Survey of bovine foamy virus infection among cattle in Japan and comparison with bovine leukemia virus infection.

The prevalence of bovine foamy virus (BFV) infections in cattle on farms in the Kanto region of Japan was determined using agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR). Six out of 20 farms contained BFV-positive cattle. Furthermore, 16.7% (91/545) of all cattle tested positive for BFV. This suggested that BFV-infected cattle are widely prevalent in Japan. Positive results for BFV infection were consistent between AGID and PCR tests. Additionally, we tested for bovine leukemia virus (BLV) infections at nine farms, primarily those containing BFV-infected cows. At each farm, the infection rate of BFV was lower than that of BLV. Further, cattle that were PCR-positive but antibody-negative, indicating immune tolerance to BFV, were not detected.

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.

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.

Isolation of an Equine Foamy Virus and Sero-Epidemiology of the Viral Infection in Horses in Japan.

An equine foamy virus (EFV) was isolated for the first time in Japan from peripheral blood mononuclear cells of a broodmare that showed wobbler syndrome after surgery for intestinal volvulus and the isolate was designated as EFVeca_LM. Complete nucleotide sequences of EFVeca_LM were determined. Nucleotide sequence analysis of the long terminal repeat (LTR) region, gag, pol, env, tas, and bel2 genes revealed that EFVeca_LM and the EFV reference strain had 97.2% to 99.1% identities. For a sero-epidemiological survey, indirect immunofluorescent antibody tests were carried out using EFVeca_LM-infected cells as an antigen against 166 sera of horses in five farms collected in 2001 to 2002 and 293 sera of horses in eight farms collected in 2014 to 2016 in Hokkaido, Japan. All of the farms had EFV antibody-positive horses, and average positive rates were 24.6% in sera obtained in 2001 to 2002 and 25.6% in sera obtained in 2014 to 2016 from broodmare farms. The positive rate in a stallion farm (Farm A) in 2002 was 10.7%, and the positive rates in two stallion farms, Farms A and B, in 2015 were 40.9% and 13.3%, respectively. The results suggested that EFV infection is maintained widely in horses in Japan.

Feline Foamy Virus is Highly Prevalent in Free-Ranging Puma concolor from Colorado, Florida and Southern California.

Feline foamy virus (FFV) is a retrovirus that has been detected in multiple feline species, including domestic cats (Felis catus) and pumas (Puma concolor). FFV results in persistent infection but is generally thought to be apathogenic. Sero-prevalence in domestic cat populations has been documented in several countries, but the extent of viral infections in nondomestic felids has not been reported. In this study, we screened sera from 348 individual pumas from Colorado, Southern California and Florida for FFV exposure by assessing sero-reactivity using an FFV anti-Gag ELISA. We documented a sero-prevalence of 78.6% across all sampled subpopulations, representing 69.1% in Southern California, 77.3% in Colorado, and 83.5% in Florida. Age was a significant risk factor for FFV infection when analyzing the combined populations. This high prevalence in geographically distinct populations reveals widespread exposure of puma to FFV and suggests efficient shedding and transmission in wild populations.

Isolation of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is endemic in many countries, but has not been reported in Japan. A syncytium-forming virus was isolated from peripheral blood leukocytes of clinically healthy cattle on a farm in Kanagawa prefecture during a periodic epidemiological survey of viral diseases. The isolate was propagated in primary fetal bovine muscle cells and subsequently passaged in Madin-Darby bovine kidney cells. Since the isolate appeared to be distinct from the viruses with syncytium-forming ability previously isolated in Japan, we attempted to identify it using genomic analyses and electron microscopy. A phylogenetic analysis revealed that the isolate belongs to the bovine foamy virus cluster and is highly similar to a BFV strain isolated in China. A sero-epidemiological survey was performed using agar gel immunodiffusion test with the isolated virus as the antigen, and five of the 57 cattle tested were found to be seropositive.

Spumaretroviruses: Updated taxonomy and nomenclature.

Spumaretroviruses, commonly referred to as foamy viruses, are complex retroviruses belonging to the subfamily Spumaretrovirinae, family Retroviridae, which naturally infect a variety of animals including nonhuman primates (NHPs). Additionally, cross-species transmissions of simian foamy viruses (SFVs) to humans have occurred following exposure to tissues of infected NHPs. Recent research has led to the identification of previously unknown exogenous foamy viruses, and to the discovery of endogenous spumaretrovirus sequences in a variety of host genomes. Here, we describe an updated spumaretrovirus taxonomy that has been recently accepted by the International Committee on Taxonomy of Viruses (ICTV) Executive Committee, and describe a virus nomenclature that is generally consistent with that used for other retroviruses, such as lentiviruses and deltaretroviruses. This taxonomy can be applied to distinguish different, but closely related, primate (e.g., human, ape, simian) foamy viruses as well as those from other hosts. This proposal accounts for host-virus co-speciation and cross-species transmission.

