Molecular epidemiology of bluetongue virus in Portugal during 2004-2006 outbreak.

Abstract

After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.

Full text

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Title: Molecular epidemiology of Bluetongue virus in Portugal
during 2004–2006 outbreak
Authors: S´ ılvia C. Barros, Fernanda Ramos, Tiago M. Lu´ ıs,
Andreia Vaz, Margarida Duarte, Margarida Henriques,
Benedita Cruz, Miguel Fevereiro
PII:
DOI:
Reference:
S0378-1135(07)00178-2
doi:10.1016/j.vetmic.2007.04.014
VETMIC 3655
To appear in:
VETMIC
Received date:
Revised date:
Accepted date:
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26-3-2007
4-4-2007
Please cite this article as: Barros S.C., Ramos F., Lu´ ıs T.M., Vaz A., Duarte
M., Henriques M., Cruz B., Fevereiro M., Molecular epidemiology of Bluetongue
virus in Portugal during 2004-2006 outbreak, Veterinary Microbiology (2007),
doi:10.1016/j.vetmic.2007.04.014
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peer-00532246, version 1 - 4 Nov 2010
Author manuscript, published in "Veterinary Microbiology 124, 1-2 (2007) 25"
DOI : 10.1016/j.vetmic.2007.04.014

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Molecular epidemiology of Bluetongue virus in Portugal
during 2004-2006 outbreak.


Sílvia C. Barros, Fernanda Ramos, Tiago M. Luís, Andreia Vaz, Margarida Duarte,
Margarida Henriques, Benedita Cruz, Miguel Fevereiro*.
Laboratório Nacional de Investigação Veterinária, Estrada de Benfica 701
1549-011 Lisboa, Portugal



Keywords: Bluetongue virus; BTV; Serotype; Phylogenetic analysis; Epidemiology














*Correspondence:
Miguel Fevereiro
Laboratório Nacional de Investigação Veteriária, Estrada de Benfica 701, 1549-011, Lisboa – Portugal
Phone: +351 217115288
Fax: +351 217115387
E.mail: miguel.fevereiro@lniv.min-agricultura.pt

Manuscript
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from inapparent infection. These species, particularity cattle, can remain viraemic for

1
Molecular epidemiology of Bluetongue virus in Portugal
during 2004-2006 outbreak.
1
2
3
4
5
6
7



Keywords: Bluetongue virus (BTV), Serotype; Phylogenetic analysis; Epidemiology

Abstract
8
After forty four years of epidemiological silence, bluetongue virus (BTV) was
9
reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue
10
disease (BT) were notified in sheep farms located in the South of Portugal, close to the
11
Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were
12
isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide
13
sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain
14
(BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2
15
(BTV2/26629/PT05) was fully determined and compared with those from other parts of
16
the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other
17
BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest
18
identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas
19
BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-
20
attenuated vaccine strain and its related field strain isolated in Italy. Since live-
21
attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may
22
represent a natural circulation of the vaccine virus used in other countries in
23
Mediterranean Europe.
24

25
1. Introduction
26
Bluetongue virus (BTV) is the prototype virus of the genus Orbivirus, family
27
Reoviridae responsible for an insect-borne disease of ruminants, transmitted between
28
vertebrate hosts by arthropods of the Culicoides species (Borden, 1971; Mellor, 1990).
29
The virus causes clinical disease in sheep, with symptoms ranging from acute to
30
subacute, mild or inapparent, depending of the virus serotype and strain, the breed of
31
sheep and certain environmental factors (Lefevre, 1991). Cattle and goats usually suffer
32
33
several months and therefore act as silent reservoirs of virus (MacLachlan, 1994).
34
The BTV virion has a double-layered icosahedral capsid that surrounds the genome of
35
10 segments of double-stranded RNA, which encode for seven structural (VP1-VP7)
36
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examined daily. Embryos that died between 36h and 5 days pi, showing hemorrhagic

