A beginner's guide for FMDV quasispecies analysis: sub-consensus variant detection and haplotype reconstruction using next-generation sequencing.

Deep sequencing of viral genomes is a powerful tool to study RNA virus complexity. However, the analysis of next-generation sequencing data might be challenging for researchers who have never approached the study of viral quasispecies by this methodology. In this work we present a suitable and affordable guide to explore the sub-consensus variability and to reconstruct viral quasispecies from Illumina sequencing data. The guide includes a complete analysis pipeline along with user-friendly descriptions of software and file formats. In addition, we assessed the feasibility of the workflow proposed by analyzing a set of foot-and-mouth disease viruses (FMDV) with different degrees of variability. This guide introduces the analysis of quasispecies of FMDV and other viruses through this kind of approach.

Antiviral efficacy of short-hairpin RNAs and artificial microRNAs targeting foot-and-mouth disease virus.

RNA interference (RNAi) is a well-conserved mechanism in eukaryotic cells that directs post-transcriptional gene silencing through small RNA molecules. RNAi has been proposed as an alternative approach for rapid and specific control of viruses including foot-and-mouth disease virus (FMDV), the causative agent of a devastating animal disease with high economic impact. The aim of this work was to assess the antiviral activity of different small RNA shuttles targeting the FMDV RNA-dependent RNA polymerase coding sequence (3D). Three target sequences were predicted within 3D considering RNA accessibility as a major criterion. The silencing efficacy of short-hairpin RNAs (shRNAs) and artificial microRNAs (amiRNAs) targeting the selected sequences was confirmed in fluorescent reporter assays. Furthermore, BHK-21 cells transiently expressing shRNAs or amiRNAs proved 70 to >95% inhibition of FMDV growth. Interestingly, dual expression of amiRNAs did not improve FMDV silencing. Lastly, stable cell lines constitutively expressing amiRNAs were established and characterized in terms of antiviral activity against FMDV. As expected, viral replication in these cell lines was delayed. These results show that the target RNA-accessibility-guided approach for RNAi design rendered efficient amiRNAs that constrain FMDV replication. The application of amiRNAs to complement FMDV vaccination in specific epidemiological scenarios shall be explored further.

ViralPlaque: a Fiji macro for automated assessment of viral plaque statistics.

Plaque assay has been used for a long time to determine infectious titers and characterize prokaryotic and eukaryotic viruses forming plaques. Indeed, plaque morphology and dimensions can provide information regarding the replication kinetics and the virulence of a particular virus. In this work, we present ViralPlaque, a fast, open-source and versatile ImageJ macro for the automated determination of viral plaque dimensions from digital images. Also, a machine learning plugin is integrated in the analysis algorithm for adaptation of ViralPlaque to the user's needs and experimental conditions. A high correlation between manual and automated measurements of plaque dimensions was demonstrated. This macro will facilitate reliable and reproducible characterization of cytolytic viruses with an increased processing speed.

Differential replication of Foot-and-mouth disease viruses in mice determine lethality.

Adult C57BL/6J mice have been used to study Foot-and-mouth disease virus (FMDV) biology. In this work, two variants of an FMDV A/Arg/01 strain exhibiting differential pathogenicity in adult mice were identified and characterized: a non-lethal virus (A01NL) caused mild signs of disease, whereas a lethal virus (A01L) caused death within 24-48h independently of the dose used. Both viruses caused a systemic infection with pathological changes in the exocrine pancreas. Virus A01L reached higher viral loads in plasma and organs of inoculated mice as well as increased replication in an ovine kidney cell line. Complete consensus sequences revealed 6 non-synonymous changes between A01L and A10NL genomes that might be linked to replication differences, as suggested by in silico prediction studies. Our results highlight the biological significance of discrete genomic variations and reinforce the usefulness of this animal model to study viral determinants of lethality.

Artificial microRNAs as antiviral strategy to FMDV: structural implications of target selection.

RNA interference (RNAi) appears as a promising strategy to control virus replication. While the antiviral power of short-hairpin RNAs or small-interfering RNAs against FMDV has been demonstrated widely, safer RNAi effectors such as artificial microRNAs (amiRs) have not been evaluated extensively. In this work, transgenic monoclonal cell lines constitutively expressing different amiRs targeting FMDV 3D-coding region or 3'UTR were established. Certain cell lines showed an effective, sequence-specific amiR-mediated silencing activity that was accomplished by degradation of the target mRNA, as demonstrated in co-transfection experiments of reporter genes fused to FMDV target sequences. However, FMDV replication in these amiR-expressing cells was affected barely. Experiments aimed at elucidating the cause of RNAi failure demonstrated limited accessibility of the targeted region in the molecular environment of the viral RNA. Since RNAi is mediated by large-dimension silencing complexes containing the siRNA and not simply by a linear oligonucleotide, we propose that target selection should consider not only the local RNA structure but also the global conformation of target RNA.