Influence of viral genes on the cell-to-cell spread of RNA silencing

The turnip crinkle virus-based vector TCV–GFPDCP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV–GFPDCP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 and p9, which were involved in the intercellular spread of virus-induced RNA silencing, were identified. TCV–GFPDCP and its p8- or p9-mutated derivatives, TCVmp8–GFPDCP and TCVmp9–GFPDCP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV–GFPDCP, TCVmp8–GFPDCP, or TCVmp9–GFPDCP was able to initiate RNA silencing that targeted and degraded recombinant viral RNAs in inoculated leaves of the GFP-expressing N. benthamiana line 16c. However, cell-to-cell spread of silencing to form silencing foci was triggered only by TCV–GFPDCP. Non-replicating TCVmp88–GFPDCP and TCVmp28mp88–GFPDCP with dysfunctional replicase genes, and single-stranded gfp RNA did not induce RNA silencing. Transient expression of the TCV p9 protein could effectively complement TCVmp9–GFPDCP to facilitate intercellular spread of silencing. These data suggest that the plant cellular trafficking machinery could hijack functional viral proteins to permit cell-to-cell movement of RNA silencing

Error correction and diversity analysis of population mixtures determined by NGS

The impetus for this work was the need to analyse nucleotide diversity in a viral mix taken from honeybees. The paper has two findings. First, a method for correction of next generation sequencing error in the distribution of nucleotides at a site is developed. Second, a package of methods for assessment of nucleotide diversity is assembled. The error correction method is statistically based and works at the level of the nucleotide distribution rather than the level of individual nucleotides. The method relies on an error model and a sample of known viral genotypes that is used for model calibration. A compendium of existing and new diversity analysis tools is also presented, allowing hypotheses about diversity and mean diversity to be tested and associated confidence intervals to be calculated. The methods are illustrated using honeybee viral samples. Software in both Excel and Matlab and a guide are available at http://www2.warwick.ac.uk/fac/sci/systemsbiology/research/software/,the Warwick University Systems Biology Centre software download site.Publisher PDFPeer reviewe

A strong immune response in young adult honeybees masks their increased susceptibility to infection compared to older bees

Honeybees, Apis mellifera, show age-related division of labor in which young adults perform maintenance ("housekeeping") tasks inside the colony before switching to outside foraging at approximately 23 days old. Disease resistance is an important feature of honeybee biology, but little is known about the interaction of pathogens and age-related division of labor. We tested a hypothesis that older forager bees and younger "house" bees differ in susceptibility to infection. We coupled an infection bioassay with a functional analysis of gene expression in individual bees using a whole genome microarray. Forager bees treated with the entomopathogenic fungus Metarhizium anisopliae s.l. survived for significantly longer than house bees. This was concomitant with substantial differences in gene expression including genes associated with immune function. In house bees, infection was associated with differential expression of 35 candidate immune genes contrasted with differential expression of only two candidate immune genes in forager bees. For control bees (i.e. not treated with M. anisopliae) the development from the house to the forager stage was associated with differential expression of 49 candidate immune genes, including up-regulation of the antimicrobial peptide gene abaecin, plus major components of the Toll pathway, serine proteases, and serpins. We infer that reduced pathogen susceptibility in forager bees was associated with age-related activation of specific immune system pathways. Our findings contrast with the view that the immunocompetence in social insects declines with the onset of foraging as a result of a trade-off in the allocation of resources for foraging. The up-regulation of immune-related genes in young adult bees in response to M. anisopliae infection was an indicator of disease susceptibility; this also challenges previous research in social insects, in which an elevated immune status has been used as a marker of increased disease resistance and fitness without considering the effects of age-related development

A virulent strain of Deformed Wing Virus (DWV) of Honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission

