BTV Segment 10

8/8/2019 BTV Segment 10

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Virus Research 55 (1998) 15–27

Phylogenetic analysis of the S10 gene of field and laboratorystrains of bluetongue virus from the United States1

Corinne M. Pierce, Udeni B.R. Balasuriya, N. James MacLachlan *

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, Uni 6ersity of California, Da6is,

CA 95616 , USA

Received 30 August 1997; received in revised form 13 February 1998; accepted 13 February 1998

Abstract

The sequence of the S10 gene segment of the United States prototype strains of BTV serotypes 10, 11, 13, and 17

obtained from the American Type Culture Collection (ATCC), the commercial modified live virus vaccine strains of

BTV serotypes 10, 11, and 17, and 20 field isolates of BTV serotypes 10, 11, 13, and 17 was determined to better

define the molecular epidemiology of BTV infection in the US. All S10 gene segments were 822 nucleotides in length

with two in-frame initiation codons (nucleotides 20 to 22 and 59 to 61) and a single termination codon (nucleotides

707 to 709), thus all S10 genes were predicted to encode two proteins (NS3, NS3A). Nucleotide differences between

the S10 genes from field isolates of BTV ranged from zero (100% identity) to 142 (81.8% identity). The sequences of

the S10 gene segments from the US prototype ATCC strains of BTV 10 and 11 were very different from the

previously published sequences of putative US prototype viruses of the same serotypes (Lee and Roy, 1986; Hwang

et al., 1992). Comparison of the predicted NS3/NS3A proteins encoded by the S10 gene showed little variationbetween the various viruses (from 93 to 100% identity). This apparent conservation of NS3/NS3A amongst different

strains and serotypes of BTV likely is a reflection of functional constraints on the protein that tolerate little variation.

The various US isolates of BTV segregate into two distinct monophyletic groups based on their S10 gene sequences

and clustering of viruses was independent of serotype, year of isolation, geographical origin, and of host species of

isolation. The S10 sequence data also show that viruses that segregated within each of these two monophyletic groups

co-circulated in the western US between 1953 and 1990, and that reassortment of the S10 gene segment likely occurs

in nature. Comparison of dendograms derived from sequence analysis of the S3 (de Mattos et al., 1996)

* Corresponding author. Tel: +1 530 7521385; fax: +1 530 7548125; e-mail: [email protected] The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and

have been assigned the accession numbers: AF004372=BTV10ATCC; AF044379=10B80Z; AF0044380=10O80Z; AF044381=

10B81U; AF044382=10B81X; AF044384=10B90Z; AF044385=10O90H; AF044376=VAC10; AF044373=BTV11ATCC;

AF044386=11O79X; AF044702=11B80Z; AF044383=11B81P; AF044703=11C81Z; AF044704=11O81X; AF044377=VAC11; AF044374=BTV13ATCC; AF044713=13O79Z; AF044711=13B80Z; AF044712=13B81K; AF044710=13B89Z;

AF044375=BTV17ATCC; AF044706=17O79Y; AF044705=17B80Z; AF044707=17B81Y; AF044708=17B90Z; AF044709=

17O90Y; AF044378=VAC17.

0168-1702/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0168-1702(98)00024-0



C .M . Pierce et al . / Virus Research 55 (1998) 15–27 16

and the S10 gene segments from the same viruses also indicates that the S10 gene segment evolves and reassorts

independently of the S3 gene segment. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Bluetongue virus; Phylogenetic analysis; S10 gene segment

1. Introduction

Bluetongue virus (BTV) is the etiological

agent of bluetongue, an arthropod-transmitted

disease of wild and domestic ruminants (Spruell,

1905; Moulton, 1961; Erasmus, 1975; Howerth

and Tyler, 1988; Howerth et al., 1988;

MacLachlan, 1994). BTV infection occurs

throughout temperate and tropical regions of the

world, and infection is dependent on the pres-

ence of competent vector Culicoides sp. (Mellor,

1990; Gibbs and Greiner, 1994). BTV is the pro-

totype virus of the genus orbivirus in the familyReoviridae. There are at least 24 serotypes of

BTV worldwide (Huismans and Cloete, 1987),

five of which have been isolated in the US

(Gibbs et al., 1983). The virus particle is com-

posed of a diffuse outer coat of viral proteins

VP2 and VP5 (Martin and Zweerink, 1972; Ver-

woerd et al., 1972) which surrounds an inner

icosahedral core that includes major proteins

VP3 and VP7 and minor proteins VP1, VP4,

and VP6 (Loudon and Roy, 1991). The viral

genome consists of 10 double stranded (ds)

