The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Complete genome sequence of a novel cytorhabdovirus infecting elderberry (Sambucus nigra L.) in the Czech Republic.

The genus Cytorhabdovirus includes plant viruses with an unsegmented, single-stranded, negative-sense RNA genome that infect various plant hosts. In this work, we report the detection of a new cytorhabdovirus infecting elderberry (Sambucus nigra L.). Total RNA was purified from infected leaves and, after ribodepletion, sequenced using an Illumina system. The RNA genome of viral isolate B15 is 12,622 nucleotides (nt) long, and that of isolate B42 is 12,621 nt long. A nearly complete sequence (12,592 nt) was also obtained for a third isolate (B160). The RNA genomes of all three isolates showed an organisation typical of cytorhabdoviruses, harbouring all six of the expected genes (3´ N-P-P3-M-G-L 5´), separated by intergenic regions. These isolates were closely related to each other (99.5-99.6% nt sequence identity) and showed the highest overall similarity to trichosanthes associated rhabdovirus 1 (63.5% identity) and Wuhan insect virus 5 (58% identity), and similar results were obtained when comparing individual coding sequences or proteins. Phylogenetic analysis confirmed that this elderberry virus, for which we propose the name "sambucus virus 1" belongs to the genus Cytorhabdovirus and fulfils the criteria to represent a novel species.

Vitamin D levels and mortality with SARS-COV-2 infection: a retrospective two-centre cohort study.

BACKGROUND: The role of vitamin D in increased mortality with SARS-COV-2 virus, namely, COVID-19, remains uncertain. We analysed all the patients who were treated as COVID-19-positive with or without a positive swab and were tested for vitamin D levels. METHODS: This was a retrospective, study involving 1226 patients swabbed for SARS-CoV-2 between the 10 February 2020 and 1 May 2020 at two hospitals of East Sussex Healthcare NHS Trust. Patients who were swab-positive for COVID-19 or treated as COVID-19-positive on clinical grounds even though swab results were negative were included in this study. We analysed the association of vitamin D levels and mortality, assessing linear and non-linear associations. RESULTS: A total of 1226 patients had SARS-CoV-2 RNA swabs in this period with age range from 1 year to 101 years. A cohort of 433 of these patients had swabs and recent vitamin D levels anytime in the previous 3 months. Mortality rates were not found to be associated with vitamin D levels (OR=1.04, 95% CI 0.96 to 1.12). CONCLUSION: Our findings suggest similar mortality risk from COVID-19 irrespective of the levels of vitamin D. Larger prospective studies will be needed to confirm these findings.

Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies.

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Complete genome sequence of a novel bromovirus infecting elderberry (Sambucus nigra L.) in the Czech Republic.

The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.

Molecular Characterization of 'Candidatus Phytoplasma prunorum' in the Czech Republic and Susceptibility of Apricot Rootstocks to the Two Most Abundant Haplotypes.

'Candidatus Phytoplasma prunorum' is one of the most destructive pathogens of Prunus species, where susceptible species render unproductive several years after infection. In epidemiology, the molecular characterization of phytoplasmas is based on sequence analysis of variable nonribosomal genes. In this study aceF, pnp, imp and secY genes were used for characterization of the 'Ca. P. prunorum' genotypes present in the Czech Republic. In total, 56 plant and 33 vector (Cacopsylla pruni) samples positive to 'Ca. P. prunorum' collected in seven localities were used in the study. Based on sequence analysis, four aceF, two pnp, six imp, and three secY genotypes were identified in analyzed samples. The most abundant in both plant and insect samples were the A6, P2, I4, and S2 genotypes. Most of the Czech 'Ca. P. prunorum' haplotypes clustered together in the haplotype network analysis. Next, two isolates representing the two most abundant Czech haplotypes (A6-P2-I4-S2 and A5-P2-I4-S2) were used in the susceptibility test of three apricot rootstock types (St. Julien A, M-VA-1, GF-305). Susceptibility was analyzed by phytoplasma quantification using quantitative real-time PCR and evaluation of symptom manifestation. Based on the results, the influence of the rootstock type on the phytoplasma titer and symptom manifestation was greater than of the phytoplasma isolate, while the year of analysis had no influence on the results. The results also showed that the phytoplasma titer is increasing in plant tissues during the vegetation period.

