Epitope Mapping of the Antibody Response Against the Envelope Proteins of the Feline Foamy Virus.

Foamy viruses (FV) are retroviruses that infect several species without pathological signs, but induce substantial antibody responses in the infected host. In the case of feline FV (FFV), antibodies against Gag, Bet, and Env have been used to indicate infection; however, it is unclear whether the response to specific epitopes correlates with immunity. Here, we investigated the epitope specificity of antibodies targeting the Env protein using peptide microarrays. Sera from naturally and experimentally FFV-infected cats and pumas and from rats immunized with FFV Env expression plasmids were analyzed. An immunodominant epitope was identified in the Env leader protein (Elp), and a strong reactivity to two epitope clusters in the transmembrane (TM) subunit of Env was observed. Moreover, a short stretch of residues in the C-terminal part of the surface (SU) protein was found to be significantly associated with FFV serotype FUV-mediated neutralization. Taken together, our results add a new level of detail on the B cell epitope repertoire induced during FFV infection. Furthermore, our results provide a basis for current attempts to modify FV vectors to express and present vaccine epitopes for the directed induction of humoral immunity.

Feline foamy virus-based vectors: advantages of an authentic animal model.

New-generation retroviral vectors have potential applications in vaccination and gene therapy. Foamy viruses are particularly interesting as vectors, because they are not associated to any disease. Vector research is mainly based on primate foamy viruses (PFV), but cats are an alternative animal model, due to their smaller size and the existence of a cognate feline foamy virus (FFV). The potential of replication-competent (RC) FFV vectors for vaccination and replication-deficient (RD) FFV-based vectors for gene delivery purposes has been studied over the past years. In this review, the key achievements and functional evaluation of the existing vectors from in vitro cell culture systems to out-bred cats will be described. The data presented here demonstrate the broad application spectrum of FFV-based vectors, especially in pathogen-specific prophylactic and therapeutic vaccination using RD vectors in cats and in classical gene delivery. In the cat-based system, FFV-based vectors provide an advantageous platform to evaluate and optimize the applicability, efficacy and safety of foamy virus (FV) vectors, especially the understudied aspect of FV cell and organ tropism.

Complete Genome Sequences of Two Novel Puma concolor Foamy Viruses from California.

We report two complete foamy retrovirus (FV) genomes isolated from Puma concolor, a large cat native to the Americas. Due to high overall genetic relatedness to known feline foamy viruses (FFVs), we propose the name Puma concolor FFV (FFVPc). The data confirm that felines are infected with distinct but closely related FVs.

Expression of multiple tfb genes in different Halobacterium salinarum strains and interaction of TFB with transcriptional activator GvpE.

Halobacterium salinarum NRC-1 contains multiple TBP and TFB proteins required for the recruitment of RNA polymerase for transcription initiation. The presence and the expression of genes encoding TFB were investigated in the two Hbt. salinarum strains NRC-1 and PHH1 and the mutant strain PHH4. The plasmid-encoded tfbC and tfbE genes of NRC-1 were lacking in PHH1 and PHH4. The 5'-end of the tfbF transcript was determined and contained a 5'-untranslated region of 39 nucleotides able to form a stem-loop structure. The expression of these tfb genes was studied in cultures growing at 15, 37°C and under heat shock conditions. Cold temperatures reduced growth and except for tfbF also the amounts of all tfb transcripts. However, the formation of gas vesicles increased in PHH1 and NRC-1. Heat shock reduced growth of PHH1 and NRC-1, but PHH4 was not affected. A 100-fold increase in tfbA and tfbB mRNA was observed in PHH1 and PHH4, whereas NRC-1 reduced the amounts of these transcripts and increased the expression of tfbG. All TFB proteins tested were able to interact with the transcription activator GvpE involved in gas vesicle formation that thus is able to recruit TFB to the gvp promoter.

Pattern of seroreactivity against feline foamy virus proteins in domestic cats from Germany.

The prevalence of feline foamy virus (FFV, spumaretrovirinae) in naturally infected domestic cats ranges between 30 and 80% FFV positive animals depending on age, sex and geographical region analyzed. Two serotypes have been reported for FFV designated FUV7-like and F17/951-like. Serotype-specific neutralization has been shown to correlate with sequence divergence in the surface (SU) domain of the envelope protein (Env). We analyzed a serum collection of 262 domestic cat sera from Germany using a GST-capture ELISA setup screening for Gag and Bet specific antibodies and identified 39% FFV positive animals. Due to the heterogeneity of the serological samples, cut-offs for Gag and Bet reactivity had to be experimentally determined since application of calculated cut-off values yielded some false-positive results; the new cut-off values turned out to be also fully applicable to a previous study. Using the already established FUV7 ElpSU antigen and the newly cloned and produced F17/951 ElpSU antigen, both consisting of the corresponding ectodomains of the envelope leader protein (Elp) and SU protein, we aimed at the detection of Env-specific antibodies and discrimination between the two known FFV serotypes within the diagnostic FFV ELISA. We validated the ElpSU antigens using cat reference sera of known serotype and screened with this assay domestic cat sera from Germany. Use of the FUV7- and F17/951 ElpSU antigens in ELISA resulted in the detection of Env-specific antibodies in both cat reference sera and sera from domestic cats in Germany, but failed to allow serotyping at the same time.

Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening.

The transmembrane envelope (TM) proteins of retroviruses are used as antigen in diagnostic immunoassays and they represent a conserved target for neutralizing antibodies. To analyze the situation in infections with the feline foamy virus (FFV), its recombinant TM protein was produced and used for ELISA and Western blot analyses. Screening sera from 404 German cats showed that 39% reacted against the TM protein, the same infection rate was determined using the Gag protein. Epitope mapping showed antibodies against the membrane proximal external region (MPER) of the TM protein in the sera from infected cats, but attempts to induce neutralizing antibodies by immunization with the recombinant TM protein failed. This is the first report demonstrating that the TM protein of the FFV is highly immunogenic and valuable for serological screening. Similar to HIV-1, but in contrast to different gammaretroviruses, immunization with the TM protein of FFV did not induce neutralizing antibodies.

Variations in the multiple tbp genes in different Halobacterium salinarum strains and their expression during growth.

The presence and expression of the multiple tbp genes encoding TATA-box binding proteins (TBPs) was investigated in various strains and mutants of the archaeon Halobacterium salinarum. Six genes, tbpA through tbpF, are present in the genome of Hbt. salinarum NRC-1 and also in the gas vesicle negative mutant strain R1. The only tbp gene located in the chromosome is tbpE, whereas all others are found in the plasmid DNA. Due to the dynamic nature of the plasmids in the Halobacterium strains, the copy numbers of the alternative tbp genes vary significantly. Five tbp genes (tbpA through tbpE) were present in the wild-type strain Hbt. salinarum PHH1. The tbpC gene of Hbt. salinarum PHH1 carried an ISH27-2 insertion element at the start of the reading frame that prevented the expression. All other tbp genes of PHH1 were expressed under aerobic and anaerobic growth conditions and quantitative RT-PCR yielded tbpE as dominant tbp transcript during the exponential growth phase. The plasmid deletion variant Hbt. salinarum PHH4 lacked all of the tbp genes except for tbpE and showed an altered growth behaviour compared to PHH1 wild-type in the stationary growth phase under anaerobic growth conditions.