Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease.

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.

Expression dynamics of self-renewal factors for spermatogonial stem cells in the mouse testis.

Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.

Variable region of the 3' UTR is a critical virulence factor in the Far-Eastern subtype of tick-borne encephalitis virus in a mouse model.

Tick-borne encephalitis virus (TBEV) is a major arbovirus that causes thousands of cases of severe neurological illness in humans annually. However, virulence factors and pathological mechanisms of TBEV remain largely unknown. To identify the virulence factors, we constructed chimeric viruses between two TBEV strains of the Far-Eastern subtype, Sofjin-HO (highly pathogenic) and Oshima 5-10 (low pathogenic). The replacement of the coding region for the structural and non-structural proteins from Sofjin into Oshima showed a partial increase of the viral pathogenicity in a mouse model. Oshima-based chimeric viruses with the variable region of the 3' UTR of Sofjin, which had a deletion of 207 nt, killed 100 % of mice and showed almost the same virulence as Sofjin. Replacement of the variable region of the 3' UTR from Sofjin into Oshima did not increase viral multiplication in cultured cells and a mouse model at the early phase of viral entry into the brain. At the terminal phase of viral infection in mice, the virus titre of the Oshima-based chimeric virus with the variable region of the 3' UTR of Sofjin reached a level identical to that of Sofjin and showed a similar histopathological change in the brain tissue. This is the first report to show that the variable region of the 3' UTR is a critical virulence factor in mice. These findings encourage further study to understand the mechanisms of the pathogenicity of TBEV, and to develop preventative and therapeutic strategies for tick-borne encephalitis.

Tick-borne flaviviruses alter membrane structure and replicate in dendrites of primary mouse neuronal cultures.

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, detailed mechanisms of viral replication in the brain and features of viral pathogenesis remain poorly understood. We carried out a comparative analysis of replication of neurotropic flaviviruses: West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus (TBEV), in primary cultures of mouse brain neurons. All the flaviviruses multiplied well in primary neuronal cultures from the hippocampus, cerebral cortex and cerebellum. The distribution of viral-specific antigen in the neurons varied: TBEV infection induced accumulation of viral antigen in the neuronal dendrites to a greater extent than infection with other viruses. Viral structural proteins, non-structural proteins and dsRNA were detected in regions in which viral antigens accumulated in dendrites after TBEV replication. Replication of a TBEV replicon after infection with virus-like particles of TBEV also induced antigen accumulation, indicating that accumulated viral antigen was the result of viral RNA replication. Furthermore, electron microscopy confirmed that TBEV replication induced characteristic ultrastructural membrane alterations in the neurites: newly formed laminal membrane structures containing virion-like structures. This is the first report describing viral replication in and ultrastructural alterations of neuronal dendrites, which may cause neuronal dysfunction. These findings encourage further work aimed at understanding the molecular mechanisms of viral replication in the brain and the pathogenicity of neurotropic flaviviruses.

N-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells.

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

Susceptibility to flavivirus-specific antiviral response of Oas1b affects the neurovirulence of the Far-Eastern subtype of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent that causes fatal encephalitis in humans. 2'-5'-oligoadenylate synthetase 1b (Oas1b) has been identified as a flavivirus resistance gene, but most inbred laboratory mice do not possess a functional Oas1b gene. In this study, a congenic strain carrying a functional Oas1b gene, B6.MSM-Oas, was used to evaluate the pathogenicity of Far-Eastern TBEV. Although intracerebral infection of B6.MSM-Oas mice by Oshima 5-10 resulted in limited signs of illness, infection by Sofjin-HO resulted in death with severe neurologic signs. While Oshima 5-10 was cleared from the brain, Sofjin-HO was not cleared despite a similar level of expression of the intact Oas1b gene. Necrotic neurons with viral antigens and inflammatory reactions were observed in the brain infected with Sofjin-HO. These data indicate that the different susceptibility to the antiviral activity of Oas1b resulted in a difference in neurovirulence in the two TBEV strains.

Dioxin pollution disrupts reproduction in male Japanese field mice.

