Saturday, 18th November 2017

Virology and Microbiology

                Genetic variability of RNA viruses




Esteban Domingo Solans




Research summary:

Our group pioneered (1978-1990) the discovery of viral quasispecies, a highly complex and dynamic population structure, with profound implications in viral evolution and pathogenesis. Our results of the last 40 years on quasispecies dynamics have been amply confirmed by application of deep sequencing methodology in several laboratories in the last three years. The genome of an RNA viral population is a weighted average of myriads of different, related sequences which are changing continuously. The consensus (or average) sequence that we write for convenience, and that fills textbooks and data banks, is an abstraction that may not even exist in the population that the sequence aims at representing. Dynamic mutant clouds are prepared to respond to changing environments by selection of subsets of sequences preferentially over others, an event crucial to disease progression.


Quasispecies dynamics demands that new approaches be investigated for the prevention and treatment of diseases associated with RNA viruses, to counteract the adaptive capacity conferred by the mutant clouds.


We are working in the development of new antiviral strategies that can avoid selection of treatment-escape mutant viruses. The work involves mainly three viruses: the picornavirus foot-and-mouth disease virus (FMDV), the arenavirus lymphocytic choriomeningitis virus (LCMV), and more recently the hepacivirus hepatitis C virus (HCV). We are collaborating with several groups to expand the scope of our work: with the group of Nuria Verdaguer (IBM, CSIC) on studies of structural alterations of the viral polymerase of mutagen-resistant mutants of FMDV; with Juan Carlos de la Torre (Scripps Research Institute, La Jolla) on lethal mutagenesis of LCMV; with Pablo Gastaminza (CNB, CSIC), Josep Quer, Celia Perales (Hospital Vall d’Hebrón), and Aurora Sánchez (IIB, CSIC) on HCV dynamics and host-virus interactions in cell culture and in the clinic.


During the last years, our emphasis has been on HCV given its magnitude as public health problem worldwide, and the direct implication of its quasispecies nature in the hepatic disease and response to treatments. The main contributions of our group to HCV have been: (i) that interferon resistance is multigenic; (ii) to make available HCV populations of different fitness level to study fitness effects on HCV biology; (iii) that viral fitness is a determinant of multi-drug resistance in absence of specific resistance mutations, (iv) the absence of population equilibrium (steady-state distribution of mutations and phenotypic stasis) even after extensive multiplication in a constant cellular environment, and (v) the design of sequential and combination treatments using both mutagenic and non-mutagenic antiviral agents for suppression of viral infectivity (summary in Figure 1). These investigations continue at the present time.

Lethal mutagenesis is an expanding field of research that has opened a new chapter in antiviral pharmacology. Our work, in collaboration with the late John Holland (UCSD), provided the first experimental evidence that an increase of mutation rate promoted by chemical mutagenesis was detrimental to RNA virus survival (1990-1997). In our current screening of mutagenic agents that have been licensed for human use, we have documented that the broad spectrum antiviral agent favipiravir (T-705) is mutagenic for FMDV and HCV, and can lead to the extinction of these viruses. We have collaborated with the group of Juan Carlos Sáiz (INIA) to show that favipiravir is also mutagenic for West Nile virus and can drive the virus towards extinction. We keep incorporating new, broad-spectrum, mutagenic and non-mutagenic agents for our antiviral designs, aimed at suppressing a broad range of RNA viral pathogens. A major unresolved issue is the integration of mutagenic and non-mutagenic inhibitors into antiviral designs to combat RNA viral diseases.

We have discovered a new mechanism of resistance to lethal mutagenesis of FMDV to ribavirin and 5-fluorouracil, consisting in selection of viral mutants that can modulate nucleotide incorporation to avoid the mutational bias produced by mutagenic nucleotides. This mechanism does not alter the amplitude of the mutant spectrum, and the corresponding adaptive flexibility. Interestingly, a joker mutation (meaning a given mutation that is repeatedly encountered when the virus is subjected to different selective constraints) in non-structural protein 2C modulated nucleotide incorporation in response to ribavirin mutagenesis, in absence of mutations in the viral polymerase. These observations emphasize the multiple adaptive resources that RNA viruses have to respond to hostile environments, and justify our ongoing efforts to search for antiviral designs to strongly suppress virus multiplication.

