Different Levels of Immunogenicity of Two Strains of Fowlpox Virus as Recombinant Vaccine Vectors Eliciting T-Cell Responses in Heterologous Prime-Boost Vaccination Strategies

Antigens and MVA recombinants.The PbCSP antigen from Plasmodium berghei and an MVA.PbCSP recombinant have been described previously (42). The GPN construct consists of a scrambled and inactivated fusion of the gag, pol, and nef genes of HIV-1 (clade B, HXB2 strain [40]). In order to abolish protein function without destroying T-cell epitopes, the genes were fused and inactivated in a redundant fashion. The p17- and p24-coding regions of gag were fused in the reverse of their natural order, and the myristoylation site of p17 was removed by a single-amino-acid substitution. The transcriptase activity of p51, encoded by pol, was ablated by splitting the sequence near the active site and swapping the order of the two resulting fragments, with duplication of a central 12-residue sequence at the novel termini to prevent loss of T-cell epitopes in this region. Finally, in the case of nef, the gene was divided into eight segments, with duplication of eight-codon sequences at the junctional regions, again to retain T-cell epitopes present at the breakpoints. In addition to these three inactivated but fully immunogenic constructs, the ∼120-kDa GPN fusion protein carries at the C terminus an eight-residue sequence from HIV gp160 (encoded by env) that is a known CD8⁺ T-cell epitope in BALB/c mice (see Table 1).

An MVA recombinant carrying this construct at the thymidine kinase insertion locus under control of the Vaccinia virus p7.5 early/late promoter with lacZ as a selectable marker was prepared using the pOPK6 shuttle vector (A. van Maurik et al., unpublished data).

Recombinant Fowlpox production.The Cyanamid Webster FPV-M strain of Fowlpox (FPW) was obtained from Solvay Animal Health and was not subjected to plaque purification prior to isolation of recombinant clones. FP9 was derived by plaque purification of the attenuated strain HP-438-Munich (27) at the Institute for Animal Health in the late 1980s (33). For introduction into Fowlpox, the antigen constructs were ligated into the SmaI site of the pEFL29 shuttle vector (38), which carries lacZ as a marker and places the antigen gene under the control of the p7.5 promoter at the fpv002 locus. Independent clones of recombinant viruses were produced by replicate transfection of primary chicken embryo fibroblasts with the shuttle vector and subsequent infection with FP9 or FPW, followed by isolation by repeated plaque purification on CEFs using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) selection as previously described (38). The viruses were amplified by three passages in CEFs and purified by centrifugation through sucrose cushions. The concentration and homogeneity of the resulting stocks were determined by titration using duplicate dilution series on two separate preparations of CEFs followed by X-Gal staining. Antigen expression was verified by Western blotting of infected CEFs.

Immunizations.Female BALB/c mice (H-2^d; 6 to 8 weeks) were used in all experiments and kept in individually ventilated cages in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Doses of 1 × 10⁶ PFU of recombinant virus were administered intravenously in 100 μl injection-grade phosphate-buffered saline (PBS). Control animals received PBS alone. Boost immunizations were performed 14 days after priming immunizations. On day 28, the animals were sacrificed by cervical dislocation, and the spleens were removed for processing.

Ex vivo IFN-γ enzyme-linked immunospot assay (ELISPOT).Splenocytes from individual mice were prepared as previously described (3). The cells were enumerated using a CASY automated cell counter (Schärfe Systems GmbH) and diluted to equalize concentrations. Appropriate numbers of splenocytes from each mouse were pipetted in duplicate into the wells of Millipore Multiscreen-IP plates previously coated with the anti-mouse IFN-γ antibody AN-18 (Mabtech). A twofold dilution series of the splenocytes was performed in the wells using a multichannel pipette. Target cells (5 × 10⁵ per well) from naïve mice were treated (1 μg/ml) with T-cell epitope peptides (obtained from NMI TT GmbH and listed in Table 1) and added to the plates, which were then agitated and placed in a CO₂ incubator at 37°C. The following day, the assay was developed by labeling with biotinylated anti-mouse IFN-γ antibody R4-6A2 (Mabtech) and alkaline phosphatase-conjugated streptavidin. The spots were counted using an Autoimmun Diagnostica automated reader. Values from duplicate wells were averaged prior to subtraction of background spot counts obtained in the absence of peptide. Statistical analyses were performed using Prism (GraphPad Software, Inc.).

