Phylogenetic analysis of Portuguese Feline Immunodeficiency
Virus sequences reveals high genetic diversity
Ana Duarte *, Luis Tavares
Centro de Investigacao Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinaria,
Avenida da Universidade Tecnica, 1300-477 Lisboa, Portugal
Received 27 July 2005; received in revised form 26 October 2005; accepted 15 November 2005
Abstract
Feline Immunodeficiency Virus (FIV) is a Lentivirus responsible for an immunodeficiency like disease in domestic cats.
Based on the genetic diversity of the V3–V5 region of env gene FIV is divided in five phylogenetic subtypes (A, B, C, D and E)
with a world-wide distribution. To understand the subtype diversity of FIV in Portugal a serological survey was conducted during
1 year in the Veterinary Faculty Hospital, Lisbon, Portugal to identify seropositive animals. Two viral genomic regions were
amplified by a nested PCR, sequenced and the phylogenetic relationships between 24 new Portuguese FIV sequences and other
previously published FIV isolates were assessed. The introduction of these sequences induced a subclustering in subtype B
including most of the new Portuguese sequences. Moreover, a new cluster emerged, with two highly divergent new sequences
that might represent a new subtype. The study of these new FIV isolates showed the presence in Portugal of a unique viral
population subclustering within subtype B and of sequences clearly divergent from the five known subtypes, providing a
contribution for the understanding of FIV’s genetic diversity.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Feline Immunodeficiency Virus; Subtypes; Phylogenetic analysis
www.elsevier.com/locate/vetmic
Veterinary Microbiology 114 (2006) 25–33
1. Introduction
Feline Immunodeficiency Virus (FIV) is a Lenti-
virus of the Family Retroviridae that causes an
immunodeficiency like disease in domestic cats,
similar to AIDS in humans (Pedersen et al., 1987).
* Corresponding author. Tel.: +351 21 3652800;
fax: +351 21 3652882.
E-mail address: [email protected] (A. Duarte).
0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved
doi:10.1016/j.vetmic.2005.11.056
Due to the common biological characteristics with
HIV, FIV has been used as a valuable model to
understand HIV and other Lentivirus pathogenesis as
well as for the development of vaccine strategies.
Based on the genetic diversity of the V3–V5 region
of the env gene FIV is currently divided in five
phylogenetic subtypes (A, B, C, D and E) with a
world-wide distribution. Subtype A is found in the
USA, Australia and Europe (Greene et al., 1993;
Rigby et al., 1993; Sodora et al., 1994; Bachmann
et al., 1997), subtype B in Japan, Europe and USA
.
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–3326
(Kakinuma et al., 1995; Bachmann et al., 1997; Pistello
et al., 1997) and subtype C in Canada, Europe, Taiwan
and Vietnam (Sodora et al., 1994; Kakinuma et al.,
1995; Inada et al., 1997; Nakamura et al., 2003).
Subtypes D and E are found in Japan, Vietnam
(Kakinuma et al., 1995; Nakamura et al., 2003) and
Argentina (Pecoraro et al., 1996). Subtype A and B
include most of the known FIV isolates. Furthermore,
several recombinant sequences have been identified and
classified between subtypes A and B, subtypes B and D
and subtypes A and C (Reggeti and Bienzle, 2004).
A dual-subtype FIV vaccine including subtype A
and D is currently available in the United States.
However, due to the continuing evolutionary pattern of
FIV, the study of the molecular epidemiology of this
virus in different geographical areas is of utmost
importance to assess the introduction of such a vaccine
in other countries, and to establish diagnostic strategies
based on the detection of viral nucleic acids, influenced
by the genetic diversity of local subtypes.
Five FIV sequences of Portuguese isolates have
been previously reported, grouping in a subcluster
within subtype B (Duarte et al., 2002). In this study,
we present 24 new sequences of Portuguese FIV
isolates, and propose the division of subtype B in four
different clusters with high values of genetic
divergence between them. The phylogenetic analysis
of our Portuguese sequences also revealed a new
cluster including two sequences that might represent
an additional sixth subtype.
