Sequence Analysis of the S10 Gene of Six Bluetongue Virus Isolates from India

ORIGINAL ARTICLE

Sequence Analysis of the S10 Gene of Six Bluetongue VirusIsolates from IndiaG. S. Desai1, M. Hosamane2, R. S. Kataria3, S. S. Patil4, K. Prabhudas4, R. K. Singh5, V. Bhanuprakash5

and B. Mondal5

1 HSADL, IVRI, Anand Nagar, Bhopal2 IVRI, Hebbal, Bangalore3 NBAGR, G.T. Road, Karnal4 PD ADMAS, Hebbal Bangalore5 Division of Virology, IVRI, Mukteswar, Kumaon

Introduction

Bluetongue (BT) is an infectious, economically important,

arthropod borne disease of domesticated and wild rumi-

nants. The disease is enzootic in many tropical and tem-

perate countries coincident with the distribution of

Culicoides midges (Mellor and Boorman, 1995). Blue-

tongue is caused by Bluetongue Virus (BTV), the proto-

type specie of the genus Orbivirus within the family

Reoviridae and about 24 serotypes of the BTV are preva-

lent world wide. The BTV particle contains 10 double

stranded RNA (dsRNA) segments as genome, surrounded

by a double layered protein capsid (Verwoerd et al.,

1972). The outer capsid proteins (VP2 and VP5, encoded

by genomic segments 2 and 3 respectively) are responsible

for serotype specificity and mediate infection of mamma-

lian cells. The inner core is made of VP1, VP3, VP4, VP6

and VP7 proteins coded by genomic segment 1 and seg-

ments 4–7. The dsRNA genomic segment 10 (S10) codes

for two proteins, NS3 and NS3A, in a single open reading

frame. These proteins localize to the plasma membrane

and intracellular vesicle membranes and mediate the

release of virus particles from BTV-infected cells (Hyatt

et al., 1993). It has been suggested that the NS3 protein

may exert an important role in determining the vector

status of individual Culicoides species for both BTV and

related Africal Horse Sickness Virus (Martin et al.,1998; :

Breard et al., 2007).

Bluetongue is a fast emerging viral disease in India and

causes mortalities up to 40% leading to huge economic

losses in native/local cross bred as well as in exotic breeds

of sheep. The disease is endemic in livestock population

in many states and many severe BT outbreaks in sheep in

recent years (2005–2008) have been reported in southern

states of Andhra Pradesh, Karnataka and Tamil Nadu. No

BTV vaccination is practiced because of the non-avaiala-

bility. Though BTV-specific antibodies have been demon-

strated in other ruminants like cattle, buffaloes and goats,

clinically overt BT has not been observed in these species

(Kakker et al., 2002). The disease was first reported in

exotic breeds of sheep in India in the state of Maharash-

tra (Sapre, 1964) and since then 21 different BTV sero-

types have been reported from India and BTV serotypes

1, 18 and 23 have been found to be associated with most

of the BT outbreaks in recent times.

Nucleotide sequencing of the BTV genes provides

genetic information for genotyping/topotyping of the

virus isolates from different geographical areas of the

Keywords:

blue tongue; BTV; India; S10 gene; sequence

Correspondence:

G. S. Desai. HSADL, IVRI, Anand Nagar, Bhopal-

462021. Tel.: +91 755 2750647; Fax: +91 755

2758842; E-mail: [email protected]

Received for publication March 22, 2009

doi:10.1111/j.1865-1682.2009.01089.x

Summary

Bluetongue Virus (BTV) genome segment 10 (S10)-based phylogenetic studies

are important in understanding the BTV evolution. S10 gene-based phyloge-

netic analysis grouped six different BTV isolates (BTV serotype 1, 18 and 23)

from India in subclade A1 and showed closer relationship with BT viruses from

Mediterranean Basin. Indian BTV serotypes 18 and 23 formed a single cluster

distinct from BTV serotype 1 isolates and were evolved from BTV from China,

Indonesia and Australia. The overall S10 sequences of BTV isolates from India

were largely conserved (>95.7% homology) and were distinct from other BT

viruses of the world.

Transboundary and Emerging Diseases

ª 2009 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 56 (2009) 329–336 329

world. For this, BTV genome segments coding VP2, VP3,

VP5, NS1 and NS3 proteins of BTV are currently relevant.

BTV S10 gene-based topotyping helps in understanding

the limitations of the virus serotypes in various locations

around the world. Previous studies have shown that phy-

logenetic analyses of the S10 genes of the global strains of

BTV tend to segregate independently of their serotype

into region-specific topotype clusters as a consequence of

negative (purifying) selection over time in each region

(Pritchard et al., 2004; Balasuriya et al., 2008). This paper

describes the analysis of the nucleotide sequences of com-

plete protein coding region of S10 of six BTV isolates that

originated from different geographic locations in India

and their epidemiological relationship with those of BTV

from around the world.

