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.

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.

Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia.

A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level (BSL)-2 laboratory. The results were similar to those obtained from the VN test using live MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment as the antigen as well as the competitive ELISA performed using a monoclonal antibody against MERS-CoV. According to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. Moreover, using the present procedure, serological tests for MERS-CoV can be conducted even in BSL 2 laboratory.

Transcriptional activation of long terminal repeat of bovine leukemia virus by bovine heat shock factor 1.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL). The BLV genome encodes Tax protein, a transcriptional activator of viral gene expression that binds to the BLV long terminal repeat (LTR). Heat shock factor 1 (HSF1) is a known regulator of the heat shock response proteins, including heat shock proteins. In the present study, the BLV LTR was investigated for interaction of heat shock element (HSE) with HSF1 and the viral Tax protein. It could be confirmed that a functional HSE is well conserved in different BLV strains. The LTR transcriptional activity, as measured by luciferase reporter assay, was upregulated by bovine HSF1 - without Tax expression - in feline CC81 cells. The HSF1 activated LTR transcription by binding to the HSE. LTR-activation was lost upon HSE removal from the LTR and upon expression of a mutant HSF1 lacking the DNA-binding domain. We conclude that BLV LTR is activated to a basal level by host transcriptional factor HSF1, but without Tax protein involvement.

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.

Mutation of the S and 3c genes in genomes of feline coronaviruses.

Feline coronavirus (FCoV) is classified into two biotypes based on its pathogenicity in cats: a feline enteric coronavirus of low pathogenicity and a highly virulent feline infectious peritonitis virus. It has been suspected that FCoV alters its biotype via mutations in the viral genome. The S and 3c genes of FCoV have been considered the candidates for viral pathogenicity conversion. In the present study, FCoVs were analyzed for the frequency and location of mutations in the S and 3c genes from faecal samples of cats in an animal shelter and the faeces, effusions, and tissues of cats that were referred to veterinary hospitals. Our results indicated that approximately 95% FCoVs in faeces did not carry mutations in the two genes. However, 80% FCoVs in effusion samples exhibited mutations in the S and 3c genes with remainder displaying a mutation in the S or 3c gene. It was also suggested that mutational analysis of the 3c gene could be useful for studying the horizontal transmission of FCoVs in multi-cat environments.

Enzootic bovine leukosis in a two-month-old calf.

A two-month-old calf was diagnosed with leukosis on the basis of the clinical sign of enlarged, superficial lymph nodes. Serological and genetic tests for bovine leukemia virus (BLV) were performed because the calf was born from a cow infected with BLV. The serum had a weakly positive BLV antibody, and the BLV provirus was detected within neoplastic cells on performing polymerase chain reaction (PCR). Analysis of the BLV provirus integration site using inverse PCR revealed that the BLV integration site location was identical on all chromosomes in all tumor tissues examined. Thus, the tumor cells monoclonally proliferated following BLV infection. The present study shows that enzootic bovine leukosis can occur in a young animal, as in the two-month-old calf in our study.

Preparation of a cell line persistently infected with maedi/visna virus and production of viral antigens.

We attempted to prepare a cell line that produces maedi/visna virus (MVV) and is free of contamination by other viruses and mycoplasmas. Three cell lines, which originated from a sheep, goat and bat, were infected with MVV and passaged approximately every 5 days. The cultured cells were then subjected to polymerase chain reaction analysis for MVV provirus. As a result, a cell line persistently infected with MVV was established from ZZ-R cells, which originated from the fetal goat tongue. The 50-fold concentrated culture fluid formed a precipitation line against reference antiserum.

Serological survey of viral diseases relating to reproductive failure among Artiodactyla in Ethiopian Camelus dromedarius.

Serological surveys were performed on Ethiopian camels with a history of abortion to investigate the presence of antibodies against viruses that infect animals classified in the order Artiodactyla. In 2013, 120 serum samples were collected from camels in various parts of Ethiopia. Several viruses related to abortion in ruminants were prevalent. In particular, antibodies against bluetongue virus, were detected at a high rate (76.7% of samples). Additionally, antibodies against Akabane virus and Japanese encephalitis virus were also detected in samples from more than 40% of the camels; however, their antibody titers were relatively low.

Survey on vertical infection of bovine viral diarrhea virus from fetal bovine sera in the field.

Bovine viral diarrhea virus (BVDV) isolation and antibody survey were performed using 2,758 fetal bovine sera (FBS) collected from slaughterhouses in New Zealand, Australia and the Dominican Republic, and then sent to Japan to manufacture commercial serum for cell culture use. FBS in the Dominican Republic were pooled for each several individuals, and those collected in other countries were separated according to each individual and subjected to the tests. BVDV was isolated from 25 (0.91%) FBS, and the BVDV antibody was detected in 44 (1.60%) FBS. The survey on 139 sets of paired sera of a dam and her fetus revealed that neither the BVDV antibody nor BVDV was detected in all FBS from BVDV antibody-positive dams.

