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Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink (Neovison vison) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.

Multiple mutations in SARS-CoV-2 variants of concern (VOCs) may increase, transmission, disease severity, immune evasion and facilitate zoonotic or anthoprozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K and N501Y, occur in the SARS-CoV-2 spike glycoprotein in combinations that allow detection of the most important VOCs. Here we present two flexible RT-qPCR platforms for small-and large-scale screening to detect these mutations, and schemes for adapting the platforms for future mutations. The large-scale RT-qPCR platform, was validated by pair-wise matching of RT-qPCR results with WGS consensus genomes, showing high specificity and sensitivity. Detection of mutations using this platform served as an important interventive measure for the Danish public health system to delay the emergence of VOCs and to gain time for vaccine administration. Both platforms are valuable tools for WGS-lean laboratories, as well for complementing WGS to support rapid control of local transmission chains worldwide.

This study aimed to elucidate the chemical response of hairy vetch (Vicia villosa Roth) to interspecific competition by rye (Secale cereale L.). Hairy vetch plants were grown as target plants in a mixture with different densities of rye plants (0, 1, 2, and 4). Twelve flavonoids in the shoot, root, and root exudate of hairy vetch plants were quantified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Kaempferol 3-O-{β-D-rhamnosyl-(1->6)-[β-D-xylosyl)-(1->2)]-β-D-galactoside} (Kaempferol-Rha-Xyl-Gal) was the predominant flavonoid found in shoots, roots, and root exudate of hairy vetch plant. Co-cultivation of hairy vetch with one rye plant increased the concentration of kaempferol-Rha-Xyl-Gal in hairy vetch root exudates. However, the concentration was reduced when co-cultivated with 2 or 4 rye plants. This study revealed that low levels of competition increased root exudation of kaempferol-Rha-Xyl-Gal, while higher levels of competition negatively affected production and root exudation of kaempferol-Rha-Xyl-Gal by hairy vetch.

Multiple SARS-CoV-2 variants have evolved increasing the transmissibility of the virus and/or cause its escape from immune response. Whole genome sequencing remains the gold standard and is implemented into national surveillance programs worldwide to detect these novel variants of concern. However, this technique can have a high turnaround time and it might not be possible to immediately be scaled up when fast action is required to limit the spread of SARS-CoV-2 variants every time a new local outbreak occurs. Four key mutations H69/V70 deletion, L452R, E484K and N501Y with multiple reassurance in the SARS-CoV-2 spike (S) glycoprotein were identified to have a distinct signature enabling to detect SARS-CoV-2 variants of worldwide concern (Alpha [B.1.1.7], Alpha [B.1.17 + E484K], Beta [B.1.351], Gamma [P1], Delta [B.1.617,2]) as well as variants of interest/notes. We developed RT-qPCRs to detect these key mutations and increased the sensitivity of the PCRs by using modified probes targeting the 452R, 484K and 501Y mutations, with a single nucleotide exchange present, leading to an amino acid substitution. Large scale screening of the SARS-CoV2 variants of concern was included into the national surveillance program in Denmark, where the RT-qPCRs run as multiplexed (H69/V70 and N501Y mutation) - or as a single reactions detecting all four key mutations in parallel in a 384-well format. Our RT-qPCR systems offer a fast and flexible testing strategy to detect SARS-CoV-2 variants of concern and of interest/note, especially needed in countries where whole genome sequencing in large scale is too cost- and labour intensive.

Entomopathogenic fungi (EPF) can display a plant-associated lifestyle as endophytes. Seed application of EPF can affect insect herbivory above ground, but the mechanisms behind this are not documented. Here we applied three EPF isolates, Beauveria bassiana, Metarhizium brunneum and M. robertsii, as seed inoculation of wheat and bean, and evaluated the effects on population growth of aphids, Rhopalosiphum padi and Aphis fabae, respectively. In wheat and bean leaves, we quantified benzoxazinoids and flavonoids, respectively, in response to EPF inoculation and aphid infestation to elucidate the role of specific plant secondary metabolites (PSMs) in plant–fungus–herbivore interactions. Inoculations of wheat and bean with M. robertsii and B. bassiana reduced aphid populations compared with control treatments, whereas M. brunneum unexpectedly increased the populations of both aphids. Concentrations of the majority of PSMs were differentially altered in EPF-treated plants infested with aphids. Changes in aphid numbers were associated with PSMs regulation rather than EPF endophytic colonisation capacity. This study links the effects of EPF seed inoculations against aphids with unique PSM accumulation patterns in planta. The understanding of PSM regulation in tri-trophic interactions is important for the future development of EPF for pest management.

Plants have evolved advanced chemical defense mechanisms, including root exudation, which enable them to respond to changes occurring in their surroundings rapidly. Yet, it remains unresolved how root exudation affects belowground plant-plant interactions. The objective of this study was to elucidate the fate of benzoxazinoids (BXs) exuded from the roots of rye (Secale cereale L.) plants grown with hairy vetch (Vicia villosa). A rapid method that allows nondestructive and reproducible chemical profiling of the root exudates was developed. Targeted chemical analysis with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to investigate the changes in the composition and concentration of BXs in the rye plant, and its root exudate in response to cocultivation with hairy vetch. Furthermore, hairy vetch plants were screened for the possible uptake of BXs from the rhizosphere and their translocation to the shoot. Rye significantly increased the production and root exudation of BXs, in particular 2-β-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) and 2-β-d-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), in response to cocultivation with hairy vetch. DIBOA-glc and DIMBOA-glc were absorbed by the roots of the cocultivated hairy vetch plants and translocated to the shoots. These findings will strongly improve our understanding of the exudation of BXs from the rye plant and their role in interaction with other plant species.