Materials and Methods

Total RNA was isolated from the infected BHK-21 cells of

the six BTV isolates (their serotype was determined by mi-

cronuetralization assay) (Fig. 1 and Table 1) using TriZol

(Invitrogen) reagent. The viral RNA was reverse tran-

scribed and the complete S10 gene protein coding region

was PCR amplified using the high fidelity Taq DNA poly-

merase (Invitrogen) and the specific primers (containing

BamHI restriction enzyme site), NS3F 5¢-cccgggatcccat

gctatccgggctg-3¢ and NS3R 5¢-cccgggatcctactgatcttaggtt

aatg-3¢ representing nt 19–34 and nt 699–717 respectively

of S10 gene sequence of BTV serotype 4 (GenBank Acces-

sion No. AJ783910). After visual confirmation of the

expected size of the PCR amplified product on agarose

gel, the amplicons were cloned in to pGEM-T easy vector

(Promega, Madison, WI, USA). The recombinant plasmids

were sequenced twice from both the directions using both

M13 primers in ABI 3130 Genetic Analyzer (ABI, Foster

City, CA, USA). The generated S10 nucleotide and trans-

lated NS3/NS3A amino acid (AA) sequences were aligned

with the corresponding sequences of different reported

BTV serotypes across the world using ClustalW software.

Phylogenetic and molecular evolutionary analyses were

conducted using MEGA version 4 (Tamura et al., 2007).

Results and Discussion

The S10 gene nucleotide sequencing revealed that coding

sequence of all six Indian BTV isolates were 690 bp in

length and contained two in-frame AUG codons. Majority

of the nucleotide changes were at the third position of

the codons and were distributed through out the entire

length of the S10 gene. Nucleotide sequence distances of

the S10 gene segment amongst all Indian BTV isolates

indicated that within the BTV 18 serotypes, BTV18BIND

was more divergent (1.0%) amongst the Indian BTV and

BTV1AIND was sharing a homology of 99% with other

two BTV 1 isolates. Both BTV23DIND and BTV23RIND

exhibited a 99.9% similarity in their nucleotide sequences.

Srinagar

Dehradun

Avikanagar

Rahuri

MadrasBangalore

Fig. 1. Map indicating the origin of BTV isolates.

Table 1. Details of the six Indian BTV isolates whose S10 gene sequences have been studied in this work

BTV isolate Serotype

Passage level in

BHK-21 cells Geographic origin

Reference/Year of

Isolation

GenBank

Accession No.

BTV1AIND 1 5 Avikanagar, Rajasthan State Prasad et al. (1994) EU131023

BTV23DIND 23 55 Dehradun Uttaranchal State Mehrotra et al. (1995) EU131022

BTV23RIND 23 6 Rahuri Maharashtra State 1988 EU131027

BTV18SIND 18 8 Srinagar Jammu and Kashmir State NA EU131026

BTV18BIND 18 14 Bangalore Karnataka State NA EU131024

BTV18MIND 18 11 Madras (Chennai) Tamil Nadu State NA EU131025

NA: not available.

Sequence Analysis of Indian BTV S10 Gene G. S. Desai et al.

330 ª 2009 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 56 (2009) 329–336

The overall nucleotide sequence divergence was 0.6–4.4%

between different Indian BTV isolates.

The deduced NS3/3A amino acid (aa) sequences of all

Indian BTV isolates showed that the two glycosylation

sites 63NS/TT65 and 150NGT152, a cysteine residue at

position 137, a tryptophan residue at position 159 and

the PPXY and PS/TAP late domain motifs between resi-

dues 36 and 44 of the proline rich region (aa 36 to aa 50)

were conserved (Fig. 2). These late domain motifs have

been indicated to usurp the vacuolar protein sorting

pathaway for the formation of multivesicular bodies to

allow BTV particles to leave host cells by a budding

mechanism (Wirblich et al., 2006), There was an A–V

substitution at position 40 located outside the first PPXY

late domain motifs in BTV1AIND, similar to that in NS3/

3A protein of two previously reported Indian BTV 1 sero-

types (BTV1f1IND and BTV1f2IND). BTVR23IND NS3/

3A had Q to R substitution just prior to the proline rich

region (aa position 35). In addition to H to Y substitu-

tion at residue 97 as in other Indian BTV1 isolates

Fig. 2. Alignment of the deduced amino acid sequences of BTV NS3/NS3A protein. PPR: Proline rich region. GI and GII: Glycosylation domain I

and II, HDI and HDII: Hydrophobic domain I and II. The ‘C’ residue identified with arrow indicates the conserved amino acid cysteine in all BTV.

