REVIEWS Viral genomics in Ebola virus research Nicholas Di Paola1, Mariano Sanchez- Lockhart1, Xiankun Zeng1, Jens H. Kuhn2 and Gustavo Palacios 1 ✉ Abstract | Filoviruses such as Ebola virus continue to pose a substantial health risk to humans. Advances in the sequencing and functional characterization of both pathogen and host genomes have provided a wealth of knowledge to clinicians, epidemiologists and public health responders during outbreaks of high- consequence viral disease. Here, we describe how genomics has been historically used to investigate Ebola virus disease outbreaks and how new technologies allow for rapid, large- scale data generation at the point of care. We highlight how genomics extends beyond consensus-level sequencing of the virus to include intra- host viral transcriptomics and the characterization of host responses in acute and persistently infected patients. Similar genomics techniques can also be applied to the characterization of non- human primate animal models and to known natural reservoirs of filoviruses, and metagenomic sequencing can be the key to the discovery of novel filoviruses. Finally , we outline the importance of reverse genetics systems that can swiftly characterize filoviruses as soon as their genome sequences are available. Next- generation sequencing Infections with viruses of the mononegaviral fam- requires more detailed genomic information than virus 6 A collection of continuously ily Filoviridae (in particular, members of the genera consensus- genome sequencing. Indeed, as predicted , evolving technologies and Ebolavirus and Marburgvirus) are an increasing threat metagenomic sequencing has become a powerful tool for techniques that allow for the to mankind. Until recently, the infrequent spillover of identifying novel viruses and, crucially, for predicting digitalization of genomic 7 material. these viruses into humans and the fact that spillover pathogen emergence . Targeted or unbiased sequencing often occurred in remote locations, coupled with a lim- of individual clinical samples aids in the identification of Metagenomic sequencing ited knowledge of non- human reservoir hosts, the use outbreaks, the determination of outbreak aetiology and The sequencing of genetic of low- output genomic sequencing, and biosafety and the definition of virus transmission chains by identify- material recovered directly biosecurity restrictions on filovirus research, contribu- ing chain- defining single nucleotide polymorphisms from an environmental or clinical sample that allows the ted to the paucity of publicly available data on filovirus (SNPs). Furthermore, field transcriptomics improves our 1 identification of all organisms genome sequences . In December 2013, complete genome understanding of host responses to virus infection and and mobile genetic elements sequences for only 29 ebolaviruses and 65 marburg- will be important in deciphering the differences between represented in the sample. viruses were available2 despite the fact that 35 outbreaks asymptomatic and symptomatic disease states and in pre- of natural filovirus disease had been recorded3. dicting whether patients with acute and chronic disease Since December 2013, atypically extensive filovirus will survive8. Functional genomics is becoming the tool disease outbreaks, from 2013 to 2016 in Western Africa of choice for the rapid characterization of patient- specific and from 2018 to present in the Democratic Republic viruses that have not been isolated in culture or that can- of the Congo, have profoundly impacted public health not be equitably shared among laboratories across bor- systems. At least 13,675 fatalities from filovirus disease ders9. Finally, the genomic analysis of patient- specific were reported between December 2013 and April 5, 2020 viruses also enables precision medicine by predicting the 1 United States Army Medical (REFS4,5). By leveraging the continued development and efficacy of available medical countermeasures (MCMs) Research Institute of next- generation sequencing Infectious Diseases, Fort improvement of technology, against these individual viruses. Detrick, Frederick, MD, USA. >800 complete filovirus genome sequences and over Here, we review how recent advances in genomic 2Integrated Research Facility 2,000 draft genomes (that is, genomes with >80% cov- technologies have shaped past and current responses at Fort Detrick, National erage) across classified and unclassified filovirus family to outbreaks of Ebola virus disease (EVD), including Institute of Allergy and members have become available since 2013 (REF.2). Indeed, insights into filovirus diversity and evolution. We empha- Infectious Diseases, National among high- consequence, Risk Group 4 viruses, the size the importance of accurate