DNA strand breaks and gaps target retroviral intasome binding and integration.

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.

PREB inhibits the replication of prototype foamy virus by affecting its transcription.

BACKGROUND: Foamy viruses (FVs) are unique nonpathogenic retroviruses, which remain latent in the host for a long time. Therefore, they may be safe, effective gene transfer vectors. In this study, were assessed FV-host cell interactions and the molecular mechanisms underlying FV latent infection. METHODS: We used the prototype FV (PFV) to infect HT1080 cells and a PFV indicator cell line (PFVL) to measure virus titers. After 48 h of infection, the culture supernatant (i.e., cell-free PFV particles) and transfected cells (i.e., cell-associated PFV particles) were harvested and incubated with PFVL. After another 48 h, the luciferase activity was used to measure virus titers. RESULTS: Through transcriptomics sequencing, we found that PREB mRNA expression was significantly upregulated. Moreover, PREB overexpression reduced PFV replication, whereas endogenous PREB knockdown increased PFV replication. PREB interacted with the Tas DNA-binding and transcriptional activation domains and interfered with its binding to the PFV long terminal repeat and internal promoter, preventing the recruitment of transcription factors and thereby inhibiting the transactivation function of Tas. PREB C-terminal 329-418 aa played a major role in inhibiting PFV replication; PREB also inhibited bovine FV replication. Therefore, PREB has a broad-spectrum inhibitory effect on FV replication. CONCLUSIONS: Our results demonstrated that PREB inhibits PFV replication by impeding its transcription.

An Outbred Calf Model for Determining Innate Immune Sensing and Evolutionary Trajectories of a Cell Culture-Adapted Bovine Foamy Virus Variant.

Bovine foamy virus (BFVbta) displays a very high degree of cell-associated replication which is unprecedented even among the other known foamy viruses. Interestingly, recent studies have shown that it can in fact adapt in vitro to high-titer (HT) cell-free transmission due to genetic changes acquired during repeated rounds of cell-free BFVbta passages in immortalized bovine MDBK cells. Molecular clones obtained from the HT BFVbta Riems cell-free variant (HT BFVbta Riems) have been thoroughly characterized in MDBK cell cultures However, during recent years, it has become increasingly clear that the source of the host cells used for virus growth and functional studies of virus replication and virus-cell interactions plays a paramount role. Established cell lines, mostly derived from tumors, but occasionally experimentally immortalized and transformed, frequently display aberrant features relating, for example. to growth, metabolism, and genetics. Even state-of-the-art organoid cultures of primary cells cannot replicate the conditions in an authentic host, especially those concerning cell diversity and the role of innate and adaptive immunity. Therefore, to determine the overall replication characteristics of the cloned wt and HT BFVbta Riems variant, we conducted a small-scale animal pilot study. The replication of the original wt BFVbta Riems isolate, as well as that of its HT variant, were analyzed. Both BFVbta variants established infection in calves, with proviruses in peripheral blood mononuclear cells and induced Gag-specific antibodies. In addition, a related pattern in the host innate immune reaction was detected in the peripheral blood leukocytes of the BFV-infected calves. Surprisingly, an analysis of the Gag sequence two weeks post-inoculation revealed that the HT BFVbta variant showed a very high level of genetic reversion to the wild type (parental BFVbta genotype).

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the ß2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Occurrence of Equine Foamy Virus Infection in Horses from Poland.

Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.

Characterization of Bovine Foamy Virus Gag Late Assembly Domain Motifs and Their Role in Recruiting ESCRT for Budding.

A large number of retroviruses, such as human immunodeficiency virus (HIV) and prototype foamy virus (PFV), recruit the endosomal sorting complex required for transport (ESCRT) through the late domain (L domain) on the Gag structural protein for virus budding. However, little is known about the molecular mechanism of bovine foamy virus (BFV) budding. In the present study, we report that BFV recruits ESCRT for budding through the L domain of Gag. Specifically, knockdown of VPS4 (encoding vacuolar protein sorting 4), ALIX (encoding ALG-2-interacting protein X), and TSG101 (encoding tumor susceptibility 101) indicated that BFV uses ESCRT for budding. Mutational analysis of BFV Gag (BGag) showed that, in contrast to the classical L domain motifs, BGag contains two motifs, P56LPI and Y103GPL, with L domain functions. In addition, the two L domains are necessary for the cytoplasmic localization of BGag, which is important for effective budding. Furthermore, we demonstrated that the functional site of Alix is V498 in the V domain and the functional site of Tsg101 is N69 in the UBC-like domain for BFV budding. Taken together, these results demonstrate that BFV recruits ESCRT for budding through the PLPI and YGPL L domain motifs in BGag.