Dioxins cause various adverse effects in animals including teratogenesis, induction of drug metabolizing enzymes, tumor promotion, and endocrine disruption. Above all, endocrine disruption is known to disturb reproduction in adult animals and may, also seriously impact their offspring. However, most previous studies have quantified the species-specific accumulation of dioxins, whereas few studies have addressed the physiological impacts of dioxins on wildlife, such as reduced reproductive function. Here we clarify an effect of endocrine disruption caused by dioxins on the Japanese field mouse, Apodemus speciosus. Japanese field mice collected from various sites polluted with dioxins accumulated high concentrations of dioxins in their livers. Some dioxin congeners, especially, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, 3,3',4,4',5-pentachloro biphenyl, 1,2,3,4,6,7,8-heptachlorodibenzofuran, and octachlorodibenzo-p-dioxin, which showed high biota-soil accumulation factors, contributed to concentration of dioxins in mouse livers with an increase of accumulation of total dioxins. As for physiological effects on the Japanese field mouse, high levels of cytochrome P450 1A1 (CYP1A1) mRNA, a drug metabolizing enzyme induced by dioxins, were found in the livers of mice captured at polluted sites. Furthermore, at such sites polluted with dioxins, increased CYP1A1 expression coincided with reduced numbers of active spermatozoa in mice. Thus, disruption in gametogenesis observed in these mice suggests that dioxins not only negatively impact reproduction among Japanese field mice, but might also act as a kind of selection pressure in a chemically polluted environment.

Dieldrin uptake and translocation in plants growing in hydroponic medium.

It has been known that the Cucurbitaceae family takes up a large amount of persistent organic pollutants from soils and that the translocation of those compounds in cucurbits is higher than those in non-cucurbits. To understand the persistent organic pollutant uptake mechanisms of plant species, we compared the dieldrin absorption and transportation potentials of several plants in hydroponic medium. Sorghum (Sorghum vulgare Moench), sunflower (Helianthus annuus L.), soybean (Glycine max), komatsuna (Brassica rapa var. peruviridis), white-flowered gourd (Lagenaria siceraria var. hispida), cucumber (Cucumis sativus L.), and zucchini (Cucurbita pepo L.) were grown in a dieldrin-added hydroponic medium for 10 d, and then the amount of dieldrin in their shoots and roots was measured. All of the roots contained dieldrin, whereas only the cucurbits (white-flowered gourd, cucumber, and zucchini) contained considerable amounts of dieldrin in their shoots. The dieldrin uptake to the roots depended on the concentration of the n-hexane soluble components in the roots, regardless of whether the dieldrin in the roots was translocated to shoots or not. The dieldrin uptake from the solution to the roots was thought to be due to a passive response, such as adsorption on the roots. The translocation of dieldrin from the roots to the shoots was probably through the xylems. The amounts of dieldrin in the shoots per transpiration rates were higher for cucurbits than for non-cucurbits. It seems likely that cucurbits have uptake mechanisms for hydrophobic organic chemicals.

Diallyl Trisulfide Enhances Benzo[a]pyrene-induced CYP1A1 Expression and Metabolic Activation in Hepatic HepG2 Cells.

BACKGROUND/AIM: Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. MATERIALS AND METHODS: The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. RESULTS: DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. CONCLUSION: DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

Identification and analysis of host proteins that interact with the 3'-untranslated region of tick-borne encephalitis virus genomic RNA.