Novel Betanucleorhabdoviruses Infecting Elderberry (Sambucus nigra L.): Genome Characterization and Genetic Variability.

The genus Betanucleorhabdovirus includes plant viruses with negative sense, non-segmented, single-stranded RNA genomes. Here, we characterized putative novel betanucleorhabdoviruses infecting a medically important plant, elderberry. Total RNA was purified from the leaves of several plants, ribodepleted and sequenced using the Illumina platform. Sequence data analysis led to the identification of thirteen contigs of approximately 13.5 kb, showing a genome structure (3'-N-P-P3-M-G-L-5') typical of plant rhabdoviruses. The detected isolates showed 69.4 to 98.9% pairwise nucleotide identity and had the highest identity among known viruses (64.7-65.9%) with tomato betanucleorhabdovirus 2. A detailed similarity analysis and a phylogenetic analysis allowed us to discriminate the elderberry isolates into five groups, each meeting the sequence-based ICTV demarcation criterion in the Betanucleorhabdovirus genus (lower than 75% identity for the complete genome). Hence, the detected viruses appear to represent five novel, closely related betanucleorhabdoviruses, tentatively named Sambucus betanucleorhabdovirus 1 to 5.

Detection of single nucleotide polymorphisms in virus genomes assembled from high-throughput sequencing data: large-scale performance testing of sequence analysis strategies.

Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.

Acute and chronic toxicity effects of silver nanoparticles (NPs) on Drosophila melanogaster.

The use of nanoscaled materials is rapidly increasing, however, their possible ecotoxicological effects are still not precisely known. This work constitutes the first complex study focused on in vivo evaluation of the acute and chronic toxic effects and toxic limits of silver nanoparticles (NPs) on the eukaryotic organism Drosophila melanogaster. For the purpose of this study, silver NPs were prepared in the form of solid dispersion using microencapsulation method, where mannitol was used as an encapsulation agent. This newly prepared solid dispersion with a high concentration of silver NPs was exploited to prepare the standard Drosophila culture medium at a silver concentration range from 10 mg·L(-1) to 100 mg·L(-1) of Ag in the case of the acute toxicity testing and at a concentration equal to 5 mg·L(-1) in the case of the chronic toxicity testing. The acute toxic effect of silver NPs on Drosophila melanogaster was observed for the silver concentration equal to 20 mg·L(-1). At this silver concentration, 50% of the tested flies were unable to leave the pupae, and they did not finish their developmental cycle. Chronic toxicity of silver NPs was assessed by a long-term exposure of overall eight filial generations of Drosophila melanogaster to silver NPs. The long-term exposure to silver NPs influenced the fertility of Drosophila during the first three filial generations, nevertheless the fecundity of flies in subsequent generations consequently increased up to the level of the flies from the control sample due to the adaptability of flies to the silver NPs exposure.

A Retrospective Observational Study: Is Absolute Lymphocyte Count a Prognostic Marker in COVID-19?

Aim Our study aimed to find a correlation between low absolute lymphocyte count and COVID-19-related mortality. Methods This study followed a retrospective observational cohort design to analyze the data of patients who presented with symptoms and signs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), at the Conquest Hospital and Eastbourne District General Hospital in East Sussex, United Kingdom, between February 10, 2020 and May 1, 2020, retrospectively. Survival and mortality for the first 30 days and comorbidities were analyzed for all patients who were tested for COVID-19 irrespective of swab results and had blood lymphocyte levels taken at the time of their visit to the ED and their data were analyzed for statistical significance. Results A total of 1226 patients had SARS-CoV-2 RNA identification swabs taken between February 10, 2020 and May 1, 2020. A cohort of 742 patients of these patients tested for COVID-19 also had blood lymphocyte levels measured. Overall, the lymphocyte count did not differ significantly between patients suspected to have COVID-19 infection with either positive or negative COVID-19 swab results. The lymphocyte count, however, was significantly lower in those who died from COVID-19 (p < 0.001) but when comorbidities were analyzed, we found an association between an increased number of comorbidities and a significantly decreased lymphocyte count. Conclusion Once adjusted for comorbidities, the lymphocyte count had no association with COVID-19 infection and mortality.

Molecular characterization of a novel Aureusvirus infecting elderberry (Sambucus nigra L.).

