2022 年 37 巻 5 号 論文ID: ME22034
Isolated RNA viruses mainly parasitize eukaryotes. RNA viruses either expand horizontally by infecting hosts (acute type) or coexist with the host and are vertically inherited (persistent type). The significance of persistent-type RNA viruses in environmental viromes (the main hosts are expected to be microbes) was only recently reported because they had previously been overlooked in virology. In this review, we summarize the host-virus relationships of eukaryotic microbial RNA viruses. Picornavirales and Reoviridae are recognized as representative acute-type virus families, and most of the microbial viruses in Narnaviridae, Totiviridae, and Partitiviridae are categorized as representative persistent-type viruses. Acute-type viruses have only been found in aquatic environments, while persistent-type viruses are present in various environments, including aquatic environments. Moreover, persistent-type viruses are potentially widely spread in the RNA viral sequence space. This emerging evidence provides novel insights into RNA viral diversity, host-virus relationships, and their history of co-evolution.
An RNA virus has single- or double-stranded RNA as its genome. The genome sizes of RNA viruses range from several kb to several tens of kb, and some harbor segmented genomes depending on the virus group/species (King et al., 2012; Koonin et al., 2020). To date, most RNA viruses have been isolated from eukaryotes, and more than 50% of isolated viruses from eukaryotes are RNA viruses (Nasir et al., 2014). Since the majority of eukaryotes are expected to be infected by RNA viruses, RNA viruses and eukaryotes likely have a long history of co-evolution (Koonin et al., 2015). In contrast, no or very few RNA virus families have been isolated from archaea and bacteria, respectively; however, the diversity of prokaryotic RNA viruses was very recently expanded by metatranscriptomics and subsequent in silico analyses (Callanan et al., 2020; Neri, U., et al. 2022. A five-fold expansion of the global RNA virome reveals multiple new clades of RNA bacteriophages. bioRxiv https://doi.org/10.1101/2022.02.15.480533).
Historically, RNA viruses have been detected as infectious causative agents in humans and economically important plants and animals (Beijerinck, 1898; Loeffler and Frosch, 1898; Reed and Carroll, 1901). However, in the last several decades, fungal RNA viruses that persistently infect their hosts without causing clear phenotypic changes have been discovered, although a few have been found in edible mushrooms and pathogenic fungi that cause phenotypic symptoms (Nuss, 2005; Roossinck, 2014). Moreover, similar “cryptic” RNA viruses are widely present in plants (Roossinck et al., 2010). To obtain a more detailed understanding of RNA viruses, Roossinck categorized the lifestyles of plant/fungal viruses into two types, persistent and acute (Roossinck, 2010, 2012, 2015). Briefly, viruses with a persistent lifestyle are transmitted vertically via cell division, while viruses with an acute lifestyle are most frequently transmitted horizontally (Fig. 1). This classification is completely different from “acute infection”, “persistent (latent) infection”, and “chronic infection” in human infections. These clinical categories indicate the course of viral dose/activity or the state of disease in humans. For example, hepatitis C virus (HCV), a positive-sense single-stranded RNA virus, sometimes establishes a chronic infection after an acute infection. However, this does not represent a change in the viral lifestyle. HCV is categorized as an acute-type RNA virus by Roossinck’s criteria because it mainly spreads by horizontal transmission.
Schematic of transmission routes of microbial RNA viruses.
The acute and persistent types have both been identified in eukaryotic microbial lineages of fungi and protists. Furthermore, viral metagenomics enables us to identify diverse RNA viruses and infer their lifestyles. To date, a large diversity of RNA viruses has been revealed in soil and aquatic microbial ecosystems (Culley et al., 2006; Suttle, 2016; Urayama et al., 2018a; Starr et al., 2019; Wolf et al., 2020; Edgar et al., 2022; Zayed et al., 2022), in addition to biological samples from organisms, such as invertebrates, deep-sea animals, and microbial consortia associated with sponges, macroalgae, and lichens (Shi et al., 2016; Urayama et al., 2018b, 2020a, 2020b; Chiba et al., 2020). In this review, we summarize eukaryotic microbial RNA viruses based on their lifestyles and discuss their significance in microbial ecosystems. This review focuses on RNA viruses excluding retroviruses. Retroviruses are distinct from other RNA viruses in their life cycle because they are embedded into the host genome, whereas other RNA viruses are not. Retroviruses have a mixed strategy of vertical and horizontal transmission and change their strategy depending on the life stage.
We define acute- and persistent-type life cycles in microbial RNA viruses based on Roossinck’s definition (Roossinck, 2010) (Fig. 1). Acute-type viruses are mainly transmitted horizontally via extracellular viral particles. These viruses have the ability to enter host cells from the outside and exit host cells to the outside. During the viral life cycle, acute-type viruses often cause “disease” as represented by cell lysis (Tomaru et al., 2004). In contrast, persistent-type viruses are mainly transmitted vertically via the cell division of their host without cell lysis (Nuss, 2005). These viruses remain associated with their hosts for many generations with nearly 100% vertical transmission, and sometimes lack the ability to enter and exit host cells. In addition, some persistent-type RNA viruses lack capsid proteins (Dolja and Koonin, 2012). No gene is shared only in persistent-type RNA viruses. Persistence and vertical transmission in viruses has been suggested to correlate with commensal or mutualistic lifestyles (Roossinck, 2011; Marquez and Roossinck, 2012).
The host cell phenotype is an attractive subject in virology, and persistent- and acute-type lifestyles overlap with symbiosis and antagonism, respectively. However, difficulties are associated with the classification of viruses according to the phenotype of the host organism because the host-virus relationship changes depending on the surrounding conditions (Xu et al., 2008). Furthermore, we cannot test all environmental conditions that may affect the host phenotype. Therefore, we defined the two types of viruses based on the viral transmission route in this review.
Heterosigma akashiwo RNA virus (HaRNAV) (Tai et al., 2003; Lang et al., 2004) is a well-known acute-type RNA virus associated with microbial eukaryotes in aquatic ecosystems. HaRNAV was isolated from a Heterosigma akashiwo culture that showed the lysis of host cells after the inoculation of a virus particle fraction from seawater. To maintain HaRNAV in lab conditions, a 0.22-μm filtered cell lysate including HaRNAV is inoculated into H. akashiwo cultures, and after cell lysis, the lysate is used as an inoculum. In this case, since the host is re-infected with the daughter virus obtained by filter filtration from the lytic solution, we defined the RNA virus as an acute-type virus. Based on the genome sequence of HaRNAV, it belongs to Marnaviridae (Lang et al., 2021), which includes several acute-type microbial RNA viral genera, such as Marnavirus and Labyrnavirus (Lang et al., 2004; Takao et al., 2006).
Among persistent-type RNA viruses, Saccharomyces cerevisiae virus L-A (ScV-L-A) (Wickner, 1996) is a representative strain. The presence of RNA viruses may be confirmed by long cellular dsRNA because it only consists of dsRNA viral genomes and/or replicative intermediates of non-retro ssRNA viruses (Morris and Dodds, 1979). ScV-L-A does not confer a detectable phenotype upon its host (yeast) cells (Schmitt and Breinig, 2002), and viral dsRNA is maintained in the host cell and transmitted to daughter cells via cell division. ScV-L-A may be maintained and amplified in S. cerevisiae cells under laboratory conditions. In this case, since the RNA virus is maintained by a host sub-culture, we define the RNA virus as a persistent-type virus. ScV-L-A belongs to the family Totiviridae, which includes other known totiviruses isolated from protists (Goodman et al., 2011).
The definitions of acute- and persistent-type viruses are not applicable in some exceptional cases. For example, fungal persistent-type RNA viruses are mainly transmitted to daughter cells, but may also be transmitted between closely related fungal strains through anastomosis (Liu et al., 2003; Vainio et al., 2011). On a geological time scale, the transmission of RNA viruses between plants and fungi may also have occurred (Nibert et al., 2014; Roossinck, 2019; Bian et al., 2020).
We herein summarized and grouped isolated microbial RNA viruses based on the infection type (Table S1). Microbial RNA viruses with acute-type lifestyles have been obtained from aquatic host strains (Sadeghi et al., 2021). Among them, ssRNA viruses that lyse host cells have been reported, such as Heterosigma akashiwo RNA virus (HaRNAV) (Picornavirales), which infects the toxic bloom-forming raphidophyte Heterosigma akashiwo (Raphidophyceae) (Tai et al., 2003); Heterocapsa circularisquama RNA virus 01 (Alvernaviridae), which infects the bivalve-killing dinoflagellate Heterocapsa circularisquama (Dinophyceae) (Tomaru et al., 2004); and Aurantiochytrium single-stranded RNA virus 01 (Picornavirales), which infects the marine fungoide Schizochytrium sp. (Thraustochytriaceae) (Takao et al., 2005). The dsRNA virus, Micromonas pusilla reovirus (Reoviridae), which infects and lyses the marine photosynthetic protist Micromonas pusilla (Mamiellophyceae) (Brussaard et al., 2004), has also been isolated.
