Feline leukemia virus subgroup C phenotype evolves through distinct alterations near the N terminus of the envelope surface glycoprotein.
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Feline leukemia viruses (FeLVs) belonging to the C subgroup induce aplastic anemia in domestic cats and have the ability, unique among FeLV strains, to proliferate in guinea pig fibroblasts in tissue culture. Previous studies have shown that the pathogenic and host range specificity of a prototype molecular clone of FeLV-C [FeLV-Sarma-C (FSC)] colocalize to a region encoding the 3' 73 amino acids of the pol gene product and the N-terminal 241 amino acids of the envelope surface glycoprotein named SU. Here, we amplified, via PCR, cloned, and sequenced the SU coding sequence from three additional anemia-inducing subgroup C FeLV isolates. Chimeric viruses were constructed by replacement of fragments of FeLV-C envelope genes into the FeLV-A prototype virus 61E. Using a modified vesicular stomatitis virus-FeLV pseudotype assay, we demonstrated that the subgroup C receptor specificity for each virus was determined by changes within the N-terminal 87-92 amino acids of SU, in which most changes occurred within the 15- to 20-amino-acid first variable region (V1). Determinants for growth in guinea pig cells colocalized to this region. Despite the consistent localization of biological determinants, the only consistent features that distinguished the deduced FeLV-A and FeLV-C proteins was one lysine-to-arginine change and a structural prediction of an alpha-helix in FeLV-A proteins versus random coil in FeLV-C proteins within V1. However, arginine in equilibrium with lysine substitutions were not sufficient to convert the subgroup A virus to the subgroup C phenotype or vice versa. Thus, certain distinct structural changes within the N-terminal region of FeLV SU can result in convergent viral phenotypes. (+info)
Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes.
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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. (+info)
Amplimers with 3'-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming.
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The 3'-->5' exonuclease activity of Vent, a thermostable polymerase from Thermococcus litoralis, enhances DNA replication fidelity but also diverts PCR primers (amplimers) from targeted amplification by degrading their 3' termini. We demonstrate that amplimers with a 3-base 3'-terminal mismatch can be efficiently truncated by Vent to prime DNA polymerizations that compete with the specific amplification reaction. However, amplimers with phosphorothioate bonds joining their 3'-terminal residues are resistant to degradation and demonstrate greatly enhanced priming specificity. Slight destabilization of base-pairing by phosphorothioate bond-linked residues also diminishes extension of mispaired 3' amplimer termini in Taq polymerase-mediated amplifications. (+info)
Pre- and postexposure chemoprophylaxis: evidence that 3'-azido-3'-dideoxythymidine inhibits feline leukemia virus disease by a drug-induced vaccine response.
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The benefits of postexposure 3'-azido-3'-dideoxythymidine (AZT) prophylaxis following human immunodeficiency virus exposure are unknown. We describe a comprehensive assessment of pre- and postexposure AZT therapy in the feline leukemia virus (FeLV)-cat model for AIDS which included in vitro testing, an in vivo dose-response titration, a postexposure treatment study, plasma drug concentration determinations, and evaluation of the immune response to FeLV. In in vitro studies, AZT prevented FeLV infection of a feline T-lymphoid cell line, giving 50 and 90% inhibition concentrations of 4.6 and 11.1 mM, respectively. In all of the in vivo efficacy studies, AZT was administered by continuous subcutaneous infusion for 28 days. AZT toxicity was excessive at a dosage of 120 mg/kg of body weight per day, causing acute anemia, but AZT was tolerable at 60 mg/kg/day. In preexposure studies, AZT was efficacious in preventing chronic antigenemia at a dosage of > or = 15 mg/kg/day, at which plasma AZT concentrations averaged between 0.51 and 0.81 micrograms/ml (2.13 and 3.03 microM). As a postexposure treatment, at 60 mg/kg/day, AZT prevented chronic FeLV antigenemia when treatment was started up to 96 h post-virus inoculation (p.i.), but not when treatment was started at 192 h p.i. The 4-day period between 96 and 192 h p.i. appears to be critical for establishing chronic viremia. It is presumed that the increase in virus load between 4 and 8 days p.i. was able to overwhelm the immunologic functions responsible for containment of FeLV infection, even though AZT therapy effectively controlled viremia during the treatment period. The antibody response to FeLV varied depending on the time of AZT treatment initiation relative to virus challenge. When AZT treatment was started 48 h before or 8 h after FeLV challenge, antibodies to FeLV were not detected until after AZT treatment was discontinued at 28 days p.i. Following AZT treatment, however, antibody titers rapidly increased at a rate suggestive of a secondary immune response. When AZT treatment was initiate at later time points relative to virus challenge (24, 48, and 96 h p.i.), antibodies to FeLV became detectable during the treatment period. These results indicate that AZT treatment does not completely prevent FeLV infection, even when treatment begins before virus challenge, and that immune sensitization to FeLV proceeds during the prophylactic drug treatment period. (+info)
Cell lines produce factors that induce fetal hemoglobin in human BFUe-derived colonies.
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Established cell lines were screened for secretion of activities than can stimulate fetal hemoglobin (HbF) production in adult burst-forming unit-erythroid (BFUe) cultures. Conditioned media from four cell lines, a human teratocarcinoma, an osteosarcoma, a bladder cell carcinoma, and feline leukemia virus (FeLV) A-infected feline fibroblasts (FEF-A cells), consistently increased the relative production of fetal globin in BFUe-derived colonies. In vitro translation of RNA from these cells in Xenopus oocytes yielded products that increased the gamma to gamma+beta ratio in adult erythroid colonies. These results demonstrate that a variety of cell lines produce factors that stimulate the production of HbF in vitro. The genes of such factors could be isolated by expression cloning of cDNA from cell lines using the Xenopus oocyte system. (+info)
T-cell lymphoma of the tympanic bulla in a feline leukemia virus-negative cat.
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This report constitutes the first description of a T-cell lymphoma of the tympanic bulla in a cat. This feline leukemia virus (FeLV)-negative cat originally presented with signs referable to middle ear disease; it deteriorated rapidly after definitive diagnosis. Lymphoma of the middle ear is extremely rare in all species. (+info)
Genomically intact endogenous feline leukemia viruses of recent origin.
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We isolated and sequenced two complete endogenous feline leukemia viruses (enFeLVs), designated enFeLV-AGTT and enFeLV-GGAG. In enFeLV-AGTT, the open reading frames are reminiscent of a functioning FeLV genome, and the 5' and 3' long terminal repeat sequences are identical. Neither endogenous provirus is genetically fixed in cats but polymorphic, with 8.9 and 15.2% prevalence for enFeLV-AGTT and enFeLV-GGAG, respectively, among a survey of domestic cats. Neither provirus was found in the genomes of related species of the Felis genus, previously shown to harbor enFeLVs. The absence of mutational divergence, polymorphic incidence in cats, and absence in related species suggest that these enFeLVs may have entered the germ line more recently than previously believed, perhaps coincident with domestication, and reopens the question of whether some enFeLVs might be replication competent. (+info)
Centrosome amplification and chromosomal instability in feline lymphoma cell lines.
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To evaluate the presence of centrosome amplification and the resulting chromosomal instability in cat tumors, a newly established feline lymphoma cell line and four already established feline lymphoma cell lines were examined using immunohistochemical analysis of centrosomes. The number of chromosomes were subsequently counted by metaphase spread. Moreover, to explore whether mutational inactivation of the p53 gene or inactivation of the P53 protein caused by mdm2 gene overexpression, occurred in the feline lymphoma cell lines, mutational analysis of the feline p53 gene was carried out. The expression of feline mdm2 mRNA was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Centrosome amplification and chromosomal instability was observed in three out of the five feline lymphoma cell lines. Of these three feline lymphoma cell lines, one had aberrations in the P53 amino-acid sequence, whereas the others had none. There was no significant difference in the expression of mdm2 mRNA between peripheral blood mononuclear cells (PBMC) obtained from a normal cat and that of the five feline lymphoma cell lines. These findings indicate that centrosome amplification also occurs in cat tumors and is strongly correlated with chromosomal instability, suggesting that the immunostaining of centrosomes could be an alternative method for the examination of the chromosomal instability. Furthermore, this study suggests the presence of unknown mechanism that leads to the centrosome amplification in feline lymphomas. (+info)