Table 1.
Characteristics of dsRNA genome segments (cDNA copy) and proteins of the bluetongue virus serotype 26 Kuwait (KUW2010/02).
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) [48]. 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 [16]. However the analyses of BTV-26 (KUW2010/02) described here indicate that it forms a new 9th 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.
Table 2.
Summary of percentage sequence identities for Seg-3/VP3[T2] between the eastern viruses, western viruses, BTV-25/SWI2008/01 and KUW2010/02.
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 [16]. Lane 2 is a negative water control. Lane M: 1 kb marker.
Table 3.
Primers for amplification of Seg-2 from BTV-26 in RT-PCR assays.