Genomic Organization and Sequence Divergence of FIV _Ple Subtypes

The LTR of FIV _Ple contains transcription and regulatory elements common to other FIV. These include the direct 2 bp repeat (IR) defining the 5' and 3' termini of LTR, AP-4, Aml-1 (EPB20), AP-1, TATA box, Poly A, and the cap transcription initiation site (Figure 1). FIV _Ple subtypes have additional transcription factors characteristic of FIV, but placed in alternate locations within the LTR U3 including NF-AT and CREBP-1/c-Jun. These and other motifs were determined by homology search with a threshold value of 85% with the Motif Search database [28] [see Additional file 1]. Overall, lion LTRs are not identical between subtypes B and E, differing by 15% in nucleotide substitutions, comparable to that observed between FIV _Fca subtypes A, B and C (Figure 1, Figure 2A).

Phylogenetic Analyses of FIV _Ple Subtypes

The evolution of FIV _Ple subtypes is defined by separate phylogenetic analyses of each viral gene as well as combined data of concatenated sequences representing the entire coding region of FIV. LTR, gag, pol, vif and orfA affirm the species-specificity of FIV both in individual gene analyses (Figure 2A–E) and in the combined concatenated data phylogeny excluding env (Figure 2F). The three subtypes of FIV _Fca from the domestic cat exhibit the least amount of genetic divergence within each viral gene phylogeny. Sharing a monophyletic lineage with distantly related FIV _Oma , the FIV _Ple subtypes B and E have intermediate levels of genetic distance with each viral gene examined. Subtypes A and B of FIV _Pco are the most divergent and have substantial differences across the viral genome. Thus, the hierarchical pattern of genetic divergence among full-length genomic analyses of FIV _Fca , FIV _Ple and FIV _Pco recapitulates earlier evolutionary studies based on portions of pol-RT and gag [4, 7, 8, 10–12, 17, 30, 31].

The relative differences in genetic diversity among FIV strains may be correlated with the amount of time since the virus entered modern felids and therefore, can be interpreted in the context of the evolutionary and phylogeographic history of each host species. The domestic cat evolved as a unique felid lineage only around 10,000 year ago [32] from subspecies of wildcat Felis silvestris inhabiting Near East Asia [33]. Preliminary results from limited seroprevalence studies, indicate that FIV appears to be absent from nearly all of the close relatives of domestic cat [(genus Felis after [34]] except for French European wildcat F. silvestris [4, 35]. Thus, the pattern of FIV _Fca divergence may represent recent emergence combined with rapid viral diversification within the domestic cat world-wide. In contrast, the puma is one of the oldest species within Felidae, sharing an evolutionary lineage with the African cheetah (Acinonyx jubatus) and the New World jaguarundi (Puma yagouaroundi) and arose approximately 4.5 MYA [34]. The extreme divergence between subtypes A and B within the FIV _Pco lineage suggests an ancient origin of FIV infection of puma, a result consistent with the published pol-RT phylogeny marked by high levels of intra-subtype divergence of FIV _Pco subtypes from throughout the host species range [4, 8, 11]. Lastly, the African lion species arose approximately 2 MYA and spread throughout Africa, Asia and the Americas [34]. However, due to episodes of population reduction followed by expansion from East Africa and recolonization, genomic diversity in modern lion populations coalesces to approximately 325,000 years ago and is confined the African continent [36]. FIV _Oma is found in wild populations of the Eurasian Pallas cat [4], a species that arose during the late Pleistocene [34]. The monophyletic lineage of Pallas cat FIV _Oma and African lion FIV _Ple observed here suggest more ancient inter-species transmission as the last time lions and Pallas cats were in geographic contact was during the Pleistocene when lion ranges spread throughout Asia, providing a possible opportunity for FIV transmission between these species [37].

