Effect of clothianidin exposure at the no-observed-adverse-effect level (NOAEL) in a mouse model of atopic dermatitis.

The effects of exposure to clothianidin (CLO), a neonicotinoid pesticide (NN), on the thymus and intestinal microbiota were recently revealed. Immune cells express nicotinic acetylcholine receptors (nAChRs), an NN target, suggesting CLO may disrupt the immune system. However, the relationship between CLO and atopic dermatitis (AD) is unknown. We administered a no-adverse-effect-level (NOAEL) dose of CLO to male NC/Nga mice with induced AD and measured, at three time points, key AD symptom indicators: epidermal thickening, mast cell number, total plasma IgE, and histamine levels. CLO increased total plasma IgE levels but reduced epidermal thickening, mast cell number, and plasma histamine levels in the early stages of AD. This demonstrates for the first time that CLO exposure inhibits AD's early symptoms.

Next-generation effects of fetal and lactational exposure to the neonicotinoid pesticide clothianidin on the immune system and gut microbiota.

Recently, the effects of exposure to clothianidin (CLO) on the thymus and gut microbiota have become clear, but no report has examined its next-generation impacts. Pregnant C57BL/6N mice were administered a no-observed-adverse-effect-level dose of CLO until weaning. We examined CLO's effects on the gut microbiota and immune organs of dams and their 3- and 10-week-old male offspring. CLO administration led to several alterations of the top 30 bacterial genera in the gut microbiota in dams and 3-week-old mice. Compared to controls, 10-week-old mice had more thymic Hassall's corpuscles, and both dams and 10-week-old mice had fewer macrophages. These results suggest that fetal and lactational CLO exposure may affect the immune system and gut microbiota of the next generation.

Differential Localization of Structural and Non-Structural Proteins during the Bluetongue Virus Replication Cycle.

Members of the Reoviridae family assemble virus factories within the cytoplasm of infected cells to replicate and assemble virus particles. Bluetongue virus (BTV) forms virus inclusion bodies (VIBs) that are aggregates of viral RNA, certain viral proteins, and host factors, and have been shown to be sites of the initial assembly of transcriptionally active virus-like particles. This study sought to characterize the formation, composition, and ultrastructure of VIBs, particularly in relation to virus replication. In this study we have utilized various microscopic techniques, including structured illumination microscopy, and virological assays to show for the first time that the outer capsid protein VP5, which is essential for virus maturation, is also associated with VIBs. The addition of VP5 to assembled virus cores exiting VIBs is required to arrest transcriptionally active core particles, facilitating virus maturation. Furthermore, we observed a time-dependent association of the glycosylated non-structural protein 3 (NS3) with VIBs, and report on the importance of the two polybasic motifs within NS3 that facilitate virus trafficking and egress from infected cells at the plasma membrane. Thus, the presence of VP5 and the dynamic nature of NS3 association with VIBs that we report here provide novel insight into these previously less well-characterized processes.

Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity.

Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

Increased expression of prion protein gene is accompanied by demethylation of CpG sites in a mouse embryonal carcinoma cell line, P19C6.

Elucidation of the processes regulating the prion protein gene (Prnp) is an important key to understanding the development of prion disorders. In this study, we explored the involvement of DNA methylation in Prnp transcriptional regulation during neuronal differentiation of embryonic carcinoma P19C6 cells. When P19C6 cells were differentiated into neuronal cells, the expression of Prnp was markedly increased, while CpG methylation was significantly demethylated at the nucleotide region between -599 and -238 from the transcription start site. In addition, when P19C6 cells were applied in a DNA methyltransferase inhibitor, RG108, Prnp transcripts were also significantly increased in relation to the decreased methylation statuses. These findings helped to elucidate the DNA methylation-mediated regulation of Prnp expression during neuronal differentiation.

CpG site DNA methylation patterns reveal a novel regulatory element in the mouse prion protein gene.

