Genomic diversity among eastern and western topotypes of bluetongue virus serotype 16 based on whole genome sequence analysis

Bluetongue (BT) is a noncontagious, vector-borne disease of domestic and wild ruminants. The causative agent bluetongue virus (BTV) is a double stranded RNA virus belonging to genus Orbivirus within family Reoviridae. Eradication of BTV from endemic regions like India is not an easy task due to the widely distributed Culicoides spp. midge vectors, the ubiquitous distribution of vertebrate hosts and existence of a large number of serotypes of the virus (at least 26 till date). The complete genomes (19,193 base-pairs) of several strains of bluetongue virus serotype 16 (BTV-16) originated from Australia, China, Indian subcontinent, Mediterranean basin, Middle East, Africa (Nigeria) and Europe, were compared. These analysis showed that all ten genome segments of a Nigerian strain are derived from a western lineage, showing only 77% 84% nt identity with the eastern topotype reference strain 'RSArrrr/16' (and its derivative 'RSAvvvv/16', a vaccine strain) that was originally isolated in Pakistan, 76.4% 83% with eastern BTV-16 strain from Australia (DPP96) and 77% 89% with a reassortant strain from India. These detailed comparisons involving global strains showed that there is a very high degree of variation (up to 24%) between BTV-16 strains from eastern and western geographical regions. These data confirm the value of whole genome sequencing for characterization of novel BTV isolates and has helped to identify representative suitable 'reference-strain' of eastern topotype (BTV-16e), western topotype (BTV-16w), as well as 'cross-topotype' reassortant strain (BTV-16r) that are generated in the field for further serological, phylogenetic and molecular epidemiology studies.

Bluetongue virus (BTV) is a double stranded serotypes in India in last two decades [8,9], illustrate RNA virus (genus Orbivirus; family Reoviridae) [1,2], the increased risks posed by BT and other emerging which can infect both domestic and wild ruminants.arboviral diseases worldwide [10].Although BTV can acquire a membrane envelope High incidence of reassortment due to segmented during budding from infected cells, it is usually genome, existence of multiple serotypes, geographical regarded as non-enveloped, consisting of a three restriction in the distribution of many of the serotypes, layered icosahedral protein capsid [3].The ten linear use of live attenuated vaccines in several parts of the dsRNA genome segments of BTV (Seg-1 to Seg-10), world and the lack of complete sequences of viruses encode a total of 7 structural proteins (VP1-VP7) and at isolated from several parts of the globe have least 4 non-structural proteins (NS1, NS2, NS3/3a and complicated our understanding of the origin, NS4) [4,5].VP2 and VP5 (encoded by Seg-2 and Segmovement and distribution of BTV.

6) are the most variable of the viral proteins and form
The nucleotide sequences of BTV isolates reflect their the outermost layer of the BTV capsid.They also represent geographic origins [11][12][13], and the majority of the a target for neutralising antibodies (in particular VP2) BTV genome segments can clearly be divided into and determine the serotype of BTV [6,7] .
'eastern' or 'western' groups / topotypes [6,[14][15][16].This The virus is transmitted between its mammalian indicates that these viruses have evolved, with little hosts via the bites of vector-competent Culicoides genetic exchange between regions, over a very long species, although it can also be transmitted vertically in period of time, allowing them to acquire multiple point ruminants, or by ingestion of infected tissues, resulting mutations and clear regional differences.in financial burden and trade restrictions.-84% nucleotide identity with the eastern topotype attenuated vaccine may be involved in the origins of all reference strain 'RSArrrr/16' that was originally of the European incursions of BTV-16.The live isolated in Pakistan, 76.4% -83% with eastern BTV-16 vaccine strain of BTV-16 was used as part of an annual strain from Australia (DPP96) and 77%-89% with vaccination campaign in Israel and could represent a reassortant strain from India.We had concluded that source for the European viruses [20].The outbreak in Nigerian strain (NIG1982/10) represented a suitable Sardinia during 2004 was caused by the reassortant 'reference-strain' of BTV-16w, for further serological, BTV-16 vaccine used in Italy during 2004 [21,22] and phylogenetic and molecular epidemiology studies [27].was not caused by the strain from Greece and Turkey.
These data showed that there is a very high degree of In November 2008, there were reports of further variation (up to 24%) between BTV-16 strains from outbreaks due to BTV-16 in Greek island of Lesvos eastern and western geographical origins.returning after ~7 years and in both Israel and Oman.
These data confirm the value of whole genome Since 2010, there were several isolation of BTV-16 sequencing (WGS) for characterization of novel BTV from India [23].
isolates and will help to identify other eastern -western Until now, four eastern BTV-16 strains, from BTV isolates, as well as 'cross-topotype' reassortant-India, China, Australia and the South African reference viruses that are generated in the field.strain have been fully sequenced [23][24][25][26].Previous distinct virus lineage, although still within the major-Authors are highly thankful to field veterinarians eastern-topotype (Fig. 1).The Indian strain of  and all international colleagues especially the colleais a reassortant, showing high levels of nt identity (92gues at the Pirbright Institute who have provided virus 97%) in the majority of its genome segments to the isolates and data for these analysis.This work was RSArrrr/16, but containing Seg-5 derived from a funded by Rastriya Krishi Vikas Yojna scheme no.western topotype (with 89% nt identity).
4011/C(g) ABT-4-0A.Our recent full genome sequence data on a Figure-1.Unrooted neighbourjoining phylogenetic tree using segment 2 sequences of BTV-16.The tree was constructed using a pdistance algorithm and pairwise deletion parameters.Texas show GenBank accession numbers and geographic origin of strains that were compared.Bootstrap values (%) are represented at each tree node.Node support was assessed with 500 bootstrap pseudoreplicates.BTV-16 strains from different geographical regions make two major eastern and western topotypic groups.

Competing interests
A.E., Anthony, S.J., Samuel, A.R., Darpel, K.E., Veronesi, Therefore, Outbreaks caused by BTV-16 occurred in Greece bluetongue (BT) represents a significant worldwide during 1999-2000, and in the Turkish province of Izmir threat to livestock industries.during 2000 [15-17].During 2002 to 2006 further The recent emergence of ten BTV serotypes in outbreaks occurred in the Mediterranean region.Europe, eight new serotypes in the USA, two new Sequence analysis of Seg-2, indicates that all of the serotypes in Australia and isolation of seven different Mediterranean / European isolates of BTV-16 belong to an eastern group of viruses and are closely related to the South African BTV-16 vaccine (<0.7% variation), suggesting a recent common ancestry [16].The BTV-16 vaccine strain can cause severe disease in some European breeds of sheep that are sufficient to allow western topotype of BTV-16 from Nigeria (NIG1982/ infection of feeding Culicoides and therefore onward 10), made it possible to carry out a detailed analysis of transmission of the virus [18].The vaccine strain was its relationships to BTV strains from other areas of the derived from the reference strain that was originally world [27].All ten genome segments of NIG1982/10 isolated from Hazara in West Pakistan during 1960 are derived from a western lineage, showing only 77% [19].This close relationship suggests that the live,