Hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. Despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. Documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) sampled from bats and shrews in China, and which are distinct from other known hantaviruses. Huangpi virus was found in Pipistrellus abramus, Lianghe virus in Anourosorex squamipes, Longquan virus in Rhinolophus affinis, Rhinolophus sinicus, and Rhinolophus monoceros, and Yakeshi virus in Sorex isodon, respectively. A phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. Notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. Our phylogenetic analysis also suggests that hantaviruses might have first appeared in Chiroptera (bats) or Soricomorpha (moles and shrews), before emerging in rodent species. Overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses.

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F 1 . Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. R ECENTLY, a simple and efficient genome editing technology, type II CRISPR/Cas9, has been developed based on the Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas9) adaptive immune system. It requires three components for effective DNA cleavage: the nuclease Cas9, a targeting CRISPR RNA (crRNA), and an additional transactivating crRNA (tracrRNA) (Gasiunas et al. 2012;Jinek et al. 2012;Cho et al. 2013;Cong et al. 2013;Hwang et al. 2013;Mali et al. 2013). Further improvement of the system was achieved by fusing the crRNA and tracrRNA to form a single guide RNA (gRNA) that is sufficient to direct Cas9-mediated target cleavage (Hwang et al. 2013). Importantly, previous studies performed in vitro (Jinek et al. 2012), in bacteria , and in human cells (Cong et al. 2013) have shown that Cas9-mediated cleavage can be abolished by single mismatch at the gRNA-target site interface, particularly in the last 10-12 nucleotides located in the 39 end of the 20-nt gRNA targeting region. Compared to the other two engineered nuclease genome-editing technologies, zinc-finger nucleases (ZFNs) (Urnov et al. 2005;Doyon et al. 2008) and transcription activator-like effector nucleases (TALENs) (Huang et al. 2011;Sander et al. 2011;Tesson et al. 2011), the CRISPR/Cas9 system is substantially less expensive and much easier to program for editing new target sites. This new approach has been widely used for genome engineering in model animals, including Caenorhabditis elegans (Dickinson et al. 2013;Friedland et al. 2013;Tzur et al. 2013), Drosophila (Bassett et al. 2013;Ren et al. 2013;Yu et al. 2013), zebrafish (Chang et al. 2013Hrusc...

Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17β and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.

Aims/hypothesis MicroRNAs (miRs) have been suggested as potential therapeutic targets for heart diseases. Inhibition of miR-195 prevents apoptosis in cardiomyocytes stimulated with palmitate and transgenic overexpression of miR-195 induces cardiac hypertrophy and heart failure. We investigated whether silencing of miR-195 reduces diabetic cardiomyopathy in a mouse model of streptozotocin (STZ)-induced type 1 diabetes. Methods Type 1 diabetes was induced in C57BL/6 mice (male, 2 months old) by injections of STZ. Results MiR-195 expression was increased and levels of its target proteins (B cell leukaemia/lymphoma 2 and sirtuin 1) were decreased in STZ-induced type 1 and db/db type 2 diabetic mouse hearts. Systemically delivering an anti-miR-195 construct knocked down miR-195 expression in the heart, reduced caspase-3 activity, decreased oxidative stress, attenuated myocardial hypertrophy and improved myocardial function in STZ-induced mice with a concurrent upregulation of B cell leukaemia/lymphoma 2 and sirtuin 1. Diabetes reduced myocardial capillary density and decreased maximal coronary blood flow in mice. Knockdown of miR-195 increased myocardial capillary density and improved maximal coronary blood flow in diabetic mice. Upregulation of miR-195 sufficiently induced apoptosis in cardiomyocytes and attenuated the angiogenesis of cardiac endothelial cells in vitro. Furthermore, inhibition of miR-195 prevented apoptosis in cardiac endothelial cells in response to NEFA, an important feature of diabetes. Conclusions/interpretation Therapeutic silencing of miR-195 reduces myocardial hypertrophy and improves coronary blood flow and myocardial function in diabetes, at least in part by reducing oxidative damage, inhibiting apoptosis and promoting angiogenesis. Thus, miR-195 may represent an alternative therapeutic target for diabetic heart diseases.

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine (PEI)2 are efficient vehicles to transduce DNA into cells and organisms. DNA/PEI nanoparticles (DNPs) also elicit rapid and systemic release of pro-inflammatory cytokines that promote anti-tumor immunity. Here we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid up-regulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells (DCs) and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFNαβ) and II (IFNγ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, while IFNγ receptor signaling was not essential for this response. Moreover, systemic IFNγ release was caused by TLR9-dependent activation of Natural Killer cells, while TLR9 signaling was not required for IFNαβ release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFNαβ production, induced IDO and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFNγ. DNP treatment to induce IDO and activate Tregs blocked antigen-specific T cell responses elicited in vivo following immunization, and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyper-immune syndromes.

Supplementary data are available at Bioinformatics online.

No abstract

It is well accepted that Forkhead box protein L2 (Foxl2) and aromatase (Cyp19a1; the enzyme responsible for estrogen synthesis) are critical for ovarian development in vertebrates. Knockouts of Foxl2 and Cyp19a1 in goat, mouse, and zebrafish have revealed similar but not identical functions across species. Functional analyses of these two genes in other animals are needed to elucidate their conserved roles in vertebrate sexual development. In this study, we established foxl2 and cyp19a1a mutant lines in Nile tilapia. Both foxl2-/- and cyp19a1a-/- XX fish displayed female-to-male sex reversal. Sf1, Dmrt1, and Gsdf were upregulated in the foxl2-/- and the cyp19a1a-/- XX gonads. Downregulation of Cyp19a1a and serum estradiol-17β level, and upregulation of Cyp11b2 and serum 11-ketotestosterone level were observed in foxl2-/- XX fish. The mutant phenotype of foxl2-/- XX individuals could be rescued by 17β-estradiol treatment from 5 to 30 days after hatching (dah). Upregulation of Star1, the enzyme involved in androgen production in tilapia, was also observed in the foxl2-/- XX gonad at 30 and 90 dah. In vitro promoter analyses consistently demonstrated that Foxl2 could suppress the transcription of star1 in a dose-dependent manner. In addition, compared with the control XX gonad, fewer germ cells were detected in the foxl2-/- XX, cyp19a1a-/- XX, and control XY gonads 10 dah. These results demonstrate that Foxl2 promotes ovarian development by upregulating Cyp19a1a expression and repressing male pathway gene expression. These results extend the study of Foxl2 and Cyp19a1a loss of function to a commercially important fish species.