Antiviral activity of bovine BST-2s against VSV.

<p>Expression plasmid for bBST-2A1, bBST-2A2, bBST-2B or hBST-2 was transfected into MDCK cells by electroporation. (A) Cells were dissolved at 16 h after transfection and then subjected to 12.5% SDS-PAGE. Intracellular expression of BST-2s was confirmed by Western blotting using anti-FLAG antibody. (B) At 24 h after transfection, cells were infected with VSV at MOI of 0.001. At 12 h after infection, culture supernatants were collected and virus production was determined by plaque assay. The experiments were conducted in triplicate, and the data are shown as the means ± standard deviation.</p

Subcellular localization of bovine BST-2s.

<p>FLAG-tagged bBST-2s expressed in MDBK cells (left two lines) and HeLa cells (right two lines) were stained with FITC-conjugated anti-FLAG antibody (showed green) and then observed by fluorescence microscopy. Nuclei stained with DAPI are shown in blue the merged panels.</p

Multiple alignment of bBST-2A1, bBST-2A2 and the corresponding regions in the bovine genome.

<p>The coloured boxes of bBST-2A1 and bBST-2A2 indicate nucleotide differences. The coloured boxes in the genomic sequences (GenBank, GCA_000003055.3) show the exon structures. The numbers at the top or bottom of the alignment indicate the relative positions or the absolute genomic positions on chromosome 7, respectively.</p

Posttranslational modifications for bovine BST-2s.

<p>(A) Expression plasmid for FLAG-tagged bBST-2A1, bBST-2A2 or bBST-2B was transfected into HEK 293T cells. Cells were dissolved 24 h after transfection and subjected to 12.5% SDS-PAGE. Western blotting was performed with anti-FLAG or anti-Actin antibody. (B) Bovine BST-2s expressed in HEK 293T cells were treated with (+) or without (−)PNGase F for de-<i>N</i>-glycosylated reaction, and then subjected to 12.5% SDS-PAGE. Reaction conditions with PNGase F were as described in the Materials and Methods. BST-2 was detected with anti-FLAG antibody by Western blotting analysis.</p

Co-localization of bovine BST-2 to the late-endosome (LE)/trans-Golgi network (TGN) compartments.

<p>FLAG-tagged bBST-2s expressed in HeLa cells were stained with FITC-conjugated anti-FLAG antibody (left panels) and then observed by confocal laser microscopy. LE or TGN was stained with anti-CD63 antibody (A) or anti-TGN46 antibody (B), respectively. Merged images of bBST-2s (green), organelle marker (red), and nuclei (blue) are shown in the right panels.</p

Antiviral activity of bovine BST-2s against BLV.

<p>(A) Standard curve of BLV-specific SYBR Green real-time RT-PCR used for the quantification of viral production was generated from the threshold cycle obtained against 10 fold serial dilutions of the plasmid as described in Materials and Methods. The assay was linear over a 7 log range from 2.4×10²–2.4×10⁹ copies. The coefficient of determination (r²) of the standard curve was 0.991. (B) Expression plasmid for bBST-2A1, bBST-2A2, bBST-2B or hBST-2 was transfected into FLK-BLV cells by electroporation. FLK-BLV cells expressing BST-2s were incubated for 24 h or 48 h. Culture supernatants were collected and virus production was determined by quantitative real-time RT-PCR. BLV productions were shown as copy numbers of viral RNA in culture supernatants (copies/ml). The experiments were conducted in triplicate, and the data are shown as the means ± standard deviation. (C) Cells were dissolved at 16 h after transfection and then subjected to 12.5% SDS-PAGE. Intracellular expression of BST-2s was confirmed by Western blotting using anti-FLAG antibody.</p

Seconde nozze e identit&#224; materna nella Firenze del tardo Medioevo,

<div><p>Feline leukemia virus (FeLV) belongs to the genus <i>Gammaretrovirus</i>, and causes a variety of neoplastic and non-neoplastic diseases in cats. Alteration of viral <i>env</i> sequences is thought to be associated with disease specificity, but the way in which genetic diversity of FeLV contributes to the generation of such variants in nature is poorly understood. We isolated FeLV <i>env</i> genes from naturally infected cats in Japan and analyzed the evolutionary dynamics of these genes. Phylogenetic reconstructions separated our FeLV samples into three distinct genetic clusters, termed Genotypes I, II, and III. Genotype I is a major genetic cluster and can be further classified into Clades 1–7 in Japan. Genotypes were correlated with geographical distribution; Genotypes I and II were distributed within Japan, whilst FeLV samples from outside Japan belonged to Genotype III. These results may be due to geographical isolation of FeLVs in Japan. The observed structural diversity of the FeLV <i>env</i> gene appears to be caused primarily by mutation, deletion, insertion and recombination, and these variants may be generated <i>de novo</i> in individual cats. FeLV interference assay revealed that FeLV genotypes did not correlate with known FeLV receptor subgroups. We have identified the genotypes which we consider to be reliable for evaluating phylogenetic relationships of FeLV, which embrace the high structural diversity observed in our sample. Overall, these findings extend our understanding of <i>Gammaretrovirus</i> evolutionary patterns in the field, and may provide a useful basis for assessing the emergence of novel strains and understanding the molecular mechanisms of FeLV transmission in cats.</p></div

Pseudotype viruses derived from FeLV recombinant <i>env</i> genes having endogenous FeLV sequences were tested for viral interference assay.

<p>“N” indicates no recombination and “R” recombination in the indicated domain.</p

Incidence of FeLV in blood samples collected from private veterinary hospitals located in each prefecture of Japan.

<p>The incidence of samples testing positive for the FeLV antigen was divided into six color-coded groups in increments of 5%. A two-letter code was assigned to each prefecture as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061009#pone.0061009.s005" target="_blank">Table S2</a>.</p

Viral interference assay using <i>Lac</i>Z pseudotyped viruses derived from FeLV <i>env</i> genes.

<p>Viral interference assay using <i>Lac</i>Z pseudotyped viruses derived from FeLV <i>env</i> genes.</p