A novel virus infecting elderberry was identified by high-throughput Illumina sequencing of double strand RNAs isolated form elderberry leaves. The complete genome sequence obtained (4512 nucleotides in length) shows an organization typical for aureusviruses, with five open reading frames (ORFs) and the typical ORF1-RT expression by the readthrough of an amber stop codon. The analysis of the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) sequences showed the highest identity (respectively 75.7% and 55%) with the corresponding amino acid sequences of Pothos latent virus. These two values, below the species demarcation criteria for the genus, indicate that the detected virus is a new member of genus Aureusvirus, family Tombusviridae, with the proposed name Elderberry aureusvirus 1 (ElAV1). A survey confirmed the wide distribution of ElAV1 in elderberry in the Czech Republic. Phylogenetic analyses of RdRp and CP sequences showed distinct microevolution of geographically separated isolates, with a tendency for isolates coming from close localities or from the same region to cluster together but heterogeneity of viral populations down to a local scale was also observed. The symptomatology of the new virus is not fully clear, but many infected trees were either asymptomatic or showed mild chlorotic mosaics. More severe symptoms, potentially impacting yields of flowers or berries, were observed in plants with mixed infections of ElAV1 and other elderberry viruses. Further efforts are now needed to determine ElAV1 prevalence outside the Czech Republic and to unravel its epidemiology.

Proteomics offers insight to the mechanism behind Pisum sativum L. response to pea seed-borne mosaic virus (PSbMV).

Pea seed-borne mosaic virus (PSbMV) significantly reduces yields in a broad spectra of legumes. The eukaryotic translation initiation factor has been shown to confer resistance to this pathogen, thus implying that translation and proteome dynamics play a role in resistance. This study presents the results of a proteome-wide analysis of Pisum sativum L. response to PSbMV infection. LC-MS profiling of two contrasting pea cultivars, resistant (B99) and susceptible (Raman) to PSbMV infection, detected >2300 proteins, 116 of which responded to PSbMV ten and/or twenty days post-inoculation. These differentially abundant proteins are involved in number of processes that have previously been reported in the plant-pathogen response, including protein and amino acid metabolism, stress signaling, redox homeostasis, carbohydrate metabolism, and lipid metabolism. We complemented our proteome-wide analysis work with targeted analyses of free amino acids and selected small molecules, fatty acid profiling, and enzyme activity assays. Data from these additional experiments support our findings and validate the biological relevance of the observed proteome changes. We found surprising similarities in the resistant and susceptible cultivars, which implies that a seemingly unaffected plant, with no detectable levels of PSbMV, actively suppresses viral replication. BIOLOGICAL SIGNIFICANCE: Plant resistance to PSbMV is connected to translation initiation factors, yet the processes involved are still poorly understood at the proteome level. To the best of our knowledge, this is the first survey of the global proteomic response to PSbMV in plants. The combination of label-free LC-MS profiling and two contrasting cultivars (resistant and susceptible) provided highly sensitive snapshots of protein abundance in response to PSbMV infection. PSbMV is a member of the largest family of plant viruses and our results are in accordance with previously characterized potyvirus-responsive proteomes. Hence, the results of this study can further extend our knowledge about these pathogens. We also show that even though no viral replication is detected in the PSbMV-resistant cultivar B99, it is still significantly affected by PSbMV inoculation.

McRAPD unlike MALDI-TOF MS is a suitable candidate for routine discrimination of new Haemophilus influenzae strain acquisition in chronic obstructive pulmonary disease (COPD) and cystic fibrosis.

BACKGROUND AND AIMS: Haemophilus influenzae new strain acquisition has been demonstrated to increase the relative risk of acute exacerbation fourfold in contrast to colonisation or chronic infection by the same strain in chronic obstructive pulmonary disease (COPD). Unfortunately, molecular typing techniques are not suitable for routine use due to cost, labour-intensity and need for special expertise. We tested two techniques potentially useful for routine typing, namely the newly available MALDI-TOF MS and the modified McRAPD compared to MLST as the gold standard. METHODS: In 10 patients (10.8%) suffering from COPD or cystic fibrosis, H. influenzae isolates were recovered repeatedly at different timepoints from the same patient during the study period. This allowed for thirteen pairwise comparisons of typing results in isolates recovered consecutively from the same patient to test the ability of the techniques to uncover new strain acquisition. RESULTS: MLST detected 9 cases of new strain acquisition among the 13 pairwise comparisons. However, MALDI-TOF MS reported all 13 pairs as different and thus new. In contrast, McRAPD was able to differentiate all the new strain acquisitions from pre-existing ones, both by visual inspection of melting profiles and by Relative Significant Difference values. CONCLUSIONS: Unlike MALDI-TOF MS, McRAPD appears to be a suitable candidate for routine discrimination of new strain acquisitions because of its accuracy and, rapid, easy and economic performance.

