Nucleotide sequence of feline immunodeficiency virus: classification of Japanese isolates into two subtypes which are distinct from non-Japanese subtypes.

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Department of Virology, Center for Basic Research, The Kitasato Institute, Tokyo 108,1and Department of Veterinary

Infectious Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Aomori-ken 034,2

Japan, and Department of Comparative and Experimental Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610-01453

Received 12 September 1994/Accepted 9 March 1995

Seven isolates of feline immunodeficiency virus (FIV), Shizuoka, Yokohama, Sendai-1, Sendai-2, Fukuoka, Aomori-1, and Aomori-2, were isolated from FIV-seropositive domestic cats in Japan, and their proviral DNAs

were amplified by PCR. The nucleotide sequences of theirenvandgaggenes were determined and compared

with those of previously described isolates: U.S. and European isolates and one Japanese isolate, TM2.

Phylogenetic analyses of completeenvgene sequences demonstrate that worldwide isolates are classified into

three subtypes: Japanese TM2, Japanese Shizuoka, and non-Japanese subtypes (U.S. and European isolates), with 20% amino acid distances from each other. This pattern indicates that an evolutionary radiation of these

three subtypes of FIV occurred at approximately the same time. The sequence data ofgaggenes also confirmed

these results. Furthermore, the Sendai-1 isolate was identified as an imported FIV isolate.

Feline immunodeficiency virus (FIV) is a member of the lentivirus genus of the family Retroviridae which includes hu-man immunodeficiency virus (HIV). On the basis of biological and morphological similarities between FIV and HIV, FIV infection of domestic cats is now considered to be an important small-animal model for studying prophylactic and therapeutic strategies against HIV infection and AIDS (4, 22). This virus was first isolated from a group of stray cats at Petaluma, Calif., in 1986 (21). Recently, its worldwide prevalence in domestic cats has been established (2) and currently, FIV isolates have been classified into three FIV subtypes (A, B, and C) based on the V3 to V5 sequence (29). Further, antigenically related viruses in wild Felidae have also been reported (19).

The nucleotide sequences of either the entire genome or partial structural genes (pol, gag, and env) have been reported for FIV isolates throughout the world (5, 6, 14–16, 23, 27). Phylogenetic analyses of gag and pol gene products of FIV and those of other lentiviruses reveal that FIV is more closely related to equine infectious anemia virus and Maedi-visna vi-rus than to primate lentivivi-ruses, HIV, and simian immunode-ficiency virus (18). However, the immunopathogenesis and early disease manifestations of FIV more closely resemble those of HIV and simian immunodeficiency virus than those of equine infectious anemia virus or Maedi-visna virus. The focus of recent epidemiologic surveys has been to determine the level of diversity observed in the FIV envelope (Env) se-quence. The Env region contains determinants important for cell tropism, cytopathogenicity, and infectivity and prominent immunoreactive domains (13, 20, 28). An alignment of pre-dicted Env amino acid sequences revealed clustered variable regions V1 to V5 (some papers reported analysis of up to nine variable regions) (29) and a high degree of conservation of

cysteine residues and N-linked glycosylation at the sites of surface Env (24). FIV epidemiology determined that viruses with specific sequence variation are also clustered within sim-ilar geographical regions.

Inactivated preparations of the Petaluma isolate have suc-cessfully protected cats against experimental infection with homologous and slightly heterologous (11% amino acid differ-ence in surface Env) FIV isolates, (8, 31, 32). However, the same vaccines were unable to protect cats against challenge infection with heterologous FIV isolates which differed by 20% in the surface Env region (12). Findings from these vaccine studies clearly demonstrate the importance of Env protein in

* Corresponding author. Mailing address: Department of Virology, Center for Basic Research, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku 108, Japan. Phone: 3444-6161, ext. 2137. Fax: 81-03-3444-6161.

