Comparison of Whole Blood and Plasma for Monitoring Cytomegalovirus and Epstein-Barr Virus.

Monitoring Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection after transplantation is recommended to enable preemptive therapy. However, the most suitable sample type remains unclear. Patients who underwent hematopoietic stem cell or liver transplantation were included in this study. Viral loads in sequential whole-blood and plasma samples were retrospectively analyzed. EBV DNA was detected more frequently in whole blood (55%) than in plasma (18%). The detection rate of CMV DNA was similar between the two sample types. The correlation of viral loads between the two sample types were 0.515 and 0.688 for EBV and CMV, respectively. Among paired samples in which EBV DNA was detected in whole blood, the plasma EBV detection rate was significantly higher in patients who underwent hematopoietic stem cell transplantation than in those who underwent liver transplantation. The viral DNA load in whole blood and plasma showed similar trends. The EBV detection rate was higher in whole blood, and a high correlation was observed between CMV DNA loads and whole blood and plasma. These results indicate that whole blood is more sensitive for monitoring both EBV and CMV, whereas plasma is a potential alternative sample for monitoring CMV.

An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Cytomegalovirus drives Vδ1+ γδ T cell expansion and clonality in common variable immunodeficiency.

The function and phenotype of γδ T cells in the context of common variable immunodeficiency (CVID) has not been explored. CVID is a primary immunodeficiency disorder characterized by impaired antibody responses resulting in increased susceptibility to infections. γδ T cells are a subset of unconventional T cells that play crucial roles in host defence against infections. In this study, we aim to determine the roles and functions of γδ T cells in CVID. We observe a higher frequency of Vδ1+ γδ T cells compared to healthy controls, particularly in older patients. We also find a higher proportion of effector-memory Vδ1+ γδ T cells and a more clonal T cell receptor (TCR) repertoire in CVID. The most significant driver of the Vδ1+ γδ T cell expansion and phenotype in CVID patients is persistent cytomegalovirus (CMV) viremia. These findings provide valuable insights into γδ T cell biology and their contribution to immune defence in CVID.

Universal and Expanded Screening Strategy for Congenital Cytomegalovirus Infection: Is Pool Testing by a Rapid Molecular Test in Saliva a New Choice in Developing Countries?

BACKGROUND: Several screening strategies for identifying congenital CMV (cCMV) have been proposed; however, the optimal solution has yet to be determined. We aimed to determine the prevalence of cCMV by universal screening with saliva pool testing and to identify the clinical variables associated with a higher risk of cCMV to optimize an expanded screening strategy. METHODS: We carried out a prospective universal cCMV screening (September/2022 to August/2023) of 2186 newborns, analyzing saliva samples in pools of five (Alethia-LAMP-CMV®) and then performed confirmatory urine CMV RT-PCR. Infants with risk factors (small for gestational age, failed hearing screening, HIV-exposed, born to immunosuppressed mothers, or <1000 g birth weight) underwent expanded screening. Multivariate analyses were used to assess the association with maternal/neonatal variables. RESULTS: We identified 10 infants with cCMV (prevalence: 0.46%, 95% CI 0.22-0.84), with significantly higher rates (2.1%, 95% CI 0.58-5.3) in the high-risk group (p = 0.04). False positives occurred in 0.09% of cases. No significant differences in maternal/neonatal characteristics were observed, except for a higher prevalence among infants born to non-Chilean mothers (p = 0.034), notably those born to Haitian mothers (1.5%, 95% CI 0.31-4.34), who had higher odds of cCMV (OR 6.82, 95% CI 1.23-37.9, p = 0.04). Incorporating maternal nationality improved predictive accuracy (AUC: 0.65 to 0.83). CONCLUSIONS: For low-prevalence diseases such as cCMV, universal screening with pool testing in saliva represents an optimal and cost-effective approach to enhance diagnosis in asymptomatic patients. An expanded screening strategy considering maternal nationality could be beneficial in resource-limited settings.

Characterization of Natural Killer Cell Profile in a Cohort of Infected Pregnant Women and Their Babies and Its Relation to CMV Transmission.