The cellular isoform of the prion protein (PrPC) plays critical roles in the development of prion disorders. Although PrP mRNA is ubiquitously present in a tissue-specific manner, the DNA methylation of PrP gene (Prnp) is still unknown. In this study, we demonstrated that the CpG island (CGI, positioned at -218 to +152 bp from the transcriptional start site) including the Prnp core promoter region was completely unmethylated in all tested tissues. On the other hand, CpG methylation in the CGI shore region (positioned at -599 to -238 bp) occurred in various tissue- and site-specific proportions. Interestingly, the correlation analysis between CpG methylation status and PrP mRNA levels showed that one CpG site methylation at -576 was negatively correlated with the PrP mRNA level (Pearson's r = -0.374, P=0.035). Taken together, our results suggest that Prnp is a typical housekeeping gene and various methylation frequencies of the CGI shore region are likely to affect Prnp expression in a tissue-specific manner.

Ontogenic and morphological study of gonadal formation in genetically-modified sex reversal XY(POS) mice.

Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sry) in the "bipotential" gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XY(POS)) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XY(POS) mice, which were generated by backcrossing of B6J-XY(POS) on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XY(POS) mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XY(POS) gonads occurred 2-3 tail somites later than those in B6N-XY(B6) gonads, but earlier than those in B6J-XY(POS), respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XY(POS). Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XY(POS) testes and a male-related gene in the XY(POS) ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development.

Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV.

Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

The combined effect of clothianidin and environmental stress on the behavioral and reproductive function in male mice.

Neonicotinoids, some of the most widely used pesticides in the world, act as agonists to the nicotinic acetylcholine receptors (nAChRs) of insects, resulting in death from abnormal excitability. Neonicotinoids unexpectedly became a major topic as a compelling cause of honeybee colony collapse disorder, which is damaging crop production that requires pollination worldwide. Mammal nAChRs appear to have a certain affinity for neonicotinoids with lower levels than those of insects; there is thus rising concern about unpredictable adverse effects of neonicotinoids on vertebrates. We hypothesized that the effects of neonicotinoids would be enhanced under a chronic stressed condition, which is known to alter the expression of targets of neonicotinoids, i.e., neuronal nAChRs. We performed immunohistochemical and behavioral analyses in male mice actively administered a neonicotinoid, clothianidin (CTD; 0, 10, 50 and 250 mg/kg/day), for 4 weeks under an unpredictable chronic stress procedure. Vacuolated seminiferous epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 were observed in the testes of the CTD+stress mice. In an open field test, although the locomotor activities were not affected, the anxiety-like behaviors of the mice were elevated by both CTD and stress. The present study demonstrates that the behavioral and reproductive effects of CTD become more serious in combination with environmental stress, which may reflect our actual situation of multiple exposure.

Structure based modification of Bluetongue virus helicase protein VP6 to produce a viable VP6-truncated BTV.

Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34-130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication.

[Reverse genetics systems for orbiviruses reveal the essential mechanisms in their replication].

The members of Orbivirus genus within the family Reoviridae cause severe arthropod-born diseases mainly in ruminants and equids. In addition, the orbiviruses, which can infect humans, have been reported. In the last decade, the molecular and structural studies for orbiviruses, including Bluetongue virus (BTV), has made a great progress. Especially, a reverse genetics system (RG) for BTV, developed soon after Orhoreovirus and Rotavirus, is a major breakthrough. Here, I introduced the recent findings in orbivirus replication, especially the function of an enzymatic protein, VP6.

Minimum requirements for bluetongue virus primary replication in vivo.

The replication mechanism of bluetongue virus (BTV) has been studied by an in vivo reverse genetics (RG) system identifying the importance of certain BTV proteins for primary replication of the virus. However, a unique in vitro cell-free virus assembly system was subsequently developed, showing that it did not require the same set of viral components, which is indicative of differences in these two systems. Here, we studied the in vivo primary replicase complex more in-depth to determine the minimum components of the complex. We showed that while NS2 is an essential component of the primary replication stage during BTV infection, NS1 is not an essential component but may play a role in enhancing BTV protein synthesis. Furthermore, we demonstrated that VP7, a major structural protein of the inner core, is not required for primary replication but appears to stabilize the replicase complex. In contrast, VP3, the other major structural core protein, is an essential component of the complex, together with the three minor enzymatic proteins (VP1, VP4, and VP6) of the core. In addition, our data have demonstrated that the smallest minor protein, VP6, which is known to possess an RNA-dependent helicase activity, may also act as an RNA translocator during assembly of the primary replicase complex.

Bluetongue virus VP1 polymerase activity in vitro: template dependency, dinucleotide priming and cap dependency.

