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.
... Other hypotheses focus on the optimal strategy for a viral genome, assuming minimal conflict between different genome segments [12]. For example, multipartitism can evolve if smaller viral capsids survive for longer periods of time in the environment [13]. ...
... This contrasts with the highest estimates from natural infections that range from 2 to 13 viral particles per host cell [14,15]. In some viral systems, mechanistic benefits, such as increased particle stability, are large enough to drive the evolution of multipartite viruses with 2 gene segments, but it is unclear whether these mechanistic advantages also exist in other groups of viruses, or other organisms that have evolved multipartitism [13]. Furthermore, evolutionary conflict is common between different viral genomes within populations; such conflict frequently destroys the kind of group optimality that these models assume, and hence we cannot use group-level advantages alone to explain the evolution of this kind of trait [16][17][18][19][20]. ...
... The values summarised in the payoff matrix of Fig 2 represent the number of successful progeny viral genomes that can infect a further host cell. Consequently, these values could reflect a number of different biological mechanisms, such as increased burst size, faster replication speed, or increased particle longevity [11][12][13]. It is possible for multipartitism to provide a group-level benefit in this model if cells infected by both types of cheat are more productive than cells infected by 2 cooperators (e > d). ...
In multipartite viruses, the genome is split into multiple segments, each of which is transmitted via a separate capsid. The existence of multipartite viruses poses a problem, because replication is only possible when all segments are present within the same host. Given this clear cost, why is multipartitism so common in viruses? Most previous hypotheses try to explain how multipartitism could provide an advantage. In so doing, they require scenarios that are unrealistic and that cannot explain viruses with more than 2 multipartite segments. We show theoretically that selection for cheats, which avoid producing a shared gene product, but still benefit from gene products produced by other genomes, can drive the evolution of both multipartite and segmented viruses. We find that multipartitism can evolve via cheating under realistic conditions and does not require unreasonably high coinfection rates or any group-level benefit. Furthermore, the cheating hypothesis is consistent with empirical patterns of cheating and multipartitism across viruses. More broadly, our results show how evolutionary conflict can drive new patterns of genome organisation in viruses and elsewhere.
... However, it remains uncertain which came first in the evolutionary history of viruses: monopartite or segmented/multipartite viral genomes (Michalakis and Blanc 2020). When compared with monopartite genomes, segmented/multipartite genomes showcase certain advantages that have already been thoroughly summarized elsewhere (Lucía-Sanz et al. 2018;Lucía-Sanz and Manrubia 2017;Michalakis and Blanc 2020), including better virion stability (Ojosnegros et al. 2011). These benefits provide a foundation for formulating a hypothesis regarding the evolutionary transition of viral genomes from monopartite to multipartite structures. ...
Viruses with split genomes are classified as being either segmented or multipartite based on whether their genomic segments occur within a single virion or across different virions. Despite variations in number and sequence during evolution, the genomic segments of many viruses are conserved within the untranslated regions (UTRs). In this study, we present a methodology that combines RNA sequencing with iterative BLASTn of UTRs (UTR-iBLASTn) (https://github.com/qq371260/Iterative-blast-v.1.0) to identify new viral genomic segments. Some novel multipartite-like viruses related to the phylum Kitrinoviricota were annotated using sequencing data from field plant samples and public databases. We identified potentially plant-infecting jingmen-related viruses (order Amarillovirales) and jivi-related viruses (order Martellivirales) with at least six genomic components. The number of RNA molecules associated with a genome of the novel viruses in the families Closteroviridae, Kitaviridae, and Virgaviridae within the order Martellivirales reached five. Several of these viruses seem to represent new taxa at the subgenus, genus, and family levels. The diversity of novel genomic components and the multiple duplication of proteins or protein domains within single or multiple genomic components emphasize the evolutionary roles of reassortment and recombination (horizontal gene transfer), and genetic deletion. The relatively conserved UTRs at the genome level might explain the relationships between monopartite and multipartite viruses, as well as how subviral agents such as defective RNAs and satellite viruses can coexist with their helper viruses.
... This fitness was linked to the higher particle thermal stability rather than a faster kinetics of RNA synthesis. Furthermore, a sufficiently high multiplicity of infection (m.o.i.) was considered to be a requirement for persistence [10]. Split genomes in complementary modules could also confer a reassortment capacity to co-infecting similar viruses, leading to the selection of reassortant progeny with potential fitness advantages [11]. ...