We have also continued collaboration with Susanna Manrubia (CNB, CSIC) that has represented a highly clarifying link between theoretical studies and experimental designs. In an interesting connection between virus expansion in the field and the clonal evolution of organisms, we have proposed for viruses a distinction between mechanistically active but inconsequential recombination, and evolutionary relevant recombination. Based on the available evidence, we think that mutation and recombination occur continuously during RNA virus replication, but that a majority of mutant and recombinant genomes do not survive (they are subjected to negative selection). However, there are subsets of mutations that combined with punctuated, biologically relevant recombination events, contribute to virus survival and evolution (Figure 2). These studies are contributing also to a deeper understanding of the molecular events underlying viral disease processes.

Our major aim is to use the new understanding of viral dynamics that has been provided by the framework of quasispecies theory to find new ways to suppress viral multiplication. It is our conviction that new antiviral designs, which are based on the understanding of the implications of quasispecies, can be used to control established, emergent, and re-emergent viral pathogens (Figure 3).


figura 1

Figure 1: Graphic summary of our recent work on quasispecies implications, and lethal mutagenesis of RNA viruses. The left panel illustrates HCV mutational waves (color-coded) quantified by molecular cloning-Sanger sequencing and deep sequencing during viral multiplication in a constant biological environment. The waves drawn here have been found by the two methods, excluding that they are due to a sampling bias. Panels on the right illustrate the conceptual basis of lethal mutagenesis, with an example of HCV extinction (top), a serial passage design (middle), and HCV extinction associated with ribavirin treatment (bottom). Detailed information is in the indicated references, and the reference list.


figura 2

Figure 2: Schematic representation of clonal evolution of viruses. From an initial infection (Origin, bottom) multiple sublineages are generated. Recombination takes place at any branch (double-headed arrows perpendicular to branches). Biologically relevant diversification is illustrated by red and blue branches. Recombination at a discontinuity point (large vertical double-headed arrow) is biologically relevant because it generates mosaic genomes with new phenotypes. Clonal evolution continues until a new discontinuity point is reached. The scheme does not imply a space or time scale. Reproduced from Perales et al. 2015, with permission from the National Academy of Sciences USA.


figura 3

Figure 3: A summary of the approach and main goal of our research.



Relevant publications (since 2012) grouped by topics :


Quasispecies implications:

  • Domingo, E., Sheldon, J. and Perales, C. (2012). Viral quasispecies evolution. Microbiology and Molecular Biology Reviews, 76, (2)159-216.
  • Andino, R. and Domingo, E. (2015). Viral quasispecies. Virology, 479-480: 46-51.
  • Domingo, E. Perales, C. (2016). Viral quasispecies and lethal mutagenesis. European Review, 24(1):39-48.
  • Domingo, E. Perales, C. (2016). Species Concepts: Viral quasispecies. In: Kliman, R.M. (ed.), Encyclopedia of Evolutionary Biology, vol.4, pp. 228-235. Oxford: Academic Press.
  • Domingo, E. (2016). Virus as Populations. Composition, complexity, dynamics and biological implications. Academic Press, Elsevier, Amsterdam.
  • Domingo, E., Schuster, P. (eds.) (2016). Quasispecies: From Theory to Experimental Systems.
  • Domingo, E., Schuster, P. (2016). What is a quasispecies? Historical origins and current scope.
  • Gregori, J., Perales, C., Rodríguez-Frías, F., Esteban, J.I., Quer, J., Domingo, E. (2016). Viral Quasispecies Complexity Measures. Virology 493: 227-237.
  • Domingo, E., de la Higuera, I., Moreno, E., de Ávila, A.I., Agudo, R., Arias, A., Perales, C. (2017). Quasispecies dynamics taught by natural and experimental evolution of foot-and-mouth disease virus. In: F. Sobrino and E. Domingo (Eds.). Foot-and-Mouth Disease Virus: Current Research and Emerging Trends, Horizon Scientific Press – Caister Academic Press, Poole, UK, pp. 147-170.
  • Moreno, E.; Gallego, I.; Gregori, J.; Lucia-Sanz, A.; Soria, M. E.; Castro, V.; Beach, N. M.; Manrubia, S.; Quer, J.; Esteban, J. I.; Rice, C. M.; Gomez, J.; Gastaminza, P.; Domingo, E.; Perales, C. (2017). Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

