Virus Replication Using the In Plant Activation (INPACT) Expression Platform.

Geminiviruses and their diseases are a considerable economic threat to a vast number of crops worldwide. Investigating how and where these viruses replicate and accumulate in their hosts may lead to novel molecular resistance strategies. In this study, we used the Rep-inducible In Plant Activation (INPACT) expression platform, based on the genome of tobacco yellow dwarf virus (TYDV), to determine where this model mastrevirus replicates in its host tobacco. By developing an infectious clone of TYDV and optimizing its delivery by agroinfiltration, we first established an efficient artificial infection process. When delivered into transgenic tobacco plants containing a TYDV-based INPACT cassette encoding the ß-glucuronidase (GUS) reporter, we showed the virus activates GUS expression. Histology revealed that reporter gene expression was limited to phloem-associated cell types suggesting TYDV replication has a restricted tissue tropism.

Pro-vitamin A carotenoids in East African highland banana and other Musa cultivars grown in Uganda.

Bananas and plantains (Musa spp.) are an important staple and food security crop in sub-Saharan Africa. In Uganda, where the consumption of East African highland banana (EAHB) is the highest in the world, the population suffers from a high incidence of vitamin A deficiency (VAD). Since the consumption of pro-vitamin A carotenoids (pVAC) made available through the food staple can help alleviate these ailments, we set out to identify the most suitable banana variety to use in future biofortification strategies through genetic engineering. The study focussed on eight popular Musa cultivars grown in the heart of banana farming communities and across the three major agricultural zones of Uganda. The fruit pVAC concentration varied considerably within and across the cultivars tested. These variations could not be explained by the altitude nor the geographical location where these fruits were grown. More than 50% of the total carotenoids present in EAHB cultivars was found to comprise of α- and ß-carotene, while the retention of these compounds following traditional processing methods was at least 70%. Storage up to 14 days postharvest improved carotenoid accumulation up to 2.4-fold in the cultivar Nakitembe. The technical challenge for a successful biofortification approach in Uganda using genetically modified EAHB lies in guaranteeing that the fruit pVAC content will invariably provide at least 50% of the estimated average requirement for vitamin A regardless of the growing conditions.

"Another tool for the tool box? I'll take it!": Feasibility and acceptability of mobile recovery outreach teams (MROT) for opioid overdose patients in the emergency room.

Drug poisoning deaths involving opioids have increased exponentially in the United States. Post-overdose outreach to patients in the emergency room (ER) is a promising strategy for increasing uptake of medication assisted treatment and reducing subsequent overdose. We conducted a mixed methods study to investigate the feasibility and acceptability of a mobile recovery outreach team (MROT) program for opioid overdose patients presenting in Nevada's ERs, which was funded by the SAMHSA Opioid State Targeted Response (STR) grant. We interviewed 25 ER staff using quantitative questions informed by Diffusion of Innovation (DOI) theory and qualitative questions regarding their experiences caring for overdose patients, perceived benefits, and concerns about the MROT program. Respondents expressed strong support and enthusiasm for the program, identified advantages of the program relative to standard of care, highlighted logistical issues that must be addressed prior to implementation, and illustrated how the MROT program is compatible with their personal and professional values. Our results suggest that the STR-funded MROT program could reduce burden and stress among ER staff and improve patient outcomes, but must be informed by formative research that addresses issues of logistical complexity and cultural compatibility.

Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform.

Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease resistance. In most cases this can only be achieved using a transgenic approach. Public and regulatory acceptance of these events would greatly increase with "clean" single copy integration events free of the selectable marker gene and extraneous vector backbone. This would also allow for the successive addition of new genes and traits as they become available. In this study, we used the pMarker Free 1 (pMF1) vector containing the green fluorescent protein (gfp) reporter gene to assess the effectiveness of steroid-inducible recombination and positive/negative dual selection to regenerate transgenic Cavendish banana plants that were potentially free of the selectable marker gene. By examining the interaction of two different Agrobacterium strains with two different cultivars of Cavendish banana, namely Williams and Grand Naine, we describe a transformation and regeneration strategy that successfully produced marker-free, single transgene copy, gfp-expressing events. The system will provide a useful means of serially improving banana into the future.

Post-overdose interventions triggered by calling 911: Centering the perspectives of people who use drugs (PWUDs).

BACKGROUND: Opioid overdose deaths have increased exponentially in the United States. Bystander response to opioid overdose ideally involves administering naloxone, providing rescue breathing, and calling 911 to summon emergency medical assistance. Recently in the US, public health and public safety agencies have begun seeking to use 911 calls as a method to identify and deliver post-overdose interventions to opioid overdose patients. Little is known about the opinions of PWUDs about the barriers, benefits, or potential harms of post-overdose interventions linked to the 911 system. We sought to understand the perspectives of PWUDs about a method for using 911 data to identify opioid overdose cases and trigger a post-overdose intervention. METHODS AND FINDINGS: We conducted three focus groups with 11 PWUDs in 2018. Results are organized into 4 categories: willingness to call 911 (benefits and risks of calling), thoughts about a technique to identify opioid overdoses in 911 data (benefits and concerns), thoughts about the proposed post-overdose intervention (benefits and concerns), and recommendations for developing an ideal post-overdose intervention. For most participants, calling 911 was synonymous with "calling the police" and law enforcement-related fears were pervasive, limiting willingness to engage with the 911 system. The technique to identify opioid overdoses and the proposed post-overdose intervention were identified as potentially lifesaving, but the benefits were balanced by concerns related to law enforcement involvement, intervention timing, and risks to privacy/reputation. Nearly universally, participants wished for a way to summon emergency medical assistance without triggering a law enforcement response. CONCLUSIONS: The fact that the 911 system in the US inextricably links emergency medical assistance with law enforcement response inherently problematizes calling 911 for PWUDs, and has implications for surveillance and intervention. It is imperative to center the perspectives of PWUDs when designing and implementing interventions that rely on the 911 system for activation.

An Improved Transformation System for Cowpea (Vigna unguiculata L. Walp) via Sonication and a Kanamycin-Geneticin Selection Regime.

An improved cowpea transformation method utilizing Agrobacterium-mediated gene delivery to explants derived from the cotyledonary nodes of imbibed cowpea seed is described. The explants were regenerated following a sonication procedure and a stringent selection comprising alternating regimes of kanamycin and geneticin. The method was reproducible and led to the recovery of independent fertile transgenic plants in the greenhouse at a level of about one per cent of starting explants. A transgene encoding an insecticidal protein from Bacillus thuringiensis was used to demonstrate the efficacy of the system.

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.