A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors

Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus—Jingmen tick virus (JMTV)—that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.Segmentation is a common form of genome organization in RNA viruses, although why it has evolved more than once and is maintained in such a diverse array of viruses, including those with both positive- and negative-sense genomes, are unclear (1). Segmented and unsegmented viruses usually belong to different viral families, such that the sequence divergence between them is often too great for any meaningful evolutionary inference. The only example of “recent” genome fragmentation in a single RNA molecule occurred in a laboratory strain of foot-and-mouth disease virus (2, 3), although that this occurred following extensive propagation in cell culture means that its relationship to segmentation in nature is uncertain. Hence, the evolutionary links between unsegmented and segmented viruses, as well as the mechanisms that underpin their genesis, are poorly understood.The Flaviviridae are a family of unsegmented positive sense RNA viruses that infect vertebrate and invertebrate species, including the important human pathogens dengue virus, yellow fever virus, and hepatitis C virus. Despite the substantial sequence divergence between the Flavivirus, Pestivirus, and Hepacivirus genera that make up the Flaviviridae, they exhibit a similar genomic structure characterized by a single ORF with distinct clusters of structural and nonstructural genes. The ORF is translated into a single polyprotein, which is subsequently cleaved by cellular and viral proteases into structural and nonstructural proteins. Among the nonstructural protein products, NS3 and NS5 possess the enzymatic domains essential for RNA capping and genome replication (4), whereas the NS3 and NS2b proteins form a two-component serine protease involved in posttranslational cleavage of the viral polyprotein (5).Herein we describe the discovery and characterization of an RNA virus with a four segment genome that encodes NS2b-NS3- and NS5-like proteins related to those found in the Flaviviridae, in turn revealing an unanticipated evolutionary link between the genomes of unsegmented and segmented RNA viruses.     

Viruses Infecting Greenhood Orchids (Pterostylidinae) in Eastern Australia

The Australasian biogeographic realm is a major centre of diversity for orchids, with every subfamily of the Orchidaceae represented and high levels of endemism at the species rank. It is hypothesised that there is a commensurate diversity of viruses infecting this group of plants. In this study, we have utilised high-throughput sequencing to survey for viruses infecting greenhood orchids (Pterostylidinae) in New South Wales and the Australian Capital Territory. The main aim of this study was to characterise Pterostylis blotch virus (PtBV), a previously reported but uncharacterised virus that had been tentatively classified in the genus Orthotospovirus. This classification was confirmed by genome sequencing, and phylogenetic analyses suggested that PtBV is representative of a new species that is possibly indigenous to Australia as it does not belong to either the American or Eurasian clades of orthotospoviruses. Apart from PtBV, putative new viruses in the genera Alphaendornavirus, Amalgavirus, Polerovirus and Totivirus were discovered, and complete genome sequences were obtained for each virus. It is concluded that the polerovirus is likely an example of an introduced virus infecting a native plant species in its natural habitat, as this virus is probably vectored by an aphid, and Australia has a depauperate native aphid fauna that does not include any species that are host-adapted to orchids.     

The Influence of Habitat on Viral Diversity in Neotropical Rodent Hosts

Rodents are important reservoirs of numerous viruses, some of which have significant impacts on public health. Ecosystem disturbances and decreased host species richness have been associated with the emergence of zoonotic diseases. In this study, we aimed at (a) characterizing the viral diversity in seven neotropical rodent species living in four types of habitats and (b) exploring how the extent of environmental disturbance influences this diversity. Through a metagenomic approach, we identified 77,767 viral sequences from spleen, kidney, and serum samples. These viral sequences were attributed to 27 viral families known to infect vertebrates, invertebrates, plants, and amoeba. Viral diversities were greater in pristine habitats compared with disturbed ones, and lowest in peri-urban areas. High viral richness was observed in savannah areas. Differences in these diversities were explained by rare viruses that were generally more frequent in pristine forest and savannah habitats. Moreover, changes in the ecology and behavior of rodent hosts, in a given habitat, such as modifications to the diet in disturbed vs. pristine forests, are major determinants of viral composition. Lastly, the phylogenetic relationships of four vertebrate-related viral families (Polyomaviridae, Flaviviridae, Togaviridae, and Phenuiviridae) highlighted the wide diversity of these viral families, and in some cases, a potential risk of transmission to humans. All these findings provide significant insights into the diversity of rodent viruses in Amazonia, and emphasize that habitats and the host’s dietary ecology may drive viral diversity. Linking viral richness and abundance to the ecology of their hosts and their responses to habitat disturbance could be the starting point for a better understanding of viral emergence and for future management of ecosystems.     