Introduction: Benzoxazinoids (BXs) are plant phytochemicals that have both defensive properties in plants and therapeutic effects in humans. The presence of BXs has been largely studied in the Poaceae family (monocots). To study the presence or absence of BXs in dicotyledons and monocotyledons outside the Poaceae family, parts of 24 plant species at several growth stages were selected for analysis, some of which were already known to contain BXs.Objectives: To devise a step-wise mass spectrometry-based approach for confirming the presence of BXs in plant samples, and to use the method to explore the status of BXs in selected plant species.Experimental: Plant samples were extracted using accelerated solvent extraction and analyzed using triple-quadrupole liquid chromatography-mass spectrometry.Results: The use of different columns, double mass transitions, and ion ratios proved to be a robust tool for confirming the presence of BXs in different plant species. By this method, the presence of BXs was confirmed in three of the 24 species. Double-hexose forms of BXs, which have not been reported before in dicotyledons, were confirmed to be present in the dicotyledon plants Acanthus mollis and Lamium galeobdolon, and the presence of BXs in the seeds of Consolida orientalis is reported for the first time here. High concentrations of BXs were found in the aerial parts of Acanthus mollis and Lamium galeobdolon, at 20 and 32 μmol/g plant dry weight, respectively.Conclusions: The stepwise approach described in this work confirmed the presence of BXs in new samples.

Multiple SARS-CoV-2 variants have evolved that increase the transmissibility of the virus and/or cause its escape from immune response. Whole genome sequencing remains the gold standard and is implemented into the national surveillance programs worldwide to detect these novel variants of concern. However, this technique cannot immediately be scaled up when a fast action is needed to limit the spread of SARS-CoV-2 variants every time a new outbreak occurs, as this can be laborious and time-consuming. Two key mutations with multiple reassurance in the SARS-CoV-2 spike glycoprotein have been identified to have a distinct signature enabling to detect five SARS-CoV-2 variants (B1.351, B1.1.7, P1 and N439K/Y453F variants). Three of them are SARS-CoV-2 variants of major concern worldwide. We developed a specific multiplexed RT-qPCR to detect these key mutations, the H69/V70deletion and the N501Y mutation and increased the sensitivity of the PCR by using LNA probes targeting the N501Y mutation carrying a single amino acid exchange. Additionally, we can exclude target failure and secure SARS-CoV-2 detection by a primer/probe pair detecting the Wt sequence (SARS-CoV-2; Wuhan lineage) at aa position 501. Our multiplexed RT-qPCR assay offers an alternative strategy for SARS-CoV-2 variant surveillance detecting five SARS-CoV-2 variants, which can build up on to detect new emerging SARS-CoV-2 variants.

A sublineage of the Delta variant, AY.4.2, recently accounted for an increase proportion of Delta cases in United Kingdom (UK), Romania, Poland, and Denmark. Several factors may account for the increased spread of AY.4.2. Here, we evaluated the sensitivity of AY.4.2 to neutralisation by sera from Pfizer/BioNTech (BNT162b2) vaccinees. AY.4.2 was not more resistant to neutralisation relative to other circulating Delta lineages or sublineages and showed only a modest 2.3-fold reduction in neutralisation relative to the vaccine strain. In contrast, the more rare B.1.617.2+E484K variant showed a 4.2-fold reduction in neutralisation that warrants surveillance of strains with the acquired E484K mutation.

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene as well as the A22920T mutation, resulting in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2., Author summary Early on, in the course of SARS-CoV-2 infections among mink in Denmark, we identified a farm, with about 15,000 mink, that had virus-infected animals. At this time, we found that a very high proportion of the mink had been infected and made antibodies against the virus. In contrast to the three previously infected farms, the mink were allowed to recover (the mink had shown few signs of disease and only low mortality) and our testing demonstrated the absence of circulating virus. Continued screening, in the following weeks, supported the absence of infection in the mink but the maintenance of antibodies against the virus. However, less than 3 months after the initial infection, we again identified the presence of virus in some dead mink from this farm and in many live mink. The viruses responsible for this second wave of infection were slightly different from those found in the first wave but were closer to each other than to the SARS-CoV-2s found on other mink farms. The antibody levels in mink during this second wave of infection were much higher than observed after the initial infection. We concluded that the initial round of infection in mink was insufficient to confer protection against re-infection.

BackgroundAlthough it is well established that plant metabolomes mediate microbiome assembly, the question of how metabolome-microbiome interactions may prevent pathogen invasion remains to be answered. To address this question, we studied microbiome and metabolome profiles of two Arabidopsis thaliana accessions, Columbia-0 (Col-0) and Landsberg erecta (Ler-0) with differential resistance profiles to the fungal pathogen Fusarium oxysporum f.sp. mathioli (FOM). We used amplicon sequencing to characterize bacterial (16S) and fungal (ITS2) communities, and we used targeted metabolite analysis across 5 stages of FOM host progression. ResultsWe found that microbiome and metabolome profiles were markedly altered in FOM-inoculated and non-inoculated samples of resistant Col-0 and susceptible Ler-0. Co-occurrence network analysis revealed robust microbial networks in the resistant Col-0 compared to the susceptible Ler-0, during FOM infection. Specific metabolites and microbial OTUs (including indicator and hub OTUs) correlated in both non-inoculated and inoculated Col-0 and Ler-0. The glucosinolates 4-hydroxyglucobrassicin, neoglucobrassicin and indole-3 carbinol, but also phenolic compounds were active in structuring the A. thaliana-microbiome. ConclusionsOur results highlight the interactive effects of host resistance and its associated microbiota on Fusarium infection and progression. These findings shed significant insights into plant inter-omics dynamics during pathogen invasion and could possibly facilitate the exploitation of microbiomes for plant disease control.

Background and objectives: Integrated weed management in commercial wheat production is urgently needed due to increasing herbicide resistance and production costs. Benzoxazinoids (BXs), which include benzoxazinones and benzoxazolinones, are unique bioactive metabolites produced by certain members of the Poaceae including maize, wheat, rye and some dicots. BXs play important roles in plant defence and are causal agents of allelopathic interference. We investigated the role of genetics, environment and crop growth stage on BX abundance in the roots and rhizoplane of selected commercial wheat cultivars, and quantified their microbial transformation products (aminophenoxazinones) in roots and rhizosphere soils. Methods: Cultivar trials of competitive wheat (Triticum aestivum L.) genotypes were conducted in two moderate to low rainfall (449–572 mm) locations in southeastern Australia in 2015 and 2016. Replicated shoot, root, rhizoplane, and rhizosphere soil samples were collected for metabolic profiling at selected crop phenological stages, extracted and further analysed for known benzoxazinoid metabolites by liquid chromatography coupled with high resolution mass spectrometry. Results: Fifteen BXs and related microbially derived aminophenoxazinones were detected in wheat shoots, roots, rhizoplanes and rhizosphere soils in both years and locations. MBOA, HMBOA and HMBOA-Glc were the three most abundant BX metabolites in wheat tissues, with the heritage cultivar Federation producing the highest levels of MBOA. The phytotoxic aminophenoxazinones APO and AMPO were the most abundant BX microbial transformation products and were detected in wheat roots, rhizoplanes and rhizospheres. Abundance varied with cultivar, growth stage, location and year. Conclusions: Microbially-produced aminophenoxazinones generated from both heritage and modern wheat root exudates were detected and quantified in rhizosphere soils, with abundance dependent on cultivar, growth stage, and season. Concentrations of microbial metabolites APO, AMPO, and AAPO were higher in the rhizosphere of young wheat seedlings in contrast to that of mature plants suggesting that phenoxazinone production was upregulated early in the season. Our findings demonstrate that BX metabolites at all life stages of wheat potentially undergo rapid biotransformation to aminophenoxazinones under field conditions, resulting in ecologically relevant concentrations sufficient for weed suppression by certain wheat cultivars.