and rapid large- scale Institutes of Health, Frederick, MD, USA. genomic diversity of filoviruses is arguably becoming data generation and its implications for the develop- ✉e- mail: gustavo.f.palacios.civ the best characterized. ment of MCMs and outbreak response. We also examine @mail.mil The impact and importance of genomics in pathogen the phenomena of Ebola virus (EBOV) persistence in https://doi.org/10.1038/ characterization is routinely demonstrated, but the rapid human hosts and provide an overview of recent genomic s41579-020-0354-7 prediction of, response to and mitigation of outbreaks advances in threat characterization, vaccine development NATURE REVIEWS | MICROBIOLOGY VOLUME 18 | JULY 2020 | 365 REVIEWS a Confirmed cases Case fatality rate Draft genome (>80% coverage) availability Guinea Western Africa 2013–2016: Democratic Republic of the Congo (Zaire until May 1997) 1856 Bonduni 1977: Guinea: 3,814 67% 1 100% 1 Exported to France, Germany, Yambuku 1976: Italy, Liberia, Mali, Netherlands, 12 Nigeria, Norway, Senegal, 318 88% Sierra Leone, Spain, Likati 2017: Switzerland, UK, USA: 8 50% 3 24,838 35% Nord-Kivu/Sud-Kivu/Ituri 2018–ongoing: 3444 66% Gabon Europe, USA Exported to Uganda: 2018–ongoing: 55 Booué 1996–1997: 1 100% 60 75% 1 Inkanamongo 2014: 19 Mendemba 2001–2002: 69 71% 65 81% 1 Bikoro 2018: 16 Mayibout II 1996: 54 61% 31 67% 1 Luebo/Mweka 2008–2009: 1 Mékouka 1994–1995: 32 47% 52 60% 1 Mweka/Luebo 2007: 264 71% 7 Republic of the Congo Kikwit 1995: 317 77% 7 Olloba/Entsiami 2001–2002: 59 73% 2 Olloba 2002: South Africa Exported to Gabon: Johannesburg 1996–1997: 11 91% Exported from Gabon (Booué): Yembelengoye 2002–2003: 2 50% 143 86% Mbandza 2003–2004: 35 83% 1 Total EVD cases by country Etoumbi 2005: 1–10 101–300 11 82% 11-100 >301 b West Germany and Yugoslavia MARV: Marburg 1967 China Hungary MARV: Belgrade 1967 MLAV: 2019 LLOV: 2018 MARV: Frankfurt am Main 1967 HUJV: 2018 Exported from Uganda: 31 23% XILV: 2018 Spain LLOV: 2011 Philippines RESTV: 1989–1990 Exported to USA: 1 0% Côte d’Ivoire RESTV: 2008 : 2015 TAFV: Parc National de RESTV Taï 1995: Exported to Switzerland Sudan 1 0% SUDV: Nzara 1976: 284 53% SUDV: Nzara 1979: 34 65% Guinea SUDV: Yambio 2004: 17 41% BOMV: 2019 Kenya Sierra Leone MARV: Nairobi 1980: 2 50% BOMV: 2018 RAVV: Kitum Cave 1987: 1 100% MARV: 2020 BOMV: 2019 Democratic Republic of the Congo Uganda MARV: Durba 1998–2000: 153 84% SUDV: Gulu 2000–2001: 425 53% RAVV: Durba 1998–2000: 1 100% BDBV: Bundibugyo 2007–2009: 149 25% BDBV: Isiro 2012: 57 51% MARV: Kitaka Mine 2007: 3 33% RAVV: Kitaka Mine 2007: 1 0% Angola Filovirus origins MARV: Maramagambo Forest 2008 MARV: Uíge 2004–2005: Exported to USA and Netherlands: 2 50% 252 90% Non-EBOV ebolavirus (BDBV, BOMV, RESTV, SUDV, TAFV) SUDV: Luweero 2011: 1 100% Marburgviruses (MARV and RAVV) SUDV: Kibaale 2012: 24 71% South Africa SUDV: Luwero/Kampala 2012: 7 57% MARV: Johannesburg 1975: Cuevaviruses (LLOV) Striaviruses (XILV) MARV: Kabale 2012: 26 58% Exported from Rhodesia MARV: Kampala 2014: 1 100% 3 33% Thamnoviruses (HUJV) Dianloviruses (MLAV) MARV: Kaptum 2014: 4 75% 366 | JULY 2020 | VOLUME 18 www.nature.com/nrmicro REVIEWS and immunotherapy. Although we focus primarily on schreibersii) in Hungary23 and in Spain24. The Ebolavirus EBOV, these practices can apply to all pathogenic filo- genus was expanded owing to the discovery (via viruses and other high- consequence viruses capable of next-generation sequencing) of Bombali virus (BOMV) sustaining human- to- human transmission. in molossid little free- tailed bats (Chaerephon pumi- lus) and Angolan free- tailed bats (Mops condylurus) Identifying filovirus reservoirs in Guinea, Kenya and Sierra Leone25–27. Finally, a new Although the global distribution and diversity of filo- filovirus genus, Dianlovirus, was recently established viruses remains largely undefined, metagenomic for a highly divergent filovirus, Měnglà virus (MLAV), sequencing is becoming a valuable tool for identifying which was discovered in unspecified rousettes in filovirus reservoirs. Until 1989, disease outbreaks owing China. Preliminary metagenomics studies indicate to infection by ebolaviruses (including EBOV, Sudan virus the existence of other, highly divergent, filoviruses (SUDV) and marburgviruses (including Marburg in the same area of China28–30. The most unexpected virus (MARV) and Ravn virus (RAVV)) had only been finding of genomics- based filovirus discovery was the recorded on the African continent (Fig. 1). As the natural genomic description of Huángjiāo virus (HUJV; genus reservoir hosts of all of these viruses remained uniden- Thamnovirus) and Xīlǎng virus (XILV; genus Striavirus) tified, despite extensive ecological studies, filoviruses in striated frogfish (Antennarius striatus)