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Evaluation of Anyplex™ II RV16 and RB5 real-time RT-PCR compared to Seeplex® RV15 OneStep ACE and PneumoBacter ACE for the simultaneous detection of upper respiratory pathogens.

This prospective study was performed to evaluate and compare the performance of the multiplex PCR Seeplex® assays and Anyplex™ II assays. From May 2014 until April 2016, a total of 247 respiratory samples were collected in Okinawa, Japan. Multiple respiratory pathogens were detected in 37% of patients with positive results. The most prevalent pathogens were influenza A virus and respiratory syncytial virus B. Despite minor differences in capabilities, both the Seeplex® assays and Anyplex™ II assays can be easily implemented in diagnostic or research laboratories to optimize the detection and management of respiratory pathogen induced diseases.

Purification of foamy viral particles.

Foamy viruses are non-pathogenic retroviruses and represent a tool for vector development. For gene therapy applications and for analyses of viral protein composition infectious particles need to be purified, which has been difficult for foamy viruses in the past. Here, we describe a novel, simple, and fast purification method for prototype foamy viruses with high purity using size exclusion and affinity chromatography. More than 99,9% of the contaminating proteins were removed. The purified viruses were used to determine the amount of the incorporated Pol protein relative to Gag. The determined Gag to Pol PR-RT ratio of 30:1 confirmed previous studies suggesting FV virions encapsidate fewer number of Pol molecules than orthoretroviruses.

Chronic progressive polyarthritis in a domestic shorthair cat.

A 6-year-old, neutered male, domestic shorthair cat was presented with shifting leg lameness and palpable effusion of the carpal and tarsal joints. Blood work, arthrocentesis, and radiographs identified an immune-mediated erosive polyarthritis. The cat was positive for feline syncytia-forming virus, and with his signalment, was diagnosed with feline chronic progressive polyarthritis.

Polyarthrite progressive chronique chez un chat commun. Un chat commun mâle stérilisé âgé de 6 ans a été présenté avec une boiterie changeante et une effusion palpable des articulations carpienne et du tarse. Une analyse sanguine, une arthrocentèse et des radiographies ont identifié une polyarthrite érosive à médiation immunitaire. Le chat s'est avéré positif pour le virus félin induisant le syncytium et, avec ce signalement, a été diagnostiqué avec la polyarthrite féline progressive chronique.(Traduit par Isabelle Vallières).

Construction of an infectious clone of simian foamy virus of Japanese macaque (SFVjm) and phylogenetic analyses of SFVjm isolates.

Foamy viruses belong to the genus Spumavirus of the family Retroviridae and have been isolated from many mammalian species. It was reported that simian foamy viruses (SFVs) have co-evolved with host species. In this study, we isolated four strains (WK1, WK2, AR1 and AR2) of SFV (named SFVjm) from Japanese macaques (Macaca fuscata) in main island Honshu of Japan. We constructed an infectious molecular clone of SFVjm strain WK1, termed pJM356. The virus derived from the clone replicated and induced syncytia in human (human embryonic kidney 293T cells), African green monkey (Vero cells) and mouse cell lines (Mus dunni tail fibroblast cells). Phylogenetic analysis also revealed that these four SFVjm strains formed two distinct SFVjm clusters. SFVjm strains WK1 and WK2 and SFV isolated from Taiwanese macaques (Macaca cyclopis) formed one cluster, whereas strains AR1 and AR2 formed the other cluster with SFV isolated from a rhesus macaque (Macaca mulatta).

Endogenous viral sequences from the Cape golden mole (Chrysochloris asiatica) reveal the presence of foamy viruses in all major placental mammal clades.

Endogenous retroviruses provide important insights into the deep history of this viral lineage. Endogenous foamy viruses are thought to be very rare and only a few cases have been identified to date. Here we report a novel endogenous foamy virus (CaEFV) within the genome of the Cape golden mole (Chrysochloris asiatica). The identification of CaEFV reveals the presence of foamy virus in the placental mammal superorder Afrotheria. Phylogenetic analyses place CaEFV basal to other foamy viruses of Eutherian origin, suggesting an ancient codivergence between foamy virus and placental mammals. These findings have implications for understanding the long-term evolution, diversity, and biology of retroviruses.

Characterization of a full-length infectious clone of bovine foamy virus 3026.