RNA segments, seven of which encode structuralproteins VP1– VP7. The three remaining gene

segments encode non-structural proteins (NS1–

NS3) that are present in virus-infected cells

(Mertens et al., 1984). Each ds RNA segment

encodes a single protein with the exception of

the S10 gene segment which encodes two

proteins, NS3 and NS3A, using two in-frame

initiation codons (Lee and Roy, 1986; Gould,

1988; Hwang et al., 1992; Wade-Evans et al.,

1992). The NS3 and NS3A proteins have char-

acteristic features that are conserved amongst

most orbiviruses (Hwang et al., 1992; Moss etal., 1992; Jensen et al., 1994; van Staden et al.,

1995), including two conserved hydrophobic do-

mains, two N-linked glycosylation sites, and a

cluster of proline residues (Hwang et al., 1992).

NS3 and NS3A localize on the surface and in-

tracellular smooth membrane vesicles of BTV-in-

fected cells, and are responsible for the extrusion

of BTV particles from the cell (Hyatt et al.,

1991, 1993; Wu et al., 1992).

Previous phylogenetic analyses of the

serotype-specific L2 gene of US field isolates of

BTV serotypes 10 and 17 demonstrated that

virus strains isolated in a restricted geographical

region had evolved by different and independent

pathways (de Mattos et al., 1994a,b). A similaranalysis of the conserved S3 gene segment,

which encodes the minor core protein VP6, from

field isolates of BTV serotypes 10, 11 13, and 17

suggested that this gene evolves independently of

the L2 gene and that it reassorts in nature (de

Mattos et al., 1996). Similarly, oligonucleotide

fingerprint analysis of US prototype strains of

BTV serotypes 2, 10, 11, 13 and 17, and 32 field

strains of BTV isolated in the US between 1979

and 1981 identified two distinct S10 constella-

tions, and that natural reassortment of these two

distinct constellations occurs amongst fieldstrains of BTV (Heidner et al., 1991). Phyloge-

netic studies of the S10 gene of putative US

prototype strains of BTV 2, 10, 11, 13 and 17

demonstrated considerable sequence homology

and conservation, and further indicated that

BTV serotypes 10 and 11 are closely related, as

are the S10 genes of serotypes 13 and 17,

whereas BTV 2 is more distantly related (Hwang

et al., 1992). In contrast, analysis of the

serotype-specific L2 gene of US strains of BTV

indicated that BTV 10, 11, and 17 are closely

related whereas both BTV 13 and BTV 2 aremore distantly related (de Mattos et al.,

1994a,b).




The objective of this study was to determine the

variability of the S10 gene segment of field strains

of BTV isolated in the US from different rumi-

nant hosts between 1979 and 1990, as compared

to that of the S10 gene segment of the US proto-

type strains of BTV serotypes 10, 11, 13, and 17

and the commercial modified live virus vaccine

strains of BTV serotypes 10, 11 and 17.

2. Materials and methods

2.1. Viruses and RNA isolation

The S10 gene segments of the US prototype

strains of BTV serotypes 10, 11, 13, and 17 ob-

tained from the American Type Culture Collec-

tion (ATCC; MD), modified live virus (MLV)

vaccine strains of BTV serotypes 10, 11, and 17

(VAC 10, VAC 11, VAC 17; Bluevac, Poultry

Health Laboratory, CA), and 20 field strains of BTV isolated from cattle, goats and sheep in

California, Idaho, Louisiana and Nebraska in the

years 1979, 1980, 1981, 1989 and 1990 were com-

pared. In addition, the S10 genes of putative

prototype US strains of BTV serotypes 10, 11, 13,

and 17 (BTV 10 UCD, BTV 11 UCD, BTV 13

UCD, and BTV 17 UCD), available in our labo-

ratory also were sequenced (Heidner et al., 1991;

Rossitto and MacLachlan, 1992). The origin of

each virus is listed in Table 1. The isolates were

processed as previously described (Heidner et al.,

1991; MacLachlan et al., 1992). The viruses were

propagated in BHK-21 cells (Heidner et al., 1991)

and the total RNA was isolated using guani-

dinium thiocyanate and phenol (RNAzolTMB;

Tel-Test, Inc., TX), according to the manufactur-

er’s instructions and essentially as previously de-

scribed (Balasuriya et al., 1995).

