Article

RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence

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Abstract

Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations.

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... The RdRp is sufficient for template switching in vitro [17]. Mutations mapping to RdRp-coding sequence can impair RNA recombination [18,19]. These recombination-defective variants replicate well in cell culture under normal conditions [18,19] but are unable to deal with high mutational loads [18] or replicate well in animals [18,19]. ...
... Mutations mapping to RdRp-coding sequence can impair RNA recombination [18,19]. These recombination-defective variants replicate well in cell culture under normal conditions [18,19] but are unable to deal with high mutational loads [18] or replicate well in animals [18,19]. Biophysical experiments revealed the existence of a stalled RdRp with its 3 0 -end of nascent RNA in a single-stranded form [20]. ...
... Mutations mapping to RdRp-coding sequence can impair RNA recombination [18,19]. These recombination-defective variants replicate well in cell culture under normal conditions [18,19] but are unable to deal with high mutational loads [18] or replicate well in animals [18,19]. Biophysical experiments revealed the existence of a stalled RdRp with its 3 0 -end of nascent RNA in a single-stranded form [20]. ...
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RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.
... 19 Compounding these challenges, picornaviruses have a propensity for recombination during RNA replication and quickly lose foreign sequences with a fitness cost. [20][21][22][23] Achieving stable expression of secreted payloads for these OVs is an important goal to enhance their therapeutic potential, particularly as innovative strategies, such as LNP delivered OV therapies termed Synthetic viruses, that enable repeat intravenous (i.v.) administration are moving toward clinical trials. 24 These so-called Synthetic viruses consider the encapsulation of a replication-competent positive-sense RNA genome (viral RNA [vRNA]) into lipid nanoparticles (LNPs) and, in contrast to their cognate viruses, retain anti-tumor activity in preclinical models in the presence of virus-neutralizing titers. ...
... 29 The resulting virus titer does not inform upon the retention of the reporter gene, as picornaviruses are capable of rapid deletion of transgenes by recombination. 20 The recovered virus was found to maintain robust expression of the reporter transgene, as microscopy images of infected NCI-H1299 and NCI-H69 cells showed clear expression of both GFP and mCherry ( Figure 1F). nLuc gene expres-sion post-infection was also confirmed by bioluminescence assay ( Figure 1G). ...
Article
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Oncolytic viruses (OVs) promote the anti-tumor immune response as their replication, and the subsequent lysis of tumor cells, triggers the activation of immune-sensing pathways. Arming OVs by expressing transgenes with the potential to promote immune cell recruitment and activation is an attractive strategy to enhance OVs’ therapeutic benefit. For picornaviruses, a family of OVs with clinical experience, the expression of a transgene is limited by multiple factors: genome physical packaging limits, high rates of recombination, and viral-mediated inhibition of transgene secretion. Here, we evaluated strategies for arming Seneca Valley virus (SVV) with relevant immunomodulatory transgenes. Specificially in the contex of arming SVV, we evaluated transgene maximum size and stabiltity, transgene secretion, and the impact of transgene inclusion on viral fitness. We find that SVV is not capable of expressing secreted payloads and has a transgene packaging capacity of ∼10% of viral genome size. To enable transgene expression, we developed SVV replicons with greater transgene size capacity and secretion capabilities. SVV replicons can be packaged in trans by virus in co-infected cells to express immunomodulatory transgenes in surrounding cells, thus providing a means to enhance the potential of this therapeutic to augment the anti-tumor immune response.
... RF-A became dominant with the worldwide spread of the D3/A CVA6 lineage, the most frequent lineage in our paediatric population (estimated proportion, 58%). This transmission pattern may indicate an advantage for the association of the D3 capsid region with the RF-A nonstructural proteins [43]. With an estimated proportion of 19%, RF-H was the second most frequent RF in our patient population. ...
... The precise factors involved in the global geographic spread of CVA6/D3 since the mid-2000s are unknown. It was hypothesized that genetic recombination was associated with increased virulence or virus transmissibility of poliovirus vaccine strains or EV-A71 subgenogroup C4a [43]. Pons-Salort and collaborators suggested that an increase in pathogenicity is the epidemiological model that best explains the change in the circulation pattern of CVA6 in Japan since 2010 [47]. ...
Article
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Coxsackievirus A6 (CVA6) emerged as the most common enterovirus of seasonal outbreaks of hand-foot-and-mouth disease (HFMD). We investigated CVA6 genetic diversity among the clinical phenotypes reported in the paediatric population during sentinel surveillance in France between 2010 and 2018. CVA6 infection was confirmed in 981 children (mean age 1.52 years [IQR 1.17–2.72]) of whom 564 (58%) were males. Atypical HFMD was reported in 705 (72%) children, followed by typical HFMD in 214 (22%) and herpangina in 57 (6%) children. Throat specimens of 245 children were processed with a target-enrichment new-generation sequencing approach, which generated 213 complete CVA6 genomes. The genomes grouped within the D1 and D3 clades (phylogeny inferred with the P1 genomic region). In total, 201 genomes were classified among the recombinant forms (RFs) A, B, F, G, H, and N, and 12 genomes were assigned to 5 previously unreported RFs (R–V). The most frequent RFs were A (58%), H (19%), G (6.1%), and F (5.2%). The yearly number of RFs ranged between 1 (in 2012 and 2013) and 6 (2018). The worldwide CVA6 epidemic transmission began between 2005 and 2007, which coincided with the global spread of the recombinant subclade D3/RF-A.
... In addition, virulent recombinants have also been observed in cases of FMDV (Abd El Rahman et al., 2020), enterovirus EV-B83 (Xiao et al., 2020), and rodent coronavirus (Li et al., 2021). In addition, RNA virus recombination can significantly accelerate adaptation and virus spread, dramatically enhancing virulence (Xiao et al., 2016). ...
... Moreover, RNA recombination can generate new viral variants that are positively selected in mosquito vectors, working as an efficient strategy to allow CHIKV to overcome "tight" genetic bottlenecks or even providing an advantage for viral replication (Filomatori et al., 2019). For poliovirus, a virus with a significantly smaller genome than coronavirus, recombination accelerates adaptation in the short time frame of acute poliovirus infection and enriches beneficial mutations while purging deleterious mutations (Xiao et al., 2016). Two high-frequency inter-lineage recombination hotspots have been located in the RdRp and GP2-GP3 regions of PRRSV-2 (Zhou et al., 2018a). ...
Article
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RNA recombination is a major driver of genetic shifts tightly linked to the evolution of RNA viruses. Genomic recombination contributes substantially to the emergence of new viral lineages, expansion in host tropism, adaptations to new environments, and virulence and pathogenesis. Here, we review some of the recent progress that has advanced our understanding of recombination in positive-strand RNA viruses, including recombination triggers and the mechanisms behind them. The study of RNA recombination aids in predicting the probability and outcome of viral recombination events, and in the design of viruses with reduced recombination frequency as candidates for the development of live attenuated vaccines. Surveillance of viral recombination should remain a priority in the detection of emergent viral strains, a goal that can only be accomplished by expanding our understanding of how these events are triggered and regulated.
... Recombination is a common process amongst positive-strand RNA viruses and is a strong driver of virus evolution through the exchange of genomic sequences that can be directly advantageous or result in the removal of deleterious mutations [1][2][3]. The mechanism of replicative recombination requires a strand transfer event in which the template copied by the RNA-dependent RNA polymerase (RdRp) changes during negativestrand synthesis [4]. ...
... As recombinants are relatively rare, when compared with the non-recombinant progeny of parental viruses, studying the process requires the ability to preferentially isolate recombinants. To this end, we developed a poliovirus-based in vitro recombination assay-the CRE-REP assay-that only allows the recovery of recombinant progeny viruses [3,[16][17][18]. The assay involves the co-transfection of two RNA templates-acceptor and donor-that are independently unable to produce infectious progeny virus. ...
Article
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Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.
... When a genome is incompletely replicated it is either left unrepaired and degraded, or it is repaired by recombination with another RNA molecule, (reviewed in [38]). Viral recombination occurs at high rates between 2-20% recombination events per 100 nucleotides [39][40][41][42][43][44], and the rate of recombination is dependent on the fidelity of the RdRp [45][46][47][48]. Specifically, RdRps with a high fidelity are associated with a low recombination rate, while RdRps with low fidelity are associated with high levels of recombination [45][46][47][48]. ...