TABLE 1. Primers for env and gag genes in PCR

Primer Sequence Residue sequencea

E7F CCTGAATTCC TGAAGGGGAT GAGT 5224–5241 E4R GGGAAGCTTT TGTCATATTA AAATG 7830–7810 E1F GGGGAATTCA GCGTTTTGTT TCTCCT 5898–5916 E2R GGCAGCTGGT GCCCAACAAT CCCA 6865–6847 E10F GGGGGATCCA ATGCAGGTAA GTTTAGAAG 6499–6518 E11R GGGAAGCTTG CAAGACCAAT TTCCAGCA 7867–7848 E18F CTCGGATCCT CCACTAGTGG TCCCAG 6804–6823 E19R GGGAAGCTTA GCTTCTCATA GCAAAGGTT 7552–7533 E3F AACCCGGGAT TGTTGGGCAC CAGA 6848–6866

E9R CATAGGATGT GGTTTTGCGG T 9263–9243

E5F GGGAGATCTT ATTGTACATT TT 7798–7814

E6R GGGAAGCTTC ATCATTCCTC CTCT 8840–8823 E17F GGGGAATTCA TATTATTCTA GCGCTTG 8105–8125 E16R CTCGGATCCC TTGTACATTA TTTTGT 8545–8526 G1F GGGGGATCCA ACAAGGTAGG AGAGATTC 605–624 G2R CTCAAGCTTA TAGGGTGTCC ATTTACAAA 2059–2040 G3F GGGAGATCTA AGGAAATTGA TATGGCAA 864–882 G4R CTCAAGCTTG CATTTTATAT CCTGGTG 1701–1682

a

The nucleotide residue numbering corresponds to that of the Petaluma isolate.

3639

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developing a FIV vaccine that is effective against a broad spectrum of existing isolates.

The FIV infection rates in Japan are approximately 12% for asymptomatic cats and 44% for symptomatic cats, and these numbers are relatively high compared with those from other countries (10). Currently, only two Japanese isolates (TM1 and TM2) have been analyzed for their nucleotide sequences and such analyses revealed that they are closely related to each other and belong to subtype B (11, 14, 15, 24). Expanded

surveys of Japanese isolates are required to determine whether FIV isolates in Japan have evolved within a single subtype. Therefore, epidemiological analysis of Japanese isolates has been undertaken by our laboratories. In this paper, the gag and

env gene sequences of seven Japanese isolates are presented

and their phylogeny and epidemiology are compared with those of isolates previously described. We have identified three FIV subtypes present in Japan, which consist of two divergent Japanese subtypes and an imported foreign subtype. The

phy-FIG. 1. Alignment of predicted env amino acid sequences of FIV isolates.p, conserved cysteine residues; – – –, conserved potential N-linked glycosylation sites; L.,SU, putative cleavage site of hydrophobic leader; SU.,TM, putative cleavage site between surface glycoprotein and transmembrane protein. The V1 to V5 regions are indicated by overlines.

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logenetic trees used to classify the subtype distribution of these Japanese isolates are based on both gag region and env region analyses.

MATERIALS AND METHODS

Virus isolates.Seven Japanese FIV isolates, Shizuoka, Yokohama, Sendai-1, Sendai-2, Fukuoka, Aomori-1, and Aomori-2, were isolated and characterized in this study. These isolates originated from naturally infected cats in the cities corresponding to their names. These cats were FIV positive and feline leukemia virus negative as determined by enzyme-linked immunosorbent assay (Pet Check ELISA; IDEXX Corp., Portland, Maine).

Virus isolation.Peripheral blood mononuclear cells were purified by Ficoll-Hypaque density gradient centrifugation of heparinized whole blood from sero-positive cats. Peripheral blood mononuclear cells (106

cells) from seropositive cats previously stimulated with concanavalin A (10mg/ml) for 72 h were cocul-tured with peripheral blood mononuclear cells (106

cells) from seronegative specific-pathogen-free cats. Cultures were maintained in RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum, 25 mM HEPES (N-2-hydroxyeth-ylpiperazine-N9-2-ethanesulfonic acid), antibiotics (100 U of penicillin per ml and 100mg of streptomycin per ml), 50 mM 2-mercaptoethanol, 2mg of Poly-brene per ml, and 100 U of human recombinant interleukin-2 per ml. The cultured cells were diluted two- to threefold every 3 or 4 days for passage. The presence of viruses in the culture was monitored by indirect fluorescent antibody assay, Mg1-dependent reverse transcriptase activity, and PCR for the gag region (9).