2.2. Amplification and sequencing

The published sequence of the S10 gene of BTV

17 (Hwang et al., 1992) was used to design syn-

thetic oligonucleotide 19-mer primers with anneal-

ing temperatures of 57°C (positive primer: 1–19;negative primer: 804–822) to amplify the entire

S10 gene segment by reverse transcription-poly-

merase chain reaction (RT-PCR) as previously

described (Akita et al., 1992). The negative primer

contained an A at position 810 to disrupt poten-

tial stem loop formation. Several different amplifi-

cations consisting of ten RT-PCR reactions were

performed for each virus to control for artifacts.

The reactions were pooled, concentrated by

ethanol precipitation or with a Centricon-30 con-

centrator (Amicon, MA), and gel-purified usinganother commercial kit (Gene-Clean, BIO101,

CA).

Both strands of the purified cDNA from each

virus were sequenced using the same primers as

those used for PCR amplification, as well as three

additional primers (positive primers: 288–308 and

422– 441; negative primer: 443– 462), and the

PRISM™ Ready Reaction DyeDeoxy™ Termina-

tor (Applied Biosystems, CA) cycle sequencing

kit. Approximately 100 ng cDNA and 10 pmol

primer were used in each reaction. Fluorescently-

labeled dideoxynucleotides were directly incorpo-rated into extension products during 25 cycles of

96°C for 30 s, 50°C for 15 s and 60°C for 4 min.

An ABI 377 Automatic Sequencer (Applied

Biosystems, CA) was used to collect and edit

sequence data according to the manufacturer’s

instructions.

2.3. Sequence and phylogenetic analysis

Computer analyses were performed using a

Power Macintosh 8100/100AV. The nucleotide

sequence of each S10 gene was translated into

amino acids, and predictions of hydrophobicity

and secondary structure were made using HIBIO

MacDNASIS Pro. Ver. 3.3 Software (Hitachi).

The ToPIR program of the Wisconsin package

(GCG Ver. 8.0 Software; Genetics Computer

Group, 1994; WI) and CLUSTAL V (Higgins and

Sharp, 1988) programs were used for multiple

sequence alignment. Phylogenetic analysis was

done with the PHYLIP (Phylogeny Inference

Package) version 3.572c for the Macintosh Power

PC (Felsenstein, 1993). The first and last 19 nucle-

otides of the 5% and 3% ends of each S10 gene were

not included in the phylogenetic analyses becauseprimers were used to amplify the segment. Dis-

tance matrices for nucleotide sequence data were




Table 1

Origin of BTV strains

Virus Year of isolation Host State/County

SheepBTV 10 ATCC CA1953

BTV 10 UCDa

Cow1980 CA/Kern10B80Z

198010O80Z Sheep CA/Solano

198110B81U Cow LA/East Baton Rouge

Cow1981 CA/Stanislaus10B81XCow CA/Riverside10B90Z 1990

Sheep1990 CA/Mendocino10O90H

VAC 10b

SheepBTV 11 ATCC TX1962

BTV 11 UCDa

197911O79X Sheep NE/Pershing

198011B80Z Cow CA/Kern

Cow1981 CA/Kern11B81P

198111C81Z Goat CA/Kern

198111O81X Sheep CA/Kern

VAC 11b

BTV 13 ATCC Cow1967 ID

BTV 13 UCDa

197913O79Z Sheep ID/Ada

Cow13B80Z ID/Wilder1980

Cow1981 CA/Stanislaus13B81K

198913B89Z Cow CA/Riverdale

1962BTV 17 ATCC Sheep WY

BTV 17 UCDa

197917O79Z Sheep CA/Merced

198017B80Z Cow CA/San Bernadino

Cow1981 CA/Kern17B81Y

199017B90Z Cow CA/Riverside

Sheep1990 CA/Linden17O90Y

VAC 17b

aThe putative prototype US strains of BTV available in our laboratory originally were obtained from Dr T.L. Barber (U.S.

Department of Agriculture, Arthropod Borne Animal Diseases Laboratory) by Dr J.L. Stott (University of California, Davis, CA),

and subsequently were passaged as described (Rossitto and MacLachlan, 1992).bModified live virus vaccine strains of BTV that were obtained from Poultry Health Laboratories, Davis, CA.

calculated for aligned sequences using the

DNADist program based on the Kimura 2

parameter model (Kimura, 1980) with a transi-

tion/transversion ratio of 2.0. The distance ma-

trices were then used in the FITCH program

(least-square method; Fitch and Margoliash,

1967) to generate phylogenetic trees. The FITCH

program was carried out with global rearrange-

ments and randomized input order. The S10 gene

from epizootic hemorrhagic disease virus (EHDV)serotype 2 was used as an outgroup for the phylo-

genetic analyses because it is distantly related to

the homologous gene segment from the BTV

serogroup, but it clearly is included in a distinct

serogroup within the orbivirus genus (Gould and

Pritchard, 1991; Jensen et al., 1994). A bootstrap

analysis with 200 replicates was done to assess

confidence limits of the branch pattern (Felsen-

stein, 1985). A confidence level of ]70% was

considered significant (de Mattos et al., 1996).

