Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

First Polycipivirus and Unmapped RNA Virus Diversity in the Yellow Crazy Ant, Anoplolepis gracilipes.

The yellow crazy ant, Anoplolepis gracilipes is a widespread invasive ant that poses significant threats to local biodiversity. Yet, compared to other global invasive ant species such as the red imported fire ant (Solenopsis invicta) or the Argentine ant (Linepithema humile), little is known about the diversity of RNA viruses in the yellow crazy ant. In the current study, we generated a transcriptomic database for A. gracilipes using a high throughput sequencing approach to identify new RNA viruses and characterize their genomes. Four virus species assigned to Dicistroviridae, two to Iflaviridae, one to Polycipiviridae, and two unclassified Riboviria viruses were identified. Detailed genomic characterization was carried out on the polycipivirus and revealed that this virus comprises 11,644 nucleotides with six open reading frames. Phylogenetic analysis and pairwise amino acid identity comparison classified this virus into the genus Sopolycivirus under Polycipiviridae, which is tentatively named "Anoplolepis gracilipes virus 3 (AgrV-3)". Evolutionary analysis showed that AgrV-3 possesses a high level of genetic diversity and elevated mutation rate, combined with the common presence of multiple viral strains within single worker individuals, suggesting AgrV-3 likely evolves following the quasispecies model. A subsequent field survey placed the viral pathogen "hotspot" of A. gracilipes in the Southeast Asian region, a pattern consistent with the region being recognized as part of the ant's native range. Lastly, infection of multiple virus species seems prevalent across field colonies and may have been linked to the ant's social organization.

Quantitative Assessment of Microbial Pathogens and Indicators of Wastewater Treatment Performance for Safe and Sustainable Water Reuse in India.

Currently, there is no data on the molecular quantification of microbial indicators of recycled water quality in India. In this study, multiple microbial pathogens and indicators of water quality were evaluated at three wastewater treatment plants located in two Indian cities (New Delhi and Jaipur) to determine the treatment performance and suitability of recycled water for safe and sustainable reuse applications. Real-time polymerase chain reaction (PCR) was used for the rapid evaluation of six human pathogens and six microbial indicators of fecal contamination. Among the microbial indicators, pepper mild mottle virus (PMMoV), F+RNA-GII bacteriophage, Bacteroides thetaiotamicron, and four human pathogens (Norovirus genogroups I & II, Giardia, and Campylobacter coli) were detected in all of the influent samples analyzed. This work suggests that the raw influents contain lower levels of noroviruses and adenoviruses and higher levels of Giardia compared to those reported from other geographic regions. Overall, the efficacy of the removal of microbial targets was over 93% in the final effluent samples, which is consistent with reports from across the world. PMMoV and Giardia were identified as the best microbial targets, from the microbial indicators spanning across bacteria, bacteriophages, DNA/RNA viruses, and protozoan parasites, by which to evaluate treatment performance and recycled water quality in Indian settings, as they were consistently present at high concentrations in untreated wastewater both within and across the sites. Also, they showed a strong correlation with other microbial agents in both the raw influent and in the final effluent. These findings provide valuable insights into the use of culture-independent molecular indicators that can be used to assess the microbial quality of recycled water in Indian settings. IMPORTANCE Wastewater treatment plants (WWTPs) have rapidly increased in India during the last decade. Nonetheless, there are only a few labs in India that can perform culture-based screening for microbial quality. In the last 2 years of the pandemic, India has witnessed a sharp increase in molecular biology labs. Therefore, it is evident that culture-independent real-time PCR will be increasingly used for the assessment of microbial indicators/pathogens in wastewater, especially in resource-limited settings. There is no data available on the molecular quantitation of microbial indicators from India. There is an urgent need to understand and evaluate the performance of widely used microbial indicators via molecular quantitation in Indian WWTPs. Our findings lay the groundwork for the molecular quantitation of microbial indicators in WWTPs in India. We have screened for 12 microbial targets (indicators and human pathogens) and have identified pepper mild mottle virus (PMMoV) and Giardia as the best molecular microbiological indicators in Indian settings.

Citizen science monitoring reveals links between honeybee health, pesticide exposure and seasonal availability of floral resources.

