A Bioinformatics Method for the Design of Live Attenuated Virus Vaccine Utilizing Host MicroRNA Response Elements
The host microRNA machinery has been employed to control viral replication. To improve safety for live attenuated virus vaccines, the binding sites of the host microRNAs, so-called microRNA response elements (MREs), were incorporated into the virus sequen
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Introduction Several studies demonstrated that the host microRNA machinery has anti-viral property [1–5]. Hence, it has been taken as a promising genetic tool for developing attenuated viruses as live vaccines [6–9]. Perez et al. generated the reassortant live attenuated influenza vaccines (LAIVs) for H1N1 and H5N1 by incorporating the non-avian microRNA response elements (MREs) into the open-reading frame (ORF) of the influenza A nucleoprotein. The MRE-based LAIVs resulted in significantly less mortality compared with controlled viruses without MREs [7]. Likewise, Barnes et al. used the poliovirus as the model and showed that the viruses harbored with MREs of the neuronal-specific miRNA were significantly attenuated in the central nervous system but still retained the replication ability in non-neuronal tissues [6]. Lee et al. demonstrated another similar restriction replication effect, incorporating MREs of the hepatic-specific miRNA into the dengue virus (DEN) replicons [8]. These results mainly focused on a specific host miRNA and a few virus sequences. Furthermore, sites for MRE insertion can be varied from 3′ UTR to the open reading frames (ORFs) with different complexities. To employ the MREs into the OPFs, for instances, the number of amino acid substitutions and the effects of changed physical properties such as hydrophobicity, polarity, and charges, due to the mutated bases in the nucleotide level need to be considered. Here, we describe a generic computational flow, which can be used to simultaneously design the effective MREs in large-scale. The flow accepts
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_47, © Springer Science+Business Media New York 2016
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the multiple or genome-wide miRNAs of interest together with the multiple sets of virus sequences. It then generates the effectives MREs for each miRNAs taking the effects of mutated bases into account.
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Materials The sequence data necessary for identifying MREs can be divided into two sets. Here, we describe how to obtain and pre-process these data sets.
2.1 Host microRNA sequences
1. Download mature microRNA (miRNA) sequences file (mature. fa) from miRBase [10, 11] (http://www.mirbase.org/). 2. Prepare a list of miRNAs of interest for a specific host (i.e., hsamiR-93), tissue, and virus (see Note 1). 3. Extract mature miRNA sequences of the specific host of interest from the mature.fa file using the extract_fasta_ sequences_separate_file.py script (Fig. 1). This script takes two parameters: (1) the mature miRNA sequence file in FASTA format (i.e., mature.fa), and (2) a list of miRBase IDs of interest (prepared from item 2). It then generates a set of FASTA files; each contains a miRBase ID and its associated sequence.
2.2
Virus Sequences
1. Download the sequences of a virus to be used for constructing a live attenuated vaccine. The influenza coding sequences (influenza.cds) and the
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