Human cytomegalovirus (CMV) is a common herpesvirus causing lifelong latent infection in most people and is a primary cause of congenital infection worldwide. Given the role of NK cells in the materno-fetal barrier, we investigated peripheral blood NK cell behavior in the context of CMV infection acquired during pregnancy. We analyzed the NK phenotype and CD107a surface mobilization on PBMCs from CMV-transmitting and non-transmitting mothers and newborns with or without congenital infection. NK cells from non-transmitting mothers showed the typical phenotype of CMV-adaptive NK cells, characterized by higher levels of NKG2C, CD57, and KIRs, with reduced NKG2A, compared to transmitting ones. A significantly higher percentage of DNAM-1+, PD-1+, and KIR+NKG2A-CD57+PD-1+ CD56dim cells was found in the non-transmitting group. Accordingly, NK cells from congenital-CMV (cCMV)-infected newborns expressed higher levels of NKG2C and CD57, with reduced NKG2A, compared to non-congenital ones. Furthermore, they showed a significant expansion of CD56dim cells co-expressing NKG2C and CD57 or with a memory-like (KIR+NKG2A-CD57+NKG2C+) phenotype, as well as a significant reduction of the CD57-NKG2C- population. Degranulation assays showed a slightly higher CD107a geomean ratio in NK cells of mothers who were non-transmitting compared to those transmitting the virus. Our findings demonstrate that both CMV-transmitting mothers and cCMV newborns show a specific NK profile. These data can guide studies on predicting virus transmission from mothers and congenital infection in infants.

Immunomonitoring via ELISPOT Assay Reveals Attenuated T-Cell Immunity to CMV in Immunocompromised Liver-Transplant Patients.

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool (p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

A UL26-PIAS1 complex antagonizes anti-viral gene expression during Human Cytomegalovirus infection.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

Association of Human Papilloma Virus, Cytomegalovirus, and Epstein-Barr Virus with Breast Cancer in Jordanian Women.

Background and Objectives: The investigation of oncogenic viruses and their potential association with breast cancer (BC) remains an intriguing area of study. The current work aims to assess evidence of three specific viruses, human papillomavirus (HPV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) in BC samples and to explore their relationship with relevant clinicopathological variables. Materials and Methods: The analysis involved BC samples from 110 Jordanian female patients diagnosed with BC and breast tissue samples from 30 control patients with no evidence of breast malignancy, investigated using real-time PCR. The findings were then correlated with various clinico-pathological characteristics of BC. Results: HPV was detected in 27 (24.5%), CMV in 15 (13.6%), and EBV in 18 (16.4%) BC patients. None of the control samples was positive for HPV or CMV while EBV was detected in only one (3.3%) sample. While (HPV/EBV), (HPV/CMV), and (EBV/CMV) co-infections were reported in 1.8%, 2.7%, and 5.5%, respectively, coinfection with the three viruses (HPV/CMV/EBV) was not reported in our cohort. A statistically significant association was observed between HPV status and age (p = 0.047), and between clinical stage and CMV infection (p = 0.015). Conclusions: Our findings indicate the presence or co-presence of HPV, CMV, and EBV in the BC subpopulation, suggesting a potential role in its development and/or progression. Further investigation is required to elucidate the underlying mechanisms that account for the exact role of oncoviruses in breast carcinogenesis.

Analytical and Clinical Performance of the NeuMoDx™ Platform for Cytomegalovirus and Epstein-Barr Virus Viral Load Testing.

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

Diagnostic Utility of Cytomegalovirus (CMV) DNA Quantitation in Ulcerative Colitis.

