Computational protocol: Geospatial and temporal associations of Getah virus circulation among pigs and horses around the perimeter of outbreaks in Japanese racehorses in 2014 and 2015

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Protocol publication

[…] Strain 15-I-752, which was isolated from a horse in 2015 [], and strain 15-I-1105, which was isolated from a pig in this study, were sequenced. Viral RNA was extracted by using a MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Mannheim, Germany). RT-PCR was performed with three primer sets (Getah F1–21 and Getah R5366–5345; Getah F5009–5028 and Getah R8649–8630; and Getah F8401–8424 and Getah R111305–11286), as described previously [], and was conducted with a PrimeScript II High Fidelity RT-PCR Kit (Takara Bio Inc., Shiga, Japan). PCR amplicons were sequenced by using Ion Torrent technology (Thermo Fisher Scientific, MA, USA). Libraries were constructed by using an Ion Xpress Plus Fragment Library Kit and an Ion Xpress Barcode Adapters Kit. Emulsion PCR, enrichment and loading onto an Ion 316 Chip were performed automatically with Ion Chef, and sequencing was conducted with the Ion PGM system according to the manufacturer’s instructions. The average depth was 6402.5 times. The raw signal data were analyzed by using Torrent Suite version 5.0.5, and 14-I-605-C1 (LC079088) was used as a reference sequence. Torrent Variant Caller version was used to call the mutated sites, and the most frequently observed alleles were selected in each of the called positions. Bases within the reference sequence were substituted with the observed alleles if frequencies exceeded 50%. The 5′- and 3′-end sequences were determined by Rapid Amplification of cDNA Ends as described previously []. Sequences were analyzed and assembled by using Geneious software (Biomatters, Auckland, New Zealand). Nucleotide sequences for 15-I-752 (LC212972) and 15-I-1105 (LC212973) were deposited in the GenBank/EMBL/DDBJ databases.Phylogenetic analysis of nucleic acid sequences was performed with MEGA 7 software []. A phylogenetic tree based on complete genome sequences was constructed by using the maximum-likelihood method, and statistical analysis was performed by using bootstrap tests (1000 replicates). Nucleotide sequence accession numbers for the Getah virus strains used in the phylogenetic analysis were as follows: MI-110-C1 (LC079086), MI-110-C2 (LC079087), 14-I-605-C1 (LC079088), and 14-I-605-C2 (LC079089) were isolated from horses; HB0234 (EU015062), YN0540 (EU015063), SC1210 (LC107870), LEIV 17741 MPR (EF631999), M1 (EU015061), LEIV 16275 Mag (EF631998), and Sagiyama (AB032553) were isolated from mosquitoes; and South Korea (AY702913) and Kochi/01/2005 (AB859822) were isolated from pigs (Fig. ).Fig. 2 […]

Pipeline specifications

Software tools Geneious, MEGA
Databases DDBJ
Application Phylogenetics
Organisms Getah virus, Sus scrofa, Equus caballus
Diseases HIV Infections
Chemicals Nucleotides