Several treeing methods were applied to the

data in an attempt to produce the dendrogramthat best reflects the nucleotide changes observed.

Among these were the distance matrix based on




maximum likelihood (Felsenstein, 1981a,b,c), Jin

and Nei analysis (Jin and Nei, 1990), and the

Kimura two parameter model (Kimura, 1980).

Distance matrices were then used in the FITCH

program (Fitch and Margoliash, 1967) or the

Neighbor-Joining/UPGMA (Saitou and Nei,

1987) program of the PHYLIP package (Felsen-

stein, 1993). Trees were also developed using the

parsimony method (PAUP Version 3.1.1; Swof-ford, 1989).

3. Results

3.1. Variation of the S 10 gene segment and

encoded NS 3 /NS 3 A proteins

The S10 gene segments from US prototype

strains of BTV 10, 11, 13 and 17 obtained from

the ATCC, putative US prototype strains of BTV

10, 11, 13 and 17 available in this laboratory,MLV vaccine strains of BTV serotypes 10, 11 and

17, and 20 field isolates of BTV from the US

(Table 1) were sequenced. Sequence analysis

showed that all S10 gene segments are 822 nucle-

otides in length (including primers; data not

shown). Two in-frame initiation codons at nucle-

otide positions 20 to 22 and 59 to 61 and a single

termination codon at nucleotide position 707 to

709 (TGA or TAA) are present in all S10 gene

segments.

The sequence of the S10 gene of US prototype

strains of BTV serotypes 10 and 11 obtained from

the ATCC differ significantly from those previ-

ously reported. The published sequence of the S10

gene segment of a putative US prototype strain of

BTV 10 (Lee and Roy, 1986) differs from that of

the ATCC strain by 142 nucleotides (81.8% iden-

tity). The sequence of the S10 gene segment of the

putative prototype strain described by Lee and

Roy (1986) differs from that of our own labora-

tory strain of BTV 10 by only two nucleotides

(99.7% identity). Similarly, the S10 gene segment

from the ATCC US prototype strain of BTV 11

differs from the published sequence of a putative

US prototype strain of BTV 11 by 148 nucleotides(81.0% identity; Hwang et al., 1992). The S10

sequence of our laboratory putative US prototype

strain of BTV 11 was identical to that of the

ATCC strain, and the sequences of the S10 genes

of our putative US prototype strains of BTV 13

and 17 each varied from the respective ATCC US

prototype strains of the same serotypes by just

one nucleotide. The sequences of the S10 genes of

the prototype ATCC strains of BTV 13 and BTV

17 each differ from those previously reported

(Hwang et al., 1992) by two nucleotides (99.7%identity).

The S10 gene segments of the three MLV vac-

cine strains (VAC 10, VAC 11, and VAC 17) are

very similar. The S10 sequences of the VAC 10

and VAC 11 viruses are identical, and differ from

VAC 17 by only six nucleotides (99.2% identity).

VAC 10 was derived from the ATCC strain of

BTV 10 and their S10 gene segments differ by

only one nucleotide (99.9% identity). The S10

sequences of VAC 11 and VAC 17 predictably are

different from those of the ATCC US prototype

strains of these serotypes (10 and 4 nucleotidedifferences respectively) because VAC 11 and

VAC 17 were derived from field isolates of BTV

and not the prototype strains (H. Gray, Poultry

Health Laboratories personal communication).

The sequence of the S10 gene segments of field

strains of BTV showed no consistent relationship

to that of the S10 gene segment of each ATCC

US prototype strain of the same serotype, thus

the S10 gene segment of all field strains of BTV

was compared to the ATCC strain of BTV 10

because this was the original isolate of BTV made

in the US. Nucleotide differences as compared to

the ATCC strain of BTV 10 are evenly dispersed

throughout the S10 gene of the field strains, with

nucleotides 1 to 48, 59 to 64, 236 to 285, 641 to

660, and 725 to 738 representing relatively con-

served regions. The field strains differ from the

ATCC strain of BTV 10 at 164 nucleotide

residues, with 46 of these changes being transver-

sions and 28 resulting in coding changes. Nucle-

otide differences between individual field strains

ranged from zero (100% identity; e.g. 10B81X and

11B81P) to 142 (81.8% identity; e.g. 10B81U and

13B80Z), and some nucleotide differences are

unique to a particular virus. Amongst the fieldisolates, nucleotides 781 in 17B90Z and 17O90Y,