DNA isolation.Genomic DNA containing FIV proviral sequences for use in PCRs was isolated from FIV-infected cells. The cells were washed in phosphate-buffered saline three times and lysed in a lysis buffer consisting of 10 mM Tris-HCl (pH 7.4), 50 mM KCl, 2 mM MgCl2, 0.45% Nonidet P-40, 0.45%

Tween 20, and 60mg of proteinase K per ml. The cells were incubated at 568C for 1 h and were then heated at 958C for 10 min to inactivate the proteinase K.

PCR.PCR (25) was performed in a Thermal cyclic reactor (Hoei Science Co. Ltd.; model TC100), with each primer at a concentration of 1 mM, 50 ng of DNA, 0.25 mM (each) deoxynucleoside triphosphates, 2 mM MgCl2, and 2.75 U

of Taq polymerase (Boehringer Mannheim) in a 50-ml total reaction volume; the reaction was continued for 27 cycles of 1 min at 948C (denaturation), 1 min at 558C (annealing), and 1 min at 728C (extension), with the exception of 3 min at each temperature on the first cycle.

The FIV genes were amplified by PCR with synthetic primers (E1F, E2R, E3F, E4R, E5F, E6R, E7F, E9R, G1F, G2R, G3F, and G4R) corresponding to the previously published sequences of clone FIV34TF10 of the Petaluma (30) and TM2 isolates (14). For nested PCR, further synthetic primers (E10F, E11R, E12F, E16R, E17F, E18F, and E19R) corresponding to the sequences deter-mined in this study were constructed. The oligonucleotide sequences of the synthetic primers and their nucleotide positions in the genomic sequence of FIV34TF10 of the Petaluma isolate are shown in Table 1.

The env genes were amplified in three overlapping cDNA fragments desig-nated L, S, and T. Fragment L (967 bp) was amplified by two rounds of PCR amplification, with primers E7F and E4R on the first PCR amplification and primers E1F and E2R on the second PCR amplification. The S fragments (1,368 bp) were amplified with primers E10F and E11R, and then nested S fragments (709 bp) were amplified with primers E18F and E19R. The T fragment (1,042 bp) was amplified with primers E3F and E9R on the first PCR amplification and primers E5F and E6R on the second PCR amplification. The nested T fragments (440 bp) were amplified with primers E17F and E16R. The gag gene was also amplified by two rounds of PCR amplification with primers G1F and G2R on the first PCR amplification and primers G3F and G4R on the second PCR ampli-fication.

PCR products were purified by ethanol precipitation with 2 M ammonium acetate and were digested with restricting endonucleases corresponding to en-zyme sites within the primers. The PCR-amplified fragments were cloned into M13mp18 and M13mp19 by using Escherichia coli JM-109. At least eight clones

of each fragment were characterized and mixed to determine the major sequence in the sample.

Sequencing.Sequencing reactions were performed by the dideoxynucleotide chain termination method with a Dye Primer cycle sequencing kit (Applied Biosystems, Foster City, Calif.). The sequence was resolved on an Applied Biosystems model 373A automated DNA sequencer.

Computer analysis.The nucleotide and protein alignments as well as the percent identity were determined with the GENETX-MAC program (Software Development Co., Ltd.).

Phylogenetic analysis.Phylogenetic analyses using the CLUSTAL V program were performed (7). Multiple sequence alignment and evolutionary distances between amino acid sequences were estimated with the PAM250 matrix (1), which compared amino acid changes according to empirically determined prob-abilities of change. Phylogenetic relationships were determined by using the neighbor-joining algorithm (26), and branching order reliability was evaluated by 1,000 replications of bootstrap resampling analysis (3).

