Seventy years after the discovery of human cytomegalovirus (HCMV), perhaps its most influential contribution to molecular biology remains the major immediate-early promoter (MIEP). A modified form of this powerful regulatory element is now used ubiquitously in biotechnology to drive protein expression, yet within the virus itself, the full promoter performs a far more complex task: governing the balance between latency and lytic replication. This review discusses our understanding of the transcription factors that regulate MIEP activation and repression and how this regulatory network shapes HCMV latency and reactivation.

After severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a minority of patients experience persistent or emerging symptoms, termed 'long coronavirus disease (COVID)' or post-acute sequelae of COVID-19. The molecular causes of long COVID remain unclear, but disrupted immune functions, such as inflammation and immune deficit, have been posited as factors. In this retrospective cohort study, we measured markers of immune function in a group of patients with long COVID up to 40 months post infection. As proxies for immune function, we measured serum antibody levels, antibody neutralizing capability and production of IFN gamma (IFN-γ) and IL-2 against SARS-CoV-2 and other viral peptides. As expected, serum antibody levels increased over time with vaccinations and reinfections with later variants of SARS-CoV-2. Patients also showed corresponding increasing SARS-CoV-2-specific IL-2 responses and stable IFN-γ responses. We observed no significant differences in immune responses among patients with ongoing long COVID, those who had recovered from it or individuals who recovered from acute COVID-19. Overall, we found no indication of a reduction in these aspects of immune function after SARS-CoV-2 infection. This study provides a valuable foundation for further research aimed at understanding the causes of long COVID.

Reactivation of latent human cytomegalovirus (CMV) can lead to severe complications in individuals with dysregulated immune systems. While antiviral therapies for CMV are approved, these compounds are limited by their toxicity and inability to specifically target the latent reservoir or prevent reactivation. Herein we show that CMV reactivation in Kasumi-3 cells, a CD34+ hematopoietic cell line, requires mitogen-activated protein kinase (MAPK) activation. Importantly, pharmacological inhibition of the MAPK signaling pathway, including MEK and ERK, restricts viral reactivation in Kasumi-3 cells. In sum, our findings show MEK-ERK signaling is critical for CMV reactivation, revealing a potential avenue for therapeutic intervention to prevent viral reactivation and downstream pathogenesis that is often detrimental for immunosuppressed and immunocompromised patients.

Cytomegaloviruses (CMVs) encode viral G-protein-coupled receptors (vGPCRs) that have diverged from their cellular homologues to perform new functions. Human cytomegalovirus (HCMV) encodes four vGPCRs: UL33, UL78, US27, and US28, which contribute to viral pathogenesis, cellular signalling, and latency. While the role of US28 in chemokine signalling and viral latency is well characterised, the functions of other vGPCRs remain incompletely understood. Rodent cytomegaloviruses only have homologues to UL33 and UL78, while primates have two to five additional GPCRs which are homologues of US27 and US28. Different CMVs appear to have evolved vGPCRs with functions specific to infection of their respective host. As non-human CMVs are used as model organisms to understand clinical cytomegalovirus disease and develop vaccines and antivirals, understanding the differences between these vGPCRs helps researchers understand critical differences between their models. This review aims to address the differences between CMV vGPCRs, and how these differences may affect models of CMV disease to facilitate future research.