Multipartite virus genomes are composed of two or more segments, each packaged into an independent viral particle. A potential advantage of multipartitism is the regulation of gene expression through changes in the segment copy number. Soil-borne beet necrotic yellow vein virus (BNYVV) is a typical example of multipartism, given its high number of genomic positive-sense RNAs (up to five). Here we analyse the relative frequencies of the four genomic RNAs of BNYVV type B during infection of different host plants (Chenopodium quinoa, Beta macrocarpa and Spinacia oleracea) and organs (leaves and roots). By successfully validating a two-step reverse-transcriptase digital droplet PCR protocol, we show that RNA1 and -2 genomic segments always replicate at low and comparable relative frequencies. In contrast, RNA3 and -4 accumulate with variable relative frequencies, resulting in distinct RNA1 : RNA2 : RNA3 : RNA4 ratios, depending on the infected host species and organ.
... The emergence of multipartitism is the focus of many studies that try to explain the condition required for the occurrence of this event; in particular, some models explain the multipartitization of a monopartite virus with the improved ability of the fragmented virus to modulate the production of the different viral proteins carried on multiple segments in response to variations of the environmental conditions (Zwart and Elena 2020). Other studies link multipartitism to a fitness benefit for the fragmented virus, leading to smaller and more stable viral particles hosting only one genome segment compared to the larger virions hosting the monopartite counterpart (Ojosnegros et al. 2011). Finally, a recent study sets up a model to explain multipartitism stating that fragmented version of the viral genome (carrying fundamental genes for the virus) could emerge without any selective pressure from the environment, simply from the production of what the authors call 'cheaters' (like defective interfering RNAs). ...
Recent advances on NGS approaches allowed a broad exploration of viromes from different fungal hosts, unveiling a great diversity of mycoviruses with interesting evolutionary features. The word mycovirus historically applies also to viruses infecting oomycetes but most studies are on viruses infecting fungi, with less mycoviruses found and characterized in oomycetes, particularly in the obligatory biotrophs. We here describe the first virome associated to Bremia lactucae the causal agent of lettuce downy mildew, which is an important biotrophic pathogen for lettuce production and a model system for the molecular aspects of the plant-oomycetes interactions. Among the identified viruses, we could detect i) two new negative sense ssRNA viruses related to the yueviruses, ii) the first example of permuted RdRp in a virus infecting fungi/oomycetes, iii) a new group of bi-segmentd dsRNA viruses showing evidence of recent bi-segmentation and concomitantly, a possible duplication event bringing a bi-segmented genome to three-segmented, iv) a first representative of a clade of viruses with evidence of recombination between distantly related viruses and v) a new ORFan virus encoding for an RdRp with low homology to known RNA viruses, vi) a new virus, belonging to riboviria but not conserved enough to provide a conclusive phylogenetic placement, that shows evidence of a recombination event between a kitrinoviricota-like and a pisuviricota-like sequence. The results obtained show a great diversity of viruses and evolutionary mechanisms previously unreported for oomycetes-infecting viruses supporting the existence of a large diversity of oomycetes-specific viral clades ancestral of many fungal and insect virus clades.
... Genome segmentation is rare among DNA viruses of animal hosts, unlike multipartite DNA viruses of fungi and plants, which package each DNA segment into a separate viral particle 33 . Experiments by Ojosnegros et al. 34 . suggested for ssRNA+ viruses that genome segmentation allows maximizing genetic content while preserving capsid stability and energetically favorable genome density. ...
Parvoviruses (family Parvoviridae) are currently defined by a linear monopartite ssDNA genome, T = 1 icosahedral capsids, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. We report the discovery of a parvovirus with a bipartite genome, Acheta domesticus segmented densovirus (AdSDV), isolated from house crickets (Acheta domesticus), in which it is pathogenic. We found that the AdSDV harbors its NS and VP cassettes on two separate genome segments. Its vp segment acquired a phospholipase A2-encoding gene, vpORF3, via inter-subfamily recombination, coding for a non-structural protein. We showed that the AdSDV evolved a highly complex transcription profile in response to its multipartite replication strategy compared to its monopartite ancestors. Our structural and molecular examinations revealed that the AdSDV packages one genome segment per particle. The cryo-EM structures of two empty- and one full-capsid population (3.3, 3.1 and 2.3 Å resolution) reveal a genome packaging mechanism, which involves an elongated C-terminal tail of the VP, “pinning” the ssDNA genome to the capsid interior at the twofold symmetry axis. This mechanism fundamentally differs from the capsid-DNA interactions previously seen in parvoviruses. This study provides new insights on the mechanism behind ssDNA genome segmentation and on the plasticity of parvovirus biology.
