NEWS FEATURE Virus images: colematt / iStock Getty Images Plus

The virome hunters diversity was—and still is to this day— mostly uncharacterized. Scientists have since Ambitious efforts to catalog viruses across the globe may facilitate expanded their analyses into other many environments, as well as animal and human our understanding of viral communities and ecology, boost viromes. Indeed, a pure metagenomic analysis infectious disease diagnostics and surveillance, and spur new of human fecal samples revealed a previously unknown virus that represents a large part therapeutics. Charles Schmidt investigates. of the dark matter—as much as 90%—of the human gut virome. Dubbed the crAssphage by Robert Edwards and collaborators from In July, scientists from UC Davis and Columbia and our questions about the viral world are San Diego State because it was pieced together University announced they had isolated a new profound,” says Edward Holmes, a virologist by tool they invented called cross assembly species of the Ebola virus from bats roosting and professor at the University of Sydney in analysis (although its origin in stool seems to inside houses in Sierra Leone. Dubbed Bombali Australia. Along with new species, investigators have been in the minds of the researchers), it after the district where the bats were captured, are turning up vast stretches of what they call was called “one of the most striking feats of this new species is the first Ebola virus to have dark matter—viral sequences unlike any seen metagenomics at that time” by Eugene Koonin its initial identification in an animal host previously. They’re using sophisticated bioin- at US National Center for Biotechnology rather than from a sick person. According to formatics to characterize viral RNA and DNA Information (NCBI). Koonin’s group Tracey Goldstein, associate director of the One and its various functions, and findings have collaborated with Edwards’ to characterize Health Institute at the University of California, shown already that viruses can play essential as this family of phage and annotate some of the Davis, who led the team behind the research, well as harmful roles in human health. Ideally, 80% of the 100kb genome that didn’t align with it isn’t yet clear whether Bombali can infect virome research will lead to biomedical payoffs, known viral known viral proteins3. people in the field, although it has been shown such as new therapies, vaccines, and opportuni- Characterizing viromes, however, is compli- to infect cultured human cells1. ties to head off new disease outbreaks. cated by the lack of a shared genetic marker The discovery of Bombali is notable for among viruses analogous to the 16S ribosomal another reason: it was detected as a result of A new high-throughput era RNA gene in bacteria. All bacteria contain a sequencing the entire virome of bats that had During the 2000s, genomic sequencing version of that gene, allowing scientists to tested positive for Ebola in a consensus PCR- combined with advances in high-resolution identify a particular species on the basis of based assay1. This new approach to virology, microscopy ushered in the modern era its unique 16S signature. Viral identification which takes advantage of high-throughput of virome research. The first uncultured relies instead on multiple markers associated genomic technologies like next genera- viral genome was sequenced in 2002 by with different taxonomic groups and on the tion sequencing, is a novel adjunct to other Forest Rowher at San Diego State University way the sequences match up with those from approaches for identifying emerging viral from seawater samples collected off the known viruses in genomic repositories, such as pathogens before they ‘spill over’ into humans. California coast2. More than 65% of the the NCBI’s Genome database. These are early days. “We know of only a viral sequences in those samples had never David Paez Espino, a bioinformaticist at minuscule fraction of the viruses out there, been seen before, reflecting how viral the US Department of Energy’s Joint Genome

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Institute (JGI) in Walnut Creek, California, 800,000 explains that scientists can isolate a viral frac- ×16-fold ×2-fold ×3-fold tion in a sample by filtering it or by extracting 700,000 and sequencing the entire microbial nucleic 600,000 acid content. Applying metagenomic methods Unique viral sequences will home in on the genomes of DNA viruses 500,000 alone, whereas metatranscriptomic methods Total viral sequences 400,000

will reveal the sequences of both viral DNA number Total and viral RNA. Paez Espino explains that the 300,000 analytical approaches are continually evolving, but as metagenomic methods came first—and 200,000 DNA is inherently more stable than RNA— viral DNA sequences still predominate in 100,000 microbial databases. But metatranscriptomic 0 analyses are taking hold at places like the JGI, August 2016 August 2017 August 2018 according to Paez Espino, because they provide Figure 1 Growth rate of virus identification and microbial host prediction. Growth over time in the so much more information about the virome— total and unique number of viral sequences in the January 2018 release of the IMG database. The not just sequences, but also expression pat- first data point represents a 16-fold increase in the number of species in comparison to the number