Bioinformatics Methods For Studying Intra-Host and Inter-Host Evolution Of Highly Mutable Viruses

Reproducibility and robustness of genomic tools are two important factors to assess the reliability of bioinformatics analysis. Such assessment based on these criteria requires repetition of experiments across lab facilities which is usually costly and time consuming. In this study we propose methods that are able to generate computational replicates, allowing the assessment of the reproducibility of genomic tools. We analyzed three different groups of genomic tools: DNA-seq read alignment tools, structural variant (SV) detection tools and RNA-seq gene expression quantification tools. We tested these tools with different technical replicate data. We observed that while some tools were impacted by the technical replicate data some remained robust. We observed the importance of the choice of read alignment tools for SV detection as well. On the other hand, we found out that the RNA-seq quantification tools (Kallisto and Salmon) that we chose were not affected by the shuffled data but were affected by reverse complement data. Using these findings, our proposed method here may help biomedical communities to advice on the robustness and reproducibility factors of genomic tools and help them to choose the most appropriate tools in terms of their needs. Furthermore, this study will give an insight to genomic tool developers about the importance of a good balance between technical improvements and reliable results

Next-generation sequencing reveals large connected networks of intra-host HCV variants

Background: Next-generation sequencing (NGS) allows for sampling numerous viral variants from infected patients. This provides a novel opportunity to represent and study the mutational landscape of Hepatitis C Virus (HCV) within a single host. Results: Intra-host variants of the HCV E1/E2 region were extensively sampled from 58 chronically infected patients. After NGS error correction, the average number of reads and variants obtained from each sample were 3202 and 464, respectively. The distance between each pair of variants was calculated and networks were created for each patient, where each node is a variant and two nodes are connected by a link if the nucleotide distance between them is 1. The work focused on large components having > 5% of all reads, which in average account for 93.7% of all reads found in a patient. The distance between any two variants calculated over the component correlated strongly with nucleotide distances (r = 0.9499; p = 0.0001), a better correlation than the one obtained with Neighbour-Joining trees (r = 0.7624; p = 0.0001). In each patient, components were well separated, with the average distance between (6.53%) being 10 times greater than within each component (0.68%). The ratio of nonsynonymous to synonymous changes was calculated and some patients (6.9%) showed a mixture of networks under strong negative and positive selection. All components were robust to in silico stochastic sampling; even after randomly removing 85% of all reads, the largest connected component in the new subsample still involved 82.4% of remaining nodes. In vitro sampling showed that 93.02% of components present in the original sample were also found in experimental replicas, with 81.6% of reads found in both. When syringe-sharing transmission events were simulated, 91.2% of all simulated transmission events seeded all components present in the source. Conclusions: Most intra-host variants are organized into distinct single-mutation components that are: well separated from each other, represent genetic distances between viral variants, robust to sampling, reproducible and likely seeded during transmission events. Facilitated by NGS, large components offer a novel evolutionary framework for genetic analysis of intra-host viral populations and understanding transmission, immune escape and drug resistance

BMC Genomics

BackgroundNext-generation sequencing (NGS) allows for sampling numerous viral variants from infected patients. This provides a novel opportunity to represent and study the mutational landscape of Hepatitis C Virus (HCV) within a single host.ResultsIntra-host variants of the HCV E1/E2 region were extensively sampled from 58 chronically infected patients. After NGS error correction, the average number of reads and variants obtained from each sample were 3202 and 464, respectively. The distance between each pair of variants was calculated and networks were created for each patient, where each node is a variant and two nodes are connected by a link if the nucleotide distance between them is 1. The work focused on large components having > 5% of all reads, which in average account for 93.7% of all reads found in a patient.ConclusionsMost intra-host variants are organized into distinct single-mutation components that are: well separated from each other, represent genetic distances between viral variants, robust to sampling, reproducible and likely seeded during transmission events. Facilitated by NGS, large components offer a novel evolutionary framework for genetic analysis of intra-host viral populations and understanding transmission, immune escape and drug resistance

Synonymous co-variation across the E1/E2 gene junction of hepatitis C virus defines virion fitness

Hepatitis C virus is a positive-sense single-stranded RNA virus. The gene junction partitioning the viral glycoproteins E1 and E2 displays concurrent sequence evolution with the 3′-end of E1 highly conserved and the 5′-end of E2 highly heterogeneous. This gene junction is also believed to contain structured RNA elements, with a growing body of evidence suggesting that such structures can act as an additional level of viral replication and transcriptional control. We have previously used ultradeep pyrosequencing to analyze an amplicon library spanning the E1/E2 gene junction from a treatment naïve patient where samples were collected over 10 years of chronic HCV infection. During this timeframe maintenance of an in-frame insertion, recombination and humoral immune targeting of discrete virus sub-populations was reported. In the current study, we present evidence of epistatic evolution across the E1/E2 gene junction and observe the development of co-varying networks of codons set against a background of a complex virome with periodic shifts in population dominance. Overtime, the number of codons actively mutating decreases for all virus groupings. We identify strong synonymous co-variation between codon sites in a group of sequences harbouring a 3 bp in-frame insertion and propose that synonymous mutation acts to stabilize the RNA structural backbone