2. Materials and methods
2.1. Serological survey
Seropositive cats were identified by immunoblot-
ting in a serological survey conducted during 1 year in
the Veterinary Faculty Hospital of Lisbon, Portugal.
FIV Petaluma strain was produced in FL4 cells.
The virus was precipitated in the presence of PEG
6000, and purified by ultracentrifugation. The viral
proteins were separated by SDS-PAGE in a 12% gel
and transferred to a nitrocellulose membrane. Animals
that reacted against the capsid protein (p24) were
considered positives.
Information regarding the 24 samples obtained is
summarized in Table 1.
2.2. gag and env PCR
Genomic DNA was extracted from whole blood
(100–1000 ml) (Wizard Genomic DNA Purification
Kit Promega), eluted in 50–100 ml of H2O and stored
at �20 8C.
A 329 bp region located in the CA amino terminus
in the gag gene (Hohdatsu et al., 1998; Kurosawa
et al., 1999) and a 550 bp fragment including the V3–
V5 region of the env gene (Sodora et al., 1994;
Bachmann et al., 1997) were amplified from the
genomic DNA by nested PCR.
Primer sequences and PCR cycling conditions are
summarized in Table 2.
Primers GagO1 and GagO2 were used in the first
round amplification of gag gene. The second round
was performed with primers GagI1 and GagI2
yielding a product of 329 bp.
Primers Env1/Env2 and Hdfor/Hdrev were used to
amplify the V3–V5 region of env gene. The second
round primers yield a product of 550 bp (Bachmann
et al., 1997). In the second round PCR the extension
period during the 30 cycles was 72 8C for 1 min.
The PCR reactions were performed in 50 ml total
volume with FideliTaq PCR Master Mix (2�)
(USB1), 100 pmol of each primer and 100 ng of
genomic DNA.
The 329 bp gag amplicons were purified and
directly cloned into p-Gem T easy vector (Promega)
according to the manufacturer instructions.
2.3. Heteroduplex mobility assay (HMA)
In order to confirm its genetic homogeneity the env
550 bp fragment was subjected to a HMA, before
cloning (Delwart et al., 1993; Bachmann et al., 1997).
Briefly 10 ml of env second round amplicons and 1 ml
of 10� annealing buffer (1 M NaCl, 100 mM Tris pH
7.8, 20 mM EDTA) were combined. The mixture was
heated to 94 8C for 5 mn, rapidly cooled on ice, 1 ml of
loading buffer (50% glycerol, 0.01% of xyleno
cyanol) was added and loaded in a 8% polyacrylamide
gel (29.2:0.8, acrylamide:bisacrylamide) poured
into plates of 15 cm � 15 cm. The electrophoresis
was carried at a constant 150 V for 5 h in a vertical
gel apparatus (Hoefer Scientific Instruments) in 1�TBE (88 mM Tris–borate, 89 mM borate, 2 mM
EDTA).
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–33 27
Table 1
Sample identification, origin and subtype assignment in the gag and env partial genes
Identification Origin gag env
Subtype Accession number Subtype Accession number
355_02CaP Canecas Subcluster B DQ072532 Subcluster B DQ072556
23_02LisP Lisbon Subtype B DQ072535 Subtype B DQ072559
43_02LisP Lisbon Subtype A DQ072536 Subtype A DQ072560
82_02LisP Lisbon Subtype A DQ072539 Subtype A DQ072563
120_02LisP Lisbon Subcluster B DQ072540 Subcluster B DQ072564
135_02LisP Lisbon Subcluster B DQ072541 Subcluster B DQ072565
150_02LisP Lisbon Subcluster B DQ072542 Subtype A DQ072566
151_02LisP Lisbon Putative F DQ072543 Putative F DQ072567
194_02LisP Lisbon Subcluster B DQ072548 Putative F DQ072572
206_02LisP Lisbon Subcluster B DQ072549 Subcluster B DQ072573
235_02LisP Lisbon Subcluster B DQ072551 Subtype A DQ072575
268_02LisP Lisbon Subcluster B DQ072552 Subcluster B DQ072576
300_02LisP Lisbon Subcluster B DQ072554 Subtype A DQ072576
350_02LisP Lisbon Subtype A DQ072555 Subcluster B DQ072579
164_02UZP Cattery Subtype B DQ072544 Subtype A DQ072568
165_02UZP Cattery Subcluster B DQ072545 Subtype B DQ072569
190_02UZP Cattery Subcluster B DQ072546 Subcluster B DQ072570
192_02UZP Cattery Putative F DQ072547 Subcluster B DQ072571
217_02UZP Cattery Subtype A DQ072550 Subtype A DQ072574
4_02MonP Montijo Subcluster B DQ072533 Subtype B DQ072557
62_02MonP Montijo Subcluster B DQ072537 Subcluster B DQ072561
63_02MonP Montijo Subtype A DQ072538 Subtype A DQ072562
14_02PalP Palmela Subcluster B DQ072534 Subcluster B DQ072558
296_02CaP Canecas Subcluster B DQ072553 Subcluster B DQ072576
The Accession number of each nucleotide sequence is indicated.
2.4. Phylogenetic analysis
The nucleotide sequences representing each geno-
mic region were sequenced and aligned with
previously characterized subtyped sequences of FIV
isolates, retrieved from their EMBL/Genbank/DDBJ
Table 2
Primers used for the amplification of the CA amino terminus in the gag gen
(Bachmann et al., 1997)
Primer Sequence (50–30)
PCR cycling conditions: 95 8C/2 min; 35 cycles of 94 8C/1 min, 50 8C/45
GagO1 AATATGACTGTACTA
GagO2 TTTTCTTCTAGAGTA
GagI1 TATTCAAACAGTAAA
GagI2 CTGCTTGTTGTTCTT
PCR cycling conditions: 5 cycles of 94 8C/1 min, 50 8C/1 min, 72 8C/2 m
72 8C/5 min
Env1 GCTCAGGTAGTATGG
Env2 ACTTCATCATTCCTC
Hdfor ATACCAAAATGTGGA
Hdrev CAAGACCAATTTCCA
aAll the nucleotide positions are in reference to FIV Petaluma (Accessio
accession numbers. The alignments were created
using ClustalW and manually corrected to maximise
genetic similarities.
The phylogenetic relationship between sequences
of each genomic region was inferred by two methods.
Genetic distance between pairs of sequences was
e (Hohdatsu et al., 1998) and the V3–V5 region within the env gene
Location (nt)a
s, 72 8C/1 min; 72 8C/5 min
CTGC 917–936
CTTTCTGG 1650–1628
TGGAG 1036–1055
GAGTT 1364–1345
in; 72 8C/5 min; 30 cycles of 94 8C/15 s, 55 8C/45 s, 72 8C/2 min;
AGACT 6788–6807
CTCTT 8836–8817
TGGTG 7316–7335
GCAAT 7866–7847
n number M25381).
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–3328
Fig. 1. Unrooted phylogenetic trees from the 329 nt gag alignment. (A) Neighbour joining tree using Kimura’s two parameter model with a
transition/transversion of 2.07 estimated from the data set. Each scale bar represents 0.10 of genetic distance. Bootstrap values are shown at the
branch points. (B) Maximum likelihood tree (quartet puzzling using the substitution model HKY85). Genbank Accession numbers: FIV Wo
(L06136), PPR (M36968), Petaluma (M25381), Sendai 1 (D37820); Sendai 2 (D37821), Aomori 2 (D37824), Aomori 1 (D37823), Yokohama
(D37819), Usil2489_7B (U11820), Italy M2 (Y13866), Italy M3 (Y13867), TI1 (AB027298), TI4 (AB027301), Fukuoka (D37818), Shizuoka
(D37822), Lp3 (AB027302), Lp20 (AB027303), Lp24 (AB027304), RP! (AJ304955), TXTG (AY139111), TX132 (AY139112), TX200
(AY139110).
calculated by the Kimura’s Two Parameter Method
(DNADIST, Phylip Package) (Kimura, 1980; Felsen-
stein, 1993) using values of transition/transversion and
nucleotide frequencies calculated from the data sets
(TREE-PUZZLE, Strimmer and von Haeseler, 1996).
