A segmented form of foot-and-mouth disease virus interferes with standard virus: A link between interference and competitive fitness

AbstractSerial passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells at high multiplicity of infection resulted in dominance of particles containing defective RNAs that were infectious by complementation in the absence of standard viral RNA. In the present study, we show that the defective FMDV particles interfere with replication of the cognate standard virus. Coinfections of defective FMDV with standard FMDV mutants that differ up to 151-fold in relative fitness have documented that the degree of interference is higher for low fitness than for high fitness standard virus. These comparisons suggest a likely overlap between those mechanisms of intracellular competition that underlie viral interference and those expressed as fitness differences between two viruses when they coinfect the same cells. Interference may contribute to the selective pressures that help maintain dominance of segmented defective RNAs over the standard FMDV genome

Viral Genome Segmentation Can Result from a Trade-Off between Genetic Content and Particle Stability

The evolutionary benefit of viral genome segmentation is a classical, yet unsolved question in evolutionary biology and RNA genetics. Theoretical studies anticipated that replication of shorter RNA segments could provide a replicative advantage over standard size genomes. However, this question has remained elusive to experimentalists because of the lack of a proper viral model system. Here we present a study with a stable segmented bipartite RNA virus and its ancestor non-segmented counterpart, in an identical genomic nucleotide sequence context. Results of RNA replication, protein expression, competition experiments, and inactivation of infectious particles point to a non-replicative trait, the particle stability, as the main driver of fitness gain of segmented genomes. Accordingly, measurements of the volume occupation of the genome inside viral capsids indicate that packaging shorter genomes involves a relaxation of the packaging density that is energetically favourable. The empirical observations are used to design a computational model that predicts the existence of a critical multiplicity of infection for domination of segmented over standard types. Our experiments suggest that viral segmented genomes may have arisen as a molecular solution for the trade-off between genome length and particle stability. Genome segmentation allows maximizing the genetic content without the detrimental effect in stability derived from incresing genome length

The structure of a protein primer-polymerase complex in the initiation of genome replication

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix α8 of the fingers domain and helix α13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction. © 2006 European Molecular Biology Organization | All Rights Reserved.Work in Barcelona was supported by Grants BIO2002-00517 and BFU2005-02376/BMC. Work in Madrid by Grants BMC 2001.1823.C02-01, BFU2005-00863/BMC and Fundación R Areces. CF and AA were supported by I3P fellowships from Ministerio de Educación y Ciencia. RA was supported by an FPI fellowship from Comunidad de Madrid. The financial support was provided by the ESRFPeer Reviewe

The foot-and-mouth disease RNA virus as a model in experimental phylogenetics

Phylogenetic reconstruction methods are subject to two types of limitations: our knowledge about the true history of organisms and the gross simplification implied in the numerical simulation models of the relationships between them. In such a situation, experimental phylogenetics provides a way to assess the accuracy of the phylogenetic reconstruction methods. Nonetheless, this capacity is only feasible for organisms in which replication and mutation rates are high enough to provide valuable data. On the other hand, experimental phylogenetics also provides insights on the main evolutionary processes acting on viral variability under different population dynamics. Our study with the foot-and-mouth disease virus (FMDV) strongly suggests that the phylogenetic reconstruction methods can infer erroneous phylogenies due to nucleotide convergences between isolates belonging to different experimental lineages. We also point out that the diverse evolutionary mechanisms acting in different experimental dynamics generate alterations and change the frequencies of genetic variants, which can lead to the misinterpretation of the real evolutionary history

New vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines

Background: New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260). Principal Finding: Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV. Conclusions: The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms. © 2010 Rodríguez-Calvo et al.Ministerio de Ciencia e Innovación, Spain, and European Union, Network of Excellence, EPIZONE (Contract # FOOD-CT-2006-016236). CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Work at Centro de Biología Molecular ‘‘Severo Ochoa’’ (CISC-UAM) was supported by an institutional grant from Fundación Ramón Arece

New Vaccine Design Based on Defective Genomes That Combines Features of Attenuated and Inactivated Vaccines

[Background] New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260).[Principal Finding] Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV.[Conclusions] The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms.This research was supported by grants AGL2004-0049, AGL2007-61374, CSD2006-07 and BFU2008-02816/BMC from Ministerio de Ciencia e Innovación, Spain, and European Union, Network of Excellence, EPIZONE (Contract # FOOD-CT-2006-016236). CIBERehd (Centro de Investigacio´n Biome´dica en Red de Enfermedades Hepa´ticas y Digestivas) is funded by Instituto de Salud Carlos III. Work at Centro de Biologı´a Molecular ‘‘Severo Ochoa’’ (CISC-UAM) was supported by an institutional grant from Fundacio´n Ramo´n Areces. T.R-C. was supported by a contract from Comunidad Auto´noma de Madrid; S.O. and M.S-R were supported by a predoctoral fellowship from the Ministerio de Educacio´n y Ciencia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure

Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

BACKGROUND: Success of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown. METHODOLOGY AND PRINCIPAL FINDINGS: we have constructed four mutant FMDVS that encode only one VPG: either VPg(1), VPg(3), or two chimeric versions containing part of VPg(1) and VPg(3). All mutants, except that encoding only VPg(1), were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and detach BHK-21 cells from the monolayer, the first stage of cell killing. CONCLUSIONS: The results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses pathogenesis are discussed

Virus emergentes. La amenaza oculta

Hace escasas décadas había un optimismo general respecto a la efectividad de las medidas para controlar la mayoría de las enfermedades infecciosas. Sin embargo, ese optimismo pronto se derrumbó debido a la aparición de nuevas enfermedades causadas por virus desconocidos hasta entonces. El SIDA o las infecciones por el virus Ébola o por nuevos tipos de hantavirus son buenos ejemplos de enfermedades que han emergido en los últimos años y para las cuales aún no existe un tratamiento efectivo. ¿Qué está sucediendo? ¿De dónde surgen estos nuevos virus? ¿Por qué es tan difícil encontrar un tratamiento frente a ellos? A lo largo de este libro se intentará responder a estas preguntas haciendo especial hincapié en que en la naturaleza casi nada es casual y muchas veces son las alteraciones en el ambiente las responsables de sacar a la luz virus desconocidos, con potencial para causar epidemias con graves impactos en la salud mundial

Nucleotide sequence of the genes 3 and 4 of bacteriophage ø29

The nucleotide sequence of an early region of the Ø29 genome has been determined. The sequenced region includes genes 3 and 4, which code for the protein covalently linked to the 5′ ends of Ø29 DNA and the protein involved in the control of late transcription, respectively. The position and nature of the mutations of mutants sus3(9l) and sus4(56) has also been determined.Peer reviewe