2
and four non-structural (NS1-NS3 and NS3A) proteins (Roy et al., 1990). VP2 induce
37
neutralising antibodies and is responsible for the serotype specificity (Gibbs and
38
Greiner, 1994). To date, twenty-four serotypes were identified, of which five (BTV-1,
39
BTV-2, BTV-4, BTV-9 and BTV-16) have been reported in the last years (1998-2005)
40
in Mediterranean countries (Mellor and Wittmann, 2002). Recently (August 2006) and
41
for the first time in Europe, BTV-8 was detected in northern European countries
42
(Netherlands, Belgium and Germany), where bluetongue (BT) had never been reported
43
before. An unusual higher incidence of infection in bovine than in sheep was observed
44
in these outbreaks where BTV-8 caused severe clinical signs and lesions in cattle
45
(Toussaint et al., 2006).
46
The first incursion of BTV in Portugal was reported in July 1956, mainly affecting
47
sheep flocks in the southern region of the country below Tagus River, excluding the
48
Algarve region (Silva, 1956). The virus was isolated in chicken embryonated eggs, and
49
identified as serotype 10 (Noronha, F. 1957, personal communication). The number of
50
outbreaks markedly decreased soon after a live attenuated vaccine was used and in 1958
51
the disease practically disappeared. The country was declared free of BTV in 1960.
52
A new introduction of the virus was registered in October 2004 in sheep flocks close to
53
the Spanish border, shortly after Spain notified an outbreak of BTV-4 in the Cadiz
54
region.
55
In this study the molecular epidemiology of BTV strains circulating in Portugal during
56
the 2004-2006 was analysed. For this purpose, the nucleotide sequences of genome
57
segments L2, S7 and S10, coding respectively for viral structural (VP2 and VP7) and
58
non-structural (NS3) proteins, were analysed to determine the serotype and explore the
59
origin of these viruses.
60

61
2. Methods
62
2.1. Virus isolation
63
For virus isolation, groups of twenty 11 day-old embryonated SPF chicken eggs (ECE)
were inoculated intravascularly with 100 µl of a 10-1 dilution of washed and lysed
64
65
blood, from sheep or cattle. The eggs were incubated for five days at 35º C and
66
67
lesions, were pooled and homogenized in Glasgow MEM BHK21 medium (Invitrogen).
68
The homogenates were clarified by centrifugation and were inoculated again in ECE.
69
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al., 1985) and quartet puzzling support values based on 1000 puzzling steps were

3
Viruses isolated in egg embryos were propagated in baby hamster kidney-clone21
70
(BHK-21) or African green monkey (Vero) cell cultures.
71

72
2.2. RNA isolation, real time RT-PCR and Sequencing
73
Total RNA was extracted from BTV infected cells using the BioSprint96 workstation
74
(Qiagen) or RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions.
75
BTV was routinely screened by real-time RT-PCR using primers and probe specific for
76
NS1 gene; forward primer 5’-GTTCTCTAGTTGGCAACCACC-3’ (BT-A, OIE),
77
reverse primer 5’-TTTAACACACATCCCATTGAATA-3’ and Taqman probe FAM
78
5’-AGTGGGGATTATGCAAATGCCAC-3’ TAMRA. The reactions were carried out
79
using the One-step RT-PCR Kit (Qiagen). The L2, S7 and S10 genes were amplified by
80
RT-PCR using group specific primers derived from the nucleotide sequences available
81
in GenBank (Table 1). Before cDNA synthesis, double-stranded RNA was denaturated
82
at 98 ºC during 5 minutes and RT-PCR reactions were carried out using the Superscript
83
One-Step RT-PCR for Long Templates (Invitrogen). Usually, the RT reaction (30 min
84
at 50 ºC) was followed by 40 cycles of the PCR amplification (15 sec at 94ºC, 30 sec at
85
50ºC, and 2 min at 68ºC) with a final elongation step of 7 min at 68ºC. The resulting
86
amplicons were cloned into pCR2.1 vector (Invitrogen) and were sequenced (Thermo
87
Sequenase Primer Cycle Sequencing Kit-Amersham Biosciences).
88