Maximum likelihood was performed with the sub-
stitution model of HKY85, assuming two rates of
variability (one variable and one invariable) along the
alignment (TREE-PUZZLE). The trees were con-
structed by neighbour joining (Saitou and Nei, 1987)
and the branching order reliability was evaluated by
bootstrap analysis of 1000 replicates (Felsenstein,
1985).
3. Results
3.1. Phylogenetic analysis of partial gag
sequences
Genetic distances and maximum likelihood were
used to assess the phylogenetic relationships of 24 new
Portuguese FIV sequences comprehending 329 nt of
gag gene. Both methods showed the same distribution
pattern for this genomic region (Fig. 1A and B).
The trees revealed six main clusters, supported by
reliable bootstrap values. Visual inspection suggested
that cluster B is divided in three branches, one of them
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–33 29
including the Argentine isolates (Lp3, Lp20 and Lp24)
previously classified as subtype E. Regarding the
distribution of the new Portuguese sequences, fifteen
grouped together with RP1, a previously reported
sequence, in a second branch of subtype B (Fig. 1A and
B). The third branch includes prototype B sequences
and two Portuguese sequences 164_01UZP and
23_02LisP. Sequence 23_02LisP although included
in subtype B, is positioned outside the main B branch,
but without significant bootstrap value.
Five Portuguese isolates were included within
subtype A (350_02LisP, 82_02LisP, 63_02MonP,
43_02LisP, 217_02UZP). None of the Portuguese
sequences analysed clustered with subtypes C and E.
Sequences 192_02UZP and 151_02LisP, grouped
together in an individual branch, supported by a
bootstrap value of 99.9%.
No individual clustering of sequences obtained
from animals of the same household or in the same
area was evident (Table 1).
To investigate the reliability of the phylogenetic
information obtained through the analysis of the
329 nt alignment, likelihood mapping was performed
using TREE-PUZZLE, indicating that 89.8% of the
analysed quartets were fully resolved assuring there-
fore its utility to assess phylogenetic relationships
between viral sequences.
Furthermore, this region was considered useful for
rapid subtyping by a RFLP assay (Hohdatsu et al.,
1998; Kurosawa et al., 1999; Duarte et al., 2002).
Based on the nucleotide sequence we confirmed the
presence of targets for six restriction endonucleases,
previously described (Table 3).
Sequence 151_02LisP revealed an extra target for
PstI endonuclease (229 bp), and sequence 192_02UZP
had an additional PvuII target (287 bp).
Table 3
Nucleotide position of restriction endonucleases sites in the 329 bp
fragment included in the gag gene, used for rapid subtype assignment
(Hohdatsu et al., 1998; Kurosawa et al., 1999; Duarte et al., 2002)
BamHI HincII PstI PvuII TaqI XbaI
A 187 bp,
286 bp
B 227 bp
B-Portuguese 222 bp 286 bp 287 bp
C 286 bp 287 bp 41 bp
D 44 bp 286 bp 240 bp
E 286 bp 287 bp 41 bp
Sequence 4_02MonP included in the Portuguese
subcluster did not share the same restriction pattern. A
unique HincII target (308 bp) positioned in a different
site was observed. Sequence 164_02UZP revealed
subtype B pattern plus an additional XbaI target
(200 bp). The unique restriction pattern observed was
in accordance with their positioning in the gag tree.
The restriction pattern of the other sequences
showed no divergence from the subtype assignment in
the phylogenetic gag tree.
