As we age, the functioning of the human immune system declines. The results of this are increases in morbidity and mortality associated with infectious diseases, cancer, cardiovascular disease, and neurodegenerative disease in elderly individuals, as well as a weakened vaccination response. The aging of the immune system is thought to affect and be affected by the human virome, the collection of all viruses present in an individual. Persistent viral infections, such as those caused by certain herpesviruses, can be present in an individual for long periods of time without any overt pathology, yet are associated with disease in states of compromised immune function. To better understand the effects on human health of such persistent viral infections, we must first understand how the human virome changes with age. We have now analyzed the composition of the whole blood virome of 317 individuals, 21–70 years old, using a metatranscriptomic approach. Use of RNA sequencing data allows for the unbiased detection of RNA viruses and active DNA viruses.The data obtained showed that Epstein-Barr virus (EBV) was the most frequently expressed virus, with other detected viruses being herpes simplex virus 1, human cytomegalovirus, torque teno viruses, and papillomaviruses. Of the 317 studied blood samples, 68 (21%) had EBV expression, whereas the other detected viruses were only detected in at most 6 samples (2%). We therefore focused on EBV in our further analyses. Frequency of EBV detection, relative EBV RNA abundance and the genetic diversity of EBV was not significantly different between age groups (21–59 and 60–70 years old). No significant correlation was seen between EBV RNA abundance and age. Deconvolution analysis revealed a significant difference in proportions of activated dendritic cells, macrophages M1, and activated mast cells between EBV expression positive and negative individuals.As it is likely that the EBV RNA quantified in this work is derived from reactivation of the latent EBV virus, these data suggest that age does not affect the rate of reactivation nor the genetic landscape of EBV. These findings offer new insight on the genetic diversity of a persistent EBV infection in the long-term.The online version contains supplementary material available at 10.1186/s12979-022-00268-x. As we age, the functioning of the human immune system declines. Resulting from this are increases in morbidity and mortality associated with infectious diseases, cancer, cardiovascular disease, and neurodegenerative disease in elderly individuals, as well as a weakened vaccination response . These aDuring the last decades, substantial evidence has accumulated demonstrating that the human body is colonized by microbial communities , which have a clear impact on human health. The human virome, the collection of all viruses present in an individual, is not limited to disease states, as chronic but asymptomatic viral infections are thought to be common . The worMany of the viruses persistently residing in humans belong to the herpesvirus family, such as Epstein-Barr virus (EBV), cytomegalovirus (CMV) and herpes simplex 1 (HSV-1), and their impact on human health may be much greater than what is currently understood about infections that do not typically have severe pathology . For exaIt seems likely that the various defense mechanisms of the body, both adaptive and innate immune mechanisms, would have a role in the modulation of the composition of the microbiomes in the various locations of the body. This should be clear e.g. in the case of the blood virome, i.e. the “antigens” are in close contact with the cells of the immune system. There are several reports about the composition of the virome in different body compartments, though the results vary between studies , 14. In One aspect of the virome that remains woefully under-explored is the diversity of viral species and subspecies in human populations as well as within individual human hosts. Viral diversity is multifaceted, as it can be studied across hosts or within-hosts, it may change over time, and it exists on different levels such as species, strain and nucleotide level. The same individual human can be infected with multiple different strains of the same virus simultaneously , while sIn the present study we have used RNA sequencing (RNA-seq) data from the Genotype-Tissue Expression (GTEx) project that has been obtained from blood samples taken from individuals of various ages. To fully understand the behavior and impacts of a virus, one must know how an infection develops in individuals over time and how the virus behaves on a population level. Our aim was therefore to investigate age-associated differences in the human virome by identifying viruses, studying their relative viral RNA abundance as well as viral diversity. Use of RNA sequencing data allows us to study RNA viruses as well as active DNA viruses.RNA-seq data obtained from blood samples was analyzed to identify viral RNA. On average, sample data consisted of 51.5 million raw read pairs . After quality control, the samples had 26.6 million quality read pairs on average . As only unambiguous mapping was accepted, the mean alignment rate to human genome was 79.3% . After human read subtraction, 2.3% of non-human reads  aligned to non-viral microbiome genomes on average.A total of 12 different virus species were observed among the 317 samples. Of these, 87 samples contained at least one virus species. Among the observed virus species, the Epstein-Barr virus was the most prevalent, identified in 68 individuals. Other prevalent viruses were herpes simplex virus 1 (HSV-1) and cytomegalovirus (CMV), that were observed in 5 and 6 individuals, respectively. Rare occurrences  included human mastadenovirus C, variety of papillomaviruses, Torque Teno viruses and betacoronavirus (Table p = 0.33) (Table p = 0.16) .Potential linear age-associated differences in EBV RNA abundance were additionally investigated. No significant correlation was seen between EBV RNA abundance and donor age in EBV positive samples . The mean total EBV RNA abundance was 1.454 and 1.360 reads per million quality reads for male and female individuals, respectively. No significant difference in abundance was observed between the sexes . Furthermore, no age-associated significant differences were seen in abundance when each sex was tested separately, as non-parametric Mann-Whitney U-test resulted in a p-value of 0.26 for male sample donors and 0.41 for female sample donors. No significant correlation was seen between EBV RNA abundance and age for men  or for women , when tested separately.As differences in aging and in virus infection have been reported between the sexes, potential differences in samples from female and male sample donors were investigated Table . There wp-value (p ≤ 0.05) for 215 of the 317 samples. A significant p-value from CIBERSORTx indicates that the results of the deconvolution are significantly different from results that would have been obtained by random chance. Only these 215 samples with high deconvolution performance were utilized in downstream analyses. Of the 68 samples that had EBV expression, 43 had a significant CIBERSORTx p-value. The cell proportions seen in these 43 samples were compared to the 172 samples that did not show EBV expression and had significant deconvolution fitting accuracy. Of the 22 different immune cell types differentiated in the CIBERSORTx LM22 data, significant differences (p ≤ 0.05) in cell proportions between EBV expression positive and negative individuals were seen with the cell types: macrophages M1, activated dendritic cells, and activated mast cells  was the most frequently expressed virus in the studied samples. Of the 317 studied blood samples, 68 (21%) had EBV expression, whereas the other viruses were only detected in at most 6 samples (2%). Therefore, for most of the viruses detected in this study, with the exception of EBV, the frequency that they appear in the studied samples was too low to be able to make meaningful statistical comparisons between age groups. We therefore focused on EBV in our further analyses. Frequency of EBV detection, relative EBV RNA abundance and the genetic diversity of EBV was not significantly different between age groups (21–59 and 60–70 years old). Neither was a significant correlation seen between EBV RNA abundance and age of sample donor. This lack of significant difference between age groups and absence of significant correlation with age was true even when testing separately for male and female sample donors.The RNA-seq data used in this work measures frequency and magnitude of EBV reactivation rather than seroprevalence of EBV, as seroprevalence of EBV is likely to be very high in the individuals studied in this work (21–70 years old). EBV seroprevalence has been reported to be as high as 89% already in 18–19 year olds , meaningEBV is known to maintain specific gene expression in latency. Latency-encoded genes include several nuclear antigens (EBNA), membrane proteins , and non-coding RNAs (EBER) . FurtherIt has been shown that genomic diversity of EBV increases during acute EBV infection, which is then followed by convergence as the infection is resolved and latency is established . It has p = 0.043), activated dendritic cells (p = 0.004), and activated mast cells (p = 0.007). Macrophages M1 and activated mast cells were present in significantly greater proportions in EBV expression negative samples. Activated dendritic cells were present in significantly greater proportions in EBV expression positive samples. Both the strongest significance and the greatest relative difference in proportions between EBV expression positive and negative samples was seen with activated dendritic cells . No significant correlation was seen between EBV RNA abundances and age. No significant differences were seen between the sexes, nor were there age-associated differences when tested separately for male and female sample donors. As it is likely that this EBV is derived from reactivation of the latent virus, these data suggest that age does not significantly affect the rate of reactivation nor the genetic landscape of EBV.The polyA-enriched RNA-sequencing data studied in this work originates from non-diseased whole blood samples taken as part of the Genotype-Tissue Expression (GTEx) Project (dbGaP accession number phs000424.v8.p2). The GTEx project as a whole is an ongoing effort to build a comprehensive public resource to study tissue-specific gene expression and regulation. As part of the project, 17,382 samples have been collected from organ and tissue donors, originating from 54 types of tissue and from 948 individuals. Samples used in the project are collected from non-diseased tissue sites and are studied using primarily molecular assays, including WGS, WES, and RNA-Seq. The whole blood samples studied in this work originate from 317 persons. Each person contributed one sample and their age varied between 21 and 70 years. All donors were surgical patients or post-mortem donors . For whoVirosaurus is a curated virus genome database, aimed at facilitating clinical metagenomics analysis . The virhttps://github.com/FelixKrueger/TrimGalore; 10.5.2021). Other quality filtering was performed with following qualifiers of PRINSEQ (lite v0.20.4) [https://www.bioinformatics.babraham.ac.uk/projects/fastqc; 10.5.2021).A Bioinformatics pipeline modified from a study by Li et al.  was run v0.20.4) : read leQuality reads were subtracted sequentially by aligning them with STAR (v2.7.1a)  against If a virus reference sequence consisted of multiple genes in Virosaurus90 database, reads mapping to different genes were summed. After this, a virus reference sequence was considered detected in a sample if its total read count in the sample was ≥5 . Virus rhttps://www.r-project.org; 10.5.2021). Difference in means was tested with non-parametric Mann-Whitney U-test and difference in frequencies was tested with two-sided Pearson’s chi-squared test (IBM SPSS Statistics version 27). To support presence of viral diversity, GFF3 annotations for each GenBank reference genome of detected viruses were downloaded from NCBI Nucleotide database and compared to both Virosaurus90 database and aligned virus reads with the help of BEDTools (version 2.29.0) [Read count data was processed in RStudio  , custom  2.29.0) .p-value to evaluate deconvolution performance. The p-value is calculated by comparing the resulting cell type fractions with fractions that would have been obtained by random chance [Deconvolution analysis of different immune cell types was done utilizing the digital cytometry tool CIBERSORTx . CIBERSOm chance . CIBERSOm chance , as the p-value is obtained, which indicates the bias corrected percentage of dendrogram variants where the specific cluster was observed.Hierarchical clustering of the samples based on EBV viral RNA abundance was performed to determine whether any statistically significant clustering along age group lines could be seen. Spearman correlation was used as the distance metric, which is robust against outliers and non-Gaussian distributions, and can capture nonlinear relationships , 52. WarAdditional file 1. Supplementary tables of virus genes. The file contains information on what viral genes are present in the reference sequences and the read counts attributed to each viral gene.