We use a national citizen science monitoring scheme to quantify how agricultural intensification affects honeybee diet breadth (number of plant species). To do this we used DNA metabarcoding to identify the plants present in 527 honey samples collected in 2019 across Great Britain. The species richness of forage plants was negatively correlated with arable cropping area, although this was only found early in the year when the abundance of flowering plants was more limited. Within intensively farmed areas, honeybee diets were dominated by Brassica crops (including oilseed rape). We demonstrate how the structure and complexity of honeybee foraging relationships with plants is negatively affected by the area of arable crops surrounding hives. Using information collected from the beekeepers on the incidence of an economically damaging bee disease (Deformed Wing Virus) we found that the occurrence of this disease increased where bees foraged in agricultural land where there was a high use of foliar insecticides. Understanding impacts of land use on resource availability is fundamental to assessing long-term viability of pollinator populations. These findings highlight the importance of supporting temporally timed resources as mitigation strategies to support wider pollinator population viability.

Silent threat in honey bee colonies: infection dynamics and molecular epidemiological assessment of black queen cell virus in Turkey.

Viruses can have devastating effects and cause epidemics in honey bee (Apis mellifera) colonies. Black queen cell virus (BQCV), which is one of the most common honey bee viruses, affects queen bee larvae and their pupae. This study provides information on the dynamics of BQCV infection in honey bees, using molecular diagnostics to investigate the effects of other pathogens and seasonal patterns that are considered relevant to the epidemiology of BQCV. The results showed a relatively high prevalence of the viruses studied. The prevalence of BQCV, acute bee paralysis virus, and deformed wing virus in worker bees was found to be 90%, 62%, and 84%, respectively. The prevalence of BQCV was 58% in larvae and pupae. Furthermore, the prevalence of Nosema ceranae was 46% in worker bees. Statistical analysis showed possible combined effects of BQCV and other examined viruses in terms of infection dynamics, while BQCV did not show seasonal variation. The BQCV isolates detected in this study were placed in a phylogenetic framework using sequence data from comprehensive sampling in previous studies. The analysis suggested that the Turkish strains of BQCV clustered together with Australian and European strains and consisted of homogeneous populations that had evolved from a common ancestor. This is the first report of BQCV infection dynamics in honey bees in Turkey.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

Sacbrood viruses cross-infection between Apis cerana and Apis mellifera: Rapid detection, viral dynamics, evolution and spillover risk assessment.

Recent outbreaks of sacbrood virus (SBV) have caused serious epizootic disease in Apis cerana populations across Asia including Taiwan. Earlier phylogenetic analyses showed that cross-infection of AcSBV and AmSBV in both A. cerana and A. mellifera seems common, raising a concern of cross-infection intensifying the risk of disease resurgence in A. cerana. In this study, we analyzed the dynamics of cross-infection in three different types of apiaries (A. mellifera-only, A. cerana-only and two species co-cultured apiaries) over one year in Taiwan. Using novel, genotype-specific primer sets, we showed that SBV infection status varies across apiaries: AmSBV-AM and AcSBV-AC were the major genotype in the A. mellifera-only and the A. cerana-only apiaries, respectively, while AmSBV-AC and AcSBV-AC were the dominant genotypes in the co-cultured apiaries. Interestingly, co-cultured apiaries were among the only apiary type that harbored all variants and dual infections (i.e., AC and AM genotype co-infection in a single sample), indicating the interactions between hosts may form a conduit for cross-infection. The cross-infection between the two honey bee species appears to occur in a regular cycle with temporal fluctuation of AmSBV-AC and AcSBV-AC prevalence synchronized to each other in the co-cultured apiaries. Artificial infection of AcSBV in A. mellifera workers showed the suppression of viral replication, suggesting the potential of A. mellifera serving as a AcSBV reservoir that may contribute to virus spillover. Furthermore, the survival rate of A. cerana larvae was significantly reduced after artificial infections of both SBVs, indicating fitness costs of cross-infection on A. cerana and thus a high risk of disease resurgence in co-cultured apiaries. Our field and laboratory data provide baseline information that facilitates understanding of the risk of SBV cross-infection, and highlights the urgent need of SBV monitoring in co-cultured apiaries.

Transcriptome-level assessment of the impact of deformed wing virus on honey bee larvae.