... It is difficult to experimentally study the competition between monopartite and bipartite viruses because most viruses exist as only one type or the other. However, a case of foot-and mouth-disease virus has been studied in which evolution from a monopartite to a bipartite virus has been found in the laboratory [10]. In this case, it was found that replication of the incomplete strands was not significantly faster than that of the complete strands. ...
RNA viruses may be monopartite (all genes on one strand), multipartite (two or more strands packaged separately) or segmented (two or more strands packaged together). In this article, we consider competition between a complete monopartite virus, A, and two defective viruses, D and E, that have complementary genes. We use stochastic models that follow gene translation, RNA replication, virus assembly, and transmission between cells. D and E multiply faster than A when stored in the same host as A or when together in the same host, but they cannot multiply alone. D and E strands are packaged as separate particles unless a mechanism evolves that allows assembly of D + E segmented particles. We show that if defective viruses assemble rapidly into separate particles, the formation of segmented particles is selected against. In this case, D and E spread as parasites of A, and the bipartite D + E combination eliminates A if the transmissibility is high. Alternatively, if defective strands do not assemble rapidly into separate particles, then a mechanism for assembly of segmented particles is selected for. In this case, the segmented virus can eliminate A if transmissibility is high. Conditions of excess protein resources favor bipartite viruses, while conditions of excess RNA resources favor segmented viruses. We study the error threshold behavior that arises when deleterious mutations are introduced. Relative to bipartite and segmented viruses, deleterious mutations favor monopartite viruses. A monopartite virus can give rise to either a bipartite or a segmented virus, but it is unlikely that both will originate from the same virus.
... Cell-to-cell transmission can also be induced for viruses that are normally transmitted in a cell-free manner when blocking the protease activity that cleaves the bond between viral glycoprotein and the host receptor; this mechanism has been observed for influenza viruses 4 in patients treated with oseltamivir (Tamiflu) (Mori et al., 2015(Mori et al., , 2011. Such cell-to-cell transmission could result in co-infection of the same cell with different viral variants or quasispecies, enabling their "cooperation" (Bordería et al., 2015;Ojosnegros et al., 2011;Shirogane et al., 2012). Cell-to-cell transmission allows a pair of viruses with resistance to a single drug to persist for a prolonged period of passages, whereas they would otherwise not be able to replicate on their own under double-drug treatment. ...
The use of multiple antivirals in a single patient increases the risk of emergence of multidrug-resistant viruses, posing a public health challenge and limiting management options. Cell-to-cell viral transmission allows a pair of viruses that are each resistant to a single drug to persist for a prolonged period of passages although neither can survive alone under double-drug treatment. This pair should then persist until they accumulate a second mutation to generate resistance to both drugs. Accordingly, we here propose a hypothesis that viruses have a much higher probability of developing double-drug resistance when they are transmitted via a cell-to-cell mode than when they are transmitted via a cell-free mode through released virions. By using a stochastic model describing the changes in the frequencies of viral genotypes over successive infections, we analytically demonstrate that the emergence probability of double resistance is approximately the square of the number of viral genomes that establish infection times greater in cell-to-cell transmission than in cell-free transmission. Our study suggests the importance of inhibiting cell-to-cell transmission during multi-drug treatment.