Discordant env phylogeny between FIV _Ple subtypes reveals ancestral FIV recombination events in the wild

The patterns of phylogenetic divergence between FIV strains from different cat species are concordant between all viral gene regions with one notable exception, the env gene. Phylogenetic analyses of the entire concatenated coding region (9391 bp) and separate analysis of the env gene (2958 bp) show the two FIV _Ple subtypes are no longer monophyletic (Figure 3A and 3B). A closer examination of the env gene shows only two shared regions of homology between FIV _Ple subtypes. The first spans the sites 1–519 of env, containing exon 1 of rev (Table 1), within the leader region exhibiting 80% nucleotide and 68% amino acid homology between FIV _Ple subtypes. The second region occurs at the terminal 3' region of env (sites 2506–2958) with 87% and 71% genetic identity for nucleotides and amino acid, respectively. Based on comparison with FIV _Fca , this region of FIV _Ple may be the rev responsive element (RRE), which is critical for targeting rev to the nucleolus of the cell [38]. As rev is conserved between lion subtypes, it is likely that RRE must remain conserved as well.

By contrast, the SU and TM regions of env differ substantially between FIV _Ple subtypes (Figure 4). A contiguous region of env, from amino acid sites 181 through 931 (green in Figure 4), shows that FIV _Ple subtype E is more similar to FIV _Fca than to FIV _Ple subtype B. Further, env of FIV _Ple subtype B, concordant with results from other gene trees (Figure 2A–E), shares more homology with FIV _Oma (blue in Figure 4). Moreover, the lack of monophyly between FIV _Ple subtype B and FIV _Oma (Figure 3) is a consequence of the recombinant env of FIV _Ple subtype E, as exclusion of this subtype from the analyses (data not shown) recovered the monophyletic relationship observed with other genome regions (Figure 2A–E).

The predicted env protein from both FIV _Ple strains were compared to other published FIV strains with respect to inferred structural elements, with particular focus on regions known to be important for receptor binding. Conserved (white in Figure 4) and variable regions (red in Figure 4) and epitope binding sites (orange in Figure 4) were identified based on their locations in the domestic cat FIV sequences [39]. The V3-V5 regions shared least homology between the two strains. In FIV _Fca , this region has been shown to contain the CXCR4 binding site [40], neutralizing antibody binding sites [41–43] and several epitopes important for cell tropism and cell line adaptation [44–46]. Within the V3-V5 region, several biochemical differences have been noted between domestic and non-domestic cat lentiviruses [39]. FIV _Ple subtype B demonstrated properties more similar to other non-domestic cat lentiviruses including a negative charge and fewer cysteine residues within this region. Conversely, FIV _Ple subtype E had a positive charge and more cysteines in V3-V5, more similar to the domestic cat lentiviruses. Both lion FIVs had similar numbers of predicted N-glycosylation sites (10 and 11 for B and E, respectively) and these numbers are intermediate to the domestic cat FIVs (8–10) and the other non-domestic FIVs (13–14). A similar trend of lower charge and more cysteine residues in B than E was noted in V3, the region implicated as receptor binding domain for FIV [44, 46, 47]. In contrast, the more conserved regions flanking V3-V5 were more positively charged in FIV _Ple subtype B than in FIV _Ple subtype E, but contained similar numbers of cysteine residues and putative N-glycosylation sites. Such differences suggest that substantial divergence may occur in secondary and tertiary structures at the receptor-binding region of these two lion lentiviruses.

Recombination in lentiviruses is not uncommon. In the ongoing global HIV pandemic, at least 34 circulating recombinant forms from HIV-1 subtypes have been so far described in patients world-wide [48]. SIV full genome sequence comparisons increasingly depict extant primate lentiviruses with mosaic structures indicative of multiple recombination events over time [49–54]. In FIV _Fca , recombination in the V3-V5 region of env was detected between subtypes A and B in feral cats [7], and different recombination frequencies occur between large regions of FIV _Pco subtype B in domestic cat experimentally infected with FIV _Pco B [31]. Whereas the frequency of FIV _Ple recombination is not yet known, our studies show that over 40% of Serengeti lions in Tanzania are multiply infected with FIV _Ple subtypes A, B and C, which circulate freely within this large population [6] and thus offer opportunities for recombination.