Cytomegalovirus (CMV) colitis is a critical condition associated with severe complications in ulcerative colitis (UC). This study aimed to investigate the diagnostic value of the presence of CMV DNA in intestinal mucosa tissue and blood samples in patients with active UC. This study included 81 patients with exacerbated symptoms of UC. Patient data were obtained from the Hospital Information Management System. CMV DNA in colorectal tissue and plasma samples were analyzed using a real-time quantitative PCR assay. CMV markers were detected using immunohistochemistry and hematoxylin-eosin staining. Immunohistochemistry positivity was observed in tissue samples from eight (9.9%) patients. Only one (1.2%) patient showed CMV-specific intranuclear inclusion bodies. CMV DNA was detected in 63.0% of the tissues (median: 113 copies/mg) and in 58.5% of the plasma samples (median: 102 copies/mL). For tissues, sensitivity and the negative predictive value (NPV) for qPCR were excellent (100.0%), whereas specificity and the positive predictive value (PPV) were low (41.9% and 15.7%, respectively). For plasma, sensitivity and NPV were high (100.0%) for qPCR, whereas specificity and PPV were low (48.6% and 24.0%, respectively). CMV DNA ≥392 copies/mg in tissue samples (sensitivity 100.0% and specificity 83.6%) and ≥578 copies/mL (895 IU/mL) in plasma samples (sensitivity 66.7% and specificity 100.0%) provided an optimal diagnosis for this test. The qPCR method improved patient management through the early detection of CMV colitis in patients with UC. However, reliance on qPCR positivity alone can lead to overdiagnosis. Quantification of CMV DNA can improve diagnostic specificity, although standardization is warranted.

Evaluation of Alinity m CMV assay performance for detecting CMV in plasma, cerebrospinal fluid, and bronchoalveolar lavage specimens.

Accurate detection and quantification of cytomegalovirus (CMV) is crucial to preventing adverse outcomes in immunocompromised individuals. Current assays were developed for use with plasma specimens, but CMV may be present in bronchoalveolar lavage (BAL) fluid and cerebrospinal fluid (CSF). We evaluated the performance of the Abbott Alinity m CMV assay compared to the Abbott RealTime CMV assay for quantification of CMV in plasma, BAL, and CSF specimens. To evaluate clinical performance, 190 plasma, 78 BAL, and 20 CSF specimens were tested with the Alinity m assay and compared to the RealTime assay. The Alinity m CMV assay showed high precision (SD <0.01 to 0.13) for all 3 specimen types. Clincal plasma and BAL specimens with quantifiable CMV DNA demonstrated strong correlation to RealTime CMV assay results (r2 = 0.9779 for plasma, r2 = 0.9373 for BAL). The Alinity m CMV assay may be useful for quantification of CMV in plasma, BAL, and CSF specimens.

Prevalence and risk factors of cytomegalovirus reactivation in critically Ill patients with COVID-19 pneumonia.

BACKGROUNDS: In critically ill patients with COVID-19, secondary infections are potentially life-threatening complications. This study aimed to determine the prevalence, clinical characteristics, and risk factors of CMV reactivation among critically ill immunocompetent patients with COVID-19 pneumonia. METHODS: A retrospective cohort study was conducted among adult patients who were admitted to ICU and screened for quantitative real-time PCR for CMV viral load in a tertiary-care hospital during the third wave of the COVID-19 outbreak in Thailand. Cox regression models were used to identify significant risk factors for developing CMV reactivation. RESULTS: A total of 185 patients were studied; 133 patients (71.9%) in the non-CMV group and 52 patients (28.1%) in the CMV group. Of all, the mean age was 64.7±13.3 years and 101 patients (54.6%) were males. The CMV group had received a significantly higher median cumulative dose of corticosteroids than the non-CMV group (301 vs 177 mg of dexamethasone, p<0.001). Other modalities of treatments for COVID-19 including anti-viral drugs, anti-cytokine drugs and hemoperfusion were not different between the two groups (p>0.05). The 90-day mortality rate for all patients was 29.1%, with a significant difference between the CMV group and the non-CMV group (42.3% vs. 24.1%, p = 0.014). Median length of stay was longer in the CMV group than non-CMV group (43 vs 24 days, p<0.001). The CMV group has detectable CMV DNA load with a median [IQR] of 4,977 [1,365-14,742] IU/mL and 24,570 [3,703-106,642] in plasma and bronchoalveolar fluid, respectively. In multivariate analysis, only a cumulative corticosteroids dose of dexamethasone ≥250 mg (HR = 2.042; 95%CI, 1.130-3.688; p = 0.018) was associated with developing CMV reactivation. CONCLUSION: In critically ill COVID-19 patients, CMV reactivation is frequent and a high cumulative corticosteroids dose is a significant risk factor for CMV reactivation, which is associated with poor outcomes. Further prospective studies are warranted to determine optimal management.

