After acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a proportion of patients experience persistent symptoms beyond 12 weeks, termed Long Covid. Understanding the mechanisms that cause this debilitating disease and identifying biomarkers for diagnostic, therapeutic, and monitoring purposes are urgently required. We detected persistently high levels of interferon-γ (IFN-γ) from peripheral blood mononuclear cells of patients with Long Covid using highly sensitive FluoroSpot assays. This IFN-γ release was seen in the absence of ex vivo peptide stimulation and remains persistently elevated in patients with Long Covid, unlike the resolution seen in patients recovering from acute SARS-CoV-2 infection. The IFN-γ release was CD8 + T cell–mediated and dependent on antigen presentation by CD14 + cells. Longitudinal follow-up of our study cohort showed that symptom improvement and resolution correlated with a decrease in IFN-γ production to baseline levels. Our study highlights a potential mechanism underlying Long Covid, enabling the search for biomarkers and therapeutics in patients with Long Covid.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused widespread morbidity and mortality since its onset in late 2019. Here, we demonstrate that prior infection with human cytomegalovirus (HCMV) substantially increases infection with SARS-CoV-2 in vitro. HCMV is a common herpesvirus carried by 40%–100% of the population, which can reactivate in the lung under inflammatory conditions, such as those resulting from SARS-CoV-2 infection. We show in both endothelial and epithelial cell types that HCMV infection upregulates ACE2, the SARS-CoV-2 cell entry receptor. These observations suggest that HCMV reactivation events in the lung of healthy HCMV carriers could exacerbate SARS-CoV-2 infection and subsequent COVID-19 symptoms. This effect could contribute to the disparity of disease severity seen in ethnic minorities and those with lower socioeconomic status, due to their higher CMV seroprevalence. Our results warrant further clinical investigation as to whether HCMV infection influences the pathogenesis of SARS-CoV-2.

This study integrates microseismic hydraulic fracture mapping with geochemical production profiling to understand the interaction between mechanical stratigraphy, fracture geometry, and effective drainage for wells landed in different benches of the STACK play in Oklahoma. Microseismic monitoring was used to map the extents of the hydraulic fracture system contacted during stimulation, while high resolution geochemical analysis or ‘fingerprinting’ was used to assess how different formations in the reservoir were draining. Microseismicity showed that hydraulic fracture growth from an Upper Meramec well rapidly cover the entire Meramec interval with some growth downward into the Woodford. Conversely, microseismicity initiating from a Woodford well clustered in that layer and grew upward into the Lower Meramec with time. Geochemical profiling closely matched the microseismic depth distributions for the associated well landing zones. Similar to the microseismic hydraulic heights from both Upper and Lower Meramec wells consistently produced from the entire Meramec, with additional recovery from the Woodford. Woodford landed wells produced Woodford oil with some production also coming from the Lower Meramec, also consistent with the microseismic depths. These production profiling trends were found to be very consistent across multiple sets of wells drilled into the same target formations. Integrating mapping of hydraulic fracture growth with geochemical assessment of the effective drainage within the hydraulically contacted zones provides unique insights into the reservoir contact and drainage. Understanding the mechanical stratigraphic controls on hydraulic fracture height growth relative to the reservoir drainage is key to informed decisions on wine-rack configurations for optimal reservoir drainage.