and 175 in 10O80Z and 10B80Z are the only




nucleotides that are unique to the different

serotypes of BTV and to individual years of isola-

tion. Most field isolates had common nucleotide

differences as compared to the ATCC strain of

BTV 10 (e.g. BTV 10 ATCC has a G at nucleotide

49 whereas an A is present in 10B81U, 10B81X,

10B90Z, 10O90H, 11O79X, 11B80Z, 11B81P,

11C81Z, 11O81X, 13B89Z, 17O79Y, 17B80Z,

17B81Y, 17B90Y and 17O90Y).The S10 gene segments of viruses isolated from

the same area in the same year were not consis-

tently similar. For instance, field isolates 10B80Z

and 11B80Z were isolated from the same ranch in

Kern County, CA, in the same year yet their S10

gene segments differ from one another by 137

nucleotides. Of the isolates derived from this same

ranch in the following year, field isolate 11B81P is

identical to 10B81X (isolated from Glenn County,

CA), and field isolate 11O81X is identical to

17B80Z (isolated from San Bernadino County,

CA). Furthermore, the S10 gene of 11B81P and11O81X differ by 12 nucleotides.

The deduced NS3 proteins encoded by the S10

gene of the ATCC prototype US strains of BTV

10, 11, 13, and 17, MLV vaccine strains of BTV

10, 11 and 17, and the 20 field isolates were

determined (data not shown). All S10 genes en-

code two proteins of 229 (NS3) and 216 (NS3A)

amino acids, as previously described (Lee and

Roy, 1986; Gould, 1988; Hwang et al., 1992).

NS3A begins at amino acid 14 of the NS3 protein

but all comparisons were made with NS3 for

convenience. As compared to one another, the

number of amino acid substitutions in the NS3

protein of the various viruses ranges from zero

(100% identity in 10B80Z and ATCC BTV 17) to

16 (93.0% identity in 13B80Z vs 11079X and

11B80Z). The deduced amino acid sequences of

the ATCC US prototype strain of BTV11 differs

from the previously published sequence of a puta-

tive US prototype strain of BTV 11 by 18 amino

acids (92.1% identity; Hwang et al., 1992). Simi-

larly, the ATCC US prototype strain of BTV 10

differs from the published sequence of a putative

US prototype strain BTV 10 by 14 amino acids

(93.9% identity; Lee and Roy, 1986). In contrast,the ATCC US prototype strains of BTV 13 and

17 differ from their respective published sequences

by only one residue (99.6% identity; Lee and Roy,

1986; Hwang et al., 1992). The deduced amino

acid sequence of the NS3 protein from the MLV

vaccine viruses VAC 10, VAC 11 and VAC 17

contain no unique amino acid substitutions as

compared to the other viruses.

The NS3 proteins from the different viruses

have many conserved characteristics. Amino acids

35–110, 127–148, 159–186 and 188–218 are con-served, as are the cysteine residues at amino acid

positions 137 and 181, a tryptophan at amino acid

position 159, and two potential N-linked glycosy-

lation sites at amino acids 63–65 (NTT) and

150–152 (NGT) (Hwang et al., 1992). A potential

O-glycosylation site (PT/SXXP; Wilson et al.,

1991) at residues 46–50 (SSMP; Jensen et al.,

1994) is also conserved amongst these viruses.

There are nine conserved phenylalanine residues

at amino acids 9, 78, 85, 147, 160, 176, 213, 216

and 224. Hwang et al. (1992) determined that the

prolines at amino acids 36, 37, 41, 44, 46, 50, 104,157, 165, 220 and 227 were conserved amongst

putative US prototype strains of BTV 10, 11, 13

and 17. These residues were also consistently pre-

dicted to be prolines in the viruses included in this

study with the single exception of amino acid 227,

which was predicted to be serine in field isolates

11O79X, 11B80Z, 11C81Z, 11O81X and 17B80Z.

Field isolates 10O90H and 10B90Z have an addi-

tional proline at amino acid 158 whereas the other

viruses have a glutamine or serine at this position.

The amino acids 114–130 and 159–175 constitute

the predicted hydrophobic domains (Lee and

Roy, 1986; Gould, 1988; Hwang et al., 1992;

Jensen et al., 1994) and also were conserved in

most of these viruses. The amino acid region

114–130, however, contained up to two amino

acid differences in many of the viruses (e.g.

13B80Z). The effect of this change is minor as

determined by comparison of the hydrophobicity

plots for 13B80Z and 10B81U (data not shown).