Human adenoviruses (HAdVs) cause diverse disease presentations as pathogens and are also used as viral vectors for vaccines and gene therapy products. Pre-existing adaptive immune responses to HAdV are known to influence symptom severity, viral clearance and the success of viral vectored products. Of note, approximately 50% of the UK's adult population has received at least one dose of a chimpanzee adenovirus vectored SARS-CoV-2 vaccine (ChAdOx1) since January 2021. We used FluoroSpot analysis to quantify the interferon-gamma (IFNγ) and interleukin-2 (IL2) responses of healthy blood donors to HAdV species A, B, C, D and F and chimpanzee adenovirus Y25, related to HAdV species E. We find that cellular immune responses to multiple species of human adenovirus are ubiquitous among healthy adult blood donors and that stimulating PBMC with whole hexon peptide libraries induces a significantly greater IFNγ and IL2 response than using selected peptide pools alone. We then compared the cellular immune responses of ChAdOx1 recipients and control donors using PBMC collected in 2021 and found that homotypic and heterotypic IFNγ responses were significantly boosted in ChAdOx1 recipients but not controls. Finally, we show that in PBMC derived from blood donors, IFNγ responses are made to both conserved and variable regions of the hexon protein. Future vaccination campaigns using adenoviral vectored vaccines will need to account for the pre-existing exposure of recipients to both circulating HAdVs and vaccines such as ChAdOx1, which convey polyfunctional antiviral T cell responses to even low seroprevalence HAdV types.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that establishes lifelong, latent infection in hematopoietic cells. Immune-competent individuals are usually asymptomatic for disease. However, immune dysregulation in latently infected individuals can result in viral reactivation, often causing further complications. Viral gene transcription during latency is restricted, although the CMV-encoded G protein-coupled receptor homologs, US28 and UL78, are expressed. We and others find US28 is critical for establishing and maintaining viral latency, in part, through regulating host cell signaling. How US28 switches from pro-latent to pro-lytic during reactivation, however, is unknown, though our findings herein reveal a role for UL78. Myeloid cells infected with a UL78 open reading frame deletion mutant maintain viral latency yet fail to efficiently reactivate. However, the UL78 G protein-coupling domain is not required for reactivation, suggesting UL78-mediated signaling is not critical for reactivation. Prior work revealed that UL78 and US28 interact, resulting in altered US28-mediated signaling. Additionally, we showed that US28 attenuates extracellular signal-regulated kinase (ERK) phosphorylation during latency, while ERK is phosphorylated upon reactivation; however, the mechanism underlying this switch is unknown. Thus, we hypothesized the UL78:US28 interaction is important for altering US28-mediated signaling upon viral reactivation. We find US28 and UL78 interact during lytic infection of fibroblasts and colocalize in myeloid cells upon their differentiation. Furthermore, reactivation in myeloid cells latently infected with wild-type virus results in upregulated ERK phosphorylation, while parallel cultures infected with the UL78-deficient virus fail to do so. Our data reveal the first function for UL78 in myeloid cells, where it influences cellular signaling during reactivation.IMPORTANCECytomegalovirus (CMV) is a ubiquitous human herpesvirus, infecting the majority of the population worldwide. As with all herpesviruses, once an individual is infected with CMV, the virus remains in the person's blood cells for life in a silenced state called latency, and this infection, for the most part, remains asymptomatic. When an infected individual's immune system fails to function properly, however, CMV can become active (termed viral reactivation), which allows the virus to replicate and cause downstream disease. Our understanding of the cellular and viral factors that dictate this switch from silenced to activated remains incomplete. Here, we show a viral protein, UL78, is required for this switch. We find that UL78 helps to reshape cellular signaling, changing the cell environment from one that favors latency to one that supports reactivation. This highlights a new avenue for therapeutic intervention to prevent CMV reactivation and downstream disease.

PURPOSE:Asymptomatic SARS-CoV-2 infections were widely reported during the COVID-19 pandemic, acting as a hidden source of infection. Many existing studies investigating asymptomatic immunity failed to recruit true asymptomatic individuals. Thus, we conducted a longitudinal cohort study to evaluate humoral- and cell-mediated responses to infection and vaccination in well-defined asymptomatic young adults (the Asymptomatic COVID-19 in Education [ACE] cohort). METHODS:Asymptomatic testing services located at three UK universities identified asymptomatic young adults who were subsequently recruited with age- and sex-matched symptomatic and uninfected controls. Blood and saliva samples were collected after SARS-CoV-2 Wuhan infection, and again after vaccination. 51 participant's anti-spike antibody titres, neutralizing antibodies, and spike-specific T-cell responses were measured, against both Wuhan and Omicron B.1.1.529.1. RESULTS:Asymptomatic participants exhibited reduced Wuhan-specific neutralization antibodies pre- and post-vaccination, as well as fewer Omicron-specific neutralization antibodies post-vaccination, compared to symptomatic participants. Lower Wuhan and Omicron-specific IgG titres in asymptomatic individuals were also observed pre- and post-vaccination, compared to symptomatic participants. There were no differences in salivary IgA levels. Conventional flow cytometry analysis and multi-dimensional clustering analysis indicated unvaccinated asymptomatic participants had significantly fewer Wuhan-specific IL-2 secreting CD4+ CD45RA+ T cells and activated CD8+ T cells than symptomatic participants, though these differences dissipated after vaccination. CONCLUSIONS:Asymptomatic infection results in decreased antibody and T cell responses to further exposure to SARS-CoV-2 variants, compared to symptomatic infection. Post-vaccination, antibody responses are still inferior, but T cell immunity increases to match symptomatic subjects, emphasising the importance of vaccination to help protect asymptomatic individuals against future variants.