Background: Given the importance of neutralising antibodies in protection against SARS-CoV-2 infection, it is critical to assess neutralisation persistence long-term following recovery. This study investigated neutralisation titres against SARS-CoV-2 up to 6 months post-symptom onset in individuals with mild COVID-19. Methods: Plasma neutralisation titres in convalescent COVID-19 individuals were determined at baseline and 6 months post-symptom onset using a cell culture infectious SARS-CoV-2 assay. Total SARS-CoV-2 spike-specific IgG and IgA binding was measured using a lectin capture ELISA and compared between timepoints and correlated to neutralising titres. Findings: All 48 convalescent COVID-19 individuals were found to have detectable SARS-CoV-2 50% inhibitory dilution neutralisation titres (ID50) at baseline and 6 months post-symptom onset with mean ID50 of 1/943 and 1/411, respectively. SARS-CoV-2 neutralisation titres peaked within 1-2 months post-symptom onset. However, 50% of individuals showed comparable ID50 at baseline and 6 months post-symptom onset. Both SARS-CoV-2 spike-specific IgG and IgA levels correlated well with neutralising titres. IgG binding was found to be sustained up to 6 months post-symptom onset, whereas IgA levels declined. Interpretation: This study demonstrates durability of SARS-CoV-2 spike-specific IgG and neutralisation responses following recovery from mild COVID-19. Thus, all subjects included in this study might potentially have protective levels of neutralising antibodies 6 months post-symptom onset. This study also demonstrates a relationship between spike-specific IgA and neutralisation decline, with implications for long-term protection against SARS-CoV-2 infection. Funding: Novo Nordisk Foundation, Independent Research Fund Denmark and Danish Agency for Science and Higher Education.

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 (HvNEP-1) protease from barley (Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. The quantitative PCR analysis of trichothecene biosynthesis genes (TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. graminearum. The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium.

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene. The infected mink recovered and after free-testing of the mink, the animals remained seropositive. During follow-up studies, after a period of more than 2 months without virus detection, over 75% of tested animals scored positive again for SARS-CoV-2 RNA. Whole genome sequencing showed that the virus circulating during this re-infection was most closely related to the virus identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies after re-infection than at free-testing. Thus, following recovery from an initial infection, seropositive mink rapidly became susceptible to re-infection by SARS-CoV-2.Article Summary LineFollowing widespread infection with SARS-CoV-2 of mink on a farm, all tested animals had seroconverted and the farm was then tested free of infection; however, less than 3 months later, a further round of infection affected more than 75% of tested animals.

Phytochemicals or phenolic compounds are important natural bioactive molecules that plants accumulate in response to environmental conditions and may exert benef icial effects on health by protecting humans against many diseases. The aim of this work was to analyze the influence of biotic and abiotic stress on the accumulation of flavonoids and phenolic acids on leaves of two cultivars of Amaranthus hypochondriacus, which are differentiated by the colour of their leaves (red or green). Phenolic compounds were extracted using the accelerated solvent extraction (ASE) method and their identif ication was carried out by LC-MS analysis. Rutin was the main flavonoid in amaranth leaves; the highest concentrations were found in green leaves when plants were subjected to stress (9715 μg g-1). Phenolic acids were minor compounds; ferulic acid was only present in red leaves (0.5 μg g-1) and p-coumaric acid only in green leaves (0.7 μg g-1). Our results indicate that leaves from A. hypochondriacus, the main species that produce edible seeds, are a good source of phytochemical compounds and their accumulation could be driven by the growing conditions.

Vaccines such as Vaccinia or BCG have non-specific effects conferring protection against other diseases than their target infection, which are likely partly mediated through induction of innate immune memory (trained immunity). MVA85A, a recombinant strain of modified Vaccinia Ankara (MVA), has been suggested as an alternative vaccine against tuberculosis, but its capacity to induce positive or negative non-specific immune effects has not been studied. This study assesses whether Vaccinia and MVA are able to induce trained innate immunity in monocytes. Human primary monocytes were primed in an in vitro model with Vaccinia or MVA for 1 day, after which the stimulus was washed off and the cells were rechallenged with unrelated microbial ligands after 1 week. Heterologous cytokine responses were assessed and the capacity of MVA to induce epigenetic changes at the level of cytokine genes was investigated using chromatin immunoprecipitation and pharmacological inhibitors. Monocytes trained with Vaccinia showed significantly increased IL-6 and TNF-α production to stimulation with non-related stimuli, compared to non-trained monocytes. In contrast, monocytes primed with MVA showed significant decreased heterologous IL-6 and TNF-α responses, an effect which was abrogated by the addition of a histone methyltransferase inhibitor. No effects on H3K4me3 were observed after priming with MVA. It can be thus concluded that Vaccinia induces trained immunity in vitro, whereas MVA induces innate immune tolerance. This suggests the induction of trained immunity as an immunological mechanism involved in the non-specific effects of Vaccinia vaccination and points to a possible explanation for the lack of effect of MVA85A against tuberculosis.

In June–November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV-2-PCR-positive and approximately 27% (95% confidence interval (CI): 25–30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.