Tick-borne encephalitis virus (TBEV) causes severe neurological disease, but the pathogenetic mechanism is unclear. The conformational structure of the 3'-untranslated region (UTR) of TBEV is associated with its virulence. We tried to identify host proteins interacting with the 3'-UTR of TBEV. Cellular proteins of HEK293T cells were co-precipitated with biotinylated RNAs of the 3'-UTR of low- and high-virulence TBEV strains and subjected to mass spectrometry analysis. Fifteen host proteins were found to bind to the 3'-UTR of TBEV, four of which-cold shock domain containing-E1 (CSDE1), spermatid perinuclear RNA binding protein (STRBP), fragile X mental retardation protein (FMRP), and interleukin enhancer binding factor 3 (ILF3)-bound specifically to that of the low-virulence strain. An RNA immunoprecipitation and pull-down assay confirmed the interactions of the complete 3'-UTRs of TBEV genomic RNA with CSDE1, FMRP, and ILF3. Partial deletion of the stem loop (SL) 3 to SL 5 structure of the variable region of the 3'-UTR did not affect interactions with the host proteins, but the interactions were markedly suppressed by deletion of the complete SL 3, 4, and 5 structures, as in the high-virulence TBEV strain. Further analysis of the roles of host proteins in the neurologic pathogenicity of TBEV is warranted.

FGF2 Has Distinct Molecular Functions from GDNF in the Mouse Germline Niche.

Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche.

Development of a serodiagnostic multi-species ELISA against tick-borne encephalitis virus using subviral particles.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A wide range of animal species could be infected with TBEV in endemic areas. A serological survey of wild animals is effective in identifying TBEV-endemic areas. Safe, simple, and reliable TBEV serodiagnostic tools are needed to test animals. In this study, ELISA was developed to detect anti-TBEV specific antibodies in multi-species of animals, using recombinant subviral particles (SPs) with an affinity tag and protein A/G. A Strep-tag was fused at the N terminus of the E protein of the plasmid coding TBEV prME. The E proteins with Strep-tag were secreted as SPs, of which Strep-tag was exposed on the surface. The tagged E proteins were associated with prM. The SPs with Strep-tag were applied as the antigen of ELISA, and TBEV-specific antibodies were detected by the protein A/G. Compared to neutralization test results, the ELISA showed 96.8% sensitivity and 97.7% specificity in rodents and 95.1% sensitivity and 96.0% specificity in humans, without cross-reactivity with antibodies to Japanese encephalitis virus. These results indicate that our ELISA would be useful to detect TBE-specific antibodies in a wide range of animal species.

Virulence of tick-borne encephalitis virus is associated with intact conformational viral RNA structures in the variable region of the 3'-UTR.

Tick-borne encephalitis virus (TBEV) is maintained between ticks and mammals in nature and causes severe neurological disease in human. However, the mechanism of viral pathogenicity is unknown. Previously, we showed that the deletion in the variable region of the 3'-untranslated region (UTR) is involved in the pathogenicity of the strains from the Far-Eastern subtype of TBEV. To investigate the detailed function of the variable region, we constructed recombinant TBEV with partial deletions in the region. In a mouse model, the partial deletions drastically increased the virulence of the virus, with no effect on virus multiplication in mouse brain. Furthermore, the mutations did not affect the production of subgenomic flavivirus RNA from the 3'-UTR, and the induction of interferon (IFN) and IFN-stimulated genes. These data suggested that the conformational structure of the variable region is associated with the pathogenicity of the Far-Eastern subtype of TBEV. These findings provide a foundation for further research to identify the pathogenic mechanisms of TBEV.

Flavivirus Antagonism of Type I Interferon Signaling Reveals Prolidase as a Regulator of IFNAR1 Surface Expression.

Type I interferon (IFN-α/ß or IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to orchestrate sterile and infectious immunity. Cellular pathways that regulate IFNAR1 are often targeted by viruses to suppress the antiviral effects of IFN-I. Here we report that encephalitic flaviviruses, including tick-borne encephalitis virus and West Nile virus, antagonize IFN-I signaling by inhibiting IFNAR1 surface expression. Loss of IFNAR1 was associated with binding of the viral IFN-I antagonist, NS5, to prolidase (PEPD), a cellular dipeptidase implicated in primary immune deficiencies in humans. Prolidase was required for IFNAR1 maturation and accumulation, activation of IFNß-stimulated gene induction, and IFN-I-dependent viral control. Human fibroblasts derived from patients with genetic prolidase deficiency exhibited decreased IFNAR1 surface expression and reduced IFNß-stimulated signaling. Thus, by understanding flavivirus IFN-I antagonism, prolidase is revealed as a central regulator of IFN-I responses.