The recombination of env in FIV infected lions has interesting evolutionary significance because the divergence in this region is extensive between the two subtypes. Therefore, subtype E recombination may represent an ancient event of recombination followed by a long period of divergence, or a more recent recombination with a highly divergent but as yet unsequenced strain either from lions or another African felid species. Although FIV _Ple subtype E env is more similar to FIV _Fca than to any other known FIV the extent of genetic divergence is still quite substantial, i.e. 64.4% nucleotide relative to FIV _Fca subtype C (Table 2), suggesting that if recombination has occurred recently, it is likely to have been with strain that has not yet been sequenced for the env gene. This recombination event may also have functional implications, as FIV _Ple subtype E env has structural features more similar to pathogenic FIV _Fca . Further investigation into complete genome analyses of FIV _Ple subtypes A, C, D and F as well as FIV from other seropositive African felids, will likely provide new insights into the role of recombination in env in the wild. Clinical studies will help to clarify the significance of these recombination events.

Cell Culture of FIV_Ple Subtype E Botswana lion Ple-1027

FIV _Ple subtype E was isolated from PBMCs (whole blood with EDTA) collected from wild lions in the Okavango Delta in Botswana, viably frozen under field conditions [23] and stored in liquid nitrogen. In preparation for cell culture, viably frozen PBMCs from Ple-1027 were thawed at 37°C, washed twice in LBT media (RPMI 1640 (Invitrogen Life Sciences, Carlsbad, Calif.) containing 20% fetal bovine serum (Atlanta Biologicals, Norcross, Ga.), 1% Glutamax I, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 5 × 10^-5 M β-2-mercaptoethanol, 100 U of penicillin/ml, 100 μg of streptomycin/ml, (all from Invitrogen Life Sciences), and 9 g of glucose (Sigma)/liter), and resuspended at a final concentration of 1–1.5 × 10⁶ cells/ml in LBT + interleukin-2 at 100 U/ml (Invitrogen Life Sciences).

Domestic cat Mya-1 naïve feeder cells [55] were prepared for co-culture by cultivation in LBT + IL-2, plated in MEM containing 10% fetal bovine serum, 100 U of penicillin, 100 ug of streptomycin/ml, and 1% glutamax, and dispensed at 2 × 10^6 cells/ml in appropriate media in a 24 well plate. Reconstituted lion PBMCs were then added to Mya-1 cells at a volume of 400 ul (4–6 × 10⁵ cells).

Media was collected biweekly and subjected to microtiter reverse transcriptase assay as follows. Briefly, 15 μl of culture supernatant in triplicate was incubated with 50 μl of 0.05 M Tris (pH 7.8) with 75 mM KCl, 5 mM MgCl₂, 0.5 mM EGTA, 2 mM dithiothreitol, 5 nM oligo(dT), 0.05% NP-40, poly(A) at 50 μg/ml, and ³²P at 20 μCi/ml for 90 to 120 minutes at 37°C. Aliquots of 2.5 μl of each reaction mixture were spotted onto a nylon filter (Wallac, Turku, Finland) and allowed to dry. Un-incorporated label was washed away with five 10 to 60 minute washes with 0.03 M sodium citrate, pH 7.0, in 0.3 M sodium chloride (SSC) buffer, and the membrane was then fixed in 100% ethanol. Counts per minute were measured using a Microbeta Counter (Wallac).

Starting on day 34 post co-culture, supernatant from lion PBMC cocultures with Mya-1 cells had reverse transcriptase (RT) values approximately 3 to 10 times naïve supernatant levels, indicating productive lentiviral replication. RT activity was not detected in any other control supernatants through 49 days of culture.

RNA was extracted from 200 μl of supernatant from positive cultures using QIAamp viral RNA mini kit (QIAGEN) and reversed transcribed to cDNA with Superscript II (Invitrogen) according to manufacturer's instructions. PCR was then performed to amplify a diagnostic region of pol as previously described [4]. Amplicons were sequenced to confirm the presence of a Botswana strain of FIV (FIV _Ple subtype E). One ml aliquots of supernatant were frozen at -70°C. Aliquots were then thawed and used to inoculate 3 × 10⁶ Mya-1 cells, which were grown 14 days to achieve positive RT values as above. Cells were supplemented with fresh media weekly and grown to 1 × 10⁷ cells at which point cells were harvested by centrifugation and cell pellets were frozen at -70°C.