Glucosinolates are biologically active secondary metabolites in Brassicaceae plants that play a critical role in positive and negative interactions between plants and root-associated microbial communities. The aim of this study was to develop a reversed-phase liquid chromatography method to quantify and identify intact glucosinolates in the root of Arabidopsis thaliana (Arabidopsis) grown in non-sterile natural soil by using liquid chromatography-hybrid triple quadruple-linear ion trap (LC-QqQ(LIT)) mass spectrometry. The Synergi Fusion C18-based column was found to be effective for sufficient retention and separation of nine intact glucosinolates without the need for time-consuming desulfation or ion-pairing steps. Method validation results showed satisfactory inter-day and intra-day precision for all glucosinolates except for 4-hydroxyglucobrassicin. Good inter-day and intra-day accuracy and recovery results were observed for glucoiberin, gluconapin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin. However, for 4-hydroxyglucobrassicin, glucoraphanin and glucoerucin corrections to quantification results might be necessary since the recovery and accuracy results were not optimal. Matrix effects were shown to have a negligible effect on the ionization of all target analytes. The established liquid chromatography&ndash, tandem mass spectrometry (LC-MS/MS) method was applied to quantify target intact glucosinolates in Arabidopsis root crude extract of four different wild-type accessions where differences in terms of concentration and composition of intact glucosinolates were observed. Employment of sensitive and selective precursor ion survey scan of m/z 97 in combination with the information-dependent acquisition (IDA) of the enhanced product ion (EPI) dependent scan (Prec97-IDA-EPI) using LC-QqQ(LIT) provided high confidence in structural characterization of diverse intact glucosinolate profiles in complex Arabidopsis root crude extract.

Although the effects of plant secondary metabolites on plant defence have been studied for decades, the exact roles of secondary metabolites in shaping plant-associated microbial and nematode communities remain elusive. We evaluated the effects of benzoxazinoids, a group of secondary metabolites present in several cereals, on root-associated nematodes. We employed 18S rRNA metabarcoding to compare maize root-associated nematode communities in a bx1 knockout maize line impaired in benzoxazinoid synthesis and in its parental wild type. Both genotype and plant age affected the composition of the nematode community in the roots, and the effects of benzoxazinoids on nematode communities were stronger in the roots than in the rhizosphere. Differential abundance analysis and quantitative PCR showed that the root lesion nematode Pratylenchus neglectus was enriched in the bx1 mutant line, while another root lesion nematode, Pratylenchus crenatus, was reduced. Correlation analysis showed that benzoxazinoid concentrations in maize roots mostly correlated negatively with the relative abundance of nematode sequence reads. However, positive correlations between benzoxazinoids and nematode taxa, including several plant-parasitic nematodes, were also identified. Our detailed nematode community analysis suggests differential and selective effects of benzoxazinoids on soil nematodes depending on both the nematode species and the benzoxazinoid compound.

Honey bees are important pollinators and are subject to numerous stressors, such as changing floral resources, parasites, and agrochemical exposure. Pesticide exposure has been linked to the decline in the global honey bee population. We have limited knowledge of the metabolic pathways and synergistic effects of xenobiotics in bees. Quercetin is one of the most abundant phytochemicals in plants and is therefore abundant in the honey bee diet. Quercetin can upregulate the detoxification system in honey bees; however, it is still unknown to what extent quercetin ingestion can reduce the content of absorbed pesticides. In this study, we investigated the effect of dietary quercetin on the contents of three pesticides in honey bees: imidacloprid (insecticide), tebuconazole (fungicide), and tau-fluvalinate (insecticide and acaricide). Bees were divided into two main groups and fed either quercetin-sucrose paste or only sucrose for 72 h. Thereafter, they were orally exposed to ∼10 ng/bee imidacloprid or contact-exposed to ∼ 0.9 μg/bee tau-fluvalinate or ∼ 5.2 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the bees were anaesthetised with CO2, sacrificed by freezing, and extracted with a validated QuEChERS method. Subsequently, the concentrations of the three pesticides and quercetin in the bees were determined with a triple quadrupole tandem mass spectrometer coupled to an HPLC system. No significant effect on the concentration of tebuconazole or tau-fluvalinate was observed in bees fed quercetin. Intake of quercetin led to a reduction in the concentration of imidacloprid in honey bees. Quercetin-rich plants may be exploited in future beekeeping.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies. ispartof: EUROSURVEILLANCE vol:26 issue:27 ispartof: location:Sweden status: published

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6-30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4-31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.

Fusarium oxysporum is a destructive root-infecting plant pathogen that causes significant yield losses in many economically important crop species. Hence, a deeper understanding of pathogen infection strategies is needed. With liquid chromatography-tandem mass spectrometry and gas chromatography-time of flight mass spectrometry platforms, we analyzed the metabolic changes in a time-course experiment with Arabidopsis accessions either resistant (Col-0) or susceptible (Ler-0) to isolates of Fusarium oxysporum forma specialis matthioli infection. We showed a concurrent effect of Fusarium-derived polyols and the mycotoxin beauvericin in the suppression of the immune response of susceptible hosts. A significant increase in oxidized glutathione in the resistant host was probably associated with effective reactive oxygen species-mediated resistance responses. Through a combination of targeted and untargeted metabolomics, we demonstrated the concurrent action of several Arabidopsis defense systems as well as the concurrent action of several virulence systems in the fungal attack of susceptible Arabidopsis.

The losses of honey bee colonies and declines of other insect pollinators have been associated with negative effects of pesticides. Honey bees as well as other pollinators are nectar and pollen foragers and thus are exposed to an extensive range of phytochemicals. Understanding the synergistic, additive, and antagonistic effects of plant secondary metabolites and pesticides in honey bees may help to protect honey bee colonies against agrochemicals. In this study, we used untargeted metabolomics to investigate the impact of dietary phytochemical composition on the residual concentration of three pesticides: imidacloprid, tau-fluvalinate and tebuconazole in exposed honey bees. Honey bees were given different diets based on pollen or nectar from four plants: Reseda odorata, Borago officinalis, Phacelia tanacetifolia, and Trifolium repens for two days. Thereafter, they were orally exposed to 10 ng/bee imidacloprid or contact-exposed to 0.9 μg/bee tau-fluvalinate or 5 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the honey bees were anaesthetised with CO2, sacrificed by freezing, extracted with a validated QuEChERS method, and residual pesticide concentrations were determined by LC-QTRAP-MS/MS. The phytochemical composition in the given diets were profiled with an UHPLC-Q Exactive-MS/MS. The results revealed that the dietary phytochemical composition has a noteworthy influence on the concentration of residual pesticides in honey bees. The correlation coefficient analysis demonstrated that flavonoids have a reducing effect on the residual concentration of imidacloprid and tau-fluvalinate in honey bees. The results also highlighted that exposure to imidacloprid impaired the metabolism of sugars in honey bees. Exploiting flavonoid-rich plants may protect honey bees against pesticides and hold promise as forage plants in future beekeeping.

