Thermal tolerance of cultured and wild Icelandic arctic charr (Salvelinus alpinus) at self-selected flow rates.

Climate change is predicted to change not only the temperature of many freshwater systems but also flow dynamics. Understanding how fishes will fare in the future requires knowing how they will respond to both extended variations of temperature and flow. Arctic charr have had their thermal tolerance measured, but never with respect to flow. Additionally, this circumpolar species has multiple populations exhibiting dramatic phenotypic plasticity which may mean that regional differences in thermal tolerance are unaccounted for. In Iceland, Arctic charr populations have experienced highly variable flow and temperature conditions over the past 10,000 years. The Icelandic climate, topography and geothermal activity have created a mosaic of freshwater habitats inhabited by charr that vary substantially in both temperature and flow. Our purpose was to test whether populations from these varied environments had altered thermal tolerance and whether phenotypic plasticity of thermal tolerance in charr depends on flow. We raised cultured Icelandic charr from hatch under a 2 X 2 matrix of flow and temperature and compared them to wild charr captured from matching flow and temperature environments. Wild fish were more thermally tolerant than cultured fish at both acclimation temperatures and were more thermally plastic. Icelandic Arctic charr were more thermally tolerant than comparison charr populations across Europe and North America, but only when acclimated to 13 °C; fish acclimated to 5 °C compared equably with comparison charr populations. Icelandic Arctic charr were also more thermally plastic than all but one other salmonine species. Neither flow of rearing or the flow selected during a thermal tolerance (CTmax) test factored into thermal tolerance. Thermal tolerance was also independent of body size, condition factor, heart and gill size. In summary, wild Icelandic Arctic charr have greater thermal tolerance and plasticity than predicted from the literature and their latitude, but artificial selection for properties like growth rate or fecundity may be breeding that increased tolerance out of cultured fish. As the world moves toward a warmer climate and increased dependence on cultured fish, this is a noteworthy result and merits further study.

Prophylactic treatment with PEGylated bovine IFNλ3 effectively bridges the gap in vaccine-induced immunity against FMD in cattle.

Foot-and-mouth disease (FMD) is a vesicular disease of cloven-hoofed animals with devastating economic implications. The current FMD vaccine, routinely used in enzootic countries, requires at least 7 days to induce protection. However, FMD vaccination is typically not recommended for use in non-enzootic areas, underscoring the need to develop new fast-acting therapies for FMD control during outbreaks. Interferons (IFNs) are among the immune system's first line of defense against viral infections. Bovine type III IFN delivered by a replication defective adenovirus (Ad) vector has effectively blocked FMD in cattle. However, the limited duration of protection-usually only 1-3 days post-treatment (dpt)-diminishes its utility as a field therapeutic. Here, we test whether polyethylene glycosylation (PEGylation) of recombinant bovine IFNλ3 (PEGboIFNλ3) can extend the duration of IFN-induced prevention of FMDV infection in both vaccinated and unvaccinated cattle. We treated groups of heifers with PEGboIFNλ3 alone or in combination with an adenovirus-based FMD O1Manisa vaccine (Adt-O1M) at either 3 or 5 days prior to challenge with homologous wild type FMDV. We found that pre-treatment with PEGboIFNλ3 was highly effective at preventing clinical FMD when administered at either time point, with or without co-administration of Adt-O1M vaccine. PEGboIFNλ3 protein was detectable systemically for >10 days and antiviral activity for 4 days following administration. Furthermore, in combination with Adt-O1M vaccine, we observed a strong induction of FMDV-specific IFNγ+ T cell response, demonstrating its adjuvanticity when co-administered with a vaccine. Our results demonstrate the promise of this modified IFN as a pre-exposure prophylactic therapy for use in emergency outbreak scenarios.

Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.

Human Cytomegalovirus UL138 Interaction with USP1 Activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquintase complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection. Importance: Human cytomegalovirus (HCMV) is one of nine herpesviruses that infect humans. Following a primary infection, HCMV establishes a life-long latent infection that is marked by sporadic, and likely frequent reactivation events. While these reactivation events are asymptomatic in the immune competent host, they pose important disease risks for the immune compromised, including solid organ or stem cell transplant recipients. Its complex interactions with host biology and deep coding capacity make it an excellent model for defining mechanisms important for viral latency and reactivation. Here we define an interaction with host proteins that commandeer typically antiviral innate immune signaling for the establishment of latency.