This work was supported by the Biotechnology and Biological Sciences Research Council, the Department for Environment, Food and Rural Affairs, the Natural Environment Research Council, the Scottish Government and the Wellcome Trust, under the Insect Pollinators Initiative (grant #BBI0008281, http://www.bbsrc.ac.uk/pollinators) and by University of Warwick HEIF5 Proof of Concept funding to DJE.The globally distributed ectoparasite Varroa destructor is a vector for viral pathogens of the Western honeybee (Apis mellifera), in particular the Iflavirus Deformed Wing Virus (DWV). In the absence of Varroa low levels DWV occur, generally causing asymptomatic infections. Conversely, Varroa-infested colonies show markedly elevated virus levels, increased overwintering colony losses, with impairment of pupal development and symptomatic workers. To determine whether changes in the virus population were due Varroa amplifying and introducing virulent virus strains and/or suppressing the host immune responses, we exposed Varroa-naive larvae to oral and Varroa-transmitted DWV. We monitored virus levels and diversity in developing pupae and associated Varroa, the resulting RNAi response and transcriptome changes in the host. Exposed pupae were stratified by Varroa association (presence/absence) and virus levels (low/high) into three groups. Varroa-free pupae all exhibited low levels of a highly diverse DWV population, with those exposed per os (group NV) exhibiting changes in the population composition. Varroa-associated pupae exhibited either low levels of a diverse DWV population (group VL) or high levels of a near-clonal virulent variant of DWV (group VH). These groups and unexposed controls (C) could be also discriminated by principal component analysis of the transcriptome changes observed, which included several genes involved in development and the immune response. All Varroa tested contained a diverse replicating DWV population implying the virulent variant present in group VH, and predominating in RNA-seq analysis of temporally and geographically separate Varroa-infested colonies, was selected upon transmission from Varroa, a conclusion supported by direct injection of pupae in vitro with mixed virus populations. Identification of a virulent variant of DWV, the role of Varroa in its transmission and the resulting host transcriptome changes furthers our understanding of this important viral pathogen of honeybees.Peer reviewe

\u3ci\u3eVarroa destructor\u3c/i\u3e Mites Vector and Transmit Pathogenic Honey Bee Viruses Acquired From an Artificial Diet

The ectoparasitic mite Varroa destructoris one of the most destructive pests of the honey bee (Apis mellifera) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) (i.e., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa-mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in ingested host tissues or the mites themselves. Here we describe a system that includes an artificial diet free of insect tissue-derived components for maintaining Varroa mites for in vitro experimentation. Using this system, together with the novel engineered cDNA clone-derived genetically tagged VDV-1 and wild-type DWV, we demonstrated for the first time that Varroa mites provided an artificial diet supplemented with engineered viruses for 36 hours could acquire and transmit sufficient numbers of virus particles to establish an infection in virus-naïve hosts. While the in vitro system described herein provides for only up to five days of mite survival, precluding study of the long-term impacts of viruses on mite health, the system allows for extensive insights into the dynamics of Varroa-mediated vectoring and transmission of honey bee viruses

Promiscuous Feeding Across Multiple Honey Bee Hosts Amplifies the Vectorial Capacity of \u3ci\u3eVarroa destructor\u3c/i\u3e

Varroa destructor is a cosmopolitan pest and leading cause of colony loss of the European honey bee. Historically described as a competent vector of honey bee viruses, this arthropod vector is the cause of a global pandemic of Deformed wing virus, now endemic in honeybee populations in all Varroa-infested regions. Our work shows that viral spread is driven by Varroa actively switching from one adult bee to another as they feed. Assays using fluorescent microspheres were used to indicate the movement of fluids in both directions between host and vector when Varroa feed. Therefore, Varroa could be in either an infectious or naïve state dependent upon the disease status of their host. We tested this and confirmed that the relative risk of a Varroa feeding depended on their previous host’s infectiousness. Varroa exhibit remarkable heterogeneity in their host-switching behavior, with some Varroa infrequently switching while others switch at least daily. As a result, relatively few of the most active Varroa parasitize the majority of bees. This multiple-feeding behavior has analogs in vectorial capacity models of other systems, where promiscuous feeding by individual vectors is a leading driver of vectorial capacity. We propose that the honeybee-Varroa relationship offers a unique opportunity to apply principles of vectorial capacity to a social organism, as virus transmission is both vectored and occurs through multiple host-to-host routes common to a crowded society

Dynamic evolution in the key honey bee pathogen deformed wing virus: Novel insights into virulence and competition using reverse genetics

