Complete genome sequences of new serotypes of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) isolated from a single alpaca in South Africa

hemorrhagic disease virus W e announce complete genome sequences of bluetongue virus and epizootic hemorrhagic disease virus isolated from a single alpaca in South Africa in 2008.Similar to recently discovered new serotypes, phylogenetic analyses suggest that both isolates are new serotypes of these orbivirus species (genus Orbivirus, family Sedoreoviri dae, order Reovirales).Orbiviruses are nonenveloped, multiprotein-layered viruses containing a 10-seg mented genome of double-stranded RNA (1).Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are transmitted by Culicoides biting midges, causing disease in ruminants and camelids (2, 3).BTV and EHDV consist of many serotypes.BTV serotypes 1-24 and EHDV cause notifiable diseases (4).Serotypes are determined by virus neutralization tests and serum neutralizing tests (serotyping) (5).VP2 protein encoded by genome segment 2, Seg-2[VP2], is the main target for serotype-specific neutralizing antibodies (6).Serotypes are grouped by some cross-neutralization (7).These groups match with the phylogenetic analysis of Seg-2[VP2] (genotyping) and are named nucleotypes "A" to "K" (8, 9).Since 2008, novel serotypes (putatively named BTV-24+) have been discovered (10-17), which is mainly based on inter-serotype genetic variation (8).Except for serotype 28, all recently discovered BTV serotypes are mem bers of nucleotype "K" (Fig. 1A).BTV-X appeared as a variant of BTV-32.Intra-serotype variation can be associated with their geographic origin (topotypes) (18).Initially, EHDV encompassed nine serotypes; however, EHDV-3 (ex-3) and EHDV-9 (318) have been regrouped into serotypes 1 and 6, respectively (19) (Fig. 1B).Detailed genotyping of Seg-2 clearly distinguished eastern and western (W) topotypes within each of the EHDV serotypes (Fig. 1B).Recently, EHDV-10E and the putative new EHDV-X were discovered in China and Japan, respectively (20,21).Serotyping of putative new serotypes is mostly impossible since virus isolates or suitable antibody-positive sera are not available.Two orbivirus isolates, ALP8 and ALP9, were isolated from a single alpaca (Vicugna pacos) in 2008 (22).The following methods have been extensively described in detail (23,24).Briefly, dsRNA was purified using TRI Reagent-LS (Molecular Research Center), LiCl precipitation of ssRNA, and extraction by MinElute Gel extraction columns used for dsDNA purification (Qiagen).A 5′-phosphorylated primer forming a loop (TibMolbiol) was ligated to dsRNA, similarly purified again (Qiagen), reverse transcribed with AMV reverse transcriptase (Invitrogen), and PCR-amplified using a second primer and Phusion polymerase (Finnzymes).cDNA was purified (PCR Purification Kit, Qiagen) and sequenced on a GS20 sequencer (Roche 454).Files were loaded into Seqman (DNASTAR, Lasergene v8.1.2) for de novo sequence assembly using default parameters.A 40-fold coverage allowed sequencing of complete genomes.Contig sequences were manually checked