Complete cap 4 formation is not required for viability in Trypanosoma brucei

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5' ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m(7)G cap, ribose 2'-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2'-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A(2) and is not required for subsequent steps; TbMT511 methylates C(3), without which U(4) methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m(7)G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A(2), C(3), and U(4) methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.     

RING-H2 protein WSSV249 from white spot syndrome virus sequesters a shrimp ubiquitin-conjugating enzyme, PvUbc, for viral pathogenesis

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.     

Designing Programs for Eliminating Canine Rabies from Islands: Bali,Indonesia as a Case Study

Background

Canine rabies is one of the most important and feared zoonotic diseases in the world. In some regions rabies elimination is being successfully coordinated, whereas in others rabies is endemic and continues to spread to uninfected areas. As epidemics emerge, both accepted and contentious control methods are used, as questions remain over the most effective strategy to eliminate rabies. The Indonesian island of Bali was rabies-free until 2008 when an epidemic in domestic dogs began, resulting in the deaths of over 100 people. Here we analyze data from the epidemic and compare the effectiveness of control methods at eliminating rabies.

Methodology/Principal Findings

Using data from Bali, we estimated the basic reproductive number, R ₀, of rabies in dogs, to be ∼1·2, almost identical to that obtained in ten–fold less dense dog populations and suggesting rabies will not be effectively controlled by reducing dog density. We then developed a model to compare options for mass dog vaccination. Comprehensive high coverage was the single most important factor for achieving elimination, with omission of even small areas (<0.5% of the dog population) jeopardizing success. Parameterizing the model with data from the 2010 and 2011 vaccination campaigns, we show that a comprehensive high coverage campaign in 2012 would likely result in elimination, saving ∼550 human lives and ∼$15 million in prophylaxis costs over the next ten years.

Conclusions/Significance

The elimination of rabies from Bali will not be achieved through achievable reductions in dog density. To ensure elimination, concerted high coverage, repeated, mass dog vaccination campaigns are necessary and the cooperation of all regions of the island is critical. Momentum is building towards development of a strategy for the global elimination of canine rabies, and this study offers valuable new insights about the dynamics and control of this disease, with immediate practical relevance.     

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

Background

Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.

Results

We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.

Conclusions

Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.     

Newcastle Disease Virus-Vectored H7 and H5 Live Vaccines Protect Chickens from Challenge with H7N9 or H5N1 Avian Influenza Viruses

Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that Newcastle disease virus (NDV)-vectored H7 (NDV-H7) and NDV-H5 vaccines are able to induce antibodies with high hemagglutination inhibition (HI) titers and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus-expressed H7 protein failed to protect chickens from H7N9 virus infection.     

Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice

Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl₄)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl₄-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis.     

Differentiation of pathogenic and nonpathogenic isolates of <Emphasis Type="Italic">Geotrichum candidum</Emphasis> sensu Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of rDNA ITS and PCR using specific primers designed in polygalacturonase genes

Pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) that affect citrus fruit are indistinguishable morphologically. In this work, differentiation of the two pathogenicity types based on the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) and polygalacturonase (PG) genes was attempted. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of rDNA ITS and PCR using specific primers to PG genes from each type could clearly differentiate the two pathogenicity types, and the profiles by PCR-RFLP of rDNA ITS genes corresponded with those by type-specific PCR of PG genes. These results indicate that the two pathogenic types can be differentiated at a molecular level and that PG genes are alternatively useful for distinguishing between the two types.     

Histopathological and ultrastructural features of enlarged cells of humpback grouper Cromileptes altivelis challenged with Megalocytivirus (family Iridoviridae) after vaccination

The genus Megalocytivirus in the family Iridoviridae encompasses isolates of red sea bream iridovirus (RSIV) and grouper sleepy disease iridovirus (GSDIV). In the present study, humpback grouper Cromileptes altivelis juveniles were challenged with GSDIV after vaccination with a commercial RSIV vaccine. The unvaccinated group (in duplicate) showed higher mortalities (59.3 to 66.7%) than the vaccination group (0% mortalitiy, in duplicate). Surviving fish in the vaccinated group displayed masses of enlarged cells in the spleen. Electron microscopy revealed that they contained hemosiderin granules within the cytoplasm. In contrast, moribund fish from the unvaccinated group exhibited large numbers of inclusion body-bearing cells (IBCs) in the spleen, while surviving fish displayed masses of enlarged cells in which a small number of GSDIV virions were assembled.     

Convergent and divergent development among M cell lineages in mouse mucosal epithelium

M cells are specialized epithelial cells mediating immune surveillance of the mucosal lumen by transepithelial delivery of Ags to underlying dendritic cells (DC). At least three M cell phenotypes are known in the airways and intestine, but their developmental relationships are unclear. We used reporter transgenic mouse strains to follow the constitutive development of M cell subsets and their acute induction by cholera toxin (CT). M cells overlying intestinal Peyer's patches (PPs), isolated lymphoid follicles, and nasal-associated lymphoid tissue are induced by distinct settings, yet show convergent phenotypes, such as expression of a peptidoglycan recognition protein-S (PGRP-S) transgene reporter. By contrast, though PP, isolated lymphoid follicle, and villous M cells are all derived from intestinal crypt stem cells, their phenotypes were clearly distinct; for example, PP M cells frequently appeared to form M cell-DC functional units, whereas villous M cells did not consistently engage underlying DC. B lymphocytes are critical to M cell function by forming a basolateral pocket and possible signaling through CD137; however, initial commitment to all M cell lineages is B lymphocyte and CD137 independent. CT causes induction of new M cells in the airway and intestine without cell division, suggesting transdifferentiation from mature epithelial cells. In contrast with intestinal PP M cells, CT-induced nasal-associated lymphoid tissue M cells appear to be generated from ciliated Foxj1(+)PGRP-S(+) cells, indicative of a possible precommitted progenitor. In summary, constitutive and inducible differentiation of M cells is toward strictly defined context-dependent phenotypes, suggesting specialized roles in surveillance of mucosal Ags.