Major histocompatibility complex (MHC) pentamer staining and FACS analysis.Splenocytes (1 × 10⁶) from individual immunized mice were stained with phycoerythrin-conjugated H2-D^d-RGPGRAFVTI pentamer (ProImmune) for 30 min at 4°C. After incubation, the cells were washed twice in fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% fetal calf serum and 0.1% sodium azide) and stained for 30 min at 4°C with fluorescein-conjugated anti-mouse CD8 (Caltag). Finally, the cells were washed twice and fixed with FACS buffer containing 2% formaldehyde. Analysis was performed using a Partec PAS flow cytometer with FloMax software.

Western blotting.CEF or 3T3 cells were infected at a multiplicity of infection of one with recombinant viruses 24 to 48 h prior to lysis in Laemmli's sample buffer, sonication, and boiling. Western blots prepared using standard techniques were labeled with appropriate mouse monoclonal antibodies: GB9 against Fpv168 (7) and Kal-1 against HIV p24 (22). The blots were developed using an ECL kit (Amersham) and exposure to film.

Sequence analysis of FPW.DNA was prepared from FPW-infected CEFs by standard methods (5). PCR amplification of selected loci was carried out as described previously (24), and purified products were subjected to capillary sequencing (MWG Biotech Ltd.). For large-scale deletions, both flanking and internal primers were used. The sequences were aligned to the published reference sequences of strains FP9 (24) and FPV-US (2) using STADEN (24).

Comparison of FP9.PbCSP and FPW.PbCSP recombinants in prime-boost immunization regimens.The superior immunogenicity of FP9.PbCSP compared to FPW.PbCSP as a component of dual-poxvirus prime-boost regimens was described by Anderson et al. (3), but the possibility remained that a clone-specific effect, rather than a strain-specific effect, was responsible for the difference. In order to investigate this question, we prepared three new independent clones each of FP9.PbCSP and FPW.PbCSP and performed a head-to-head comparison of the abilities of these six recombinants to elicit murine T-cell responses when given in combination with MVA.PbCSP as a heterologous prime-boost immunization. We employed a prime-boost regimen, rather than single immunizations, in order to improve the signal-to-noise ratio in immune readouts and because Fowlpox is used as a combination vaccine in clinical settings. Both orders of administration, Fowlpox-MVA (FM) and MVA-Fowlpox (MF), were tested, using an intravenous dose of 10⁶ PFU and a 14-day interval between the prime and boost. The cellular immune responses to the PbCSP antigen were determined at day 28 by ex vivo IFN-γ ELISPOT of splenocytes stimulated with the known immunodominant H2-K^d-restricted peptide Pb9 (Table 1) (41, 42).

Figure 1 shows the results, expressed as spot-forming cells (SFC) per million splenocytes, of the Fowlpox-MVA (FM) prime-boost vaccinations, in comparison to a control treatment consisting only of MVA.PbCSP. The data were analyzed using a one-way analysis of variance (ANOVA) followed by a Newman-Keuls post hoc test for each pairwise comparison. While the responses produced by FP9 priming were similar between independent clones (P > 0.05 for all FP9-FP9 pairwise comparisons), there was considerable variability in those produced by FPW. In particular, clone 2 of FPW.PbCSP was as immunogenic as any clone of FP9.PbCSP (P > 0.05); however, in contrast, the response to clone 3 was as low as that elicited by MVA.PbCSP alone (P > 0.05) and was significantly lower than the responses elicited by clone 2 of FPW.PbCSP (P < 0.001) or any clone of FP9.PbCSP (0.001 < P < 0.01). Presenting an intermediate immunogenicity, clone 1 of FPW.PbCSP is more effective than clone 3 (P < 0.05), but not as effective as clone 2 of FPW.PbCSP (P < 0.05).

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FIG. 1.

Frequencies of Pb9-specific SFC per million splenocytes determined by IFN-γ ELISPOT following prime-boost immunization with three independent clones of FP9.PbCSP or FPW.PbCSP in combination with MVA.PbCSP. The order of administration was either Fowlpox-MVA (FM) or MVA-Fowlpox (MF). Left-hand panels show individual data points (one circle per mouse), with horizontal bars indicating the mean values. Right-hand panels show pooled data for all three clones of each strain, with error bars indicating standard errors of the means. Dashed lines indicate the mean response observed after immunization with MVA.PbCSP alone.