In contrast, microbial RNA viruses with persistent-type lifestyles have mainly been identified using intracellular dsRNA (a molecular biomarker) or RNA-seq technology. This type of RNA virus has been exclusively studied and identified in fungi (Ghabrial et al., 2015; Kotta-Loizou and Coutts, 2017). To date, identified dsRNA viruses include unclassified RNA viruses that infect the cultivated mushroom Agaricus bisporus (van der Lende et al., 1994); Penicillium chrysogenum virus (Chrysoviridae), which infects the penicillin-producing strains of Penicillium chrysogenum (Banks et al., 1969); and Saccharomyces cerevisiae virus L-A (Totiviridae), which infects the budding yeast S. cerevisiae (Wickner, 1996). Oomycetes (Oomycota) have also been subject to dsRNA-based surveillance, and the plant pathogens Phytophthora infestans and Asparagus officinalis are known to harbor Phytophthora infestans RNA virus 3 (unclassified) and Phytophthora endornavirus 2 (and 3) (Endornaviridae), respectively (Cai et al., 2013; Uchida et al., 2021). In addition, protozoans, such as Trichomonas, Leishmania, and Giardia, harbor persistent-type RNA viruses (Wang and Wang, 1986; Stuart et al., 1992; Khoshnan and Alderete, 1994); however, the presence of extracellular infection routes has been suggested (Wang and Wang, 1986; Torrecilhas et al., 2020).
RNA viruses with acute-type lifestyles have been detected in aquatic environments, while those with persistent-type lifestyles have been found in terrestrial environments. However, ssRNA and dsRNA viruses with persistent-type lifestyles have both been recently identified from a marine oomycete strain in the genus Halophytophthora (Botella et al., 2020) and a marine fungal strain isolated from the seagrass Posidonia oceanica (Nerva et al., 2016). The identification of these strains was based on high-throughput sequencing methods, while culture-dependent isolation has typically been used to identify viruses in aquatic research. However, in terrestrial environments, few attempts have been made to obtain acute-type RNA viruses. We cannot exclude the possibility that the different distribution pattern observed between the two lifestyle types is a result of methodological bias in virus detection and isolation; however, acute-type RNA viruses may have advantages in dispersal via viral particles to access new host cells in aquatic environments. In the case of DNA phages, a number of theories have been proposed, including Kill-the-Winner and Piggyback-the-Winner (Obeng et al., 2016; Pratama and van Elsas, 2018). Theoretical modeling in addition to further studies will provide more detailed insights into the distribution of acute- and persistent-type RNA viruses under diverse environmental conditions.
To visualize the richness of persistent-type RNA viruses in the sequence space, RNA viruses isolated from eukaryotic microorganisms were classified into acute- or persistent-types based on culture-dependent laboratory observations (Table S1), as described above. In total, 96% (304/314) of eukaryotic microbial RNA viruses were recognized as persistent types (Fig. 2), while we were unable to exclude the possibility of experimental biases in culture-dependent analyses to obtain acute-type RNA viruses in eukaryotic microorganisms. To predict the distribution of persistent-type RNA viruses in the total RNA virome sequence space, we used this list as an operational reference virus list because of the lack of knowledge on persistent-type RNA viruses in other host organisms and limited metagenomic data. Based on the operational reference virus list, RNA viral clusters including all known viral RdRp sequences were analyzed, and clusters including RNA viruses related to microbial persistent-type viruses (>50% amino acid similarity) were distinguished from those of acute-type viruses (Fig. 3). In our current analysis, more than 1/3 of the clusters (including five or more sequences) were predicted to include persistent-type viruses. Since the number of isolated persistent-type RNA viruses is limited, the true richness of persistent-type RNA viruses needs to be higher than that estimated.
A total of 293 isolated acute- and persistent-type microbial RNA viruses in sequence-based clusters. Colored circles indicate the lifestyle of each RNA virus: red, acute; blue, persistent. In total, 315 isolated microbial eukaryotic RNA viruses were collected from the manually curated Virus-Host database (Mihara et al., 2016) downloaded on 2021.12.09. Sequences were clustered at 70% amino acid identity. Representative sequences were applied to a network analysis with MOCASSIN-prot (Keel et al., 2018).
RdRp sequences of known RNA viruses obtained from the Identical Protein Groups resource (https://www.ncbi.nlm.nih.gov/ipg) with keywords “rna dependent rna polymerase” and “viruses”. After the removal of short (<200 aa) sequences, RdRp sequences were clustered at 70% using CD-HIT (Huang et al., 2010). Representative sequences were applied to a network analysis with MOCASSIN-prot (Keel et al., 2018). Colored circles indicate percent identity to persistent- or acute-type microbial RNA viruses: blue 100% to persistent; sky blue >70% to persistent; green >50% to persistent; red 100% to acute (>70 and 50% to acute were not identified).
