Molecular Characterization of Human Pathogenic Bunyaviruses of the Nyando and Bwamba/Pongola Virus Groups Leads to the Genetic Identification of Mojuí dos Campos and Kaeng Khoi Virus

<div><p>Background</p><p>Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease.</p><p>Methodology/Principal Findings</p><p>In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types.</p><p>Conclusions/Significance</p><p>On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated.</p></div

Phylogenetic relationships among BWAV/PGAV and NDV/MDCV/KKV group viruses.

<p>Maximum likelihood trees were constructed based on the nucleotide sequences of the S segment, M segment and L segment, as indicated. Bootstrap values based on 1,000 replicates are also indicated. Viruses lineages added based on sequences determined as a part of this study are indicated in color: Bwamba virus (orange), Pongola virus (red), Nyando virus (blue), Mojuí dos Campos virus (purple), Kaeng Khoi virus (pink).</p

Comparison of virus growth in various cell lines.

<p>Cell lines derived from bats (i.e. Tb 1 Lu and RE05), mosquito (i.e. C6/36) or non-human primates (i.e. VeroE6) were infected with the indicated viruses at a multiplicity of infection of 0.1. Supernatants were harvested either immediately after infection (0 h) or after 72 h incubation and titres were determined using plaque assay.</p

Comparison of virus genome structures.

<p>The genomes of the various virus groups sequenced in this study are shown to scale. The non-coding regions are shown in grey while the major open reading frames encoded by each segment [S segment: N (ORF), M segment: GPC (ORF) and L segment: LORF)] are shown in colored boxes. The NSs protein, which is produced from a downstream ATG in the S segment, is shown in a lighter shade of the corresponding color for each virus. The total genome length of each segment is indicated at right.</p

Virus strains used in this study.

<p>Virus strains used in this study.</p

Geographic distribution of viruses used in this study.

<p>Distribution of (A) Bwamba virus, (B) Pongola virus, (C) Nyando virus, (D) Mojuí dos Campos virus, and (E) Kaeng Khoi virus. Countries in which these viruses have been isolated (dark colors) and/or where specific antibodies to these viruses have been detected (light colors) are shown in orange (BWAV), red (PGAV), blue (NDV), purple (MDCV), and pink (KKV), respectively. The geographic location from which the virus strains used in the present study were isolated, are indicated with black dots. The strain names for these isolates are also indicated.</p

NASA’s in-situ materials challenge: team ISU final report

Transforming in situ materials such as regolith or basalt into useful structural elements is a significant way to reduce the mass of materials launched as payload from Earth. Considering exploration on Mars, for every kilogram of native materials used, one saves 11 kg of transportation propellant and spacecraft mass required to launch to Low Earth Orbit (LEO). Given the cost for LEO is US$10,000/kg, one avoids at least US$110,000/kg of cost by using 1 kg of in situ materials, making space pioneering on Mars more affordable and feasible. One could use surface-based materials such as regolith or basalt to produce structural elements that can be interconnected to create launch/landing pads; blast protection berms; roads and walkways; radiation, thermal, and micro-meteorite shielding insulation and structures; equipment shelters; pressure vessels for fluids storage; ablative atmospheric entry heat shields; construction foundations; and other useful structures

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment

<div><p>Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/β receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNAR^fl/flxFoxp3^YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNAR^fl/flxFoxp3^YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8⁺ T cells and tumor infiltrating lymphocytes from IFNAR^fl/flxFoxp3^YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8⁺ T cells were decreased in IFNAR^fl/flxFoxp3^YFP-Cre mice and the effector CD4⁺ and CD8⁺ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNAR^fl/flxFoxp3^YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNAR^fl/flxFoxp3^YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.</p></div

Absence of IFNAR signaling in Tregs results in decreased virus-specific CD8⁺ T cells and cytokine production.

<p>(<b>A</b> and <b>B</b>) Spleen cells of chronic LCMV infected (days 25 and 46) IFNAR^fl/fl and IFNAR^fl/fl x Foxp3^YFP-Cre mice were analyzed for GP33 Tet⁺ and NP396 Tet⁺ T cells within CD8⁺CD44⁺ T cells. (<b>C</b> and <b>D</b>) Spleen cells from (days 25 and 46) Cl-13 infected mice were stimulated with GP33, NP396, and Golgi stop for 5 hours at 37 ^oC. Frequencies and absolute numbers of IFN-γ⁺ and TNF-α⁺ cytokine producing cells within CD8⁺CD44⁺GP33 Tet⁺ and CD8⁺CD44⁺NP396 Tet⁺ T cells are shown. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, and *** <i>P</i> < 0.001 (unpaired two-tailed Student’s <i>t</i>-test). Data shown (<b>A-D</b>) from a representative and two experiments involving three to eight mice per group (Mean±SEM).</p

Transcriptome analysis of Foxp3⁺ Tregs from LCMV infected Treg-specific IFNAR-deficient mice.

<p>(<b>A</b>) PCA was performed on day 5 LCMV Armstrong infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice sorted CD4⁺YFP⁺ Treg cells RNA-seq samples (4 samples in each group). (<b>B</b>) Scatter plot showing the comparison of global gene expression profiles of Tregs between Armstrong infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice. Total of 586 genes were significantly differentially expressed and colored, 249 genes (red) were down regulated, and 337 genes (blue) upregulated in IFNAR^fl/fl x Foxp3^YFP-Cre mice (fold change 1.5 and above, adjusted <i>P</i> < 0.05). (<b>C</b>) GSEA for non-IFN related genes, showing enrichment plot for natural Treg vs. T conv DN gene set (36 out 42 genes were enriched in core, Enrichment score: 0.566, <i>P</i> < 0.01, FDR: 0.0) with positive enrichment in IFNAR^fl/fl x Foxp3^YFP-Cre mice Tregs relative to Foxp3^YFP-Cre mice Tregs. (<b>D</b>) Heat map showing the significant differential expression of 32 Treg-signature genes (fold change 1.5 and above, adjusted <i>P</i> < 0.05), as previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006985#ppat.1006985.ref049" target="_blank">49</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006985#ppat.1006985.ref050" target="_blank">50</a>], differentially expressed genes were normalized by z-score. (<b>E</b>) PCA was performed on day 25 LCMV Cl-13 infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice sorted CD4⁺YFP⁺ Treg cells RNA-seq samples (5 samples in each group). (<b>F</b>) Scatter plot showing the comparison of global gene expression profiles of Tregs between Cl-13 infected Foxp3^YFP-Cre and IFNAR^fl/fl x Foxp3^YFP-Cre mice. Total of 36 genes were significantly differentially expressed and colored, 23 genes (red) were down regulated, and 13 genes (blue) upregulated in IFNAR^fl/fl x Foxp3^YFP-Cre mice (fold change 1.5 and above, adjusted <i>P</i> < 0.05). (<b>G</b>) Heat map showing the significant differential expression of 22 non-IFN related genes (fold change 1.5 and above, adjusted <i>P</i> < 0.05, normalized by z-score). (<b>H</b>) Heat map showing the significant differential expression of 14 genes from LCMV Armstrong infected Foxp3^YFP-Cre mice and IFNAR^fl/fl x Foxp3^YFP-Cre mice Tregs, which were in consistent with transcriptome from LCMV Cl-13 infected mice Tregs (fold change 1.5 and above, adjusted <i>P</i> < 0.05, normalized by z-score). Armstrong infection transcriptome data obtained from an experiment involving four mice per group (<b>A-D</b> and <b>H</b>), and transcriptome data from Cl-13 infection, involved an experiment with five mice per group (<b>E-G</b>).</p