Evaluation of the stability and intratumoral delivery of foreign transgenes encoded by an oncolytic Foamy Virus vector.

Foamy Viruses are cell cycle-dependent retroviruses capable of persisting unintegrated in quiescent cells until cell division occurs. This unique ability allows them to target slowly dividing human tumor cells which remains an unmet need in oncolytic virotherapy. We have previously reported the generation of oncolytic Foamy Virus (oFV) vector system and demonstrated its superiority over oncolytic Murine Leukemia Virus vectors in infecting slowly dividing cancer cells. In the present study we evaluated (i) the ability of oFV to carry foreign transgenes and (ii) the genetic stability of these vectors upon serial passage. The thymidine kinase (TK) and inducible caspase 9 (iCasp9) cDNAs could be detected in the oFV backbone for up to 3 in vitro passages. In vivo, GFP-, TK- and iCasp9- carrying oFV vectors propagated efficiently in subcutaneous xenograft glioblastoma tumors and drove transgene expression for up to 66 days. However, in vivo oFV vector spread eventually resulted in complete loss of the iCasp9 cDNA, minor loss of the TK cDNA and negligible loss of the GFP. Our results suggest that oFV is a promising gene delivery platform and that transgenes smaller than 1 kb might be most suitable for oFV arming.

Molecular Epidemiology and Whole-Genome Analysis of Bovine Foamy Virus in Japan.

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan.

Foamy Viruses, Bet, and APOBEC3 Restriction.

Non-human primates (NHP) are an important source of viruses that can spillover to humans and, after adaptation, spread through the host population. Whereas HIV-1 and HTLV-1 emerged as retroviral pathogens in humans, a unique class of retroviruses called foamy viruses (FV) with zoonotic potential are occasionally detected in bushmeat hunters or zookeepers. Various FVs are endemic in numerous mammalian natural hosts, such as primates, felines, bovines, and equines, and other animals, but not in humans. They are apathogenic, and significant differences exist between the viral life cycles of FV and other retroviruses. Importantly, FVs replicate in the presence of many well-defined retroviral restriction factors such as TRIM5α, BST2 (Tetherin), MX2, and APOBEC3 (A3). While the interaction of A3s with HIV-1 is well studied, the escape mechanisms of FVs from restriction by A3 is much less explored. Here we review the current knowledge of FV biology, host restriction factors, and FV-host interactions with an emphasis on the consequences of FV regulatory protein Bet binding to A3s and outline crucial open questions for future studies.

Genomic characterization and distribution of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.

Genome Analysis and Replication Studies of the African Green Monkey Simian Foamy Virus Serotype 3 Strain FV2014.

African green monkey (AGM) spumaretroviruses have been less well-studied than other simian foamy viruses (SFVs). We report the biological and genomic characterization of SFVcae_FV2014, which was the first foamy virus isolated from an African green monkey (AGM) and was found to be serotype 3. Infectivity studies in various cell lines from different species (mouse, dog, rhesus monkey, AGM, and human) indicated that like other SFVs, SFVcae_FV2014 had broad species and cell tropism, and in vitro cell culture infection resulted in cytopathic effect (CPE). In Mus dunni (a wild mouse fibroblast cell line), MDCK (Madin-Darby canine kidney cell line), FRhK-4 (a fetal rhesus kidney cell line), and MRC-5 (a human fetal lung cell line), SFVcae_FV2014 infection was productive resulting in CPE, and had delayed or similar replication kinetics compared with SFVmcy_FV21 and SFVmcy_FV34[RF], which are two Taiwanese macaque isolates, designated as serotypes 1 and 2, respectively. However, in Vero (AGM kidney cell line) and A549 (a human lung carcinoma cell line), the replication kinetics of SFVcae_FV2014 and the SFVmcy viruses were discordant: In Vero, SFVcae_FV2014 showed rapid replication kinetics and extensive CPE, and a persistent infection was seen in A549, with delayed, low CPE, which did not progress even upon extended culture (day 55). Nucleotide sequence analysis of the assembled SFVcae_FV2014 genome, obtained by high-throughput sequencing, indicated an overall 80-90% nucleotide sequence identity with SFVcae_LK3, the only available full-length genome sequence of an AGM SFV, and was distinct phylogenetically from other AGM spumaretroviruses, corroborating previous results based on analysis of partial env sequences. Our study confirmed that SFVcae_FV2014 and SFVcae_LK3 are genetically distinct AGM foamy virus (FV) isolates. Furthermore, comparative infectivity studies of SFVcae_FV2014 and SFVmcy isolates showed that although SFVs have a wide host range and cell tropism, regulation of virus replication is complex and depends on the virus strain and cell-specific factors.