Background: Cardiovascular complications of COVID-19 are numerous and aspects of this phenomenon are not well known. The main objective of this manuscript is a systematic review of the acute and chronic cardiovascular complications secondary to COVID-19. Methods: A systematic review of the literature through Medline via PubMed was conducted (2020–2024). Results: There is a plethora of effects of COVID-19 on the heart in the acute setting. Here we discuss pathophysiology, myocardial infarctions, heart failure, Takotsubo Cardiomyopathy, myocardial injury, myocarditis and arrhythmias that are caused by COVID-19. Additionally, these cardiovascular injuries can linger and may be an underlying cause of some Long COVID symptoms. Conclusions: Cardiovascular complications of COVID-19 are numerous and life-threatening. Long COVID can affect cardiovascular health. Microclotting induced by SARS-CoV-2 infection could be a therapeutic target for some aspects of Long Covid.

It is important to be able to retrospectively determine severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections with high accuracy, both for post-coronavirus disease 2019 (COVID-19) epidemiological studies, and to distinguish between Long COVID and other multi-syndromic diseases that have overlapping symptoms. Although serum antibody levels can be measured to retrospectively diagnose SARS-CoV-2 infections, peptide stimulation of memory T cell responses is a more sensitive approach. This is because robust memory T cells are generated after SARS-CoV-2 infection and persist even after antibodies wane below detectability thresholds. In this study, we compare T cell responses using FluoroSpot-based methods and overnight stimulation of whole blood with SARS-CoV-2 peptides followed by an ELISA. Both approaches have comparable sensitivity and specificity but require different equipment and samples to be used. Furthermore, the elimination of peptides that cross-react with other coronaviruses increases the assay specificity but trades off some sensitivity. Finally, this approach can be used on archival, cryopreserved PBMCs. This work shows comparative advantages for several methods to measure SARS-CoV-2 T cell responses that could be utilized by any laboratory studying the effects of the coronavirus disease 2019 pandemic.

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.

Age is a major risk factor for hospitalization and death after SARS-CoV-2 infection, even in vaccinees. Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here we show that individuals 70 or older who received a primary two dose schedule with AZD1222 and booster third dose with mRNA vaccine achieved significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared to those younger than 70. One month after the booster neither the concentration of serum binding anti spike IgG antibody, nor the frequency of spike-specific B cells showed differences by age grouping. However, the impaired neutralization potency and breadth post-third dose in the elderly was associated with enrichment of circulating “atypical” spike-specific B cells expressing CD11c and FCRL5. Single cell RNA sequencing confirmed an expansion of TBX21-, ITGAX -expressing B cells in the elderly that enriched for B cell activation/receptor signalling pathway genes. Importantly we also observed impaired T cell responses to SARS-CoV-2 spike peptides in the elderly post-booster, both in terms of IFNgamma and IL2 secretion, as well as a decrease in T cell receptor signalling pathway genes. This expansion of atypical B cells and impaired T cell responses may contribute to the generation of less affinity-matured antibodies, with lower neutralizing capacity post-third dose in the elderly. Altogether, our data reveal the extent and potential mechanistic underpinning of the impaired vaccine responses present in the elderly after a booster dose, contributing to their increased susceptibility to COVID-19 infection.