Some amino acids substitutions are unique to an

individual virus (e.g. glycine at amino acid 41 in

13B80Z), and some of these substitutions are not

conservative (e.g. glutamic acid at position 4 in

10O90H).The hydrophobicity plots (Kyte and Doolittle,

1982) of the deduced NS3 protein from these




viruses are virtually identical. Slight fluctuations

in hydrophobicity occur around amino acid 25

in 11B80Z (0.5 vs 1.5 in other viruses), and at

residue 158 in viruses 10B80Z, 10O80Z,VAC 10/

11, BTV 11, BTV 13, 13O79Z, 13B80Z, BTV 17

and VAC 17 (−1.0 vs −2.0 in other viruses;

data not shown). The secondary structure maps

(Chou et al., 1978) suggest that two distinct

structures of the NS3 protein occur in theseviruses, with the proline at amino acid residue

158 in 10O90H and 10B90Z being predicted to

form an additional turn in the NS3 protein of

these viruses (data not shown).

3.2. Phylogenetic analysis

Phylogenetic analysis of the S10 gene seg-

ments of ATCC prototype US strains of BTV

serotypes 10, 11, 13 and 17, MLV vaccine

strains VAC 10, VAC 11 and VAC 17, the 20field isolates of BTV isolated in the US, as well

as previously published sequences of the US

prototype strain of BTV 2 (Hwang et al., 1992),

putative US prototype strains of BTV 10 (Lee

and Roy, 1986) and BTV 11 (Hwang et al.,

1992), and EHDV 2 (Jensen et al., 1994) was

done using several methods. These methods gave

similar groupings but not identical trees (dendo-

grams; data not shown). The dendogram de-

picted in Fig. 1 was obtained using a distance

matrix method based on the Kimura two

parameter model (Kimura, 1980) and imple-

mented in the Fitch program (Fitch and Margo-

liash, 1967) of the PHYLIP package

(Felsenstein, 1993). The viruses cluster into two

distinct monophyletic lineages with a confidence

level of 100%. The segregation of these viruses

into two monophyletic groups based on their

S10 sequences corresponds exactly with results

previously determined by oligonucleotide finger-

print analysis of the same viruses (Heidner et

al., 1991).

The first monophyletic group (A; confidence

level of 100%) contains viruses isolated from cat-

tle and sheep between the years 1953 and 1981in California, Idaho, Texas, and Wyoming. The

members of this group include ATCC prototype

US strains of BTV 10, 11, 13 and 17, the previ-

ously published sequences of the US prototype

strain of BTV 2 (Hwang et al., 1992), all MLV

vaccine strains of BTV, two field isolates of

BTV 10 and three of BTV 13. The viruses in

this group have a nucleotide identity that ranges

from 100% (VAC 10 and VAC 11) to 93.2% (53

nucleotide differences; BTV 2 and ATCC BTV

11), with a genetic distance range from zero to0.0704. This monophyletic group is further sub-

divided into additional clades. The first clade

contains the ATCC prototype US strains of

BTV 11 and BTV 10, the MLV vaccine strains

VAC 10 and VAC 11, and two field isolates of

BTV 10. The second clade includes the MLV

vaccine strain VAC 17, the ATCC US prototype

strains of BTV 13 and BTV 17, and three field

isolates of BTV 13. The third clade contains

only the US prototype strain of BTV 2.

The second monophyletic group (B) contains

viruses with a nucleotide identity that rangesfrom 100% (11O81X vs 17B80Z) to 91.5% (66

nucleotide differences; BTV 11 (Hwang et al.,

1992) vs 10B90Z), with a genetic distance mini-

mum of zero and a maximum of 0.0918. The

viruses in this group were isolated from three

different animal species (cattle, sheep, and goats)

between the years 1979 and 1990 in California,

Nebraska and Louisiana. This large group is

further subdivided into three distinct clades. One

clade contains only a putative prototype US

strain of BTV 11 (Hwang et al., 1992). A second

small clade contains a putative prototype USstrain of BTV 10 (Lee and Roy, 1986), our pu-

tative US prototype strain of BTV 10 (data not

shown), and the 10B81U field isolate. The third

large clade contains all field isolates of BTV 17

and BTV 11, one field isolate of BTV 13, and

three field isolates of BTV 10. Some clustering

between the two monophyletic groups (A and B)

occurs based on serotype and/or year of isola-

tion as demonstrated by 13B80Z, 13B81K, and

13O79Y and 10B90Z and 10O90H. The presence

of viruses representing the different serotypes of

BTV endemic in the US in each monophyleticgroup, independently of serotype, strongly sug-

gests that reassortment of the S10 gene segment




F i g .

