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Human cytomegalovirus (HCMV) actively manipulates cellular signaling pathways to benefit viral replication. Phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling is an important negative regulator of HCMV replication, and during lytic infection the virus utilizes pUL38 to limit Akt phosphorylation and activity. During latency, PI3K/Akt signaling also limits virus replication, but how this is overcome at the time of reactivation is unknown. Virally encoded microRNAs (miRNAs) are a key component of the virus arsenal used to alter signaling during latency and reactivation. In the present study we show that three HCMV miRNAs (miR-UL36, miR-UL112 and miR-UL148D) downregulate Akt expression and attenuate downstream signaling, resulting in the activation of FOXO3a and enhanced internal promoter-driven IE transcription. A virus lacking expression of all three miRNAs is unable to reactivate from latency both in CD34 + hematopoietic progenitor cells and in a humanized mouse model of HCMV infection, however downregulating Akt restores the ability of the mutant virus to replicate. These findings highlight the negative role Akt signaling plays in HCMV replication in lytic and latent infection and how the virus has evolved miRNA-mediated countermeasures to promote successful reactivation.

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes. We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)). MCMV/ZEBOV-NP(CTL) induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection. This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34 + hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34 + hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 –RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity.

West Nile Virus (WNV) can cause severe and long-lasting neurological disease and results in some neuropathology and neuroinflammation seen in Alzheimer's disease (AD). Exposure to WNV might impact AD-relevant behavioral and cognitive performance and neuropathology via AD-susceptibility genes (i.e., E4) and by inducing neuroinflammation (i.e., increases in TCR-α, IFN-γ, TNF-α, and CXCL- 10). There are three human apolipoprotein E (E) isoforms, which play a role in cholesterol metabolism: E2, E3, and E4. Compared to E3, E4 is an AD risk factor. We crossed knock-in (KI) mice expressing human amyloid precursor protein (APP) containing the dominant NL-G-F mutations with human apoE targeted replacement (TR) mice and used middle-aged NL-G-F/E3 and NL-G-F/E4 mice to assess the role of prior WNV (subtype Kunjin virus) (KUNV) exposure on hAPP/Aβ-induced behavioral alterations, cognitive injury, circadian body temperatures, viral loads, neuropathology, and transcript levels of four immune measures important in the detrimental effects of KUNV on brain function. KUNV affected physiological, behavioral, cognitive, amyloid pathology, viral load, and immune measures in middle aged NL-G-F mice in an apoE isoform-dependent fashion. NL-G-F/E4 mice were more susceptible to KUNV induced cognitive injury and prolonged viral load in the cortex. These results support an important apoE isoform-dependent role in modulating phenotypes in the NL-G-F AD mouse model following WNV exposure.

Zika virus (ZIKV) infection during pregnancy can cause a broad range of neurological birth defects, collectively named Congenital Zika Syndrome (CZS). We have previously shown that infection with the Puerto Rican isolate PRVABC59 (ZIKV-PR) results in abnormal oxygen transport in the placenta due to villous damage and uterine vasculitis in a nonhuman primate model. To investigate whether this type of damage occurs with endemically circulating strains in Thailand, we investigated a CZS case isolate, MU1-2017 (ZIKV-TH), in pregnant rhesus macaques. Pregnant animals (n = 3 per group) were infected subcutaneously with either ZIKV-PR or ZIKV-TH at ~50 days gestation (GD) and monitored for 40 days post-infection (GD90). Similar courses of viremia and immune activation were observed for both viruses when compared to uninfected controls. In addition, both viruses induced changes to the placental architecture, including spiral artery remodeling and the development of infarctions. Similar levels of viral RNA were detected at necropsy in maternal and fetal tissues. Overall, our results show that the ZIKV-TH strain MU1-2017 behaves similarly to the ZIKV-PR strain, and, importantly, provide evidence of in-utero infection with an additional contemporary strain of ZIKV.

Powassan virus (POWV) is a pathogenic tick-borne flavivirus that causes fatal neuroinvasive disease in humans. There are currently no approved therapies or vaccines for POWV infection. Here, we develop a POW virus-like particle (POW-VLP) based vaccine adjuvanted with the novel synthetic Toll-like receptor 7/8 agonist INI-4001. We demonstrate that INI-4001 outperforms both alum and the Toll-like receptor 4 agonist INI-2002 in enhancing the immunogenicity of a dose-sparing POW-VLP vaccine in mice. INI-4001 increases the magnitude and breadth of the antibody response as measured by whole-virus ELISA, induces neutralizing antibodies measured by FRNT, reduces viral burden in the brain of infected mice measured by RT-qPCR, and confers 100% protection from lethal challenge with both lineages of POWV. We show that the antibody response induced by INI-4001 is more durable than standard alum, and 80% of mice remain protected from lethal challenge 9-months post-vaccination. Lastly, we show that the protection elicited by INI-4001 adjuvanted POW-VLP vaccine is unaffected by either CD4 + or CD8 + T cell depletion and can be passively transferred to unvaccinated mice indicating that protection is mediated through humoral immunity. This study highlights the utility of novel synthetic adjuvants in VLP-based vaccines.

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.

The current commercially available vaccine used to prevent tetanus disease following infection with the anaerobic bacterium Clostridium tetani is safe and effective. However, tetanus remains a major source of mortality in developing countries. In 2008, neonatal tetanus was estimated to have caused >59,000 deaths, accounting for 1% of worldwide infant mortality, primarily in poorer nations. The cost of multiple vaccine doses administered by injection necessary to achieve protective levels of anti-tetanus toxoid antibodies is the primary reason for low vaccine coverage. Herein, we show that a novel vaccine strategy using a cytomegalovirus (CMV)-based vaccine platform induces protective levels of anti-tetanus antibodies that are durable (lasting >13 months) in mice following only a single dose. This study demonstrates the ability of a ‘single-dose’ CMV-based vaccine strategy to induce durable protection, and supports the potential for a tetanus vaccine based on CMV to impact the incidence of tetanus in developing countries.

[This corrects the article DOI: 10.1371/journal.ppat.1006219.].