... Viral recombination occurs at high rates between 2-20% recombination events per 100 nucleotides [39][40][41][42][43][44], and the rate of recombination is dependent on the fidelity of the RdRp [45][46][47][48]. Specifically, RdRps with a high fidelity are associated with a low recombination rate, while RdRps with low fidelity are associated with high levels of recombination [45][46][47][48]. This is due to the major RNA viral recombination mechanism being initiated by faulty, or incomplete, viral replication. ...
Article
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The current SARS-CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS-CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions.
... Mutation or recombination of the viral genome can increase its diversity, thereby allowing viruses to easily adapt to a new host and to mediate immune escape and exhibit resistance to gene therapy (Pilipenko et al., 1992;Miras et al., 2014;Xiao et al., 2016;Ngoveni et al., 2019;Patino-Galindo et al., 2021). This evolutionary pattern makes the viral genome more prone to prey on ribosomes. ...
Article
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Viruses are strictly intracellular parasites requiring host cellular functions to complete their reproduction cycle involving virus infection of host cell, viral genome replication, viral protein translation, and virion release. Ribosomes are protein synthesis factories in cells, and viruses need to manipulate ribosomes to complete their protein synthesis. Viruses use translation initiation factors through their own RNA structures or cap structures, thereby inducing ribosomes to synthesize viral proteins. Viruses also affect ribosome production and the assembly of mature ribosomes, and regulate the recognition of mRNA by ribosomes, thereby promoting viral protein synthesis and inhibiting the synthesis of host antiviral immune proteins. Here, we review the remarkable mechanisms used by RNA viruses to regulate ribosomes, in particular, the mechanisms by which RNA viruses induce the formation of specific heterogeneous ribosomes required for viral protein translation. This review provides valuable insights into the control of viral infection and diseases from the perspective of viral protein synthesis.
... RNA recombination can repair damage to the viral genome during transcription and replication, eliminate harmful mutations, and maintain the genetic characteristics of the virus population (11). Recombinant viruses may be highly virulent and transmissible (10,12,13), promoting viral escape from immune protection (14,15), expanding the host range, and affecting the viral disease epidemic (16). Live attenuated vaccine viruses can acquire pathogenicity via recombination (17). ...
Article
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Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the pig-raising industry. The PRRS virus (PRRSV) has high genetic diversity, partly owing to viral recombination. Some individual recombinant type 2 PRRSV (PRRSV-2) strains have been detected; however, the sequence composition characteristics of recombination hot spots and potential driving forces for recombinant PRRSV-2 are still unreported. Therefore, all available genomic sequences of PRRSV-2 (n = 949, including 29 genomes sequenced in this study) from 11 countries from 1991 to 2021 were collected and analyzed. The results revealed that the dominant major recombinant parent has been converted from lineage 3 (L3) to L1 since 2012. The recombination hot spots were located at nucleotides (nt) 7900 to 8200 (in NSP9, encoding viral RNA-dependent RNA polymerase) and nt 12500 to nt 13300 (in ORF2-ORF4, mean ORF2 to ORF4); no AU-rich characteristics were found in the recombination hot spots. Based on infectious clones of L1 and L8 PRRSV-2, recombinant PRRSVs were generated by switching complete or partial NSP9 (harboring the recombination hot spot). The results showed that recombinant PRRSVs based on the L1 backbone, but not the L8 backbone, acquired a higher replication capacity in pig primary alveolar macrophages. These findings will help to understand the reason behind the dominance of L1-based recombination in PRRSV-2 strains and provide new clues for an in-depth study of the recombination mechanism of PRRSV-2.
... Data processing scripts are available openly in https://github.com/ mouneem/IDbSV and the extracted list of strains can be found in Supplementary Table 1 of more virulent strains, and drug resistance (24,25). Figures 2A,E. ...
Article
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Ending COVID-19 pandemic requires a collaborative understanding of SARS-CoV-2 and COVID-19 mechanisms. Yet, the evolving nature of coronaviruses results in a continuous emergence of new variants of the virus. Central to this is the need for a continuous monitoring system able to detect potentially harmful variants of the virus in real-time. In this manuscript, we present the International Database of SARS-CoV-2 Variations (IDbSV), the result of ongoing efforts in curating, analyzing, and sharing comprehensive interpretation of SARS-CoV-2's genetic variations and variants. Through user-friendly interactive data visualizations, we aim to provide a novel surveillance tool to the scientific and public health communities. The database is regularly updated with new records through a 4-step workflow (1—Quality control of curated sequences, 2—Call of variations, 3—Functional annotation, and 4—Metadata association). To the best of our knowledge, IDbSV provides access to the largest repository of SARS-CoV-2 variations and the largest analysis of SARS-CoV-2 genomes with over 60 thousand annotated variations curated from the 1,808,613 genomes alongside their functional annotations, first known appearance, and associated genetic lineages, enabling a robust interpretation tool for SARS-CoV-2 variations to help understanding SARS-CoV-2 dynamics across the world.
... Our understanding of the effects of genomic recombination on SARS-CoV-2 fitness and transmission dynamics are still limited, but genetic exchange has been previously associated with evolutionary adaptation in viruses under experimental conditions (e.g. Poliovirus; Xiao et al., 2016), in individual hosts (e.g. HIV; Song et al., 2018) and in nature (e.g. ...
Preprint
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Genetic recombination is an important driving force of coronavirus evolution. While some degree of virus recombination has been reported during the COVID-19 pandemic, previously detected recombinant lineages of SARS-CoV-2 have shown limited circulation and been observed only in restricted areas. Prompted by reports of unusual genetic similarities among several Pango lineages detected mainly in North and Central America, we present a detailed phylogenetic analysis of four SARS-CoV-2 lineages (B.1.627, B.1.628, B.1.631 and B.1.634) in order to investigate the possibility of virus recombination among them. Two of these lineages, B.1.628 and B.1.631, are split into two distinct clusters (here named major and minor). Our phylogenetic and recombination analyses of these lineages find well-supported phylogenetic differences between the Orf1ab region and the rest of the genome (S protein and remaining reading frames). The lineages also contain several deletions in the NSP6, Orf3a and S proteins that can augment reconstruction of reliable evolutionary histories. By reconciling the deletions and phylogenetic data, we conclude that the B.1.628 major cluster originated from a recombination event between a B.1.631 major virus and a lineage B.1.634 virus. This scenario inferred from genetic data is supported by the spatial and temporal distribution of the three lineages, which all co-circulated in the USA and Mexico during 2021, suggesting this region is where the recombination event took place. We therefore support the designation of the B.1.628 major cluster as recombinant lineage XB in the Pango nomenclature. The widespread circulation of lineage XB across multiple countries over a longer timespan than the previously designated recombinant XA lineage raises important questions regarding the role and potential effects of recombination on the evolution of SARS-CoV-2 during the ongoing COVID-19 pandemic.
... In many RNA viruses, the process of recombination generates additional diversity within the virus population. Recombination allows for a larger exchange of genetic material between closely related viruses and is proposed to be a mechanism by which genomes can be rescued from error catastrophe, which would otherwise have resulted from the accumulation of deleterious mutations introduced by the RdRp [1][2][3]. Recombination is widely accepted to occur via a copy-choice mechanism involving the RdRp switching from one template (the donor) to another (the acceptor) during negative-strand synthesis [4]. Sequence identity and RNA structure have been proposed to influence the template-switching event, although evidence is often contradictory and may reflect analysis of highly selected populations (reviewed in [5]). ...
Article
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Recombination is a common feature of many positive-strand RNA viruses, playing an important role in virus evolution. However, to date, there is limited understanding of the mechanisms behind the process. Utilising in vitro assays, we have previously shown that the template-switching event of recombination is a random and ubiquitous process that often leads to recombinant viruses with imprecise genomes containing sequence duplications. Subsequently, a process termed resolution, that has yet to be mechanistically studied, removes these duplicated sequences resulting in a virus population of wild type length genomes. Using defined imprecise recombinant viruses together with Oxford Nanopore and Illumina high throughput next generation sequencing technologies we have investigated the process of resolution. We show that genome resolution involves subsequent rounds of template-switching recombination with viral fitness resulting in the survival of a small subset of recombinant genomes. This alters our previously held understanding that recombination and resolution are independent steps of the process, and instead demonstrates that viruses undergo frequent and continuous recombination events over a prolonged period until the fittest viruses, predominantly those with wild type length genomes, dominate the population.
... As for genomic recombination, sequence variation brings about heterogeneous sequences that may recombine during replications in single cells, and this therefore makes the recombinations between divergent sequences predictable, even though recombinations will negatively regulate the divergences (Lai, 1992). In our data, higher sequence variations were associated with more genomic recombinations, probably because variable populations require recombinations for purging their deleterious mutations (Xiao et al., 2016). However, in this sense, the lower sequence variation and negative recombination prediction results of the GSPIdV RNAs do not mean the absence of recombinations; it is possible that these could not be predicted using a computer. ...