Usefulness of PCR-HRMA in identification of non-fermentative Gram-negative rods recovered from patients suffering from cystic fibrosis or chronic obstructive pulmonary disease.

Adequate treatment of microbial infections requires rapid and accurate identification of the etiological agent. In routine diagnostics, identification of bacteria conventionally relies on phenotypic testing, which can be hindered by phenotypic variations. Therefore, genotyping techniques should perform faster and more accurately. Recently, the technique of high-resolution melting analysis (HRMA) of PCR amplicons promises to provide a convenient and economic tool of genotypic identification. In our study, we performed prospective routine testing of a PCR-HRMA system that was recently published in a proof-of-the-principle study. The system was evaluated by analysing 275 clinical isolates of bacteria acquired from 65 patients suffering from cystic fibrosis or chronic obstructive pulmonary disease. Our results show that its routine use may result in partial worsening of its discriminatory power; however, it still outmatched conventional phenotyping in the group of non-fermentative Gram-negative rods. Moreover, when supplemented by rapid, simple and economic oxidase test, it can be even simplified for more economic performance.

Geographical gradient of the eIF4E alleles conferring resistance to potyviruses in pea (Pisum) germplasm.

BACKGROUND: The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. METHODOLOGY/PRINCIPAL FINDINGS: Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4E(A-B-C-S) variants, whose distribution was geographically structured. The eIF4E(A) variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4E(B), was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4E(C) variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4E(S) variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4E(A-1-2-3-4-5-6-7), eIF4E(B-1), eIF4E(C-2)) conferred resistance to the P1 PSbMV pathotype. CONCLUSIONS/SIGNIFICANCE: This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4E(S1) allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the pea host.

Tuf and secY PCR amplification and genotyping of phytoplasmas.

Tuf and secY genotyping techniques have been developed to distinguish phytoplasma strains. Tuf polymerase chain reaction sequence analyses are available for phytoplasma taxonomic groups 16SrI, 16SrV, 16SrXII-A, and XII-B. In addition to their use to confirm the taxonomic status of phytoplasma strains, they allow the spread of phytoplasma strains in host plants and insect vectors to be traced. SecY is more variable than tuf and is therefore more discriminatory than tuf, but secY and tuf phylogenies show congruence.

Photosynthetic alterations of pea leaves infected systemically by pea enation mosaic virus: A coordinated decrease in efficiencies of CO(2) assimilation and photosystem II photochemistry.

We have investigated photosynthetic changes of fully expanded pea leaves infected systemically by pea enation mosaic virus (PEMV) that often attacks legumes particularly in northern temperate regions. A typical compatible virus-host interaction was monitored during 40 post-inoculation days (dpi). An initial PEMV-induced decrease in photosynthetic CO(2) assimilation was detected at 15 dpi, when the virus appeared in the measured leaves. This decrease was not induced by stomata closure and corresponded with a decrease in the efficiency of photosystem II photochemistry (Φ(PSII)). Despite of a slight impairment of oxygen evolution at this stage, PSII function was not primarily responsible for the decrease in Φ(PSII). Chlorophyll fluorescence imaging revealed that Φ(PSII) started to decrease from the leaf tip to the base. More pronounced symptoms of PEMV disease appeared at later stages, when a typical mosaic and enations appeared in the infected leaves and oxidative damage of cell membranes was detected. From 30 dpi, a degradation of photosynthetic pigments accelerated, stomata were closing and corresponding pronounced decline in CO(2) assimilation was observed. A concomitant photoprotective responses, i.e. an increase in non-photochemical quenching and accumulation of de-epoxidized xanthophylls, were also detected. Interestingly, alternative electron sinks in chloroplasts were not stimulated by PEMV infection, which is in contradiction to earlier reports dealing with virus-induced plant stresses. The presented results show that the PEMV-induced alterations in mature pea leaves accelerated leaf senescence during which a decrease in Φ(PSII) took place in coordinated manner with an inhibition of CO(2) assimilation.