De novo assembly and transcriptome analysis of Plasmodium gallinaceum identifies the Rh5 interacting protein (ripr), and reveals a lack of EBL and RH gene family diversification

BACKGROUND: Malaria parasites that infect birds can have narrow or broad host-tropisms. These differences in host specificity make avian malaria a useful model for studying the evolution and transmission of parasite assemblages across geographic ranges. The molecular mechanisms involved in host-specificity and the biology of avian malaria parasites in general are important aspects of malaria pathogenesis that warrant further examination. Here, the transcriptome of the malaria parasite Plasmodium gallinaceum was characterized to investigate the biology and the conservation of genes across various malaria parasite species. METHODS: The P. gallinaceum transcriptome was annotated and KEGG pathway mapping was performed. The ripr gene and orthologous genes that play critical roles in the purine salvage pathway were identified and characterized using bioinformatics and phylogenetic methods. RESULTS: Analysis of the transcriptome sequence database identified essential genes of the purine salvage pathway in P. gallinaceum that shared high sequence similarity to Plasmodium falciparum when compared to other mammalian Plasmodium spp. However, based on the current sequence data, there was a lack of orthologous genes that belonged to the erythrocyte-binding-like (EBL) and reticulocyte-binding-like homologue (RH) family in P. gallinaceum. In addition, an orthologue of the Rh5 interacting protein (ripr) was identified. CONCLUSIONS: These findings suggest that the pathways involved in parasite red blood cell invasion are significantly different in avian Plasmodium parasites, but critical metabolic pathways are conserved throughout divergent Plasmodium taxa

Rhoptry proteins ROP5 and ROP18 are major murine virulence factors in genetically divergent South American strains of Toxoplasma gondii

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection

A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion

ABSTRACT The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties. </jats:p

HIF1α is required for NK cell metabolic adaptation during virus infection

Natural killer (NK) cells are essential for early protection against virus infection and must metabolically adapt to the energy demands of activation. Here, we found upregulation of the metabolic adaptor hypoxia-inducible factor-1α (HIF1α) is a feature of mouse NK cells during murine cytomegalovirus (MCMV) infection in vivo. HIF1α-deficient NK cells failed to control viral load, causing increased morbidity. No defects were found in effector functions of HIF1αKO NK cells; however, their numbers were significantly reduced. Loss of HIF1α did not affect NK cell proliferation during in vivo infection and in vitro cytokine stimulation. Instead, we found that HIF1α-deficient NK cells showed increased expression of the pro-apoptotic protein Bim and glucose metabolism was impaired during cytokine stimulation in vitro. Similarly, during MCMV infection HIF1α-deficient NK cells upregulated Bim and had increased caspase activity. Thus, NK cells require HIF1α-dependent metabolic functions to repress Bim expression and sustain cell numbers for an optimal virus response

Viral MHCI inhibition evades tissue-resident memory T cell formation and responses

Tissue-resident memory CD

Cross-priming induces immunodomination in the presence of viral MHC class I inhibition.

Viruses have evolved mechanisms of MHCI inhibition in order to evade recognition by cytotoxic CD8+ T cells (CTLs), which is well-illustrated by our prior studies on cowpox virus (CPXV) that encodes potent MHCI inhibitors. Deletion of CPXV viral MHCI inhibitors markedly attenuated in vivo infection due to effects on CTL effector function, not priming. However, the CTL response to CPXV in C57BL/6 mice is dominated by a single peptide antigen presented by H-2Kb. Here we evaluated the effect of viral MHCI inhibition on immunodominant (IDE) and subdominant epitopes (SDE) as this has not been thoroughly examined. We found that cross-priming, but not cross-dressing, is the main mechanism driving IDE and SDE CTL responses following CPXV infection. Secretion of the immunodominant antigen was not required for immunodominance. Instead, immunodominance was caused by CTL interference, known as immunodomination. Both immunodomination and cross-priming of SDEs were not affected by MHCI inhibition. SDE-specific CTLs were also capable of exerting immunodomination during primary and secondary responses, which was in part dependent on antigen abundance. Furthermore, CTL responses directed solely against SDEs protected against lethal CPXV infection, but only in the absence of the CPXV MHCI inhibitors. Thus, both SDE and IDE responses can contribute to protective immunity against poxviruses, implying that these principles apply to poxvirus-based vaccines

CPXV subdominant epitope-specific memory CTLs immunodominate responses by naïve CD8⁺ T cells.

<p>(A) Schematic of i.n. prime/boost experiment. (B and C) Immunodomination of naïve CD8⁺ T cells. B6 mice (n = 5–6) were primed i.n. with 5 x 10³ pfu, i.n. boosted at 25 dpi with 5 x 10³ pfu (B) or 5 x 10⁴ pfu (C), and sacrificed 8 days after boosting. CD8⁺ T cell responses in the lungs (top) and spleens (bottom) were determined by ICS. Data are the combined results from two independent experiments. (D) Generation of memory CD8⁺ T cells. i.n. primed mice were sacrificed at 25 dpi and memory CD8⁺ T cells were measured in the spleen by ICS. (E) Antibody-independent memory CTL immunodomination. μmT mice were primed by s.s. with 1 x 10⁵ and i.n. boosted with 1 x 10⁵ pfu at 25 dpi. CD8⁺ T cell responses in the spleens were determined 7 days after boost. Data are the combined results from two independent experiments. (F) Memory CTLs cross-compete for peptide-MHCI complexes on APCs. Peptide-pulsed BMDCs were adoptively transferred by tail vein injection into ΔB8_19-26-primed B6 mice (n = 4) and CD8⁺ T cell responses in the spleen were evaluated by ICS 6 days after transfer. (G) Naïve CD8⁺ T cells do not cross-compete for peptide-MHCI complexes on APCs. Peptide-pulsed BMDCs were transferred into naïve B6 mice and CD8⁺ T cell responses were evaluated by ICS as in the experimental setup of F. Data are representative of two independent experiments.</p