Myeloid cell tropism enables MHC-E-restricted CD8+ T cell priming and vaccine efficacy by the RhCMV/SIV vaccine.

The strain 68-1 rhesus cytomegalovirus (RhCMV)-based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8+ T cells that recognize epitopes presented by major histocompatibility complex (MHC)-II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II- and/or MHC-Ia-restricted CD8+ T cells do not protect against SIV, it remains unclear whether MHC-E-restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II-restricted component. Using host microRNA (miR)-mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope-targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142-mediated tropism restriction eliminated MHC-E epitope-targeted CD8+ T cell priming, yielding an exclusively MHC-II epitope-targeted response. Inhibition with the endothelial cell-selective miR-126 eliminated MHC-II epitope-targeted CD8+ T cell priming, yielding an exclusively MHC-E epitope-targeted response. Dual miR-142 + miR-126-mediated tropism restriction reverted CD8+ T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8+ T cell responses were generally similar, only the vectors programmed to elicit MHC-E-restricted CD8+ T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

Thermal tolerance of cyprinids along an urban-rural gradient: Plasticity, repeatability and effects of swimming and temperature shock.

Urbanization changes the thermal profile of streams in much the same way that climate change is predicted to with higher temperatures, more varied flow and rapid temperature pulses with precipitation events. Whether exceptional tolerance to these altered thermal conditions is a pre-requisite for a fish species to inhabit urban streams or if urbanization has changed the thermal physiology of those fish species that persist in urban streams is unknown, but could help predict the outcome of future climate disruption. To test whether residence in urban streams is associated with altered thermal tolerance, we compared thermal tolerance (CTMax) and phenotypic plasticity of thermal tolerance (ΔCTMax/Δ acclimation temperature) in five populations of an urban-tolerant cyprinid, the blacknose dace (Rhinichthys atratulus), from multiple watersheds along an urban/rural gradient. Thermal tolerance of these stream fish was tested while swimming at 10 cm*s-1 but also in static water and after thermal shocks of 4°-6 °C simulating precipitation events. To test whether blacknose dace as a species has unusual thermal tolerance or thermal plasticity, we also compared two blacknose dace populations with two co-resident, co-familiars (creek chub (Semotilus atromaculatus) and rosyside dace (Clinostomus funduloides), that don't persist in urban streams at three different acclimation temperatures. Thermal tolerance of blacknose dace, as measured by a critical thermal maximum test (CTMax), was independent of size and activity level, i.e. individuals had identical thermal tolerance whether swimming or resting and CTMax was significantly repeatable across two levels of activity. Although there was some variance among populations, blacknose dace from streams of varied urbanization generally exhibited comparable thermal tolerances, ability to acclimate to different temperatures and were unaffected by thermal shocks. Rosyside dace had significantly lower thermal tolerance than the other two species but plasticity of thermal tolerance was uniform across the three cyprinid species. Our conclusions are that exceptional thermal tolerance or ability to thermally acclimate are not pre-requisite characters for a given cyprinid species to survive in urban streams, nor has thermal tolerance undergone directional selection in this urban environment.

Unusual Presentation of Widely Metastatic Extraskeletal Osteosarcoma: Case Report.

Extraskeletal osteosarcoma is a highly aggressive malignant osteoid forming mesenchymal neoplasm arising from soft tissues which accounts for 1% of all soft tissue sarcomas. We report the case of a 46-year-old female with no significant past medical history presenting to an emergency department with a right lateral thigh mass following minor trauma. She was eventually found to have high grade extraskeletal osteosarcoma with rapid progression of disease resulting in patient demise. Differentiation of these lesions from alternative processes relies on specific imaging and pathologic features. Differential diagnoses include both benign and malignant etiologies such as myositis ossificans, soft tissue hemangiomas, and other malignant soft tissue neoplasms such as epithelial and synovial sarcoma.

Changing Enhancement Pattern and Tumor Volume of Vestibular Schwannomas After Subtotal Resection.