Background: The case presented here illustrates that interdisciplinary teamwork can be essential for the understanding of the COVID-19 disease presentation and enlightening of the pathophysiology. Case presentation: A 60-years-old overweight woman without any comorbidities was found dead in her apartment after 14 days of home isolation due to suspicion on the Coronavirus disease 2019 (COVID-19). She had reported symptoms of tachycardia, fever, and increasing respiratory difficulty one day before her death. Due to the Danish legal act on sudden deaths a forensic autopsy was performed including a thorough examination and biosampling. The results of the forensic autopsy displayed sever densified, almost airless, firm lungs, and an unspecific reactive minimal focal perivascular inflammation consisting of macrophages of the brain tissue. The final diagnosis, COVID-19 with involvement of the central nervous system was established by use of the RT-RNA analysis on cerebrospinal fluid, as well as by serologic detection of the specific antibodies for SARS-CoV-2 in cerebrospinal fluid and serum. The genetic analysis displayed a 2 % variation between SARS-CoV-2 isolates recovered from the tracheal sample, cerebrospinal fluid, and tissues from both lungs.Conclusion: The combination of all available results revealed that the cause of death was COVID-19 with severe pulmonary disease and neuroinvasion, as well as renal affection resulting in hyponatremia. To our knowledge, it was not shown previously that neuroinvasion could be confirmed by the detection of specific antibodies for SARS-CoV-2 and SARS-CoV-2 specific RNA in cerebrospinal fluid. This case supports hypotheses that SARS-CoV-2 may cause central nervous system infection. The genetic distinction between SARS-CoV-2 isolates was done by whole-genome sequencing, where the isolate recovered from the cerebrospinal fluid was the most different.

Due to urgency and demand, numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. Thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of SARS-CoV-2 infection, confirmation of molecular results, contact tracing, and epidemiological studies. This study evaluated the sensitivity and specificity of nine commercially available serological tests. These included three enzyme-linked immunosorbent assays (ELISAs) and six point-of-care (POC) lateral flow tests. The assays were validated using serum samples from: i) SARS-CoV-2 PCR-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China; iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or non-coronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and Epstein Barr virus. The results showed 100% specificity for the Wantai SARS-CoV-2 Total Antibody ELISA, 93% for the Euroimmun IgA ELISA, and 96% for the Euroimmun IgG ELISA with sensitivities of 90%, 90%, and 65%, respectively. The overall performance of the POC tests according to manufacturer were in the rank order of AutoBio Diagnostics > Dynamiker Biotechnology = CTK Biotech > Artron Laboratories > Acro Biotech ≥ Hangzhou Alltest Biotech. Overall, these findings will facilitate selection of serological assays for the detection SARS-CoV-2-specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies.

Plants may utilize bioactive chemical compounds which exude from their root to shape their associated beneficial microorganism in the rhizosphere to acquire resistance against pathogen infection [Fig. 1]. However, the underlying mechanism regulating this interaction has been poorly elucidated. The aim of the present study was to investigate the functional role of root exudate (from different host genotypes) in mediating belowground indirect plant defense. To elucidate the molecular mechanism underlying plant interaction with health-promoting microorganism, Arabidopsis thaliana (Col-0 and Ler-0 accessions) were grown for three weeks in field soil (non-sterile). Subsequently, the roots were exposed to Fusarium oxysporum matthioli (FOM). Sampling was done every five days after the infection for 25 days. The collected samples were subjected to both targeted (aliphatic and indole glucosinolates, plant hormones, and mycotoxins) and untargeted analysis by employing LC-MS/MS and GC-TOF/MS platforms as well as metabarcoding (Illumina MiSeq). Phenotypic characterization of the plants displayed that the Col-0 accessions had developed a resistance to FOM exposure whereas the Ler-0 ecotype was susceptible. The disease progression was also evaluated on a molecular level and resulted in the identification of beauvericin in both root and shoot tissues of the susceptible plants which likely was associated with promoting pathogenicity in FOM. The result from targeted and untargeted metabolomics as well as analysis of the root microbial community composition (bacteria and fungi) revealed the role of novel natural products in shaping the distinct member of the microbial community leading to diseases suppression. Plants may utilize bioactive chemical compounds which exude from their root to shape their associated beneficial microorganism in the rhizosphere to acquire resistance against pathogen infection [Fig. 1]. However, the underlying mechanism regulating this interaction has been poorly elucidated. The aim of the present study was to investigate the functional role of root exudate (from different host genotypes) in mediating belowground indirect plant defense. To elucidate the molecular mechanism underlying plant interaction with health-promoting microorganism, Arabidopsis thaliana (Col-0 and Ler-0 accessions) were grown for three weeks in field soil (non-sterile). Subsequently, the roots were exposed to Fusarium oxysporum matthioli (FOM). Sampling was done every five days after the infection for 25 days. The collected samples were subjected to both targeted (aliphatic and indole glucosinolates, plant hormones, and mycotoxins) and untargeted analysis by employing LC-MS/MS and GC-TOF/MS platforms as well as metabarcoding (Illumina MiSeq). Phenotypic characterization of the plants displayed that the Col-0 accessions had developed a resistance to FOM exposure whereas the Ler-0 ecotype was susceptible. The disease progression was also evaluated on a molecular level and resulted in the identification of beauvericin in both root and shoot tissues of the susceptible plants which likely was associated with promoting pathogenicity in FOM. The result from targeted and untargeted metabolomics as well as analysis of the root microbial community composition (bacteria and fungi) revealed the role of novel natural products in shaping the distinct member of the microbial community leading to diseases suppression.