Genetic and biological characterization of tick-borne encephalitis virus isolated from wild rodents in southern Hokkaido, Japan in 2008.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A recent epizootiological survey indicated that endemic foci of TBEV have been maintained in the southern part of Hokkaido until recently. In this study, we sought to isolate TBEV from wild rodents in the area. One virus, designated Oshima 08-As, was isolated from an Apodemus speciosus captured in Hokuto in 2008. Oshima 08-As was classified as the Far Eastern subtype of TBEV and formed a cluster with the other strains isolated in Hokkaido from 1995 to 1996. Thirty-six nucleotide differences resulted in 12 amino acid changes between Oshima 08-As and Oshima 5-10 isolated in 1995. Oshima 08-As caused high mortality and morbidity in a mouse model compared with Oshima 5-10. Although similar transient viral multiplication in the spleen was observed in the mice infected with Oshima 08-As and Oshima 5-10, greater viral multiplication with an inflammatory response was noted in the brains of mice infected with Oshima 08-As than those infected with Oshima 5-10. These data indicate that a few naturally occurring mutations affect the pathogenicity of the Oshima strains endemic in the southern part of Hokkaido.

Relationship between dieldrin uptake in cucumber and solvent-extractable residue in soil.

To prevent the distribution of cucumbers with dieldrin contamination exceeding the limit set by the Japanese Food Sanitation Law, the extraction solvent for dieldrin-contaminated soil was selected prior to cultivation so that the dieldrin residue level in cucumber could be predicted. The exhaustive extraction from soil could not explain the dieldrin uptake by cucumber plants. However, significant correlation (R(2) = 0.966, P < 0.001) was observed between dieldrin concentrations in cucumber and dieldrin concentrations extracted with 50% (v/v) methanol-water solution from soils. This was a result of the phytoavailability of dieldrin to the cucumber plants. The extractability of soil dieldrin with the methanol-water solution decreased as the organic carbon content in the soils increased. This suggested that a 50% (v/v) methanol-water solution is the optimal solution for predicting dieldrin concentrations in cucumbers by soil analysis.

Temporal trends and sources of PCDD/Fs, pentachlorophenol and chlornitrofen in paddy field soils along the Yoneshiro River basin, Japan.

In order to understand the long-term behaviors of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), pentachlorophenol (PCP), and 2,4,6-trichlorophenyl-4'-nitrophenyl ether (chlornitrofen, CNP) in paddy soil, we measured their concentrations in paddy soil samples collected in 1982 and 1984 (1980s) and in 2000 and 2002 (2000s) from the Yoneshiro River basin, Japan. The concentrations of PCP and CNP decreased from the 1980s to the 2000s, whereas the concentrations of PCDD/Fs and their toxic equivalency (WHO2006-TEQ) remained. The major sources of PCDD/Fs in the paddy soil samples were attributed to impurities in PCP and CNP as a result of comparisons of homologue and congener profiles and principal component analysis. Based on the results of comparison of total input and remaining amount, it is estimated that more than 99% of PCP and CNP applied to the paddy fields had disappeared, whereas most of the applied PCDD/Fs and TEQ remained.

Mass balance and long-term fate of PCDD/Fs in a lagoon sediment and paddy soil, Niigata, Japan.

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in a sediment core and in samples of surface sediment and paddy soil collected from the Toyano lagoon and Kameda basin in Niigata, Japan, were analyzed to elucidate the temporal trends of their concentrations in the lagoon sediment and the relationship between the sediment and the paddy soil. The mass balance of these pollutants was also estimated to determine their long-term fate in surface waters. An analysis by chemical mass balance identified the agrochemicals pentachlorophenol and 2,4,6-trichlorophenyl 4-nitrophenyl ether as the major sources of PCDD/Fs. On the basis of the findings regarding the mass balance in the Kameda basin over the last 40 years, we estimate that more than half the input of PCDD/Fs to the Kameda basin has disappeared. We suggest that the PCDD/Fs that flowed out from the paddy fields have been transferred to the lower basin.