BACKGROUND: Surgical strategy in vestibular schwannomas may require subtotal resection to preserve neurologic function. Residual tumor growth pattern and contrast enhancement in the immediate postresection period remain uncertain. We sought to evaluate changes in the enhancement pattern and volume of vestibular schwannomas after subtotal resection in the immediate postoperative period. METHODS: Volumetric analysis of tumor size and enhancement patterns of vestibular schwannomas were measured on magnetic resonance imaging (MRI) scans obtained within 3 days of surgery, 3 months after surgery, and 1 year after surgery. RESULTS: Nineteen patients were eligible for inclusion in the study (9 male and 10 female) with an average age of 47 years. Contrast enhancement was absent in 6 of 19 (32%) of cases on the immediate postresection MRI with return of expected enhancement on subsequent studies. Volumetric analysis identified that tumors decreased in size by an average of 35% in the first 3 months (P = 0.025) after resection and 46% in the first year after resection (P < 0.01). CONCLUSIONS: Vestibular schwannomas that undergo subtotal resection tend to decrease in size over the first 3 months after resection. Residual tumor volume may fail to enhance on the immediate postresection MRI. Both of these findings could lead surgeons to misinterpret degree of resection after surgery and have implications for clinical decision making and research reporting in the scientific literature for vestibular schwannomas after subtotal resection.

Human Cytomegalovirus miR-US25-1 Targets the GTPase RhoA To Inhibit CD34+ Hematopoietic Progenitor Cell Proliferation To Maintain the Latent Viral Genome.

Human cytomegalovirus (HCMV) microRNAs play essential roles in latency and reactivation in CD34+ hematopoietic progenitor cells (HPCs) via regulation of viral and cellular gene expression. In the present study, we show that HCMV miR-US25-1 targets RhoA, a small GTPase required for CD34+ HPC self-renewal, proliferation, and hematopoiesis. Expression of miR-US25-1 impairs signaling through the nonmuscle myosin II light chain, which leads to a block in cytokinesis and an inhibition of proliferation. Moreover, infection with an HCMV mutant lacking miR-US25-1 resulted in increased proliferation of CD34+ HPCs and a decrease in the proportion of genome-containing cells at the end of latency culture. These observations provide a mechanism by which HCMV limits proliferation to maintain latent viral genomes in CD34+ HPCs.IMPORTANCE Each herpesvirus family establishes latency in a unique cell type. Since herpesvirus genomes are maintained as episomes, the virus needs to devise mechanisms to retain the latent genome during cell division. Alphaherpesviruses overcome this obstacle by infecting nondividing neurons, while gammaherpesviruses tether their genome to the host chromosome in dividing B cells. The betaherpesvirus human cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs), but the mechanism used to maintain the viral genome is unknown. In this report, we demonstrate that HCMV miR-US25-1 downregulates expression of RhoA, a key cell cycle regulator, which results in inhibition of CD34+ HPC proliferation by blocking mitosis. Mutation of miR-US25-1 during viral infection results in enhanced cellular proliferation and a decreased frequency of genome-containing CD34+ HPCs. These results reveal a novel mechanism through which HCMV is able to regulate cell division to prevent viral genome loss during proliferation.

Cytomegaloviral determinants of CD8+ T cell programming and RhCMV/SIV vaccine efficacy.

Simian immunodeficiency virus (SIV) insert-expressing, 68-1 rhesus cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex E (MHC-E)- and MHC-II-restricted, SIV-specific CD8+ T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) have not been characterized. We show that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/Rh157.4 and Rh158-161), revealing three patterns of unconventional response inhibition. Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences (UL128/UL130; UL146/UL147) leads to either of two distinct CD8+ T cell response types-MHC-Ia-restricted only or a mix of MHC-II- and MHC-Ia-restricted CD8+ T cells. Response magnitude and functional differentiation are similar to RhCMV 68-1, but neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8+ T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all genes that inhibit these responses from the HCMV/HIV vector.

CD34+ Hematopoietic Progenitor Cell Subsets Exhibit Differential Ability To Maintain Human Cytomegalovirus Latency and Persistence.

In human cytomegalovirus (HCMV)-seropositive patients, CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study, we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.

Human Cytomegalovirus miR-US5-2 Downregulation of GAB1 Regulates Cellular Proliferation and UL138 Expression through Modulation of Epidermal Growth Factor Receptor Signaling Pathways.