Nucleotide sequence accession numbers.The nucleotide sequence data re-ported in this paper have been deposited in the DDBJ database under accession numbers D37811 through D37824. Other FIV sequences used in this study are as follows (the names of isolates and GenBank accession numbers are listed): TM2, M59418; Petaluma, M25729; Dixon, L00608; PPR, M36968; UK2, X69494; UK8, X69496; Dutch113, X60725; Dutch19K, M73964; SwissZ2, X57001; FranceWo, L06136; Netherlands, X68019; CABCpady02C, U02392; CABCpbar03C, U02395; CABCpbar07C, U02397; USILbrny03B, U02418; USMAsboy03B, U02419; USMOglwd03B, U02420; USOKlgrl02B, U02421; USTXmtex03B, U02422; USCAhnky11A, U02402; USCAlemy01A, U02404; USCAsam_01A, U02410; USCAtt_10A, U02413; and USCAzepy01A, U02417.

RESULTS

Sequence alignments. The complete env nucleotide

se-quences of the seven Japanese isolates (Shizuoka, Yokohama, Sendai-1, Sendai-2, Fukuoka, Aomori-1, and Aomori-2) were determined by PCR amplification and cloning in an M13 phage vector. The predicted Env amino acid sequences of these iso-lates are aligned with a consensus sequence and with those of nine previously reported FIV isolates (one Japanese isolate, TM2, and three U.S. and five European isolates) in Fig. 1. Except for some minor differences, the clustering of variable and conserved regions is basically the same as that previously reported (24). Variable regions, V1 to V2 and V3 to V5, are located in the leader and surface (SU) Env regions, respec-tively (Fig. 1). The sequences of the Japanese isolates pre-sented have revealed a V1 region which is 11 residues larger than those in the previously reported non-Japanese isolates (i.e., residues 50 to 76 instead of 51 to 66). In the V2 region, SwissZ2 and the Japanese isolates, excluding the Sendai-1 iso-late, had limited length variation related to the single base deletions in these isolates. These deletions led new sequence patterns in this region (namely, two Japanese patterns: TM2 isolate type and Shizuoka and Fukuoka isolate type). Hetero-geneities of substantial length were found in the V5 region, where Sendai-1 and Fukuoka were shorter than the longest isolates, Shizuoka and SwissZ2, by 5 amino acid residues. The large deletion caused markedly less amino acid variation in the Japanese isolates than in the U.S. and European isolates. Fur-thermore, two patterns of amino acid sequence heterogeneity

FIG. 2. ORF D-encoded amino acid sequence alignment.p, stop codon; L.,SU, position of cleavage site in env frame (11 relative to ORF D).

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the sequence for the full-length env gene is not available). All 22 cysteines in the SU glycoprotein and 6 in the transmem-brane (TM) protein were conserved, and they are represented by an asterisk under the alignment in Fig. 1. Of the potential N-linked glycosylation sites, seven in SU and four in TM are conserved (represented by dashes under the full alignment). Two of these sites are located in the V5 region immediately adjacent to the N-terminal end of a region where a large number of FIV isolates frequently have amino acid deletions of various lengths.

Rigby et al. reported that all isolates, except Japan TM2, have a small open reading frame called ORF D (24). ORF D, which consists of 70 codons, overlaps the leader-surface junc-tion of the env region and is associated with no known func-tional activity. Japanese TM2 contains three stop codons in this region and consequently has no ORF D. To examine if such termination within ORF D is common among the Japanese isolates, this region of our seven new isolates was sequenced and aligned with previously reported sequences. Like isolate TM2 (subtype B), the Yokohama, Sendai-2, Aomori-1, and Aomori-2 isolates contain three stop codons within this region (Fig. 2). Furthermore, Aomori-1 and Sendai-2 have an addi-tional stop codon at the usual site for termination in those isolates that include ORF D. The rest of the Japanese isolates (Shizuoka, Fukuoka, and Sendai-1) contain no stop codon within the ORF D region, and ORF D is retained in these isolates. The ORF D sequence of Sendai-1 is similar to those of the U.S. and European type A isolates, whereas the Shi-zuoka and Fukuoka sequences are very similar to each other but differ significantly from subtype A and B isolates. Interest-ingly, the Dixon isolate, a California isolate closely related to the first discovered FIV isolate, has the largest ORF D with five additional amino acids at the N terminus.

