Replicative difference in early-passage feline brain cells among feline immunodeficiency virus isolates

Summary The susceptibility of early-passage feline brain cells and Crandell feline kidney (CRFK) cells to infection with three isolates of feline immunodeficiency virus (FIV) was investigated. The Petaluma strain of FIV could well infect both the feline brain cells and CRFK cells. The KYO-1 strain could well infect the feline brain cells but the replication in CRFK cells was demonstrated only by coculturing fresh feline T-lymphoblastoid cells with the infected cells. On the other hand, the TM1 strain could infect the feline brain cells but not CRFK cells. Moreover, the replicative ability of the TM1 strain in the feline brain cells was much less than the KYO-1 and Petaluma strains. These results indicate that biological differences can be detected among the FIV isolates.     

Molecular characterization and heterogeneity of feline immunodeficiency virus isolates

Summary We have molecularly cloned the complete genomic DNA of TM2 strain of feline immunodeficiency virus (FIV) isolated in Japan and compared its nucleotide and the deduced amino acid sequence with those of previously described U.S. isolates, FIV Petaluma and FIV PPR. The infectious molecular clone of FIV TM2 is different from FIV Petaluma in host cell range; the clone can not infect Crandell feline kidney cells which were permissive for FIV Petaluma. The amino acid sequence homologies, ingag, pol, andenv genes between FIV TM2 and Petaluma were 90%, 87%, and 81%, respectively. On the other hand, comparative analysis of each gene between FIV Petaluma and PPR showed 96, 95, and 85%, respectively. These results suggested that the genomic diversity was present among FIV strains isolated from geographically distant areas. Interestingly,tat- andrev-like short open reading frames contained in-frame stop codons in the FIV Petaluma but not in the FIV TM2.     

Expression of feline immunodeficiency virusgag gene inEscherichia coli

Summary Thegag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed inEscherichia coli as a fusion protein with TrpE. Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIVgag protein in feline sera. With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.     

Establishment of a feline astrocyte-derived cell line (G355-5 cells) expressing feline CD134 and a rapid quantitative assay for T-lymphotropic feline immunodeficiency viruses

Few laboratory strains of feline immunodeficiency virus (FIV) can infect Crandell feline kidney cells (an epithelial-type of cells), however, most primary isolates are T-lymphotropic. T-lymphotropic FIV requires both feline CD134 (an activation marker of helper T-lymphocytes) and CXCR4 (a chemokine receptor) in infection as primary and secondary receptors, respectively. Using feline T-lymphoblastoid cell lines, titration of primary FIV isolates was carried out, however the titration assay was laborious and time-consuming. In this study, using G355-5 cells (a feline astrocyte-derived cell line) transduced with a cDNA of feline CD134 as target cells, an assay system was developed to quantitate primary FIV isolates. With a previous method using a feline T-lymphoblastoid cell line (MYA-1 cells) highly sensitive to FIV, it took 12 days to complete the assay, however, it took only 2 days with the new method. The FIV-infected cells became in a state of persistent infection, producing a large amount of FIV, indicating that the cells will be useful for propagation of T-lymphotropic FIV strains.     

A feline CD2 homologue interacts with human red blood cells

A cDNA encoding a feline homologue of CD2 (fCD2) was identified. Several amino acids (aa) important for ligand interaction, molecular folding or signal transduction, found in other mammalian CD2, were found to be highly conserved in the predicted fCD2 aa sequence. fCD2-expressing cells were able to form rosettes with human red blood cells (probably via human CD58), and the rosette formation was inhibited by an anti-fCD2 monoclonal antibody. These results are indicative of the similarity of feline and human CD2 structures. fCD2 was found to be expressed in feline peripheral blood T lymphocytes, monocytes and cultured lymphoid cells.     

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus

Summary.  We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301ddlTK- gag was confirmed to express the FIV Gag precursor protein in the cells. Accepted November 12, 1997 Received August 26, 1997     

The feline immunodeficiency virus vif gene is required for productive infection of feline peripheral blood mononuclear cells and monocyte-derived macrophages.