Adequate antiviral treatment lowers overall complications of cytomegalovirus colitis among inpatients with inflammatory bowel diseases.

BACKGROUND: Cytomegalovirus (CMV) colitis significantly complicates the course of inflammatory bowel disease (IBD), frequently leading to severe flare-ups and poor outcomes. The role of antiviral therapy in hospitalized IBD patients with CMV colitis is currently under debate. This retrospective analysis seeks to clarify the influence of antiviral treatment on these patients. METHODS: We retrospectively reviewed IBD patients diagnosed with CMV colitis via immunohistochemistry staining from colonic biopsies at a major tertiary center from January 2000 to May 2021. The study focused on patient demographics, clinical features, risk factors, prognostic indicators, and antiviral treatment outcomes. RESULTS: Among 118 inpatients, 42 had CMV colitis. Risk factors included hypoalbuminemia and antibiotic use. IBD patients with CMV colitis receiving < 14 days of antiviral therapy had higher complication (72% vs. 43%, p = 0.028) and surgery rates (56% vs. 26%, p = 0.017) compared to those without CMV. Adequate antiviral therapy (≥ 14 days) significantly reduced complications in the CMV group (29% vs. 72%, p = 0.006), especially in Crohn's disease (20% vs. 100%, p = 0.015). Independent predictors of IBD-related complications were CMV colitis (Odds Ratio [OR] 3.532, 90% Confidence Interval [CI] 1.012-12.331, p = 0.048), biological treatment failure (OR 4.953, 95% CI 1.91-12.842, p = 0.001), and adequate antiviral therapy (OR 0.108, 95% CI 0.023-0.512, p = 0.005). CONCLUSION: CMV colitis and a history of biological treatment failure increase complication risks in IBD patients. Adequate antiviral therapy significantly mitigates these risks, highlighting its importance in managing IBD patients with CMV colitis.

Mesenchymal Stem Cell-Derived Exosomes Attenuate Murine Cytomegalovirus-Infected Pneumonia via NF-κB/NLRP3 Signaling Pathway.

Reactivation and infection with cytomegalovirus (CMV) are frequently observed in recipients of solid organ transplants, bone marrow transplants, and individuals with HIV infection. This presents an increasing risk of allograft rejection, opportunistic infection, graft failure, and patient mortality. Among immunocompromised hosts, interstitial pneumonia is the most critical clinical manifestation of CMV infection. Recent studies have demonstrated the potential therapeutic benefits of exosomes derived from mesenchymal stem cells (MSC-exos) in preclinical models of acute lung injury, including pneumonia, ARDS, and sepsis. However, the role of MSC-exos in the pathogenesis of infectious viral diseases, such as CMV pneumonia, remains unclear. In a mouse model of murine CMV-induced pneumonia, we observed that intravenous administration of mouse MSC (mMSC)-exos reduced lung damage, decreased the hyperinflammatory response, and shifted macrophage polarization from the M1 to the M2 phenotype. Treatment with mMSC-exos also significantly reduced the infiltration of inflammatory cells and pulmonary fibrosis. Furthermore, in vitro studies revealed that mMSC-exos reversed the hyperinflammatory phenotype of bone marrow-derived macrophages infected with murine CMV. Mechanistically, mMSC-exos treatment decreased activation of the NF-κB/NLRP3 signaling pathway both in vivo and in vitro. In summary, our findings indicate that mMSC-exo treatment is effective in severe CMV pneumonia by reducing lung inflammation and fibrosis through the NF-κB/NLRP3 signaling pathway, thus providing promising therapeutic potential for clinical CMV infection.

Non-Detection of HCMV Total Genomic DNA in Human Glioma Cells Genome.