Following infection, the human cytomegalovirus (HCMV) genome becomes rapidly associated with host histones which can contribute to the regulation of viral gene expression. This can be seen clearly during HCMV latency where silencing of the major immediate early promoter (MIEP), normally responsible for expression of the key lytic proteins IE72 and IE86, is mediated by histone methylation and recruitment of heterochromatin protein 1. Crucially, reversal of these histone modifications coupled with histone acetylation drives viral reactivation which can be blocked with specific histone acetyltransferase inhibitors (HATi). In lytic infection, a role for HATi is less clear despite the well- established enhancement of viral replication observed with histone deacetylase inhibitors. Here we report that a number of different broad- acting HATi have a minor impact on viral infection and replication during lytic infection with the more overt phenotypes observed at lower multiplicities of infection. However, specific analyses of the regulation of major immediate early (MIE) gene expression reveal that the HATi C646, which targets p300/ CBP, transiently repressed MIE gene expression via inhibition of the MIEP but by 24 h post- infection MIE gene expression was rescued due to compensatory activation of an alternative IE promoter, ip2. This suggested that silencing of the MIEP promoted alternative ip2 promoter activity in lytic infection and, consistent with this, ip2 transcription is impaired in cells infected with a recombinant HCMV that does not auto- repress the MIEP at late times of infection. Furthermore, inhibition of the histone methyltransferases known to be responsible for auto- repression is similarly inhibitory to ip2 transcription in wild- type infected cells. We also observe that these discrete transcriptional activities of the MIEP and ip2 promoter are also reflected in reactivation; essentially in cells where the MIEP is silenced, ip2 activity is easier to detect at very early times post- reactivation whereas in cells where robust activation of the MIEP is observed ip2 transcription is reduced or delayed. Finally, we observe that inhibition of pathways demonstrated to be important for reactivation of HCMV in dendritic cells, e.g. in response to IL- 6, are preferentially important for activation of the MIEP and not the ip2 promoter. Together, these data add to the hypothesis that the existence of multiple promoters within the MIE region of HCMV can drive reactivation in a cell type- and ligand- specific manner and also suggest that inter- dependent regulatory activity between the two promoters exists.

Human cytomegalovirus (HCMV) can undergo either a latent or a lytic infection in cells of the myeloid lineage. Whilst the molecular mechanisms which determine the outcome of infection are far from clear, it is well established that a key factor is the differential regulation of the major immediate early promoter (MIEP) responsible for driving lytic immediate early gene expression. Using a myelomonocytic cell line stably transduced with a GFP reporter under the control of the MIEP, which recapitulates MIEP regulation in the context of virus infection, we have used an unbiased CRISPR-Cas9 sub-genomic, epigenetic library screen to identify novel cellular factors involved in MIEP repression during establishment and maintenance of latency in myeloid cells. One such cellular factor identified was MORC3. Consistent with MORC3 being a robust repressor of the MIEP, we show that THP1 cells devoid of MORC3 fail to establish latency. We also show that MORC3 is induced during latent infection, recruited to the MIEP and forms MORC3 nuclear bodies (MORC3-NBs) which, interestingly, co-localize with viral genomes. Finally, we show that the latency-associated functions of MORC3 are regulated by the deSUMOylase activity of the viral latency-associated LUNA protein likely to prevent untimely HCMV reactivation.

BACKGROUND: Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfu-sion (EVLP). METHODS: HCMV seropositive human lungs were placed on EVLP alone or EVLP + 1mg/L of F49A-FTP for 6 hours (n = 6, each). CD14+ monocytes isolated from biopsies pre and post EVLP underwent HCMV reactivation assay designed to evaluate viral reactivation capacity. Off-target effects of F49A-FTP were studied evaluating cell death markers of CD34+ and CD14+ cells using flow cytometry. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. RESULTS: We demonstrate that lungs treated ex-vivo with F49A-FTP had a significant reduction in HCMV reactivation compared to controls, suggesting successful targeting of latent virus (76% median reduction in F49A-FTP vs 15% increase in controls, p = 0.0087). Furthermore, there was comparable cell death rates of the targeted cells between both groups, suggesting no off-target effects. Ex-vivo lung function was stable over 6 hours and no differences in key inflammatory cytokines were observed demonstrating safety of this novel treatment.

HCMV-specific CD8+ T-cells are potent anti-viral effector cells in HCMV infected individuals, but evidence from other viral infections suggests that CD8+ T-cells can also produce the immunomodulatory cytokine IL-10. In this work we show that there are HCMV-specific IL-10 CD8+ T-cell responses in a cohort of individuals aged 23–76 years of age, predominantly directed against the HCMV proteins known to be expressed during latent infections as well as towards the proteins US3 and pp71. The analysis of HCMV-specific responses established during primary infection has shown that the IL-10 responses to US3 and pp71 HCMV proteins are detectable in the first weeks post infection, but not the responses to latency-associated proteins, and this IL-10 response is produced by both CD8+ and CD4+ T-cells. Phenotyping studies of HCMV-specific IL-10+ CD8+ T-cells show that these are CD45RA+ effector memory cells and co-express CD28 and CD57, however, the expression of the inhibitory receptor PD-1 varied from 90% to 30% between donors. In this study we have described for the first time the HCMV-specific IL-10 CD8+ T-cell responses and have demonstrated their broad specificity and the potential immune modulatory role of the immune response to HCMV latent carriage and periodic reactivation.