The role of the feline immunodeficiency virus (FIV) vif gene in establishing productive infection in feline peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) was examined in cell culture systems. A 375-bp deletion was introduced into the vif gene of the wild-type FIV-pPPR infectious molecular clone to produce Vif deletion mutant FIV-pPPRDeltavif. This mutant FIV proviral construct expressed FIV proteins p24gag and gp100env in transfected Crandell feline kidney cells as measured by immunoprecipitation and Western blot analysis as well as immunocytochemical analysis; these cultures produced very low levels of virus by cocultivation of transfected cells with PBMCs and K-258 cells, as measured by production of p24gag. Replication kinetics of wild-type and vif-deleted virus were compared in PBMCs and monocyte-derived macrophages (MDMs) by infection with cell-free virus preparations. Similar to findings with other lentiviruses, the vif gene was found to be essential for establishment of productive FIV infection in both PBMCs and MDMs. This study indicates that vif is essential for productive FIV infection of host target cells in vitro and that FIV-pPPRDeltavif may be an excellent candidate viral mutant for attenuated virus vaccine studies.     

Sequence analysis of the CXCR4 gene derived from cells surviving feline lentivirus infection

The feline 3201-S cell line was established from cells that survived productive infection with feline immunodeficiency virus (FIV). We have recently shown that 3201-S cells are free of FIV DNA and are refractory to FIV reinfection. In addition, while the cells express CXCR4, a co-receptor for FIV infection, they are unresponsive to the CXCR4 ligand. In the present study, we show that 3201-S cells encode distinct mutations in the CXCR4 gene. It appears that 3201 cells are heterogeneous, consisting of phenotypically diverse mixed populations resulting from genetic mutations, suggesting that this defect can render the CXCR4 receptor expressed in 3201-S cells biologically dysfunctional.     

Cats are protected against feline immunodeficiency virus infection following vaccination with a homologous AP-1 binding site-deleted mutant

Summary.  Following establishment, via the vaginal route, of infection with an AP-1 binding-site deleted mutant (ΔAP-1) of feline immunodeficiency virus (FIV), cats were challenged with a homologous intact strain (TM2) of FIV. The cats were observed for 23 weeks to evaluate the efficacy of the ΔAP-1 against the homologous TM2 strain challenge. These two viruses were differentiated by Southern blotting after amplification of proviral DNA by semi-nested polymerase chain reaction in DNAs of peripheral blood mononuclear cells and tissues. A TM2-specific band was detected in one cat exposed to but not infected with ΔAP-1, but not in two ΔAP-1-infected. These results indicate that ΔAP-1 could protect against subsequent challenge with homologous FIV TM2 strain. Received December 23, 1998 Accepted March 31, 1998     

Establishment of a feline T-lymphoblastoid cell line highly sensitive for replication of feline immunodeficiency virus

Summary Interleukin-2 dependent feline T-lymphoblastoid cells designated as MYA-1 cells were established. The cells were free from exogenous retroviruses and sensitive for replication of feline immunodeficiency virus (FIV). FIV can grow more efficiently in MYA-1 cells than in feline primary peripheral blood mononuclear cells. This line of cells will be useful not only for isolation and propagation of FIV, but also for further investigation of properties of FIV.     

Sequences within the feline immunodeficiency virus long terminal repeat that regulate gene expression and respond to activation by feline herpesvirus type 1.

We constructed a series of deletion mutants of feline immunodeficiency virus (FIV) long terminal repeat (LTR) to identify the regions that regulate gene expression and are responsive to trans-activation of FIV LTR by feline herpes-virus type 1 (FHV-1). We demonstrated that sequences between -124 and -79, and between -21 and -32 (relative to the cap site) are essential for gene expression of FIV in Felis catus whole fetus 4 (fcwf-4) cells. Further, we demonstrated that the sequence between -63 and -23 responds to trans-activation of FIV LTR by FHV-1 in fcwf-4 cells.     

Evaluation of feline immunodeficiency virus and feline leukemia virus transmembrane peptides for serological diagnosis.