When Fowlpox was used as a boost, rather than a prime, in combination with MVA (MF), a somewhat different picture emerged, as shown in Fig. 1 (six mice per group, instead of four, were used in this experiment). In this case, no clone performs significantly differently from any other clone of the same strain (P > 0.05, Newman-Keuls), and every recombinant FP9.PbCSP clone performs significantly better than every FPW.PbCSP clone (0.001 < P < 0.01). To clarify, using MF compared to FM prime-boost, the intrastrain (clone-specific) differences are less pronounced, and the interstrain differences are more pronounced. There appear to be two reasons for this: (i) the variable priming performances of FPW.PbCSP clones and (ii) the superior boosting performance of FP9.PbCSP, which produces markedly higher responses (average about 6,500 SFC/million compared to 2,500) when given MF compared to FM, unlike FPW.PbCSP (for which the responses are only slightly increased using MF).

To aid interpretation of the results, Fig. 1 also presents the same data pooled by strain. From these graphs, it is clear that, overall, the FP9 strain elicits higher Pb9-specific T-cell responses than FPW, whether used as a prime or boost in combination with MVA.PbCSP. As revealed by Table 2, which shows the difference in immunogenicity as a ratio (FP9 response/FPW response) together with the 95% confidence intervals for the difference, both of these comparisons are significant in t tests (P = 0.04 for FM; P < 0.0001 for MF).

The magnitude of the difference between FP9 and FPW immunogenicity is comparable to that previously reported using single PbCSP clones in the two backgrounds (3). The FPW.PbCSP recombinant described by Anderson et al. (3) behaved similarly to clone 1 of FPW.PbCSP (which showed intermediate immunogenicity) when used as a prime, and for FM administration, the overall difference in efficacy presented here is very similar to that previously reported. Using the reverse order of administration (MF), however, the magnitude of the strain efficacy difference observed in the current study is somewhat greater than that previously reported. Nevertheless, the available data taken together clearly demonstrate that the virus strain is a key determinant of the immunogenicity of Fowlpox PbCSP recombinant vaccines, though there is also evidence for clonal variation, especially in the FPW strain.

Comparison of multiple clones of FP9.GPN and FPW.GPN recombinants.The next objective was to ascertain whether the superior immunogenicity of strain FP9 is observed when the virus carries a different recombinant antigen. In order to increase the number of T-cell epitopes available for analysis and to include a CD4⁺ epitope, we selected an artificial polyprotein derived from HIV as the alternative antigen. Briefly, this construct, abbreviated GPN, consists of an inactivated fusion of the gag, pol, and nef genes from HIV-1 (van Maurik et al., unpublished). Further information can be found in Materials and Methods.

In a series of experiments analogous to those carried out using PbCSP recombinants, we analyzed the abilities of FP9.GPN and FPW.GPN recombinants to elicit T-cell responses in heterologous prime-boost regimens with MVA.GPN. In this case, four independent clones of FPW.GPN and FP9.GPN were tested. These were given either as prime (FM) or boost (MF) immunizations using an intravenous dose of 10⁶ PFU and a 14-day interval between the prime and boost as before. Splenocytes from immunized mice were analyzed at day 28 for T-cell responses by ex vivo IFN-γ ELISPOT and additionally by FACS using a fluorescent MHC pentamer.

The immune assays for T-cell responses to GPN were conducted using peptides corresponding to three T-cell epitopes present in the construct. The amino acid sequences and MHC restriction of these peptides, abbreviated AMQ, RGP, and NPP, are given in Table 1. These sequences have been previously identified as immunodominant HIV-1 T-cell epitopes in BALB/c mice (26, 48) and are present in the scrambled and inactivated GPN construct. Experiments using overlapping peptides have confirmed the immunodominance of the CD8⁺ epitopes in the BALB/c T-cell response to the GPN construct (van Maurik et al., unpublished). Responses to all three peptides were determined in IFN-γ ELISPOTs, and RGP was additionally used as an MHC pentamer for FACS assays. RGP is somewhat unusual: in H-2^d mice, it is a dual CD8⁺ and CD4⁺ epitope, and the peptide is presented by MHC class I (H2-D^d) and class II (H2-IA^d) (48). As mentioned above, RGP is encoded by env and was included in the GPN (gag-pol-nef) construct as a control marker epitope for BALB/c mice. In contrast, AMQ and NPP (both encoded by gag) are conventional CD8⁺ and CD4⁺ T-cell epitopes, respectively (26).

