RESUMEN
Single-cell RNA sequencing (scRNA-seq) is currently one of the most powerful techniques available to study the transcriptional response of thousands of cells to an external perturbation. Here, we perform a pseudotime analysis of SARS-CoV-2 infection using publicly available scRNA-seq data from human bronchial epithelial cells and colon and ileum organoids. Our results reveal that, for most genes, the transcriptional response to SARS-CoV-2 infection follows a non-linear pattern characterized by an initial and a final down-regulatory phase separated by an intermediate up-regulatory stage. A correlation analysis of transcriptional profiles suggests a common mechanism regulating the mRNA levels of most genes. Interestingly, genes encoded in the mitochondria or involved in translation exhibited distinct pseudotime profiles. To explain our results, we propose a simple model where nuclear export inhibition of nsp1-sensitive transcripts will be sufficient to explain the transcriptional shutdown of SARS-CoV-2 infected cells.
Asunto(s)
COVID-19 , Humanos , COVID-19/genética , Análisis de Datos , SARS-CoV-2/genética , ARN Mensajero/genética , Células EpitelialesRESUMEN
Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the family Betaflexiviridae in the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.
Asunto(s)
Flexiviridae , Virus , Flexiviridae/genética , Genoma Viral , Virus/genética , Filogenia , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Here, public scRNA-seq datasets from Drosophila melanogaster midgut cells were used to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication.
Asunto(s)
Drosophila melanogaster/virología , Virus ARN/clasificación , Virus ARN/fisiología , Animales , Heterogeneidad Genética , Filogenia , Virus ARN/aislamiento & purificación , ARN Viral/química , ARN Viral/clasificación , ARN Viral/genética , Virosis/veterinaria , Replicación ViralRESUMEN
BACKGROUND: Pepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured. RESULTS: A collection of 25 PepMV clones each containing a single nucleotide nonsynonymous substitution was created by site-directed mutagenesis and the fitness of each mutant was determined. PepMV-P22 genome showed a high degree of robustness against point mutations, with 80% of mutations being either neutral or even beneficial and only 20% being deleterious or lethal. We found that the effect of mutations strongly depended on the gene in which they were introduced. Mutations with the largest average beneficial effects were those affecting the RdRp gene, in contrast to mutations affecting TGB1 and CP genes, for which the average effects were deleterious. Moreover, significant epistatic interactions were observed between nonsynonymous mutations and the genetic background, meaning that the effect of a given nucleotide substitution on a particular genomic context cannot be predicted by knowing its effect in a different one. CONCLUSIONS: Our results indicated that PepMV genome has a surprisingly high robustness against mutations. We also found that fitness consequences of a given mutation differ between the two strains analyzed. This discovery suggests that the strength of selection, and thus the rates of evolution, vary among PepMV strains.
Asunto(s)
Enfermedades de las Plantas/virología , Potexvirus/genética , Solanum lycopersicum , Evolución Biológica , Proteínas de la Cápside/genética , Chile , Epistasis Genética , Virus del Mosaico/clasificación , Virus del Mosaico/genética , Mutagénesis Sitio-Dirigida , Mutación , Polimorfismo de Nucleótido Simple , Potexvirus/clasificación , Transcripción GenéticaRESUMEN
Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.