Nucleotide and amino acid distances.Genetic distances

be-tween sequences of the env and gag regions were estimated by means of the CLUSTAL V program, and they are presented in Tables 2 and 3, respectively. Nucleotide distances are shown in the lower triangular matrix, and amino acid distances are shown in the upper triangular matrix of Tables 2 and 3. The amino acid distances of the Env sequences are 7 to 15% among non-Japanese isolates and 17 to 20% between Japanese iso-lates excluding the Sendai-1 isolate and non-Japanese isoiso-lates. This observation suggests that a large distance exists between Japanese and the non-Japanese isolates. On the other hand, the amino acid distances among the eight Japanese isolates are 2 to 20%, which suggests that more than one subtype exists for the Japanese isolates. The Sendai-1, Shizuoka, and Fukuoka isolates differ from the rest of the Japanese isolates. The amino acid distance between Sendai-1 and the other Japanese isolates is 20%, and that between Sendai-1 and the non-Japanese iso-lates of subtype A is 8 to 13%. This isolate shows a distance pattern opposite to that of the rest of Japanese isolates and is more closely related to non-Japanese subtype A isolates. Fur-ther, the amino acid distance between Shizuoka and the rest of the Japanese isolates, excluding the Sendai-1 and Fukuoka isolates, is 17 to 20%. This result suggests that the Shizuoka

TABLE 2. Pairwise distances between env sequences of FIV isolates a Sendai-1 Sendai-2 Fukuoka Aomori-1 Aomori-2 TM2 Petaluma Dixon PPR UK2 UK8 Dutch113 Dutch19K SwissZ2 0.2104 0.1819 0.1150 0.1725 0.1960 0.1913 0.1991 0.2019 0.2078 0.1960 0.2043 0.2061 0.2014 0.2038 0.1777 0.0562 0.1355 0.0656 0.0574 0.0504 0.1584 0.1619 0.1785 0.1870 0.1822 0.1806 0.1842 0.1785 0.1941 0.2036 0.1976 0.1976 0.1917 0.1034 0.0858 0.1322 0.0939 0.0861 0.1235 0.1153 0.0833 0.2030 0.1271 0.0480 0.0503 0.0433 0.1913 0.1806 0.1889 0.2009 0.1998 0.1969 0.1958 0.1913 0.2107 0.1096 0.1012 0.1318 0.1341 0.1953 0.1903 0.1927 0.1964 0.2012 0.1986 0.1939 0.2012 0.2054 0.0410 0.0895 0.0573 0.0585 0.1960 0.1877 0.1924 0.2021 0.2045 0.2017 0.2052 0.1948 0.2073 0.0569 0.1265 0.0534 0.0234 0.1854 0.1806 0.1865 0.1986 0.1962 0.1899 0.1910 0.1948 0.2010 0.0476 0.1273 0.0566 0.0211 0.1830 0.1747 0.1854 0.1903 0.1915 0.1851 0.1910 0.1877 0.0689 0.1978 0.2042 0.2038 0.2033 0.1966 0.0985 0.1462 0.1137 0.1129 0.1279 0.1338 0.1193 0.0575 0.1976 0.2019 0.2024 0.2015 0.1944 0.0558 0.1307 0.0938 0.0859 0.1115 0.1092 0.0878 0.1244 0.1947 0.2009 0.1991 0.1932 0.1908 0.1363 0.1230 0.1306 0.1208 0.1506 0.1459 0.1331 0.0705 0.2030 0.2072 0.2054 0.2030 0.1967 0.0821 0.0699 0.1187 0.0881 0.1291 0.1291 0.0890 0.0692 0.2039 0.2072 0.2064 0.2047 0.1991 0.0830 0.0694 0.1090 0.0698 0.1197 0.1185 0.0940 0.0878 0.2025 0.1993 0.2053 0.2021 0.1998 0.0955 0.0848 0.1284 0.0935 0.0878 0.0678 0.1254 0.0889 0.2017 0.1985 0.2026 0.2002 0.1966 0.0931 0.0833 0.1327 0.0947 0.0902 0.0382 0.1184 0.0646 0.2040 0.2064 0.2084 0.2079 0.2016 0.0773 0.0672 0.1226 0.0664 0.0741 0.0852 0.0887 as percentages in a triangular matrix. Nucleotide distances are presented in the lower half and amino acid distances in the upper half of each matrix.

