Dothistroma septosporum and Dothistroma pini, the causal agents of Dothistroma Needle Blight, are infected by multiple viruses.

Dothistroma septosporum and Dothistroma pini are severe foliar pathogens of conifers. They infect a broad spectrum of hosts (mainly Pinus spp.), causing chlorosis, defoliation of needles, and eventually the death of pine trees in extreme cases. Mycoviruses represent a novel and innovative avenue for controlling pathogens. To search for possible viruses hosted by Dothistroma spp. we screened a subset of isolates (20 strains of D. septosporum and one D. pini) originating from the Czech Republic, Slovenia, Italy and Ireland for viral dsRNA segments. Only five of them showed the presence of dsRNA segments. A total of 21 fungal isolates were prepared for total RNA extractions. RNA samples were pooled, and two separate RNA libraries were constructed for stranded total RNA sequencing. RNA-Seq data processing, pairwise sequence comparisons (PASC) and phylogenetic analyses revealed the presence of thirteen novel putative viruses with varying genome types: seven negative-sense single-stranded RNA viruses, including six bunya-like viruses and one new member of the order Mononegavirales; three positive-sense single-stranded RNA viruses, two of which are similar to those of the family Narnaviridae, while the genome of the third correspond to those of the family Gammaflexiviridae; and three double-stranded RNA viruses, comprising two novel members of the family Chrysoviridae and a potentially new species of gammapartitivirus. The results were confirmed with RT-PCR screening that the fungal pathogens hosted all the viruses and showed that particular fungal strains harbour multiple virus infections and that they are transmitted vertically. In this study, we described the narnavirus infecting D. pini. To our knowledge, this is the first virus discovered in D. pini.

Exploring Virus Diversity in the Potato leafhopper (Empoasca fabae), an Economically Important Agricultural Pest.

The potato leafhopper (Empoasca fabae, PLH) is a serious pest that feeds on a wide range of agricultural crops and is found throughout the United States but is not known to be a vector for plant-infecting viruses. We probed the diversity of virus sequences in field populations of PLH collected from four Midwestern states: Illinois, Indiana, Iowa, and Minnesota. High-throughput sequencing data from total RNAs extracted from PLH were used to assemble sequences of fifteen positive-stranded RNA viruses, two negative-stranded RNA viruses, and one DNA virus. These sequences included ten previously described plant viruses and eight putative insect-infecting viruses. All but one of the insect-specific viruses were novel and included three solemoviruses, one iflavirus, one phenuivirus, one lispivirus, and one ambidensovirus. Detailed analyses of the novel genome sequences and their evolutionary relationships with related family members were conducted. Our study revealed a diverse group of plant viruses circulating in the PLH population and discovered novel insect viruses, expanding knowledge on the untapped virus diversity in economically important crop pests. Our findings also highlight the importance of monitoring the emergence and circulation of plant-infecting viruses in agriculturally important arthropod pests.

Prediction of viral spillover risk based on the mass action principle.

Infectious zoonotic disease emergence, through spillover events, is of global concern and has the potential to cause significant harm to society, as recently demonstrated by COVID-19. More than 70% of the 400 infectious diseases that emerged in the past five decades have a zoonotic origin, including all recent pandemics. There have been several approaches used to predict the risk of spillover through some of the known or suspected infectious disease emergence drivers, largely using correlative approaches. Here, we predict the spatial distribution of spillover risk by approximating general transmission through animal and human interactions. These mass action interactions are approximated through the multiplication of the spatial distribution of zoonotic virus diversity and human population density. Although our results indicate higher risk in regions along the equator and in Southeast Asia where both virus diversity and human population density are high, it should be noted that this is primarily a conceptual exercise. We compared our spillover risk map to key factors, including the model inputs of zoonotic virus diversity estimate map, human population density map, and the spatial distribution of species richness. Despite the limitations of this approach, this viral spillover map is a step towards developing a more comprehensive spillover risk prediction system to inform global monitoring.

Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making.

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous, inactive or activatable sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV in Rubus germplasm, we sequenced the genomes of various cultivars and mined eight raspberry whole genome datasets. Bioinformatics analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, short sequence fragments. We developed assays to genotype the main lineages as well as the only known episomal lineage present in the United States. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding efforts have focused on a limited pool of germplasm that harbored endoRYNVs.

Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems.

Brine shrimp (Artemia) has existed on Earth for 400 million years and has major ecological importance in hypersaline ecosystems. As a crucial live food in aquaculture, brine shrimp cysts have become one of the most important aquatic products traded worldwide. However, our understanding of the biodiversity, prevalence and global connectedness of viruses in brine shrimp is still very limited. A total of 143 batches of brine shrimp (belonging to seven species) cysts were collected from six continents including 21 countries and more than 100 geographic locations worldwide during 1977-2019. In total, 55 novel RNA viruses were identified, which could be assigned to 18 different viral families and related clades. Eleven viruses were dsRNA viruses, 16 were +ssRNA viruses, and 28 were-ssRNA viruses. Phylogenetic analyses of the RNA-directed RNA polymerase (RdRp) showed that brine shrimp viruses were often grouped with viruses isolated from other invertebrates and fungi. Remarkably, most brine shrimp viruses were related to those from different hosts that might feed on brine shrimp or share the same ecological niche. A notable case was the novel brine shrimp noda-like virus 3, which shared 79.25% (RdRp) and 63.88% (capsid proteins) amino acid identity with covert mortality nodavirus (CMNV) that may cause losses in aquaculture. In addition, both virome composition and phylogenetic analyses revealed global connectedness in certain brine shrimp viruses, particularly among Asia and Northern America. This highlights the incredible species diversity of viruses in these ancient species and provides essential data for the prevalence of RNA viruses in the global aquaculture industry. More broadly, these findings provide novel insights into the previously unrecognized RNA virosphere in hypersaline ecosystems worldwide and demonstrate that human activity might have driven the global connectedness of brine shrimp viruses.