FV Vectors as Alternative Gene Vehicles for Gene Transfer in HSCs.

Hematopoietic Stem Cells (HSCs) are a unique population of cells, capable of reconstituting the blood system of an organism through orchestrated self-renewal and differentiation. They play a pivotal role in stem cell therapies, both autologous and allogeneic. In the field of gene and cell therapy, HSCs, genetically modified or otherwise, are used to alleviate or correct a genetic defect. In this concise review, we discuss the use of SFVpsc_huHSRV.13, formerly known as Prototype Foamy Viral (PFV or FV) vectors, as vehicles for gene delivery in HSCs. We present the properties of the FV vectors that make them ideal for HSC delivery vehicles, we review their record in HSC gene marking studies and their potential as therapeutic vectors for monogenic disorders in preclinical animal models. FVs are a safe and efficient tool for delivering genes in HSCs compared to other retroviral gene delivery systems. Novel technological advancements in their production and purification in closed systems, have allowed their production under cGMP compliant conditions. It may only be a matter of time before they find their way into the clinic.

Persistent Infection of Simian Foamy Virus Derived from the Japanese Macaque Leads to the High-Level Expression of microRNA that Resembles the miR-1 microRNA Precursor Family.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that suppress the expression of target mRNAs. The seed sequence of miRNA plays a crucial role in recognizing the 3'-untranslated region of the target mRNA. Cells infected with a simian foamy virus (SFV) isolated from an African green monkey (Chlorocebus aethiops) (SFVcae) showed high expression levels of viral miRNAs encoded in the long terminal repeat of SFVcae. In the present study, we investigated the roles and expression of miRNAs derived from an SFV isolated from a Japanese macaque (Macaca fuscata) (SFVmfu) using next-generation sequencing technologies. The results obtained showed that SFVmfu also expressed viral miRNAs; however, the seed sequences of most miRNAs derived from SFVmfu differed from those reported previously from SFVcae. Cells persistently infected with SFVmfu strongly expressed an miRNA with the same seed sequence as the miR-1 microRNA precursor family. Luciferase reporter assays indicated that this miRNA down-regulates the expression of adenylyl cyclase-associated protein 1, which is up-regulated in several solid tumors. The present results suggest that SFVmfu utilizes viral miRNAs to establish long-term co-existence with the Japanese macaque.

Infection with Foamy Virus in Wild Ruminants-Evidence for a New Virus Reservoir?

Foamy viruses (FVs) are widely distributed and infect many animal species including non-human primates, horses, cattle, and cats. Several reports also suggest that other species can be FV hosts. Since most of such studies involved livestock or companion animals, we aimed to test blood samples from wild ruminants for the presence of FV-specific antibodies and, subsequently, genetic material. Out of 269 serum samples tested by ELISA with the bovine foamy virus (BFV) Gag and Bet antigens, 23 sera showed increased reactivity to at least one of them. High reactive sera represented 30% of bison samples and 7.5% of deer specimens. Eleven of the ELISA-positives were also strongly positive in immunoblot analyses. The peripheral blood DNA of seroreactive animals was tested by semi-nested PCR. The specific 275 bp fragment of the pol gene was amplified only in one sample collected from a red deer and the analysis of its sequence showed the highest homology for European BFV isolates. Such results may suggest the existence of a new FV reservoir in bison as well as in deer populations. Whether the origin of such infections stems from a new FV or is the result of BFV inter-species transmission remains to be clarified.

Restoration of Functional Full-Length Dystrophin After Intramuscular Transplantation of Foamy Virus-Transduced Myoblasts.