89
2.3. Sequence and phylogenetic analysis
90
The nucleotide sequences of L2, S7 and S10 genes of the isolates were submitted to
91
GenBank under the accession numbers EF434175 to EF434180. These sequences were
92
compared with sequences representative of serotypes most commonly detected in
93
Europe namely serotypes 1, 2, 4, 9 and 16 (Table 2). Multiple alignments of the
94
nucleotide sequences were generated by the CLUSTALW program, version 1.6
95
(Thompson et al., 1994) and phylogenetic analysis was carried out by maximum
96
likelihood analysis in TREE-PUZZLE program, version 5.1 (Strimmer and von
97
Haeseler, 1996), using a quartet puzzling algorithm to generate the tree. The analysis
98
was run with Hasegawa-Kishino-Yano (HKY-85) model of substitution (Hasegawa et
99
100
calculated. The overall tree topology was identical when the phylogenetic analysis was
101
repeated using the neighbor-joining method.
102

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topotype inference can be obtained through the nucleotide variation of this gene

4
3. Results
104
Virus and Phylogenetic analysis of the L2 gene segment
105
BTV was not detected in Portugal for almost 50 years. To investigate the origin of the
106
viruses isolated during the recent outbreaks of 2004-2006, nucleotide sequence analysis
107
of the L2, S7 and S10 genes was performed.
108
At the beginning of the outbreak three strains of BTV of serotype 4 were isolated
109
(BTV4/22043/PT04, BTV4/22045/PT04, and BTV4/22126/PT04). These viruses were
110
obtained from sheep with clinical signs and lesions of BT disease that included fever,
111
submandibular oedema, hyperaemia and petechial haemorrhages in the oral cavity and
112
pulmonary artery. The affected sheep originated from neighbouring farms located in the
113
south of the country, close to the Spanish border (Fig. 1). Since then two more strains of
114
BTV-4 were isolated, one from an asymptomatic cow in 2005 (BTV4/28207/PT05) and
115
the other in 2006 (BTV4/37069/PT06) from sheep with symptoms and lesions of BT.
116
To date all the BTV-4 strains analysed showed nearly 100% nucleotide identity for the
117
L2, S7 and S10 gene segments and therefore strain BTV4/22045/PT04 from the initial
118
outbreak was used in this study as a prototype of BTV-4. In July 2005, one BTV of
119
serotype 2 (BTV2/26629/PT05) was isolated from the blood of an asymptomatic cow
120
belonging to a farm located in the southwest of Portugal (Fig. 1).
121
The phylogenetic analysis of L2 gene segment show that the BTV4/22045/PT04 and
122
BTV2/26629/PT05 strains clearly segregated according to serotype (Fig. 2).
123
BTV4/22045/PT04 strain showed the highest sequence identity (99%) with the Sardinia
124
and Corsica BTV-4 isolates of 2003. With respect to Portuguese BTV-2, isolate
125
BTV2/26629/PT05 clustered with the South Africa vaccine strain and its related field
126
strain isolated in Italy with a nucleotide sequence identity of 99.2% and 99.4%,
127
respectively. The diversity found among the L2 gene of the BTV serotypes analysed
128
ranged between 58%-97% (Table 3). Between strains of BTV-4 and BTV-2 serotypes
129
ranges of respectively 1%-11% and 0,5%-19% were observed.
130

Phylogenetic analysis of the S10 gene segment
131
132
The S10 gene was analysed since it has been suggested that meaningful phylogenetic
133
134
(Bonneau et al., 1999). Figure 3 shows that the various BTV serotypes clustered into
135
three different lineages, with high levels of bootstrap support. Lineage A includes the
136
four Chinese serotypes, two Italian serotypes (BTV-9 and BTV-16), the Australian
137
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asymptomatic cows. In November 2006 one outbreak of BTV4 was reported in sheep

5
BTV-1, the Greek serotypes 1, 9 and 16, and the Israelian BTV-16. Lineage B groups
138
the majority of South African serotypes, BTV-1 from Honduras, the Portuguese isolate
139
BTV2/26629/PT05, the South Africa BTV-2 vaccine and its related Italian strain.
140
Lineage C includes mainly strains of BTV-2 and BTV-4 serotypes isolated in
141
Mediterranean countries, the Dominican Republic BTV-4 and South African BTV-1.
142
As with the L2, the S10 gene segment of BTV4/22045/PT04 and BTV2/26629/PT05
143
isolates show a high degree of identity (99% and 100%) respectively, with Corsica 2003
144
isolate and South Africa BTV-2 vaccine strain (Fig. 3).
145
The S10 phylogenetic tree revealed an overall branching pattern similar to that
146
previously described for some BTV isolates included in the present study (Nikolakaki et
147
al., 2005). According to our study, the lineages A and C correspond respectively to
148
Greek II and I clusters referred by these authors. Lineage A also corresponds to Asian
149
group as defined by Bonneau et al. (1999).
150