Genomic Analysis and Taxonomic Characterization of Seven Bacteriophage Genomes Metagenomic-Assembled from the Dishui Lake.

Viruses in aquatic ecosystems exhibit remarkable abundance and diversity. However, scattered studies have been conducted to mine uncultured viruses and identify them taxonomically in lake water. Here, whole genomes (29-173 kbp) of seven uncultured dsDNA bacteriophages were discovered in Dishui Lake, the largest artificial lake in Shanghai. We analyzed their genomic signatures and found a series of viral auxiliary metabolic genes closely associated with protein synthesis and host metabolism. Dishui Lake phages shared more genes with uncultivated environmental viruses than with reference viruses based on the gene-sharing network classification. Phylogeny of proteomes and comparative genomics delineated three new genera within two known viral families of Kyanoviridae and Autographiviridae, and four new families in Caudoviricetes for these seven novel phages. Their potential hosts appeared to be from the dominant bacterial phyla in Dishui Lake. Altogether, our study provides initial insights into the composition and diversity of bacteriophage communities in Dishui Lake, contributing valuable knowledge to the ongoing research on the roles played by viruses in freshwater ecosystems.

Virome analysis of an ectomycorrhizal fungus Suillus luteus revealing potential evolutionary implications.

Suillus luteus is a widespread edible ectomycorrhizal fungus that holds significant importance in both ecological and economic value. Mycoviruses are ubiquitous infectious agents hosted in different fungi, with some known to exert beneficial or detrimental effects on their hosts. However, mycoviruses hosted in ectomycorrhizal fungi remain poorly studied. To address this gap in knowledge, we employed next-generation sequencing (NGS) to investigate the virome of S. luteus. Using BLASTp analysis and phylogenetic tree construction, we identified 33 mycovirus species, with over half of them belonging to the phylum Lenarviricota, and 29 of these viruses were novel. These mycoviruses were further grouped into 11 lineages, with the discovery of a new negative-sense single-stranded RNA viral family in the order Bunyavirales. In addition, our findings suggest the occurrence of cross-species transmission (CST) between the fungus and ticks, shedding light on potential evolutionary events that have shaped the viral community in different hosts. This study is not only the first study to characterize mycoviruses in S. luteus but highlights the enormous diversity of mycoviruses and their implications for virus evolution.

Whole genome sequencing reveals insights into hepatitis E virus genome diversity, and virus compartmentalization in chronic hepatitis E.

BACKGROUND: Hepatitis E virus (HEV) is a leading cause of acute hepatitis and can cause chronic infections in immunocompromised patients. Although HEV infections can be treated with ribavirin, antiviral efficacy is hampered by resistance mutations, normally detected by virus sequencing. OBJECTIVES: High-throughput sequencing (HTS) allows for cost-effective complete viral genome sequencing. This enables the discovery and delineation of new subtypes, and revised the recognition of quasispecies and putative resistance mutations. However, HTS is challenged by factors including low viral load, sample degradation, high host background, and high viral diversity. STUDY DESIGN: We apply complete genome sequencing strategies for HEV, including a targeted enrichment approach. These approaches were used to investigate sequence diversity in HEV RNA-positive animal and human samples and intra-host diversity in a chronically infected patient. RESULTS: Here, we describe the identification of potential novel subtypes in a blood donation (genotype 3) and in an ancient livestock sample (genotype 7). In a chronically infected patient, we successfully investigated intra-host virus diversity, including the presence of ribavirin resistance mutations. Furthermore, we found convincing evidence for HEV compartmentalization, including the central nervous system, in this patient. CONCLUSIONS: Targeted enrichment of viral sequences enables the generation of complete genome sequences from a variety of difficult sample materials. Moreover, it enables the generation of greater sequence coverage allowing more advanced analyses. This is key for a better understanding of virus diversity. Investigation of existing ribavirin resistance, in the context of minorities or compartmentalization, may be critical in treatment strategies of HEV patients.

The Unique Genome of the Virus and Alternative Strategies for its Realization.

Dedicated to the 130th anniversary of Dmitry Ivanovsky's discovery of the virus kingdom as a new form of biological life. The genome of some RNA-containing viruses comprises ambipolar genes that are arranged in stacks (one above the other) encoding proteins in opposite directions. Ambipolar genes provide a new approach for developing viral diversity when virions possessing an identical genome may differ in its expression scheme (strategy) and have distinct types of progeny virions varying in the genomic RNA polarity and the composition of proteins expressed by positive- or negative-sense genes, the so-called ambipolar virions. So far, this pathway of viral genome expression remains hypothetical and hidden from us, like the dark side of the Moon, and deserves a detailed study.