Article
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Zanthoxylum armatum is an important woody crop with multiple applications in pharmaceutics, cosmetics, and food industries. With continuous increases in the plantation area, integrated pest management is required for scale production when diseases caused by biotic factors such as pests and pathogens have become new problems, one of which is the infectious flower yellowing disease (FYD). Here, isolates of a new illarvirus (3) and a new nepovirus-associated subviral satellite RNA (12) were identified in Z. armatum , in addition to 38 new isolates of four previously reported RNA viruses. Sequence variation can be observed in viral/subviral quasispecies and among predominant isolates from the same or different samples and geographic origins. Intriguingly, RNA sequencing of different diseased trees invariably showed an extraordinary pattern of particularly high reads accumulation of the green Sichuan pepper-nepovirus (GSPNeV) and the satellite RNA in symptomatic tissues. In addition, we also examined small RNAs of the satellite RNA, which show similar patterns to those of coinfecting viruses. This study provides further evidence to support association of the FYD with viral/subviral infections and deepens our understanding of the diversity and molecular characteristics of the viruses and satellite, as well as their interactions with the host.
... Many viruses produce diverse genetically linked variants that can be defined as viral populations. These populations are maintained by mutation-selection equilibrium, (41,42) and have the potential to generate beneficial interactions and cooperation which increase viral fitness and adaptability to the host (43)(44)(45)(46). ...
Article
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The Pacific oyster (Crassostreae gigas) has been introduced from Asia to numerous countries around the world during the 20th century. C. gigas is the main oyster species farmed worldwide and represents more than 98% of oyster production. The severity of disease outbreaks that affect C. gigas, which primarily impact juvenile oysters, has increased dramatically since 2008. The most prevalent disease, Pacific oyster mortality syndrome (POMS), has become panzootic and represents a threat to the oyster industry. Recently, major steps towards understanding POMS have been achieved through integrative molecular approaches. These studies demonstrated that infection by Ostreid herpesvirus type 1 µVar (OsHV-1 µvar) is the first critical step in the infectious process and leads to an immunocompromised state by altering hemocyte physiology. This is followed by dysbiosis of the microbiota, which leads to a secondary colonization by opportunistic bacterial pathogens, which in turn results in oyster death. Host and environmental factors (e.g. oyster genetics and age, temperature, food availability, and microbiota) have been shown to influence POMS permissiveness. However, we still do not understand the mechanisms by which these different factors control disease expression. The present review discusses current knowledge of this polymicrobial and multifactorial disease process and explores the research avenues that must be investigated to fully elucidate the complexity of POMS. These discoveries will help in decision-making and will facilitate the development of tools and applied innovations for the sustainable and integrated management of oyster aquaculture.
... While there is 94.0% to 99.7% sequence identity among the Chinese APPVs in genotype 3, Anna/2020 showed 87.0% to 89.3% identity to these Chinese APPVs in genotype 3 (Table 1). Recombination events are an important mechanism by which viruses acquire genetic diversity, and they have been identified in APPVs (Guo et al., 2020;Xiao et al., 2016). Thus, to find crossover sites between Anna/2020 and other APPVs, similarity plot analysis was performed using SimPlot software. ...
Preprint
Atypical porcine pestivirus (APPV), which has been confirmed to be associated with congenital tremor (CT) in pigs, is a newly discovered porcine virus that has been found in the Americas, Europe, and Asia; however, no report of APPV in Japan has been published. We identified an APPV in the central nervous system of Japanese piglets with CT, and firstly determined and analyzed the complete genome sequence. Phylogenetic analysis using the complete genome nucleotide sequence of the Japanese APPV, named Anna/2020, and those of APPVs from the NCBI database showed that APPVs were divided into three genotypes (genotypes 1 to 3), and that Anna/2020 clustered with the genotype 3 APPV strains, but distantly branched from these strains. Pairwise complete coding region nucleotide sequence comparisons revealed that there was 94.0% to 99.7% sequence identity among the genotype 3 strains, while Anna/2020 showed 87.0% to 89.3% identity to those genotype 3 strains, suggesting that Anna/2020 represents a novel APPV lineage within genotype 3. Retrospective examinations using RT-PCR revealed one genotype 1 and two novel genotype 3 APPVs from pigs without CT, and that novel genotype 3 APPVs have been prevalent in Japan since at least 2007.
... CoV genomes encode for a methyltransferase with 3'-5' exonuclease activity; this constitutes a proofreading and mismatch correction system, resulting in lower mutation rates compared to other RNA viruses [3][4][5]. This feature might ultimately limit the mutagenic variability of the virus, and enhance the prominence of recombination in its evolution and expansion of species and cellular tropism [6,7]. Recombination is an important factor driving viral diversity [2,[8][9][10][11][12][13], and it has been reported as a possible mechanism favoring cross-species CoVs transmission as well as increasing their adaptability to a new host [2,8]. ...
Article
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Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.
... As to a certain recombinant virus that is able to express a heterologous protein, the genetic stability of foreign sequence is an easily overlooked but important aspect, which impacts on further application of the recombinant virus. Compared with some positive-sense and single-stranded RNA viruses (27,28), from which non-self sequences can be easily deleted due to their genetic instabilities, paramyxoviruses are able to retain foreign sequences in their genomes with serial passaging in cells. We simultaneously constructed another recombinant CDV that contained a GFP marker, which could be stably expressed during serial passages even under the continuous selective pressure of mutagen. ...
Article
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Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae , is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro . The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro . Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.
... The virus adaptation rate, which is the rate at which Maltusian fitness changes per unit time, V = dlog R 0 /dt, where R 0 is the basic reproduction number, is evaluated based on a model of evolution. In the past, the value of V has been expressed, in the general form, in terms of the evolutionary model parameters for various models [34,35,39,42,44,[60][61][62]. In the basic scenario when fitness benefit of a mutation is fixed, and in the absence of recombination [34], the adaptation rate is given by ...
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The time to the onset of AIDS symptoms in an HIV infected individual is known to correlate inversely with viremia and the level of immune activation. The correlation exists against the background of strong individual fluctuations demonstrating the existence of hidden variables depending on patient and virus parameters. At the moment, prognosis of the time to AIDS based on patient parameters is not possible. In addition, it is of paramount importance to understand the reason of progression to AIDS in untreated patients to be able to learn to control it by means other than anti-retroviral therapy. Here we develop a mechanistic mathematical model to predict the speed of progression to AIDS in individual untreated patients and patients treated with suboptimal therapy, based on a single-time measurement of several virological and immunological parameters. We show that the gradual increase in virus fitness during a chronic infection causes slow gradual depletion of CD4 T cells. Using the existing evolution models of HIV, we obtain general expressions predicting the time to the onset of AIDS symptoms in terms of the patient parameters, for low-viremia and high-viremia patients separately. We show that the evolution model of AIDS fits the existing data on virus-time correlations better than the alternative model of the deregulation of homeostatic response.
... It seems that many viruses have the ability to produce diverse genetically linked mutants that can be defined as viral populations or quasispecies maintained by mutation-selection equilibrium (Perales et al., 2015;Poirier and Vignuzzi, 2017). This concept hypothesizes that dynamic viral populations generate beneficial interactions and cooperation that allows them to rapidly adapt to changing environments, including various hosts and their different tissues, relevant for viral fitness and virulence (Xiao et al., 2016;Brooke, 2017). Studies have shown that the level of genetic diversity within viral populations likely influences viral pathogenicity, dissemination and host immune evasion (Mao et al., 2007). ...
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Juvenile Pacific oysters (Crassostrea gigas) are subjected to recurrent episodes of mass mortalities that constitute a threat for the oyster industry. This mortality syndrome named “Pacific Oyster Mortality Syndrome” (POMS) is a polymicrobial disease whose pathogenesis is initiated by a primary infection by a variant of an Ostreid herpes virus named OsHV-1 μVar. The characterization of the OsHV-1 genome during different disease outbreaks occurring in different geographic areas has revealed the existence of a genomic diversity for OsHV-1 μVar. However, the biological significance of this diversity is still poorly understood. To go further in understanding the consequences of OsHV-1 diversity on POMS, we challenged five biparental families of oysters to two different infectious environments on the French coasts (Atlantic and Mediterranean). We observed that the susceptibility to POMS can be different among families within the same environment but also for the same family between the two environments. Viral diversity analysis revealed that Atlantic and Mediterranean POMS are caused by two distinct viral populations. Moreover, we observed that different oyster families are infected by distinct viral populations within a same infectious environment. Altogether these results suggest that the co-evolutionary processes at play between OsHV-1 μVar and oyster populations have selected a viral diversity that could facilitate the infection process and the transmission in oyster populations. These new data must be taken into account in the development of novel selective breeding programs better adapted to the oyster culture environment.