The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1–1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host–pathogen interactions

Advances in Plant Virus Evolution: Translating Evolutionary Insights into Better Disease Management

Recent studies in plant virus evolution are revealing that genetic structure and behavior of virus and viroid populations can explain important pathogenic properties of these agents, such as host resistance breakdown, disease severity, and host shifting, among others. Genetic variation is essential for the survival of organisms. The exploration of how these subcellular parasites generate and maintain a certain frequency of mutations at the intra- and inter-host levels is revealing novel molecular virus–plant interactions. They emphasize the role of host environment in the dynamic genetic composition of virus populations. Functional genomics has identified host factors that are transcriptionally altered after virus infections. The analyses of these data by means of systems biology approaches are uncovering critical plant genes specifically targeted by viruses during host adaptation. Also, a next-generation resequencing approach of a whole virus genome is opening new avenues to study virus recombination and the relationships between intra-host virus composition and pathogenesis. Altogether, the analyzed data indicate that systematic disruption of some specific parameters of evolving virus populations could lead to more efficient ways of disease prevention, eradication, or tolerable virus–plant coexistence.SD was supported by the NJ Agricultural Experiment Station. SFE was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2009-06993) and Generalitat Valenciana (PROMETEO2010/019). Work on CTV was supported by funding from USDA grants 2003-34399-13764 and 2005-34399-16070 to ZX. Work on BNYVV was funded by The Minnesota-North Dakota Research and Education Board, and The Beet Sugar Development Foundation. RAL thanks Ramon L. Jordan (USDA-ARS, MPPL), Rayapati A. Naidu(Washington State University), and Scott Adkins (USDA ARS USHRL) for their logistic support in the realization of the originating symposium.Peer reviewe

Advances in plant virus evolution: translating evolutionary insights into better disease management

[EN] Recent studies in plant virus evolution are revealing that genetic structure and behavior of virus and viroid populations can explain important pathogenic properties of these agents, such as host resistance breakdown, disease severity, and host shifting, among others. Genetic variation is essential for the survival of organisms. The exploration of how these subcellular parasites generate and maintain a certain frequency of mutations at the intra- and inter-host levels is revealing novel molecular virus plant interactions. They emphasize the role of host environment in the dynamic genetic composition of virus populations. Functional genomics has identified host factors that are transcriptionally altered after virus infections. The analyses of these data by means of systems biology approaches are uncovering critical plant genes specifically targeted by viruses during host adaptation. Also, a next-generation re-sequencing approach of a whole virus genome is opening new avenues to study virus recombination and the relationships between intra-host virus composition and pathogenesis. Altogether, the analyzed data indicate that systematic disruption of some specific parameters of evolving virus populations could lead to more efficient ways of disease prevention, eradication, or tolerable virus plant coexistence.Acosta-Leal, R.; Duffy, S.; Xiong, Z.; Hammond, R.; Elena Fito, SF. (2011). Advances in plant virus evolution: translating evolutionary insights into better disease management. Phytopathology. 101(10):1136-1148. doi:10.1094/ PHYTO-01-11-0017S113611481011