Deformed wing virus (DWV) prevalence is high in honey bee (Apis mellifera) populations. The virus infects honey bees through vertical and horizontal transmission, leading to behavioural changes, wing deformity, and early mortality. To better understand the impacts of viral infection in the larval stage of honey bees, artificially reared honey bee larvae were infected with DWV (1.55 × 1010 copies/per larva). No significant mortality occurred in infected honey bee larvae, while the survival rates decreased significantly at the pupal stage. Examination of DWV replication revealed that viral replication began at 2 days post inoculation (d.p.i.), increased dramatically to 4 d.p.i., and then continuously increased in the pupal stage. To better understand the impact of DWV on the larval stage, DWV-infected and control groups were subjected to transcriptomic analysis at 4 d.p.i. Two hundred fifty-five differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified. Of these DEGs, 168 genes were downregulated, and 87 genes were upregulated. Gene Ontology (GO) analysis showed that 141 DEGs (55.3%) were categorized into molecular functions, cellular components and biological processes. One hundred eleven genes (38 upregulated and 73 downregulated) were annotated by KO (KEGG Orthology) pathway mapping and involved metabolic pathways, biosynthesis of secondary metabolites and glycine, serine and threonine metabolism pathways. Validation of DEGs was performed, and the related gene expression levels showed a similar tendency to the DEG predictions at 4 d.p.i.; cell wall integrity and stress response component 1 (wsc1), cuticular protein and myo-inositol 2-dehydrogenase (iolG) were significantly upregulated, and small conductance calcium-activated potassium channel protein (SK) was significantly downregulated at 4 d.p.i. Related gene expression levels at different d.p.i. revealed that these DEGs were significantly regulated from the larval stage to the pupal stage, indicating the potential impacts of gene expression levels from the larval to the pupal stages. Taken together, DWV infection in the honey bee larval stage potentially influences the gene expression levels from larvae to pupae and reduces the survival rate of the pupal stage. This information emphasizes the consequences of DWV prevalence in honey bee larvae for apiculture.

X-ray inactivation of RNA viruses without loss of biological characteristics.

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log10 decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Characterization of a Novel Mitovirus of the Sand Fly Lutzomyia longipalpis Using Genomic and Virus-Host Interaction Signatures.

Hematophagous insects act as the major reservoirs of infectious agents due to their intimate contact with a large variety of vertebrate hosts. Lutzomyia longipalpis is the main vector of Leishmania chagasi in the New World, but its role as a host of viruses is poorly understood. In this work, Lu. longipalpis RNA libraries were subjected to progressive assembly using viral profile HMMs as seeds. A sequence phylogenetically related to fungal viruses of the genus Mitovirus was identified and this novel virus was named Lul-MV-1. The 2697-base genome presents a single gene coding for an RNA-directed RNA polymerase with an organellar genetic code. To determine the possible host of Lul-MV-1, we analyzed the molecular characteristics of the viral genome. Dinucleotide composition and codon usage showed profiles similar to mitochondrial DNA of invertebrate hosts. Also, the virus-derived small RNA profile was consistent with the activation of the siRNA pathway, with size distribution and 5' base enrichment analogous to those observed in viruses of sand flies, reinforcing Lu. longipalpis as a putative host. Finally, RT-PCR of different insect pools and sequences of public Lu. longipalpis RNA libraries confirmed the high prevalence of Lul-MV-1. This is the first report of a mitovirus infecting an insect host.

Utility of Proteomics in Emerging and Re-Emerging Infectious Diseases Caused by RNA Viruses.

Emerging and re-emerging infectious diseases due to RNA viruses cause major negative consequences for the quality of life, public health, and overall economic development. Most of the RNA viruses causing illnesses in humans are of zoonotic origin. Zoonotic viruses can directly be transferred from animals to humans through adaptation, followed by human-to-human transmission, such as in human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and, more recently, SARS coronavirus 2 (SARS-CoV-2), or they can be transferred through insects or vectors, as in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), Zika virus (ZIKV), and dengue virus (DENV). At the present, there are no vaccines or antiviral compounds against most of these viruses. Because proteins possess a vast array of functions in all known biological systems, proteomics-based strategies can provide important insights into the investigation of disease pathogenesis and the identification of promising antiviral drug targets during an epidemic or pandemic. Mass spectrometry technology has provided the capacity required for the precise identification and the sensitive and high-throughput analysis of proteins on a large scale and has contributed greatly to unravelling key protein-protein interactions, discovering signaling networks, and understanding disease mechanisms. In this Review, we present an account of quantitative proteomics and its application in some prominent recent examples of emerging and re-emerging RNA virus diseases like HIV-1, CCHFV, ZIKV, and DENV, with more detail with respect to coronaviruses (MERS-CoV and SARS-CoV) as well as the recent SARS-CoV-2 pandemic.