Foot-and-mouth disease virus and the viral polymerase:

  • Ferrer-Orta, C., de la Higuera, I., Caridi, F., Sanchez-Aparicio, M.T., Moreno, E. Perales, C., Singh, K., Sarafianos, S.G., Sobrino, F. Domingo, E., Verdaguer, N. (2015). Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.
  • Sobrino, F. and Domingo, E. (eds.) (2017). “Foot-and-Mouth Disease Virus: Current Research and Emerging Trends”, Horizon Scientific Press – Caister Academic Press, Poole, UK.

HCV virus-host interactions:

  • Madejón, A., Sheldon, J., Francisco-Recuero, I., Perales, C., Dominguez-Beato, M., Lasa, M., Sanchez-Perez, I., Muntané, J., Domingo, E., Garcia-Samaniego, J., Sanchez-Pacheco, A. (2015). Hepatitis C virus-mediated Aurora B kinase inhibition modulates inflammatory pathway and viral infectivity. Journal of Hepatology, 63(2): 312-9.
  • Perez-del-Pulgar, S., Gregori, J., Rodriguez-Frias, F., Gonzalez, P., García-Cehic, D., Ramirez, S., Casillas, R., Domingo, E., Esteban, J. I., Forns, X., Quer, J. (2015). Quasispecies dynamics in hepatitis C liver transplant recipients receiving grafts from hepatitis C virus infected donors.
  • Valero, M.L., Sabariegos, R., Cimas, F., Perales, C., Domingo, E., Sánchez-Prieto, R., Mas, A. (2016). HCV RNA-dependent RNA polymerase interacts with Akt/PKB inducing its subcellular re-localization.

Drug resistance:

  • Perales, C., Beach, N. M., Gallego, I., Soria, M. E., Quer, J., Esteban, J. I., Rice, C., Domingo, E., and Sheldon, J. (2013). Response of hepatitis C virus to long-term passage in the presence of interferon-α. Multiple mutations and a common phenotype. J. Virol., 87(13), 7593-7607.
  • Sheldon, J., Beach, NM., Moreno, E., Gallego, I., Piñeiro, D., Martínez-Salas, E., Gregori, J., Quer, J., Esteban, JI., Rice, CM., Domingo, E., Perales, C. (2014). Increased replicative fitness can lead to decreased drug sensitivity of hepatitis C virus. J. Virol., 88(20):12098-12111.
  • Perales, C., Quer, J., Gregori, J., Esteban, J.I., Domingo, E. (2015). Resistance of hepatitis C virus to inhibitors: complexity and clinical implications. Viruses, 7:5746-5766.
  • Gallego, I., Sheldon, J., Moreno, E., Gregori, J., Quer, J., Esteban. J.I., Rice, C.M., Domingo, E., Perales, C. (2016). Barrier-Independent, Fitness-Associated Diferences in Sofosbuvir Efficacy against Hepatitis C virus.
  • Martín, V., Perales, C., Fernández-Algar, M., Dos Santos, HG., Garrido, P., Pernas, M., Parro, V., Moreno, M., García-Pérez, J., Alcamí, J., Torán, JL., Abia, D., Domingo, E., Briones, C. (2016). An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies. PLoS ONE, 11(12):e0166902.

Lethal mutagenesis:

  • Ortega-Prieto, A.M., Sheldon, J., Grande-Pérez, A., Tejero, H., Gregori, J., Quer, J., Esteban, J.I., Domingo, E. and Perales, C. (2013). Extinction of hepatitis c virus by ribavirin in hepatoma cells involves lethal mutagenesis. PLoS ONE, 8(8): e71039.
  • Perales, C., Domingo, E. (2016). Antiviral strategies based on lethal mutagenesis and error threshold. Curr. Top. Microbiol. Immunol., 392:323-339.
  • Agudo, R., de la Higuera, I., Arias, A., Grande-Perez, A., Domingo, E. (2016). Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism. Virology 494:257-266.
  • De Ávila, A.I., Gallego, I., Soria, M.E., Gregori, J., Quer, J., Esteban, J.I., Rice, C.M., Domingo, E., Perales, C. (2016). Lethal mutagenesis of hepatitis C virus induced by favipiravir. PLoS ONE, 11 (10): e0164691.
  • de Ávila, A. I.; Moreno, E.; Perales, C.; Domingo, E. (2017). Favipiravir can evoke lethal mutagenesis and extinction of foot-and-mouth disease virus. Virus Res., 233, 105-112.
  • de la Higuera, I.; Ferrer-Orta, C.; de Avila, A. I.; Perales, C.; Sierra, M.; Singh, K.; Sarafianos, S. G.; Dehouck, Y.; Bastolla, U.; Verdaguer, N.; Domingo, E. (2017). Molecular and Functional Bases of Selection against a Mutation Bias in an RNA Virus. Genome Biol. Evol., 9 (5), 1212-1228.
  • Escribano-Romero, E., Jiménez de Oya, N., Domingo, E., Saiz, J.C. (2017). Extinction of West Nile virus by favipiravir through lethal mutagenesis.

General viral evolution:

  • Perales, C. Moreno, E. Domingo, E. (2015). Clonality and intracellular polyploidy in virus evolution and pathogenesis. Proc Natl Acad Sci USA, 112(29):8887-92.




  • N. Sevilla, E. Domingo, C. Escarmís, S. Ojosnegros, J. García-Arriaza, M. Sanz-Rojo, T. Rodríguez. "Vacuna atenuada para la fiebre aftosa". Nº DE SOLICITUD: P200801583. Patente concedida en España el 16/06/2011. Nº PUB: ES2344875.



Doctoral Theses:

  • Héctor Moreno Borrego (2012).  Dinámica poblacional del virus de la coriomeningitis linfocitaria del ratón en su interacción con agentes mutagénicos. Universidad Autónoma de Madrid. Directors: Esteban Domingo y Verónica Martín.
  • Ignacio de la Higuera Hernández (2014). Factores determinantes del reconocimiento de nucleótidos en el virus de la fiebre aftosa. Universidad Autónoma de Madrid. Director: Esteban Domingo.
  • Ana Mª Ortega Prieto (2014). Mutagénesis letal del virus de la Hepatitis C. Universidad Autónoma de Madrid. Directors: Esteban Domingo y Celia Perales.
  • Elena Moreno del Olmo (2017). Evolución a largo plazo de virus RNA en ambiente biológico constante. Universidad Autónoma de Madrid. Directors: Esteban Domingo y Celia Perales.



Other Activities:

  • Académico Numerario de la Real Academia de Ciencias Exactas, Físicas y Naturales, adscrito a la Sección de Ciencias Naturales, (2011).
  • Miembro de la Red Española de Biofísica, coordinada por el Dr. David Reguera, desde 2011.
  • Miembro del Global Virology Network, coordinado por el Dr. Robert Gallo, desde 2011.
  • Miembro del Comité Organizador del Congreso FEMS 2011 (Ginebra, Suiza, 2011).
  • Editor asociado de la revista Virus Research desde 2012.