G6PC c.247C>T and c.1039C>T). Six previously unreported variants were identified . The proportion of GSD IX was higher in our cohort compared to other studies. Brain imaging abnormalities were more frequent among patients with GSD I, early-symptom onset, longer hospitalization, and inadequate metabolic control. The frequency of mutations was similar to that observed among the North American and European populations. None of the mutations observed in PHKA2 have been described previously. Therefore, current study reports six GSD variants previously unknown, and neurological consequences of GSD I. The principal neurological impact of GSD appeared to be related to inadequate metabolic control, especially hypoglycemia.Glycogen storage diseases (GSD) encompass a group of rare inherited diseases due dysfunction of glycogen metabolism. Hypoglycemia is the most common primary manifestation of GSD, and disturbances in glucose metabolism can cause neurological damage. The aims of this study were to first investigate the metabolic, genetic, and neurological profiles of children with GSD, and to test the hypothesis whether GSD type I would have greater neurological impact than GSD type IX. A cross-sectional study was conducted with 12 children diagnosed with GSD . Genetic testing was conducted for the following genes using multigene panel analysis. The biochemical data and magnetic resonance imaging of the brain presented by the patients were evaluated. The criteria of adequate metabolic control were adopted based on the European Study on Glycogen Storage Disease type I consensus. Pathogenic mutations were identified using multigene panel analyses. The mutations and clinical chronology were related to the disease course and neuroimaging findings. Adequate metabolic control was achieved in 67% of patients . Fourteen different mutations were detected, and only two co-occurring mutations were observed across families ( Glucose is stored in the form of glycogen, primarily in the cytoplasm of liver and muscle cells, and to a lesser degree, in brain tissues , 2. HepaTwelve types of GSD have been identified, which are classified according to their associated enzyme deficiencies , 5. The via glycogenolysis and gluconeogenesis. This enzyme is anchored onto the ER and catalyzes the conversion of G6P to glucose and inorganic phosphate. Two main subtypes of GSD I are recognized: GSD type Ia  and GSD type Ib . GSD Ia results from a mutation in the G6PC gene (MIM *613742), which encodes the catalytic subunit of G6Pase α. GSD Ib results from a biallelic pathogenic variant in the SLC37A4 gene (MIM *602671), which codes for a G6P translocase. A defect in this translocase’s activity accounts for about 10% of the cases (GSD type I occurs due to a deficiency of the enzyme glucose-6-phosphatase α (G6Pase α), which impairs the ability to produce glucose  MIM *61342, whichhe cases .PHKA1, PHKA2, PHKB, and PHKG2. GSD IXa (MIM #306000) is an X-linked recessive disorder caused by a pathogenic mutation in the PHKA2 gene (MIM *300798); GSD IXb (MIM #261750) is caused by a compound heterozygous mutation in the PHKB gene (MIM *172490) (GSD type IX results from a deficiency of hepatic phosphorylase kinase (PhK), the enzyme responsible for the activation of glycogen phosphorylase and a key controller in the mobilization of glucose from glycogen. The PhK enzyme is comprised of four copies of each of the four subunits , encoded respectively by the genes MIM *30078; GSD IX*172490) . The mos*172490) .The dysregulation of glucose metabolism affects the entire central nervous system and can cause serious damage, because glucose is essential for normal neuronal function . Low braDisorders of glycogen metabolism are associated with liver and muscle disorders. Although the presence of glycogen in the brain has been recognized for decades, its functional roles in the brain have been discovered only recently , 7, inclHospital Infantil Pequeno Príncipe, Curitiba, PR, Brazil.This cross-sectional, observational, descriptive study enrolled children diagnosed with GSD who were being followed-up at the rare diseases outpatient clinic of the The study was conducted between January 2020 and January 2021. A convenience sampling strategy was used. All participants were assessed by the same researcher, who conducted targeted history taking and physical examination. The variables of interest included sex, current age, age at diagnosis, duration of hospitalization due to GSD (in days), and current laboratory serum levels. All patients had their diet with energy needs assessed individually and proportions of diet components introduced once the diagnosis was established.The inclusion criteria were as follows: patients aged 2–14 years old, with a clinical diagnosis of GSD, requiring ≥12 months of specialized care, who presented with the clinical manifestations of GSD, including hypoglycemia, hyperlactatemia, hypertriglyceridemia, hyperuricemia, hepatomegaly, and/or a growth deficit (short stature for age) at the time of diagnosis or inclusion in the study. Patients with associated neurodegenerative diseases were excluded.Serum levels were recorded from each patient’s most recent medical record. The biochemical blood parameters evaluated included blood glucose, triglycerides, high-density lipoprotein, low-density lipoprotein, cholesterol, uric acid, and lactate. Since, in their totality, patients showed adequate adherence to the diet, based on direct recall or use of food diaries, the evaluation of dietary control was carried out through metabolic criteria. The following criteria of adequate metabolic control were adopted based on the European Study on Glycogen Storage Disease type I (ESGSD I) consensus: glucose >63 mg/dL, triglycerides <530 mg/dL, uric acid <7 mg/dL, lactate <2.5 mg/dL, and body mass index within 2 standard deviations from the population mean .AGL, FBP1, G6PC, GAA, GBE1, GYS2, PHKA2, PHKB, PYGL, SLC2A2, and SLC37A4. Buccal swab samples were collected, and DNA was extracted for genetic analyses of the target genomic regions. Next-generation sequencing was performed using Illumina technology: alignment and variant identification was performed based on bioinformatics protocols using the GRCh38 human genome as a reference. The potential pathogenic variants and regions with inadequate sequencing depth were confirmed using automated Sanger sequencing, which was conducted with a genetic analyzer. The variants were described according to the nomenclature recommended by the Human Genomic Variation Society.Genetic testing was conducted for the following genes using multigene panel analysis: https://www.hgmd.cf.ac.uk/) and ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). Mutations were grouped according to type (missense or non‐missense). Mutations resulting in a frameshift or splicing modifications were considered to be potentially pathogenic. The pathogenicity of novel missense mutations was predicted using in-silico analyses.Novel variants were classified according to the guidelines of the American College of Medical Genetics and Genomics  on the bBrain MRI datasets obtained within the 5 years prior to the study were analyzed retrospectively. If the patient had more than one neuroimaging scan, the images were compared to assess the appearance of new lesions. MRI was requested for patients who had not undergone neuroimaging during the past 5 years. Brain MRI was performed with a 1.5-T magnetic resonance unit . T1-weighted , T2-weighted , fluid-attenuated inversion recovery , and diffusion-weighted  imaging was performed.p < 0.05.Data were stored and analyzed using Microsoft Excel 2016 and SPSS for Windows . Descriptive analyses were performed by calculating the summary measures. Inferential analyses were performed using the Chi-squared, Fisher’s exact (non-parametric variables), and Student’s t tests (parametric variables), with a significance level of G6PC (17q21.31), 2 in SLC37A4 (11.q23.3), 5 in PHKA2 (Xp22.13), and 2 in PHKB (16q12.1). The majority of the mutations were of the missense/nonsense type and located in the exon regions. All mutations, except for two, were observed in a single family; two G6P6 mutations were identified in two families each (c.247C>T and c.1039C>T). All five PHKA2 variants and one of the two PHKB variants have not been reported previously.p = 0.010), longer hospitalization (p = 0.002), and inadequate metabolic control, and were significantly associated with uricemia (p = 0.043), hyperlactatemia (p = 0.001), hypertriglyceridemia (p < 0.001), and elevated low-density lipoprotein levels (p = 0.031)  Table 4.PHKA2, which is located on the X chromosome  of the 5 patients with GSD IX in our sample had  pattern , 23. Mis pattern .Several factors are related to neuronal death induced by hypoglycemia, and not just related to energy failure. There is an increase in glutamate induced by hypoglycemia, with a reduction in astrocytic glutamate reuptake and increased activation of aspartate receptors . In respDespite the difficulty associated with identifying the exact topography of lesions caused by hypoglycemia, lesions in the frontal lobes, temporal lobes, and basal ganglia have often been described in patients with diabetes type I . PossiblThe characterizations of the natural history of rare diseases and their impact on patients and their families is always hampered by small sample sizes, which is among the noteworthy limitations of this study. Accordingly, the present characterizations were limited by our small sample population of 12 patients, which included only 7 patients with GSD I and 5 patients with GSD IX. Nevertheless, we were able to identify GSD-associated mutations even in this small cohort that have not been described in the literature previously, and one-third of the cases in our sample were diagnosed with GSD IXa, which is generally considered to be very rare subtype of GSD , suggestG6PC mutations was similar to that reported previously for North American and European Caucasian populations. Notably, we detected two PHKB2 mutations that have not been described previously, suggesting the probability of inadequate identification and underdiagnosis of GSD IXa in Brazil. The observations of MRI alterations in regions of high metabolism, such as the frontal lobes, temporal lobes, and basal ganglia, are consistent with the hypoglycemia-induced origin of the lesions. Thus, the main neurological impact of glycogenosis is apparently related to inadequate metabolic control, especially that of hypoglycemia.In conclusion, GSD Ia was the most prevalent form of GSD, affecting half of the patients in the present cross-sectional, observational, descriptive study. Notwithstanding, we observed a higher than typically reported prevalence of GSD IX, probably because GSD IXa may be oligosymptomatic with infrequent hospitalizations and few laboratory alterations, which would result in underdiagnosis. The frequency of The data presented in the study are deposited in the BioProject repository, accession number PRJNA725850 and the new mutations detected were uploaded to the ClinVar database.All procedures performed involving human participants were conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by this hospital’s Human Research Ethics Committee of Hospital Pequeno Principe (n. CAAE: 31888620.6.0000.0097). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article. Informed consent was also obtained for the publication of the images in JM conceptualized the research, gathered and analyzed the data, and wrote the initial manuscript. DV helped with the data analyses and draft of the initial manuscript. BT reviewed the neuroimaging scans. MC coordinated the study, and revised and conducted critical reviews of the manuscript for key intellectual content. MS helped with the data analyses. All authors contributed to the article and approved the submitted version.This study was funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior-Brazil (CAPES) conferred to JM and DV (Finance Code 001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Defective interfering particles arise spontaneously during a viral infection as mutants lacking essential parts of the viral genome. Their ability to replicate in the presence of the wild-type (WT) virus  is mimicked and exploited by therapeutic interfering particles. We propose a strategy for the design of therapeutic interfering RNAs (tiRNAs) against positive-sense single-stranded RNA viruses that assemble via packaging signal-mediated assembly. These tiRNAs contain both an optimised version of the virus assembly manual that is encoded by multiple dispersed RNA packaging signals and a replication signal for viral polymerase, but lack any protein coding information. We use an intracellular model for hepatitis C viral (HCV) infection that captures key aspects of the competition dynamics between tiRNAs and viral genomes for virally produced capsid protein and polymerase. We show that only a small increase in the assembly and replication efficiency of the tiRNAs compared with WT virus is required in order to achieve a treatment efficacy greater than 99%. This demonstrates that the proposed tiRNA design could be a promising treatment option for RNA viral infections. Many DVGs are well-known to have a replicative advantage over WT and to play a role in interference with WT virus4, virus persistence5 as well as specific6 and unspecific immune activation7. The exploitation of DIPs is a promising recent approach for therapy9. DIPs are selected and amplified for therapeutic use facilitated by advanced cloning techniques14, and have progressed to the clinic17.Viruses are a major burden for public health and economy, yet our repertoire of antiviral options is still very limited. This is, in part, due to the high frequency with which viral genomes mutate and thus evade treatment. On the other hand, these mutations can sometimes lead to the production of defective viral genomes (DVGs) which are shed in defective interfering particles (DIPs). DVGs are spontaneously occurring mutants in a viral infection that lack essential genetic information, e.g. through deletion mutations, but are capable of replicating in the presence of, and indeed at the expense of, resources produced by viruses18. Subsequently, in a prolonged persistence of vesicular stomatitis virus infections, mediated by WT DIPs, mutants -mediated assembly in single-stranded RNA (ssRNA) viruses for the design of therapeutic interfering RNAs (tiRNAs) which are packaged into therapeutic interfering particles (TIPs) mimicking essential features of these DIPs. The genomes of many RNA viruses, including bacteriophages, plant viruses, and human pathogens present multiple dispersed sequence/structure motifs (PSs) that share a core recognition motif for the viral coat protein (Cp). Sequence variation around that core motif confers differential levels of affinity for Cp to the PSs, creating a hierarchy of affinities across the genome-embedded PS ensemble. This enables PSs to recruit Cp to the growing capsid shell, collectively promoting virus assembly32 Fig. a.Figure 33. In some cases, such as in the Picornavirus human parechoviruses (HPeV), the PSs and their Cp binding sites have been characterised to atomistic detail32. They have also been identified in hepatitis B virus34, a DNA virus that packages its genome into its capsid in the form of pre-genomic RNA. Our detailed mechanistic understanding of PS-mediated assembly has enabled us to optimise the assembly code in satellite tobacco necrosis virus (STNV), creating RNA mutants that outcompete viral genomes in a competition assay35. Many of the best-studied examples of DVGs are found in (-)ssRNA viruses, which are presently known not to assemble via multiple dispersed PSs. For viruses assembling via multiple dispersed PSs, indeed DIPs that are assembly competent and competitive with WT may not spontaneously arise through the standard mechanisms5; however, using our new mechanistic understanding they could be engineered. Here we propose to decouple the assembly code from the genetic message, and create synthetic assembly substrates containing only the PS-encoded virus assembly instructions allowing them to be efficiently encapsulated by viral Cp. To mimic the naturally occurring DIPs, we also include a recognition signal for viral replicase into our de novo designed tiRNAs, so that they are replicated by viral replicase , we deliberately exclude such IRESs here in order to give tiRNAs another competitive advantage over vRNAs by not spending time on translation. An added advantage is that the presence of IRESs, and consequently interactions between ribosomes and IRES containing tiRNAs, would trigger an immune response that could lead to the removal of the cell, unnecessarily increasing tissue damage.Stochastic simulations of virus assembly reveal that the differences in affinity at distinct PS sites are important, because they bias the number of possible assembly pathways to a small number of dominant ones with very similar geometric properties, resulting in higher yields of fully assembled capsids compared with PS distributions lacking this variation. They also ensure specific encapsidation in the context of cellular competitor RNAs. This means that PSs enable the virus to overcome the equivalent of Levinthal’s Paradox in protein folding. The latter refers to the complexity of protein folding, which would require an unrealistically long time to complete if it solely relied on a random exploration of all combinatorially possible pathways. Similarly, the assembly of a viral capsid would be too slow in the arms race against host defence mechanisms if it were exploring all geometrically possible pathways, rather than the subset favoured due to interaction with the PSsase Fig. b. Althou36. The population dynamics of DIPs interacting with WT virus  can be complicated40 with chaotic or predator-prey dynamics, but often the parasitic relationship between DIPs and helper virus results in the attenuation or clearance of the original infection. Thus, we use HCV (an ssRNA virus) as a model system and we develop an extension of an intracellular model of HCV presented by Aunins et al.41 that now includes also the dynamic competition between tiRNAs and viral RNAs (vRNAs).Mathematical models of a viral infection can be used to assess the merits of novel antiviral strategieset al. presented a detailed, parameterised intracellular model for hepatitis C viral (HCV) infection based on experimental data41. The model consists of 6 steps Step 2; the cleavage of PPs at a rate Step 3; NS5BStep 4; the imported RNA forms a double-strand RNA (dsRNA) and releases Step 5; dsRNA binds to the Step 6; (+)41.Recently Aunins 35, enabling them to potentially assemble more efficiently than the virus 35, suggesting that In this paper, we extend this model to include the dynamics of tiRNAs Fig. c. Both trus Fig. a. We conrus Fig. b. As thementclass2pt{minimet al.41 used ordinary differential equations (ODE) to model the production of virions over a relatively short timescale (50 hours). In this work we derive a continuous-time Markov chain (CTMC) model from the ODE model45 and use the Gillespie algorithm as a discrete stochastic method for solving the CTMC model and plot stochastic trajectories46. The mean over the stochastic trajectories plotted using the Gillespie algorithm is in excellent agreement with the ODE dynamics . However, using the Gillespie method will also enable the tracking of individual particles, which is of particular interest at low concentrations during the initial kinetic phase34. As the average life span of adult hepatocytes ranges from 200 to 300 days47 and since the half-life of HCV infected cells is estimated to be between 1.4 and 700 days48, we ran our simulations for 100 days.Aunins 46 implemented in Fortran and using parameter values from Table 6. Figure Here we study the viral dynamics in the absence and presence of tiRNAs. The dynamics are computed as an average over 250 stochastic simulations of the reaction network using the Gillespie algorithmmentclass2pt{minimmentclass2pt{minimThe impact of the tiRNAs relies on two characteristic features: their relative replication  higher than the MOI of vRNA (V), the number of released virions decreases while increasing the ratio of TIPs/Virions Fig. , with TIin vivo. Figure 52. Recent experiments for influenza DIPs have established that prophylactic intranasal treatment can achieve delivery of around one DIP per susceptible cell, where they can stay present for several weeks6. Although evaluation of the full impact of TIPs as a treatment option during a chronic infection needs to be studied in the context of a within-host model, our model provides the foundation for studying such aspects by coupling the intracellular model presented here with an intercellular model in a multiscale approach54.The results presented above are based on the assumption that both the vRNAs and the tiRNAs begin the infection at the same time. However, this would not necessarily be the case acy Fig. . Howevercle Fig. . This su25 with the benefits of PS-mediated assembly. This novel design for TIPs opens up unprecedented therapeutic potential to interfere with viral replication and assembly, and misdirect viral resources. We have demonstrated the benefits of this combined strategy through stochastic simulations.In this paper we have exploited recent insights into PS-mediated assembly in order to propose a novel design for TIPs that combines the replicative advantages of existing DIP/TIP strategies over viruses55). Some viruses have been repurposed by nature for vital functions in the host organism. For instance, captured retroviruses and retrotransposons can be expressed in different tissues and at different points during life cycles, such as in neuronal functioning57 or placentation61. Any PS-targeting drugs interfering with virus assembly in related viruses would therefore have to be monitored carefully with regards to their effect on other vital functions. tiRNAs, on the other hand, would not pose that risk.The tiRNA designs proposed here are alternatives to other therapeutic strategies exploiting PS-mediated assembly, such as small molecular weight compounds targeting the PS:RNA contacts. PS motifs are shared by viruses from the same family  of the PSs, which would result in a significant intermittent fitness loss due to the multiple dispersed nature of the PSsde novo-engineer tiRNAs that not only are assembly-competent but are more assembly-efficient than WT, as well as having a replicative advantage, therefore constitutes a step-change for antiviral approaches for this group of viruses. As TIPs are indistinguishable from WT virus on the particle exterior, they can elicit an immune response (increasing antigen levels), thus priming the immune system against a subsequent infection. Multiscale within-host models of the immune response will be required to study the impact of this within infected patients, and analyse the impact of TIPs on both acute and chronic infections. Furthermore, models of between-host infection dynamics will reveal the consequences of any TIP transmission between hosts, and thus potentially reveal additional benefits to the host population arising from this. Such models would also enable evaluation of risks associated with the use of TIPs39.As we demonstrate here, tiRNAs show promise as an effective treatment at the intracellular level, especially as a prophylactic treatment. Our intracellular model shows that in this case the tiRNA only needs to be as effective at packaging and replicating as vRNA to dominate the population of particles released from the infected cell. DVGs arising naturally in viruses that assemble via multiple dispersed packaging signals may have only a subset of the PS sites and therefore not be optimal at assembly; the possibility to The first step is the production of polyprotein (PP) using the host cell ribosomes (Rib) from the (+)RNA in cytoplasm [(+)et al.41 and are presented in Table We use parameter values that have been reported in Aunins Supplementary Information.