We have previously shown that tomato apex necrosis virus that cannot express the RNA2-ORF1 protein (P21) is not able to systemically infect plant hosts but is not affected in cell autonomous aspects of virus replication/accumulation. Here we attempted to provide P21 in trans by co-agroinfiltrating the RNA2-ORF1 null constructs (a stop codon mutant and a deletion mutant) with a P21-expressing construct under control of the 35S promoter and containing the 5’ and 3’ UTRs of wild type (WT) RNA2. Such construct when co-agroinfiltrated with the stop codon mutant originates a WT bipartite virus through homologous recombination. More surprisingly, when co-agroinfiltrated with the P21 deletion mutant it cannot immediately complement the mutant, but it serendipitously originates a tripartite virus with an actively replicating P21-expressing RNA3 only after this replicating RNA3 accumulates deletions in a small region inside the original 3’-UTR provided by the cDNA clone. Such virus can be transmitted mechanically and by whiteflies, is competent for virion formation, and its RNA3 is encapsidated. The tripartite virus can be mechanically transferred for eleven generations without losing its infectivity or show major genomic rearrangements. Furthermore, mixing equal amounts of WT and tripartite virus inocula in the same leaf originated plants systemically infected only with the WT virus, showing that the tripartite virus has lower fitness than the WT. To our knowledge this is the first example of a stable virus evolving in vitro from bipartite to tripartite genomic structure from a synthetic construct in a plant virus.
Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-α), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV.
IMPORTANCE Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness.
Fitness of viruses has become a standard parameter to quantify their adaptation to a biological environment. Fitness determinations for RNA viruses (and some highly variable DNA viruses) meet with several uncertainties. Of particular interest are those that arise from mutant spectrum complexity, absence of population equilibrium, and internal interactions among components of a mutant spectrum. Here, concepts, fitness measurements, limitations, and current views on experimental viral fitness landscapes are discussed. The effect of viral fitness on resistance to antiviral agents is covered in some detail since it constitutes a widespread problem in antiviral pharmacology, and a challenge for the design of effective antiviral treatments. Recent evidence with hepatitis C virus suggests the operation of mechanisms of antiviral resistance additional to the standard selection of drug-escape mutants. The possibility that high replicative fitness may be the driver of such alternative mechanisms is considered. New broad-spectrum antiviral designs that target viral fitness may curtail the impact of drug-escape mutants in treatment failures. We consider to what extent fitness-related concepts apply to coronaviruses and how they may affect strategies for COVID-19 prevention and treatment.
The genomes of most virus species have overlapping genes--two or more proteins coded for by the same nucleotide sequence. Several explanations have been proposed for the evolution of this phenomenon, and we test these by comparing the amount of gene overlap in all known virus species. We conclude that gene overlap is unlikely to have evolved as a way of compressing the genome in response to the harmful effect of mutation because RNA viruses, despite having generally higher mutation rates, have less gene overlap on average than DNA viruses of comparable genome length. However, we do find a negative relationship between overlap proportion and genome length among viruses with icosahedral capsids, but not among those with other capsid types that we consider easier to enlarge in size. Our interpretation is that a physical constraint on genome length by the capsid has led to gene overlap evolving as a mechanism for producing more proteins from the same genome length. We consider that these patterns cannot be explained by other factors, namely the possible roles of overlap in transcription regulation, generating more divergent proteins and the relationship between gene length and genome length.
Dispersal mechanisms play a main role in the dynamics of infection spread. Recent experimental results with in vitro infections of foot-and-mouth disease virus reveal that the time needed for the virus to kill a cellular monolayer depends qualitatively on the number of viral particles required to initiate infection in a susceptible cell. A two-dimensional susceptible-infected-removed (SIR) model based on the experimental setting agrees with the observations only when viral particles are subject to long-range transport. Numerical and analytical results show that this long-range transport plays a role when a single particle causes infection, while it is inefficient when complementation between two or more particles is necessary.
Author Summary
RNA viruses are associated with many important human and animal diseases such as AIDS, influenza, hemorrhagic fevers and several forms of hepatitis. RNA viruses mutate at very high rates and, therefore, can adapt easily to environmental changes. Viral mutants resistant to antiviral inhibitors are readily selected, resulting in treatment failure. The simultaneous administration of three or more inhibitors is a means to prevent or delay selection of resistant mutants. A new antiviral strategy termed lethal mutagenesis is presently under investigation. It consists of the administration of mutagenic agents to elevate the mutation rate of the virus above the maximum level compatible with virus infectivity, without mutagenizing the host cells. Since low amounts of virus are extinguished more easily, the combination of a mutagen and inhibitor was more efficient than a mutagen alone in driving virus to extinction. Here we show that foot-and-mouth disease virus replicating in cell culture can be extinguished more easily when the inhibitor guanidine is administered first, followed by the mutagenic agent ribavirin, than when both drugs are administered simultaneously. Interfering mutants that contribute to extinction were active in the presence of ribavirin but not in the presence of guanidine. This observation provides a mechanism for the advantage of the sequential versus the combination treatment. This unexpected effectiveness of a sequential treatment is supported by a theoretical model of virus evolution in the presence of the inhibitor and the mutagen. The results can have an application for future lethal mutagenesis protocols and for current antiviral treatments that involve the antiviral agent ribavirin when it acts as a mutagen.
Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones
of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21
cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the
amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing
of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced
at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic
site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure
of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition
to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical
level the biological defects that arise when viruses are subjected to multiple bottleneck events.
RNA viruses provide unique insights into the patterns and processes of evolutionary change in real time. The study of viral evolution is especially topical given the growing awareness that emerging and re-emerging diseases (most of which are caused by RNA viruses) represent a major threat to public health. However, while the study of viral evolution has developed rapidly in the last 30 years, relatively little attention has been directed toward linking work on the mechanisms of viral evolution within cells or individual hosts, to the epidemiological outcomes of these processes. This novel book fills this gap by considering the patterns and processes of viral evolution across their entire range of spatial and temporal scales. The Evolution and Emergence of RNA Viruses provides a comprehensive overview of RNA virus evolution, with a particular focus on genomic and phylogenetic approaches. This is the first book to link mechanisms of viral evolution with disease dynamics, using high-profile examples in emergence and evolution such as influenza, HIV, dengue fever, and rabies. It also reveals the underlying evolutionary processes by which emerging viruses cross species boundaries and spread in new hosts.
Bacteriophage Qβ minus strands substituted in position 15 with N4-hydroxyCMP were used as template for Qβ replicase. About 33% of the plus strands generated after one round of transcription had a G→A mutation at position 16 (G−16→A) from the 3′ end (Flavell et al., 1974). Such plus strand preparations, as well as preparations resulting from multiple rounds of replication in which the mutant RNA had been enriched, were used to infect spheroplasts, and clones of progeny phage were isolated. Analysis of the 3′ terminal region of the RNA of 120 isolates revealed that all were wild-type. Thus, the G−16→A mutation in the 3′ terminal extracistronic region diminished the plaque-forming capacity of Qβ RNA to less than 5% of wild-type, as measured in the spheroplast assay.
A biochemical procedure, selective elongation, was developed to prepare mutant G−16→A RNA free from wild-type RNA. Using ATP and GTP as the only substrates, and a mixture of mutant and wild-type plus strands as template, the first 14 nucleotides of mutant and wild-type minus strands were synthesized: pppGpGpGpApGpGpApGpApGpApGpGpGOH. Further elongation of wild-type minus strands required CTP (followed by ATP), whereas elongation of mutant minus strands required UTP (followed by ATP). Incubation with CTP and the chain-terminating ATP analog 3′-amino-3′-deoxyATP led specifically to the termination of wild-type but not of mutant minus strands. Subsequent elongation with ATP, GTP, CTP and UTP resulted in the completion of mutant minus strands, which were purified free of plus strand template. Purified mutant minus strands were used as template to prepare mutant plus strands, which had a specific infectivity 0·5% or less than that of wild-type RNA. Reversion of the mutation at position – 16 was undertaken by site-directed mutagenesis: purified mutant plus strands were used to direct the stepwise synthesis of minus strands and N4-hydroxyCMP was incorporated into position 15 in place of UMP. Replication of the substituted minus strands in vitro gave rise to a mixture of RNAs, about 30% of which had the wild-type sequence at position 16. Following the reversion step, the specific infectivity of the RNA was increased significantly, albeit only to about 0·6 to 6% of the value expected. The incomplete recovery of infectivity was due to the accumulation of mutations at various sites during the extensive amplification in vitro to which the preparation had been subjected.