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isolate distinctly differs from the non-Japanese and TM2-type Japanese isolates. The amino acid distance between Fukuoka and the rest of the Japanese isolates excluding Sendai-1 is 10 to 14%. It is difficult to determine whether the Fukuoka isolate is similar to the Shizuoka isolate or TM2-type Japanese isolates. Similar results were derived by nucleotide distance analysis.

Distances in the gag region analyzed by the method de-scribed above are shown in Table 3. The amino acid distances of Gag are 2.6 to 7.5% among non-Japanese isolates, 1.1 to 9.4% among the Japanese isolates, and 6.4 to 9.4% between the Japanese and the non-Japanese isolates. Further, the nu-cleic acid distances are 4.8 to 16.9%, 0.88 to 16.3%, and 14.9 to 17.2%, respectively. Both nucleotide and amino acid distance analyses for the gag gene or Gag polypeptide support the findings from nucleotide-amino acid distance analyses of the

env gene and Env glycoprotein.

Phylogenetic analysis. Phylogenetic analyses by

neighbor-joining and bootstrap resampling methods were performed on the full-length env region (2,556 bp), and the unrooted phylo-genetic tree is shown in Fig. 3a. Since this analysis was per-formed without an outgroup sequence, it includes no informa-tion about the direcinforma-tion of the evoluinforma-tionary changes and therefore this tree is unrooted. On the basis of the derived topology, FIV isolates can be classified into three major branches, non-Japanese, Japanese TM2, and Japanese Shi-zuoka subtypes, with approximately 20% diversity between each group. The Sendai-1 isolate, previously determined by nucleotide-amino acid distance and ORF D sequence analyses to belong to non-Japanese subtype A, clusters with other sub-type A viruses.

To compare our new data with that for the three subtypes (A, B, and C) previously classified, additional phylogenetic analysis based on the V3 to V5 env region was performed. The phylogenetic tree (Fig. 3b) shows that worldwide 29 FIV iso-lates can be classified into four major branches, subtypes A, B, and C and a fourth subtype containing the Shizuoka and Fukuoka isolates, with 20 to 25% diversity between subtypes. An unrooted tree based on the gag region (nucleic acid residues 883 to 1681), which is more conserved than the env region, is shown in Fig. 3c. The two trees from the env and gag regions show similar relationships among FIV isolates, with a slight discrepancy in that the distance between Japanese and non-Japanese isolates is longer than that between the Shizuoka and TM2 isolates.

DISCUSSION

Previous studies based only on V3 to V5 phylogenetic anal-ysis have reported three FIV subtypes consisting of subtype A (California and Europe), B (non-California and Japan), and C (British Columbia) (29). In this study, we have analyzed 16 FIV isolates, including 9 previously reported subtype A and B iso-lates and 7 new Japanese isoiso-lates, using the Gag, entire Env, and ORF D regions. On the basis of amino acid and nucleotide alignment and distance and phylogenetic analyses, our findings establish the existence of Japanese isolates that belong to FIV subtypes A and B and to a new Shizuoka subtype, with 20% divergence between each pair of subtypes. Additional phylo-genetic analysis of the V3 to V5 regions of 29 worldwide FIV isolates classified the isolates into four major branches: sub-types A, B, and C and a new subtype, with 20 to 25% diver-gence. Bootstrap values for each diverged point were relatively low, but phylogenetic trees based on three of four subtypes (data not shown) show a pattern similar to that shown in Fig. 3a, with high bootstrap values (85 to 100%). Thus, we establish new subtype D consisting of the Shizuoka and Fukuoka iso-lates. Further, the clustering of isolates from similar geograph-ical sources observed for Europe appears to also exist for Japan. The two major Japanese subtypes, B (Aomori-1, Ao-mori-2, Yokohama, Sendai-2, and TM2) and D (Fukuoka and Shizuoka), are evolving separately in eastern and western Ja-pan, respectively. Sequence analyses of additional Japanese isolates are needed to confirm such an observation. Our anal-ysis of env and gag genes suggests that the four subtypes (A, B, C, and D) diverged at approximately the same time. This deduction is based on the assumption that mutation occurred in each subtype at the same rate.