Cell Culture of FIV _Ple Subtype B: Serengeti lion Ple-458

Isolation and culture methods for FIV _Ple Subtype B are similar to the methods described for Subtype E (above) with the following exceptions. FIV _Ple Subtype B was isolated from PBMCs from a wild, sero-positive lion (Ple-458, Serengeti National Park), separated from heparinized whole blood by sucrose gradient centrifugation using Histopaque (Sigma). Cells were mixed with 10% DMSO with 90% fetal calf serum and viably frozen in nitrogen vapor in aliquots of ~10⁶ cells per ml. Post-freezing, thawed PBMCs (10⁶ cells) from the wild lion were co-cultivated with an equal number of lion donor cells (Ple-73, captive, National Zoological Park, Wash., D.C.; this lion was sero-positive but had repeatedly tested negative for virus isolation). All PBMCs were mitogen stimulated with concanavalin A (5 ug/ml) for 72 hrs. Co-cultures were propagated in RPMI 1640 with 10% bovine serum and 10% human interleukin-2 (Gibco-BRL). Fresh media was added every 72 hours and new donor cells (10⁶ cells) were added every 14 days. Replicating virus was confirmed in the supernatant by demonstrating both positive Mg₂+ – dependent reverse transcriptase (RT) and the presence of typical lentiviral particles seen by electron microscopy [10]. Virus rich supernatants were clarified by slow speed centrifugation and stored in liquid nitrogen freezers.

In order to expand the culture sufficiently to harvest viral supernatant for Western blot assays and to conduct the genetic analysis, 1 ml RT positive supernatant (LLV-2, SV lab) was used to inoculate 3201 cells (5 ml at 2 × 10⁶/ml), FeLV negative lymphosarcoma cells [56]. Cells were maintained in equal parts Leibovitz's L-15 media and RPMI 1640 with 20% fetal calf serum with glutamine (2x) and penicillin/streptomycin (1x). Initially, this culture was difficult to maintain in 3201 cells because it caused rapid cell death thus, in order to keep the culture alive, fresh media and naive 3201 cells had to be added every 3–4 days. After 21 days post infection (dPI), fresh media continued to be added to the culture every 3–4 days, but the addition of naïve 3201 cells was stopped and the % viability was allowed to decline (in the hope that a cell adapted virus could emerge that would enhance our ability to grow up viral stocks for use in Western blot assays). From dPI 28 to 49 the culture viability hovered between 18–24%, but after dPI 52 it was clear that both the viability and cell numbers began to improve (viability from 46 to 86%). By dPI 71 the cell viability was holding at >90% and the culture was growing at 40–50% per day. Infected cells for DNA extraction and genetic analysis of subtype B virus were harvested on dPI 88, centrifuged, and the pellets frozen at -70C.

DNA extraction, Cloning and Sequencing of FIV _Ple Subtypes B and E

DNA was extracted and purified from frozen cell culture pellets following the manufacturer's protocols established for blood products (Quiagen). Following extraction, DNA quality was checked by gel electrophoresis, and quantified by spectrophotometer (NanoDrop).

FIV _Ple proviral DNA was amplified using long PCR to generate overlapping proviral genome regions of approximately 5 kb (Roche's Expand PCR kit). For FIV _Ple subtype B, three over-lapping regions were amplified using the followoing primer pairs: FSHltr2F and FIVpol6R (LTR-pol); FIVgag2aF and FIVpol5R (gag-pol); and FIVpol5F and FIVltr4R (pol-LTR) (see Additional file 2). For subtype E, two over-lapping regions were amplified using the two primer pairs FSHltr2F and FIVpol5R (LTR-pol), and FIVgag2aF and FIVltr4R (gag-LTR) (see Additional file 2). PCR reactions used 0.2–2.0 ug DNA with the following thermocycling conditions: 94°C for 2 minutes; ten cycles of 94°C for 10 seconds, 52°C for 30 seconds and 68°C for 4 minutes; 25 cycles of 94°C for 10 seconds, 52°C for 30 seconds and 68°C for 4 minutes and 20 seconds, with each having an extension time 20 seconds longer than the one before it; followed by 68°C for 7 minutes and 4°C hold. Additional "internal" primers were developed to fill in sequence gaps within each subtype (see Additional file 2) using the same PCR conditions listed above. Biometra T1 thermocyclers were used for all PCR reactions and amplicons were visualized on a 1% agarose gel.

The PCR products were cloned using TOPO TA XL cloning kits (Invitrogen). The resultant colonies were grown on LB agar plates with kanamycin and mini-prepped using Qiagen's REAL Prep 96. A restriction digest with EcoRI was performed to confirm successful cloning. The ends of the inserted PCR product were sequenced using the primers provided with the cloning kit for additional verification of FIV _Ple cloned products.