AIM: To demonstrate if the human cytomegalovirus (HCMV) genome, that is involved in the pathogenesis of gliomas, is part of the genomic DNA of glioma cells or not. MATERIAL AND METHODS: The study included U87MG glioblastoma cell culture and tumor samples from glioma patients. The genomic DNA of tumor samples and U87MG cells were extracted and real-time quantitative PCR was used to assess the presence of the human cytomegalovirus genomic DNA. RESULTS: Consequently, HCMV positivity was not detected in the tumor and cell line genomic DNA under the aforementioned experimental conditions. CONCLUSION: We found that the genomic DNA of all the samples was negative for HCMV genomic DNA. Thus, HCMV could not be detected in human glioma tumors and we put forward that HCMV genomic DNA was not incorporated into the genomic DNA of glioma cells. Thus, total viral DNA is not involved in the pathogenesis of glioma; however, small viral particles or specific genes might be incorporated into the genomic DNA of glioma cells, leading to cancer development. This prompts further studies for verification.

The association of Torque Teno viral load with CMV and BKV infection in pediatric and adolescent kidney transplant patients.

BACKGROUND: Long-term allograft and patient survival after kidney transplantation (KTX) depends on the balance between over- and under-immunosuppression (IS). High levels of IS predispose to opportunistic infections. Plasma load of Torque Teno Virus (TTV), a non-pathogenic highly prevalent Annellovirus, is associated with its hosts immune status, especially after solid organ transplantation. OBJECTIVES: To investigate the association of plasma TTV load and opportunistic viral infections after pediatric KTX. STUDY DESIGN: This retrospective study includes all pediatric KTX patients followed at the Medical University of Vienna 2014-2020. PCR for Cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), and TTV was performed every 4-8 weeks at routine follow-up visits. RESULTS: 71 pediatric KTX patients were followed with TTV measurements for a median of 2.7 years. TTV plasma load was associated with CMV DNAemia at the next visit with an OR of 2.37 (95 % CI 1.15-4.87; p = 0.03) after adjustment for time after KTX and recipient age. For a cut-off of 7.68 log10 c/mL TTV a sensitivity of 100 %, a specificity of 61 %, a NPV 100 %, and a PPV of 46 % to detect CMV DNAemia at the next visit was calculated. TTV plasma loads were also associated with BKV DNAuria and BKV DNAemia at the next visit, but not with EBV DNAemia. CONCLUSIONS: This is the first study to analyse associations between TTV plasma loads and opportunistic viral infections in pediatric KTX. We were able to present a TTV cut-off for the prediction of clinically relevant CMV DNAemia that might be useful in clinical care.

SARS-CoV-2 superinfection in CD14+ monocytes with latent human cytomegalovirus (HCMV) promotes inflammatory cascade.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), has posed significant challenges to global health. While much attention has been directed towards understanding the primary mechanisms of SARS-CoV-2 infection, emerging evidence suggests co-infections or superinfections with other viruses may contribute to increased morbidity and mortality, particularly in severe cases of COVID-19. Among viruses that have been reported in patients with SARS-CoV-2, seropositivity for Human cytomegalovirus (HCMV) is associated with increased COVID-19 risk and hospitalization. HCMV is a ubiquitous beta-herpesvirus with a seroprevalence of 60-90 % worldwide and one of the leading causes of mortality in immunocompromised individuals. The primary sites of latency for HCMV include CD14+ monocytes and CD34+ hematopoietic cells. In this study, we sought to investigate SARS-CoV-2 infection of CD14+ monocytes latently infected with HCMV. We demonstrate that CD14+ cells are susceptible and permissive to SARS-CoV-2 infection and detect subgenomic transcripts indicative of replication. To further investigate the molecular changes triggered by SARS-CoV-2 infection in HCMV-latent CD14+ monocytes, we conducted RNA sequencing coupled with bioinformatic differential gene analysis. The results revealed significant differences in cytokine-cytokine receptor interactions and inflammatory pathways in cells superinfected with replication-competent SARS-CoV-2 compared to the heat-inactivated and mock controls. Notably, there was a significant upregulation in transcripts associated with pro-inflammatory response factors and a decrease in anti-inflammatory factors. Taken together, these findings provide a basis for the heightened inflammatory response, offering potential avenues for targeted therapeutic interventions among HCMV-infected severe cases of COVID-19. SUMMARY: COVID-19 patients infected with secondary viruses have been associated with a higher prevalence of severe symptoms. Individuals seropositive for human cytomegalovirus (HCMV) infection are at an increased risk for severe COVID-19 disease and hospitalization. HCMV reactivation has been reported in severe COVID-19 cases with respiratory failure and could be the result of co-infection with SARS-CoV-2 and HCMV. In a cell culture model of superinfection, HCMV has previously been shown to increase infection of SARS-CoV-2 of epithelial cells by upregulating the human angiotensin-converting enzyme-2 (ACE2) receptor. In this study, we utilize CD14+ monocytes, a major cell type that harbors latent HCMV, to investigate co-infection of SARS-CoV-2 and HCMV. This study is a first step toward understanding the mechanism that may facilitate increased COVID-19 disease severity in patients infected with SARS-CoV-2 and HCMV.