Understanding the mechanisms which regulate HCMV latency could allow therapeutic targeting of the latent virus reservoir from where virus reactivation can cause severe disease. We show that the BMPR2/TGFbeta receptor/YY1 signaling axis is crucial to maintain HCMV latency in undifferentiated cells and that pharmacological reduction of BMPR2 in latently infected cells leads to reactivation of the viral lytic transcription program, which renders the infected cell open to immune detection and clearance in infected individuals.

Human cytomegalovirus (HCMV) infection is not cleared by the initial immune response but persists for the lifetime of the host, in part due to its ability to establish a latent infection in cells of the myeloid lineage. HCMV has been shown to manipulate the secretion of cellular proteins during both lytic and latent infection; with changes caused by latent infection mainly investigated in CD34+ progenitor cells. Whilst CD34+ cells are generally bone marrow resident, their derivative CD14+ monocytes migrate to the periphery where they briefly circulate until extravasation into tissue sites. We have analyzed the effect of HCMV latent infection on the secretome of CD14+ monocytes, identifying an upregulation of both CCL8 and CXCL10 chemokines in the CD14+ latency-associated secretome. Unlike CD34+ cells, the CD14+ latency-associated secretome did not induce migration of resting immune cell subsets but did induce migration of activated NK and T cells expressing CXCR3 in a CXCL10 dependent manner. As reported in CD34+ latent infection, the CD14+ latency-associated secretome also suppressed the anti-viral activity of stimulated CD4+ T cells. Surprisingly, however, co-culture of activated autologous CD4+ T cells with latently infected monocytes resulted in reactivation of HCMV at levels comparable to those observed using M-CSF and IL-1β cytokines. We propose that these events represent a potential strategy to enable HCMV reactivation and local dissemination of the virus at peripheral tissue sites.

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.

Purpose Post-transplant CMV reactivation from latently-infected lung allografts is extremely prevalent and results in inferior long-term outcomes. CMV latently infected cells ubiquitously express a viral surface protein, US28. We aimed to evaluate the therapeutic effects of a novel immunotoxin (F49A-FTP) that targets US28 in human lungs during EVLP, with the ultimate goal of minimizing or preventing CMV transmission and reactivation post-transplantation. Methods Human lungs rejected for transplantation were randomly placed on EVLP alone or EVLP+1mg/L of F49A-FTP (n=6 each) for 6 hours. Biopsies were collected pre and post EVLP and subjected to an in vitro assay designed to evaluate viral reactivation capacity (Fig 1A). Based on US28's homology to the human CX3CR1 chemokine receptor, potential off-target effects of the toxin were studied evaluating cell death markers of CD34+ and CD14+ using flow cytometry, and the ratio of cells post:pre perfusion were calculated. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. Results Delivery of F49A-FTP during EVLP showed no acute toxic events demonstrated by no significant differences in lung physiology and levels of IL-8, IL1-b and IFN-γ between the groups. Furthermore, while control lung tissue demonstrated a median increase from baseline of 32% (Range -16% to +112.5%) in viral reactivation, treated lungs demonstrated a significant median reduction in CMV reactivation of 76% (Range -15% to -99.9%) (Fig 1B, p=0.0087). No off-target effects were noted upon F49A-FTP delivery, demonstrated by similar amounts of cell death between the groups (Fig 1C). Conclusion We demonstrate that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation and could potentially be used to treat donor organs prior to transplant. Ongoing in vivo reactivation experiments using the EVLP treated samples in a humanized mouse will further validate these findings.

The extensive tropism of human cytomegalovirus (HCMV) results in the productive infection of multiple cell types within the human host. However, infection of other cell types, such as undifferentiated cells of the myeloid lineage, give rise to nonpermissive infections. This aspect has been used experimentally to model latent infection, which is known to be established in the pluripotent CD34+ hematopoietic progenitor cell population resident in the bone marrow in vivo. The absence of a tractable animal model for studies of HCMV has resulted in a number of laboratories employing experimental infection of cells in vitro to simulate both HCMV lytic and latent infection. Herein, we will focus on the techniques used in our laboratory for the isolation and use of primary cells to study aspects of HCMV latency, reactivation, and lytic infection.

Human Cytomegalovirus (HCMV) can cause a variety of health disorders that can lead to death in immunocompromised individuals and neonates. The HCMV lifecycle comprises both a lytic (productive) and a latent (non-productive) phase. HCMV lytic infection occurs in a wide range of terminally differentiated cell types. HCMV latency has been less well-studied, but one characterized site of latency is in precursor cells of the myeloid lineage. All known viral genes are expressed during a lytic infection and a subset of these are also transcribed during latency. The UL111A gene which encodes the viral IL-10, a homolog of the human IL-10, is one of these genes. During infection, different transcript isoforms of UL111A are generated by alternative splicing. The most studied of the UL111A isoforms are cmvIL-10 (also termed the “A” transcript) and LAcmvIL-10 (also termed the “B” transcript), the latter being a well-characterized latency associated transcript. Both isoforms can downregulate MHC class II, however they differ in a number of other immunomodulatory properties, such as the ability to bind the IL10 receptor and induce signaling through STAT3. There are also a number of other isoforms which have been identified which are expressed by differential splicing during lytic infection termed C, D, E, F, and G, although these have been less extensively studied. HCMV uses the viral IL-10 proteins to manipulate the immune system during lytic and latent phases of infection. In this review, we will discuss the literature on the viral IL-10 transcripts identified to date, their encoded proteins and the structures of these proteins as well as the functional properties of all the different isoforms of viral IL-10.

Our study demonstrates that ex vivo F49A-FTP treatment of human lungs on EVLP markedly attenuates CMV reactivation. Ongoing ex vivo and in vivo reactivation experiments will confirm these findings.

Human cytomegalovirus (HCMV) establishes either a latent (non-productive) or lytic (productive) infection depending upon cell type, cytokine milieu and the differentiation status of the infected cell. Undifferentiated cells, such as precursor cells of the myeloid lineage, support a latent infection whereas terminally differentiated cells, such as monocytes or dendritic cells are an environment conducive to reactivation and support a lytic infection. The mechanisms which regulate HCMV in either a latent or lytic infection have been the focus of intense investigation with a view to developing novel treatments for HCMV-associated disease which can have a heavy clinical burden after reactivation or primary infection in, especially, the immune compromised. To this end, a number of studies have been carried out in an unbiased manner to address global changes occurring within the latently infected cell to address the molecular changes associated with HCMV latency. In this review, we will concentrate on the proteomic analyses which have been carried out in undifferentiated myeloid cells which either stably express specific viral latency associated genes in isolation or on cells which have been latently infected with virus.

One site of latency of human cytomegalovirus (HCMV) in vivo is in undifferentiated cells of the myeloid lineage. Although latently infected cells are known to evade host T cell responses by suppression of T cell effector functions, it is not known if they must also evade surveillance by other host immune cells. Here we show that cells latently infected with HCMV can, indeed, be killed by host neutrophils but only in a serum-dependent manner. Specifically, antibodies to the viral latency-associated US28 protein mediate neutrophil killing of latently infected cells. To address this mechanistically, a full proteomic screen was carried out on latently infected monocytes. This showed that latent infection downregulates the neutrophil chemoattractants S100A8/A9, thus suppressing neutrophil recruitment to latently infected cells. The ability of latently infected cells to inhibit neutrophil recruitment represents an immune evasion strategy of this persistent human pathogen, helping to prevent clearance of the latent viral reservoir.

Background Human cytomegalovirus (HCMV) is a threat to immunologically weak patients. HCMV cannot yet be eliminated with a vaccine, despite recent advances. Sources of data Sources of data are recently published research papers and reviews about HCMV treatments. Areas of agreement Current antivirals target the UL54 DNA polymerase and are limited by nephrotoxicity and viral resistance. Promisingly, letermovir targets the HCMV terminase complex and has been recently approved by the FDA and EMA. Areas of controversy Should we screen newborns for HCMV, and use antivirals to treat sensorineural hearing loss after congenital HCMV infection? Growing points Growing points are developing drugs against latently infected cells. In addition to small molecule inhibitors, a chemokine-based fusion toxin protein, F49A-FTP, has shown promise in killing both lytically and latently infected cells. Areas timely for developing research We need to understand what immune responses are required to control HCMV, and how best to raise these immune responses with a vaccine.