The late motif domains PPXY and PS/TAP have been underlined.

G. S. Desai et al. Sequence Analysis of Indian BTV S10 Gene


(BTV1f1IND and BTV1f2IND), BTV1AIND also con-

tained aa change 128 A to V in the first hydrophobic

domain (aa 119 to 133) and substitution of leucine for

proline at position 220. In the second hydrophobic

domain of BTV18BIND NS3/3A (aa 167–183), the cyste-

ine residue (position 191) was replaced by serine and at

position 177 methionine was the constituent aa of NS3/

3A protein. Hence, in spite of some characteristic amino

acid changes, the basic architecture of different domains/

regions of the NS3/3A protein and the overall protein

profile was found to be conserved. There was overall con-

servation of (>96.5%) between different NS3/3A aa

sequence of BTV isolates from India. This high level of

conservation is very much essential since NS3 protein is

important in determining the vector status of individual

species of Culicoides midges for BTV as well as for the

virus assembly and release from the infected cells (Han

and Harty, 2004).

Though many serotypes of BT viruses have been iso-

lated from different parts of India, only a small number

of isolates have been genetically characterized. Earlier

studies have clearly shown that the BTV segment 10 gene

is very much suitable for BTV topotype analysis (Pierce

et al., 1998; Van Niekerk et al., 2003; MacLachlan et al.,

2007; Balasuriya et al., 2008). The S10 gene-based phylo-

genetic analysis grouped global BT viruses in to two

Fig. 2. Continued.



major clades, clade A and B (Fig. 3) as previously

reported (MacLachlan et al., 2007; Balasuriya et al., 2008).

All BTV S10 gene sequences from India segregated in to

subclade A1 along with BT viruses of Asia (China, Indo-

nesia) and South Africa (BTV16vSA), Israel (BTV16ISR),

Italy and Greece (Mediterranean basin). Indian BTV sero-

type 1 cluster was closer to BTV serotype 16 from South

Africa, Italy, Israel and China. The totpotypic cluster

formed by Indian BTV serotype 18 and 23 isolates was

nearer to recent BTV isolates (serotype 1, 9 and 16) from

Greece. Based on the closer relationship between the S10

genes of BTV isolates from India and Greece, Italy and

Israel, the origin of BTV isolates of the recent outbreaks

in the Mediterranean Basin have been indicated to be the

spread from Asia through the Middle East (Nikolakaki

et al., 2004; Potgieter et al., 2005; Purse et al., 2005). BTV

Fig. 2. Continued.



VP2 gene-based phylogenetic relationship has indicated

that the strain of BTV-1 recorded in 2001 from Greece is

most closely related to BTV isolates originating from

India, suggesting virus movement from East to West

(Maan et al., 2003).

Wind-borne transmission of infected midges can occur

over considerable long distances. It has been demon-

strated that BTV and Akabane viruses might have been

carried to western Turkey by midges from Cyprus and to

eastern Turkey from Syria (Sellers and Pedgley, 1985) and

from Cuba to Florida (Sellers and Maarouf, 1989). Also,

it has been reported that recent changes in the European

climate has resulted in the spread of six strains of BTV

across 12 countries in Europe due to expansion of the

activity of the Culicoides vector (Purse et al., 2005).

Although the present study strengthens the earlier obser-

vations of Asian origin of few of the European BTV iso-

lates, comprehensive scientific data about S10 gene

sequences of all 21 prevalent BTV serotypes from India,

epidemiological information about the active BTV sero-

types in different West Asian countries along with ento-

mological studies of the competent BTV vectors in entire

Asia are needed to clearly ascertain the exact pattern of

Western spread of the BT and Culicoides vector/s from

Asia/Australasia.

