Computational protocol: Recent US bluetongue virus serotype 3 isolates found outside of Florida indicate evidence of reassortment with co-circulating endemic serotypes

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Protocol publication

[…] A total of 27 bluetongue isolates of multiple serotypes (17 from BTV-3) from the United States, Central America and the Caribbean were sequenced for this study (see ). BTVs not isolated in the United States were obtained from the Inter-American Bluetongue Project and the Onderstepoort Veterinary Institute virus library. Isolates from the United States were obtained from the National Veterinary Services Laboratories or the Arthropod-borne Animal Disease Research Unit reference collection. Viruses were typically isolated in embryonated chicken eggs or cattle pulmonary artery endothelial (CPAE) cells (ATCC CCL-209), followed by one to as many as seven passages in baby hamster kidney (BHK-21) (ATCC CCL-10) or CPAE cells. Total RNA was extracted from cells as previously described []. Viral double-stranded RNA was then purified by lithium chloride differential precipitation as described in [] and subjected to whole genome sequencing using the sequence-independent amplification procedure described by [] with modifications as described previously [].Library preparation and sequencing was performed using the Ion Torrent OneTouch ES, Personal Genome Machine with the Ion Xpress Plus fragmentation library kit, Xpress barcode adapters, Ion library quantitation kit, OneTouch 200 template kit v2, Ion PGM sequencing 400 kit and Ion 314 chip following the protocols recommended by the manufacturer (Life Technologies, Grand Island, NY). Briefly, approximately 1 µg of viral cDNA was fragmented, barcoded and quantitated. Template generation, enrichment and sequencing were performed on the appropriate Ion OneTouch instruments (Life Technologies, Grand Island, NY).Standard Flow-gram Format files were imported into Geneious 6.0 (Biomatters) for contig creation. Partial contigs were assembled and blasted against the NCBI nucleotide database to determine reference sequences that were then used for reference-based assemblies. [...] In order to encompass the currently known diversity of isolates from the United States, Central America and Caribbean regions additional BTV segment sequences were downloaded from GenBank. The genome segments from serotypes 1 and 2 of the closely related orbivirus, epizootic haemorrhagic disease virus (EHDV) were used to root the trees. The newly sequenced BTV genomes have been deposited in GenBank, KY091901-KY092170 (). GenBank accession numbers for all viral segments included in the study are located in Table S2.Alignments of each BTV segment were made in Geneious 8.0 (, []) using the MAFFT alignment option with default values. The ORF for each segment was determined using the Find ORFs option and the UTRs at the 5′ and 3′ ends were removed.Phylogenetic trees for each segment were estimated by the neighbour-joining method under the Jukes–Cantor distance model in Geneious 8.0. The homologous segments of EHDV-1 and EHDV-2 were used as outgroups to root the trees. Support values for nodes were estimated by 1000 bootstrap pseudo-replicates. Nodes with bootstrap values less than 70 were considered to be unsupported. Four BTV-3 strains, Mississippi 2006, Arkansas 2008, Mississippi 2009 and South Dakota 2012 were evaluated for potential reassortment events by comparison of nearest neighbours in phylogenetic trees for each individual segment. Reassortment can be demonstrated by the degree of topological congruence between phylogenetic trees of individual genome segments. When all of the segments for a viral strain share the same ancestor–descendent relationships the topologies of the phylogenetic trees of each segment will be very similar or identical. A segment from a co-infecting strain will have different ancestor–descendent relationships and therefore the closest relatives will differ in the phylogenetic tree for that segment. […]

Pipeline specifications

Software tools Geneious, MAFFT
Applications Phylogenetics, WGS analysis
Diseases HIV Infections