Hierarchical natural move Monte Carlo refines flexible RNA structures into cryo-EM densities.

Ribonucleic acids (RNAs) play essential roles in living cells. Many of them fold into defined three-dimensional (3D) structures to perform functions. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have enabled structure determinations of RNA to atomic resolutions. However, most RNA molecules are structurally flexible, limiting the resolution of their structures solved by cryo-EM. In modeling these molecules, several computational methods are limited by the requirement of massive computational resources and/or the low efficiency in exploring large-scale structural variations. Here we use hierarchical natural move Monte Carlo (HNMMC), which takes advantage of collective motions for groups of nucleic acid residues, to refine RNA structures into their cryo-EM maps, preserving atomic details in the models. After validating the method on a simulated density map of tRNA, we applied it to objectively obtain the model of the folding intermediate for the specificity domain of ribonuclease P from Bacillus subtilis and refine a flexible ribosomal RNA (rRNA) expansion segment from the Mycobacterium tuberculosis (Mtb) ribosome in different conformational states. Finally, we used HNMMC to model atomic details and flexibility for two distinct conformations of the complete genomic RNA (gRNA) inside MS2, a single-stranded RNA virus, revealing multiple pathways for its capsid assembly.

A model of the economic benefits of global hepatitis C elimination: an investment case.

Major gains in reducing the burden of hepatitis C are now possible because of the discovery of a cure. The prevention of premature deaths and increased workforce participation among people who are cured are likely to provide substantial indirect economic benefits. We developed an investment case for hepatitis C for the six WHO world regions, which, to our knowledge, is the first to consider both indirect and direct economic benefits in this context. Scaling up of testing and treatment to reach the 2030 WHO hepatitis C elimination targets was estimated to prevent 2·1 million (95% credible interval 1·3-3·2 million) hepatitis C-related deaths and 10 million (4-14 million) new hepatitis C virus infections globally between 2018 and 2030. This elimination strategy was estimated to cost US$41·5 billion (33·1-48·7 billion) in testing, treatment, and health care between 2018 and 2030 ($23·4 billion more than the status quo scenario of no testing or treatment scale up), with a global average of $885 (654-1189) per disability-adjusted life-year averted at 2030. Compared with the status quo scenario, the elimination scenario generated $46·1 billion (35·9-53·8 billion) in cumulative productivity gains by 2030. These indirect costs made elimination cost-saving by 2027, with a net economic benefit of $22·7 billion (17·1-27·9 billion) by 2030. This model shows that countries might be underestimating the true burden of hepatitis C and will benefit from investing in elimination.

Immune related genes as markers for monitoring health status of honey bee colonies.

BACKGROUND: Honey bee population decline threatens the beekeeping sector, agriculture and global biodiversity. Early detection of colony mortality may facilitate rapid interventions to contain and prevent mortality spread. Among others, deformed wing virus (DWV) is capable of inducing colony losses, especially when combined with Varroa destructor mite. Since the bee immune system plays a crucial role in ensuring that bees are able to face these pathogens, we explored whether expression of immune genes could serve as biomarkers of colony health. RESULTS: Herein, we describe a preliminary immunological marker composed of two immune genes (relish and defensin), which provide insight on honey bee antiviral defense mechanism. Of the tested genes, relish expression correlated with the presence of DWV-Varroa complex, while decreased defensin expression correlated with poor resistance to this complex. CONCLUSIONS: The monitoring of these genes may help us to better understand the complex physiology of honey bees's immune system and to develop new approaches for managing the health impacts of DWV infection and varroa infestation in the field.

Heterobasidion Partitivirus 13 Mediates Severe Growth Debilitation and Major Alterations in the Gene Expression of a Fungal Forest Pathogen.

The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of €800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

Assessment of a cricket, Acheta domesticus, bioassay for Chequa Iflavirus and bunya-like virus from redclaw crayfish Cherax quadricarinatus.