The biological features of most foamy viruses (FVs) are poorly understood, including bovine foamy virus (BFV). BFV strain 3026 (BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid (AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Horizontal acquisition and a broad biodistribution typify simian foamy virus infection in a cohort of Macaca fascicularis.

BACKGROUND: Foamy viruses are non-pathogenic in vivo and naturally infect all species of non-human primates (NHP). Simian foamy viruses (SFV) are highly prevalent in both free ranging and captive NHP but few longitudinal studies have been performed to assess the prevalence and biodistribution of SFV within captive NHP. METHOD: LTR and pol gene along with Gag antibody detection were undertaken to identify infection in a cohort of over 80 captive macaques. RESULTS: The prevalence of SFV was between 64% and 94% in different groups. Access to 23 dam-infant pairs allowed us to reveal horizontal transfer as the dominant route of SFV transmission in our cohort. Further, analysis of SFV from a range of tissues and blood revealed that macaques as young as six months old can be infected and that proviral biodistribution increases with age. CONCLUSIONS: These are the first data of this type for a captive cohort of cynomolgus macaques.

Non-simian foamy viruses: molecular virology, tropism and prevalence and zoonotic/interspecies transmission.

Within the field of retrovirus, our knowledge of foamy viruses (FV) is still limited. Their unique replication strategy and mechanism of viral persistency needs further research to gain understanding of the virus-host interactions, especially in the light of the recent findings suggesting their ancient origin and long co-evolution with their nonhuman hosts. Unquestionably, the most studied member is the primate/prototype foamy virus (PFV) which was originally isolated from a human (designated as human foamy virus, HFV), but later identified as chimpanzee origin; phylogenetic analysis clearly places it among other Old World primates. Additionally, the study of non-simian animal FVs can contribute to a deeper understanding of FV-host interactions and development of other animal models. The review aims at highlighting areas of special interest regarding the structure, biology, virus-host interactions and interspecies transmission potential of primate as well as non-primate foamy viruses for gaining new insights into FV biology.

A new indicator cell line established to monitor bovine foamy virus infection.

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Production of foamy virus vector and transduction of hematopoietic cells.

Foamy viruses (FVs), or spumaviruses, are nonpathogenic retroviruses that have been developed as integrating viral vectors. This protocol presents methods for producing high-titer FV vector stocks, free of contaminating replication-competent retrovirus, to be used for transducing hematopoietic stem cells. FV vector stocks are produced by transfecting 293 cells, harvesting and filtering the culture medium, and concentrating vector virions by ultracentrifugation. The resulting stocks are free of replication-competent helper virus, as indicated by a sensitive marker rescue assay. A typical stock made from 23 10-cm dishes has a final volume of 2 mL with a titer of 10(7) to 10(8) transducing units/mL. Potential advantages of FV vectors include a lack of pathogenicity of the wild-type virus, a wide host range, stable virions that can be concentrated by centrifugation, a double-stranded DNA genome that is reverse-transcribed in the vector-producing cells, and the largest packaging capacity of any retrovirus. FV vectors are especially useful for transducing hematopoietic cells. Because hematopoietic stem cells have the ability to self-renew, proliferate, and repopulate the bone marrow after transplantation, efficient transduction of these cells offers the promise to cure many inherited and acquired diseases.

Role of neutralizing antibodies in controlling simian foamy virus transmission and infection.

BACKGROUND: Human infections with simian foamy viruses (SFVs) have been reported after occupational and nonoccupational exposure to infected animals and their tissues, blood, and body fluids, although there is no evidence for human-to-human transmission. We previously demonstrated SFV transmission in monkeys by blood transfusion with whole blood from one donor animal that had a low neutralizing antibody (NAb) endpoint titer, whereas blood transfusion from a second donor monkey that had a high NAb titer failed to transmit virus. These results suggested a role for NAbs in SFV transmission and establishment of infection. STUDY DESIGN AND METHODS: Whole blood and antibody-reduced blood were transfused into SFV-negative rhesus macaques. SFV infection in recipient animals was monitored by detection of virus sequences using polymerase chain reaction assays with nucleotide sequence confirmation, by analysis for antibody development in Western blots, and by virus isolation in coculture assays. NAb titer was evaluated by endpoint dilution assays. RESULTS: SFV transmission by whole blood transfusion from a donor monkey with high NAb endpoint titer failed to establish infection in SFV-negative monkeys, whereas virus transmission was successful with transfer of antibody-reduced blood cells. CONCLUSIONS: Passive transfer of high-titer NAbs blocked SFV cell-associated transmission, indicating that NAbs may play a role in virus transmission to individuals exposed to SFV-infected blood and tissues.