Virological, innate, and adaptive immune profiles shaped by variation in route and age of host in murine cytomegalovirus infection.

Human cytomegalovirus (hCMV) is a ubiquitous facultative pathogen, which establishes a characteristic latent and reactivating lifelong infection in immunocompetent hosts. Murine CMV (mCMV) infection is widely used as an experimental model of hCMV infection, employed to investigate the causal nature and extent of CMV's contribution to inflammatory, immunological, and health disturbances in humans. Therefore, mimicking natural human infection in mice would be advantageous to hCMV research. To assess the role of route and age at infection in modeling hCMV in mice, we infected prepubescent and young sexually mature C57BL/6 (B6) mice intranasally (i.n., a likely physiological route in humans) and intraperitoneally (i.p., a frequently used experimental route, possibly akin to transplant-mediated infection). In our hands, both routes led to comparable early viral loads and tissue spreads. However, they yielded differential profiles of innate and adaptive systemic immune activation. Specifically, the younger, prepubescent mice exhibited the strongest natural killer cell activation in the blood in response to i.p. infection. Further, the i.p. infected animals (particularly those infected at 12 weeks) exhibited larger anti-mCMV IgG and greater expansion of circulating CD8+ T cells specific for both acute (non-inflationary) and latent phase (inflationary) mCMV epitopes. By contrast, tissue immune responses were comparable between i.n. and i.p. groups. Our results illustrate a distinction in the bloodborne immune response profiles across infection routes and ages and are discussed in light of physiological parameters of interaction between CMV, immunity, inflammation, and health over the lifespan. IMPORTANCE: The majority of experiments modeling human cytomegalovirus (hCMV) infection in mice have been carried out using intraperitoneal infection in sexually mature adult mice, which stands in contrast to the large number of humans being infected with human CMV at a young age, most likely via bodily fluids through the nasopharyngeal/oral route. This study examined the impact of the choice of age and route of infection in modeling CMV infection in mice. By comparing young, prepubescent to older sexually mature counterparts, infected either via the intranasal or intraperitoneal route, we discovered substantial differences in deployment and response intensity of different arms of the immune system in systemic control of the virus; tissue responses, by contrast, appeared similar between ages and infection routes.

Lingering Effects of Early Institutional Rearing and Cytomegalovirus Infection on the Natural Killer Cell Repertoire of Adopted Adolescents.

Adversity during infancy can affect neurobehavioral development and perturb the maturation of physiological systems. Dysregulated immune and inflammatory responses contribute to many of the later effects on health. Whether normalization can occur following a transition to more nurturing, benevolent conditions is unclear. To assess the potential for recovery, blood samples were obtained from 45 adolescents adopted by supportive families after impoverished infancies in institutional settings (post-institutionalized, PI). Their immune profiles were compared to 39 age-matched controls raised by their biological parents (non-adopted, NA). Leukocytes were immunophenotyped, and this analysis focuses on natural killer (NK) cell populations in circulation. Cytomegalovirus (CMV) seropositivity was evaluated to determine if early infection contributed to the impact of an atypical rearing. Associations with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), two cytokines released by activated NK cells, were examined. Compared to the NA controls, PI adolescents had a lower percent of CD56bright NK cells in circulation, higher TNF-α levels, and were more likely to be infected with CMV. PI adolescents who were latent carriers of CMV expressed NKG2C and CD57 surface markers on more NK cells, including CD56dim lineages. The NK cell repertoire revealed lingering immune effects of early rearing while still maintaining an overall integrity and resilience.