of terns. Moreover, scientists are motivated to previously identified viruses. Subsequent points are twofold and threefold higher, respectively (image sequence RNA viruses because they account by David Paez Espino, JGI). for roughly half the entire viral world. “Most of big infectious diseases, such as Zika, Ebola Microbial Genomes (IMG) and Microbiomes pathogenicity begins. In her own laboratory, and influenza, are caused by RNA viruses,” database. Those viral contigs currently add up Goldstein starts by evaluating whether pro- Paez Espino adds. to 750,000 total sequences (428,000 unique) teins from a particular virus bind to human cell Simon Roux, a research scientist at the JGI’s owing to the continuous addition of new receptors, as was demonstrated with Bombali. facility in Berkeley, California, adds that ana- samples submitted by the JGI and the NCBI If they do, then researchers will try to grow the lytical methods used in virome research each and are deposited into the IMG/VR, a database virus in cell culture, test whether it causes dis- have their inherent limitations. For instance, of cultured and uncultured DNA viruses and ease symptoms in experimental animals, and scientists still can’t purify the viral content in retroviruses. look for antibodies against the virus in people a given sample completely. Some may be lost who live near where it was discovered. during filtration, for example. And the short Into the wild Several other large-scale efforts are now reads that one gets from sequencing often Goldstein’s team and many other groups are expanding on PREDICT. The Global Virome have uncertain origins: they could be viral or leveraging virome research with the aim of Project (GVP) is setting out to discover derived from some other microbe. To con- intercepting disease pandemics before they roughly 1.2 million new zoonotic viruses in firm their sources, scientists stitch the reads occur. The new Bombali finding is only the animals over the next ten years6. The project together into ‘contigs’, or longer sequences that latest to emerge from the PREDICT project, will depend heavily on the development and may have recognizable functions. According a global effort to discover new viral threats use of low-cost sequencing tools designed for to Roux, that process requires bioinformatic in wildlife with the potential to spill over developing countries. Peter Daszak, president algorithms that look for features unique to into human populations. Funded by the US of the New York–based EcoHealth Alliance, a viruses that other microbes don’t share. For Agency for International Development and nonprofit organization that works on global instance, newly formed viruses—but not other based at the University of California, Davis, infectious disease issues, is among those direct- microbes—come wrapped in a protein capsid PREDICT—which launched in 2009—relies ing the project. The GVP’s goal, he says, is to that can give away their identity. And microbial on PCR and next-generation sequencing to go from being reactive to proactive in the way sequences that look completely unlike anything characterize viromes in bats, rodents and pri- health officials confront zoonotic pathogens. seen previously are often assumed to be viral mates, three taxonomic groups that account Should the GVP succeed, he adds, then it will simply because they are so novel. “Say you’ve for a high proportion of zoonotic viral dis- have accumulated a database of all the high- gotten ten genes in your contig, and one looks eases. According to Goldstein, PREDICT risk viruses that threaten human populations. like it encodes for a capsid, and the other nine scientists have sampled more than 70,000 Run by the US Department of Defense, genes are totally new and we have no idea what animals and people in over 30 countries the PREEMPT project has a complementary they’re doing,” Roux says. “That’s what you’d with high zoonotic disease risks, and the focus on the biological mechanisms under- expect with a new virus genome.” researchers have reported on the discovery lying viral spillover into humans. According Although bacterial genomes greatly out- of 1,000 virus species with the potential to to Jim Gimlett, a program manager in the numbered their viral counterparts in micro- infect human beings5. To further character- Department of Defense’s Defense Advanced bial databases until recently, the number of ize those viruses, PREDICT researchers will Research Projects Agency, PREEMPT focuses published viral sequences is rapidly grow- isolate them or, if need be, synthesize them to on known classes of dangerous viruses, such as ing (Fig. 1). In 2016, JGI scientists unveiled see how they behave and replicate in a cellular Ebola, Lassa fever, Rift Valley fever and avian roughly 125,000 partial and complete DNA host. However, given the potential danger, sub- influenza. Project scientists are modeling virus genomes from samples taken around sequent step in the research will have to take viral evolution and zoonotic potential, “and the world, including oceans, freshwater sys- place only in level 4 biosafety facilities. we’re testing scalable methods to prevent viral tems, soils, plants, animals and humans4, by Goldstein says that after a new viral threat species from jumping to humans in the first mining the Department of Energy’s Integrated is identified, the hard work of determining place,” Gimlett says.