Fishing for the Virome of Tropical Tuna

While planktonic viruses have received much attention in recent decades, knowledge of the virome of marine organisms, especially fish, still remains rudimentary. This is notably the case with tuna, which are among the most consumed fish worldwide and represent considerable economic, social and nutritional value. Yet the composition of the tuna virome and its biological and environmental determinants remain unknown. To begin to address this gap, we investigated the taxonomic diversity of viral communities inhabiting the skin mucus, gut and liver of two major tropical tuna species (skipjack and yellowfin) in individuals fished in the Atlantic and Indian Oceans. While we found significant differences in the virome composition between the organs, this was totally independent of the tuna species or sex. The tuna virome was mainly dominated by eukaryotic viruses in the digestive organs (gut and liver), while bacteriophages were predominant in the mucus. We observed the presence of specific viral families in each organ, some previously identified as fish or human pathogens (e.g., Iridoviridae, Parvoviridae, Alloherpesviridae, Papillomaviridae). Interestingly, we also detected a ‘core virome’ that was shared by all the organs and was mainly composed of Caudovirales, Microviridae and Circoviridae. These results show that tuna host a mosaic of viral niches, whose establishment, role and circulation remain to be elucidated.     

New World Cactaceae Plants Harbor Diverse Geminiviruses

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.     

Host Range and Coding Potential of Eukaryotic Giant Viruses

Giant viruses are a group of eukaryotic double-stranded DNA viruses with large virion and genome size that challenged the traditional view of virus. Newly isolated strains and sequenced genomes in the last two decades have substantially advanced our knowledge of their host diversity, gene functions, and evolutionary history. Giant viruses are now known to infect hosts from all major supergroups in the eukaryotic tree of life, which predominantly comprises microbial organisms. The seven well-recognized viral clades (taxonomic families) have drastically different host range. Mimiviridae and Phycodnaviridae, both with notable intrafamilial genome variation and high abundance in environmental samples, have members that infect the most diverse eukaryotic lineages. Laboratory experiments and comparative genomics have shed light on the unprecedented functional potential of giant viruses, encoding proteins for genetic information flow, energy metabolism, synthesis of biomolecules, membrane transport, and sensing that allow for sophisticated control of intracellular conditions and cell-environment interactions. Evolutionary genomics can illuminate how current and past hosts shape viral gene repertoires, although it becomes more obscure with divergent sequences and deep phylogenies. Continued works to characterize giant viruses from marine and other environments will further contribute to our understanding of their host range, coding potential, and virus-host coevolution.     

Diversity and Distribution of Viruses Infecting Wild and Domesticated Phaseolus spp. in the Mesoamerican Center of Domestication

Viruses are an important disease source for beans. In order to evaluate the impact of virus disease on Phaseolus biodiversity, we determined the identity and distribution of viruses infecting wild and domesticated Phaseolus spp. in the Mesoamerican Center of Domestication (MCD) and the western state of Nayarit, Mexico. We used small RNA sequencing and assembly to identify complete or near-complete sequences of forty-seven genomes belonging to nine viral species of five genera, as well as partial sequences of two putative new endornaviruses and five badnavirus- and pararetrovirus-like sequences. The prevalence of viruses in domesticated beans was significantly higher than in wild beans (97% vs. 19%; p < 0.001), and all samples from domesticated beans were positive for at least one virus species. In contrast, no viruses were detected in 80–83% of the samples from wild beans. The Bean common mosaic virus and Bean common mosaic necrosis virus were the most prevalent viruses in wild and domesticated beans. Nevertheless, Cowpea mild mottle virus, transmitted by the whitefly Bemisia tabaci, has the potential to emerge as an important pathogen because it is both seed-borne and a non-persistently transmitted virus. Our results provide insights into the distribution of viruses in cultivated and wild Phaseolus spp. and will be useful for the identification of emerging viruses and the development of strategies for bean viral disease management in a center of diversity.     

Discovery of Known and Novel Viruses in Wild and Cultivated Blueberry in Florida through Viral Metagenomic Approaches

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum. In this study, viral diversity in wild and cultivated blueberries (V. corymbosum) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families (Amalgaviridae, Caulimoviridae, Endornaviridae, Ophioviridae, Phenuiviridae, and Virgaviridae). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach.     

RNA Viruses in Aquatic Unicellular Eukaryotes

Increasing sequence information indicates that RNA viruses constitute a major fraction of marine virus assemblages. However, only 12 RNA virus species have been described, infecting known host species of marine single-celled eukaryotes. Eight of these use diatoms as hosts, while four are resident in dinoflagellate, raphidophyte, thraustochytrid, or prasinophyte species. Most of these belong to the order Picornavirales, while two are divergent and fall into the families Alvernaviridae and Reoviridae. However, a very recent study has suggested that there is extraordinary diversity in aquatic RNA viromes, describing thousands of viruses, many of which likely use protist hosts. Thus, RNA viruses are expected to play a major ecological role for marine unicellular eukaryotic hosts. In this review, we describe in detail what has to date been discovered concerning viruses with RNA genomes that infect aquatic unicellular eukaryotes.     

