doi:10.5455/vetworld.2013.244-248 Segment 2 based characterization of a novel Indian

Aim: The study was conducted to characterize and serotype the novel isolate of bluetongue virus (BTV) isolated from India. 
 
Materials and Methods: The BTV isolate was propagated in BHK-21 cell line. Nucleic acid (dsRNA) was extracted using Trizol method and cDNA was prepared using a process called reverse transcription. The cDNA was subjected to group specific PCR using ns1 gene specific primer to confirm the isolate as BTV. The type specific PCR was conducted to confirm the serotype of the virus using vp2 gene specific primers for all the BTV serotype including BTV10. The vp2 gene specific PCR amplicon was sequenced and in-silico restriction enzyme analysis and phylogenetic analysis was conducted. 
 
Results: Group specific PCR using ns1 gene specific primers showed a single 274bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The type specific PCR using BTV10 vp2 gene specific primer showed a single amplicon of 647bp. Remaining BTV serotype specific primers didn\'t show any amplification. The vp2 gene PCR amplicon was sequenced. The in-silico restriction enzyme analysis of vp2 gene of Indian BTV10 isolate along with other isolates from GenBank database using HindIII, XhoII and ApoI showed a common pattern between Indian and USA isolates. Similarly, phylogenetic analyses using vp2 gene nucleotide as well as deduced amino acid sequence of Indian BTV10 isolate and global isolates 
showed that Indian and most of the USA isolates placed in a single clad. 
 
Conclusion: A novel BTV isolate was isolated and confirmed as BTV serotype 10. Upon molecular analysis Indian BTV10 isolate was found closer to that of USA isolates than other global isolates.


Introduction
concrete shells i.e., the inner and outer capsid and 4 non-structural proteins (NS1, NS2, NS3 and NS3a).Bluetongue (BT) is an infectious, non contagious Till 2008, twenty four distinct serotypes of BTV arthropod borne viral disease of wild and domestic have been isolated and characterised worldwide [1].ruminants especially sheep.The disease is caused by However, recently two more proposed serotypes i.e. bluetongue virus (BTV) the type species of the genus BTV-25 from Switzerland [8] and BTV-26 from Orbivirus and belongs to family Reoviridae [1].BTV Kuwait [9] have been reported.A total of 21 different infects most of the domestic and wild ruminant and BTV serotypes have been reported from different parts causes BT disease primarily in sheep characterised by of Indian subcontinent based upon serology and virus severe clinical signs such as fever, lameness isolation [10].However, in recent past BTV serotype (coronitis), swelling and cyanosis of lips and tongue 21 has been isolated from West Bengal state of India leading to death.It is listed as a 'notifiable disease' by [11].In the present study a virus isolated from sheep the Office International des Epizooties [2].
suspected for BT disease from Andhra Pradesh was The disease occurs as more severe form in sheep undertaken for typing and characterization studies.[3].Occasionally Severe forms of clinical signs and

Materials and Methods
mortalities can be observed in goats and camelids [4].

Cattle act as reservoirs for virus and remain viraemic
Sample preparation: The BTV isolate used in present for several months and spread virus through insect study was isolated from Andhra Pradesh state.The vector [5].Subclinical form of infection can also cause virus was grown in confluent monolayer of BHK 21 loss of condition, reduced milk yield, abortion leading cell line to raise the stock.After appearance of to infertility [6].Therefore mandatory export restricomplete CPE in infected BHK-21 cell line, the ctions and the surveillance requirements are imposed infected cells were harvested and pelleted at 3,000X g on movement of ruminants and other animal products for 10 minutes in table top centrifuge (REMI, India). of BT endemic countries to BT free countries [7].
The supernatant was decanted carefully and viral BTV consists of double-stranded RNA (dsRNA) dsRNA was extracted from pelleted material using ten segmented linear genome which encode 7 Guanidinium isothiocynate method [12].The extracted structural proteins (VP1 to VP7) organised into two viral RNA was screened by RNA-PAGE followed by silver staining to visualize the 10 dsRNA segments for minute.All the PCR products were visualised by confirmation as BTV.
agarose gel electrophoresis under UV light in gel documentation system (Biovis, USA).along with 6% DMSO and 30 pM of random primer Terminator sequencing kit (ABI, USA) as per then finally 400µM each dNTPs and 500 U of Momanufacturers' instructions.The amplified product MuLV reverse transcriptase (Promega) for reverse was purified, precipitated and dissolved in Hi-Di transcription was added.The primer was subjected to formamide (ABI, USA).The dissolved product was anneal at 25°C for 10 min, reverse transcription at 37°C transferred to sequencing plate and subjected to TM for 60 min and finally heat inactivation at 90°C for 10 sequencing in DNA analyzer (ABI PRISM 3130 min. Version 3.0) in the Department.The sequencing data The group specific PCR was carried out to was collected and subjected to sequence analysis using confirm the presence of BT viral genome in the sample.
identity calculation of partial vp2 gene nucleotide as well as deduced amino acid sequences along with other