151
Phylogenetic analysis of the S7 gene segment
152
As with L2 and S10 genes, the phylogenetic analysis based on the S7 gene segment of
153
BTV4/22045/PT04 and BTV2/26629/PT05 isolates showed that they are more closely
154
related, respectively, to the Corsica 2003 (99.3%) and South Africa BTV-2 vaccine
155
(99.9%) strains (Fig. 4). A nucleotide sequence identity of 74% was observed between
156
the S7 genes of the two Portuguese strains.
157

4. Discussion
158
159
The present study reports on the outbreak of BTV occurring in Portugal since October
160
2004. The first clinical cases of BT were notified in sheep farms located in the
161
Southeast of Portugal, close to the Spanish border. The disease was confirmed by virus
162
isolation, detection of viral genome by RT-PCR and specific antibodies by ELISA and
163
seroneutralisation.
164
In 2004 three BTV of serotype 4 were isolated from blood or organs of sheep with
165
clinical signs or victimised by the disease in the initial outbreak. During 2005 the
166
disease was not reported in sheep but one BTV-4 and one BTV-2 were isolated from
167
168
located in the centre of the country.
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2005).

6
The areas of the country affected by the present BT outbreak match with the predicted
170
geographic distribution of culicoides vectors (Tatem et al., 2003) and also with the areas
171
affected by the BT epizootics of 1956.
172
Phylogenetic analysis of L2, S7 or S10 genes showed that BTV4/22045/PT04 virus
173
clustered closely with the BTV-4 viruses circulating in Italy and Corsica in 2003. This
174
suggests that the Portuguese BTV-4 viruses have the same origin as the BTV-4 strains
175
circulating in these countries. In this study it was not possible to include sequences of
176
L2, S7 and S10 genes from Spanish BTV-4 strains since they are not yet available in the
177
GenBank database. However, due to the close geographic proximity and the time frame
178
between the occurrence of the Andalusia outbreak and the first episode of BT in
179
Portugal it is pertinent to consider that BTV4/22045/PT04 is most likely related to the
180
Spanish BTV-4 outbreak. This consideration is also supported by the absolute identity
181
found between the M6 gene of Portuguese and Spanish (SPA2004) isolates while when
182
compared with the Corsica BTV-4 the identity is lower (99%).
183
Beside Italy and Corsica, BTV of serotype 4 has been detected in Greece, Bulgaria and
184
Turkey, during the years 1998-1999 and in Spain and Morocco in 2004. Also BTV-4
185
has established both in South Africa and in the Caribbean area (Mo et al., 1994).
186
Previous studies indicated a western origin, probably North African, for Corsica and
187
The Balearics 2003 BTV-4 strains (Potgieter et al., 2005; Purse et al., 2005). According
188
to these authors, the European BTV serotypes have arrived from at least two sources.
189
The BTV-1, BTV-9 and BTV-16 serotypes belong to an eastern (Asian/Australian)
190
group of viruses, whereas BTV-2 and BTV-4 belong to a western (African) group. A
191
recent study suggest that BTV-4 strains of the Mediterranean basin have three different
192
lineages, the first includes the Greek isolates, the second groups strains from Italy,
193
Morocco, Spain, and Corsica, and the third lineage contains Turkish isolates (Breard et
194
al., 2007).
195
BTV-2 epidemics occurred in Tunisia in December 1999 and in the following summer
196
the virus was spread to northeast Algeria, Sicily and Calabria, Corsica and Balearic
197
Islands (Mellor and Wittmann, 2002). BTV-2 is also enzootic in the Southeast United
198
States and in some African countries (Aradaib et al., 2005; Mecham and Johnson,
199
200
The Scientific committee of European Union recommends the vaccination as an
201
emergency action, based on a cost/risk benefit analysis and the epidemiological
202
situation (Sanco/C3/AH/R19/2000). During 2000-2002 vaccination campaigns were
203
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Field strains of BTV from around the world vary markedly, as a consequence of both

7
carried out in the Balearic Islands, Corsica and Italy, using the attenuated monovalent
204
serotype 2 vaccine produced by Onderstepoort Biological Products - South Africa
205
(Agüero et al., 2002; Breard et al., 2004; Ferrari et al., 2005). Attenuated vaccines have
206
been widely used to control the disease in southern Africa and USA. The circulation of
207
BTV vaccine strains in the field via active vectors among susceptible animals was
208
suggested before (Murray and Eaton, 1996). Nevertheless, only in 2005 the
209
transmission of BTV vaccine strain from vaccinated to non-vaccinated animals was
210
confirmed (Ferrari et al., 2005).
211
Regarding the genes analysed, the Portuguese BTV2/26629/PT05 showed the highest
212
nucleotide identity (>99%) with the South Africa BTV-2 vaccine strain and its related
213
field strain isolated in Italy (Ferrari et al. 2005) suggesting a common origin.
214
BTV2/26629/PT05 strain is apparently of low pathogenicity for sheep since it was
215
isolated from an asymptomatic cow belonging to a farm located in an area where the
216
disease in sheep had not been reported, and because in all the few clinical cases of BT
217
disease registered to date in the country only BTV-4 has been detected.
218
The BTV-2 vaccine strain used in the vaccination campaigns in Italy and Spain may
219
have been introduced in Portugal through imported viraemic animals or by aerial
220
dispersion of Culicoides from infected areas. Evidence of the role of Culicoides vectors
221
in spreading BTV-2 vaccine strain in areas where vaccination is not practised have been
222
reported before (Ferrari et al. 2005). BTV2/26629/PT05 could have also been
223
introduced as a contaminant of the BTV-4 live vaccine used in the vaccination
224
campaign in early 2005 in Portugal, though vaccine vials belonging to the same batches
225
of the BTV-4 vaccine tested negative for BTV-2 by RT-PCR.
226
Preliminary sequence analysis revealed that BTV-4 viruses of 2006 do not differ
227
significantly from those isolated in 2004 therefore suggesting that the initial strains still
228
circulate in Portugal. In contrast, BTV-2 has not been detected since July 2005.
229
Concerning the genes analysed in this study, evidence of gene reassortment between
230
BTV-2 and BTV-4 was not found. However, it cannot be ruled out, as BTV genes may
231
reassort independently of serotype amongst field strains of BTV that co-circulate in the
232
same area (Bonneau et al., 2001).
233
234
genetic drift and reassortment (Heidner et al. 1991; de Mattos et al., 1994; Bonneau et
235
al., 1999). It has been suggested that genetic typing of BTV based on the genetic
236
diversity of the NS3 gene give meaningful phylogenetic topotype inference (Bonneau et
237
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Bonneau, K.R., Zhang, N.Z., Wilson, W.C., Zhu, J.B., Zhang, F.Q., Li,Z. H., Zhang, K.L., Xiao, L.,

8
al., 1999). However, considering the reassortment probability, the definition of a
238
topotype based on any single genome segment might be inadequate (White et al., 2005).
239
This report presents the results of a study on the first wave of BTV-4 outbreak which
240
started in 2004 in Portugal. The prompt confirmation of the virus serotype at the
241
beginning of the outbreak allowed the Portuguese Veterinary Authorities to rapidly
242
implement a vaccination campaign in sheep using a BTV-4 live vaccine (Onderstepoort
243
Biological Products, South Africa) in the spring of 2005. Later this vaccine was
244
replaced by an inactivated vaccine (Merial) administered in both cattle and sheep in
245
winter of 2005.
246
In conclusion, assessment of the genetic variation of viruses in the field is therefore not
247
only useful for determining the origin of outbreaks, but also impacts the selection of
248
appropriate vaccine strains and the accurate design of molecular diagnostic tools.
249
Acknowledgements
The authors are thankful to the Portuguese Veterinary Authorities involved in the BTV
250
251
eradication and surveillance program. We thank R. Ferreira, A. Batista, A. Almeida, F.
252
Cordeiro and M. R. Ferreira, for technical assistance.
253
S. Barros was supported by FCT-Grant no.18760/2004.
254


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Fig. 1. Location of the farms and year where BTV-2 and BTV-4 were isolated.
Alandroal, BTV4/22043/PT04 and BTV4/22045/PT04; Campo Maior,
BTV4/22126/PT04; Barrancos, BTV4/28207/PT05; Grândola, BTV2/26629/PT05 and
Alenquer, BTV4/37069/PT06.
BTV4/22043/PT04
BTV4/22045/PT04BTV4/22045/PT04BTV4/22045/PT04
BTV4/28207/PT05BTV4/28207/PT05BTV4/28207/PT05
BTV4/22126/PT04 BTV4/22126/PT04 BTV4/22126/PT04
BTV4/37069/PT06BTV4/37069/PT06BTV4/37069/PT06
BTV2/26629/PT05BTV2/26629/PT05BTV2/26629/PT05
BTV4/22043/PT04BTV4/22043/PT04
Figures 1-4
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Fig. 2. Phylogenetic analysis of the nucleotide sequences of the L2 gene segment.
Maximun likelihood tree performed by PUZZLE, version 5.1 with the HKY-85 model
was generated by using a transition/transversion of 1.57, a nucleotide frequency of A
=0.308; C =0.172; G =0.246; T =0.274. Log Likelihood = -21544.30. Branch lengths
represent genetic distances between sequences. The branch values represent the support
based on quartet-puzzling steps of 1000 replicates. The GenBank accession numbers of
the published sequences of the BTV isolates are listed in Table 2. Only a single
representative of 100% identical isolates was included in the tree.
1-Australia1-Australia
2-USA
2-Sardinia
2-Corsica
2-Sicily
2-S.Africa-vaccine
2/26629/PT05
2-Italy-vaccine
2-S-Africa
2/26629/PT05
100 100
100100
9-Greece
9-Greece
9-Greece
9-Calabria9-Calabria9-Calabria
100 100
4-S.Africa-vaccine
4-Calabria
100100
100100
16-Italy
16-S.Africa
16-Israel
16-S.Africa
VP2VP2
BTV-2BTV-2
BTV-9 BTV-9
BTV-4BTV-4
BTV-16BTV-16
BTV-1 BTV-1
1-S.Africa 1-S.Africa
9-S.Africa9-S.Africa
4-Sardinia
100
4/22045/PT04
100
4-Corsica
4-Turkey
4-Greece
4-Israel
4-Calabria
2-USA
2-Sardinia
2-Corsica
2-Sicily
2-S.Africa-vaccine
2-Italy-vaccine
2-S-Africa
4-S.Africa-vaccine
16-Italy
16-Israel
4-Sardinia
4/22045/PT04
4-Corsica
4-Turkey
4-Greece
4-Israel
4-Turkey
4-Greece
4-Israel
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9-S.Africa-vaccine
4-S.Africa-vaccine
100
1-Honduras
2-S.Africa
4-S.Africa
2-Italy-vaccine
2/26629/PT05
2-S.Africa-vaccine
100
99
100
96
100
4-Dom.Republic
1-S.Africa
4-Israel
4-Greece
88
2-Tunisia
2-Corsica
4/22045/PT04
4-Corsica
4-Balearic Isl.
4-Sardinia
100
100
100
1-Australia
4-China
2-China
1-China
70
1-Greece
16-Greece
16-Italy
9-Greece
100 16-China
9-Italy
100
16-Israel
9596
97
NS3
A
B
C


Fig. 3. Phylogenetic analysis of the nucleotide sequences of the S10 gene segment.
Maximun likelihood tree performed by PUZZLE, version 5.1 with the HKY-85 model
was generated by using a transition/transversion of 4.75, a nucleotide frequency of A
=0.318; C =0.185; G =0.252; T =0.246. Log Likelihood = -3467.74. Branch lengths
represent genetic distances between sequences. The branch values represent the support
based on quartet-puzzling steps of 1000 replicates. The GenBank accession numbers of
the published sequences of the BTV isolates are listed in Table 2.



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