Plants are consistently releasing root exudates into the rhizosphere which have the potential to influence the growth and development of neighboring plant species. Root exudates contain low and high molecular weight compounds such as sugars, amino acids, secondary metabolites, phytohormones, proteins, enzymes, and polysaccharides. Although aboveground plant interactions through volatile compounds has been extensively studied, only few studies investigated role of the belowground chemical interactions between plant species. The aim of this study is to elucidate the belowground chemical interactions between rye and hairy vetch grown together as cover crop mixture. Rye and hairy vetch were cultivated alone and together in pots filled with micro glasssbeads. Plant were grown for three weeks in climate chamber and their root exudates were collected for the chemical analysis. Targeted analysis with LC-MS/MS was done to identify the changes occurring in secondary metabolite profile of the rye and hairy vetch’s root exudates as result of co-cultivation. Quantification results displayed that both hairy vetch and rye are altering their root exudation in response to rye-hairy vetch co cultivation. Hairy vetch significantly increased concentration of root exuded flavonoids such as kaempferol and pratensein in response to presence of rye. Concentrations of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and BOA (1,3-benzoxazol-2-one) in rye’s root exudate were increased significantly as result of co cultivation with hairy vetch. The results from this study will increase our understanding about root exuded natural products with growth suppressive effects, which finally leads us to more sustainable plant protection system. Plants are consistently releasing root exudates into the rhizosphere which have the potential to influence the growth and development of neighboring plant species. Root exudates contain low and high molecular weight compounds such as sugars, amino acids, secondary metabolites, phytohormones, proteins, enzymes, and polysaccharides. Although aboveground plant interactions through volatile compounds has been extensively studied, only few studies investigated role of the belowground chemical interactions between plant species. The aim of this study is to elucidate the belowground chemical interactions between rye and hairy vetch grown together as cover crop mixture. Rye and hairy vetch were cultivated alone and together in pots filled with micro glasssbeads. Plant were grown for three weeks in climate chamber and their root exudates were collected for the chemical analysis. Targeted analysis with LC-MS/MS was done to identify the changes occurring in secondary metabolite profile of the rye and hairy vetch’s root exudates as result of co-cultivation. Quantification results displayed that both hairy vetch and rye are altering their root exudation in response to rye-hairy vetch co cultivation. Hairy vetch significantly increased concentration of root exuded flavonoids such as kaempferol and pratensein in response to presence of rye. Concentrations of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and BOA (1,3-benzoxazol-2-one) in rye’s root exudate were increased significantly as result of co cultivation with hairy vetch. The results from this study will increase our understanding about root exuded natural products with growth suppressive effects, which finally leads us to more sustainable plant protection system.

Background: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. Objectives: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. Methods: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500 μg and 800 μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. Results: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800 μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500 μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) − as well as crossreactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dosedependent manner. Conclusion: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination. info:eu-repo/semantics/publishedVersion

Background Sensitization to both inhalant and food allergens has been shown to be risk factors for development of asthma and rhinoconjunctivitis (RC). However, few studies have addressed the role of transient or persistent IgE sensitization to specific allergens in early life for later development of allergic diseases. The aim of this study was to explore the association between transient and persistent sensitization in early life and the development of asthma and RC at 6 and 14 years. Methods The Danish Allergy Research Center (DARC) cohort is a prospective non-interventional birth cohort study comprising 562 children. For the purpose of this study, we examined a subgroup of the original cohort with specific IgE measured at, at least 3 of 4 follow-ups between 3 and 18 months of age (n = 366). Multiple logistic regression models were used to investigate the association between transient and persistent early-life sensitization to groups of and to individual allergens and asthma and RC at 6 and 14 years compared to a reference group with no sensitization. Results Both transient early-life sensitization and persistent early-life sensitization to cow's milk or hen's egg proteins were associated with asthma (aOR 3.99[1.41-11.32] and 5.95[1.78-19.92]) and RC (aOR 2.94[1.19-7.28] and 6.18[1.86-20.53]) at 14 years, this association being driven mainly by sensitization to hen's egg. Transient early-life sensitization to house dust mite (HDM) had increased risk of asthma (aOR 3.80[1.17-12.41]) at 14 years. Conclusions Early transient IgE sensitization and persistent IgE sensitization to hen's egg were associated with asthma and RC at 14 years. Furthermore, sensitization to HDM was associated with asthma at 14 years.

Crop management practices such as tillage can influence the dissipation of herbicides in soil. This study aimed to determine the effects of tillage systems on soil dissipation of prosulfocarb (PSC) using two assessment methods: bioassay and high-performance liquid chromatography (HPLC) analysis. PSC was applied on plots cultivated under three tillage systems (moldboard plowing, tine tillage at 8- to 10-cm soil depth, and direct drilling) at different rates (0, 500, 1,000, 2,000, 4,000 and 8,000 g ai ha−1) and two spraying times, representing early and late sowing time of winter cereals in Denmark. The experiment was conducted over 2 yr. The soil was analyzed for PSC residues by HPLC and a bioassay, using silky windgrass as the indicator plant. Neither technique revealed an effect of tillage systems on PSC dissipation, but the LD50values estimated based on the bioassays were generally lower under direct drilling (11.7 d) than with plowing (17.5 d). Moreover, LD50estimates based on bioassay results were generally lower than those estimated with HPLC analyses. Half-life values estimated with HPLC were low and not within the range of values reported in the literature (ca. 20 d), suggesting enhanced degradation of PSC. In addition to influencing the performance of PSC against problematic weed species, an enhanced dissipation rate could also hamper the benefits of PSC in an antiresistance strategy.

Background Plants actively shape their associated microbial communities by synthesizing bio-active substances. Plant secondary metabolites are known for their signaling and plant defense functions, yet little is known about their overall effect on the plant microbiome. In this work, we studied the effects of benzoxazinoids (BXs), a group of secondary metabolites present in maize, on the host-associated microbial structure. Using BX knock-out mutants and their W22 parental lines, we employed 16S and ITS2 rRNA gene amplicon analysis to characterize the maize microbiome at early growth stages. Results Rhizo-box experiment showed that BXs affected microbial communities not only in roots and shoots, but also in the rhizosphere. Fungal richness in roots was more affected by BXs than root bacterial richness. Maize genotype (BX mutants and their parental lines) as well as plant age explained both fungal and bacterial community structure. Genotypic effect on microbial communities was stronger in roots than in rhizosphere. Diverse, but specific, microbial taxa were affected by BX in both roots and shoots, for instance, many plant pathogens were negatively correlated to BX content. In addition, a co-occurrence analysis of the root microbiome revealed that BXs affected specific groups of the microbiome. Conclusions This study provides insights into the role of BXs for microbial community assembly in the rhizosphere and in roots and shoots. Coupling the quantification of BX metabolites with bacterial and fungal communities, we were able to suggest a gatekeeper role of BX by showing its correlation with specific microbial taxa and thus providing insights into effects on specific fungal and bacterial taxa in maize roots and shoots. Root microbial co-occurrence networks revealed that BXs affect specific microbial clusters. Electronic supplementary material The online version of this article (10.1186/s40168-019-0677-7) contains supplementary material, which is available to authorized users.