INTRODUCTION:Long COVID is a chronic condition that follows after acute COVID-19 and is characterized by a wide range of persistent, cyclic symptoms. SOURCES OF DATA:PubMed search for publications featuring 'Long COVID' or 'post-acute sequelae of COVID-19'. AREAS OF AGREEMENT:Long COVID occurs frequently post-acute COVID-19, with a majority of people experiencing at least one symptom (such as cough, fatigue, myalgia, anosmia and dyspnoea) 4 weeks after infection. AREAS OF CONTROVERSY:The specific symptoms and the minimum duration of symptoms required to be defined as Long COVID. GROWING POINTS:There is a consistent reduction in Long COVID incidence amongst vaccinated individuals, although the extent of this effect remains unclear. AREAS TIMELY FOR DEVELOPING RESEARCH:There is an urgent need to understand the causes of Long COVID, especially extreme fatigue more than 6 months after infection. We must understand who is at risk and whether reinfections similarly risk Long COVID.

Cytomegalovirus (CMV) reactivation from latency following immune dysregulation remains a serious risk for patients, often causing substantial morbidity and mortality. Here, we demonstrate the CMV-encoded G protein-coupled receptor, US28, in coordination with cellular Ephrin receptor A2, attenuates mitogen-activated protein kinase signaling, thereby limiting viral replication in latently infected primary monocytes. Furthermore, treatment of latently infected primary monocytes with dasatinib, a Food and Drug Association-approved kinase inhibitor used to treat a subset of leukemias, results in CMV reactivation. These ex vivo data correlate with our retrospective analyses of the Explorys electronic health record database, where we find dasatinib treatment is associated with a significant risk of CMV-associated disease (odds ratio 1.58, P = 0.0004). Collectively, our findings elucidate a signaling pathway that plays a central role in the balance between CMV latency and reactivation and identifies a common therapeutic cancer treatment that elevates the risk of CMV-associated disease.

Long coronavirus disease (COVID [LC]) constitutes a potential health emergency as millions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections lead to chronic symptoms. We must understand whether vaccines reduce LC because this has major implications for health policy. We report a 79% reduction in LC referrals correlating with vaccination in the United Kingdom.

Summary Background There is currently no consensus on the diagnosis, definition, symptoms, or duration of COVID-19 illness. The diagnostic complexity of Long COVID is compounded in many patients who were or might have been infected with SARS-CoV-2 but not tested during the acute illness and/or are SARS-CoV-2 antibody negative. Methods Given the diagnostic conundrum of Long COVID, we set out to investigate SARS-CoV-2-specific T cell responses in patients with confirmed SARS-CoV-2 infection and/or Long COVID from a cohort of mostly non-hospitalised patients. Findings We discovered that IL-2 release (but not IFN-γ release) from T cells in response to SARS-CoV-2 peptides is both sensitive (75% +/−13%) and specific (88%+/−7%) for previous SARS-CoV-2 infection >6 months after a positive PCR test. We identified that 42–53% of patients with Long COVID, but without detectable SARS-CoV-2 antibodies, nonetheless have detectable SARS-CoV-2 specific T cell responses. Interpretation Our study reveals evidence (detectable T cell mediated IL-2 release) of previous SARS-CoV-2 infection in seronegative patients with Long COVID. Funding This work was funded by the Addenbrooke's Charitable Trust (900276 to NS), NIHR award (G112259 to NS) and supported by the NIHR Cambridge Biomedical Research Centre. NJM is supported by the MRC (TSF MR/T032413/1) and NHSBT (WPA15-02). PJL is supported by the Wellcome Trust (PRF 210688/Z/18/Z, 084957/Z/08/Z), a Medical Research Council research grant MR/V011561/1 and the United Kingdom Research and a Innovation COVID Immunology Consortium grant (MR/V028448/1).