Determination of serotype of BT isolate by using type
global sequences of BTV10 obtained from GenBank specific primers: The cDNA was further subjected to database.type specific PCR using all 24 serotype specific vp2 Phylogenetic tree was constructed based on gene based primers.A total of 24 PCR reactions were nucleotide as well as deduced amino acid sequences for carried out using serotype 1-24 type specific primers the Indian BT isolate with other global isolates using individually in 20 µl reaction mixture.Each reaction Mega 5 software [16].Restriction mapper version 3.0 mixture contained 2 µl cDNA, 3% DMSO, 20 µM of (http://www.restrictionmapper.org/)software was type specific primer pair along with 0.4 µl of 10mM used for in-silico restriction analysis of Partial vp2 dNTPs mix (Finnzyme), 4 µl 5X HF buffer and 0.  country [17].Different serotype specific antibodies of as BTV10, a novel serotype not isolated in BHK21 cell BTV that have been reported so far from various parts line earlier at BTV typing centre Hisar so far. of India are 1, 2, 3, 4, 5, 6,7, 8,9, 10., 11, 12, 13, 14, 15, The PCR product of BTV 10 was allowed for 16, 17, 18, 19, 20, 21 and 23 [10].The virus isolated in direct sequencing and the nucleotide sequence cell culture have been listed as BTV serotypes 1, 2, 3, 4, obtained was deposited to GenBank database and an 8, 9, 12, 16, 17, 18, 21, 23 but not 5, 6, 7, 10, 11, 13, 14, accession no.JF303890.1 was assigned.The sequence 15, 19, 20, 22 and 24.In the present study, a BTV data was further analysed to determine the identity of isolate of sheep origin adapted in BHK21 cell line was this isolate with rest of the global isolated in the isolated from sheep of Andhra Pradesh was typed and genBank using Bioedit v7.0.8 software programme characterized.
(Table -1).The data analysis showed that novel BTV10 The dsRNA extracted from cell culture grown isolate (JF303890.1_BTV10IND) share 100% virus was screened by RNA-PAGE and silver staining nucleotide sequence identity with four USA isolates showed presence of 10 dsRNA segments arranged in (GenBank accession nos.U06786.1,L29027.1, 3:3:3:1 pattern typical of BTV (data not shown).The L29026.1andM11787.1) and one of the Indian isolate viral RNA was further subjected to ns1 gene specific (GenBank accession nos.JQ740772.1).However, it RT-PCR.This group specific RT-PCR showed a single showed more than 95% nucleotide identity with six 274 bp amplicon of expected size confirmed the other USA isolates (GenBank accession nos.sample as positive for BTV (Fig. 1).After confirming U06780.1,U06781.1,U06782.1,U06783.1,U06784.1 the sample as BTV isolate, the sample was subjected to and U06785.1)and one Indian isolate (GenBank typing PCR to determine the serotype it possessed accession nos.JF727655.1).While, novel BTV10 using 24 serotype specific primer pairs based on vp2 isolate showed only 88.7% and 89.5% nucleotide gene.A single expected 648bp amplicon size was identity with South Africa and France BTV 10 isolate.observed without any nonspecific amplification in 1% The nucleotide sequences of the BTV10 isolate agarose gel electrophoresis (LifeTech.USA) (Fig. 2).
was deduced into corresponding amino acid sequences There was no amplification with remaining twenty according to open reading frame (ORF).The percent three serotype specific primers.The isolate was typed amino acid sequence identity was determined using    Bioedit v7.0.8 software programme and the result was for BTV10 serotype [18,19] but no reports of virus coincidental with nucleotide based identity (Table 1).It isolation have been observed till 2011.However, was observed that novel BTV 10 isolate showed 100% recently BTV10 was reported from Andhra Pradesh.amino acid identity with seven of USA isolates (Gen The partial nucleotide sequence analysis revealed that Bank accession nos.U06786.1,L29027.1,L29026.1, the isolate in study showed 99.7% identity with one of M11787.1,U06783.1,U06782.1, and U06780.1)and the Indian isolate [20] and 100% identity with one two Indian isolate (GenBank accession nos. another Indian isolate [21] as well as four USA isolates.JQ740772.1andJF727655.1).However, it showed Similarly deduced partial amino acid sequence based more than 99% identity with three other USA isolates analysis revealed 100% identity with above two (GenBank accession nos.U06781.1,U06785.1 and isolates and USA isolates.The BTV 10 is prevalent in U06784.1).However, Indian isolate showed 91.3%American continent since 1970s [22] but it is recently and 93.2% identity with South Africa and France isolate.
reported from India.Therefore, the present study Phylogenetic tree was constructed based on suggested that BTV 10 serotype could have been nucleotides as well as deduced amino acids sequences migrated to India from the USA through illegal import of novel BTV 10 isolate (JF303890.1_BTV10IND) of live vaccines or animals infected with low doses of with other isolates available in GenBank using Mega 5 BTV10 or vaccinated with live virus vaccines.software programme (Fig. 3 and 4).The novel BTV10 However it is difficult to say how and when this had isolate formed close cluster with Indian isolate happened.The 100% identity of the Indian BTV10 (GenBank accession nos.JQ740772.1andJF727655.1)isolate used under present study with USA vaccine and most of the USA isolates and was closer to minor isolate (GenBank accession no.U06786.1)along with cluster obtained from three other USA isolates few other BTV10 isolates of sheep origin in USA is (GenBank accession nos.U06781.1,U06785.1 and supportive of the hypothesis.These findings speculate U06784.1).However, novel isolate BTV10 was found the increased incidences and severity of the disease in distantly related to South African (AJ585131.1 local Indian sheep breeds in near future._BTV10 R.S.A.) and France (HQ222821.1_BTV10

FRA) isolates. The phylogenetic trees generated on the
A novel BT virus isolate from in India was typed basis of analysis of nucleotide sequences and amino and characterized as BTV serotype 10.Since India is acid sequences generated similar type of findings endemic for BTV a large number of BTV serotypes are except the amino acid generated tree showed more circulating in the country.Many of these viruses closeness of the isolates in major clad than that of including novel BTV10 might have get entry into nucleotide generated tree (Fig3 and 4).
India though import of animals (infected or vaccinated The in-silico restriction analysis of novel BTV 10 with live virus vaccine) alone or along with vectors (JF303890.1_BTV10IND) and other isolates available directly from USA to India or indirectly from USA to in GenBank with Hind III, XhoII and ApoI restriction the neighbouring countries and then in India.. enzyme was done using online software programme Therefore to control the disease in the country suitable restriction mapper version 3.0 (http://www.restriction measures should be adopted having facility for control mapper.org/)(Table -2).The in-silico restriction on importation of animal and animal's products and enzyme analysis revealed a single restriction site for live vaccines.Hind III and XhoII at 2148 nt and 2478 nt respectively in all the isolates from India and USA.However, in

Figure- 3 .
Figure-3.Phylogenetic tree of BTV10 isolates based on partial vp2 gene nucleotide sequences.Tree was constructed from partial nucleotide sequences of vp2 gene using the neighbour joining method in Mega5 software programme with default parameters.Numbers at the major nodes indicate the bootstrap values.

Figure- 4 .
Figure-4.Phylogenetic tree of BTV10 isolates based on partial vp2 gene amino acid sequences.Tree was constructed from partial amino acid sequences of vp2 gene using the neighbour joining method in Mega5 software programme with default parameters.Numbers at the major nodes indicate the bootstrap values.

Amplification of BT viral genes by RT-PCR for confirmation of BT isolate: The BT viral genomic Nucleotide sequence based characterization of BT dsRNA
was used as template for cDNA synthesis using isolate: The serotype 10 specific PCR product was

Table - 1
. Percent nucleotide and amino acid sequence identity of vp2 gene of BTV 10 (2124-2611bp nucleotides and its corresponding amino acids)

Table - 2
. In-silico restriction analysis of BTV 10 isolates from different parts of the world IND=India, RSA= South Africa, FRA=France