This work was funded in part by the Spanish Government, Ministerio de Econom?a y Competitividad de España (MINECO), project: AGL2013-48923-C2-2-R, and by the collaborative infrastructure project funded by the European Comission (EC) under Horizon 2020, project Transvac2-730964-INFRAIA-2016-1. IRTA is supported by CERCA Programme/ Generalitat de Catalunya. M.S.O. is supported by MINECO (scholarship n BES-2014-068506). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections.

During summer 2019, three patients residing by Tisvilde Hegn, Denmark were hospitalised with tick-borne encephalitis (TBE) after tick bites. A new TBE virus (TBEV) micro-focus was identified in tick nymphs collected around a playground in Tisvilde Hegn forest. Estimated TBEV prevalence was 8%, higher than in endemic areas around Europe. Whole genome sequencing showed clustering to a TBEV strain from Norway. This is the second time TBEV is found in Ixodes ricinus outside Bornholm, Denmark.

BACKGROUND: While much is known on childhood atopic diseases, knowledge about persistence of atopic diseases from childhood to adulthood is limited. We therefore aimed to study the clinical course of atopic diseases and type I sensitization prospectively in an unselected cohort of adolescents followed into adulthood.METHODS: A cohort of unselected 8th-grade school children (mean age 14 years) established in 1995 and followed up in 2010 were evaluated with questionnaire, clinical examination, skin prick tests and measurements of specific IgE.RESULTS: The lifetime prevalence of atopic diseases was high and increased significantly from adolescence (31%) to adulthood (57%); particularly allergic rhinitis increased with an incidence rate of 17.5/1000 person-years. Childhood predictors for adult allergic rhinitis were atopic dermatitis, asthma and asymptomatic sensitization to pollen and house dust mite. Among those with asymptomatic sensitization in adolescence, 53%-78% developed allergic rhinitis in adulthood. Furthermore, type I sensitization increased significantly from adolescence to adulthood mostly due to increased sensitization to pollen. Type I sensitization was found mainly in those with allergic rhinitis. A high number of adults had oral allergy symptoms due to the high number of birch pollen allergic individuals.CONCLUSION: Persistence of atopic diseases in adulthood is common, and a high proportion of the adult population is sensitized giving a high prevalence of allergic rhinitis. Many with asymptomatic sensitization in adolescence will develop allergic rhinitis in adult life. The focus should be on prevention of atopic diseases and sensitization already in childhood.

Integrated and more sustainable weed management practices are in great demand all around the globe. The adoption of more weed-suppressive cereal cultivars could be part of an integrated weed management strategy. Recently, a study was published analysing the relative contribution of above-ground competition and allelopathy to weed suppression of Scandinavian winter cereals at the field level. The present study used the same approach with Canadian spring wheat and triticale cultivars, and confirmed the results of the previous study. The competitive traits such as leaf area index, crop height and early vigour, and the allelochemicals belonging to the chemical group of benzoxazinoids measured in the root zone were of equal importance to explain the variance of weed biomass at the field level. In addition, a dendogram showed large genetic variability for competitive and allelopathic traits in the Canadian spring cereals, providing the genetic basis for the initiation of breeding programmes for more weed-suppressive cultivars.

The ongoing SARS-CoV-2 pandemic has highlighted both the importance of One Health, i.e., the interactions and transmission of pathogens between animals and humans, and the potential power of gene-based vaccines, specifically nucleic acid vaccines. This review will highlight key aspects of the development of plasmid DNA Nucleic Acid (NA) vaccines, which have been licensed for several veterinary uses, and tested for a number of human diseases, and will explain how an understanding of their immunological and real-world attributes are important for their efficacy, and how they helped pave the way for mRNA vaccines. The review highlights how combining efforts for vaccine development for both animals and humans is crucial for advancing new technologies and for combatting emerging diseases.

Background Atopic diseases are among the most common chronic diseases in adolescents and it is uncertain whether the prevalence of atopic diseases has reached a plateau or is still increasing. The use of the ISAAC (International Study of Asthma and Allergy in Childhood) questionnaire has provided comparable prevalence rates from many countries; whereas studies including clinical examinations and strict diagnostic criteria are scarce. We aimed to investigate the prevalence of atopic diseases, the pattern of sensitization and comorbidities at 14 years in a prospective birth cohort. Methods The children were examined eight times from birth to 14 years. Visits included questionnaire-based interviews, clinical examination, skin prick test and specific IgE. Results Follow-up rate at 14-years was 66.2%. The 12-month prevalence of any atopic disease was high (40.3%) mostly due to a high prevalence of rhinoconjunctivitis (32.8%) whereas the prevalence of asthma was 12.9% and of atopic dermatitis 8.1%. In children with at least one atopic disease 60% were sensitized while only 16% of those without atopic diseases were sensitized. The frequency of sensitization depended on the phenotype. Among children with rhinoconjunctivitis only, rhinoconjunctivitis with concomitant astma or atopic dermatitis or both 62.5%, 81.5%, 70% and 100% respectively were sensitized, whereas it was 7.7% and 33.3% of children with only asthma or atopic dermatitis. Conclusion The prevalence of rhinoconjunctivitis was high in adolescence. Children with rhinoconjunctivitis with and without comorbidities were frequently sensitized. Children with asthma without concomitant allergic rhinoconjunctivitis were rarely sensitized. This article is protected by copyright. All rights reserved.

BACKGROUND: Pigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable.OBJECTIVES: To investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach.METHODS: The humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase.RESULTS: Needle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200μg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains.CONCLUSION: The results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.