The general model for retrovirus transmembrane (TM) proteins proposed by Gallaher et al. (W. R. Gallaher, J. M. Ball, R. F. Garry, M. C. Griffin, and R. C. Montelaro, AIDS Res. Hum. Retroviruses 5:431-440, 1989) suggests that all retrovirus TM proteins may contain an immunodominant domain (Imd-TM peptide) located at the apex of the TM polypeptide. Although this Imd-TM peptide has been shown to be immunodominant in a variety of lentivirus infections, there has not been a detailed serological analysis of an oncovirus Imd-TM peptide as a diagnostic agent. We describe here an analysis of the antigenic properties and diagnostic potentials of the predicted Imd-TM peptides of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in serological assays of sera from infected cats. The results of these studies demonstrate that antibodies specific to the FIV Imd-TM peptide are detected within 2 weeks postinfection, are maintained at high levels for extended periods, and are not detectable in uninfected or FeLV-infected cats. In marked contrast, the FeLV Imd-TM peptide displayed only negligible levels of serological reactivity in FeLV-infected cats. These studies indicate that the peptide is a useful reagent for the detection of antibodies to FIV.     

Molecular characterization of feline immunodeficiency virus genome obtained directly from organs of a naturally infected cat with marked neurological symptoms and encephalitis

Summary Feline immunodeficiency virus (FIV) was first isolated from cats with immunodeficiency syndrome. Recently, neurological abnormalities and brain lesions were shown in cats infected with FIV. To investigate the FIV genome associated with central nervous system (CNS) lesions, proviral DNA sequences from the V3–V6 region of the FIVenv gene were directly amplified from uncultured necropsy tissues of a 2-year-old naturally FIV-infected cat with marked neurological symptoms and encephalitis. By in situ hybridization, FIV RNA was detected mainly in the astrocytes. Fifteen clones isolated from cerebrum, bone marrow and lymph node samples showed only a small number of mutations or deletions in this region. A representative clone, JN-BR1, was distantly related to the previous Japanese strain (TM2) belonging to the subtype B. However, it was relatively close to the Petaluma strain which is known to infect feline brain-derived culture cells and induce brain lesions in inoculated cats. By phylogenetic analysis, the JN-BR1 strain was placed in subtype A that included Petaluma strain and several other American and European strains. The JN-BR1 strain derived from brain with encephalitis in this study and the Petaluma strain may share a common genetic structure that is related to their neuropathogenicity.     

Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus

Summary.  We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats. Received January 14, 2000 Accepted August 4, 2000     

Feline peripheral blood mononuclear cells express message for both CXC and CC type chemokine receptors

Summary.  The feline immunodeficiency virus (FIV) causes a disease in cats similar in clinical presentation and disease progression to that of HIV and AIDS. It is now known that, for HIV infection, as well as primary binding of virus to the CD4 receptor, entry and infection of cells requires coreceptors which are members of the chemokine group of G-protein coupled receptors. Because of the similarity of HIV and FIV, we hypothesised that coreceptors are required for the entry and infection of cells by FIV. Using a feline cDNA library derived from a feline IL-2 sensitive lymphocyte cell line, we identified the presence of message for both CC and CXC chemokine receptors. The feline CXCR4 has been shown to facilitate fusion by FIV [44] and we suggest that the feline CCR5 receptor mediates infection of feline cells by M-tropic strains of FIV. Accepted September 7, 1998 Received November 12, 1998     

Mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion

Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU(1) failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation.     

Expression of CXCR4 in the brain of feline immunodeficiency virus infected cat

Advances in understanding the mechanisms of human immunodeficiency virus (HIV)-1 entry have revealed that the cell surface CD4 expression alone is insufficient and needs an additional molecule on its surface for the viral entry. These are G-protein coupled seven transmembrane (7-TM) family molecules (chemokine receptor) and amongst them one is CXCR4. Feline homologue of CXCR4 acting as a co-receptor for feline immunodeficiency virus (FIV) entry is already reported for the Crandle feline kidney cells strain (CrFK) of FIV. An experiment was carried out to search the expression of CXCR4 retrospectively in FIV (CrFK) infected cat brain tissues using immunohistochemically in the formalin fixed paraffin sections against 12G5, a mouse monoclonal antibody to CXCR4. We observed the expression of this receptor in feline neurons, astrocytes and in some vascular endothelial cells. The study of expression of CXCR4 in the brain, which is one of the many chemokine receptors in the central nervous system, may provide further insight into the interactions between brain cells, pathogens, and the immune system, and help understand the pathogenesis of HIV dementia.