Metagenomics is a powerful tool for identifying DNA viromes in nature (Breitbart et al., 2002; Edwards and Rohwer, 2005; Suttle, 2016), and its use for RNA viromes has markedly increased (Culley et al., 2006, 2010; Steward et al., 2013; Wolf et al., 2020; Edgar et al., 2022; Zayed et al., 2022). RNA virome research is conducted as either metagenomics (metatranscriptomics) of RNA virus particles or as cellular transcriptomics. RNA virus particles in extracellular environments are expected to predominantly be acute-type viruses, and cellular RNA may harbor viral RNA from both acute- and persistent-type viruses (Fig. 1). Accordingly, cellular RNA-specific viruses are expected to be of the persistent type. In addition, dsRNA-seq for cellular RNA has also been used to detect cellular viral RNA sequences in some studies in order to more efficiently retrieve RNA viral sequences (Decker and Parker, 2014; Decker et al., 2019; Izumi et al., 2021).
In aquatic environments, most of the acute-type RNA viruses identified from extracellular viral particles belong to specific RNA viral lineages, such as Picornavirales (ssRNA) and Reoviridae (dsRNA) (Culley et al., 2006, 2014; Djikeng et al., 2009; Steward et al., 2013; Culley, 2017; Urayama et al., 2018a; Wolf et al., 2020). In addition, Picobirnaviridae (dsRNA) viruses that may infect bacterial hosts were identified as a dominant RNA virus population (Ghosh and Malik, 2021; Neri, U., et al. 2022. bioRxiv https://doi.org/10.1101/2022.02.15.480533.). The contribution of Picornavirales to viral lysis via their acute lifestyle and subsequent release of organic matter (previously defined as the virus shunt in DNA virus studies [Wilhelm and Suttle, 1999; Zimmerman et al., 2020]) was suggested based on the correlation between the relative abundance of transcripts related to Picornavirales and the amount of particulate organic carbon in pelagic ecosystems (Kaneko et al., 2021). These findings are also consistent with the results of culture-dependent isolation experiments as described above.
Based on pelagic microbial cellular RNA, in addition to Picornavirales, Reoviridae, and Picobirnaviridae, a wide range of RNA virus groups has been identified (Urayama et al., 2016, 2018a). Among them, the dominant members belong to Narnaviridae (ssRNA) and Partitiviridae (dsRNA), which have been recognized as persistent-type RNA viruses associated with fungi and plants/eukaryotic microorganisms, respectively (Hillman and Cai, 2013; Nibert et al., 2014). Moreover, RNA virome studies have been conducted on macrocolonies of eukaryotic microorganisms, such as Delisea pulchra (red algae) and Scytosiphon lomentaria (brown algae). An association with Totiviridae (dsRNA) viral genomes, which are a persistent type, is generally observed; however, diverse RNA virus groups constitute the viromes (Lachnit et al., 2016; Urayama et al., 2016; Chiba et al., 2020). In contrast, Leviviridae (ssRNA bacteriophage) and Narnaviridae virus operational taxonomic units (OTUs) have been identified as the predominant populations in soil RNA viromes, although the physical separation of cellular and viral particles has not been examined (Starr et al., 2019).
We previously compared viral dsRNA in extracellular virus particle fractions and cellular dsRNA from surface seawater (Urayama et al., 2018a). We showed 3.7- to 14.9-fold more dsRNA viral OTUs in cellular dsRNA metatranscriptomes than in extracellular dsRNA metatranscriptomes. Although we cannot rule out the possibility that some dsRNA virus particles were lost during sample processing, cellular dsRNA viruses, presumed to be of the persistent type, were abundant in the aquatic environment. In the present and related studies, we used a combination of fragmented and primer-ligated dsRNA sequencing (FLDS) and genome reconstruction in silico to identify viral sequences that are distinct from those in public databases. FLDS is a sequencing method that is applicable to long dsRNAs and enables the retrieval of the complete genomic sequences of both dsRNA and ssRNA viruses (Urayama et al., 2016; Fukasawa et al., 2020; Hirai et al., 2021; Uehara-Ichiki et al., 2021). In this technique, the terminal sequences of an RNA virus genome or genome segment may both be identified by sequence read mapping (Fig. 4). Therefore, it may be used to reconstruct multi-segmented RNA viral genomes based on terminally conserved sequences among segments (Fig. 5). In other words, a group of contigs sharing either/both terminal end sequences may represent a multi-segmented RNA virus genome. Moreover, if an RdRp gene or other virus proteins in any one of the segments in a reconstructed genome is identified based on sequence-dependent methods, all of the segments in the reconstructed genome may subsequently be identified as viral genome segments. We previously identified approximately 800 novel RNA viral genes that did not show significant (e-value less than 1×10–5) similarities to known protein sequences (Urayama et al., 2018a). Therefore, FLDS provides opportunities for deep surveys of RdRp and the identification of novel genes that are distinct from the viral genes in public databases.