... In other words, an increase in the mutation rate may not be sufficient by itself to "knock down" the population, but if it is combined with the reduction in the recombination rate, the effect is amplified. Although it is unknown the extent to which both factors are functionally independent of each other in coronaviruses, it has been shown that some mutations can affect the rate of recombination without altering replication fidelity, for example, in polioviruses (Xiao et al. 2016). ...
... This is unsurprising as altering fidelity also changes the speed and processivity of the RdRp [39][40][41][42][43][44][45], which has been linked to changes in polymerase stuttering and recombination frequency [46]. However, there is evidence that at least some fidelity mutants do not exhibit large changes in recombination frequency or vice-versa [32,47], although this is important to reexamine using updated techniques. In the future, using ClickSeq to analyze virus sequence diversity and recombination would allow both of these aspects to be examined using the same dataset, which will clarify the link between replication fidelity and recombination. ...
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Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.
... Recombination, defined as the exchange of genetic elements between parental genomes to generate novel chimeras, occurs in divers positive-sense RNA viruses [1]. It can involve replicative or non-replicative mechanisms [2,3] and potentially serves functions that include the purging of deleterious mutations and the creation of advantageous novel genetic combinations to evade host immunity, gain resistance to antiviral agents, alter virulence and expand the host range [1,4,5]. Molecular epidemiological studies, for example, of circulating enteroviruses, have shown that recombination is a frequent occurrence in picornaviruses (e.g., [6][7][8]), and analysis of viral metagenomic data has emphasized the importance of recombination in the acquisition of novel sequences and, consequently, in the evolution of viruses [9,10]. ...
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Members of Picornaviridae and of the Hepacivirus, Pegivirus and Pestivirus genera of Flaviviridae all contain an internal ribosomal entry site (IRES) in the 5′-untranslated region (5′UTR) of their genomes. Each class of IRES has a conserved structure and promotes 5′-end-independent initiation of translation by a different mechanism. Picornavirus 5′UTRs, including the IRES, evolve independently of other parts of the genome and can move between genomes, most commonly by intratypic recombination. We review accumulating evidence that IRESs are genetic entities that can also move between members of different genera and even between families. Type IV IRESs, first identified in the Hepacivirus genus, have subsequently been identified in over 25 genera of Picornaviridae, juxtaposed against diverse coding sequences. In several genera, members have either type IV IRES or an IRES of type I, II or III. Similarly, in the genus Pegivirus, members contain either a type IV IRES or an unrelated type; both classes of IRES also occur in members of the genus Hepacivirus. IRESs utilize different mechanisms, have different factor requirements and contain determinants of viral growth, pathogenesis and cell type specificity. Their dissemination between viruses by horizontal gene transfer has unexpectedly emerged as an important facet of viral evolution.
... The eGFP-expressing picornaviruses are generally unstable and prone to losing their foreign sequences by a recombination mechanism [20,38]. In this study, the eGFP could be stably expressed in BSR cells up to approximately eight virus passages. ...
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Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus Senecavirus in the family Picornaviridae. The SVA CH-LX-01-2016 was isolated from Guangdong Province of China in 2016. In this study, a recombinant SVA CH-LX-01-2016 was constructed using reverse genetics, and proven to be able to express efficiently an enhanced green fluorescent protein (eGFP) in vitro. This eGFP-tagged recombinant SVA (rSVA-eGFP) exhibited a high capacity for viral replication. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay. The rSVA-eGFP-based VNT was used to detect eight porcine serum samples, out of which four were determined to be neutralization titer-positive. Subsequently, two antiviral drugs, ribavirin and apigenin, were assayed for evaluating both effects against the rSVA-eGFP in vitro. The result showed that only the ribavirin exhibited an anti-SVA activity.
... truncated or mutated viral RNA genomes, thus increasing viral fitness (5). A major role for RNA recombination in the emergence of new viruses or virus strains is well documented for numerous human, plant, animal, bacterial, insect, and fungal viruses (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). ...
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Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca ²⁺ /Mn ²⁺ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca ²⁺ /Mn ²⁺ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.
... The quasispecies model provides a framework to understand fitness and evolvability of a population Stern et al., 2014;Xiao et al., 2016). Fitness is determined not by the genetic characteristic of a single, isolated, static species or gene, but by the collective distribution of the members of the quasispecies whose clonal expansion reflects their underlying genotypic diversity and their fitness with respect to a particular environment or selection pressure including antibiotics, other medications, and treatments (Hu and Zhu, 2016;Qin et al., 2010). ...
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Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.
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Using a non-pathogenic pseudotyped virus as a surrogate for a wide-type virus in scientific research complies with the recent requirements for biosafety. Enterovirus (EV) contains many species of viruses, which are a type of nonenveloped virus. The preparation of its corresponding pseudotyped virus often needs customized construction compared to some enveloped viruses. This article describes the procedures and challenges in the construction of pseudotyped virus for enterovirus (pseudotyped enterovirus, EVpv) and also introduces the application of EVpv in basic virological research, serological monitoring, and the detection of neutralizing antibody (NtAb).
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The first bovine parechovirus (Bo_ParV) was reported in 2021, and currently, only two nearly complete genome sequences of Bo_ParV are available. In this study, we detected Bo_ParVs in 10 out of 158 bovine fecal samples tested using real-time RT-PCR, and Bo_ParVs were isolated from three of these samples using MA104 cells. Analysis of the P1 region revealed that Bo_ParVs shared high pairwise amino acid sequence similarity (≥ 95.7% identity), suggesting antigenic similarity among Bo_ParVs, whereas nucleotide sequence identity values (≥ 84.8%) indicated more variability. A recombination breakpoint was identified in the 2B region, which may influence the evolution of this virus.
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Background Enterovirus (EV) infections are being increasingly seen in younger infants, often being more severe than in older children. The risk factors of EV infection in infants have been inadequately investigated till date. Methods We conducted a retrospective study on hospitalized children with laboratory-confirmed EV infection (50 infants aged 0–3 months and 65 older than 3 months) at a tertiary care center in China. Prevalence, clinical characteristics, and genetic features of the virus were analyzed, and independent predictors for severe infection were assessed. Results Clinical findings showed that severe infection was more common in infants aged 0–3 months than in older children (78.0% vs. 35.4%, p < 0.001), with higher morbidity of pneumonia, meningitis, and sepsis (p < 0.01). EV-B types were detected more frequently in infants aged 0–3 months than in older children (88.0% vs. 7.7%, p < 0.001). Echovirus 11 was the most identified EV-B, and it recombined with E6 in P2 and P3 regions. Risk factors for severe EV infection included EV-B types infection, age less than 3 months, elevated alanine aminotransferase level, abnormal platelet count, and abnormal cerebrospinal fluid characteristics. Conclusions Our data indicated that EV-B types mainly cause severe infection in infants aged 0–3 months. Therefore, knowledge about EV-B types could have implications in designing effective intervention and prevention strategies for young infants with severe EV infection.
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The human microbiome containing bacteria, fungi, and viruses, is a community that coexists peacefully with humans most of the time, but with the potential to cause disease under certain conditions. When the environment changes or certain stimuli are received, microbes may interact with each other, causing or increasing the severity of disease in a host. With the appropriate methods, we can make these microbiota work for us, creating new applications for human health. This review discusses the wide range of interactions between microorganisms that result in an increase in susceptibility to, severity of, and mortality of diseases and also briefly introduces how microorganisms interact with each other directly or indirectly. The study of microbial interactions and their mechanisms has revealed a new world of treatments for infectious disease. The regulation of the balance between intestinal flora, the correct application of probiotics, and the development of effective drugs by symbiosis all demonstrate the great contributions of the microbiota to human health and its powerful potential value. Consequently, the study of interactions between microorganisms plays an essential role in identifying the causes of diseases and the development of treatments.
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Background Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18–22 weeks. Methods In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18–22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model. Findings Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5ʹ untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 log10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007 (95% CI 0·002–0·023; p<0·0001) indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients. Interpretation The data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses. Funding Bill & Melinda Gates Foundation.