Phylogenetic clustering of BT viruses within the subc-

lade A1 suggested the origin of Indian BT viruses from

China, Indonesia and Australia. RNA segment 3-based

molecular epidemiology has designated BTV isolates of

Indonesia as Malaysia A group. This Malaysia A group

overlapped the genetic groupings within isolates from

India as well as with those identified in Australia, indicat-

ing the movement of BTV from Australia. Accordingly,

the gradation in the virulence of BTV isolates increased

from those in Australia to India through South-East Asia

(Daniels et al., 2004). Study based on the nucleotide

sequence analyses of VP7 gene have also indicated that

Indian BTV serotype 1, 18 and 23 shared closer relation-

ship with BTV isolates from China and Australia (Kataria

et al., 2006). Identical geographic origin strengthened by

BTV18BIND

0.02

10032

53

53100

100

100

100

100

100100

100

100

100100

100

100

100

60

70

96

8696

96

78

45

75

4552

58

99

99

99

97

30

2799

9886

83

42

35

69

A2

A

B

Subclade B1

Subclade B2

Subclade B3

Subclade A4

Subclade A3

Subclade A1

BTV23DINDBTV23RINDBTV18MINDBTV18fIND

BTV18SINDBTV1GRC

BTV1GRC01BTV9GRCBTV16GRC

BTV16vSABTV16ITLBTV16ISR

BTV16CHBTV1AINDBTV1f1INDBTV1f2IND

BTV23f2WJBTV2CH

BTV1CHBTV1AU

BTV1WJBTV12CH

BTV3WJBTV23f1WJ

BTV15CHBTV4GRC

BTV1rSABTV4PRTGBTV4FRA

BTV3rSABTV11vSA

BTV11rSABTV11USBTV10USBTV17vUS

BTV1vSABTV9vSABTV9ITL

BTV3vSABTV3rSA

BTV3TRNDBTV4vSA

BTV4rSABTV2ITLBTV2PRTG

BTV18rSABTV1HNRBTV13US

BTV17fUS

Fig. 3. Phylogenetic tree of the S10 genomic sequences of different

BTV isolates of the world. For comparison, S10 gene sequences of fol-

lowing BTV isolates obtained from GenBank: BTV10US (AF044372),

BTV11US (AF044373), BTV13US (AF044710), BTV17vUS (AF044378),

BTV17fUS (AF044707), BTV1CH (AF135223), BTV2CH (AF135224),

BTV12CH (AF135224), BTV15CH (AF135228), BTV16CH (AF135229),

BTV1vSA (AF512910), BTV1rSA (AF512911), BTV3rSA (AF512917),

BTV3vSA (AF512918), BTV4vSA (AF512908), BTV4rSA (AF512909),

BTV8rSA (AF512924), BTV9vSA (AY823224), BTV11vSA (AF512922),

BTV11rSA (AF512923), BTV16vSA (AY775164), BTV18rSA

(AF512915), BTV1WJ (AF529049), BTV3WJ (AF529050) BTV23f1WJ

(AF529051), BTV23f2WJ (AF529059), BTV4FRA (AY857503),

BTV2PRTG (EF434179), BTV4PRTG (EF434180), BTV1GRC

(AY449657), BTV1GRC01 (AY677628), BTV4GRC (AY449653),

BTV9GRC (AY823223), BTV16GRC (AY449652), BTV2ITL (AY823222),

BTV9ITL (AY438034), BTV16ITL (AY775162), BTV16ISR (AY775163),

BTV1AU (D00253), BTV1HNR (AY426598), BTV3TRND (AY426601),

BTV1f1IND (AF512913), BTV1f2IND (AF512912) and BTV18fIND

(AF512914) were included along with the six Indian BTV isolates. (f:

Field isolate, v: Vaccine isolate, r: Reference Strain, AU: Australia, CH:

China, FRA: France, GRC: Greece, HNR; Honduras, IND: India, ISR:

Israel, ITL: Italy, PRTG: Portugal, US: United States, TRND: Trinidad and

WJ: Indonesia). The reliability of the tree was estimated from 1000

bootstrap replicates.



closer serological relationship must have influenced the

S10 gene-based single topotypic cluster formation of

Indian BTV serotype 23 and 18 isolates. Based on the

sequence data of the VP2 genes of BTV reference strains

of all 24 serotypes, it is shown that both BTV serotype 18

and 23 belong to nucleotype D because of cross- or het-

erotypic antibody responses in cross-protection assays and

BTV serotype 1 isolates formed H nucleotype with the

serological relationship to serotypes 1 and 2 (Maan et al.,

2007). However, BTV serotype 23 of subclade A1 from

Indonesia (BTV23f2WJ) was far distant from Indian iso-

lates and shared more homology with Chinese isolates.

In conclusion, the S10 gene sequence-based phyloge-

netic clustering indicated that Indian BTV isolates have

evolved independently from BTV ancestors from China,

South-Eastern countries and Australia. And, amongst the

Indian BTV isolates, the pattern of evolution of S10 genes

of BTV 18 and 23 is distinct from S10 genes of BTV 1

isolates. The varied geographical origin of Indian BTV

isolates has no effect on the segregation of their S10 gene

sequences.

Acknowledgements

The authors wish to thank Director, Indian Veterinary

Research Institute for providing necessary facilities to

carry out the present study.

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Sequence Analysis of the S10 Gene of Six Bluetongue Virus Isolates from India

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Transcript of Sequence Analysis of the S10 Gene of Six Bluetongue Virus Isolates from India