Virus particle concentration is a critical piece of information for virology, viral vaccines and gene therapy research. We tested a novel nanoparticle counting device, “Videodrop”, for its efficacy in titering and characterization of virus particles. The Videodrop nanoparticle counter is based on interferometric light microscopy (ILM). The method allows the detection of particles under the diffraction limit capabilities of conventional light microscopy. We analyzed lenti-, adeno-, and baculovirus samples in different concentrations and compared the readings against traditional titering and characterization methods. The tested Videodrop particle counter is especially useful when measuring high-concentration purified virus preparations. Certain non-purified sample types or small viruses may be impossible to characterize or may require the use of standard curve or background subtraction methods, which increases the duration of the analysis. Together, our testing shows that Videodrop is a reasonable option for virus particle counting in situations where a moderate number of samples need to be analyzed quickly. Virus particle concentration is critical for most research involving viruses, including virus-based gene therapy vectors, vaccines or virus-like particles. Several methods already exist to assess the total (full and empty) virus particle concentration, including enzyme-linked immunosorbent assay (ELISA) ,2, high-https://www.myriadelab.com/, accessed on 19 May 2021) and is marketed as the Videodrop particle analyzer. Videodrop is able to measure >70 nm biological nanoparticles in solution from a single drop of a sample. h) by using the Stokes–Einstein equation. The attractiveness of this method lies in its simplicity: the measurement is label-free, filtration-free, non-destructive and fast.In 2016, Boccara et al.  developescatt) scattered by the NPs depending on the material  and the size of the particle. The device records short video blocs  of particle movement and the user can record as many blocs as needed. Recording of a single bloc takes approximately 15 s and in the accumulation mode the device keeps recording until the user stops the measurement or the maximum number of blocs defined in the settings has been recorded. In our case, 5–15 blocs were generally recorded depending on the particle concentration. Increasing the number of blocs allows more particles to be analyzed and thus increases the precision of the measurement. As with any optical microscope, shot noise can degrade the image quality. Videodrop adjusts the saturation automatically, but the user needs to follow the saturation level indicator and start the measurements only once 90–95% saturation has been reached. Usually, this step does not considerably delay the start of measurements.A Videodrop measurement consists of pipetting 5–10 µL of a sample on a glass coverslip, lifting the stage to the measurement position, operating the software and finally cleaning the device . The sample is illuminated by a single light-emitting diode (LED) light source. A camera connected to the optical system records the particle movement. The user can adjust the particle detection threshold (TH) and various other settings—for example, specifying the tracked particle track length, adjusting the sample diluent viscosity, or selecting to discard macro particles from analysis. Adjustment may be necessary due to different intensity-signal levels  or was followed for a longer period (tracked). Both detected and tracked particles contribute to the physical particles per milliliter (pp/mL) reading. The D8–7 × 109 pp/mL, or 5–100 pp/frame, according to manufacturer). In the visual threshold adjustment method, no prior information regarding the sample is needed. The threshold is tuned visually until all particles are detected and the result can be read from this single measurement . Generally, the target concentration method is suitable for defined assays, where high precision and reproducibility are needed, whereas the visual threshold method is more suitable for fast approximations of particle content, especially for comparing between samples without the need for absolute concentrations.The manufacturer recommends two alternative virus characterization techniques: the target concentration method or the visual threshold adjustment method. In the target concentration method, the threshold is pre-set beforehand during the assay development, the sample is serially diluted and the final result is calculated as an average from the results which fall on the linear part of the results curve , adenoviruses (Ad) and baculoviruses (BV), were used in our tests. Our primary interest was to test the precision and reproducibility of the Videodrop system for a LV particle assay. Therefore, we first describe setting up an assay for lentivirus vectors, discuss its comparability with traditional titering methods, and then present brief examples on how the Videodrop performs with Ad and BV samples.The used viruses were manufactured as described in previous publications ,24,25,26® 500 bioreactor as described by Leinonen et al. [® transfection reagent from Polyplus. Before starting the virus collection, 24 h post-transfection, a complete media change using serum-free media was performed. Bioreactor runs were ended 72 h post-transfection. Bioreactors were drained and combined with the harvested media. The bulk harvest was endonuclease-treated with 30 U/mL benzonase (Merck) for 2 h at 37 °C in the presence of 2 mM MgCl2. The harvested material was treated as described by Valkama et al. [® Pod CE50, 0.77 m2 depth filter (Millipore) and a peristaltic pump. The LV product was further processed using the Mobius FlexReady tangential flow filtration (TFF) system (Merck) with the Sartocon® Slice 100-kDa cassette (Sartorius). The virus was first concentrated 13-fold and then diafiltered into TFF buffer . No further processing was performed for the LV internal control and the material was aliquoted and frozen.The LV internal control (LV-GFP) was manufactured in 293T cells using the iCELLisn et al. . Shortlya et al. . BrieflyThe Ad internal control is a replication-defective recombinant adenovirus vector. It is produced in iCELLis 500 bioreactor and clarified after harvest. Its downstream processing consists of crossflow ultrafiltration, two consecutive anion exchange (AEX) chromatography steps and a final tangential flow filtration. In addition to the internal Ad control, a wild-type adenovirus )  was alsog for 20 min at room temperature to remove cell debris. BV was purified and concentrated in two steps—first by overnight centrifugation  through a 25% sucrose layer, and then by ultracentrifugation  through a sucrose gradient (20–50%). The virus band was collected and dialyzed (Pierce 10 K MWCO cassette) by PBS at 4 °C.The BV samples were produced in Spodoptera frugiperda 9 (Sf9) suspension culture . Sf9 cells were grown for 3 days before infection with the stock virus. Infected cultures were grown for 4 days, after which the cell culture medium was clarified by centrifugation at 5000× n = 41) LV samples used in For the various . For the LV serial dilution analysis and for testing against the traditional titering methods, the target concentration method was used: 3–5× PBS  dilution series was performed, and 7–8 µL sample was measured on the Videodrop device  to a final concentration of 0.1% ,32,33. TAd and BV analysis were performed using varying TH values, as shown in the figures. Some Ad results are shown as raw values (indicated the figure caption). Ad heat-treatment was performed on a +47 °C heat block, on which separate tubes were placed for different incubation durations.Ad ammonium sulfate aggregation was performed by mixing the Ad stock 1:8 with PBS containing 40% (NH₄)₂SO₄ . Samples were measured  after 5, 20 and 40 min.10 lysed cells/mL. The final sample therefore contained 5.48 × 1010 vg/mL of lysate from 5.5 × 109 cells/mL.For the buffer effect test, Ads were diluted 1:8 in the studied buffers. Buffers A and B are typical high- and low-salt HEPES-based buffers used during Ad downstream processing for a 300–600 mM NaCl gradient, according to Burova and Ioffe . Buffer Normal device cleaning between the measurements consisted of sample removal using a lint-free wipe. The sample-contact parts (objective glass and the lens protective window) were then cleaned using 70% ethanol.t-tests (parametric test) were used to determine potentially significant differences between two datasets. Pearson’s correlation coefficient was used to measure the correlation between Videodrop results and traditional methods. Confidence levels and confidence intervals were set to 95%. The ROUT method [After measurements, the majority of the results were saved both as a PDF report and as a CSV file, of which the former was a back-up copy and the latter was used for the analysis. Results were analyzed using GraphPad Prism 8.4.2. Unpaired T method  was used8 pp/mL (before blank subtraction) based on the PBS blank intermediate average and standard deviation ((SD) average + 3SD). It was also noted that the background signal from the used cell culture media  and 2.67 × 107 (CV% 115) pp/mL, respectively. It should be noted that some media measurement replicates fell below the PBS blank average, thus resulting in a zero pp/mL result.The preliminary work for LV particle assay set-up is described in 9 vg/mL) was analyzed for its particle titers. The aim was to set the working range for the LV assay. Two independent dilution series preparations and measurement rounds were performed on different weeks . Based on this test, dilutions leading to raw LV concentrations between 1.1 × 108 and 3 × 109 were confirmed suitable for the target concentration method. With the preliminary assay limits in place, a 2-fold LV dilution series made of internal LV-control .The repeatability of an LV concentration measurement was assessed from a full LV assay as well as from a single dilution. For the single dilution repeatability, the 8× dilution preparations and measurements were repeated by two operators on a single day b. Other 10 pp/mL , was higher than either the virus particle  or virus genome  titer. The internal LV control is a depth filter-clarified and TFF-processed virus. It has not been chromatographically purified and thus still contains cell-derived impurities. However, due to the nature of the LV and the relatively high number of non-virus material in LV preparations, a matching result with existing titers was not expected. Additionally, a drop in the mean internal control titer from 2.1 × 1010 vp/mL to 4.55 × 109 vp/mL was seen when the earlier p24 ELISA kit by another manufacturer was changed to the current one. The 29 weeks timepoint in Intermediate precision results were gathered over several months by repeated assays of the LV internal control by two operators c. The LVWith the main LV assay specifications  in place, various LV samples with different transgenes and pseudotypes were analyzed using the Videodrop system. The samples included upstream and downstream materials harvested from cell culture flasks as well as from adherent and suspension bioreactor runs. The full titer set  was available for 27 out of 41 samples. Videodrop results were plotted against the in-house titering results from p24 ELISA vp/mL particle titer d, functiTimewise, the Videodrop device offers superior time to results compared to traditional ELISA or PCR assays when a low or moderate number of samples (approx. 