Open access
5 March 2020

Complete Coding Sequence of a Novel Bluetongue Virus Isolated from a Commercial Sheeppox Vaccine


The full genome sequences of two isolates of bluetongue virus (BTV) from a commercial sheeppox vaccine were determined. Strain SPvvvv/02 shows low sequence identity to its closest relative, strain BTV-26 KUW2010/02, indicating the probable detection of a novel BTV genotype, whereas strain SPvvvv/03 shows high sequence identity to strain BTV-28/1537/14.


Bluetongue (BT) is a disease of ruminants which is transmitted by blood-feeding Culicoides midges (1). BT is one of the major diseases of ruminants listed by the World Organisation for Animal Health (OIE), and suspicion of disease needs to be reported to veterinary authorities. BT virus (BTV) belongs to the Orbivirus genus, and its genome consists of 10 linear double-stranded RNA segments encoding seven structural (VP1 to VP7) and five nonstructural (NS1 to NS5) proteins (2, 3). While the typical BTV genotypes (BTV-1 through BTV-24) are noncontagious and almost exclusively transmitted via their biological insect vector, Culicoides biting midges, direct transmission between infected animals has been documented for the atypical genotypes BTV-25, BTV-26, BTV-27, and BTV-28 (47).
BTV contamination of commercial batches of sheeppox and lumpy skin disease vaccines was previously reported (8). However, full-genome sequencing data were incomplete in this study and were limited to only a few segments. A subsequent study released the full genome sequence and demonstrated that the sheeppox vaccine-derived BTV-28 strain (BTV-28/1537/14) caused clinical signs in experimentally infected ewes and could be directly transmitted between infected and healthy sheep (7).
The commercial sheeppox vaccine (JOVAC, batch number 200214/01) was resuspended in 1 ml phosphate-buffered saline (PBS; pH 7.20) and used as the inoculum for virus propagation in baby hamster kidney (BHK) cells. Total RNA was extracted from the cell pellets using TRIzol reagent (Life Technologies, UK). Single-stranded RNA (ssRNA) was removed by RNase T1 digestion, and then double-stranded DNA (dsDNA) synthesis was performed using SuperScript III reverse transcriptase (RT) (Life Technologies) and the NEBNext Ultra II nondirectional RNA second-strand synthesis module (New England Biolabs, UK) according to the manufacturer’s instructions. Library preparation was performed using the Nextera XT DNA library kit (Illumina, USA), and paired-end read sequencing (2 × 150 bp) was carried out using an Illumina MiSeq instrument. The raw data were quality (with the parameter -q 25) and adapter trimmed along with the removal of short sequences (<50 bp) using Trim Galore (9). Paired-end reads were mapped to a set of reference genomes using Bowtie2 version 2.2.9 (10) with the “relax” setting to increase sensitivity. Local alignment was performed using a short seed (–15), allowing for one mismatch in the seed. The DiversiTools software (11) was used to generate the consensus sequence. Subsequently, the best consensus sequences (without any gaps) were used as the reference sequence (Table 1) for each segment, and the reads were mapped using BWA-MEM version 0.7.12-r1039 (12). In addition, the consensus sequences generated from BWA-MEM mapping were compared to those generated using in-house de novo mapping (unpublished protocol), but no changes were identified.
TABLE 1 Sequencing data for isolates SPvvvv/02 and SPvvvv/03
IsolateaGenBank accession no.Segment no.Length (bp)No. of mapped readsAvg depth (bp)Reference sequence nameReference sequence accession no.Nucleotide identity (%)
SPvvvv/02MN72387013,944202,3617,104BTV-26 KUW2010/02JN25515692.7
MN72387122,928142,9926,746BTV-26 KUW2010/02HM59064272.5
MN72387561,62973,3536,157BTV-26 KUW2010/02JN25515988.0
MN72387671,15725,4742,912BTV-26 KUW2010/02HM59064491.8
MN723879b108227,9991,272BTV-26 KUW2010/02JN25516287.2
The entire genome of SPvvvv/02 (segments 1 through 10) has a segment length of 19,181 bp, 790,759 mapped reads, and an average coverage depth of 4,824 bp, and the entire genome of SPvvvv/03 (segments 1 through 10) has a segment length of 19,193 bp, 1,960,587 mapped reads, and an average coverage depth of 12,448 bp.
BTV segment 10 (GenBank accession number KT946752) was first sequenced directly using RNA extracted from a commercial sheeppox vaccine (8); it had 100% nucleotide identity with SPvvvv/02 and 98.39% identity with both SPvvvv/03 and BTV-28/1537/14.
The full genome sequences of the two isolates, SPvvvv/02 and SPvvvv/03, were obtained from the sheeppox vaccine. SPvvvv/02 showed only 72.5% and 88.0% nucleotide identity to its closest relative, BTV-26 KUW2010/02, in segments 2 and 6, respectively (Table 1). This finding indicates the detection of a putative novel genotype of BTV. In contrast, SPvvvv/03 was highly identical to strain BTV-28/1537/14 across all 10 segments (99.86% to 100%), but its segment 1 was slightly shorter (3,944 bp) in comparison with that of BTV-28/1537/14 (3,985 bp). Our study indicates that the commercial sheeppox vaccine was contaminated with more than one novel BTV genotype.

Data availability.

The full genome sequences of isolates SPvvvv/02 and SPvvvv/03 have been deposited in GenBank under accession numbers MN723870 through MN723879 and MN723880 through MN723889, respectively. The raw sequencing reads have been deposited in the NCBI SRA under BioProject accession number PRJNA599340.


This research was funded by the Department for Environment, Food and Rural Affairs, grant number SE2621, and the Biotechnology and Biological Science Research Council (BBSRC) through projects BBS/E/I/00007030, BBS/E/I/00007033, BBS/E/I/00007036, and BBS/E/I/00007037. This publication was also supported by the European Virus Archive Goes Global (EVAg) project (research and innovation program grant agreement 653316) and PALE-Blu, both funded within the European Union’s Horizon 2020 framework.


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Information & Contributors


Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 9Number 105 March 2020
eLocator: 10.1128/mra.01539-19
Editor: Kenneth M. Stedman, Portland State University


Received: 9 January 2020
Accepted: 11 February 2020
Published online: 5 March 2020



Paulina Rajko-Nenow
The Pirbright Institute, Pirbright, Surrey, United Kingdom
Natalia Golender
Department of Virology, Kimron Veterinary Institute, Bet Dagan, Israel
Velizar Bumbarov
Department of Virology, Kimron Veterinary Institute, Bet Dagan, Israel
Hannah Brown
The Pirbright Institute, Pirbright, Surrey, United Kingdom
Lorraine Frost
The Pirbright Institute, Pirbright, Surrey, United Kingdom
Karin Darpel
The Pirbright Institute, Pirbright, Surrey, United Kingdom
Chandana Tennakoon
The Pirbright Institute, Pirbright, Surrey, United Kingdom
John Flannery
The Pirbright Institute, Pirbright, Surrey, United Kingdom
Carrie Batten
The Pirbright Institute, Pirbright, Surrey, United Kingdom


Kenneth M. Stedman
Portland State University


Address correspondence to Paulina Rajko-Nenow, [email protected].

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