Figure 2 shows IFN-γ ELISPOT analysis of the frequencies of antigen-specific T-cell responses to these epitopes elicited by immunization with the four independent clones of FP9.GPN and FPW.GPN as a prime-boost with MVA.GPN, using both orders of administration. It is immediately apparent from the data pooled by strain (see also Table 2) that FP9 elicits a greater response against all three epitopes than FPW, establishing that the superior immunogenicity of FP9 is not solely restricted to PbCSP. For both orders of administration, a two-way ANOVA (strain versus peptide) indicates that this difference is highly significant (P < 0.0001, FM; P < 0.003, MF). Bonferroni post-tests on these comparisons reveal that there is a strain-specific difference in the responses against RGP for both FM (P < 0.001) and MF administration (P < 0.01) but that the responses against AMQ are significantly different only using FM (P < 0.01) and not MF (P > 0.05), though a consistent trend is observed. In the case of NPP, it is justifiable to consider the responses against this class II-restricted peptide as an independent measure of immunogenicity and to conduct separate statistical analysis (such that the variance in the other two peptide groups is not taken into account). t tests reveal a significant difference in the NPP responses using MF administration (P < 0.03) but not FM (though the pooled data reflect a trend consistent with the CD8⁺ responses). The increased immunogenicity of FP9 compared to FPW therefore extends across both types of T-cell response.

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FIG. 2.

Frequencies of AMQ-, RGP-, and NPP-specific spot-forming cells per million splenocytes determined by IFN-γ ELISPOT following prime-boost immunization with four independent clones of FP9.GPN or FPW.GPN. Fowlpox was given either as a prime (FM) or boost (MF) immunization in combination with MVA.GPN. Left-hand panels show individual data points (one circle per mouse), with horizontal bars indicating the mean values. Right-hand panels show pooled data for all four clones of each strain, with error bars indicating standard errors of the means. Dashed lines indicate the mean response observed after immunization with MVA.GPN alone.

In comparison with PbCSP, the effect of the order of administration upon the strain-specific difference in immunogenicity of Fowlpox GPN recombinants is not as pronounced. For PbCSP, FP9 was about 1.6 times more immunogenic than FPW when given as a prime, but it was about 2.7 times more immunogenic when given as a boost (Table 2) as discussed above; however, for GPN, the difference in immunogenicity of FP9 relative to FPW is similar (about 1.6-fold on average, with some dependence upon peptide; Table 2) regardless of the order of administration. In other words, the relative priming efficacy of the strains is independent of antigen, but the difference in the relative boosting efficacy of FP9 and FPW is greater when the antigen is PbCSP than when it is GPN. The conclusion is therefore that differential strain efficacy is a general phenomenon but that its magnitude under specific circumstances depends upon the antigen.

Statistical analyses of pairwise comparisons of the eight GPN recombinants are complex due to the multiple peptide-specific responses measured. An exhaustive approach is not necessary to perceive that it is not the case that all clones of FP9.GPN recombinant are more immunogenic than all clones of FPW.GPN and that this is the result of variation among clones of the same background. For example, clone B of FPW.GPN is as immunogenic as any clone of FP9.GPN when used as a prime (except for the RGP-specific response elicited by clone C of FP9.GPN), and when used as a boost, it is as immunogenic as three of the four FP9.GPN clones (A, B, and C). Indeed, the highest CD8⁺ responses were observed in mice boosted with clone C of FPW.GPN, though the scatter in this group is exceptionally high (it also contains two of the lowest responders). On the other hand, clone A of FPW.GPN is inferior to all clones of FP9.GPN both as a priming and boosting vector. This contrasts with the situation observed using PbCSP, where equal strain efficacy (for one FPW clone) was observed only using FM prime-boost. Furthermore, the within-strain variation observed using GPN is not confined to the FPW strain (as it was using PbCSP) and is apparent both in MF and FM prime-boosts (also unlike PbCSP). To exemplify this, clone D of FP9.GPN is a relatively rather poor priming agent, but it elicits the highest CD8⁺ responses when used as a boost: it is the only GPN clone of any strain for which there is a higher response using MF versus FM administration (as was the case for FP9.PbCSP). In addition, clone A of FP9.GPN produces outstandingly high responses against NPP when used as a prime, even though it elicits average CD8⁺ responses. To summarize, in addition to the overall difference in strain efficacy, there are (in both strains) clone-specific differences, which may be apparent only using a particular antigen or order of administration. However, the variation inherent in the experimental approach makes it harder to characterize these “secondary” viral phenomena than the clearer overarching effects of the strain background.

An additional correlate of immunogenicity was obtained by using an RGP H2-D^d pentamer in FACS analysis of the CD8⁺ T-cell responses in the mice given FPW.GPN and FP9.GPN prime-boost immunizations. It is simplest to consider only the pooled data (shown in Fig. 3) due to the lower signal-to-noise ratio compared to that of ELISPOTs. The mean differences in frequencies of RGP-binding CD8⁺ cells elicited using FP9.GPN compared to FPW.GPN are 2.5-fold (FM) or 1.7-fold (MF), and these differences are highly significant (P < 0.0001, ANOVA). These values are comparable to the mean ratios of frequencies of RGP-specific IFN-γ-secreting T cells shown in Table 2 (2.1 for FM; 1.6 for MF), despite the lack of discrimination between CD4⁺ and CD8⁺ responses in the ELISPOT (RGP is an epitope of both populations of T cells [26]). The pentamer data provide a useful corroboration of the ELISPOT responses by a different readout parameter.

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FIG. 3.

Frequencies of RGP-pentamer-binding splenocytes measured by FACS as a percentage of CD8⁺ splenocytes from mice vaccinated with four individual clones of FP9.GPN or FPW.GPN recombinant given either as a prime (FM) or boost (MF) immunization in combination with MVA.GPN. The data are shown pooled (A), such that each circle corresponds to a single mouse immunized with one of the eight viruses. Horizontal bars indicate mean values, and dashed lines indicate mean responses observed after immunization with MVA.GPN alone. Two example FACS scattergrams corresponding to the data points indicated by the arrows in panel A are shown below (B).

Antigen expression levels in FP9- and FPW-infected cells.In order to verify that the increased immunogenicity of FP9 compared to FPW is not attributable to an inequality of expression of antigen in murine cells by the two strains, we conducted Western blotting of infected mouse fibroblast (3T3) and chicken embryo fibroblast cells. The blots were probed using antibodies against the recombinant antigen and the viral protein Fpv168, an essential virion core protein (the orthologue of Vaccinia virus A4L) that is produced abundantly during late gene expression (7). As shown previously, this protein has a different molecular weight in FP9 and FPW due to diversity in the copy number of a nonapeptide repeat sequence (8).

Figure 4 shows the Western blots obtained when an equal multiplicity of infection with the eight Fowlpox GPN recombinants, plus MVA.GPN for comparison, was used; similar results were obtained using the PbCSP recombinants (not shown). In CEFs (in which Fowlpox can replicate), the four FP9 clones produce large and equal amounts of Fpv168, but FPW generally produces less protein and exhibits greater variability in the amount expressed by the individual clones. Since fpv168 is a late gene, its expression levels at the time of lysis (24 h postinfection) are indicative of the replication rates of the viruses, and indeed the relative abundance of Fpv168 shows some correlation with the virus yield obtained during productive infections (data not shown). The clonal levels of GPN polyprotein correlate well with those of the viral protein, but the recombinant antigen is expressed more evenly by the two strains, since its expression is driven by an early/late promoter. These observations have no bearing on immunogenicity in mammals but rather reflect (i) the very different behaviors of the two strains in cell cultures derived from the natural host (a result of the extensive tissue culture adaptation of FP9) and (ii) variation in growth rates of individual clones derived from the non-plaque-purified FPW strain.

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FIG. 4.

(A) Western blots of chicken (CEF) and murine (3T3) fibroblast cells infected at an equal multiplicity of infection (1.0) with clones A to D of FP9.GPN or FPW.GPN, with MVA.GPN or mock infected (−), immunolabeled for the GPN fusion protein (∼120 kDa) and the endogenous viral protein Fpv168 (168). The molecular mass of Fpv168 is ∼39 kDa in strain FP9 and it is ∼36 kDa in strain FPW due to differences in the copy number of a repeat sequence (8). Cells were lysed for analysis 24 h postinfection or after 48 h for detection of Fpv168 in 3T3 cells. Note that GPN expression is controlled by a synthetic early/late promoter but that fpv168 is under control of its endogenous late promoter. (B) ImageJ (1) quantitation of bands in GPN-labeled immunoblots of infected 3T3 cells. The graph shows the mean area of the GPN band in the blot shown in panel A and in an additional blot (not shown). Error bars show standard errors of the means. The table to the right shows the individual values obtained for each GPN band in these two immunoblots.

When infected 3T3 cells (in which viral replication is blocked) are analyzed, a rather different picture emerges. Here, despite their very different behaviors in permissive CEFs, FPW and FP9 produce nearly equal levels of Fpv168, and there is less variability among the FPW clones. Most importantly, the amount of GPN produced is similar across all eight clones: while small variations are evident in both strains, it is not the case that FP9 produces larger amounts of recombinant antigen than FPW does in mouse cells in vitro. Quantitation of these GPN bands using ImageJ (1) confirms that there is no significant difference (P > 0.4) in the mean labeling intensity obtained following infection with FP9 and FPW clones (Fig. 4B). Unlike the situation in CEFs, there does not appear to be a correlation between the levels of GPN and Fpv168 (compare clones A and B of both strains); furthermore, there is no obvious clonal correlation with the immunogenicity observed in the experiments described above. Nevertheless, the key observation is that, even though FP9 and FPW produce different amounts of viral and recombinant protein in CEFs as a result of their different replication rates in the permissive cells to which FP9 growth is highly adapted, they produce similar quantities of recombinant antigen (and viral protein) in nonpermissive mouse cells, at least within the limitations of a semiquantitative Western blot readout. The different levels of immunogenicity of the two strains are not therefore attributable merely to inequality of antigen expression.

Limited screen for FP9-specific mutations present in FPW.The genome sequences of the FP9 and FPV-US strains of Fowlpox have been determined, but that of FPW remains unknown. In a comparative analysis, Laidlaw and Skinner (24) categorized the differences in FP9 relative to the FPV-US strain into lineage- and passage-specific mutations by sequencing the HP-1 strain (the FP9 progenitor) at those loci. The passage-specific mutations arose during the attenuation of HP-1 by extensive passage in CEFs (27), whereas the lineage-specific mutations are a reflection of the different origins of the strains. We have conducted a partial screen of FPW for these passage-specific mutations using restriction mapping and sequencing of selected PCR-amplified regions.

The most striking FP9 passage-specific mutations are seven multikilobase deletions, cumulatively responsible for the removal of more than 25 kb of coding sequence and the elimination or severe disruption of 17 open reading frames (out of 260 in FPV-US). These dramatic mutations are amenable to analysis by restriction mapping. Table 3 presents a comparison of PstI-digested Fowlpox genome fragment sizes, using both actual agarose gel migration data for strains FPW and FP9 (13, 33) or data generated in silico from the genomic sequences for FP9 and FPV-US (2, 24). The discrepancies between the two FP9 maps are indicative of the inaccuracy inherent in the traditional method, but it is clear that FPW does not possess any of the large-scale deletions. There is no reduction in the sizes of fragments D, L, and C with deletions 9, 11, and 13, respectively. Fragment A, which has deletions 22 and 24, is accounted for in full by the two FPW fragments A and D′ (FPW having an extra PstI site here). Fragment G, which contains the inverted terminal repeats (ITRs) and diploid deletions 2 and 25, is not reduced in size compared to the corresponding sequence in FP9, though the situation is complicated by the presence of a shorter noncoding repeat region in the ITR of FPV-US than in FP9 (2, 24). To verify this analysis, we generated PCR products spanning the junctional regions to confirm an FPV-US-type, nondeleted genotype at all six loci and in three cases (deletions 9, 11, and 24) obtained sequence demonstrating identity with FPV-US at these regions.

The restriction map data also suggest that strain FPW is similar, though not identical to strain FPV-US. There is an extra PstI site in fragment A, and there are possibly significant differences in the sizes of fragments B and F (the size difference of fragment G is due to the shorter repeat region of the ITR of FPV-US, which is not representative of the FPV-US population but rather of the individual isolate sequenced [2]). FPV-US is also known as the USDA challenge strain, and while it is correct to describe it as pathogenic (2), it is, according to the U.S. Department of Agriculture, derived from a commercial vaccine strain. FPW is also pathogenic, though partially attenuated (10), and like FPV-US, it lacks the full-length reticuloendotheliosis provirus characteristic of wild-type field isolates (18). Without conducting a full-scale genomic analysis, it is not possible to deduce whether FPW has a lineage distinct from that of FPV-US, as might be expected from their respective Australian and North American origins.

The remaining FP9 passage-specific mutations consist of small-scale deletions (1 to 27 bp), insertions (1 to 108 bp), and single-nucleotide substitutions. By PCR amplification and sequencing, we determined the FPW genotype at approximately two-thirds of these loci (Table 4). In all cases investigated, the sequence was identical to that of FPV-US (and HP-1). In combination with the observation that FPW lacks FP9's characteristic large-scale deletions, these results show that FPW has not undergone an extensive adaptation to growth in culture, since it has a genotype identical to pathogenic strains at loci where mutations were acquired by FP9 during its multiple CEF passages.