1 . P h

y l o g e n e t i c t r e e b a s e d o n s e q u e n c e a n a l y s i s o f t h e S 1 0 g e n e s e g m e n t o f l a b o r a t o r y a n d fi e l d s t r a i n s o f B T V .

T h e S 1 0 g e n e f r o m

E H D V s e r o t y p e 2 w a s u s e d

a s a n o u t g r o u p ( J e n s e n e t a l . ,

1 9 9 4 ) t o r o o t t h e t r e e

. H o r i z o n t a l b r a n c h l e n g t h s r e fl e c t g e n e t i c

d i s t a n c e s . S l a s h e s i n t h e b r a n c h e s r e p r e s e

n t l a r g e r d i s t a n c e s . B o o t s t r a p

p e r c e n t a g e s a r e i n d i c a t e d a t s e l e c t e d b r a n c h i n g p o i n

t s .

S e q u e n c e o f t h e S 1 0 g e n e o f B T V s e r o

t y p e s 2 a n d 1 1 a c c o r d i n g t o H w a n g e t a l .

( 1 9 9 2 ) ; B T V 1 0 a c c o r d i n g t o

L e e a n d R o y ( 1 9 8 6 ) .




occurs in nature. Some viruses isolated from the

same ranch in Kern County, CA, clustered

within the same group (e.g. 11B80Z, 11C81Z,

and 11O81X), whereas others (e.g. 10B80Z) were

placed in the other monophyletic group. Fur-

thermore, sequence analysis of viruses isolated in

1980 from this ranch clearly shows the co-circu-

lation of viruses with S10 gene segments that

represent the two different monophyletic groups

(e.g. 11B80Z from group B and 10B80Z from

group A). Four strains of BTV were isolated

from ruminants on the same ranch in the fol-

lowing year and none of these viruses had an

S10 gene segment that was identical to that of

any of the previous isolates from 1980. All the

viruses isolated from this ranch in 1981 were

placed in monophyletic group B.

Eleven of the BTV strains included in this

study are common to a previous phylogenetic

analysis based on the sequence of the S3 genesegment (de Mattos et al., 1996). A comparison

of the phylogenetic trees derived from the se-

quence of the S3 and S10 gene segments from

these viruses was done using the methods de-

scribed for the S10 gene segment, except with

500 boot strap replicates and that the published

sequences of the S3 and S10 gene segments of

BTV 1 were used to root the trees (Gould, 1988;

Wade-Evans et al., 1992). The various strains of

BTV 10, 11, 13 and 17 segregate into two or

three major monophyletic groups in each tree

with no correlation to serotype (Fig. 2), howeverthese viruses do not consistently segregate into

the same monophyletic groups or clades based

on analyses of their S10 and S3 genes (e.g.

13B89Z and 10O80Z, and 17B90Z and

10O90H). The genetic distances of these viruses

also differ significantly based on the S10 gene

segment as compared to those based on their S3

genes. Differences in the genetic distances and

tree topologies of the common viruses, as deter-

mined by comparative sequence analysis of their

S3 and S10 gene segments, indicates that these

gene segments reassort independently and im-plies that these gene segments also evolve inde-

pendently.

4. Discussion

The objective of this study was to define bet-

ter the molecular epidemiology of BTV infection

in the US by determining the variability and

reassortment frequency of the S10 gene segment

amongst field strains of BTV isolated from rumi-

nants predominantly in the western US over a

period of 37 years. The results of this studyindicate that the S10 gene segments of the field

strains of BTV group within two distinct but

related monophyletic groups, and that segrega-

tion of the viruses into these two groups can be

independent of serotype.

The evolution of the S10 gene is distinct from

that of either the serotype-specific L2 gene or

the S3 gene, which encodes the minor core

protein VP6. Previous studies of the serotype-

specific L2 gene from different strains of BTV

10 and BTV 17 clearly demonstrated that field

isolates of BTV segregate into monophyleticgroups according to serotype (de Mattos et al.,

1994a,b). A similar study of the conserved S3

gene segment of US prototype and field strains

BTV showed that these viruses segregated into

two monophyletic groups with no correlation to

serotype (de Mattos et al., 1996). Hence the S3

gene, like the S10 gene, evolves and reassorts

independently of the L2 gene, however, different

tree topologies resulted from the sequence and

phylogenetic analysis of the S10 and S3 genes

from eleven viruses that were included in both

studies suggesting that these two genes evolveindependently of one another and at different

rates.