3.2. Phylogenetic analysis of env V3–V5 region
Regarding the V3–V5 region of the env gene, two
phylogenetic trees were constructed by neighbour
joining, using genetic distances and maximum like-
lihood. Both trees showed the same rearrangement
observed in the gag tree (Fig. 2A and B), however
subtype A was divided in two subclusters and subtype
B in four subclusters with high bootstrap values to
support them. Additionally, several Portuguese
sequences change their positioning in the env tree.
The following sequences included in the Portu-
guese B subcluster in the gag tree behaved differently.
Sequences 235_02LisP, 300_02LisP and 150_02LisP
were included in subtype A. Sequence 165_02UZP
grouped in subtype B and sequence 194_02LisP
grouped in an individual branch together with
151_02LisP. Sequence 150_02LisP moved to a branch
within subtype A in the env tree.
Sequence 164_02UZP classified as subtype B in
the gag tree, was located in subtype A. Sequence
350_02LisP was included in subtype A in the gag tree
and moved to the Portuguese B subcluster in the env
tree. Sequence 192_02UZP clustered with sequence
151_02LisP in the gag tree also moved to Portuguese
B subcluster.
Moreover a subclustering of subtype A was
observed, including sequences 150_02LisP and
164_01UZP grouping in a single branch with high
reliability (100% of bootstrap).
Three main clusters with 100–95% of bootstrap
emerged in parallel with subtype B, assuming similar
values of genetic distance between them, namely
subtype E, three Texas isolates (Weaver et al., 2004)
and sequences 4_02MonP and 23_02LisP.
Sequence 151_02LisP group with 194_02LisP in a
unique branch also observed in the gag tree.
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–3330
Fig. 2. Unrooted phylogenetic trees from the 550 nt env alignment. (A) Neighbour joining tree using Kimura’s two parameter model with a
transition/transversion of 2.16 estimated from the data set. (B) Maximum likelihood tree (quartet puzzling using the substitution model HKY85).
Genbank Accession numbers: FIV Wo (L06312), PPR (M36968), Petaluma (M25381), Sendai 1 (D37813); Sendai 2 (D37814), Aomori 2
(D37817), Aomori 1 (D37816), Yokohama (D37812), Usil2489_7B (U11820), Italy M2 (X69501), Italy M3 (X69502), TI1 (AB016025), TI4
(AB016028), Fukuoka (D37815), Shizuoka (D37811), Lp3 (D84496), Lp20 (D84498), Lp24 (D84500), RP1 (AJ304986), TXTG (AY139101),
TX132 (AY138099), TX200 (AY139096).
Due to the discordance in the subtype assignment in
gag and env trees, observed for several Portuguese
sequences, a bootscanning was performed. SIMPLOT
program was used in order to test for possible
recombination points along the alignment (Lole
et al., 1999). A window of 100 nt with 40 nt overlaps
was used to calculate similarity values, using maximum
likelihood with 100 replicates. Petaluma (subtype A),
Usil2489_7B (subtype B), TI1 (subtype C), Fukuoka
(subtype D) and Lp3 (subtype E) were used as reference
subtypes and each discordant sequence was tested
individually (235_02LisP, 300_02LisP, 150_02LisP,
165_02UZP, 194_02Lis, 192_02UZP, 164_02UZP,
4_02MonP and 350_02LisP). However, no recombi-
nant sequences were detected (data not shown).
To further clarify the subtype assignment of
different sequences in the env tree, genetic distance
between and among subtypes was calculated
by maximum likelihood (Table 4) (Sodora et al.,
1994).
The genetic distance within subtypes range from
0.034 to 0.10 (Table 4). The minimal value between
subtypes did not exceed 0.208, but between sub-
clusters of subtype B, higher values were observed
(Table 4). The observed divergences are in accordance
with previously reported data (Sodora et al., 1994;
Bachmann et al., 1997). The genetic distance between
sequences 194_02LisP/151_02LisP and the major
subtypes was always superior to 0.24.
