Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2

Abstract

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Figure 1. Electrophoretic analysis of cDNA products from Seg-2 of BTV reference strains using ‘type-specific’ primer-pairs for individual serotypes (Panels A–F).

Panel A: PCR amplicons were generated from Seg-2 of BTV-1/RSArrrr/01 using primer-pairs ‘1A1’ −1621 bp, ‘1A2’ −864 bp, and ‘1W1’ −1743 bp (lanes 1, 2, and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-2/RSArrrr/02 using primer-pairs ‘2A1’ −1800 bp, ‘2A2’ −2343 bp and ‘2W2’ −1246 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-3/RSArrrr/03 using primer-pairs ‘3W1’ −648 bp, ‘3W2’ −480 bp and ‘3W3’ −652 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-4/RSArrrr/04 using primer-pairs ‘4W1’ −2045 bp, ‘4W4’ −1071 bp and ‘4W5’ −929 bp (lanes 10, 11 and 12 respectively). Panel B: PCR amplicons were generated using primer-pairs ‘5W1’ −1362 bp, ‘5W2’ −1280 bp and ‘5W3’ −2124 bp from Seg-2 of BTV-5/RSArrrr/05 (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-6/RSArrrr/06 using primer-pairs ‘6W1’ −1724 bp, ‘6W3’ −1303 bp and ‘6W4’ −2051 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-7/RSArrrr/07 using primer-pairs ‘7W1’ −1798 bp, ‘7W2’ −1577 bp and ‘7W3’ −1609 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-8/RSArrrr/08 using primer-pairs ‘8W4’ −363 bp, ‘8W5’ −2216 bp and ‘8W6’ −562 bp (lanes 10, 11 and 12 respectively). Panel C: PCR amplicons were generated using primer-pairs ‘9W1’ −1093 bp, ‘9W2’ −961 bp from Seg-2 of BTV-9/RSArrrr/09 (lanes 1 and 2 respectively), and from Seg-2 of BTV-9/BUL1999/01 using primer-pair ‘9E1’ −1105 bp (lane 3). PCR amplicons were generated from Seg-2 of BTV-10/RSArrrr/10 using primer-pairs ‘10W4’ −964 bp, ‘10W5’ −1094 bp and ‘10W6’ −1109 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-11/RSArrrr/11 using primer-pairs ‘11W4’ −1077 bp, ‘11W5’ −1096 bp and ‘11W6’ −355 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-12/RSArrrr/12 using primer-pairs ‘12W1’ −1613 bp, ‘12W2’ −892 bp and ‘12W3’ −1326 bp (lanes 10, 11 and 12 respectively). Panel D: PCR amplicons were generated from Seg-2 of BTV-13/RSArrrr/13 using primer-pairs ‘13W2’ −1323 bp, ‘13W3’ −1236 bp and ‘13W4’ −1655 bp (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-14/RSArrrr/14 using primer-pairs ‘14W1’ −850 bp, ‘14W2’ −1581 bp and ‘14W3’ −849 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-15/RSArrrr/15 using primer-pairs ‘15W1’ −1823 bp, ‘15W2’ −991 bp and ‘15W3’ −1067 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-16/RSArrrr/16 using primer-pairs ‘16A3’ −851 bp, ‘16E2’ −1288 bp (lanes 10 and 11 respectively) and from Seg-2 of BTV-16/NIG1982/10 using primer-pair ‘16W2’ −726 bp (lane 12). Panel E: PCR amplicons were generated from Seg-2 of BTV-17/RSArrrr/17 using primer-pairs ‘17W1’ −1256 bp, ‘17W4’ −689 bp and ‘17W3’ −1090 bp (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-18/RSArrrr/18 using primer-pairs ‘18W1’ −1021 bp, ‘18W4’ −1556 bp and ‘18W3’ −1381 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-19/RSArrrr/19 using primer-pairs ‘19W1’ −1787 bp, ‘19W2’ −1680 bp and ‘19W3’ −1494 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-20/RSArrrr/20 using primer-pairs ‘20E1’ −1324 bp, ‘20E2’ −1257 bp and ‘20E3’ −827 bp (lanes 10, 11 and 12 respectively).Panel F: PCR amplicons were generated from Seg-2 of BTV-21/RSArrrr/21 using primer-pairs, and ‘21E3’ −1524 bp, ‘21E2’ −1726 bp and ‘21E1’ −1320 bp (lanes 1, 2 and 3 respectively). PCR amplicons were generated from Seg-2 of BTV-22/RSArrrr/22 using primer-pairs ‘22W1’ −1074 bp, ‘22W2’ −2034 bp and ‘22W3’ −2211 bp (lanes 4, 5 and 6 respectively). PCR amplicons were generated from Seg-2 of BTV-23/RSArrrr/23 using primer-pairs ’23E1’ −1548 bp, ‘23E2’ −1623 bp and ‘23E3’ −1421 bp (lanes 7, 8 and 9 respectively). PCR amplicons were generated from Seg-2 of BTV-24/RSArrrr/24 using primer-pairs ‘24W1’ −1776 bp, ‘24W2’ −1557 bp and ‘24W3’ −2021 bp (lanes 10, 11 and 12 respectively).Lane M: 1 Kb marker. +C is a positive control using RNA from BTV-6/RSArrrr/06, with primer-pair BTV-6/2/301F & BTV-6/2/790R −1631 bp – . −C is a negative water control. For primer position and sequence see Table S1 and S2. The use of type specific primers for BTV-26 was recently published by , .

Figure 2. Neighbour-joining tree, showing relationships in Seg-2 between reference and field strains of BTV-1w, 4w, 8w and 16w from Oman.

The tree was constructed using distance matrices, generated using the p-distance determination algorithm in MEGA5 (500 bootstrap replicates) . BTV split into 26 distinct groups based on Seg-2 sequences, reflecting serological relationships between virus strains . Reference strains of BTV are shown in black and field isolates from Oman are shown in blue font. Isolate designations: IAH ‘dsRNA virus reference collection number’ composed of country code, year, and the number of the isolate in that year from that country . Full length Seg-2 sequences of BTV-1/OMN2009/01, BTV-4/OMN2009/09, BTV-8/OMN2009/03 and 16/OMN2009/02 showed 95%, 94%, 93% and 73.6% nucleotide (nt) identity to the reference strains of respective serotype, confirming their types and topotypic groups.Scale represents number of substitutions per site. Values at major branching points represent NJ bootstraps.