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The World Health Organization Western Pacific Region (WPR) has maintained the polio-free status for more than two decades. At the global level, there were only 6 confirmed polio cases due to wild type 1 poliovirus in Pakistan, Afghanistan, and Malawi in 2021, therefore, the risk of the importation of wild poliovirus from the endemic countries to the WPR is considerably lower than ever before. On the other hand, the risk of polio outbreaks associated with circulating vaccine-derived polioviruses (cVDPVs) still cannot be ignored even in the WPR. Since late 2010s, cVDPV outbreaks in the WPR have appeared to be more extensive in frequency and magnitude. Moreover, the emergence of concomitant polio outbreaks of type 1 and type 2 cVDPVs in the Philippines and Malaysia during 2019–2020 has highlighted the remaining risk of cVDPV outbreaks in high-risk areas and/or communities in the WPR. The previous cVDPV outbreaks in the WPR have been rapidly and effectively controlled, however, the future risk of polio outbreaks associated with cVDPVs needs to be reconsidered and polio immunization and surveillance strategies should be updated accordingly.
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Co-infection of RNA viruses may contribute to their recombination and cause severe clinical symptoms. However, the tracking and identification of SARS-CoV-2 co-infection persist as challenges. Due to the lack of methods for detecting co-infected samples in a large amount of deep sequencing data, the lineage composition, spatial-temporal distribution, and frequency of SARS-CoV-2 co-infection events in the population remains unclear. Here, we propose a hypergeometric distribution–based method named Cov2Coinfect with the ability to decode the lineage composition from 50,809 deep sequencing data. By resolving the mutational patterns in each sample, Cov2Coinfect can precisely determine the co-infected SARS-CoV-2 variants from deep sequencing data. Results from two independent and parallel projects in the United States achieved a similar co-infection rate of 0.3%∼0.5% in SARS-CoV-2 positive samples. Notably, all co-infected variants were highly consistent with the co-circulating SARS-CoV-2 lineages in the regional epidemiology, demonstrating that the co-circulation of different variants is an essential prerequisite for co-infection. Overall, our study not only provides a robust method to identify the co-infected SARS-CoV-2 variants from sequencing samples, but also highlights the urgent need to pay more attention to co-infected patients for better disease prevention and control.
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While recombination is a feature of coronavirus evolution, previously detected recombinant lineages of SARS-CoV-2 have shown limited circulation thus far. Here, we present a detailed phylogenetic analysis of four SARS-CoV-2 lineages to investigate the possibility of virus recombination among them. Our analyses reveal well-supported phylogenetic differences between the Orf1ab region encoding viral non-structural proteins and the rest of the genome, including Spike (S) protein and remaining reading frames. By accounting for several deletions in NSP6, Orf3a and S, we conclude that the B.1.628 major cluster, now designated as lineage XB, originated from a recombination event between viruses of B.1.631 and B.1.634 lineages. This scenario is supported by the spatiotemporal distribution of these lineages across the USA and Mexico during 2021, suggesting this recombination event originated in this geographical region. This event raises important questions regarding the role and potential effects of recombination on SARS-CoV-2 evolution.
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Background Coronavirus disease 2019 (COVID-19) became pandemic in 2020 and recently, mutated coronaviruses have emerged in many countries. The aim of this study was to identify the clinical characteristics and risk factors for critical illness in hospitalized COVID-19 patients in Zhengzhou for clinical prevention and management. Materials and methods A total of 70 patients hospitalized with COVID-19 were enrolled between 21 January and 29 February 2020, in Zhengzhou, China. Clinical characteristics, hematological findings, neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), and inflammatory index on admission were obtained from medical records, COVID-19 patients with different outcomes were compared. Results The median age was 55 years. Forty-three (61.0%) patients were classified as having severe or critical cases. Eighteen (25.7%) patients died in hospital and the remaining 52 were discharged. Patients who died tend to be old with expectoration and chronic obstructive pulmonary disease. Compared to survivor, non-survivor had significantly higher numbers of leucocytes and neutrophils, NLR, aspartate aminotransferase (AST), γ-glutamyl transpeptidase, total bilirubin, direct bilirubin, lactate dehydrogenase (LDH), prothrombin time, D-dimer, C-reactive protein, and decreased platelets, lymphocytes, uric acid, and albumin (ALB). Logistic regression analysis identified leucocytes, platelets, PLR, NLR, AST, and ALB as independent predictive factors for poor outcomes. The area under curve of the combination of leucocytes, PLR, NLR, and AST was 0.87, with a sensitivity of 0.83 and specificity of 0.81. Conclusion Our results identified risk factors among COVID-19 patients for in-hospital mortality. Leucocytes, PLR, NLR, and AST could have important reference value for predicting prognosis, especially in low-resource countries.
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Background The factors driving the late phase of COVID-19 are still poorly understood. However, autoimmunity is an evolving theme in COVID-19’s pathogenesis. Additionally, deregulation of human retroelements (RE) is found in many viral infections, and has also been reported in COVID-19. Results Unexpectedly, coronaviruses (CoV) – including SARS-CoV-2 – harbour many RE-identical sequences (up to 35 base pairs), and some of these sequences are part of SARS-CoV-2 epitopes associated to COVID-19 severity. Furthermore, RE are expressed in healthy controls and human cells and become deregulated after SARS-CoV-2 infection, showing mainly changes in long interspersed nuclear element (LINE1) expression, but also in endogenous retroviruses. Conclusion CoV and human RE share coding sequences, which are targeted by antibodies in COVID-19 and thus could induce an autoimmune loop by molecular mimicry.
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Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3–5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human–animal contact. The presence of porcine deltacoronavirus has been detected in three children from Haiti that could have originated from zoonotic spillover.
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Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
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Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.
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Parainfluenza virus 5 (PIV5) belongs to the genus Orthorubulavirus in the family Paramyxoviridae. PIV5 can infect a range of mammals, but induce mild or even unobservable clinical signs in some animals, except kennel cough in dogs. It is also able to infect a variety of cell lines, but causes minimal or even invisible cytopathic effects on many cells. Sometimes, owing to neither observable cytopathic effects in vitro nor typical clinical signs in vivo, the PIV5 is not easily usable for screening antiviral drugs. To solve this issue, we used reverse genetics to recover a dual reporter-tagged recombinant PIV5 that could simultaneously express enhanced green fluorescence protein (eGFP) and NanoLuc® luciferase (NLuc) in virus-infected cells. Both reporters were genetically stable during twenty serial passages of virus in MDBK cells. The eGFP allowed us to observe virus-infected MDBK cells in real time, and moreover the NLuc made it possible to quantify the degree of viral replication for determining antiviral activity of a given drug. Subsequently, the recombinant PIV5 was used for antiviral assays on five common drugs, i.e., ribavirin, apigenin, 1-adamantylamine hydrochloride, moroxydine hydrochloride and tea polyphenol. The results showed that only the ribavirin had an anti-PIV5 effect in MDBK cells. This study proposed a novel method for rapid screening (or prescreening) of anti-PIV5 drugs.
Chapter
Enterovirus 71 (EV-A71) is one of the main etiological agents of hand, foot and mouth disease (HFMD). EV-A71 mainly infects infants and young children below six years of age. Clinical manifestations of HFMD include fever, rashes with the presence of vesicles on the hand, feet and mouth. However, the onset of severe HFMD can lead to neurological complications such as acute flaccid paralysis, brainstem encephalitis and cardiac pulmonary failure that can be fatal. This chapter addresses how genetic events such as recombination and spontaneous mutations could change the genomic organization of EV-A71, leading to strains with higher virulence. An understanding of the recombination mechanism of the poliovirus and non-polio enteroviruses provides evidence for the emergence of novel EV-A71 strains responsible for fatal HFMD outbreaks. Currently, it is unknown if the virulence of EV-A71 is contributed by the events of recombination between EV-A71 and other enteroviruses or it is due to the presence of spontaneous mutations that effects its virulence.
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Atypical porcine pestivirus (APPV), which has been confirmed to be associated with congenital tremor (CT) in pigs, is a newly discovered porcine virus that has been found in the Americas, Europe, and Asia; however, no report of APPV in Japan has been published. We identified an APPV in the central nervous system of Japanese piglets with CT, and firstly determined and analyzed the complete genome sequence. Phylogenetic analysis using the complete genome nucleotide sequence of the Japanese APPV, named Anna/2020, and those of APPVs from the NCBI database showed that APPVs were divided into three genotypes (genotypes 1 to 3), and that Anna/2020 clustered with the genotype 3 APPV strains, but distantly branched from these strains. Pairwise complete coding region nucleotide sequence comparisons revealed that there was 94.0% to 99.7% sequence identity among the genotype 3 strains, while Anna/2020 showed 87.0% to 89.3% identity to those genotype 3 strains, suggesting that Anna/2020 represents a novel APPV lineage within genotype 3. Retrospective examinations using RT‐PCR revealed one genotype 1 and two novel genotype 3 APPVs from pigs without CT, and that novel genotype 3 APPVs have been prevalent in Japan since at least 2007.