The impacts of invertebrate RNA virus population dynamics on virulence and infection out- comes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previ- ous reports on the reduction of DWV diversity following the arrival of the parasitic mite Var- roa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus qua- sispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complemen- tation between genotypes was not required to replicate to high levels. Mixed clone–derived infections showed a lack of strong competitive exclusion, suggesting that the DWV geno- types were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversifi- cation. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative fre- quency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population

Semaglutide and cardiovascular outcomes in patients with obesity and prevalent heart failure: a prespecified analysis of the SELECT trial

Background: Semaglutide, a GLP-1 receptor agonist, reduces the risk of major adverse cardiovascular events (MACE) in people with overweight or obesity, but the effects of this drug on outcomes in patients with atherosclerotic cardiovascular disease and heart failure are unknown. We report a prespecified analysis of the effect of once-weekly subcutaneous semaglutide 2·4 mg on ischaemic and heart failure cardiovascular outcomes. We aimed to investigate if semaglutide was beneficial in patients with atherosclerotic cardiovascular disease with a history of heart failure compared with placebo; if there was a difference in outcome in patients designated as having heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction; and if the efficacy and safety of semaglutide in patients with heart failure was related to baseline characteristics or subtype of heart failure. Methods: The SELECT trial was a randomised, double-blind, multicentre, placebo-controlled, event-driven phase 3 trial in 41 countries. Adults aged 45 years and older, with a BMI of 27 kg/m2 or greater and established cardiovascular disease were eligible for the study. Patients were randomly assigned (1:1) with a block size of four using an interactive web response system in a double-blind manner to escalating doses of once-weekly subcutaneous semaglutide over 16 weeks to a target dose of 2·4 mg, or placebo. In a prespecified analysis, we examined the effect of semaglutide compared with placebo in patients with and without a history of heart failure at enrolment, subclassified as heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, or unclassified heart failure. Endpoints comprised MACE (a composite of non-fatal myocardial infarction, non-fatal stroke, and cardiovascular death); a composite heart failure outcome (cardiovascular death or hospitalisation or urgent hospital visit for heart failure); cardiovascular death; and all-cause death. The study is registered with ClinicalTrials.gov, NCT03574597. Findings: Between Oct 31, 2018, and March 31, 2021, 17 604 patients with a mean age of 61·6 years (SD 8·9) and a mean BMI of 33·4 kg/m2 (5·0) were randomly assigned to receive semaglutide (8803 [50·0%] patients) or placebo (8801 [50·0%] patients). 4286 (24·3%) of 17 604 patients had a history of investigator-defined heart failure at enrolment: 2273 (53·0%) of 4286 patients had heart failure with preserved ejection fraction, 1347 (31·4%) had heart failure with reduced ejection fraction, and 666 (15·5%) had unclassified heart failure. Baseline characteristics were similar between patients with and without heart failure. Patients with heart failure had a higher incidence of clinical events. Semaglutide improved all outcome measures in patients with heart failure at random assignment compared with those without heart failure (hazard ratio [HR] 0·72, 95% CI 0·60-0·87 for MACE; 0·79, 0·64-0·98 for the heart failure composite endpoint; 0·76, 0·59-0·97 for cardiovascular death; and 0·81, 0·66-1·00 for all-cause death; all pinteraction>0·19). Treatment with semaglutide resulted in improved outcomes in both the heart failure with reduced ejection fraction (HR 0·65, 95% CI 0·49-0·87 for MACE; 0·79, 0·58-1·08 for the composite heart failure endpoint) and heart failure with preserved ejection fraction groups (0·69, 0·51-0·91 for MACE; 0·75, 0·52-1·07 for the composite heart failure endpoint), although patients with heart failure with reduced ejection fraction had higher absolute event rates than those with heart failure with preserved ejection fraction. For MACE and the heart failure composite, there were no significant differences in benefits across baseline age, sex, BMI, New York Heart Association status, and diuretic use. Serious adverse events were less frequent with semaglutide versus placebo, regardless of heart failure subtype. Interpretation: In patients with atherosclerotic cardiovascular diease and overweight or obesity, treatment with semaglutide 2·4 mg reduced MACE and composite heart failure endpoints compared with placebo in those with and without clinical heart failure, regardless of heart failure subtype. Our findings could facilitate prescribing and result in improved clinical outcomes for this patient group. Funding: Novo Nordisk