Viral metagenomics

Viral metagenomics is the study of viral genetic material sourced directly from the environment rather than from a host or natural reservoir. The goal is to ascertain the viral diversity in the environment that is often missed in studies targeting specific potential reservoirs. It reveals important information on virus evolution and the genetic diversity of the viral community without the need for isolating viral species and cultivating them in the laboratory. With the new techniques available that exploit next-generation sequencing (NGS), it is possible to study the virome of some ecosystems, even if the analysis still has some issues, in particular the lack of universal markers. Some of the first metagenomic studies of viruses were done with ocean samples, and revealed that most of the sequences of DNA and RNA viruses had no matches in databases.[1][2] Subsequently, some studies about the soil virome were performed with a particular interest on bacteriophages, and it was discovered that there are almost the same number of viruses and bacteria.[3] This approach has created improvements in molecular epidemiology and accelerated the discovery of novel viruses.[4][5]

Environmental Shotgun Sequencing (ESS)
         (A) Sampling from habitat
         (B) filtering particles, typically by size
         (C) Lysis and DNA extraction
         (D) cloning and library construction
         (E) sequencing the clones
         (F) sequence assembly into contigs and scaffolds

Acknowledging the importance of viral metagenomics, the International Committee on Taxonomy of Viruses (ICTV) recognizes that genomes assembled from metagenomic data represent actual viruses and encourages their official classification following the same procedures as those used for viruses isolated and characterized using classical virology approaches.[6] The IMG/VR system[7] and the IMG/VR v.2.0[8] – the largest interactive public virus database with over 760,000 metagenomic viral sequences and isolate viruses – serves as a starting point for the sequence analysis of viral fragments derived from metagenomic samples. The virus detection method and host assignment approach in IMG/VR is described in a paper discussing Earth's virome[9] and is fully presented as a protocol.[10]

The Global Virome Project

The aim of the Global Virome Project (GVP) is to expand the viral discovery to reduce the risk of harm for new viral large-scale outbreak. It is centered on the massive collection and sequencing of the majority of the planet’s unknown viruses. In fact, it was estimated that between 631,000 to 827,000 yet-to-be-discovered viral species in animal reservoirs (as mammal and bird host) have a zoonotic potential. Total transparency of the data acquired, and the correlated possible development of some medical products (vaccine) are two main benefits of this project.[11]

The main limit of this project is the cost. To analyze the majority of viruses with a zoonotic potential, the total cost has been estimated around $1.2 billion. Another GVP’s aim is also to improve the possibility to detect with low cost sequencing viruses also in developing countries in order to avoid possible outbreaks. For a worldwide sample collection some network between different agencies and nations must be created. For example, the USAID (agency for international development) EPT (Emerging Pandemic Threats) PREDICT project is included in this plan and it is focused on the study of the biology of some dangerous viruses, such as Ebola, Lassa fever, Rift Valley fever and avian influenza. PREDICT project was founded also to discover new viral species in the animal reservoir host and individuate the main characteristics that can cause the viral transmission to human, to avoid the viral outbreak in the population. Next-generation sequencing can help the massive sequencing of this viral genome samples collected, allowing the increase of speed and efficiency and moreover reducing the cost of sequencing. To validate the possibility for these new-discovered viruses to be transmitted from animal to human, new approaches must be developed for the study of their pathogenicity.[12]

The Global Virome Project could help the current pandemic surveillance, diagnosis techniques and prevention strategies, as far as helping for pre-emptive production of vaccine and other countermeasures for candidate high-risk viruses. Another benefit of this research could be a deeper comprehension of the viral biology. Its discovers can be applied not only for medical need, but also in other field as the agricultural and food one, for example to enhance biosecurity of food.[11]

Methods
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DNA barcoding
 
DNA barcoding
Environmental DNA (eDNA)
 
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Metagenomic approach

It is used to sequence all the microbial genomes in a sample by using the Shotgun approach. Its aim is to identify the nucleic acid diversity present in the sample (either DNA, RNA or both, depending on the sequencing method), in order to provide information about features of the viruses within the samples such as drug resistance, viral genotypes and virus epidemiology. The sensitivity of this method is affected by the presence of contaminating nucleic acids from the host and other microorganisms. This method has been used for the sequencing of viruses like Epstein-Barr virus (EBV) and HCV. It may be used also to provide information about cancer evolution and integrated virus genomes in cases of virus-associated cancers. This method requires a low number of PCR cycles, so the consequent risk of contamination is decreased. Although no primers or probes are required, the cost to obtain enough data is high.[13] Because this method is agnostic to expected viral content of a sample, it can be used to identify new virus species or divergent members of known species. It therefore has a role in clinical diagnostics, such as identification of pathogens causing encephalitis.