Preprint
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Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, coronavirus emergence in our species has been associated with zoonotic transmissions from animal reservoirs, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae (Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus), human infections reported to date have been limited to alpha and betacoronaviruses. We identify, for the first time, porcine deltacoronavirus (PDCoV) strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the nsp15 and the spike glycoprotein genes by convergent evolution. In particular, structural analysis predicts that one of the changes in the Spike S1 subunit, which contains the receptor-binding domain, may affect flexibity of the protein and binding to the host cell receptor. Our findings not only underscore the ability of deltacoronaviruses to adapt and potentially lead to human-to-human transmission, but also raise questions about the role of such transmissions in development of pre-existing immunity to other coronaviruses, such as SARS-CoV-2.
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Natural language predicts viral escape Viral mutations that evade neutralizing antibodies, an occurrence known as viral escape, can occur and may impede the development of vaccines. To predict which mutations may lead to viral escape, Hie et al. used a machine learning technique for natural language processing with two components: grammar (or syntax) and meaning (or semantics) (see the Perspective by Kim and Przytycka). Three different unsupervised language models were constructed for influenza A hemagglutinin, HIV-1 envelope glycoprotein, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein. Semantic landscapes for these viruses predicted viral escape mutations that produce sequences that are syntactically and/or grammatically correct but effectively different in semantics and thus able to evade the immune system. Science , this issue p. 284 ; see also p. 233
Article
Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently co-located with TRS-B sites than expected.
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Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.
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Porcine epidemic diarrhea virus (PEDV) is a fatal epizootic swine coronavirus that presents a financial threat to the global swine industry. Since the discovery of the low-pathogenic genotype 1b (G1b) in 2014, it has been responsible for sporadic outbreaks in South Korea. In this study, we identified novel G1b variants arising from the natural recombination of a major pandemic-like G2b virus and a minor G1b virus currently circulating in the domestic field. The whole-genome sequences of two 2018–19 G1b recombinants, KNU-1808 and KNU-1909, were determined. A genomic comparison showed that these two viruses share the highest nucleotide sequence similarity with the 2017 G1b strain but share less similarity with the 2014 G1b emergent strain KNU-1406. However, the putative recombination breakpoints spanning the first 1,170 nucleotides of the spike (S) gene were almost identical among the emergent and contemporary G1b strains. Recombination detection indicated that the inter-subgroup G1b recombinant first emerged in 2017 by introducing the N-terminal domain of S from KNU-1406 into the backbone of KNU-1703, possibly leading to antigenic shift. It then evolved into KNU-1808 and KNU-1909 through genetic drift, moving toward a more G2b-like genotype. Phylogenetic analysis revealed that the 2018–2019 G1b recombinants belong to a cluster containing other G1b strains but form a new branch. This study provides an important advance warning in regard to the emergence and prevalence of new genotypes or variants that can result from genetic recombination between two different PEDV genotypes circulating in endemic areas and continuous non-lethal mutations essential for viral fitness in the host environment.
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During the 1990s, a group of virologists and physicists began development of a quantitative theory to explain the rapid evolution of human immunodeficiency virus type 1 (HIV-1). This theory also proved to be instrumental in understanding the rapid emergence of drug resistance in patients. Over the past two decades, this theory expanded to account for a broad array of factors important to viral evolution and propelled development of a generalized theory applicable to a broad range of asexual and partly sexual populations with many evolving sites. Here, we discuss the conceptual and theoretical tools developed to calculate the speed and other parameters of evolution, with a particular focus on the concept of 'clonal interference' and its applications to untreated patients.
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When mutation rates are low, natural selection remains effective, and increasing the mutation rate can give rise to an increase in adaptation rate. When mutation rates are high to begin with, however, increasing the mutation rate may have a detrimental effect because of the overwhelming presence of deleterious mutations. Indeed, if mutation rates are high enough: (i) adaptive evolution may be neutralized, resulting in a zero (or negative) adaptation rate despite the continued availability of adaptive and/or compensatory mutations, or (ii) natural selection may be neutralized, because the fitness of lineages bearing adaptive and/or compensatory mutations-whether established or newly arising-is eroded by excessive mutation, causing such lineages to decline in frequency. We apply these two criteria to a standard model of asexual adaptive evolution and derive mathematical expressions-some new, some old in new guise-delineating the mutation rates under which either adaptive evolution or natural selection is neutralized. The expressions are simple and require no a priori knowledge of organism- and/or environment-specific parameters. Our discussion connects these results to each other and to previous theory, showing convergence or equivalence of the different results in most cases.
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The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific "hotspots," including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains.
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When large asexual populations adapt, competition between simultaneously segregating mutations slows the rate of adaptation and restricts the set of mutations that eventually fix. This phenomenon of interference arises from competition between mutations of different strengths as well as competition between mutations that arise on different fitness backgrounds. Previous work has explored each of these effects in isolation, but the way they combine to influence the dynamics of adaptation remains largely unknown. Here, we describe a theoretical model to treat both aspects of interference in large populations. We calculate the rate of adaptation and the distribution of fixed mutational effects accumulated by the population. We focus particular attention on the case when the effects of beneficial mutations are exponentially distributed, as well as on a more general class of exponential-like distributions. In both cases, we show that the rate of adaptation and the influence of genetic background on the fixation of new mutants is equivalent to an effective model with a single selection coefficient and rescaled mutation rate, and we explicitly calculate these effective parameters. We find that the effective selection coefficient exactly coincides with the most common fixed mutational effect. This equivalence leads to an intuitive picture of the relative importance of different types of interference effects, which can shift dramatically as a function of the population size, mutation rate, and the underlying distribution of fitness effects.
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In nonrecombining genomes, genetic linkage can be an important evolutionary force. Linkage generates interference interactions, by which simultaneously occurring mutations affect each other's chance of fixation. Here, we develop a comprehensive model of adaptive evolution in linked genomes, which integrates interference interactions between multiple beneficial and deleterious mutations into a unified framework. By an approximate analytical solution, we predict the fixation rates of these mutations, as well as the probabilities of beneficial and deleterious alleles at fixed genomic sites. We find that interference interactions generate a regime of emergent neutrality: all genomic sites with selection coefficients smaller in magnitude than a characteristic threshold have nearly random fixed alleles, and both beneficial and deleterious mutations at these sites have nearly neutral fixation rates. We show that this dynamic limits not only the speed of adaptation, but also a population's degree of adaptation in its current environment. We apply the model to different scenarios: stationary adaptation in a time-dependent environment and approach to equilibrium in a fixed environment. In both cases, the analytical predictions are in good agreement with numerical simulations. Our results suggest that interference can severely compromise biological functions in an adapting population, which sets viability limits on adaptive evolution under linkage.
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RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand.
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HIV adaptation to a host in chronic infection is simulated by means of a Monte-Carlo algorithm that includes the evolutionary factors of mutation, positive selection with varying strength among sites, random genetic drift, linkage, and recombination. By comparing two sensitive measures of linkage disequilibrium (LD) and the number of diverse sites measured in simulation to patient data from one-time samples of pol gene obtained by single-genome sequencing from representative untreated patients, we estimate the effective recombination rate and the average selection coefficient to be on the order of 1% per genome per generation (10(-5) per base per generation) and 0.5%, respectively. The adaptation rate is twofold higher and fourfold lower than predicted in the absence of recombination and in the limit of very frequent recombination, respectively. The level of LD and the number of diverse sites observed in data also range between the values predicted in simulation for these two limiting cases. These results demonstrate the critical importance of finite population size, linkage, and recombination in HIV evolution.