BACKGROUND: Bluetongue virus (BTV) protein, VP1, is known to possess an intrinsic polymerase function, unlike rotavirus VP1, which requires the capsid protein VP2 for its catalytic activity. However, compared with the polymerases of other members of the Reoviridae family, BTV VP1 has not been characterized in detail. METHODS AND FINDINGS: Using an in vitro polymerase assay system, we demonstrated that BTV VP1 could synthesize the ten dsRNAs simultaneously from BTV core-derived ssRNA templates in a single in vitro reaction as well as genomic dsRNA segments from rotavirus core-derived ssRNA templates that possess no sequence similarity with BTV. In contrast, dsRNAs were not synthesized from non-viral ssRNA templates by VP1, unless they were fused with specific BTV sequences. Further, we showed that synthesis of dsRNAs from capped ssRNA templates was significantly higher than that from uncapped ssRNA templates and the addition of dinucleotides enhanced activity as long as the last base of the dinucleotide complemented the 3' -terminal nucleotide of the ssRNA template. CONCLUSIONS: We showed that the polymerase activity was stimulated by two different factors: cap structure, likely due to allosteric effect, and dinucleotides due to priming. Our results also suggested the possible presence of cis-acting elements shared by ssRNAs in the members of family Reoviridae.

Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge.

The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.

Decreased intake of sucrose solutions in orexin knockout mice.

Orexins are synthesized by lateral hypothalamic neurons and are suggested to be implicated in feeding behavior. Recent studies have shown that intracerebroventricular administration of orexin-A increases intake of sweet-tasting solution. Effects of suppressing the orexin system on consumption of sweet-tasting solution and sensory processing with sweet taste inputs, however, have yet to be examined. We examined the effects of orexin deficiency on sucrose solution intake, locomotor activity, and preference for sweet solution using male orexin knockout (OxKO) and littermate wild-type (WT) mice. In the dark and over 24-h periods, OxKO mice showed significantly less sucrose intake and lower locomotor activity than WT mice without alteration in food intake whereas preferences for 100 mM sucrose were not different between the genotypes. Moreover, sucrose intake of OxKO mice was significantly less than sucrose intake of a subgroup of WT mice with similar locomotor activity compared to that of OxKO mice. These results suggest that factors other than the lower energy expenditure due to lower locomotor activity are likely responsible for the decreased sucrose intake of OxKO mice. Orexin deficiency may lower the satiety threshold resulting in reduced sucrose intake, without altering food intake.

A reverse genetics system of African horse sickness virus reveals existence of primary replication.

African horse sickness virus (AHSV), a member of the orbivirus genus of the family Reoviridae, is an insect-vectored pathogen of horses of concern to the equine industry. Studies on AHSV replication and pathogenesis have been hampered by the lack of reverse genetics allowing targeted mutation of viral genomes. We demonstrate that AHSV single-stranded RNA synthesized in vitro (core transcripts) is infectious and that there are distinct primary and secondary stages of the replication cycle. Transfection with a mixture of core transcripts from two different serotypes or a mixture of core transcripts and a T7 derived transcript resulted in the recovery of reassortant viruses. Recovery of infectious ASHV from nucleic acid will benefit investigation of the virus and the generation of attenuated vaccines.

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death.

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-alpha caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized.

Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation.

A minor core protein, VP6, of bluetongue virus (BTV) possesses nucleoside triphosphatase, RNA binding, and helicase activities. Although the enzymatic functions of VP6 have been documented in vitro using purified protein, its definitive role in BTV replication remains unclear. In this study, using a recently developed T7 transcript-based reverse genetics system for BTV, we examined the importance of VP6 in virus replication. We show that VP6 is active early in replication, consistent with a role as part of the transcriptase or packaging complex, and that its action can be provided in trans by a newly developed complementary cell line. Furthermore, the genomic segment encoding VP6 was mutated to reveal the cis-acting sequences required for replication or packaging, which subsequently enabled the construction of a chimeric BTV expressing enhanced green fluorescent protein. These data confirm that one of the 10 genome segments of BTV can be replaced with a chimeric RNA containing the essential packaging and replication signals of BTV and the coding sequence of a foreign gene.

Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins.

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.

Oligomerization of hepatitis C virus core protein is crucial for interaction with the cytoplasmic domain of E1 envelope protein.

Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein.