Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma

Abstract

Background: In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses.

Results: The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence.

Conclusions: Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

Figure 1

Time course of FeLV infection in cat #261. A) FIV transmembrane (TM) specific antibody levels as determined by ELISA. B) Total anti-FeLV antibodies (black squares) and anti-FeLV p45 antibodies (black triangles) as determined by ELISA. C) Plasma FeLV p27 antigen levels as determined by ELISA. D) FeLV provirus loads as determined by real-time PCR. Two samples, indicated by asterisks, were positive for FeLV by non-quantitative PCR. Time points of FIV infection (at the age of 17 weeks), FeLV vaccinations (41 weeks, 4 years) and FeLV exposure with FeLV-A/Glasgow-1 (59 weeks) are indicated.

Figure 2

Time course of hematological parameters for cat #261. A) White blood cell counts. B) Neutrophil granulocyte counts. C) Lymphocyte counts. D) CD4⁺ T-cell counts. E) Packed cell volume. The reference ranges (5th to 95th percentiles) are indicated by the shaded areas (A to C, and E). No reference range was available for the absolute numbers of CD4⁺ T cells. In panel D, the dotted line indicates a CD4⁺ T-cell count of 200 CD4⁺ T cells/μL. No differential was possible at the time of sacrifice due to the low WBC number (100 cells/μL).

Figure 3

FeLV and FIV provirus and viral loads in blood and tissue samples from cat #261. FeLV and FIV provirus and viral loads in cat #261, quantified in the various tissues collected upon necropsy and in blood samples collected over the course of the last 14 months prior to sacrifice. Provirus and cDNA loads were determined using TaqMan real-time PCR and viral RNA loads were measured by TaqMan real-time RT-PCR. The tissues were classified according to the absence (n = 9) or presence (n = 18) of apparent lymphoma, as determined by histological examination. A) FeLV provirus loads. B) FIV provirus loads. C) FeLV viral (cDNA) loads in the tissues. D) FIV viral loads in the tissues. Viral tissue loads were normalized using GAPDH. Provirus loads (A and B) were tested for statistical differences by Kruskal-Wallis one-way ANOVA by Ranks (p_KW as indicated) and subsequently by Dunn's post test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Viral loads (C and D) were tested for statistically significant differences using the Mann-Whitney U-test (p_MWU as indicated).

Figure 4

Evolutionary relationship of the three SU variants found in cat #261. Phylogenetic trees were constructed by the MP method. Trees were drawn to scale, with the length being relative to the number of changes over the entire sequence. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. A) Relationships at the DNA level. MP tree length: 765. The codon positions included were 1st + 2nd + 3rd + noncoding. There were a total of 1,363 base positions in the final dataset of which 349 were parsimony-informative. B) Relationships at the protein level. MP tree length: 329. There were a total of 453 amino acid positions in the final dataset of which 121 were parsimony-informative. GenBank accession numbers of the sequences included in the phylogenetic analyses are noted in square brackets following the virus identity. SP261-III, KI261-I and KI261-II (depicted in bold) were derived from cat #261.

Figure 5

FeLV-A/Glasgow-1 and env variant loads in blood and tissue samples from cat #261. Loads of FeLV-A/Glasgow-1 and the env variants of cat #261 quantified by real-time PCR in the tissues collected upon necropsy (A to D) and in blood samples collected over the course of the last 14 months prior to sacrifice (E). A) Provirus loads of FeLV-A/Glasgow-1 and env variants in all tissues. B) env variant provirus loads in tissues without (n = 9) and with (n = 18) apparent lymphoma. C) Viral (cDNA) FeLV-A/Glasgow-1 and env variant loads in all tissues. D) Viral (cDNA) env variant loads in tissues with and without apparent lymphoma. E) Time course of provirus loads of FeLV-A/Glasgow-1 and env variants in the blood of cat #261. Viral loads (C and D) were normalized using GAPDH. Provirus and viral loads (A to D) were tested for statistical significance using the Mann-Whitney U-test (p_MWU as indicated).