The final full-length sequence for each over-lapping long PCR product generated for each FIV _Ple subtype was obtained using transposon bombing [GPS-1 kit (New England BioLabs)]. In this method, transposons were randomly inserted into one of the successfully transformed plasmids for each primer combination for each sample. The results were used to transform OneShot Chemically Competent E. coli cells (Invitrogen) and grown on LB agar plates with kanamycin and chloramphenicol and were mini-prepped for DNA extraction using REAL Prep 96 (Quiagen). Restriction digest with EcoRI was performed to confirm successful insertion of the transposon. Using sequencing primers provided in the GPS-1 kit, 48 transposon fragments/PCR reaction were sequenced using an automated sequencer model ABI 3730.

Sequences (average read was approximately 600 bp) randomly generated by transposon bombing of individual clones defined multiple overlapping regions and were assembled into the full-length viral genome using Sequencher version 4.1 (Gene Codes Corporation) and submitted to GenBank [accession number EU117991 (Subtype B) and EU117992 (Subtype E)].

Genetic and Phylogenetic Analyses of FIV _Ple Subtypes B and E

Gene annotation of gag, pol, env, orfA, vif and rev from open reading frames in lion subtypes B and E used translation into amino acids and comparison with existing full-length FIV strains. The following sequences were used from GenBank: for FIV _Pco Subtype B in pumas strain PLV-1695 accession number DQ192583[27]; for FIV _Pco Subtype A in pumas strain PLV-14 accession number U03982[14]; for FIV _Oma in Pallas cat accession number AY713445[16]; for FIV _Fca subtype C in domestic cat strain C36 accession number AY600517[24]; for FIV _Fca subtype B, strain usil2489, accession number U11820; for FIV _Fca [57]; Subtype A, strain PPR accession number M36968 and strain Petaluma accession number M25381[15, 25, 58]. Open reading frames were determined and regions of homology between FIV _Ple with other FIV strains using pair-wise comparisons implemented by BLAST of two sequences [59]. The boundaries of both the 5'LTR and 3'LTR regions were identified by the conserved polypurine tract (PPT) shared by all FIV [60] and the primer binding site (PBS) which mark the boundary between the 3'LTR and the 5'LTR, respectively.

The genome of lion FIV _Ple was compared with existing full-length FIV by multiple sequence alignments of each viral gene. LTR regions were aligned using Clustal X [61] and verified and edited by eye using Se-Al ver 2.0 [62]. Due to large genetic divergence between FIV from different species, alignment for coding regions of FIV used the program REVTRANS ver 1.4 [29] which takes a multiple sequence file, translates that file into amino acid residues, aligns the amino acids, and uses this alignment as the scaffold for nucleotide alignment. Aligned multiple sequence files were imported into Modeltest ver 3.7 [63] and the optimal model of nucleotide substitution was selected using the AIC criterion (see Additional file 3).

Viral genes were analyzed separately, as well as combined, for genome comparison and phylogenetic reconstruction. Phylogenetic trees based on nucleotide data were obtained using a heuristic search with three different optimality criteria of maximum likelihood (ML), minimum evolution (ME) and maximum parsimony (MP) as implemented in PAUP* ver 4.0b10 [64]. Conditions for the ML analysis included starting trees obtained by stepwise addition, and branch swapping using the tree-bisection-reconnection (TBR) algorithm. Specific conditions for the ME search included starting trees obtained by neighbor – joining, TBR branch-swapping algorithm, and no collapsing of zero-length branches. The MP analyses coded gaps as "missing", with step-wise addition of taxa and TBR branch swapping. Support for nodes within the phylogeny used bootstrap analysis with identical settings established for each method of phylogenetic reconstruction and retention of node bootstrap values greater than 50%. The number of bootstrap iterations consisted of 1000 for ME and MP methods and 100 for ML. Additional analyses were conducted on FIV coding sequences after translation into amino acids. Genetic distances between strains were derived using the Pam-Dayhoff model of amino acid substitution as implemented in MEGA verson 3.1 [65] with gamma-correction (alpha = 2.5) and pairwise deletion of missing data.