The development of therapeutic and prophylactic HIV vaccines for African countries is urgently needed, but the question of what immunogens to use needs to be answered. One approach is to include HIV envelope immunogens derived from HIV-positive individuals from a geographically concentrated epidemic with more limited viral genetic diversity for a region-based vaccine. To address if there is a basis for a regional selected antibody vaccine, we have screened two regionally separate cohorts from Guinea-Bissau and Denmark for neutralizing antibody activity and antibody-dependent cellular cytotoxicity (ADCC) against local and nonlocal circulating HIV-1 strains. The neutralizing activity did not demonstrate higher potential against local circulating strains according to geography and subtype determination, but the plasma from Danish individuals demonstrated significantly higher inhibitory activity than that from Guinea-Bissau individuals against both local and nonlocal virus strains. Interestingly, an opposite pattern was observed with ADCC activity, where Guinea-Bissau individual plasma demonstrated higher activity than Danish plasma and was specifically against the local circulating subtype. Thus, on basis of samples from these two cohorts, no local-specific neutralizing activity was detected, but a local ADCC response was identified in the Guinea-Bissau samples, suggesting potential use of regional immunogens for an ADCC-inducing vaccine.

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.

Fusarium head blight (FHB) causes substantial losses of yield and quality in grains, both in the field and in post-harvest storage. To date, adequate natural genetic resistance is not available for the control of FHB. This study reports the cloning and overexpression of a barley (Hordeum vulgare L.) antifungal gene, nepenthesin 1 (HvNEP-1), in the endosperm of barley grains. Transgenic barley lines overexpressing HvNEP-1 substantially reduced FHB severity and disease progression after inoculation with Fusarium graminearum or Fusarium culmorum. The transgenic barley also showed reduced accumulation of the mycotoxin deoxynivalenol (DON) in grain, far below the minimum value allowable for food. Semi-field evaluation of four HvNEP-1 transgenic lines revealed substantial reduction of FHB severity and progression as compared with the control H. vulgare cultivar Golden promise (GP) plants. Our study demonstrated the utility of HvNEP-1 for the control of FHB in barley, and possibly other grains such as wheat and maize.

In 2008–2009 a tick-borne encephalitis virus (TBEV) micro-focus was detected in Northern Zealand, Denmark. No new cases of TBE with an epidemiological link to Northern Zealand has been reported since. Here we undertook to investigate Ixodes ricinus ticks from this endemic micro-focus in 2016 and 2017. In addition to TBEV, I. ricinus ticks may host other pathogens that include Borrelia spp., Babesia spp., Rickettsia spp. and Neoehrlichia mikurensis, together with various endosymbiont microorganisms. To detect multiple organisms we used a metagenomics PanVirus microarray and next-generation sequencing to examine the persistence and evolution of other emerging viruses, bacteria and parasites. Here we report the rise and fall of the Danish TBEV micro-focus in Northern Zealand. However, we identify for the first time in Danish I. ricinus ticks the presence of Uukuniemi virus in addition to a tick-borne phlebovirus and a range of bacteria.

Disease progression of human immunodeficiency virus type 1 (HIV-1) is delayed by HIV type 2 (HIV-2) in individuals with dual HIV-1/HIV-2 infection. The protective mechanisms, however, are still to be revealed. In the current study we examined type-specific and cross-reactive antibody-dependent cellular cytotoxicity (ADCC) in HIV-1 and HIV-2 monoinfection or dual infection. Of note, intertype cross-reactive antibodies that mediated HIV-1 envelope glycoprotein (Env)–targeted ADCC were frequently identified in HIV-2–infected individuals. Furthermore, the magnitude of HIV-1 cross-reactive ADCC activity during HIV-2 infections depended on the HIV-1 Env origin and was associated with the duration of infection. These results suggest that preexisting antibodies against HIV-2, which mediate intertype ADCC, might contribute to control of HIV-1 during dual infection., Antibody-dependent cellular cytotoxicity (ADCC) was analyzed in human immunodeficiency virus (HIV) 1 and 2 infections. Cross-reactive antibodies that mediated HIV-1 envelope glycoprotein–targeted ADCC were identified in HIV-2–infected individuals, suggesting that preexisting HIV-2 antibodies might help control HIV-1 during dual infection.

Mast cell activation disorders is a term proposed to cover diseases and conditions related to activation of mast cells and effects of mast cell mediators. In its broadest sense, the term encompasses a wide range of diseases from allergic asthma to rhinoconjunctivitis, urticaria, food allergy, anaphylaxis, mastocytosis, and other conditions where MC activation is contributing to the pathogenesis. This article focuses on clinical presentations, challenges, and controversies in pediatric mastocytosis and gives an overview of current knowledge and areas in need of further research.

Hairy roots induced by Agrobacterium rhizogenes are well established models to study the metabolism of xenobiotics in plants for phytoremediation purposes. However, the model requires special skills and resources for growing and is a time-consuming process. The roots induction process alters the genetic construct of a plant and is known to express genes that are normally absent from the non-transgenic plants. In this study, we propose and establish a non-transgenic maize root model to study xenobiotic metabolism in plants for phytoremediation purpose using azoxystrobin as a xenobiotic compound. Maize roots were grown aseptically in Murashige and Skoog medium for two weeks and were incubated in 100 μM azoxystrobin solution. Azoxystrobin was taken up by the roots to the highest concentration within 15 min of treatment and its phase I metabolites were also detected at the same time. Conjugated metabolites of azoxystrobin were detected and their identities were confirmed by enzymatic and mass spectrometric methods. Further, azoxystrobin metabolites identified in maize root culture were compared against azoxystrobin metabolites in azoxystrobin sprayed lettuce grown in green house. A very close similarity between metabolites identified in maize root culture and lettuce plant was obtained. The results from this study establish that non-transgenic maize roots can be used for xenobiotic metabolism studies instead of genetically transformed hairy roots due to the ease of growing and handling.

The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone, and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human peripheral blood mononuclear cells as effector cells and CEM.NKRCCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.

Weed suppressive potential of 33 winter wheat, 24 winter rye and 11 winter triticale cultivars recently introduced to the Scandinavian market was investigated. Competitive traits recorded were early vigor, leaf area index and crop height. Allelopathic potential was assessed by analyzing plant root and shoot material by LC-MS/MS for their content of 12 phytotoxic metabolites of the chemical group of benzoxazinoids (BX). Total BX content was highest in rye, followed by triticale and wheat. Benzoxazinoid composition varied between species with non-methoxy substituted BX dominating in rye and methoxy substituted BX dominating in wheat and triticale. Principal component analysis exhibited a clear relationship of allelopathic and competitive traits, together explaining 62% of the variance in the data set. This result underlines the need for further investigations of the relative contribution of allelopathy and competition to weed suppression in future studies.