Chirohepevirus from Bats: Insights into Hepatitis E Virus Diversity and Evolution

Homologs of the human hepatitis E virus (HEV) have been identified in more than a dozen animal species. Some of them have been evidenced to cross species barriers and infect humans. Zoonotic HEV infections cause chronic liver diseases as well as a broad range of extrahepatic manifestations, which increasingly become significant clinical problems. Bats comprise approximately one-fifth of all named mammal species and are unique in their distinct immune response to viral infection. Most importantly, they are natural reservoirs of several highly pathogenic viruses, which have induced severe human diseases. Since the first discovery of HEV-related viruses in bats in 2012, multiple genetically divergent HEV variants have been reported in a total of 12 bat species over the last decade, which markedly expanded the host range of the HEV family and shed light on the evolutionary origin of human HEV. Meanwhile, bat-borne HEV also raised critical public health concerns about its zoonotic potential. Bat HEV strains resemble genomic features but exhibit considerable heterogeneity. Due to the close evolutionary relationships, bat HEV altogether has been recently assigned to an independent genus, Chirohepevirus. This review focuses on the current state of bat HEV and provides novel insights into HEV genetic diversity and molecular evolution.     

A High Rate Algal Pond Hosting a Dynamic Community of RNA Viruses

Despite a surge of RNA virome sequencing in recent years, there are still many RNA viruses to uncover—as indicated by the relevance of viral dark matter to RNA virome studies (i.e., putative viruses that do not match to taxonomically identified viruses). This study explores a unique site, a high-rate algal pond (HRAP), for culturing industrially microalgae, to elucidate new RNA viruses. The importance of viral-host interactions in aquatic systems are well documented, and the ever-expanding microalgae industry is no exception. As the industry becomes a more important source of sustainable plastic manufacturing, a producer of cosmetic pigments and alternative protein sources, and a means of CO₂ remediation in the face of climate change, studying microalgal viruses becomes a vital practice for proactive management of microalgae cultures at the industrial level. This study provides evidence of RNA microalgal viruses persisting in a CO₂ remediation pilot project HRAP and uncovers the diversity of the RNA virosphere contained within it. Evidence shows that family Marnaviridae is cultured in the basin, alongside other potential microalgal infecting viruses (e.g., family Narnaviridae, family Totitiviridae, and family Yueviridae). Finally, we demonstrate that the RNA viral diversity of the HRAP is temporally dynamic across two successive culturing seasons.     

Evidence of a Possible Viral Host Switch Event in an Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi)

Avipoxviruses have been characterized from many avian species. Two recent studies have reported avipoxvirus-like viruses with varying pathogenicity in reptiles. Avipoxviruses are considered to be restricted to avian hosts. However, reports of avipoxvirus-like viruses from reptiles such as the green sea turtle (Chelonia mydas) and crocodile tegu (Crocodilurus amazonicus) suggest that cross-species transmission, within avian species and beyond, may be possible. Here we report evidence for a possible host switching event with a fowlpox-like virus recovered from an endangered northern royal albatross (Diomodea sanfordi)—a species of Procellariiformes, unrelated to Galliformes, not previously known to have been infected with fowlpox-like viruses. Complete genome sequencing of this virus, tentatively designated albatrosspox virus 2 (ALPV2), contained many fowlpox virus-like genes, but also 63 unique genes that are not reported in any other poxvirus. The ALPV2 genome contained 296 predicted genes homologous to different avipoxviruses, 260 of which were homologous to an American strain of fowlpox virus (FWPV). Subsequent phylogenetic analyses indicate that ALPV2 likely originated from a fowlpox virus-like progenitor. These findings highlight the importance of host-switching events where viruses cross species barriers with the risk of disease in close and distantly related host populations.     

The Viral Susceptibility of the Haloferax Species

Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus–host model systems. Euryarchaea belonging to the genus Haloferax are interesting models, as a reliable genetic system and versatile microscopy methods are available. However, only one virus infecting the Haloferax species is currently available. In this study, we tested ~100 haloarchaeal virus isolates for their infectivity on 14 Haloferax strains. From this, we identified 10 virus isolates in total capable of infecting Haloferax strains, which represented myovirus or siphovirus morphotypes. Surprisingly, the only susceptible strain of all 14 tested was Haloferax gibbonsii LR2-5, which serves as an auspicious host for all of these 10 viruses. By applying comparative genomics, we shed light on factors determining the host range of haloarchaeal viruses on Haloferax. We anticipate our study to be a starting point in the study of haloarchaeal virus–host interactions.     

Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.     

Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish

The zebrafish Danio rerio is a new model system for studying the genetics of hematopoiesis. To define naturally occurring viruses which could infect and replicate within hematopoietic precursors of the zebrafish, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were studied. Infection of whole fish with viral supernatants demonstrated infectious replicants for both viruses, indicating that the virus host range includes the zebrafish. In other species, infection with these viruses leads to prominent hematopoietic necrosis of the head kidney, the major site of adult hematopoiesis. We detected a transient toxicity of the virus to hematopoietic precursors and terminally differentiated red cells after viral infections. The kinetics of hematopoietic defects between IHNV and IPNV infection differed; fish infected with either virus, however, recovered by 6 days postinfection. In contrast to other fish infected with the virus, hematocrit did not change appreciably during this time. These studies are the first to demonstrate IHNV and IPNV infection of the zebrafish and reveal the potential for use of such viruses for gene transfer experiments to infect zebrafish hematopoietic cells.     

Comparative Molecular Characterization of Novel and Known Piscine Toti-Like Viruses

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5′ or 3′ ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3′ end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3′ end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.