Differences in conventional RNA-seq and FLDS for resultant contigs. In a de novo analysis, terminal sequence positions were not defined by RNA-seq data. However, FLDS data enabled us to identify terminal sequence positions because FLDS sequence reads included RACE (Rapid Amplification of cDNA Ends), which is widely used to assess the terminal sequences of RNA molecules (Urayama et al., 2016).
Concept of RNA viral genome reconstruction based on conserved terminal sequences in segmented RNA virus genomes. Many segmented RNA viruses have conserved 5′- and 3′-terminal sequences (colored boxes). FLDS enabled us to obtain full-length RNA sequences, which are difficult to obtain with conventional RNA-seq technologies (Fig. 2). Based on terminal sequences, we reconstructed RNA viral genomes. If RdRp is identified in a potential RNA viral genome, we predict that other RNA sequences, which do not show significant similarity to known RNA viruses, will be segments of the RNA virus.
Knowledge on the ecological functions of acute-type microbial RNA viruses is based on the effects of host cell lysis. For example, previous studies suggested the contribution of RNA viruses to the virus shunt in pelagic zones (Kaneko et al., 2021) and soil environments (Starr et al., 2019). In addition, a sequence match between a CRISPR spacer and a bacterial RNA virus was reported (Wolf et al., 2020). In contrast, their impacts on the evolution of microbial hosts have not yet been revealed. In the case of animals, RNA viral sequences incorporated into the host genome protect the host organism from related RNA viral infections (Suzuki et al., 2020). However, a similar system has not been reported for microbes infected with RNA viruses. The ability to mediate horizontal gene transfer, which is often reported for DNA viruses, has also not been reported.
In contrast, persistent-type microbial RNA viruses have a wide range of effects through the manipulation of their host organisms. For example, fungi with an RNA virus may induce thermal tolerance to plants, while those without the RNA virus do not (Marquez et al., 2007). Persistent-type RNA viruses also influence biological interactions between hosts and other organisms by changing secondary metabolites and pathogenicity (Chiba et al., 2009; Zhang et al., 2014; Aihara et al., 2018; Okada et al., 2018; Takahashi-Nakaguchi et al., 2019; Ninomiya et al., 2020). In some cases, persistent-type RNA viruses provide advantages to their hosts under specific conditions (Okada et al., 2018). Therefore, these viruses may affect the adaptation of microbes to environmental challenges (Bao and Roossinck, 2013). Many of these are examples in fungi, which serve as model systems for persistent-type RNA viral research; however, their functions in aquatic environments remain largely unknown. Nevertheless, the host-virus relationship and the impact of persistent-type RNA viral infection on the physiology of their hosts in terrestrial ecosystems imply that persistent-type RNA viruses in aquatic ecosystems also influence host physiology and subsequent ecological functions, including the niche adaptation of their host organisms.
Climate change due to global warming and ocean acidification, is an important issue for our planet. These environmental changes are expected to affect pelagic microbial ecosystems, and other impacts on marine microbial ecosystems and subsequent biogeochemical cycles may also accelerate environmental changes. Therefore, observations of the marine microbial community are essential for understanding microbial responses to a changing ocean. Furthermore, current updates in our understanding of the RNA virome suggest that acute- and persistent-type microbial RNA viruses both play a significant role in biogeochemical cycles via the viral shunt and the regulation of host physiology. Accordingly, we need to pay close attention to marine RNA viromes in the changing ocean even though pelagic RNA viromes have so far been overlooked in marine microbial ecology.
Urayama, S., Takaki, Y., Chiba, Y., Zhao, Y., Kuroki, M., Hagiwara, D., and Nunoura, T. (2022) Eukaryotic Microbial RNA Viruses—Acute or Persistent? Insights into Their Function in the Aquatic Ecosystem. Microbes Environ 37: ME22034.
https://doi.org/10.1264/jsme2.ME22034
This study was supported by a grant from the Institute for Fermentation, Osaka, by JSPS KAKENHI (Grant No. 18K19358, 20K20377, 20H05579, 21K18217, 21K20567, 22H04879, and 22J40078), and by Grants‐in‐Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan (Grant Nos. 16H06429, 16K21723, and 16H06437).