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Accurate estimates of virus mutation rates are important to understand the evolution of the viruses and to combat them. However, methods of estimation are varied and often complex. Here, we critically review over 40 original studies and establish criteria to facilitate comparative analyses. The mutation rates of 23 viruses are presented as substitutions per nucleotide per cell infection (s/n/c) and corrected for selection bias where necessary, using a new statistical method. The resulting rates range from 10−8 to10−6 s/n/c for DNA viruses and from 10−6 to 10−4 s/n/c for RNA viruses. Similar to what has been shown previously for DNA viruses, there appears to be a negative correlation between mutation rate and genome size among RNA viruses, but this result requires further experimental testing. Contrary to some suggestions, the mutation rate of retroviruses is not lower than that of other RNA viruses. We also show that nucleotide substitutions are on average four times more common than insertions/deletions (indels). Finally, we provide estimates of the mutation rate per nucleotide per strand copying, which tends to be lower than that per cell infection because some viruses undergo several rounds of copying per cell, particularly double-stranded DNA viruses. A regularly updated virus mutation rate data set will be available at www.uv.es/rsanjuan/virmut.
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The adverse effect of co-inheritance linkage of a large number of sites on adaptation has been studied extensively for asexual populations. However, it is insufficiently understood for multi-site populations in the presence of recombination. In the present work, motivated by our studies of HIV evolution in infected patients, we consider a model of haploid populations with infrequent recombination. We assume that small quantities of beneficial alleles preexist at a large number of sites and neglect new mutation. Using a generalized form of the traveling wave method, we show that the effectiveness of recombination is impeded and the adaptation rate is decreased by inter-sequence correlations, arising due to the fact that some pairs of homologous sites have common ancestors existing after the onset of adaptation. As the recombination rate per individual becomes smaller, site pairs with common ancestors become more frequent, making recombination even less effective. In addition, an increasing number of sites become identical by descent across large samples of sequences, causing reversion of the direction of evolution and the loss of beneficial alleles at these sites. As a result, within a 10-fold range of the recombination rate, the average adaptation rate falls from 90% of the infinite-recombination value down to 10%. The entire transition from almost maximum to almost zero may occur at very small recombination rates. Interestingly, the strong effect of linkage on the adaptation rate is predicted in the absence of average linkage disequilibrium (Lewontin's measure).
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Although the evolution of recombination is still a major problem in evolutionary genetics, recent theoretical studies have shown that recombination can evolve by breaking down interference ("Hill-Robertson effects") among multiple loci. This leads to selection on a recombination modifier in a population subject to recurrent deleterious mutation. Here, we use computer simulations to investigate the evolution of a recombination modifier under three different scenarios of recurrent mutation in a finite population: (1) mutations are deleterious only, (2) mutations are advantageous only, and (3) there is a mixture of deleterious and advantageous mutations. We also investigate how linkage disequilibrium, the strength of selection acting on a modifier, and effective population size change under the different scenarios. We observe that adding even a small number of advantageous mutations increases the fixation rate of modifiers that increase recombination, especially if the effects of deleterious mutations are weak. However, the strength of selection on a modifier is less than the summed strengths had there been deleterious mutations only and advantageous mutations only.
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The evolutionary dynamics of HIV during the chronic phase of infection is driven by the host immune response and by selective pressures exerted through drug treatment. To understand and model the evolution of HIV quantitatively, the parameters governing genetic diversification and the strength of selection need to be known. While mutation rates can be measured in single replication cycles, the relevant effective recombination rate depends on the probability of coinfection of a cell with more than one virus and can only be inferred from population data. However, most population genetic estimators for recombination rates assume absence of selection and are hence of limited applicability to HIV, since positive and purifying selection are important in HIV evolution. Yet, little is known about the distribution of selection differentials between individual viruses and the impact of single polymorphisms on viral fitness. Here, we estimate the rate of recombination and the distribution of selection coefficients from time series sequence data tracking the evolution of HIV within single patients. By examining temporal changes in the genetic composition of the population, we estimate the effective recombination to be rho = 1.4+/-0.6 x 10(-5) recombinations per site and generation. Furthermore, we provide evidence that the selection coefficients of at least 15% of the observed non-synonymous polymorphisms exceed 0.8% per generation. These results provide a basis for a more detailed understanding of the evolution of HIV. A particularly interesting case is evolution in response to drug treatment, where recombination can facilitate the rapid acquisition of multiple resistance mutations. With the methods developed here, more precise and more detailed studies will be possible as soon as data with higher time resolution and greater sample sizes are available.
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Adaptation often involves the acquisition of a large number of genomic changes that arise as mutations in single individuals. In asexual populations, combinations of mutations can fix only when they arise in the same lineage, but for populations in which genetic information is exchanged, beneficial mutations can arise in different individuals and be combined later. In large populations, when the product of the population size N and the total beneficial mutation rate U(b) is large, many new beneficial alleles can be segregating in the population simultaneously. We calculate the rate of adaptation, v, in several models of such sexual populations and show that v is linear in NU(b) only in sufficiently small populations. In large populations, v increases much more slowly as log NU(b). The prefactor of this logarithm, however, increases as the square of the recombination rate. This acceleration of adaptation by recombination implies a strong evolutionary advantage of sex.
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The rate of mutation is central to evolution. Mutations are required for adaptation, yet most mutations with phenotypic effects are deleterious. As a consequence, the mutation rate that maximizes adaptation will be some intermediate value. Here, we used digital organisms to investigate the ability of natural selection to adjust and optimize mutation rates. We assessed the optimal mutation rate by empirically determining what mutation rate produced the highest rate of adaptation. Then, we allowed mutation rates to evolve, and we evaluated the proximity to the optimum. Although we chose conditions favorable for mutation rate optimization, the evolved rates were invariably far below the optimum across a wide range of experimental parameter settings. We hypothesized that the reason that mutation rates evolved to be suboptimal was the ruggedness of fitness landscapes. To test this hypothesis, we created a simplified landscape without any fitness valleys and found that, in such conditions, populations evolved near-optimal mutation rates. In contrast, when fitness valleys were added to this simple landscape, the ability of evolving populations to find the optimal mutation rate was lost. We conclude that rugged fitness landscapes can prevent the evolution of mutation rates that are optimal for long-term adaptation. This finding has important implications for applied evolutionary research in both biological and computational realms.
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Based on the FISHER-MULLER theory of the evolution of recombination, an argument can be constructed predicting that a recessive allele favoring recombination will be favored, if there are either favorable or deleterious mutants occurring at other loci. In this case there is no clear distinction between individual and group selection. Computer simulation of populations segregating for recessive or dominant recombination alleles showed selection favoring recombination, except in the case of a dominant recombination allele with deleterious background mutants. The relationship of this work to parallel investigations by WILLIAMS and by STROBECK, MAYNARD SMITH, and CHARLESWORTH is explored. All seem to rely on the same phenomenon. There seems no reason to assume that the evolution of recombination must have occurred by group selection.
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The high error rate inherent in all RNA synthesis provides RNA virus genomes with extremely high mutation rates. Thus nearly all large RNA virus clonal populations are quasispecies collections of differing, related genomes (14, 49). These rapidly mutating populations can remain remarkably stable under certain conditions of replication. Under other conditions, virus-population equilibria become disturbed, and extremely rapid evolution can result. This extreme variability and rapid evolution can cause severe problems with previously unknown virus diseases (such as AIDS). It also presents daunting challenges for the design of effective vaccines for the control of diseases caused by rapidly evolving RNA virus populations.
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RNA viruses show high mutation frequencies partly because of a lack of the proofreading enzymes that assure fidelity of DNA replication. This high mutation frequency is coupled with high rates of replication reflected in rates of RNA genome evolution which can be more than a millionfold greater than the rates of the DNA chromosome evolution of their hosts. There are some disease implications for the DNA-based biosphere of this rapidly evolving RNA biosphere.
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Two clonal populations of vesicular stomatitis virus of approximately equal relative fitness were mixed together and allowed to compete during many transfers in vitro as large virus populations. Eventually, one or the other population suddenly excluded its competitor population, yet both the winners and losers exhibited absolute gains in fitness. Our results agree with the predictions of two major theories of classical population biology; the Competitive Exclusion Principle and the Red Queen's Hypothesis, where (in Lewis Carroll's words) "it takes all the running you can do to keep in the same place."
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A hallmark of RNA genomes is the error-prone nature of their replication and retrotranscription. The major biochemical basis of the limited replication fidelity is the absence of proofreading/repair and postreplicative error correction mechanisms that normally operate during replication of cellular DNA. In spite of this unique feature of RNA replicons, the dynamics of viral populations seems to follow the same basic principles that classical population genetics has established for higher organisms. Here we review recent evidence of the profound effects that genetic bottlenecks have in enhancing the deleterious effects of Muller's ratchet during RNA virus evolution. The validity of the Red Queen hypothesis and of the competitive exclusion principle for RNA viruses are viewed as the expected result of the highly variable and adaptable nature of viral quasispecies. Viral fitness, or ability to replicate infectious progeny, can vary a million-fold within short time intervals. Paradoxically, functional and structural studies suggest extreme limitations to virus variation. Adaptability of RNA viruses appears to be based on the occupation of very narrow portions of sequence space at any given time.