The considerable discrepancy between the se-

quences obtained from the S10 gene segment of

the ATCC US prototype strains of BTV

serotypes 10 and 11 and those of previously

published sequences of putative prototype US

strains of BTV 10 and 11 was unexpected. De-

termination of the serotype of a BTV strain is

dependent only on the L2 gene segment and its

encoded VP2 protein, whereas all BTV gene seg-

ments are important in the identification of avirus strain. Designation of a BTV strain as a

prototype, therefore, should not be based solely




on the sequence of the L2 gene but on that of all

the gene segments. Results from this study

strongly suggest that reassortment of gene seg-

ments has occurred amongst putative prototype

US laboratory strains of BTV. To avoid such

confusion in the future, it is most logical that the

ATCC strains of BTV always be used as the US

prototype strains.

Previous phylogenetic analyses of the USstrains of BTV based on the L2 gene segment

indicated that BTV 2 and 13 are more closely

related, and that these serotypes are different

from BTV 10, 11 and 17 which cluster closely (de

Mattos et al., 1994a,b). Both BTV 13 and BTV 2

are considered recent introductions into the US,

however BTV 13 became endemic whereas BTV 2

did not. Based on their S10 sequences, the ATCC

US prototype strain of BTV serotype 13 and the

early field isolates of that serotype (13O79Z,

13B80Z, and 13B81K) segregate into a different

monophyletic group than the more recent isolate13B89Z. Furthermore, the ATCC prototype BTV

13 virus and the three early field isolates of BTV

13 cluster with the MLV VAC17 virus. These

data might indicate that a reassortment event

which provided a replication and transmission

advantage occurred between the strain of BTV 13

that originally incurred into North America, and

the established endemic serotypes and/or the

MLV vaccine strains. de Mattos et al. (1996) also

suggested that a reassortment event may have

occurred between the prototype BTV 13 virus

and VAC 10, based on sequence and phylogenetic

analysis of the S3 gene segment of field isolates of

BTV.

Phylogenetic analysis of the S10 gene segment

from 20 field isolates of BTV shows clusterings

were independent of the host species of isolation.

Viruses co-cluster that were isolated from rumi-

nants with either clinical bluetongue disease or

undergoing asymptomatic infection, and there

was no obvious correlation beween the sequence

of the S10 gene segment and virulence as appar-

ently occurs with African Horse Sickness virus

(Laegried et al., 1995). For example, 10O90H was

isolated from a sheep with severe clinical diseaseand it clustered with 10B90Z which was isolated

from a healthy cow. However, bluetongue disease

is common in BTV-infected sheep (Erasmus,

1975) whereas infection of cattle typically is

asymptomatic (MacLachlan, 1994; Barratt-Boyes

and MacLachlan, 1995). It also is appreciated

that the adaption of the virus strains evaluated to

cell culture also likely introduces a selection bias.

Despite the considerable sequence divergence

of the field isolates of BTV, the deduced NS3

amino acid sequences do not differ greatly andmost of the characteristic features of the protein

are highly conserved. Hyatt et al. (1993) used a

baculovirus expression system to prove that the

NS3 protein is necessary for extrusion of virus-

like particles from cells, and this crucial role of

NS3/NS3A during BTV infection apparently tol-

erates little variation in the protein. Site-specific

and deletion mutations of the glycosylation and

hydrophobic domains of NS3 expressed in a bac-

ulovirus expression system did not alter the intra-

cellular processing of the protein from the

endoplasmic reticulum through the golgi to thecellular surface (Bansal et al., 1996), but the con-

servation of the NS3 protein amongst US field

strains of BTV suggests the conservation of these

features is critical to its biological function and to

fitness of the virus in this ecosystem.

In summary, sequence comparison of the

ATCC US prototype strain of BTV serotypes 10,

11, 13, and 17, MLV vaccines strains VAC 10,

VAC 11, and VAC 17, and 20 field isolates of

BTV shows that two distinct monophyletic lin-

eages of S10 gene segments co-circulated in the

US over a period of 37 years, that reassortment

of the S10 gene segment likely occurred in nature

that was independent of serotype and of the gene

segment encoding core protein VP6, and that the

characteristic features of the NS3 protein are

highly conserved amongst field strains of BTV

present in the US.

Acknowledgements

The authors gratefully acknowledge Jodi Hedges

for information and assistance regarding the se-quence analysis and computer support. These stud-

ies were supported by USDA-NRI Competitive




Grant 91-37204-6407 and funds provided by the

Center for Food Animal Health and the USDA

under the Animal Health Act 1977, Public Law

95-113.

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