1–40 tubes) need to be measured. A single visual threshold measurement takes only a couple of minutes. On the other hand, analyzing a large number of samples becomes a slow process with a long hands-on time. As an example: a target concentration method session, during which 40 tubes  were carefully analyzed, took a total of 4 h. The time consumption was the following: 30 min for planning, set-up and sample retrieval; 30 min for tube marking, documentation and dilution series; 3 h for sample measurements and handling the data. Each dilution was measured in less than 3 min, with an additional 1.5 min for data saving and cleaning between the tubes.In addition to LV assay development, Videodrop was also tested using BV and Ad samples. A clarified BV stock was analyzed using a ddPCR virus genome assay for its virus genome content and the sample was later measured with Videodrop a. It was10 pp/mL . The lowest dilutions with well-correlating TH 3.2 would yield results very close (up to 4.12 × 1011 pp/mL) to the expected value (5.48 × 1011 vg/mL), but the raw particle concentration was under the LOD level and background subtraction led to zero pp/mL. In order to make sure that the difference is not due to the internal Ad control, data from another experiment  using the well-characterized ARM adenovirus reference material (ATCC® VR-1516™) [11 vp/mL, Videodrop 2.83 × 1010 pp/mL).Due to its smaller size, and resulting lower intensity values, setting a threshold for adenovirus detection required a compromise c. Based R-1516™)  was chech through the Stokes–Einstein equation. Ad samples were mixed with different buffers and measured  are available, the mean, CI and CV% are 1.56 × 109 pp/mL, 7.72 × 108–2.35 × 109 and 40.6, respectively. LOD for the Ad and BV samples (before background subtraction) with TH3.5 is 1.3 × 109. The particle median diameter from Videodrop for an Ad sample  agrees with the dynamic light scattering diameter values for the same virus .Ad intermediate precision was determined over several months by performing single-dilution measurements e. For THh was also seen, possibly due to aggregation of the broken virus debris. Here, the drop in the tracked particle value appeared as a useful measure of the virus breakage, as the observed particle value seems prone to background signals from broken viruses.We wanted to test if Videodrop could be used to detect virus breakage and aggregation. A heat-sensitive (pIX-deleted) adenovirus preparation was subjected to heat-treatment at +47 °C a. Based A similar virus inactivation experiment was performed using LV b. The weThe ability to analyze the extent of virus aggregation is an important factor in virus vector research and development. To test how Videodrop results react to Ad precipitation and aggregation, we used ammonium sulfate  to precin = 10,742, CI = 75.4–75.6, CV% = 7.71).As Ads are commonly harvested by lysing the cells, we used the Videodrop to study the diameter a and intAdditionally, experimentally purified lentiviruses (data combined from two differently processed samples) and growth media after 2–3 days on cells were studied for the diameter c and intIn this manuscript, the Videodrop nanoparticle analyzer was evaluated. To our knowledge, this is the first manuscript describing Videodrop’s performance in virus vector sample titering and characterization.Despite the Videodrop method being non-specific for viruses, the obtained results are generally in line or correlating with the traditional virus-specific titering methods. More specifically, we have shown herein that the Videodrop can be successfully used for the analysis of LV samples provided that the background signal level is carefully controlled. Due to the large concentration of extracellular vesicles released by mammalian cells, the background signal correction is especially important for low concentration upstream samples. The intermediate precision  for the LV particle concentration assay was 19.5% (CV%). The repeatability (intra-day precision) for the LV particle concentration assay was 14.0%. Repeatability was also tested by repeated sample preparations and measurements as a single dilution in a “worst case scenario”, with device adjustments, a cover glass change and other samples being measured between the test samples. The single dilution repeatability (35.7%) and intermediate precision (19.4%) values are therefore not comparable in this case, as the repeatability should usually give the smallest variation.A wide variety of LV sample types were successfully tested. Due to the nature of the LV, virus preparations always also contain non-viral particles and therefore fully corresponding results were not expected, nor did we see the need to finetune the TH value for LV at this stage. The correlation between Videodrop and the traditional p24 ELISA assay results was very good. Furthermore, this manuscript demonstrates Videodrop’s ability to measure BV and Ad concentrations, yet the pp/mL titers are underestimated and the threshold value needs careful adjustment. Videodrop can be used to measure >70 nm physical particles from biological samples. The minimum virus particle observable with Videodrop is thus slightly above that of the NTA, TRPS or flow virometry. TEM or super-resolution imaging-based systems reach considerably better resolutions but are not suitable for fast virus quantification. In principle, the virus size is a major determinant of the optimal TH value. Smaller viruses should generally require lower threshold settings, yet we also saw a need to reduce the threshold from its default setting when analyzing larger BV, with enveloped, rod-shaped capsids with a length of 230–385 nm and a diameter of 40–60 nm. The threshold adjustment raises an obvious concern. Lowering the threshold increases the number of detected and tracked particles (background), even from pure buffer samples. In theory, this will lead to lower threshold measurements appearing less pure due to the increased background signal in the intensity and diameter histograms, and therefore a background subtraction option for automatic concentration, intensity and density correction would be a useful feature. Virus diameter and intensity values can be used to further characterize the sample. The variation coefficients were lower for the diameter values than for the particle concentration, making the median diameter results quite stable. After measuring Ad mixed with cell lysate from a HEK293 cell pellet in PBS, it became evident that excluding the cell lysate signal based on the diameter or intensity value would not be a trivial task due to the overlap of the peaks and the variability of the results. The lysate, however, contained an excess of cells compared to a typical Ad lysate, and therefore the signal separation could be easier with the real samples. The shape and location of the peaks can be used to estimate the purity of the virus preparation. The sample buffer composition affects the diameter . Therefore, a minimum dilution into a standard buffer may be a practical solution.scatt, not Iscatt, as in non-interferometric techniques [6 ratio (fold difference) in their scattered light, the signal ratio in ILM is only √106 = 103. The camera used in ILM also differs from that used in dark field microscopy , giving it a high dynamic range and reduced saturation likelihood. However, our studies show that too concentrated samples lead to underestimated virus titers (due to screen effect or not), highlighting the importance of setting the working concentration limits. Low-concentration samples with impurities are also problematic to analyze, due to the high background signal level and because the majority of the measured particles may be non-viral . Analyzing only a specific population of particles, and thus excluding most of the non-viral material, based on the intensity or diameter values may be possible in some cases, but we did not see that as a viable option for our LV preparations. Because of the background noise from cell debris, the device has not been used to analyze Ad from crude cell lysates. According to our estimates, the cell debris-derived particles would require diluting the sample more and lead to highly variable results. The techniques utilizing virus-specific staining, such as flow virometry [Any microscopic object can screen nanoparticles behind it. Particles with significant size differences can create signals with too high differences in amplitude for the camera dynamics to resolve. Together, these two phenomena are the main factors behind the “screen effect”, which becomes significant when the concentration of microscopic objects and nanoparticles is high enough. The data obtained here are not sufficient to determine the level of screen effect for Videodrop measurements. In theory, ILM is less affected by the screen effect than various other techniques relying on NPs’ scattered light . In an interferometric observation method, the detected signal is proportional to √Ichniques . Therefoirometry  or NTA [8 pp/mL) based on detected particles, despite the fact that the number of tracked  particles may remain zero, whereas a low-concentration virus preparation may have practically the same pp/mL titer with tracked particles found. This begs the question as to whether the tracked particle vs. detected particle ratio should remain within a specified range in order to report the result as “approved”. The high background noise is a significant limitation for the device usage as certain sample types fall under the set LOD. According to our communication with the manufacturer, these issues have been recognized and are being remedied.The dual mode of detection (tracked and counted particles) leads to some obvious problems, especially regarding the very high background signal level in a situation where a low threshold  is being used. Pure PBS may give a fairly high titer  using Videodrop was still faster than a p24 assay, but anything more may have taken longer overall. When comparing the costs of the different systems, the price of the device, its maintenance and consumable costs should be considered. The Videodrop device has a reasonable price and price-per-sample is very low, as there are no stains or consumables that would need to be exchanged after each measurement and the diluent price is generally negligible.Various analytical techniques exist to quantify the total physical particle or virus concentration. See We have evaluated the Videodrop nanoparticle counter by means of developing an LV particle concentration assay. Furthermore, we have tested the system using Ad and BV. Following these tests, we conclude that ILM and the Videodrop device are suitable tools for analyzing the particle concentrations of several different types of lentivirus preparations, of clarified or purified BV preparations, and purified adenovirus preparations.


Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named “ambiviruses” with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.Species of  Armillaria (Fr.) Staude includes more than 40 described species1. They are mainly known as notorious plant pathogens of managed natural forests and plantations of non-native tree species that infect hundreds of different plants, including economically important conifers  and agronomic plants 2. Most Armillaria species are facultative necrotrophs capable of colonizing living roots, killing root tissues and utilizing dead wood as their source of nutrition. Some species produce edible fruiting bodies, generally known as honey mushrooms. Vegetative diploid individuals (clones) of A. ostoyae and A. gallica are amongst the largest and oldest organisms on earth, and single clones have been estimated to occupy up to 965 ha and be 1900–8650 years of age6.The fungal genus Armillaria lineages causing most damage to trees and woody plants are A. mellea and A. solidipes/ostoyae5, both of which have very broad host ranges7. The A. mellea lineage has a transcontinental distribution in Europe, North America, Asia and Middle East, and it also occurs as an alien species after being introduced by human activities beyond its natural distribution range. This fungus was introduced into South Africa by the Dutch settlers more than 300 years ago8 and has escaped from the planted environment to the sensitive and ecologically important natural woody habitat of the surrounding Table Mountain9. In central and northern Europe, A. ostoyae occurs sympatrically with A. borealis and A. cepistipes (syn. A. lutea). A. gallica is also native to the northern hemisphere , where it is considered as a weak pathogen12. The species also occurs in Kirstenbosch Botanical Gardens in South Africa, presumably being introduced in the country from Asia.In Europe, the two Armillaria diseases by root collar excavation or chemical pesticides. Stump removal has been shown as an effective method for controlling A. ostoyae in the boreal forest region13. However, none of the methods that are presently used can fully eradicate an established Armillaria mycelium from a contaminated site.Agricultural crops may be protected against 17. They are also commonly found in edible mushrooms like the cultivated button mushroom (Agaricus bisporus) and the shiitake (Lentinula edodes) as well as true truffles (Tuber spp.)20. On the other hand, root rot pathogens such as Heterobasidion spp. infecting conifers and the white root rot fungus Rosellinia necatrix infecting fruit trees have been extensively investigated for the occurrence of mycoviruses23 in attempts to find viruses suitable for biological control. However, currently the only mycoviral biocontrol agent used in field conditions is the hypovirus CHV1 that significantly reduces the pathogenicity of the ascomycetous Chestnut blight fungus (Cryphonectria parasitica) in Europe25.Fungal viruses (mycoviruses) infect hosts representing diverse fungal taxa and various lifestyles including ascomycetous and basidiomycetous micro- and macrofungi, as well as early-diverging fungal lineages such as Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Zoopagomycota and MucoromycotaBunyavirales, Serpentovirales, Hepelivirales, Martellivirales, Tolivirales and Tymovirales, many of which have traditionally known to include only viruses that infect animals or plants . Furthermore, metagenomic studies have revealed the common occurrence of newly discovered evolutionary lineages of mycoviruses, such as the viral family Mymonaviridae in order Mononegavirales26, the viral family Botourmiaviridae in order Ourlivirales27, and the unclassified virus group tentatively named “ambiviruses”29.Our understanding of the virosphere has recently been revolutionized by high-throughput sequencing (HTS) studies, which have revealed many novel viruses representing yet unassigned virus groups related to but phylogenetically distinct from classified members of the orders Armillaria spp. have not been molecularly characterized prior to this study. Early studies by Blattný30 and Reaves et al.31 reported the occurrence of virus-like particles in A. mellea and A. ostoyae, but these studies were not followed by viral genome sequence characterization. In this study, we used cellulose chromatography and HTS (RNA-Seq) complemented with Sanger sequencing to investigate the occurrence of virus infections in isolates of A. mellea and A. gallica from South Africa, A. borealis from Finland and Siberia and A. cepistipes from Finland. In addition, we examined transcriptomic datasets of 12 Armillaria species available in GenBank for the presence of viruses using the same bioinformatics pipeline.Despite their importance as forest and agricultural pathogens, viruses of Armillaria strains for dsRNA virus screening . For this reason, we employed an RNA-Seq approach as an alternative virus detection strategy as it enables the detection all viruses with RNA genomes as well as transcribed DNA viruses. Nine Armillaria strains from three different geographical regions  and three host species  were included in the analysis. The RNA-Seq library of ~ 130 million paired-end reads can be accessed through the BioProject accession number PRJNA685612 in the SRA archive of NCBI.Initially, we selected 63 The de novo contig assembly using Trinity produced a total of 187,685 contigs, of which 64 . The 5′ and 3′ untranslated region (UTR) sequences of AbMV1 were determined with Sanger sequencing, resulting in a complete genome sequence of 11,238 nt without the poly(A) tail and four predicted ORFs  from China32. The 5′ UTR of AbMV1 includes repeat sequences of UUCCA and UUCAA, which resemble those of the AhMV1. The largest ORF was predicted to encode a replication protein (predicted Mr = 272.504 kDa) including an RNA-dependent RNA polymerase (RdRP) domain, a methyltransferase and a helicase  homologous to virgavirus movement proteins (MPs), and the fourth ORF resembles a coat protein (predicted Mr = 34.078 kDa) similar to those in AhMV1. Interestingly, the second ORF showed some homology with the protease domains of Setosphaeria turcica hypovirus 1 and Phomopsis longicolla hypovirus (GenBank accessions AZT88613 and AIG94930). A recombination event between distantly related viruses (a dsRNA megabirnavirus and a positive-sense ssRNA hypovirus) has been reported earlier by Wang et al.33.Two nearly identical Trinity contigs shared the highest Blastx similarity with virga-like viruses .A new mymona-like virus was identified in the RNA-Seq library of gs Table . Mappinggs Table , Fig. 1Cgs Table  online. A. mellea ELDO17  . The in silico detected virus sequence was named as Armillaria mellea negative strand RNA virus 2  and 731 aa (Mr = 81.976 kDa), respectively. Based on read mapping, there were seven and five polymorphic nucleotide sites in the coding region of AmOlV1 and AmOlV2 with variant frequencies of ca. 6–30%, respectively  , and were designated as Armillaria borealis ambi-like virus 1 (AbAlV1), Armillaria borealis ambi-like virus 2 (AbAlV2) and Armillaria sp. ambi-like virus 3 (AsAlV3).Three distinct ambi-like viruses were retrieved from our RNA-Seq library, and were all found to be hosted by more than one te Table , Fig. 6AArmillaria RNA-Seq libraries deposited in the NCBI SRA database . The RNA-Seq library of A. mellea ELDO17 (BioProject PRJNA568830) analysed in silico hosted another mymona-like virus and an ambi-like virus. Only ambi-like viruses were found in the Finnish isolates, of which A. borealis Ab9A hosted all three ambi-like viruses. The occurrence of the different ambi-like viruses in a single host isolate further supports the notion that they represent distinct virus species. This is because mutual exclusion of conspecific virus strains is considered a species delimitation criterion in many viral taxa.All the other viruses except for the ambi-like viruses occurred in one host isolate only. However, some of them were found in mixed infections. For example, A. borealis isolates shared more than 90% nt sequence identities  than between Finnish variants Ab9A and Al65A from two different host species   hosted by different ies Fig. A, even ties Fig. . MoreoveIn order to assess possible effects of viruses on the growth rate of their host, isogenic fungal strains with and without viral infections were generated using temperature treatment. Thermal treatment was successfully used to cure isolates N40, CMW3973, CMW50256, Al65A and Ab9A of viral infections. The highest incubation temperature used for the thermal treatment was 35 °C for isolates CMW50256 and Al65A and 36 °C for isolates N40, CMW3973 and Ab9A.Armillaria strains was compared by recording their growth on 2% malt extract agar (MEA) plates during a three-week period , whereas three hosts were cured of multiple virus infections  and Rosellinia necatrix , form large and long-living clonal individuals, which can be considered as optimal targets for viral biocontrol agents. Therefore, the viromes of both Heterobasidion spp. and R. necatrix have been extensively studied, which has led to the discovery of numerous mycoviruses, some of which cause host debilitation23. Interestingly, the persistent lifestyle of these root-rot pathogens seems to lead to the accumulation of viruses in the clonal individuals (via anastomosis contacts and spores)39 and even occasional virus transmission from other fungal species occurring in the same habitat42. In the case of Armillaria spp., there is no prior knowledge regarding the possible accumulation of viruses in ancient clones. Dvořák43 investigated the presence of dsRNA elements in 40 strains of A. gallica, A. cepistipes and A. ostoyae from the Czech Republic but none were detected. This is consistent with our