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

Maan S, Maan NS, Nomikou K, Veronesi E, Bachanek-Bankowska K, Belaganahalli MN, Attoui H, Mertens PP - PLoS ONE (2011)

Bottom Line: Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV.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.

View Article: PubMed Central - PubMed

Affiliation: Vector-Borne Diseases Programme, Institute for Animal Health, Pirbright, Woking Surrey, United Kingdom.

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.

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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.
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pone-0026147-g005: 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.

Mentions: Sequence data generated for Seg-2 of KUW2010/02, and comparisons to other BTV types, were used to design four sets of oligonucleotide primers for conventional RT-PCR assays (Table 3). All four primer-pairs (1 to 4) worked well, generating products of the expected sizes from the original blood sample (KUW2010/01) and both passage levels of BTV-26 (KUW2010/02 and KU2010/03) (Figure 5). Although other combinations of these forward and reverse primers also appeared to be effective, they were not widely evaluated (data not shown). Primer-pairs 1 to 4 were also tested with reference strains of the most closely related heterologous serotypes (BTV-4, 10, 11, 17, 20 and 24, belonging to nucleotype ‘A’ [15]. Primer-pairs 1 and 2 generated faint bands that were near to the ‘predicted’ size, with some strains from nucleotype ‘A’ (Primer-pair 1 with BTV-4, 10, 17, 20 and 24; Primer-pair 2 with BTV-4, 10 and 17). Therefore primer-pairs 1 and 2 are not considered to be entirely BTV-26 specific. However, any cDNA amplicons generated can be sequenced using the same primer sets, helping to identify both the virus strain and its relationships to other isolates.


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

Maan S, Maan NS, Nomikou K, Veronesi E, Bachanek-Bankowska K, Belaganahalli MN, Attoui H, Mertens PP - PLoS ONE (2011)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198726&req=5

pone-0026147-g005: 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.
Mentions: Sequence data generated for Seg-2 of KUW2010/02, and comparisons to other BTV types, were used to design four sets of oligonucleotide primers for conventional RT-PCR assays (Table 3). All four primer-pairs (1 to 4) worked well, generating products of the expected sizes from the original blood sample (KUW2010/01) and both passage levels of BTV-26 (KUW2010/02 and KU2010/03) (Figure 5). Although other combinations of these forward and reverse primers also appeared to be effective, they were not widely evaluated (data not shown). Primer-pairs 1 to 4 were also tested with reference strains of the most closely related heterologous serotypes (BTV-4, 10, 11, 17, 20 and 24, belonging to nucleotype ‘A’ [15]. Primer-pairs 1 and 2 generated faint bands that were near to the ‘predicted’ size, with some strains from nucleotype ‘A’ (Primer-pair 1 with BTV-4, 10, 17, 20 and 24; Primer-pair 2 with BTV-4, 10 and 17). Therefore primer-pairs 1 and 2 are not considered to be entirely BTV-26 specific. However, any cDNA amplicons generated can be sequenced using the same primer sets, helping to identify both the virus strain and its relationships to other isolates.

Bottom Line: Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV.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.

View Article: PubMed Central - PubMed

Affiliation: Vector-Borne Diseases Programme, Institute for Animal Health, Pirbright, Woking Surrey, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus