Mark A. Rigby

Feline leukaemia virus: generation of pathogenic and oncogenic variants.

Although feline leukaemia virus (FeLV) is a common and potent pathogen of the domestic cat, neoplastic disease is a relatively rare outcome of the virus-host interaction and generally occurs after a long latent period (Hardy et al. 1976; Jarrett 1984). For these reasons, it has been customary to classify FeLV as a chronic leukaemogenic retrovirus and to place the acute transforming C-type retroviruses in a separate subclass. In our view, this classification has been unhelpful since it obscures the fact that acute transforming retroviruses invariably arise from the chronic leukaemogenic variety. Moreover, there is mounting evidence that terminal diseases which develop in FeLV-infected cats are induced by variant genomes that arise de novo. In this review, we discuss the origins of FeLV variants and briefly consider their roles in disease.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.

VIRUS NEUTRALIZATION REVEALS ANTIGENIC VARIATION AMONG FELINE IMMUNODEFICIENCY VIRUS ISOLATES

By using a focus reduction assay in CrFK feline fibroblast cells, virus neutralizing antibodies (VNA) to feline immunodeficiency virus (FIV) were demonstrated in cats that had been naturally or experimentally infected with FIV. The antigenic relatedness of four strains of FIV, divergent in nucleotide sequence within the env gene, was investigated by neutralization following adaptation of each virus for growth in CrFK cells. Two of the viruses were from The Netherlands (FIV/AM-4 and AM-6), one was from the U.K. (FIV/GL-8) and one was from the U.S.A. (FIV/PET). Reaction of the viruses in the neutralization assay with cat antibodies to homologous or heterologous strains indicated that while there was a degree of cross-reactivity between all four, there were consistent differences suggesting the existence of FIV neutralization subtypes. In particular, FIV/PET and FIV/AM-6 were closely related but FIV/PET and FIV/GL-8 were clearly distinct. VNA from naturally infected cats in the field showed a pattern of reactivity against FIV/PET and FIV/GL-8 that confirmed the antigenic diversity of FIV.

Immunodiagnosis of feline immunodeficiency virus infection using recombinant viral p17 and p24.

The coding sequences of p17 and p24 of the Glasgow-8 strain of feline immunodeficiency virus (FIV) were amplified using the polymerase chain reaction and cloned into plasmid vectors. The predicted amino-acid sequences of FIV/Glasgow-8 p17 and p24 were compared with those of the Petaluma and PPR isolates of FIV. As seen with other retroviruses, these gag gene products are highly conserved, indicating that the protein products would be suitable antigens to detect anti-FIV antibodies in an immunoassay. Both p17 and p24 were stably expressed in Escherichia coli as fusion proteins with glutathione S transferase. A pure preparation of each fusion protein was obtained from induced bacterial lysates by affinity chromatography using glutathione-agarose beads. These recombinant proteins were used in an enzyme-linked immunosorbent assay to detect antibodies directed against FIV p17 and p24 in cat sera. This assay allows the identification of seropositive cats following infection with FIV and has greater sensitivity and specificity than a currently available immunodiagnostic test.

Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection.

Immunogenicity of a peptide from a major neutralising determinant of the feline immunodeficiency virus surface glycoprotein

The third variable region (V3) of the feline immunodeficiency virus (FIV) surface glycoprotein is predicted to have similar physical properties to that of HIV and has been shown to contain immunodominant and neutralizing epitopes. Immunological characteristics of this region were investigated further using a peptide corresponding to the middle of the putative FIV V3 loop. The peptide was recognized in ELISA by sera from the majority of naturally FIV-infected cats, and absorbed a significant fraction of the virus neutralizing activity from a pool of sera of cats naturally infected with FIV, confirming the immunogenic nature of this region. A sheep immunized with an octameric form of the peptide (multiple antigenic peptide; MAP) in Freunds complete adjuvant generated neutralizing antibody to a higher titre than infected cats. However, immunization of cats with the same MAP in an acceptable adjuvant formulation (Quil A) induced antibody and cytotoxic T-cell responses to the immunizing peptides but only minimal neutralizing activity. These responses did not significantly alter the kinetics of infection or the proviral load after challenge with a homologous strain of FIV, compared with naive controls. While the potential efficacy of peptide vaccines to lentiviruses remains to be determined, this study shows that the immune response evoked may be highly dependent on the delivery and adjuvant regime used.

A recombinant feline immunodeficiency virus envelope fusion protein stimulates peripheral blood lymphocytes from naive cats to proliferate in vitro

A region of feline immunodeficiency virus (FIV)/Glasgow-8 external envelope glycoprotein (env) incorporating the third and fourth variable regions (V3/V4) was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase. The fusion protein V3/V4GST was used in lymphocyte proliferation assays, where it consistently caused peripheral blood lymphocytes from naive cats to proliferate in a dose-dependent manner. Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and glutathione S-transferase alone did not cause proliferation in this system. The monoclonal antibody vpg15, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST. Human peripheral blood lymphocytes did not proliferate in response to V3/V4GST.