Stem cell therapy is a promising strategy to treat muscle diseases such as Duchenne muscular dystrophy (DMD). To avoid immune rejection of donor cells or donor-derived muscle, autologous cells, which have been genetically modified to express dystrophin, are preferable to cells derived from healthy donors. Restoration of full-length dystrophin (FL-dys) using viral vectors is extremely challenging, due to the limited packaging capacity of the vectors, but we have recently shown that either a foamy viral or lentiviral vector is able to package FL-dys open-reading frame and transduce myoblasts derived from a DMD patient. Differentiated myotubes derived from these transduced cells produced FL-dys. Here, we transplanted the foamy viral dystrophin-corrected DMD myoblasts intramuscularly into mdx nude mice, and showed that the transduced cells contributed to muscle regeneration, expressing FL-dys in nearly all the muscle fibers of donor origin. Furthermore, we showed that the restored FL-dys recruited members of the dystrophin-associated protein complex and neuronal nitric oxide synthase within donor-derived muscle fibers, evidence that the restored dystrophin protein is functional. Dystrophin-expressing donor-derived muscle fibers expressed lower levels of utrophin than host muscle fibers, providing additional evidence of functional improvement of donor-derived myofibers. This is the first in vivo evidence that foamy virus vector-transduced DMD myoblasts can contribute to muscle regeneration and mediate functional dystrophin restoration following their intramuscular transplantation, representing a promising therapeutic strategy for individual small muscles in DMD.

In-Vivo Gene Therapy with Foamy Virus Vectors.

Foamy viruses (FVs) are nonpathogenic retroviruses that infect various animals including bovines, felines, nonhuman primates (NHPs), and can be transmitted to humans through zoonotic infection. Due to their non-pathogenic nature, broad tissue tropism and relatively safe integration profile, FVs have been engineered as novel vectors (foamy virus vector, FVV) for stable gene transfer into different cells and tissues. FVVs have emerged as an alternative platform to contemporary viral vectors (e.g., adeno associated and lentiviral vectors) for experimental and therapeutic gene therapy of a variety of monogenetic diseases. Some of the important features of FVVs include the ability to efficiently transduce hematopoietic stem and progenitor cells (HSPCs) from humans, NHPs, canines and rodents. We have successfully used FVV for proof of concept studies to demonstrate safety and efficacy following in-vivo delivery in large animal models. In this review, we will comprehensively discuss FVV based in-vivo gene therapy approaches established in the X-linked severe combined immunodeficiency (SCID-X1) canine model.

Bovine Foamy Virus: Shared and Unique Molecular Features In Vitro and In Vivo.

The retroviral subfamily of Spumaretrovirinae consists of five genera of foamy (spuma) viruses (FVs) that are endemic in some mammalian hosts [1]. Closely related species may be susceptible to the same or highly related FVs. FVs are not known to induce overt disease and thus do not pose medical problems to humans and livestock or companion animals. A robust lab animal model is not available or is a lab animal a natural host of a FV. Due to this, research is limited and often focused on the simian FVs with their well-established zoonotic potential. The authors of this review and their groups have conducted several studies on bovine FV (BFV) in the past with the intention of (i) exploring the risk of zoonotic infection via beef and raw cattle products, (ii) studying a co-factorial role of BFV in different cattle diseases with unclear etiology, (iii) exploring unique features of FV molecular biology and replication strategies in non-simian FVs, and (iv) conducting animal studies and functional virology in BFV-infected calves as a model for corresponding studies in primates or small lab animals. These studies gained new insights into FV-host interactions, mechanisms of gene expression, and transcriptional regulation, including miRNA biology, host-directed restriction of FV replication, spread and distribution in the infected animal, and at the population level. The current review attempts to summarize these findings in BFV and tries to connect them to findings from other FVs.

The free energy landscape of retroviral integration.

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

First report on the prevalence and genetic relatedness of Feline Foamy Virus (FFV) from Turkish domestic cats.

Feline Foamy Virus (FFV) is an important retroviral agent affecting domestic cats in Turkey that has been studied less intensively than Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV). Accordingly, we aimed to investigate the presence and prevalence of FFV among domestic cats by molecular techniques. PCR was used to amplify the gag-pol gene overlap in order to detect the presence of FFV. The gene encoding bet, an important accessory gene, was also characterized. Molecular characteristics were analyzed and phylogenetic trees were constructed. We determined the positivity rate as 10% in all samples (20/200) based on the gag-pol test. The phylogenetic analysis indicated that the Turkish FFV sequences form a separate cluster among other isolates in the constructed maximum likelihood (ML) tree. bet-based products were obtained for two samples (1%; 2/200) that were also positive for gag-pol. These bet gene sequences confirm the presence of a separate cluster for the Turkish FFV isolates. The results suggest that FFV is prevalent and widespread in Turkish domestic cats. Additionally, these new FFV sequences represent the first FFV sequences from Turkey to be submitted to GenBank. This study paves the way for studies on the pathogenicity of FFV.