Regulation of epidermal growth factor (EGF) receptor (EGFR) signaling is critical for the replication of human cytomegalovirus (HCMV) as well as latency and reactivation in CD34+ hematopoietic progenitor cells. HCMV microRNAs (miRNAs) provide a means to modulate the signaling activated by EGF through targeting components of the EGFR signaling pathways. Here, we demonstrate that HCMV miR-US5-2 directly downregulates the critical EGFR adaptor protein GAB1 that mediates activation and sustained signaling through the phosphatidylinositol 3-kinase (PI3K) and MEK/extracellular signal-regulated kinase (ERK) pathways and cellular proliferation in response to EGF. Expression of HCMV UL138 is regulated by the transcription factor early growth response gene 1 (EGR1) downstream of EGFR-induced MEK/ERK signaling. We show that by targeting GAB1 and attenuating MEK/ERK signaling, miR-US5-2 indirectly regulates EGR1 and UL138 expression, which implicates the miRNA in critical regulation of HCMV latency.IMPORTANCE Human cytomegalovirus (HCMV) causes significant disease in immunocompromised individuals, including transplant patients. HCMV establishes latency in hematopoietic stem cells in the bone marrow. The mechanisms governing latency and reactivation of viral replication are complex and not fully understood. HCMV-encoded miRNAs are small regulatory RNAs that reduce protein expression. In this study, we found that the HCMV miRNA miR-US5-2 targets the epidermal growth factor receptor (EGFR) adaptor protein GAB1 which directly affects downstream cellular signaling pathways activated by EGF. Consequently, miR-US5-2 blocks the EGF-mediated proliferation of human fibroblasts. Early growth response gene 1 (EGR1) is a transcription factor activated by EGFR signaling that regulates expression of HCMV UL138. We show that miR-US5-2 regulates UL138 expression through GAB1-mediated downregulation of the signaling pathways that lead to EGR1 expression. These data suggest that miR-US5-2, through downregulation of GAB1, could play a critical role during reactivation from latency by reducing proliferation and UL138 expression.

Human Cytomegalovirus Infection Suppresses CD34+ Progenitor Cell Engraftment in Humanized Mice.

Human cytomegalovirus (HCMV) infection is a serious complication in hematopoietic stem cell transplant (HSCT) recipients due to virus-induced myelosuppression and impairment of stem cell engraftment. Despite the clear clinical link between myelosuppression and HCMV infection, little is known about the mechanism(s) by which the virus inhibits normal hematopoiesis because of the strict species specificity and the lack of surrogate animal models. In this study, we developed a novel humanized mouse model system that recapitulates the HCMV-mediated engraftment failure after hematopoietic cell transplantation. We observed significant alterations in the hematopoietic populations in peripheral lymphoid tissues following engraftment of a subset of HCMV+ CD34+ hematopoietic progenitor cells (HPCs) within the transplant, suggesting that a small proportion of HCMV-infected CD34+ HPCs can profoundly affect HPC differentiation in the bone marrow microenvironment. This model will be instrumental to gain insight into the fundamental mechanisms of HCMV myelosuppression after HSCT and provides a platform to assess novel treatment strategies.

Hypoxia tolerance is unrelated to swimming metabolism of wild, juvenile striped bass (Morone saxatilis).

Juvenile striped bass residing in Chesapeake Bay are likely to encounter hypoxia that could affect their metabolism and performance. The ecological success of this economically valuable species may depend on their ability to tolerate hypoxia and perform fitness-dependent activities in hypoxic waters. We tested whether there is a link between hypoxia tolerance (HT) and oxygen consumption rate (MO2 ) of juvenile striped bass measured while swimming in normoxic and hypoxic water, and to identify the interindividual variation and repeatability of these measurements. HT (loss of equilibrium) of fish (N=18) was measured twice collectively, 11 weeks apart, between which MO2  was measured individually for each fish while swimming in low flow (10.2 cm s-1) and high flow (∼67% of critical swimming speed, Ucrit) under normoxia and hypoxia. Both HT and MO2  varied substantially among individuals. HT increased across 11 weeks while the rank order of individual HT was significantly repeatable. Similarly, MO2  increased in fish swimming at high flow in a repeatable fashion, but only within a given level of oxygenation. MO2  was significantly lower when fish were swimming against high flow under hypoxia. There were no clear relationships between HT and MO2  while fish were swimming under any conditions. Only the magnitude of increase in HT over 11 weeks and an individual's MO2  under low flow were correlated. The results suggest that responses to the interacting stressors of hypoxia and exercise vary among individuals, and that HT and change in HT are not simple functions of aerobic metabolic rate.