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In sexual populations, beneficial mutations that occur in different lineages may be recombined into a single lineage. In asexual populations, however, clones that carry such alternative beneficial mutations compete with one another and, thereby, interfere with the expected progression of a given mutation to fixation. From theoretical exploration of such 'clonal interference', we have derived (1) a fixation probability for beneficial mutations, (2) an expected substitution rate, (3) an expected coefficient of selection for realized substitutions, (4) an expected rate of fitness increase, (5) the probability that a beneficial mutation transiently achieves polymorphic frequency (> or = 1%), and (6) the probability that a beneficial mutation transiently achieves majority status. Based on (2) and (3), we were able to estimate the beneficial mutation rate and the distribution of mutational effects from changes in mean fitness in an evolving E. coli population.
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The controversy over the evolutionary advantage of recombination initially discovered by Fisher and by Muller is reviewed. Those authors whose models had finite-population effects found an advantage of recombination, and those whose models had infinite populations found none. The advantage of recombination is that it breaks down random linkage disequilibrium generated by genetic drift. Hill and Robertson found that the average effect of this randomly-generated linkage disequilibrium was to cause linked loci to interfere with each other's response to selection, even where there was no gene interaction between the loci. This effect is shown to be identical to the original argument of Fisher and Muller. It also predicts the "ratchet mechanism" discovered by Muller, who pointed out that deleterious mutants would more readily increase in a population without recombination. Computer simulations of substitution of favorable mutants and of the long-term increase of deleterious mutants verified the essential correctness of the original Fisher-Muller argument and the reality of the Muller ratchet mechanism. It is argued that these constitute an intrinsic advantage of recombination capable of accounting for its persistence in the face of selection for tighter linkage between interacting polymorphisms, and possibly capable of accounting for its origin.
Article
Sex is a general feature of the life cycle of eukaryotes. It is not universal, however, as many organisms seem to lack sex entirely¹. The widespread occurrence of sex is puzzling, both because meiotic recombination can disrupt co-adapted combinations of genes, and because it halves the potential rate of reproduction in organisms with strongly differentiated male and female gametes². Most attempts to explain the maintenance of sexuality invoke differences between parents and sexual offspring. These differences may be advantageous in novel or changing environments if new gene combinations are favoured from time to time¹. Sex would then serve to concentrate beneficial mutations that have arisen independently into the same line of descent. But in a stable environment sex might serve to concentrate deleterious mutations, so that they will be more effectively purged from the population by selection³. We have studied the effect of sex on mean fitness in experimental populations of the budding yeast Saccharomyces cerevisiae. Our results show that sex increases mean fitness in an environment to which the populations were well adapted, but not in an environment to which new adaptation occurred, supporting the hypothesis that the advantage of sexuality lay in the removal of deleterious mutations.
Article
The complete 7410 nucleotide sequence of the poliovirus type I genome was obtained from cloned cDNA. Double-stranded poliovirus cDNA was synthesized and inserted into the Pst I site of plasmid pBR322, and three clones were derived that together provided DNA copies of the entire poliovirus genome. Two of the clones contained inserts of 2.5 and 6.5 kilobases and represented all but the 5' 115 bases of poliovirus RNA. A third clone was generated from primer-extended DNA and contained sequences from the 5' end of the viral RNA. An open reading frame that was identified in the nucleotide sequence starting 743 bases from the 5' end of the RNA and extending to a termination codon 71 bases from the 3' end contained known poliovirus polypeptide sequence.
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Circular resequencing (CirSeq) is a novel technique for efficient and highly accurate next-generation sequencing (NGS) of RNA virus populations. The foundation of this approach is the circularization of fragmented viral RNAs, which are then redundantly encoded into tandem repeats by 'rolling-circle' reverse transcription. When sequenced, the redundant copies within each read are aligned to derive a consensus sequence of their initial RNA template. This process yields sequencing data with error rates far below the variant frequencies observed for RNA viruses, facilitating ultra-rare variant detection and accurate measurement of low-frequency variants. Although library preparation takes ∼5 d, the high-quality data generated by CirSeq simplifies downstream data analysis, making this approach substantially more tractable for experimentalists.
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RNA viruses exist as genetically diverse populations. It is thought that diversity and genetic structure of viral populations determine the rapid adaptation observed in RNA viruses and hence their pathogenesis. However, our understanding of the mechanisms underlying virus evolution has been limited by the inability to accurately describe the genetic structure of virus populations. Next-generation sequencing technologies generate data of sufficient depth to characterize virus populations, but are limited in their utility because most variants are present at very low frequencies and are thus indistinguishable from next-generation sequencing errors. Here we present an approach that reduces next-generation sequencing errors and allows the description of virus populations with unprecedented accuracy. Using this approach, we define the mutation rates of poliovirus and uncover the mutation landscape of the population. Furthermore, by monitoring changes in variant frequencies on serially passaged populations, we determined fitness values for thousands of mutations across the viral genome. Mapping of these fitness values onto three-dimensional structures of viral proteins offers a powerful approach for exploring structure-function relationships and potentially uncovering new functions. To our knowledge, our study provides the first single-nucleotide fitness landscape of an evolving RNA virus and establishes a general experimental platform for studying the genetic changes underlying the evolution of virus populations.
Article
The controversy over the evolutionary advantage of recombination initially discovered by Fisher and by Muller is reviewed. Those authors whose models had finite-population effects found an advantage of recombination, and those whose models had infinite populations found none. The advantage of recombination is that it breaks down random linkage disequilibrium generated by genetic drift. Hill and Robertson found that the average effect of this randomly-generated linkage disequilibrium was to cause linked loci to interfere with each other's response to selection, even where there was no gene interaction between the loci. This effect is shown to be identical to the original argument of Fisher and Muller. It also predicts the ''ratchet mechanism'' discovered by Muller, who pointed out that deleterious mutants would more readily increase in a population without recombination. Computer simulations of substitution of favorable mutants and of the long-term increase of deleterious mutants verified the essential correctness of the original Fisher-Muller argument and the reality of the Muller ratchet mechanism. It is argued that these constitute an intrinsic advantage of recombination capable of accounting for its persistence in the face of selection for tighter linkage between interacting polymorphisms, and possibly capable of accounting for its origin.
Article
1. A distinction is made between opportunistic and equilibrium species. 2. There is little ecological interest in the relative abundances of opportunistic species, but such species abundances should frequently have a log-normal distribution. 3. The relative abundances of equilibrium species are of considerable ecological interest and frequently can be deduced from the assumption that increase in one species population results in a roughly equal decrease in the populations of other species. To make the formulae well-defined it is necessary to assume that the census-taker has sampled a small area and thus achieved a certain sort of randomness. 4. For bird populations, at least, discrepancies between observations and predictions are negligible except when the censused area is compounded from different habitats. The discrepancy is then partly due to the fact that common species in one habitat are more likely to be present in adjacent habitats than are rate ones.
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RNA viruses are the main agents of emerging disease. To understand how RNA viruses are able to jump species boundaries and spread in new hosts it is essential to determine the basic processes of evolutionary change in these infectious agents. RNA virus evolution is largely shaped by very high rates of mutation. This, coupled with potentially enormous intra- and interhost population sizes and continual replication, allows the rapid production of genetic diversity, including those mutations that facilitate host adaptation. However, high mutation rates also act to constrain aspects of RNA virus evolution, as the majority of mutations, including many at synonymous sites, are deleterious, which in turn places an upper limit on genome size. Ironically, although RNA viruses are characterized by their mutation rates, these rates may not be high enough to allow the onset of quasispecies dynamics, in which natural selection acts on the viral population as a whole.
Article
RNA viruses exist as dynamic and diverse populations shaped by constant mutation and selection. Yet little is known about how the mutant spectrum contributes to virus evolvability and pathogenesis. Because several codon choices are available for a given amino acid, a central question concerns whether viral sequences have evolved to optimize not only the protein coding consensus, but also the DNA/RNA sequences accessible through mutation. Here we directly test this hypothesis by comparing wild-type poliovirus to synthetic viruses carrying re-engineered capsid sequences with hundreds of synonymous mutations. Strikingly, such rewiring of the population's mutant network reduced its robustness and attenuated the virus in an animal model of infection. We conclude that the position of a virus in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenicity. This organizing principle for RNA virus populations confers tolerance to mutations and facilitates replication and spread within the dynamic host environment.