Overexpression of feline tripartite motif-containing 25 interferes with the late stage of feline leukemia virus replication.

Tripartite motif-containing 25 (TRIM25) regulates various cellular processes through E3 ubiquitin ligase activity. Previous studies have revealed that the expression of TRIM25 is induced by type I interferon and that TRIM25 is involved in the host cellular innate immune response against retroviral infection. Although retroviral infection is prevalent in domestic cats, the roles of feline TRIM25 in the immune response against these viral infections are poorly understood. Because feline TRIM25 is expected to modulate the infection of feline leukemia virus (FeLV), we investigated its effects on early- and late-stage FeLV replication. This study revealed that ectopic expression of feline TRIM25 in HEK293T cells reduced viral protein levels leading to the inhibition of FeLV release. Our findings show that feline TRIM25 has a potent antiviral activity and implicate an antiviral mechanism whereby feline TRIM25 interferes with late-stage FeLV replication.

Isolation of maedi/visna virus from a sheep in Japan.

Maedi/visna (MV) is a lentiviral disease of sheep caused by the maedi/visna virus (MVV). Although MV is prevalent in many countries, it had not been reported in Japan. In 2011, however, three sheep in northern Japan were reported to be seropositive against the MVV antigen, indicating a persistent MVV infection. In the present study, we isolated MVV from one sheep to confirm MVV infection and conducted genomic classification of the virus. The co-culture of leukocytes from a seropositive sheep with fetal goat lung cells resulted in the formation of syncytial cells and the amplification of a long terminal repeat sequence of MVV by polymerase chain reaction. The isolate was confirmed as being MVV, rather than the caprine arthritis-encephalitis virus based on phylogenetic analysis of the gag gene sequence. Although the sheep was asymptomatic, nonpurulent meningitis and demyelination were found in the spinal cord. These were considered to be early lesions associated with pathogenic MVV infection. Therefore, the present study demonstrated that MVV is distributed in Japan.

Efficient propagation of equine viruses in a newly established equine cell line, FHK-13.1 cells.

Equine cells are required for isolation of viruses that infect the horse. However, only a few equine cell lines and cell cultures are available so far. Fetal horse kidney (FHK)-Tcl3.1 cell is a novel cell line established by introducing simian virus 40 (SV40) large T antigen. In the present study, the ability to propagate equine viruses was compared between FHK-Tcl3.1 cells and other equine cells. FHK-Tcl3.1 cells efficiently increased many viruses derived from or having pathogenicity to horses and produced high infective titers in culture fluids. These results indicate that FHK-Tcl3.1 cells would be useful for propagation and serological tests of viruses that affect Equidae.

Characterization of feline TRIM genes: molecular cloning, expression in tissues, and response to type I interferon.

Members of the tripartite motif (TRIM) protein family in mammals are responsible for various cellular processes. Previous studies have revealed that several TRIM proteins were induced by interferons (IFN) and that these proteins were involved in innate immune response against retroviral infection. Although retroviral infection is prevalent in domestic cats, the expression profiles and roles of feline TRIM genes against these viral infections are not well understood. In the present study, we examined tissue expression and IFN inducibility of nine feline TRIM genes. In addition, the complete coding sequences of six cloned TRIM genes were determined, and their structures were analyzed. Nine TRIM genes were expressed in feline tissues and five were up-regulated by type I IFN. The predicted amino acid sequence of six feline TRIM proteins showed high sequence similarities to other mammalian TRIM proteins, and suggest that feline TRIM genes are potentially involved in antiviral reactivity in IFN-mediated immune response.

Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates.

In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward primers and one reverse primer in the polymerase chain reaction enabled the amplification of all EIAV strains used in this study. However, further study is required to confirm these primers as universal for all EIAV strains. The nucleotide sequence of gag is considered highly conserved, as evidenced by the use of gag-encoded capsid proteins as a common antigen for the detection of EIAV in serological tests. However, significant sequence variation in the gag genes of different EIAV strains was found in the current study.

Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis through prostaglandin E(2) (PGE(2)) biosynthesis. It has been shown by in vitro studies that PGE(2) signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE(2)-induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE(2) pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin-binding EGF-like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE(2) pathway-dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE(2) receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE(2) in these cells, suggesting indirect mechanism possibly through PGE(2)-associated inflammatory responses. These results suggest that PGE(2) signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX-2 inhibitors. Therefore, it is possible that inhibition of both COX-2/PGE(2) and EGFR pathways represents an effective strategy for preventing gastric cancer.