The distribution of deleterious mutations in a population of organisms is determined by the opposing effects of two forces, mutation pressure and selection. If mutation rates are high, the resulting mutation-selection balance can generate a substantial mutational load in the population. Sex can be advantageous to organisms experiencing high mutation rates because it can either buffer the mutation-selection balance from genetic drift, thus preventing any increases in the mutational load (Muller, 1964: Mut. Res.1, 2), or decrease the mutational load by increasing the efficiency of selection (Crow, 1970: Biomathematics1, 128). Muller's hypothesis assumes that deleterious mutations act independently, whereas Crow's hypothesis assumes that deleterious mutations interact synergistically, i.e., the acquisition of a deleterious mutation is proportionately more harmful to a genome with many mutations than it is to a genome with a few mutations. RNA viruses provide a test for these two hypotheses because they have extremely high mutation rates and appear to have evolved specific adaptations to reproduce sexually. Population genetic models for RNA viruses show that Muller's and Crow's hypotheses are also possible explanations for why sex is advantageous to these viruses. A re-analysis of published data on RNA viruses that are cultured by undiluted passage suggests that deleterious mutations in such viruses interact synergistically and that sex evolved there as a mechanism to reduce the mutational load.
Viruses can reproduce sexually. Sex in some RNA viruses is so different from sex in eukaryotes that it may have evolved independently. Yet, recent research indicates that sex in both groups can be accounted for by models of either positive or purifying selection. This review appraises the role that these types of selection may have played in the evolution of sex in RNA viruses.
![Schematic representation of the segmented and ST virus.
The illustration shows the requirement of double infection for complementation and progeny production by the segmented FMDV population C-S8p260, but not by its ST derivative C-S8p260p3d [1], [2]. A scheme of the FMDV genome with indication of the four main coding regions (L, P1, P2, P3) and the position of the internal deletions (Δ, black boxes) is drawn for each genotype.](https://www.researchgate.net/profile/Juan-Garcia-Arriaza/publication/50851600/figure/fig3/AS:340060240138248@1458088087199/Schematic-representation-of-the-segmented-and-ST-virus-The-illustration-shows-the_Q320.jpg)
![Replication kinetics of C-S8p260 (segmented) and C-S8p260p3d (ST) FMDV in BHK-21 cells.
Cells were infected at a MOI of 20 PFU/cell. A to D) At different times after infection, the intracellular or extracellular concentration of genomic viral RNA (normalized to the number of cells) was determined. A) Intracellular concentration of viral RNA in two independent infections carried out in parallel; each value represents the average of two determinations. The data have been fitted to an exponential curve: C-S8p260: 4.8·102·e0.065×, R2 = 0.94; C-S8p260p3d :8.9·102·6e0.054×; R2 = 0.92. In B to D, BHK-21 cells were coinfected with the two viruses (C-S8p260 and C-S8p260p3d), at a MOI of 20 PFU/cell, and viral RNA was quantified as follows (symbols are as in A): B) Intracellular concentration of viral RNA in the course of virus entry into the cell. C) Intracellular viral RNA concentration during the exponential replication phase. D) Extracellular concentration of RNA measured in the cell culture supernatant obtained in the infection represented in C). In B–D the determinations were carried out from triplicate experiments (average values and standard deviation are shown). E) Electrophoretic analysis of 35S-labeled proteins extracted from BHK-21 cells electroporated with FMDV RNAs. BHK-21 cells were either mock-electroporated (BHK lanes) or electroporated with transcripts from either pMT260p3d or a mixture of viral transcripts from pMT260Δ417ns and pMT260Δ999ns (which give rise to C-S8p260p3d and C-S8p260, respectively; see Figure S2). Parallel cultures were pulse-labeled with [35S]Met/Cys for 30 min., at different times after 1 h post-electroporation, as indicated above each lane, and analyzed by PAGE, as described in Materials and Methods. The amount of cellular proteins was monitored by the relative amount of actin, visualized by Western-blot using a specific monoclonal antibody (actin panels). F) The amount of viral proteins VP3, VP1, 3D and 3CD (in arbitrary units) at each time point was determined by densitometric scanning of the corresponding protein bands, and normalized to the concentration of actin (top panels). Values were added sequentially at each time point to obtain the accumulated level of viral protein (bottom panels).](https://www.researchgate.net/profile/Juan-Garcia-Arriaza/publication/50851600/figure/fig5/AS:341395068997632@1458406335067/Replication-kinetics-of-C-S8p260-segmented-and-C-S8p260p3d-ST-FMDV-in-BHK-21_Q320.jpg)