The Fukuoka isolate is at similar distances from Shizuoka and the rest of the Japanese isolates excluding the Sendai-1 isolate. Distance analyses using the limited regions leader, SU, V3 to V5, V3, V4, V5, and TM were performed, and on the basis of the TM region, this isolate was classified as subtype B (data not shown). Thus, the N-terminal half is similar to that of the Shizuoka isolate but the transmembrane regions are simi-lar to those of the subtype B isolates. The analyses of the gag gene, which is more conserved than the env gene, indicate that Fukuoka and Shizuoka isolates have similar origins and differ significantly from other isolates. The limited similarities of Fukuoka and the subtype B isolates in the SU and TM regions suggest that either the mutational direction in these regions was similar in these isolates or a recombination between these

TABLE 3. Pairwise distances between gag sequences of FIV isolatesa

Isolate Shizuoka Yokohama Sendai-1 Sendai-2 Fukuoka Aomori-1 Aomori-2 TM2 PPR Petaluma FranceWo Netherlands

Shizuoka 0.0455 0.0947 0.0530 0.0151 0.0492 0.0606 0.0455 0.0833 0.0871 0.0947 0.0795 Yokohama 0.1566 0.0642 0.0075 0.0455 0.0075 0.0151 0.0038 0.0642 0.0642 0.0755 0.0566 Sendai-1 0.1633 0.1570 0.0717 0.0871 0.0679 0.0755 0.0642 0.0340 0.0340 0.0491 0.0453 Sendai-2 0.1541 0.0138 0.1570 0.0530 0.0151 0.0226 0.0113 0.0717 0.0717 0.0830 0.0642 Fukuoka 0.0313 0.1629 0.1621 0.1604 0.0492 0.0606 0.0455 0.0795 0.0833 0.0909 0.0758 Aomori-1 0.1516 0.0175 0.1608 0.0163 0.1566 0.0226 0.0113 0.0642 0.0717 0.0792 0.0642 Aomori-2 0.1491 0.0175 0.1633 0.0163 0.1566 0.0138 0.0189 0.0755 0.0755 0.0868 0.0679 TM2 0.1491 0.0113 0.1608 0.0100 0.1566 0.0100 0.0088 0.0642 0.0642 0.0755 0.0566 Petaluma 0.1619 0.1656 0.0439 0.1656 0.1593 0.1694 0.1706 0.1694 0.0302 0.0340 0.0340 PPR 0.1694 0.1593 0.0439 0.1593 0.1694 0.1681 0.1656 0.1631 0.0488 0.0264 0.0189 FranceWo 0.1694 0.1656 0.0565 0.1681 0.1669 0.1706 0.1719 0.1694 0.0489 0.0338 0.0302 Netherlands 0.1669 0.1493 0.0602 0.1506 0.1719 0.1568 0.1543 0.1518 0.0651 0.0501 0.0576

a

Pairwise distances are presented as percentages in a triangular matrix. Nucleotide distances are presented in the lower half and amino acid distances in the upper half of each matrix.

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isolates occurred during coinfection of a cat. In light of recent findings that demonstrate superinfection of cats experimentally infected with subtypes A and B and subtypes A and D (17), it is tempting to speculate that the Fukuoka isolate derived its TM2-like SU and TM regions through recombination. Further studies to determine the mutation rate of each subtype in cats are needed to clarify this issue.