Chequa iflavirus and a bunya-like virus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks after a stress event. To complete River's postulates for viruses, virus-free animals are needed. Due to a lack of chequa iflavirus and bunya-like virus-free crayfish (testing shows>85% infection rate) coupled with the facts that iflavirus and bunyaviruses are found in insects and that crickets had been successful alternate hosts for crustacean viruses before, Acheta domesticus was trialled asa bioassay animal. There was no significant difference (P>0.05) in mortality rates between uninfected control crickets and infected crickets. Reverse transcriptase polymerase chain reaction for both viruses failed to find any trace of the RNA viruses in fed or inoculated crickets after 30days. The search for an alternative bioassay host will have to be widened.

A metagenomic assessment of viral contamination on fresh parsley plants irrigated with fecally tainted river water.

Microbial food-borne diseases are still frequently reported despite the implementation of microbial quality legislation to improve food safety. Among all the microbial agents, viruses are the most important causative agents of food-borne outbreaks. The development and application of a new generation of sequencing techniques to test for viral contaminants in fresh produce is an unexplored field that allows for the study of the viral populations that might be transmitted by the fecal-oral route through the consumption of contaminated food. To advance this promising field, parsley was planted and grown under controlled conditions and irrigated using contaminated river water. Viruses polluting the irrigation water and the parsley leaves were studied by using metagenomics. To address possible contamination due to sample manipulation, library preparation, and other sources, parsley plants irrigated with nutritive solution were used as a negative control. In parallel, viruses present in the river water used for plant irrigation were analyzed using the same methodology. It was possible to assign viral taxons from 2.4 to 74.88% of the total reads sequenced depending on the sample. Most of the viral reads detected in the river water were related to the plant viral families Tymoviridae (66.13%) and Virgaviridae (14.45%) and the phage viral families Myoviridae (5.70%), Siphoviridae (5.06%), and Microviridae (2.89%). Less than 1% of the viral reads were related to viral families that infect humans, including members of the Adenoviridae, Reoviridae, Picornaviridae and Astroviridae families. On the surface of the parsley plants, most of the viral reads that were detected were assigned to the Dicistroviridae family (41.52%). Sequences related to important viral pathogens, such as the hepatitis E virus, several picornaviruses from species A and B as well as human sapoviruses and GIV noroviruses were detected. The high diversity of viral sequences found in the parsley plants suggests that irrigation on fecally-tainted food may have a role in the transmission of a wide diversity of viral families. This finding reinforces the idea that the best way to avoid food-borne viral diseases is to introduce good field irrigation and production practices. New strains have been identified that are related to the Picornaviridae and distantly related to the Hepeviridae family. However, the detection of a viral genome alone does not necessarily indicate there is a risk of infection or disease development. Thus, further investigation is crucial for correlating the detection of viral metagenomes in samples with the risk of infection. There is also an urgent need to develop new methods to improve the sensitivity of current Next Generation Sequencing (NGS) techniques in the food safety area.

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses.

BACKGROUND: Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools. METHODS: In this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform. RESULTS: Twenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25-30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2-3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample. CONCLUSIONS: This work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies.

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ∼319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.

Role of RNA Branchedness in the Competition for Viral Capsid Proteins.

To optimize binding-and packaging-by their capsid proteins (CP), single-stranded (ss) RNA viral genomes often have local secondary/tertiary structures with high CP affinity, with these "packaging signals" serving as heterogeneous nucleation sites for the formation of capsids. Under typical in vitro self-assembly conditions, however, and in particular for the case of many ssRNA viruses whose CP have cationic N-termini, the adsorption of CP by RNA is nonspecific because the CP concentration exceeds the largest dissociation constant for CP-RNA binding. Consequently, the RNA is saturated by bound protein before lateral interactions between CP drive the homogeneous nucleation of capsids. But, before capsids are formed, the binding of protein remains reversible and introduction of another RNA species-with a different length and/or sequence-is found experimentally to result in significant redistribution of protein. Here we argue that, for a given RNA mass, the sequence with the highest affinity for protein is the one with the most compact secondary structure arising from self-complementarity; similarly, a long RNA steals protein from an equal mass of shorter ones. In both cases, it is the lateral attractions between bound proteins that determines the relative CP affinities of the RNA templates, even though the individual binding sites are identical. We demonstrate this with Monte Carlo simulations, generalizing the Rosenbluth method for excluded-volume polymers to include branching of the polymers and their reversible binding by protein.