Human Cytomegalovirus miRNAs Regulate TGF-ß to Mediate Myelosuppression while Maintaining Viral Latency in CD34+ Hematopoietic Progenitor Cells.

Infection with human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality following hematopoietic stem cell transplant (HSCT) because of various hematologic problems, including myelosuppression. Here, we demonstrate that latently expressed HCMV miR-US5-2 downregulates the transcriptional repressor NGFI-A binding protein (NAB1) to induce myelosuppression of uninfected CD34+ hematopoietic progenitor cells (HPCs) through an increase in TGF-ß production. Infection of HPCs with an HCMVΔmiR-US5-2 mutant resulted in decreased TGF-ß expression and restoration of myelopoiesis. In contrast, we show that infected HPCs are refractory to TGF-ß signaling as another HCMV miRNA, miR-UL22A, downregulates SMAD3, which is required for maintenance of latency. Our data suggest that latently expressed viral miRNAs manipulate stem cell homeostasis by inducing secretion of TGF-ß while protecting infected HPCs from TGF-ß-mediated effects on viral latency and reactivation. These observations provide a mechanism through which HCMV induces global myelosuppression following HSCT while maintaining lifelong infection in myeloid lineage cells.

Characterization of a live-attenuated HCMV-based vaccine platform.

Vaccines based on cytomegalovirus (CMV) demonstrate protection in animal models of infectious disease and cancer. Vaccine efficacy is associated with the ability of CMV to elicit and indefinitely maintain high frequencies of circulating effector memory T cells (TEM) providing continuous, life-long anti-pathogen immune activity. To allow for the clinical testing of human CMV (HCMV)-based vaccines we constructed and characterized as a vector backbone the recombinant molecular clone TR3 representing a wildtype genome. We demonstrate that TR3 can be stably propagated in vitro and that, despite species incompatibility, recombinant TR3 vectors elicit high frequencies of TEM to inserted antigens in rhesus macaques (RM). Live-attenuated versions of TR3 were generated by deleting viral genes required to counteract intrinsic and innate immune responses. In addition, we eliminated subunits of a viral pentameric glycoprotein complex thus limiting cell tropism. We show in a humanized mouse model that such modified vectors were able to establish persistent infection but lost their ability to reactivate from latency. Nevertheless, attenuated TR3 vectors preserved the ability to elicit and maintain TEM to inserted antigens in RM. We further demonstrate that attenuated TR3 can be grown in approved cell lines upon elimination of an anti-viral host factor using small interfering RNA, thus obviating the need for a complementing cell line. In sum, we have established a versatile platform for the clinical development of live attenuated HCMV-vectored vaccines and immunotherapies.

HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation.

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.

Host signaling and EGR1 transcriptional control of human cytomegalovirus replication and latency.

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of alpha and beta herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. How signaling downstream of EGFR is regulated and how this impacts CMV infection and latency is not fully understood. We demonstrate that CMV downregulates EGFR early in the productive infection, which blunts the activation of EGFR and its downstream pathways in response to stimuli. However, CMV infection sustains basal levels of EGFR and downstream pathway activity in the context of latency in CD34+ hematopoietic progenitor cells (HPCs). Inhibition of MEK/ERK, STAT or PI3K/AKT pathways downstream of EGFR increases viral reactivation from latently infected CD34+ HPCs, defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription important to latency. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through the MEK/ERK pathway and is important for the maintenance of hematopoietic stemness. We demonstrate that EGR1 binds the viral genome upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevents the establishment of latency in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 gene expression and suppression of replication for latency. By this mechanism, the virus has hardwired itself into host cell biology to sense and respond to changes in homeostatic host cell signaling.

Human Cytomegalovirus US28 Ligand Binding Activity Is Required for Latency in CD34+ Hematopoietic Progenitor Cells and Humanized NSG Mice.