Our phylogenetic analyses of the Sendai-1 isolate suggest that it belongs to non-Japanese subtype A. The geographical isolation of the Japanese islands from the American and Eu-ropean continents leads to a theory that this isolate was im-ported into Japan recently by an infected cat prior to the discovery of FIV. In the phylogenetic tree, Sendai-1 isolate is more closely related to European SwissZ2 (8% amino acid diversity) than to the U.S. Petaluma isolate (10.3% diversity). This isolate is unlikely to be a recent variant of the Petaluma

isolate, because the distance between Sendai-1 and Petaluma is beyond the mutation rate of FIV (6). In either case, this isolate should be considered one of the first imported FIV isolates that belongs in subtype A.

The development of an HIV vaccine for worldwide use is difficult and presents a major challenge because of the high mutation rate and large degree of env variability between HIV isolates observed throughout the world. Similarly, our analysis of the FIV env region has shown a high level of variability in certain parts of the gene. Also, a significant rate of variant development within individual cats has been reported (6). These similarities make the FIV cat model an important model for studying vaccine approaches against HIV infection. Re-cently, inactivated Petaluma virus vaccine has successfully pro-tected cats against homologous and slightly heterologous (,11% diversity) FIV isolates of subtype A. However, vaccine

FIG. 3. Unrooted neighbor-joining trees of FIV structural gene sequences. (a) Complete env gene (859-amino-acid) tree of 16 FIV isolates; (b) region V3 to V5 (210-amino-acid) tree of env of 29 FIV isolates; (c) tree for p17 to p24 region of the gag gene (266 amino-acids) of 12 FIV sequences. All branch lengths are drawn to scale for each individual tree. To evaluate the consistency of the phylogenetic groupings, the data were subjected to bootstrap analysis. The numbers at each point indicate percent probabilities obtained with 1,000 replications of bootstrap resampling.

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protection against a distinctly heterologous (20% diversity) isolate of subtype D was not achieved. Further, we are studying cell tropism relating to our seven Japanese isolates and the Petaluma isolate, using various types of cells in vitro. Our major objective for the current epidemiological survey is to construct and use phylogenetic trees of the env regions of isolates worldwide as a method for identifying candidate strains for FIV vaccine development. Our current studies es-tablish the existence of a new subtype (D) in addition to the three previously reported. Hence, an FIV vaccine for world-wide use needs to be effective against all four subtypes. Addi-tional env gene sequences of isolates throughout the world combined with information on their neutralizing epitopes should help construct phylogenetic trees useful for the predic-tion of vaccine efficacy. Nevertheless, experimental vaccine trials with additional FIV isolates, for use as both a vaccine immunogen and a challenge inoculum, are needed to confirm their prophylactic value.

ACKNOWLEDGMENT

This study was supported in part by a grant from Japan Keirin Association.

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Figure

TABLE 1. Primers for env and gag genes in PCR

TABLE 1.

Primers for env and gag genes in PCR p.1
FIG. 1. Alignment of predicted envL�� amino acid sequences of FIV isolates. �, conserved cysteine residues; – – –, conserved potential N-linked glycosylation sites;SU, putative cleavage site of hydrophobic leader; SU��TM, putative cleavage site between sur
FIG. 1. Alignment of predicted envL�� amino acid sequences of FIV isolates. �, conserved cysteine residues; – – –, conserved potential N-linked glycosylation sites;SU, putative cleavage site of hydrophobic leader; SU��TM, putative cleavage site between sur p.3
FIG. 2. ORF D-encoded amino acid sequence alignment. �, stop codon; L��SU, position of cleavage site in env frame (�1 relative to ORF D).
FIG. 2. ORF D-encoded amino acid sequence alignment. �, stop codon; L��SU, position of cleavage site in env frame (�1 relative to ORF D). p.4
TABLE 3. Pairwise distances between gag sequences of FIV isolatesa

TABLE 3.

Pairwise distances between gag sequences of FIV isolatesa p.6
FIG. 3. Unrooted neighbor-joining trees of FIV structural gene sequences. (a) Complete env(210-amino-acid) tree ofto scale for each individual tree
FIG. 3. Unrooted neighbor-joining trees of FIV structural gene sequences. (a) Complete env(210-amino-acid) tree ofto scale for each individual tree p.7
Related subjects : phylogenetic analyses non-Japanese