findings that revealed only ssRNA viruses in the strains of Armillaria investigated in this study. Furthermore, only one negative-strand ssRNA virus was identified by in silico mining of transcriptomic datasets of several Armillaria species. Although more Armillaria isolates and species should be investigated for viral infections, our results showed that the virus diversity detected in Armillaria spp. is moderate compared to many other fungal species.In this study, we describe the first genome sequences of mycoviruses hosted by the notorious, globally distributed plant pathogens of genus A. borealis. Classified members of family Virgaviridae infect plants and have rod-shaped, non-enveloped particles of about 20 nm in diameter and up to about 300 nm long44. However, virga-like viruses have been recently described in the basidiomycete Auricularia heimuer32, the ascomycetes Macrophomina phaseolina36 and Podosphaera prunicola45 and the oomycete Plasmopara viticola 46. They have also been provisionally identified by metatranscriptomic analysis in the basidiomycetous Leucocoprinus sp., and the ascomycetous Acidomyces richmondensis47 and Mycosphaerella sp. . Members of family Virgaviridae are classified in seven genera, four of which include viruses transmitted by soilborne organisms: members of the genera Furovirus, Pecluvirus, Pomovirus are vectored by plasmodiophorids , for example Polymyxa graminis and Spongospora subterranea, whereas members of the genus Tobravirus are transmitted by nematodes44. The vast majority of virga-like viruses found in fungi are monopartite with a similar predicted ORF structure and genome organization as plant viruses of genus Tobamovirus, which are readily transmitted mechanically but lack known vectors. However, a tripartite virga-like virus related to unclassified invertebrate-derived viruses was recently discovered in Aspergillus fumigatus48. Notably, fungal virga-like viruses appear to encode a MP, a typical feature for plant viruses but usually lacking in mycoviruses. This supports the hypothesis that fungal viruses in the class Alsuviricetes could originate from plant viruses transmitted horizontally to plant-pathogenic fungi49. However, most mycoviruses in the class Alsuviricetes seem to have lost the MP and coat protein. In contrast to plant viruses, the persistent lifestyle and intracellular mode of transmission (via porous septa within a mycelium and anastomosis contacts between mycelia) generally enables mycoviruses to be transmitted without a MP, and the genes encoding such proteins may represent evolutionary remnants in the genomes of fungal virga-like viruses52.In this study, we identified a new virga-like virus in a Siberian strain of A. mellea from South Africa also hosted positive-sense RNA viruses resembling members of family Botourmiaviridae. The evolutionary history of these positive-sense RNA viruses is intriguing and appears to follow an opposite trend compared to virgaviruses that seem to have been transmitted from plants to fungi. Thus, plant viruses in the genus Ourmiavirus have a genome segment encoding an RdRP resembling those of fungal narnaviruses, another segment encoding a MP related to of plant tombusviruses, and a third genome segment encoding a CP53. In contrast to the plant viruses, fungal ourmia-like viruses that are currently classified in four different genera have only one genome segment encoding the RdRP. Therefore, it appears that the plant ourmiaviruses have evolved from a narnavirus that has gained the proteins encoded by the two extra genome segments from plants to be able to move between plant cells54. Recently, a taxonomic proposal was submitted to accommodate two new genera and 25 new species in family Botourmiaviridae . This suggests that this previously unknown virus group is highly common among fungi. The two ourmia-like viruses in Armillaria resemble viruses found in Rhizoctonia solani that are within the genus Magoulivirus, and proposed genus Rhizoulivirus, as well as several unclassified fungal and plant botourmiaviruses. Based on criteria for the classification of members of Botourmiaviridae by the ICTV27 and virus classification guidelines in the age of metagenomics55, coding complete sequences are sufficient for the classification of new virus species.Two isolates of A. mellea from South Africa hosted a negative-strand RNA virus related to members of family Mymonaviridae. In addition, another mymona-like virus was detected in silico from a North American strain of A. mellea. As expected, based on the origin of the host isolates, the two mymona-like viruses in A. mellea were relatively distantly related. Mymonaviridae is a relatively new family of negative-sense RNA viruses that have been found only in fungi26. They have enveloped filamentous particles of 25–50 nm in diameter and about 1000 nm in length. Mymonaviruses have been found in the fungal pathogens Sclerotinia sclerotiorum and Fusarium graminearum, and the basidiomycetous wood decay fungi Lentinula edodes20 and Bondarzewia berkeleyi34, as well as in metatranscriptomic datasets from soybean leaves and arthropods60. Currently there are nine classified species in two genera in family Mymonaviridae . However, a recent proposal accepted by the ICTV Executive Committee (2020.004F.Ac.v1.Mymona) suggests the introduction of seven more genera in the family, including the genus Lentimonavirus where the newly characterized AmNSRV1 also groups based on phylogenetic analysis. AmNSRV2 was more closely associated with the genus Auriculariamonavirus. The elongated viral particles discovered earlier in North American strains of pathogenic A. ostoyae isolates seem to be at least 600–800 nm in length, and resemble those of mymonaviruses31.One isolate of A. mellea isolates from Cape Town were different in their viral composition, although both represent the same introduced pathogen of European origin. Strain CMW3973 isolated in 1996 from Quercus robur in the Company Gardens hosted a mymonavirus and a botourmiavirus, whereas isolate CMW50256 isolated in 2015 from Leucadendron conocarpodendron in the Kirstenbosch National Botanical Garden hosted another putative species of Botourmiaviridae. The two botourmiaviruses shared only ca. 20% aa sequence identity, therefore indicating that they are separate infections. Isolate CMW3973 represents A. mellea genotype A2 and isolate CMW50256 genotype A9, which are somatically compatible. It is considered that these closely related genotypes originate from a single introduction event of the pathogen into South Africa in the 1600s8.It was notable that two Armillaria and occurred in almost all isolates from the Northern hemisphere. “Ambiviruses” constitute a new group of putative viruses that was recently identified by HTS in endomycorrhizal fungi28 as well as in Cryphonectria parasitica and Rhizoctonia solani29. Based on our ongoing investigations this virus group seems to be very common among asco- and basidiomycetes, but so far there is no information on their morphology, biology or population structure. The virus-like particles described by Blattný et al.30 in A. mellea strains from the Czech Republic were described as either rod-shaped (22–28 × 119 nm) or isometric (30 nm). As botourmiaviruses are capsidless, these two viral particle types suggest the presence of a yet uncharacterized virus group, but at this point we cannot verify whether one of them represents “ambiviruses”. Moreover, Blattný and colleagues considered their results preliminary as no negative staining was performed. Very similar viral strains occurred in A. borealis isolates from two different sites in Finland as well as Siberia, and only one A. borealis isolate (Ab3B) was free of ambivirus infections. All three ambivirus types co-infected the Siberian A. borealis isolate N40 and the Finnish A. borealis isolate Ab9A. Furthermore, AsAlV3 occurred in one strain of A. cepistipes in addition to these two A. borealis isolates. Interestingly, the sequence identity of the AsAlV3 strain from A. cepistipes and A. borealis from Finland was lower than that between the two A. borealis strains originating far from each other, suggesting that the ambivirus population might have differentiated based on its host species. However, further studies are needed to decipher whether the Armillaria viruses are species-specific. The Finnish A. borealis isolates infected with ambiviruses were all somatically incompatible and were therefore deemed to represent different clonal individuals, although their collection sites were relatively closely located in Southern Finland. Korhonen61 reported that the clone size of A. borealis (at that time designated as “species A”) was 120–150 m in diameter, and suggested their age to be about 100 years.Ambi-like viruses were found to be very common in isolates of Armillaria isolates from viruses and made a pilot growth experiment to investigate the possibility that viruses could affect the phenotype of their host. No major growth differences were observed between the virus-free and virus-infected isogenic strains, but it should be noted that the laboratory growth rate of virus-infected fungal strains is not necessarily reduced even if they have a lower virulence in natural conditions62. Moreover, mycovirus effects have been shown to be dependent on environmental and ecological conditions, such as temperature and fungal competitors present in the substrate64, and infections by multiple co-infecting viruses may have a different phenotypic outcome than single virus infections65. Future studies should determine whether viral infections are transmitted to Armillaria basidiospores and if they are present in actively growing hyphal tips. In this study, the virus-infected fungal isolates originated from fruiting body tissue (Finnish isolates), from wood  or from diploid mycelia from rhizomorphs, mycelial fans or A. mellea mycelium growing on the roots and root collars (South African isolates). This shows that the viruses are carried into different types of mycelia of Armillaria, and potentially have a possibility to spread into the mycelial network formed by large clonal individuals.In this study, we were able to cure the Armillaria sp. strains were isolated from fruiting body tissue samples collected mostly from Southern Finland in September 2017  of Forestry and Agricultural Biotechnology Institute (FABI) and V.N. Sukachev Institute  in 15% ethanol concentration. The presence and size of dsRNA was estimated by agarose gel electrophoresis. A virus-infected Heterobasidion isolate  as described earlier28. RNA quality and quantity was determined using NanoDrop (Thermo Scientific) and by analysing an aliquot of each RNA by an agarose gel electrophoresis.For the extraction of total RNA, Armillaria strains: N40, CMW3973, CMW50256, Ab2B, Ab3B, Ab4B, Ab9A, Al65A and MUS36. The RNA pool included 1 µg of total RNA from each fungal strain, except for strain N40 that was represented by two separate RNA extractions (2 µg). The RNA pool was sent in ethanol precipitation to Macrogen Korea for further quality determination, library construction and HTS. The library was generated using TruSeq Stranded Total RNA Library prep kit with Human/Mouse/Rat Gold for rRNA removal (Illumina) and 101 bp paired-end reads were obtained using Novaseq (Illumina).An RNA-Seq library was constructed using pooled RNA samples from nine 72 and de novo assembly was conducted using Trinity (version 2.8.4 or 2.8.5)73 in R74. Host-specific contigs were determined with a Blastx run  using a custom Armillaria protein database (db), generated by combining the A. solidipes, A. gallica and A. ostoyae protein models75. Contigs showing significant Blastx similarity with Armillaria protein models were omitted and a subsequent Blastx run  was performed against a custom viral protein db. Contigs longer than 500 nt and resembling viral proteins were then re-examined using Blastn and Blastx searches against complete nucleotide and non-redundant protein sequences. Geneious 10.2.6 (Biomatters Ltd) was used for further analysis of the putative virus contigs and mapping of reads. Additionally, fifteen publicly available RNA-Seq libraries representing 13 different Armillaria species  ≥ 1500 nt in length, having ORF ≥ 400 nt were selected and examined with Blastn, Blastp and Blastx searches against complete nucleotide and non-redundant protein sequences. Finally, the raw reads of the Armillaria library were mapped against contigs with no hits  and random hexamer primers. Determination of host strains was performed using RT-PCR with DreamTaq DNA Polymerase (Thermo Fisher Scientific) and virus-specific primers . Diluted nucleic acid samples were used as templates in standard PCR with DreamTaq DNA Polymerase and virus-specific primers (the same as used for host screening). The quality of DNA was verified by using it as a template for PCR amplification of ITS rDNA and M13 minisatellite markers77 as described earlier78. Putative virus contigs DN471, DN562 and DN2538 were confirmed to occur in more than one Armillaria strain. For contig DN471 genomic integration was tested using DNA of N40. DNA of N40 and DNA of MUS36 were used in testing the integration of contig DN562 and DNA of N40 and DNA of Al65A for DN2538.To investigate the possibility of genomic integration of the viral sequences, two nucleic acid extractions were performed for 79 was used for attempts to determine the viral 5′ and 3′ UTRs. The T4 adapter or primer A blocked at the 3´end by an NH2 group81 was ligated to viral dsRNA or total RNA using the T4 RNA ligase (Thermo Fisher Scientific), followed by purification of RNA by agarose gel electrophoresis and extraction using the RNAid kit  and reverse transcription. Initial denaturation was conducted for 3–6 min at 99 °C, and reverse transcription (1 h 30 min) at 55 °C, 60 °C and 50 °C for Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific), SuperScript IV (Invitrogen) and RevertAid H minus Reverse Transcriptase (Thermo Fisher Scientific), respectively. Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) was used for PCRs together with a primer complementary to the T4 RNA adapter or primer A and a virus specific primer  to prevent amplification from DNA templates. The same RNA extract without DNase treatment yielded ITS rDNA/Tef-1a amplification products, whereas the DNase-treated template without reverse transcription did not, hence validating the functionality of the DNase treatment.Armillaria strains were compared by inoculating each fungal strain on 2% MEA plates and recording their growth during three weeks at room temperature (20 °C) in the dark. The inoculum was an agar piece of 0.5 mm in diameter from the actively growing part of the mycelium. There were six replicate cultures for each fungal strain. The ImageJ program of the Fiji Platform84 was used to measure the area of mycelial growth, and the statistical difference between isogenic virus-infected and virus-free fungal strains was tested using t-test in Microsoft Excel 2016 .The growth rates of isogenic virus infected and cured (virus-free) Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


In August 2018, a fatal autochthonous case of Crimean-Congo hemorrhagic fever was confirmed in western Spain. The complete sequence of the viral genome revealed circulation of a new virus because the genotype differs from that of the virus responsible for another case in 2016. Practitioners should be alert to possible new cases. A fatal case of Crimean-Congo hemorrhagic fever (CCHF) detected in Spain in 2018 was caused by a different genotype, a reassortant virus, than the genotype of a previous case detected in 2016. This unexpected variability contrasts with the situation in other CCHF-endemic countries. Because CCHF is a zoonotic disease and animal migratory routes between Europe and Africa usually pass through Spain, data about genetic sequences are crucial for monitoring infections in humans, developing suitable detection tools, and providing information about the dynamics of virus circulation and spread. On July 31, 2018, a 74-year-old man sought care at Nuestra Señora de Sonsoles Hospital  with fever (39.2°C), pain in the sacroiliac area, chills, shivering, and a feeling of dizziness without loss of consciousness. No relevant physical findings or analytical parameters were detected . While iOn August 4, the man returned to the hospital with general discomfort and no improvement. He reported increased stools that he (a retired physician) assumed were associated with the antimicrobial drug and reported probably having been bitten by a tick while participating in boar hunting on July 24 in Helechosa de los Montes  . The pathttps://www.qiagen.com) and after addition of ethanol was sent to the Biosafety Level 2 facility. Diagnostic testing was performed by using a RealStar CCHFV RT-PCR Kit 1.0 , and CCHFV infection was confirmed by 2 methods: reverse transcription PCR .On August 7, infection with CCHF virus (CCHFV) was considered. The patient progressively worsened and died at the end of the day. On August 8, a blood sample was collected into an EDTA tube and sent to the National Center for Microbiology  for CCHFV diagnostic testing. The sample was inactivated in the Biosafety Level 3 facility by using a QIAamp viral RNA kit , according to the manufacturer’s instructions  was >10ructions . IgM wasSequencing of small (S), medium (M), and large (L) segments was performed by using primers previously described (https://www.megasoftware.net) and the Beauti/Beast 1.75 package (https://beast.community) programs show similar results. The strain, Badajoz 2018, belongs to genotype III if the L and M segments are analyzed; however, the S segment is closely related to sequences of genotype IV and shares the highest identity with the strains BT 958 (92.62%) from Central African Republic and IbAn7620 (92.58%) from Nigeria. Phylogenetic analyses with neighbor-joining and Bayesian  approachPhylogenetic analysis of the virus responsible for a fatal case of CCHF in 2018 showed reassortment, indicating a new CCHFV circulating in Spain. The patient was probably infected by a tick bite obtained while hunting. The incubation period for this patient was longer (7 days) than that typical after a tick bite (1–3 days) . After rSequence analysis revealed circulation of a CCHFV very different genetically than the one previously described in Spain in humans or ticks (The sequencing results showing 2 virus genetic lineages circulating in Spain indicate that at least 2 introductions have occurred. This situation seems to be distinct from that of the Balkans region, where only 1 virus was introduced from Asia and the virus causing human cases has remained genetically stable for decades  in SpainBecause of the high pathogenicity of CCHFV, a detailed medical history of the patient, including travel history and possible risk factors, is crucial for prompt diagnosis to ensure that appropriate infection control measures can be implemented in a timely manner. For the patient that we report, lack of immediate information regarding the tick bite in combination with the nonspecific initial symptoms meant that CCHF was not suspected until day 8 of illness. The public health services performed contact tracing to identify all persons exposed, and none contracted symptomatic CCHF. This case and the description of a new virus do not modify the risk for infection by CCHFV in Spain. Risk is still considered low, although clinicians at hospitals and general practitioners need to be alert to the possibility of new cases.