PCR amplicon enrichment

Its aim is to identify the organism and in order to do that, it enriches a portion of the genome of the virus before sequencing. For the amplification it uses specific primers for a highly-conserved target sequence. This method has been used to track Ebola virus and Zika virus during their outbreaks or to sequence the whole genome of HCMV. Another possible application is the monitoring of mutations associated to drug-resistance in order to administer the more efficient drug to the patient. Although this method is cheaper than the metagenomic approach and has a great specificity and sensitivity, it has some limits: it requires many PCR cycles so it can introduce mutations and contaminants and the primers may be subjected to mismatches.[13] Clinical samples may lack sufficient nucleic acid to enable many PCR reactions; this makes PCR amplicon sequencing of viruses more appropriate if the viral genome is small (eg influenza, norovirus or HIV), or if the virus has been cultured to increase the available genomic material.

Target enrichment

It is an overlapping PCR method. It does not require a culture step because it sequences the whole viral genome directly from the clinical sample. Small oligonucleotides, complementary to the target, are used as probes for a hybridization reaction. The probes can be bound to a solid phase or to magnetic beads in liquid phase. Capture is followed by a small number of PCR cycles and shotgun sequencing. This method can be used to compare the genome of healthy cells and of tumor cells in cases of virus-associated cancer. It has been used to characterize HCV, HSV-1, HCMV and other viruses. The presence of overlapping probes increases the tolerance for primer mismatches but their design requires high cost and time so a rapid response is limited.[13]

Applications
  • Finding ways to use modified viruses as therapeutic agents for plants
  • Analysis of viruses
  • Analysis on how viruses can affect other organisms (for example bacteria)
  • Discover if viruses can shape the microbiome [14]
  • Detection of all the drug-resistance variants in one test
  • Contribution to the viral classification, providing a new criteria based on the information obtained from their genomes and not on their biological characteristics. This would allow the possibility to classify also new-discovered viruses that are still unknown by the biological point of view [15]
  • In clinics for difficult-to-diagnose cases[16]
  • Used to help better understand the virome [16]