Human cytomegalovirus (HCMV) infection of CD34+ hematopoietic progenitor cells (CD34+ HPCs) provides a critical reservoir of virus in stem cell transplant patients, and viral reactivation remains a significant cause of morbidity and mortality. The HCMV chemokine receptor US28 is implicated in the regulation of viral latency and reactivation. To explore the role of US28 signaling in latency and reactivation, we analyzed protein tyrosine kinase signaling in CD34+ HPCs expressing US28. US28-ligand signaling in CD34+ HPCs induced changes in key regulators of cellular activation and differentiation. In vitro latency and reactivation assays utilizing CD34+ HPCs indicated that US28 was required for viral reactivation but not latency establishment or maintenance. Similarly, humanized NSG mice (huNSG) infected with TB40E-GFP-US28stop failed to reactivate upon treatment with granulocyte-colony-stimulating factor, but viral genome levels were maintained. Interestingly, HCMV-mediated changes in hematopoiesis during latency in vivo and in vitro was also dependent upon US28, as US28 directly promoted differentiation toward the myeloid lineage. To determine whether US28 constitutive activity and/or ligand-binding activity were required for latency and reactivation, we infected both huNSG mice and CD34+ HPCs in vitro with HCMV TB40E-GFP containing the US28-R129A mutation (no CA) or Y16F mutation (no ligand binding). TB40E-GFP-US28-R129A was maintained during latency and exhibited normal reactivation kinetics. In contrast, TB40E-GFP-US28-Y16F exhibited high levels of viral genome during latency and reactivation, indicating that the virus did not establish latency. These data indicate that US28 is necessary for viral reactivation and ligand binding activity is required for viral latency, highlighting the complex role of US28 during HCMV latency and reactivation.IMPORTANCE Human cytomegalovirus (HCMV) can establish latency following infection of CD34+ hematopoietic progenitor cells (HPCs), and reactivation from latency is a significant cause of viral disease and accelerated graft failure in bone marrow and solid-organ transplant patients. The precise molecular mechanisms of HCMV infection in HPCs are not well defined; however, select viral gene products are known to regulate aspects of latency and reactivation. The HCMV-encoded chemokine receptor US28, which binds multiple CC chemokines as well as CX3CR1, is expressed both during latent and lytic phases of the virus life cycle and plays a role in latency and reactivation. However, the specific timing of US28 expression and the role of ligand binding in these processes are not well defined. In this report, we determined that US28 is required for reactivation but not for maintaining latency. However, when present during latency, US28 ligand binding activity is critical to maintaining the virus in a quiescent state. We attribute the regulation of both latency and reactivation to the role of US28 in promoting myeloid lineage cell differentiation. These data highlight the dynamic and multifunctional nature of US28 during HCMV latency and reactivation.

Repeatability of Hypoxia Tolerance of Individual Juvenile Striped Bass Morone saxatilis and Effects of Social Status.

Chesapeake Bay is the primary nursery for striped bass (Morone saxatilis), which are increasingly being exposed to hypoxic waters. Tolerance to hypoxia in fish is generally determined by a single exposure of an isolated individual or by exposing large groups of conspecifics to hypoxia without regard to social status. The importance of social context in determining physiological responses to stressors is being increasingly recognized. To determine whether social interactions influence hypoxia tolerance (HT) in striped bass, loss of equilibrium HT was assessed in the same fish while manipulating the social environment around it. Small group settings were used to be more representative of the normal sociality experienced by this species than the paired encounters typically used. After establishing the dominance hierarchy within a group of fish, HT was determined collectively for the individuals in that group, and then new groups were constructed from the same pool of fish. Individuals could then be followed across multiple settings for both repeatability of HT and hierarchy position ( X¯=4.2±0.91 SD groups per individual). HT increased with repeated exposures to hypoxia ( P<0.001 ), with a significant increase by a third exposure ( P=0.004 ). Despite this changing HT, rank order of HT was significantly repeatable across trials for 6 mo ( P=0.012 ). Social status was significantly repeatable across trials of different group composition ( P=0.02 ) and unrelated to growth rate but affected HT weakly in a complex interaction with size. Final HT was significantly correlated with blood [hemoglobin] and hematocrit. The repeatability and large intraspecific variance of HT in juvenile striped bass suggest that HT is potentially an important determinant of Darwinian fitness in an increasingly hypoxic Chesapeake Bay.