Identification of Feline Foamy Virus-derived MicroRNAs.

MicroRNAs (miRNAs) classified as non-coding RNAs regulate various metabolic systems and viral life cycles. To date, numerous DNA viruses, many of which are members of the herpesvirus family, and a relatively small number of RNA viruses, including retroviruses, have been reported to encode and express miRNAs in infected cells. A few retroviruses have been shown to express miRNAs, and foamy viruses (FVs) were initially predicted by computational analyses to possess miRNA-coding regions. Subsequent studies on simian and bovine FVs confirmed the presence of functional and biologically active miRNA expression cassettes. We herein identified feline FV-derived miRNAs using a small RNA deep sequencing ana-lysis. We confirmed their repressive functions on gene expression by dual-luciferase reporter assays. We found that the seed sequences of the miRNAs identified in the present study were conserved among all previously reported FFV isolates. These results suggest that FFV-derived miRNAs play a pivotal role in FFV infection.

Phylogenetic analyses reveal that simian foamy virus isolated from Japanese Yakushima macaques (Macaca fuscata yakui) is distinct from most of Japanese Hondo macaques (Macaca fuscata fuscata).

Japanese macaque (Macaca fuscata) is an indigenous Old World monkey (OWM) species that inhabits the Japanese archipelago. There are two subspecies of Japanese macaque: Yakushima macaque (M. f. yakui) which inhabits Yakushima Island exclusively, and Hondo macaque (M. f. fuscata) which inhabits the mainland of Japan. Yakushima macaque is considered to be branched off from a certain parental macaque group that had inhabited the mainland of Japan. However, the process of sub-speciation of the Yakushima macaque is still unclear at present. In this study, to gain new insight into the process of sub-speciation of Japanese macaque, we utilized the simian foamy virus (SFV) as a marker. SFVs are found in virtually all primates except humans and undergo species-specific cospeciation with the hosts. The phylogenetic analysis of conserved regions of the env gene in SFVs remarkably resembled that of the OWMs with high statistical confidence. The phylogenetic analyses also indicated that there are four (1-4) genotypes among Asian OWMs investigated. SFVs derived from Asian OWMs except Yakushima macaque were classified as genotypes 1-3, whereas SFVs isolated from all Yakushima macaques and one Hondo macaque were classified as genotype 4. Interestingly, genotype 4 was firstly branched off from the rest of the genotypes, which might indicate that the macaques infected with genotype 4 SFV were derived from the "older" population of Japanese macaques. The high prevalence of genotype 4 SFVs among Yakushima macaque might reflect the possibility that they are a descendant of the population settled earlier, which has been geographically isolated in Yakushima Island.

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.

Susceptibility of domestic animals to a pseudotype virus bearing RD-114 virus envelope protein.

Retroviral vectors are used for gene transduction into cells and have been applied to gene therapy. Retroviral vectors using envelope protein (Env) of RD-114 virus, a feline endogenous retrovirus, have been used for gene transduction. In this study, we investigated the susceptibility to RD-114 Env-pseudotyped virus in twelve domestic animals including cattle, sheep, horse, pig, dog, cat, ferret, mink, rabbit, rat, mouse, and quail. Comparison of nucleotide sequences of ASCT2 (SLC1A5), a receptor of RD-114 virus, in 10 mammalian and 2 avian species revealed that insertion and deletion events at the region C of ASCT2 where RD-114 viral Env interacts occurred independently in the mouse and rat lineage and in the chicken and quail lineage. By the pseudotype virus infection assay, we found that RD-114 Env-pseudotyped virus could efficiently infect all cell lines except those from mouse and rat. Furthermore, we confirmed that bovine ASCT2 (bASCT2) functions as a receptor for RD-114 virus infection. We also investigated bASCT2 mRNA expression in cattle tissues and found that it is expressed in various tissues including lung, spleen and kidney. These results indicate that retrovirus vectors with RD-114 virus Env can be used for gene therapy in large domestic animals in addition to companion animals such as cat and dog.

Evaluation of membrane filtration system using The "Pore Diffusion" for eliminating viruses.

Here, we report a first study of virus removal by a novel membrane filtration system, named the "Pore Diffusion". The "Pore Diffusion" manipulated the direction of circulating flow from vertical to parallel to the membrane, thereby achieved to alter the trans-membrane pressure as low as possible. We compared the viral activity between before and after filtration by both infectivity assay and real-time reverse transcription-PCR. Among 4 "Pore Diffusion" modules tested, the big module with average pore size of 80 nm showed the highest log reduction value of viral activity. Our study shows the possibility of "The Pore Diffusion" to filtrate viruses from bioproducts without increasing the trans-membrane pressure, so that the filtration process can be carried out effectively and economically.