Variability and host-dependency of RNA virus mutation rates

Los virus de ARN pueden infectar todo tipo de organismos, desde los procariotas a los eucariotas superiores, y estos agentes infecciosos parecen particularmente propensos a causar enfermedades emergentes tanto en humanos, animales, como en plantas. Su habilidad para escapar del sistema inmunitario, evadir estrategias antivirales o infectar a nuevas especies son aspecto más de su rápida evolución. Por lo tanto, comprender los procesos básicos del la evolución de los virus de ARN podría ayudar en el diseño de nuevas estrategias antivirales. Una de las principales características de los virus de ARN es su tasa de mutación extremadamente alta. De hecho, la alta diversidad genética de las poblaciones virales da lugar a una nube de variantes que interactúan y contribuyen colectivamente, conocido también por el término de cuasiespecies, y que permite a las poblaciones virales adaptarse rápidamente a entornos dinámicos. Estudios anteriores han demostrado que la tasa de mutaciones espontáneas de los virus de ARN varía de 10-6 a 10-4 sustituciones por nucleótido por célula infectada (s/n/c) y puede variar considerablemente, incluso para el mismo virus, aunque se sabe poco sobre las causas de esta variabilidad. Consecuentemente, el conocimiento de la tasa de mutación y el espectro molecular de mutaciones espontáneas son importantes para entender la evolución de la composición genética de las poblaciones virales. En los virus de ARN las tasas de mutación están determinadas por factores codificados por el virus, como la fidelidad de la polimerasa viral, la presencia/ausencia de mecanismos correctores, o el modo de replicación viral. Sin embargo, sólo unos pocos estudios se han centrado en las características celulares que podrían explicar la variabilidad en la producción de la diversidad genética viral, o si esta variabilidad pudiera ser distribuida heterogéneamente entre células únicas. En este estudio, se propuso analizar algunos de los factores subyacentes a la variabilidad en las tasas de mutación de los virus de ARN. En primer lugar, se estudió el efecto del tipo de célula que se infecta, y la variación en función del huésped en el que el virus se replica. A continuación, nos centramos en la variabilidad que ocurre a nivel de una célula única, mediante la caracterización de la diversidad genética de los virus liberados a partir de células individuales. Por último, nos fijamos en el potencial efecto de factores celulares de tipo ADAR, mediante la determinación del tipo de mutaciones espontáneas que se acumulan en el genoma de un norovirus. Se utilizó la prueba de fluctuación de Luria-Delbrück para comprobar la variabilidad en la tasa de mutación del virus de la estomatitis vesicular (VSV) entre diferentes células de mamíferos, así como entre diferentes condiciones de cultivo. Se encontró una tasa de mutación similar entre las células BHK-21, así como en células embrionarias de ratón (MEF), células MEF inmortalizadas mediante deleción del gen p53, células de cáncer de colon murino (CT26) y de neuroblastoma (Neuro-2A), sugiriendo que VSV se replica con la misma fidelidad en estos diferentes tipos de células de mamíferos. Por otra parte, debido a que el ciclo de vida de VSV no sólo implica su replicación en mamíferos, sino también en insectos, comprobamos su tasa de mutación en células de Drosophila melanogaster (S2), de Spodoptera frugiperda (sf21) y del mosquito Aedes albopictus (C6/36). Curiosamente, la tasa de mutación de VSV fue cuatro veces mas baja en células de insectos en comparación con las células de mamíferos probadas. Curiosamente, los arbovirus parecen tener una evolución más lenta que los virus que no estan transmitidos por un vector, y nuestros resultados sugieren que en los insectos esto puede ser en parte debido a una tasa de mutación más baja. Se desarrolló un enfoque que combina micromanipulación y secuenciación masiva para estudiar la diversidad genética de los virus producidos y liberados por células únicas, usando de nuevo VSV como sistema modelo. Demostramos que el virus presenta gran diversidad genética en células únicas, aunque esta diversidad no fue homogéneamente contribuida por todas las células. Llama la atención que la variabilidad existente en el inóculo viral demostró que la unidad infecciosa mínima (PFU) también puede transmitir diversidad genética viral. En efecto, no sólo se observó complementación genética entre alelos de las variantes albergadas dentro de la misma PFU, sino también que el efecto de una mutación se determinó colectivamente por otras variantes durante su co-transmisión, un proceso que podría seguir durante varias generaciones. Esta co-transmisión de diversidad genética dentro de la misma unidad infecciosa tiene implicaciones importantes para la evolución viral, como por ejemplo la de mantener la diversidad genética durante fuertes cuellos de botella de las poblaciones virales, o crear una asociación espacial entre los alelos, y sugiere que la selección natural actúa a nivel de conjuntos de partículas virales diversas en lugar de sobre genotipos individuales. Por último, hemos visto que la variabilidad en la diversidad genética producida entre células únicas estaba estrechamente asociada con el rendimiento viral, sugiriendo un compromiso entre la eficacia y la fidelidad de la replicación del virus, que puede ser determinado por un modelo de replicación geométrica. Aunque este tipo de replicación alimenta la aparición de nuevas mutaciones, también aumenta la carga genética. Para equilibrar ambos mecanismos, hemos demostrado que los mutantes que muestran un fenotipo mutador deberían ser encontrados en una baja frecuencia en las poblaciones virales. Se usó un clon infeccioso del virus Norwalk (NV). Puesto que NV no es capaz de iniciar subsiguientes ciclos de infección en las células aquí utilizadas (HEK293T), tuvimos la posibilidad de observar la aparición de mutaciones espontáneas en un único ciclo, descartando así posibles efectos de la selección o la acumulación de mutaciones a lo largo de varias generaciones. Obtuvimos así una tasa de mutación de 9 × 10-5 s/n/c consistente con las estimaciones publicadas anteriormente para otros virus de ARN. Curiosamente, se observó que de los 128 clones moleculares secuenciados con el Sanger, dos mostraron múltiples cambios T -> C. Sugerimos que estas hipermutaciones podrían ser el resultado de la edición del RNA viral por parte de enzimas celulares de tipo ADAR. Esta hipótesis fue confirmada por secuenciación masiva, sugiriendo que los factores celulares de tipo ADAR también pueden actuar en la variabilidad de la tasa de mutación de los virus de ARN