The value observed between sequences
150_02LisP/164_01UZP and subtypes A, B and the
Portuguese main group was unexpected, due to the
sequences positioning in the env tree (Fig. 2A and B).
The visual inspection and the bootstrap value
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–33 31
Fig. 3. Neighbour joining tree using Dayhoff PAM001 matrix based
on the predicted amino-acid sequences of FIV isolates.
Tab
le4
Aver
age
gen
etic
div
erg
ence
bet
wee
nan
dam
on
gsu
bty
pes
inth
een
vg
ene
Su
bty
pe
AB
Su
bB
CD
EF
A0
.10
(0.1
4–0
.02)
0.2
7(0
.35
–0
.19)
0.3
0(0
.35
–0
.26)
0.2
8(0
.32
–0
.25)
0.3
1(0
.28
–0
.35)
0.2
6(0
.30
–0
.23)
0.2
7(0
33
–0
.25
)
B0
.09
(0.1
7–0
.05)
0.1
6(0
.24
–0
.13)
0.2
1(0
.26
–0
.12)
0.2
6(0
.31
–0
.23)
0.2
1(0
.26
–0
.12)
0.2
6(0
.29
–0
.22)
Su
bB
0.0
6(0
.09
–0
.004
)0
.19
(0.2
0–
0.1
6)
0.2
7(0
.29
–0
.25)
0.2
1(0
.22
–0
.19)
0.2
7(0
.29
–0
.24)
E0
.05
(0.0
6–
0.0
4)
0.3
0(0
.31
–0
.28)
0.2
1(0
.22
–0
.20)
0.2
6(0
.28
–0
.25)
C0
.03
0.2
6(0
.28
–0
.25)
0.2
8(0
.28
–0
.27)
D0
.06
0.2
4(0
.25
–0
.22)
F0
.21
15
0_
02
Lis
P/1
64
_0
1U
ZP
0.2
3(0
.31
–0
.09)
0.2
1(0
.26
–0
.18)
0.1
4(0
.16
–0
.13)
0.2
9(0
.29
–0
.28)
0.2
4(0
.25
–0
.23)
0.2
2(0
.23
–0
.20)
0.2
9(0
.32
–0
.26)
The
max
imum
and
min
imum
val
ues
are
indic
ated
inea
chce
ll.
suggested an additional branch in subtype A but this
was not supported by genetic distance.
To clarify this observation one additional tree was
calculated (Fig. 3) based on the alignment (ClustalW)
of the predicted amino-acid sequence of the isolates,
using Dayhoff PAM001 matrix of distances. The
sequences distribution were similar to the one
obtained with the nucleotide alignment. Nevertheless,
the branch including sequences 150_02LisP and
164_01UZP emerged outside subtype A much closer
to the B Portuguese subcluster. This observation is in
accordance with the genetic relationship between
these sequences. Sequences 194_02LisP and
151_02LisP maintained the individual clustering,
although with lower bootstrap consistency.
4. Discussion
In the last few years, an effort has been made to
understand the molecular epidemiology of FIV, due to
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–3332
the implications of viral genetic diversity on vaccine
development and diagnosis of FIV infection (Sodora
et al., 1994; Bachmann et al., 1997; Steinrigl and
Klein, 2004; Reggeti and Bienzle, 2004).
FIV has been divided in five subtypes (A, B, C, D
and E) based on the phylogenetic analysis of two main
genomic regions, the V3–V5 regions of the SU
glycoprotein in the env gene and CA amino terminus
in the gag gene (Rigby et al., 1993; Sodora et al., 1994;
Bachmann et al., 1997; Pistello et al., 1997; Hohdatsu
et al., 1998).
The V3–V5 env region is responsible for cell
tropism, cytopathogenity and effective host immuno
response (Vahlenkamp et al., 1999). The observed
nucleotide variability of different isolates strongly
suggests a positive evolutionary pressure in this region
(Sodora et al., 1994; Steinrigl and Klein, 2004) and the
presence of recombination hot spots. Through the
analysis of the variation pattern within gag gene the
region coding the CA amino terminus was considered
useful for the genetic subtyping of FIV (Rigby et al.,
1993; Pistello et al., 1997). This region was also tested
for rapid subtyping by a RFLP assay (Cammarota
et al., 1996; Hohdatsu et al., 1998).
Based on two genomic regions, we analysed 24 new
Portuguese FIV sequences and found a higher viral
diversity with the introduction of these sequences. Most
of them grouped within a subcluster of subtype B along
with previously reported Portuguese sequences (Duarte
et al., 2002). We also found Portuguese sequences
belonging to subtype A and B, confirming the presence
of these subtypes in Europe reported by other authors
(Rigby et al., 1993; Pistello et al., 1997; Bachmann
et al., 1997; Steinrigl and Klein, 2004).
In several sequences the subtype assignment
differed with the genomic region used for phyloge-
netic analysis. In the V3–V5 region, all amplicons
were cloned after confirming the absence of viral
quasispecies within the same animal by HMA. No
evidence of recombination was observed between the
suspected sequences in the same genomic region. This
rearrangement may be due to the occurrence of
recombinant events between different viral genes.
However, to clarify this statement, the analysis of full
length genes or full FIV genomes would be necessary.
In both genomic regions we also described two
sequences (151_02LisP and 194_02LisP/192_UZP)
grouping in an individual cluster supported by high
bootstrap values and by different phylogenetic
methodologies. Sequence 151_02LisP also revealed
a unique restriction pattern in the partial gag region
studied. Considering the proposed use of this region
for rapid subtyping by RFLP of new FIV sequences,
this finding was consistent with the individual
branching observed in the gag tree (Cammarota
et al., 1996; Hohdatsu et al., 1998). The assignment of
a new subtype should be supported by a higher number
of isolates and by the analysis of a larger genomic
region, similarly to what has been done for HIV.
Nevertheless, we propose that sequence 151_02LisP
might represent the prototype of a new subtype termed
subtype F. Sequences 194_02LisP and 192_02UZP
with different subtype assignment in both genomic
regions may be representatives of mosaic virus
between the Portuguese B subcluster and the putative
new subtype, present in Portugal. Sequence
151_02LisP 194_02LisP, and 192_02UZP have no
epidemiological linkage between them. However, we
found high subtype diversity in the same geographical
area (Table 1). The presence of different circulating
subtypes within the same population might contribute
to recombinant events.
The overall conclusion of this study is the
increasing viral diversity within and between subtypes
and the need for the assignment of subclusters within
subtype B, represented by the main Portuguese
sequences and the Texas isolates, already proposed
by Weaver et al. (2004). The genetic heterogeneity
within subtype B, stated by other authors (Sodora
et al., 1994; Bachmann et al., 1997), may justify its
future division in subtypes or sub groups according to
genetic divergence and branch reliability.
The observation of a restricted FIV group in Lisbon
(Portugal) enhances the need for a more comprehen-
sive epidemiological survey in different areas. This
information would contribute for the correct evalua-
tion of a FIV vaccine in Portugal and development of
new diagnostic methodologies.
Acknowledgments
This work was sponsored by the Research
Interdisciplinary Center in Animal Health, Veterinary
Faculty of Lisbon. FL4 cells were gently provided by
Dr. Janet Yamamoto.
A. Duarte, L. Tavares / Veterinary Microbiology 114 (2006) 25–33 33
We are grateful to our colleagues from the Hospital
of the Veterinary Faculty of Lisbon for their
collaboration in biologic sample collections. The
availability of the Bioinformatics programs was made
possible through the Portuguese EmbNet Node (Pen)
at the Gulbenkian Institute of Science, Portugal. We
would also like to thank Dr. Isabel Marques (Science
Gulbenkian Institute) for the revision of this work.
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