References
  1. Angly FE; Felts B; Breitbart M; Salamon P; Edwards RA; Carlson C; Chan AM; Haynes M; Kelley S; Liu H; Mahaffy JM; Mueller JE; Nulton J; Olson R; Parsons R; Rayhawk S; Suttle CA; Rohwer F (2006). "The marine viromes of four oceanic regions". PLOS Biology. 4 (11): e368. doi:10.1371/journal.pbio.0040368. PMC 1634881. PMID 17090214.
  2. Culley, A. I.; Lang, A. S.; Suttle, C. A. (2006). "Metagenomic analysis of coastal RNA virus communities". Science. 312 (5781): 1795–1798. Bibcode:2006Sci...312.1795C. doi:10.1126/science.1127404. PMID 16794078. S2CID 20194876.
  3. Pratama, Akbar Adjie; van Elsas, Jan Dirk (August 2018). "The 'Neglected' Soil Virome – Potential Role and Impact". Trends in Microbiology. 26 (8): 649–662. doi:10.1016/j.tim.2017.12.004. ISSN 0966-842X. PMID 29306554.
  4. Kristensen, David M.; Mushegian, Arcady R.; Dolja, Valerian V.; Koonin, Eugene V. (2010). "New dimensions of the virus world discovered through metagenomics". Trends in Microbiology. 18 (1): 11–19. doi:10.1016/j.tim.2009.11.003. PMC 3293453. PMID 19942437.
  5. Bernardo, P; Albina, E; Eloit, M; Roumagnac, P (May 2013). "Pathology and viral metagenomics, a recent history". Med Sci (Paris). (in French). 29 (5): 501–8. doi:10.1051/medsci/2013295013. PMID 23732099.
  6. Simmonds P, Adams MJ, Benkő M, Breitbart M, Brister JR, Carstens EB, Davison AJ, Delwart E, Gorbalenya AE, Harrach B, Hull R, King AMQ, Koonin EV, Krupovic M, Kuhn JH, Lefkowitz EJ, Nibert ML, Orton R, Roossinck MJ, Sabanadzovic S, Sullivan MB, Suttle CA, Tesh RB, van der Vlugt RA, Varsani A, Zerbini FM (2017). "Consensus statement: Virus taxonomy in the age of metagenomics" (PDF). Nature Reviews Microbiology. 15 (3): 161–168. doi:10.1038/nrmicro.2016.177. PMID 28134265. S2CID 1478314.CS1 maint: multiple names: authors list (link)
  7. Paez-Espino D, Chen AI, Palaniappan K, Ratner A, Chu K, Szeto E, Pillay M, Huang J, Markowitz VM, Nielsen T, Huntemann M, Reddy TBK, Pavlopoulos GA, Sullivan MB, Campbell BJ, Chen F, McMahon K, Hallam SJ, Denef V, Cavicchioli R, Caffrey SM, Streit WR, Webster J, Handley KM, Salekdeh GH, Tsesmetzis N, Setubal JC, Pope PB, Liu W, Rivers AR, Ivanova NN, Kyrpides NC (2016). "IMG/VR: A database of cultured and uncultured DNA Viruses and Retroviruses". Nucleic Acids Research. 45 (D1): D457–D465. doi:10.1093/nar/gkw1030. PMC 5210529. PMID 27799466.CS1 maint: multiple names: authors list (link)
  8. Paez-Espino D, Roux S, Chen IA, Palaniappan K, Ratner A, Chu K, et al. (2018). "IMG/VR v.2.0: an integrated data management and analysis system for cultivated and environmental viral genomes". Nucleic Acids Res. 47 (D1): D678–D686. doi:10.1093/nar/gky1127. PMC 6323928. PMID 30407573.
  9. Paez-Espino D, Eloe-Fadrosh EA, Pavlopoulos GA, Thomas AD, Huntemann M, Mikhailova N, Rubin E, Ivanova NN, Kyrpides NC (2016). "Uncovering Earth's Virome". Nature. 536 (7617): 425–30. Bibcode:2016Natur.536..425P. doi:10.1038/nature19094. PMID 27533034. S2CID 4466854.CS1 maint: multiple names: authors list (link)
  10. Paez-Espino D, Pavlopoulos GA, Ivanova NN, Kyrpides NC (2016). "Non-targeted virus sequence discovery pipeline and virus clustering for metagenomic data". Nature Protocols. 12 (8): 1673–1682. doi:10.1038/nprot.2017.063. PMID 28749930. S2CID 2127494.CS1 maint: multiple names: authors list (link)
  11. Carroll, Dennis; Daszak, Peter; Wolfe, Nathan D.; Gao, George F.; Morel, Carlos M.; Morzaria, Subhash; Pablos-Méndez, Ariel; Tomori, Oyewale; Mazet, Jonna A. K. (2018-02-23). "The Global Virome Project". Science. 359 (6378): 872–874. Bibcode:2018Sci...359..872C. doi:10.1126/science.aap7463. ISSN 0036-8075. PMID 29472471. S2CID 3543474.
  12. Schmidt, Charles (2018-10-11). "The virome hunters". Nature Biotechnology. 36 (10): 916–919. doi:10.1038/nbt.4268. ISSN 1087-0156. PMC 7097093. PMID 30307913.
  13. Houldcroft, Charlotte J.; Beale, Mathew A.; Breuer, Judith (2017-01-16). "Clinical and biological insights from viral genome sequencing". Nature Reviews Microbiology. 15 (3): 183–192. doi:10.1038/nrmicro.2016.182. ISSN 1740-1526. PMC 7097211. PMID 28090077.
  14. Cesar Ignacio-Espinoza, J; Solonenko, Sergei A.; Sullivan, Matthew B (October 2013). "The global virome: not as big as we thought?". Current Opinion in Virology. 3 (5): 566–571. doi:10.1016/j.coviro.2013.07.004. ISSN 1879-6257. PMID 23896279.
  15. Simmonds, Peter; Adams, Mike J.; Benkő, Mária; Breitbart, Mya; Brister, J. Rodney; Carstens, Eric B.; Davison, Andrew J.; Delwart, Eric; Gorbalenya, Alexander E. (2017-01-03). "Virus taxonomy in the age of metagenomics". Nature Reviews Microbiology. 15 (3): 161–168. doi:10.1038/nrmicro.2016.177. ISSN 1740-1526. PMID 28134265.
  16. Dutilh, Bas; Reyes, Alejandro; Hall, Richard; Whiteson, Katrine (September 2017). "Editorial: Virus Discovery by Metagenomics: The (Im)possibilities". Frontiers in Microbiology. 8 (1710): 1710. doi:10.3389/fmicb.2017.01710. PMC 5596103. PMID 28943867.

See also
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