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Still, the notion that scientists could prevent However, Frederic Bushman and colleagues which include species from the Herpesviridae new zoonotic pandemics strikes some scien- at the University of Pennsylvania’s Perelman (herpesviruses) and Anelloviridae (for exam- tists as unrealistic. Holmes, for instance, argues School of Medicine found that in stool sam- ple, the torque teno virus), may reside perma- such efforts amount to “an absolute waste of ples isolated from the same person repeat- nently in healthy blood. “Sometimes viruses time” because new disease outbreaks occur edly for two-and-a-half years, 80% of the viral will attack us, but we also have to embrace the infrequently relative to the virome’s immen- sequences were unchanged7. Temperate phages fact there’s probably much more symbiosis sity. “If the goal is to better understand viral were stable, but lytic phages—which battle than we anticipated,” Telenti says. “Everyone diversity, evolution and ecology, then great— constantly with bacterial defenses—evolved seems to have a herpesvirus, so you have to that’s what we should do,” he says. “But there rapidly: “It was as if some of them had become wonder why they might be useful.” One pos- are just an enormous number of viruses in completely different species,” Bushman says. sibility, Telenti adds, is that persistent, latent wildlife. And to try to predict which of them A controversial finding is that some viruses herpesviruses have roles in modulating and will emerge in humans is totally infeasible. It’s can be detected that reside in body fluids— “educating the immune system.” using rare data to predict rare events, and that body fluids that were once considered sterile, The view that some viruses help to keep just won’t work.” such as healthy blood and cerebrospinal fluid. the immune system nimble and responsive The GVP’s Daszak responds that skeptics Amalio Telenti, a computational biologist by stimulating low-level reactions—even as were once similarly dubious about weather at Scripps Research in La Jolla, California, the immune system regulates viral behavior prediction. But as meteorological data accu- had detected what he suspected were blood- to keep illness in check—is gaining support. mulated with time, he says, weather reports borne viral fragments in healthy people years Mounting evidence suggests that phages, for became increasingly reliable, and the same ago with PCR. Newer sequencing tools, he instance, contribute to normal gut function- might prove true for predicting new outbreaks says, provided an opportunity to investigate ing by pruning the commensal bacteria we of disease. “More data to get to better predic- whether these fragments were indeed viral, ordinarily live with, as well as by killing off tions is exactly what we’re trying to provide,” as opposed to bacterial, human “or just low- bacterial pathogens9. he says. “Because if we don’t understand what’s quality reads.” Others had found that 95% of However, changes in phage distribution have going on out there, then we’re just stuck in the DNA sequences in blood were from human also been linked with disease. Herbert Virgin, same situation: discovering viruses the hard cells, and they typically ignored the rest. now executive vice president and chief sci- way, which is what we want to avoid.” Telenti wanted to characterize those residual ence officer at San Francisco–based Vir sequences, since the presence of pathogenic Biotechnology (headed by George Scangos), The human virome viruses in transfusion blood products (for was the first to associate the phage virome Apart from cataloging new viruses in the envi- example, red cells, platelets and plasma) with human illness. While at the Washington ronment, researchers are also trying to under- constitutes a major hazard, particularly for University in St. Louis, Missouri, he and his stand what viruses are doing in their hosts immunocompromised patients. So, with fund- colleagues reported in 2015 that people with and where—in what exact cell types—they ing from Human Longevity in San Diego, a ulcerative colitis and Crohn’s disease have ele- are doing it (Table 1). The human virome dif- company cofounded by J. Craig Venter (who vated numbers of Caudovirales phages in their fers from one person to the next, but within was a coauthor on the subsequent publica- gut. The increased abundance of these viruses an individual it is remarkably stable over time. tion but has since retired), he led a team that led him to speculate that an unbalanced phage The viral composition in the gut, for instance, sequenced blood samples from more than virome contributes to these illnesses, and pos- which is dominated by temperate DNA bac- 8,000 healthy participants in a large-scale sibly to others as well10. teriophages (which can shift between being investigation of the whole human genome. Eukaryotic viruses that infect human temperate, or dormant, and replicating, or The findings revealed 94 different phages and cells are rare in the gut by comparison, but lytic), not only varies with age, health status, eukaryotic viruses8. Although Telenti says knowledge is also increasing about their role geography and especially diet, but also has a some were likely introduced as contaminants in health and disease. Virgin and his academic major influence on the gut’s bacterial make-up. in sequence reagents, he posits that the others, collaborators also reported that they had

Table 1 Known virotypes according to culturomics and metagenomics Virus type Genome type Environment Associated disease Eukaryotic virus Rotavirus, Astrovirus, Calicivirus, Norovirus, hepatitis E virus, Coronavirus, All RNA except Human small bowel and colon Gastroenteritis Torovirus, Adenovirus (serotypes 40,41) adenovirus (DNA) Adenoviridae, Picornaviridae and Reoviridae (genus Enterovirus) RNA Human intestine Unknown Plant-derived virus Pepper mild mottle virus (PMMV), oat blue dwarf virus, grapevine asteroid RNA Plants and human feces Pathogenic for plants mosaic-associated virus, maize chlorotic mottle virus, oat chlorotic stunt Nonpathogenic for humans virus, Panicum mosaic virus, tobacco mosaic virus Giant virus (>300 kb) Mimiviridae, Mamaviridae, Marseilleviridae, Poxviridae, Iridoviridae, DNA Human fecal protists; amoebae Pneumonitis, childhood diarrhea Ascoviridae, Phycodnaviridae, Asfaviridae in lakes, rivers and seawater (Mimiviridae only) Prophages Myoviridae, Siphoviridae, Podoviridae, Tectiviridae, Leviviridae, Inoviridae dsDNA Human feces Unknown Virus (<145 kb) Microviridae (Microvirus, Gokushovirinae, Alpavirinae, Pichovirinae) ssDNA Seawater, human gut bacteria Unknown dsDNA, double-stranded DNA; ssDNA, single-stranded DNA (modified from Scarpellini, E. et al. Dig. Liver Dis. 47, 1007–1012, 2015).

918 VOLUME 36 NUMBER 10 OCTOBER 2018 NATURE BIOTECHNOLOGY NEWS FEATURE detected several eukaryotic viruses—including of Washington University in St. Louis. The we are closer than ever before to achieving this species from the Circoviridae, Anelloviridae, field of virome research is a decade behind the reality. “We will have to properly identify the and Picobirnaviridae—in stool samples from study of the microbiome, according to Wang, ‘killer’ application for human virome-based healthy children11. In his view, the evidence and many of the tools that are being used to do therapeutics—one where the risk of infecting points to these viruses as members of a normal functional testing of elements of the microbi- people with a virus is worth the risk. This is gut virome, even though they can also make ome (fecal transplantation, gnotobiotic mice, not straightforward, since there are multiple people sick. Furthermore, Virgin discovered and even antibiotics) are simply not available ethical and safety concerns,” he says. that viral diversity was remarkably low in the for the virome. “The first [thing we have to Vir Biotechnology’s Virgin also cautions stool of children with type 1 diabetes, whereas do] is to develop culture systems for any virus that we’re still “substantially behind in drawing stool samples from healthy children were that we find an association for. If we don’t have statistical associations between the virome and enriched for eukaryotic Circoviridae viruses a culture system, we can’t begin to try to do disease.” And the road to turning these associa- that seemed to protect against the disease. infection experiments in different settings,” tions into treatments will be even longer. If the he says. Wang has developed one of the few aim is to design approaches that would have an Translating the virome systems to date for culturing a novel virus immediate therapeutic effect, he says, “we are Scott Plevy, a gastroenterologist and expert detected in human stool. In a 2017 paper, his further behind still in our ability to manipulate on inflammatory bowel disease (IBD) at group described a Caco-2 culture system for the virome in a predictable manner.” Janssen Research & Development in Raritan, astrovirus VA1/HMO-C, which is prevalent in But after spending several decades doing New Jersey, emphasizes that the virome offers a human encephalitis12. But, he points out, this is seminal research on viruses at Washington wealth of untapped diagnostic and therapeutic only the first step. Once it is established that a University before joining Vir, he says he’s opti- opportunities. He’s now exploring how phage novel virus infects human cells, animal models mistic about the field’s emerging prospects. “I composition varies in response to new IBD will have to be established to study pathogenic- think there’s enormous potential,” he says. treatments. That research was prompted by ity. According to Wang, until these systems are Charles Schmidt, Portland, Maine findings showing that the gut’s bacterial com- developed—and they are both challenging to 1. Goldstein, T. et al. Nat. Microbiol. https://doi. position varies in tandem with IBD flareups implement and difficult to get funding for—the org/10.1038/s41564-018-0227-2 (2018). and remissions. Plevy describes those bacte- field will be limited to association studies. 2. Breitbart, M. et al. Proc. Natl. Acad. Sci. USA 99, rial changes as merely “the tip of the iceberg Aleks Radovic-Moreno, vice president at 14250–14255 (2002). 3. Yutin, N. et al. Nat. Microbiol. 3, 38-46 (2018). in terms of the diagnostic information we can PureTech Health, which is affiliated with the 4. Paez-Espino, D. et al. Nature 536, 425–430 (2016). extract from the gut.” Corresponding phage microbiome company Vedanta Biosciences, 5. Olival, K.J. et al. Nature 546, 646–650 (2017). changes, he says, could offer even deeper agrees that until these tools are available, com- 6. Carroll, D. et al. Science 359, 872–874 (2018). 7. Minot, S. et al. Proc. Natl. Acad. Sci. USA 110, insights into how patients respond to therapeu- panies will sit on the sidelines. “Solving these 12450–12455 (2013). tic interventions. And ideally, that could pave two challenges will be necessary for the field 8. Moustafa, A. et al. PLoS Pathog. https://doi. org/10.1371/journal.ppat.1006292 (2017). the way toward phage therapy—using phages to accelerate,” he says. And it’s going to take 9. Metgzer, R.N. et al. Viruses 10, 146 (2018). as medical treatments to kill the bacteria that a dedicated effort, not something a microbi- 10. Norman, J.M. et al. Cell 160, 447–460 (2015). might cause or exacerbate IBD. ome company can do by having one or two 11. Zhao, G. et al. Proc. Natl. Acad. Sci. USA 114, E6166–E6175 (2017). How to go from association to causation is scientists looking at viruses, according to 12. Janowski, A.B. et al. J. Virol. https://doi.org/10.1128/ “the million dollar question,” says David Wang Radovic-Moreno. Nonetheless, he believes that JVI.00740-17 (2017).

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