Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait

Abstract

Bluetongue virus is the "type" species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing "bluetongue" (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen "VP7" showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein "VP2" identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other "eastern" or "western" BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.

Figure 1. Neighbour-joining tree showing relationships between VP3[T2] of KUW2010/02 with other orbiviruses.

KUW2010/02 showed up to 76.6%/88.9% nt/aa identity in Seg-3/VP3[T2] with other BTV strains confirming that it is an isolate of BTV. Accession numbers and further detail of the sequence and viruses used are included in Table 1. The tree was constructed using distance matrices, generated using the p-distance determination algorithm in MEGA 5 (500 bootstrap replicates) . The trees shown in Figures 2 and 3 were drawn using same parameters. The scale bar indicates the number of substitutions per site. Values at the nodes indicate bootstrap confidence. Epizootic haemorrhagic disease virus (EHDV), Bluetongue virus (BTV), Equine encephalosis virus (EEV), African horse sickness virus (AHSV), Chuzan virus (CHUV), St. Croix River virus (SCRV), Yunnan orbivirus (YUOV), Middle point orbivirus (MPOV), Peruvian horsesickness virus (PHSV), Broadhaven virus (BRDV), Stretch Lagoon Orbivirus (SLOV). Eastern and western isolates of EHDV and BTV are shown in blue and yellow respectively. Seg-3 accession numbers used for comparative analyses: AM745079, AM745029, AM745039, AM745049, AM745059, AM744979, AM744999, AM745019, AM745069, NC_005989, AF021236, FJ183386, M87875, NC_012755, NC_007749, NC_007657, EF591620, NC_005998, DQ186827, DQ186797, DQ186822, DQ186811, DQ186816, AF529047, AY493688, DQ186790, AM498052, DQ186792, DQ186826, DQ186819, DQ186817, L19969, L19968, NC 006014, AF017281, L19967.

Figure 2. Neighbour-joining tree showing relationships between Seg-6 from KUW2010/02 with the twenty five reference strains of different BTV serotypes.

The eight evolutionary branching points are indicated by black dots on the tree (along with their bootstrap values), dividing the sequences into nine ‘Seg-6 nucleotypes’ designated ‘A–I’. In previous studies, eight Seg-6 nucleotypes were identified. Members of the same nucleotype show >76% nt identity in Seg-6, while members of different nucleotypes show <76% nt identity . However the analyses of BTV-26 (KUW2010/02) described here indicate that it forms a new 9^th Seg-6 nucleotype (I), as it shows a maximum of 73.0%/79.3% nt/aa identity with previously existing BTV serotypes. Seg-6 accession numbers used for comparative analyses: AJ586695 - AJ586699, AJ586700, AJ586703 - AJ586711, AJ586713, AJ586714, AJ586716, AJ586719, AJ586720 - AJ586725, AJ586727, AJ586730, EU839842.

Figure 3. Neighbour-joining tree showing relationships between VP7[T13] from KUW2010/02 with other orbiviruses.

KUW2010/02 showed between 69.2%/80.8% to 81.2%/97.7% nt/aa identity in Seg-7/VP7[T13] to other BTV isolates, confirming its identity as a member of the Bluetongue virus species. Accession numbers and further detail of the sequence and viruses used are included in Table 1. Epizootic haemorrhagic disease virus (EHDV), Bluetongue virus (BTV), Equine encephalosis virus (EEV), African horse sickness virus (AHSV), Chuzan virus (CHUV), St. Croix River virus (SCRV), Yunnan orbivirus (YUOV), Peruvian horsesickness virus (PHSV), Broadhaven virus (BRDV) and California mosquito pool virus (CMPV). Seg-7 accession numbers used for comparative analyses: AM745023, AM744983, AM745013, AM745063, AM745033, AM745043, AM745073, AM745003, AM744993, AM745053, AM745083, FJ183391, AY078469, FJ183371, HM035361, HM035392, AF545433, M87876, NC 007754, NC 007663, NC 006004, ACF22097, AY485667, AM498057, FJ437558, AY841352, GQ506542, GQ506502, AF172829, AF188660, X53740, AY493692, M63417, AJ277802, AF172826, AF172825, AF188674, AF188673, AF172831, EU839843, L11724, DQ465027, DQ465028, DQ465026.

Figure 4. Examples of contiguous repeats found in the aa sequence of KUW2010/02 VP6.

Evidence was detected for repeated contiguous aa sequences in VP6 of KUW2010/02. The aa positions, as indicated, are between residues 205 to 232. The region 213 to 223 is shown as the target sequence, with matching repeats 205–211 (upstream) and 225–232 (downstream), shown in the upper and lower lines respectively. + similar residue: * identical residue.

Figure 5. Electrophoretic analysis of cDNA products generated from Seg-2 of BTV-26 (KUW2010/02) using primer-pairs designed from the homologous sequence.

PCR amplicons were generated from Seg-2 of BTV-26, isolate KUW2010/02 using primer- pairs 1 – 4 - Table 3 (lanes 3 to 6 respectively). Primer-pairs 3 and 4 are BTV-26 specific, while primer-pairs 1 and 2 also amplifies certain other serotypes in Seg-2 nucleotype ‘A’. Lane 1 is a positive control using RNA from BTV-6/RSArrrr/06, with primer-pair BTV-6/2/301F & BTV-6/2/790R – 1631 bp . Lane 2 is a negative water control. Lane M: 1 kb marker.