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

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

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

Role of Hydroxyl Groups in the Basic Reactivity of MgO: a Theoretical and Experimental Study

Clean (i.e., carbonate- and hydroxyl-free) and hydroxylated MgO surfaces of various morphologies were used to study the correlation between thermodynamic Brønsted basicity (evaluated by the deprotonation of methanol and propyne followed by FTIR) and basic reactivity (evaluated by the conversion of 2-methyl-but-3-yn-2-ol (MBOH)). A correlation is evidenced for clean surfaces, but for hydroxylated surfaces, OH groups, though weak Brønsted bases, are much more active than oxide O2- ions. Some of the hydroxyl groups whose VOH infra-red stretching band is included in the narrow high-frequency OH band were shown to be responsible for such activity. In order to identify them, the irregularities of surfaces involving low coordination (LC) ions were modeled by DFT calculations (mono- and di-atomic steps for 4C ions, corners, kinks and divacancies for 3C ions). All of them dissociate water: except for kinks and divancancies where the OH formed can be considered as isolated, in all the other cases, OH groups are H-bonded. The í OH infra-red frequencies obtained by calculations are consistent with the experimental spectrum which exhibits two domains: the broad low-frequency (${\rm V}_{\rm OH} 3700$ cm-1) corresponds to isolated OH (O2CH, O3CH and O4CH in kinks and divacancies) and H-bond acceptor OH groups (O1CH and O2CH). The latter are inferred to be implied in basic reactivity of hydroxylated surfaces toward MBOH

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

Genetic mapping of the virulence phenotype identified the ROP5 locus as the single major QTL.

<p>A) The parents and progeny of the genetic cross between type 2 ME49-FUDR^r and type 10 VAND-SNF^r strains were assessed for virulence in 5 outbred CD-1 mice per strain. (B) A QTL scan of the virulence phenotype generates one significant peak on the left end of chromosome XII with a LOD score of 6.95 and effect size 71%. Genome-wide LOD significance thresholds were obtained from 10,000 permutation tests, α .05 ≥ LOD 2.7. (C) A secondary scan of the virulence phenotype with the locus on chromosome XII run as an additive covariate. (D) A two-dimensional genome scan with a full model testing for interacting QTLs (top half) and an additive model testing for multiple QTLs without interaction (bottom half). Genome-wide LOD significance thresholds were obtained from 1,000 permutation tests.</p

Conventional cross-priming, but not cross-dressing, is the main mechanism driving CTL responses during CPXV infection.

<p>(A) Schematic of bone marrow chimera cross-dressing experiment. B) CTLs are not activated by cross-dressed APCs. <i>Batf3</i>^<i>-/-</i>-F₁ mice (n = 5–6) were depleted of NK cells, lethally irradiated 2 days after NK cell depletion, and reconstituted with 1 x 10⁷ T cell depleted bone marrow cells from B6 or BALB/c mice. 8 weeks later, chimeric mice were infected i.n. with 5 x 10³ pfu WT CPXV and CD8⁺ T cell responses in the lungs were determined by ICS at 8 dpi. (C) Memory CTLs are not activated by cross-dressed APCs. Chimeric mice (n = 3–5) previously infected for 25 days were boosted with 5 x 10⁴ pfu WT CPXV and CD8⁺ T cell responses in the lungs were determined by ICS at 8 days after boost. The data are the combined results of three independent experiments.</p

Cryptic subdominant epitopes can compensate for the loss of the CPXV immunodominant epitope-specific CTL response, revealing immunodomination.

<p>CTL immunodomination occurs during primary responses against CPXV. (A and B) B6 mice (n = 11–13) were infected i.n with 5 x 10³ pfu of WT, ΔB8_19-26, ΔMHCIi, or ΔMHCIiΔB8_19-26 and were sacrificed at 8dpi. CD8⁺ T cells in the lungs were restimulated with B8_19-26 peptide or DC2.4 cells infected with ΔMHCIi or ΔMHCIiΔB8_19-26. The legend to B indicates the viruses used for infections and the X-axis indicates the stimuli used for <i>ex vivo</i> restimulation and ICS. Data are the combined results of five independent experiments. (C) WT and mutant viral strains replicate to similar titers. Viral titers in the lungs of infected B6 mice were determined at 8 dpi by plaque assay. (D and E) Comparable CTL responses against all viral strains tested. B6 mice (n = 6) were infected by i.p. and splenic CD8⁺ T cells were restimulated with peptides (D) or with DC2.4 cells infected with ΔMHCIi or ΔMHCIiΔB8_19-26 (E). Data are the combined results of four independent experiments.</p

Subdominant epitope-specific CTL responses protect against CPXV infection.

<p>(A) Schematic of adoptive transfer experiment. B6 mice were primed with ΔB8_19-26 or MCMV by subcutaneous (s.c.) or i.p. routes, respectively. At 7 dpi, splenic CD8⁺ T cells were isolated by positive selection and adoptively transferred into naive B6 mice (n = 11–13) by tail vein injection. After ~1 day, mice were infected by i.n. inoculation with ΔB8_19-26 or ΔMHCIiΔB8_19-26 and monitored for survival (B) and weight loss (C). Data are the combined results from two independent experiments.</p