Prostaglandin E2 signaling and bacterial infection recruit tumor-promoting macrophages to mouse gastric tumors.

BACKGROUND & AIMS: Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS: We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS: Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS: Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Inflammation, tumor necrosis factor and Wnt promotion in gastric cancer development.

Infection-associated chronic inflammation plays an important role in tumorigenesis, and macrophages are a key player in both inflammation and tumorigenesis. Tumor-associated macrophages accelerate tumorigenesis through the enhancement of angiogenesis, remodeling and the suppression of antitumor immunity. Helicobacter pylori infection induces inflammatory responses, which are closely associated with gastric cancer development. Recent studies using mouse models indicate that activated macrophages in the infected and inflamed gastric mucosa express TNF-alpha, which stimulates the surrounding epithelial cells to promote Wnt signaling activity. Such a promotion of Wnt signaling activity beyond the threshold for tumorigenesis may, therefore contribute to gastric cancer development. Accordingly, it is possible that the TNF-alpha-induced promotion of Wnt signaling is a novel protumorigenic mechanism of inflammation in gastric carcinogenesis.

Prostaglandin E2, Wnt, and BMP in gastric tumor mouse models.

The development of gastric cancer is closely associated with Helicobacter pylori (H. pylori) infection. The expression of cylooxigenase-2 (COX-2), a rate-limiting enzyme for prostaglandin biosynthesis, is induced in H. pylori-associated chronic gastritis, which thus results in the induction of proinflammatory prostaglandin, PGE(2). The COX-2/PGE(2) pathway plays a key role in gastric tumorigenesis. On the other hand, several oncogenic pathways have been shown to trigger gastric tumorigenesis. The activation of Wnt/beta-catenin signaling is found in 30-50% of gastric cancers, thus suggesting that Wnt signaling plays a causal role in gastric cancer development. Mutations in the bone morphogenetic protein (BMP) signaling pathway are responsible for the subset of juvenile polyposis syndrome (JPS) that develops hamartomas in the gastrointestinal tract. BMP suppression appears to contribute to gastric cancer development because gastric cancer risk is increased in JPS. Wnt signaling is important for the maintenance of gastrointestinal stem cells, while BMP promotes epithelial cell differentiation. Accordingly, it is possible that both Wnt activation and BMP suppression can cause gastric tumorigenesis through enhancement of the undifferentiated status of epithelial cells. Recent mouse model studies have indicated that induction of the PGE(2) pathway is required for the development of both gastric adenocarcinoma and hamartoma in the Wnt-activated and BMP-suppressed gastric mucosa, respectively. This article reviews the involvement of the PGE(2), Wnt, and BMP pathways in the development of gastric cancer, and gastric phenotypes that are found in transgenic mouse models of PGE(2) induction, Wnt activation, BMP suppression, or a combination of these pathways.

Induction and down-regulation of Sox17 and its possible roles during the course of gastrointestinal tumorigenesis.

BACKGROUND & AIMS: The activation of Wnt/beta-catenin signaling causes the development of gastric and colon cancers. Sox17 represses Wnt/beta-catenin signaling and is down-regulated in colon cancer. This study was designed to elucidate the role of Sox17 during the course of gastrointestinal tumorigenesis. METHODS: Sox17 expression was examined in gastrointestinal tumors of mouse models and humans. The roles of Sox17 in gastric tumorigenesis were examined by cell culture experiments and by construction of Sox17 transgenic mice. RESULTS: Sox17 was induced in K19-Wnt1/C2mE mouse gastric tumors and K19-Wnt1 preneoplastic lesions, where Wnt/beta-catenin signaling was activated. Consistently, Wnt activation induced Sox17 expression in gastric cancer cells. In contrast, Sox17 was rarely detected by immunohistochemistry in gastric and colon cancers, whereas strong nuclear staining of Sox17 was found in >70% of benign gastric and intestinal tumors. Treatment with a demethylating agent induced Sox17 expression in gastric cancer cells, thus indicating the down-regulation of Sox17 by methylation. Moreover, transfection of Sox17 in gastric cancer cells suppressed both the Wnt activity and colony formation efficiency. Finally, transgenic expression of Sox17 suppressed dysplastic tumor development in K19-Wnt1/C2mE mouse stomach. CONCLUSIONS: Sox17 plays a tumor suppressor role through suppression of Wnt signaling. However, Sox17 is induced by Wnt activation in the early stage of gastrointestinal tumorigenesis, and Sox17 is down-regulated by methylation during malignant progression. It is therefore conceivable that Sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis, and down-regulation of Sox17 contributes to malignant progression through promotion of Wnt activity.