Human cytomegalovirus (CMV) is a ubiquitous pathogen that latently resides in hematopoietic cells. Latently infected individuals with dysfunctional immune systems often experience CMV reactivation, which can cause devastating disease and mortality. While factors dictating the balance between latency and reactivation are not completely understood, CMV US28 is required for maintaining latent infection, and viral mutants that alter US28 function result in a lytic-like, rather than latent, infection in hematopoietic cells. In turn, viral lytic factors alter the host cell, making it challenging to characterize the US28-specific changes in the cellular milieu. To circumvent this, we generated a temperature-sensitive TB40/E recombinant virus, TB40/EgfpC510G (tsC510G), into which we engineered an amino acid change at position 510 (C510G) of IE2, as previously described in the CMV Towne strain. Using tsC510G, we then deleted the US28 ORF, termed tsC510G-US28Δ. Consistent with previous findings, tsC510G-US28Δ fails to undergo latency in Kasumi-3 cells at the permissive temperature. However, parallel cultures maintained at the non-permissive temperature showed a significant reduction in infectious center frequency, as measured by limiting dilution assay. Thus, we generated a new US28 mutant virus for use as a tool to study US28-specific changes in latently infected hematopoietic cells in the absence of induced lytic replication.

Human cytomegalovirus (HCMV) establishes life-long latent infection in hematopoietic progenitor cells and circulating monocytes in infected individuals. Myeloid differentiation coupled with immune dysregulation leads to viral reactivation, which can cause severe disease and mortality. Reactivation of latent virus requires chromatin reorganization and the removal of transcriptional repressors in exchange for transcriptional activators. While some factors involved in these processes are identified, a complete characterization of the viral and cellular factors involved in their upstream regulation remains elusive. Herein we show the HCMV-encoded G protein-coupled receptor (GPCR), UL33, is expressed during latency. While this viral GPCR is not required to maintain latent infection, our data reveal UL33-mediated signaling is important for efficient viral reactivation. Additionally, UL33 signaling induces cellular cAMP response element binding protein (CREB) phosphorylation, a transcription factor whose recruitment to the major immediate early (MIE) enhancer/promoter promotes reactivation. Finally, targeted pharmacological inhibition of CREB activity reverses the reactivation phenotype of the UL33 signaling deficient mutant. In sum, our data reveal UL33-mediated signaling functions to activate CREB, resulting in successful viral reactivation.

National Institute for Health and Care Excellence (NICE) guidelines define Long COVID as signs and symptoms that develop during or after an infection consistent with COVID-19, that continue for more than 12 weeks and are not explained by an alternative diagnosis. Long COVID is as yet poorly understood and difficult to diagnose. The diagnostic complexity of Long COVID is compounded in many patients who were infected with SARS-CoV-2 but not tested at acute presentation and are antibody negative. Given the diagnostic conundrum of Long COVID, we set out to design a SARS-CoV-2 specific T cell assay, to follow up a cohort of undifferentiated mostly non-hospitalised patients with Long COVID for up to 13 months. Here, we show that IL-2 release from SARS-CoV-2-specific memory T cells shows >75% sensitivity and >88% specificity in identifying individuals with confirmed SARS-CoV-2 infection >6 months after a positive PCR test.

Viral latency is an active process during which the host cell environment is optimized for latent carriage and reactivation. This requires control of both viral and host gene promoters and enhancers often at the level of chromatin, and several viruses co-opt the chromatin organiser CTCF to control gene expression during latency. While CTCF has a role in the latencies of alpha- and gamma-herpesviruses, it was not known whether CTCF played a role in the latency of the beta-herpesvirus human cytomegalovirus (HCMV). Here, we show that HCMV latency is associated with increased CTCF expression and CTCF binding to the viral major lytic promoter, the major immediate early promoter (MIEP). This increase in CTCF binding is dependent on the virally encoded G protein coupled receptor, US28, and contributes to suppression of MIEP-driven transcription, a hallmark of latency. Furthermore, we show that latency-associated upregulation of CTCF represses expression of the neutrophil chemoattractants S100A8 and S100A9 which we have previously shown are downregulated during HCMV latency. As with downregulation of the MIEP, CTCF binding to the enhancer region of S100A8/A9 drives their suppression, again in a US28-dependent manner. Taken together, we identify CTCF upregulation as an important mechanism for optimizing latent carriage of HCMV at both the levels of viral and cellular gene expression.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continues to evolve. We previously showed the HCMV-encoded G proteincoupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to WT. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Furthermore, AP-1 is critical for derepression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency.