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Human cytomegalovirus persists in myeloid progenitors and is passed to the myeloid progeny in a latent form
British Journal of Haematology
Abstract
CD34+ progenitor cells can harbour latent human cytomegalovirus (HCMV); however, the mechanisms of HCMV latency remain unclear. We have investigated the effects of the haematopoietic lineage restriction on the establishment and spread of the latent HCMV to progeny cells. In vitro-infected and latently-infected haematopoietic progenitor cells derived from HCMV seropositive donors were studied. The presence of HCMV DNA in bone marrow progenitor (BMP) cells was determined by single colony polymerase chain reaction and fluorescent in situ hybridization (FISH). The presence of CMV DNA was found to be restricted to myeloid progenitors and the percentage of HCMV-infected cells was lower in naturally-infected cells than in in vitro-infected cells. Erythroid differentiation resulted in an abortive infection with persistence of the viral nucleic acids in red cell precursors. In BMP cells from HCMV seronegative donors, HCMV DNA was localized in the nucleus. Bone marrow progenitors in the presence of granulocyte-macrophage colony stimulating factor (GMCSF) maintained HCMV DNA for extended periods of time. No viral production could be detected throughout the culture but the comparison of the numbers of latently-infected cells prior to and after the culture suggests that proliferation of haematopoietic progenitor cells may lead to the expansion of latently-infected cells.
... Generally, the virus is widespread (Cannon et al., 2010), and serum positive for infection in the general population has been estimated to be 83% (Zuhair et al., 2019). Moreover, seronegativity does not show a complete correlation with negativity for HCMV DNA in CD34 + hematopoietic progenitor cells (HPCs) (Khaiboullina et al., 2004). Transmission of primary infection usually occurs via intrauterine (Boppana and Britt, 1995), breast milk (Hayes et al., 1972), and contaminations exposure routes (such as saliva or genital secretions) (Khaiboullina et al., 2004;Murata et al., 2009). ...
... Moreover, seronegativity does not show a complete correlation with negativity for HCMV DNA in CD34 + hematopoietic progenitor cells (HPCs) (Khaiboullina et al., 2004). Transmission of primary infection usually occurs via intrauterine (Boppana and Britt, 1995), breast milk (Hayes et al., 1972), and contaminations exposure routes (such as saliva or genital secretions) (Khaiboullina et al., 2004;Murata et al., 2009). Reactivation occurs from latency, the sources of which are CD34 + HPCs and CD14 + monocytes (Mendelson et al., 1996), often after blood transfusion and organ transplantation (Zhao et al., 2017). ...
... Viral abilities to re-express lytic genes from the quiescent form in response to certain stimuli are associated with complications such as severe pneumonia and gastrointestinal disease following transplantation (Zhao et al., 2017). CD34 + HPCs and CD14 + monocytes do not provide a platform for viral replication, and undifferentiated cells are considered to be the latent reservoirs of HCMV (Mendelson et al., 1996;Khaiboullina et al., 2004). Nuclear pp71 can be detected by immunofluorescence in normal human dermal fibroblasts (NHDFs), the permissive cells for HCMV lytic replication. ...
Human cytomegalovirus (HCMV), a ubiquitous beta-herpesvirus, is able to establish lifelong latency after initial infection. Periodical reactivation occurs after immunosuppression, remaining a major cause of death in immunocompromised patients. HCMV has to reach a structural and functional balance with the host at its earliest entry. Virion-carried mediators are considered to play pivotal roles in viral adaptation into a new cellular environment upon entry. Additionally, one clear difference between primary infection and reactivation is the idea that virion-packaged factors are already formed such that those molecules can be used swiftly by the virus. In contrast, virion-carried mediators have to be transcribed and translated; thus, they are not readily available during reactivation. Hence, understanding virion-carried molecules helps to elucidate HCMV reactivation. In this article, the impact of virion-packaged molecules on viral structure, biological behavior, and viral life cycle will be reviewed.
... effects on haematopoiesis may reflect a direct inhibition of haematopoietic stem cells and progenitor cells (HSPCs), or alternatively damage to bone marrow (BM) stromal cells that play a supportive role in haematopoiesis [2][3][4]. CD34+ HSPCs constitute a critical reservoir of latent HCMV in seropositive individuals, and latency is maintained when these HSPCs differentiate along the myeloid lineage into monocytes [5][6][7][8][9]. Reactivation of latent viral genomes occur when infected HSPCs and monocytes differentiate into mature dendritic cells or macrophages, and is characterized by the expression of lytic genes and production of infectious viral progeny [10][11][12][13][14]. Due to the species-specificity of HCMV infection, and the limitations of studying molecular mechanisms responsible for reactivation in vivo in human subjects, we and others have utilized murine models to study CMV reactivation in vivo, while HCMV studies remain restricted to cell and tissue culture in vitro models. ...
... Multiple studies have demonstrated that reactivation from latency of HCMV in haematopoietic cells is intrinsically tied to differentiation. While remaining latent in CD34+ HSPCs and monocytes, reactivation occurs when these cells further differentiate into mature macrophages or DCs [7,[10][11][12][13][14]. To test whether MCMV follows a similar pattern of reactivation in mouse BM haematopoietic cells, BM haematopoietic cells were induced to differentiate to cBMMs in the presence of M-CSF or cBMDCe cells in the presence of GM-CSF and IL-4. ...
... In addition, latently infected cells similar to our sBMDCe cells capable of reactivation have been previously derived following acute infection of common precursor cells [58][59][60][61][62][63]. Thus, taken together, our data on the cellular profiles observed for both acute and latent MCMV infection in the murine BM haematopoietic system appear consistent with a number of observations in HCMV models [6,7,52,64,65]. ...
CD34+ myeloid lineage progenitor cells are an important reservoir of latent human cytomegalovirus (HCMV), and differentiation to macrophages or dendritic cells (DCs) is known to cause reactivation of latent virus. Due to its species-specificity, murine models have been used to study mouse CMV (MCMV) latency and reactivation in vivo. While previous studies have shown that MCMV genomic DNA can be detected in the bone marrow (BM) of latently infected mice, the identity of these cells has not been defined. Therefore, we sought to identify and enrich for cellular sites of MCMV latency in the BM haematopoietic system, and to explore the potential for establishing an in vitro model for reactivation of latent MCMV. We studied the kinetics and cellular characteristics of acute infection and establishment of latency in the BM of mice. We found that while MCMV can infect a broad range of haematopoietic BM cells (BMCs), latent virus is only detectable in haematopoietic stem cells (HSCs), myeloid progenitor cells, monocytes and DC-enriched cell subsets. Using three separate approaches, MCMV reactivation was detected in association with differentiation into DC-enriched BMCs cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) followed by lipopolysaccharide (LPS) treatment. In summary, we have defined the kinetics and cellular profile of MCMV infection followed by the natural establishment of latency in vivo in the mouse BM haematopoietic system, including the haematopoietic phenotypes of cells that are permissive to acute infection, establish and harbour detectable latent virus, and can be stimulated to reactivate following DC enrichment and differentiation, followed by treatment with LPS.
... Moreover, while most studies have focused on single viruses (Baranski et al., 1988;Cantin et al., 1994;Cassinotti et al., 1997;Eis-Hübinger et al., 2001;Heegaard et al., 2002;Khaiboullina et al., 2004;Lundqvist et al., 2005;Manning et al., 2007;Rollıń et al., 2007;Corcioli et al., 2008;Tan et al., 2009;Garcia et al., 2011;Matsushita et al., 2013;Pambrun et al., 2014), a comprehensive analysis of the different types concomitantly persisting in the BM of an individual is lacking. This is however important, as their chronic effects, either concerted or combined, may contribute to BM dysfunction. ...
... Our study is limited by the lack of distinction of the specific host-cell types. To date, the cellular reservoirs of most viruses in the BM are actually unknown, although in vitro a wide range of constituent and memory cells have been shown permissive for one or more (Chiara et al., 1996;Movassagh et al., 1996;Bahner et al., 1997;Babcock et al., 1998;Okamoto et al., 2000;Khaiboullina et al., 2004;Luppi et al., 2005;Sundin et al., 2006;Wu et al., 2006;Rollıń et al., 2007;Smirnov et al., 2007;Alexaki and Wigdahl, 2008;Rong et al., 2008;Sundin et al., 2008;Ma et al., 2011;Bua et al., 2016;Furuta et al., 2017;Pyöriä et al., 2017). ...
The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation.
... Studying whether there is a source, and the site, of latent infection within the brain is a novel concept, as most latency studies have focused on hematopoietic progenitors in bone marrow and monocytes in peripheral blood, which are known to be primary sites harboring HCMV in vivo (19,20). In vitro infection studies using primary CD34 ϩ cells have shed light on the establishment, maintenance, and reactivation of HCMV from latency (21)(22)(23)(24)(25)(26)(27)(28). However, due to the difficulty of obtaining and culturing homogeneous populations of these cells, several cell lines have also been utilized. ...
... There are currently several models used to study HCMV latency, including hematopoietic progenitors from bone marrow and peripheral blood monocytes (both known to be primary sites harboring HCMV in vivo) (19,20). Although using primary CD34 ϩ cells for all studies of HCMV latency might be the ideal and much has been learned from these cells (21)(22)(23)(24)(25)(26), there is a very real difficulty in obtaining and culturing large homogeneous populations of these cells. In addition, they cannot be maintained for long periods in culture; neither do they maintain very high copy numbers of viral genomes (72). ...
Unlabelled:
After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection, a leading cause of central nervous system birth defects. We previously reported long-term HCMV infection in the T98G glioblastoma cell line (1). HCMV infection has been further characterized in T98Gs, emphasizing the presence of HCMV DNA over an extended time frame. T98Gs were infected with either HCMV Towne or AD169-IE2-enhanced green fluorescent protein (eGFP) strains. Towne infections yielded mixed IE1 antigen-positive and -negative (Ag(+)/Ag(-)) populations. AD169-IE2-eGFP infections also yielded mixed populations, which were sorted to obtain an IE2(-) (Ag(-)) population. Viral gene expression over the course of infection was determined by immunofluorescent analysis (IFA) and reverse transcription-PCR (RT-PCR). The presence of HCMV genomes was determined by PCR, nested PCR (n-PCR), and fluorescence in situ hybridization (FISH). Compared to the HCMV latency model, THP-1, Towne-infected T98Gs expressed IE1 and latency-associated transcripts for longer periods, contained many more HCMV genomes during early passages, and carried genomes for a greatly extended period of passaging. Large numbers of HCMV genomes were also found in purified Ag(-) AD169-infected cells for the first several passages. Interestingly, latency transcripts were observed from very early times in the Towne-infected cells, even when IE1 was expressed at low levels. Although AD169-infected Ag(-) cells expressed no detectable levels of either IE1 or latency transcripts, they also maintained large numbers of genomes within the cell nuclei for several passages. These results identify HCMV-infected T98Gs as an attractive new model in the study of the long-term maintenance of virus genomes in the context of neural cell types.
Importance:
Our previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.
... The reservoir for latent HCMV is commonly accepted to reside within hematopoietic stem cells within the bone marrow, particularly in undifferentiated cells of the myeloid lineage and monocytes (15,26,28,32,39,55). Several hallmarks define herpesvirus latency, including long-term maintenance of the viral genome coupled with limited viral transcript expression and a lack of detectable productive viral replication (reviewed in reference 8). ...
... Ex vivo latency models that utilize primary CD34 ϩ /CD38 Ϫ hematopoietic progenitor cells (HPCs) isolated from bone marrow or umbilical cord blood (13,14,26,35,42), as well as peripheral blood monocytes (7,16,27,33,50,52,56,58,67), represent perhaps a more complete assessment of HCMV latency. These model systems support HCMV latent infection, and importantly, the latent virus can be reactivated, producing infectious progeny (12,14,16,50,55,56). ...
Human cytomegalovirus (HCMV) is a herpesvirus that establishes a lifelong, latent infection within a host. At times when the immune system is compromised, the virus undergoes a lytic reactivation producing infectious progeny. The identification and understanding of the biological mechanisms underlying HCMV latency and reactivation are not completely defined. To this end, we have developed a tractable in vitro model system to investigate these phases of viral infection using a clonal population of myeloid progenitor cells (Kasumi-3 cells). Infection of these cells results in maintenance of the viral genome with restricted viral RNA expression that is reversed with the addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, also known as PMA). Additionally, a latent viral transcript (LUNA) is expressed at times where viral lytic transcription is suppressed. Infected Kasumi-3 cells initiate production of infectious virus following TPA treatment, which requires cell-to-cell contact for efficient transfer of virus to other cell types. Importantly, lytically infected fibroblast, endothelial, or epithelial cells can transfer virus to Kasumi-3 cells, which fail to initiate lytic replication until stimulated with TPA. Finally, inflammatory cytokines, in addition to the pharmacological agent TPA, are sufficient for transcription of immediate-early (IE) genes following latent infection. Taken together, our findings argue that the Kasumi-3 cell line is a tractable in vitro model system with which to study HCMV latency and reactivation.
... Clearance of infected and virus-producing cells resolves the primary infection, but in some cells viral genomes remain in a quiescent state. In particular, bone marrowresident CD34 pos hematopoietic stem and progenitor cells are a critical reservoir of latent human CMV (HCMV) infection, which is maintained during differentiation into CD14 pos monocytes [13][14][15]. Viral reactivation occurs upon further cellular differentiation and/or under pro-inflammatory conditions [16,17]. Repetitively occurring reactivation over time ensures that the levels of anti-CMV T cells do not contract but are rather maintained and accumulate at high frequencies. ...
Simple Summary
Numerous studies have developed strategies to utilize anti-cytomegalovirus (CMV) T cells for cancer treatment, as they have many beneficial characteristics including extraordinarily high numbers and frequent presence in cancer tissues. In this review, we present multiple strategies that exploit anti-CMV T cells for cancer (immuno)therapy in various ways. We aim to advance the understanding of how anti-CMV T cells can be applied best to further improve treatment outcomes for cancer patients. For this purpose, we identify similarities and discuss benefits, disadvantages, and challenges of each strategy. Finally, we comment on the future directions of this new promising field of cancer (immuno)therapy.
Abstract
Infection with cytomegalovirus (CMV) is highly prevalent in the general population and largely controlled by CD8pos T cells. Intriguingly, anti-CMV T cells accumulate over time to extraordinarily high numbers, are frequently present as tumor-resident ‘bystander’ T cells, and remain functional in cancer patients. Consequently, various strategies for redirecting anti-CMV CD8pos T cells to eliminate cancer cells are currently being developed. Here, we provide an overview of these strategies including immunogenic CMV peptide-loading onto endogenous HLA complexes on cancer cells and the use of tumor-directed fusion proteins containing a preassembled CMV peptide/HLA-I complex. Additionally, we discuss conveying the advantageous characteristics of anti-CMV T cells in adoptive cell therapy. Utilization of anti-CMV CD8pos T cells to generate CAR T cells promotes their in vivo persistence and expansion due to appropriate co-stimulation through the endogenous (CMV-)TCR signaling complex. Designing TCR-engineered T cells is more challenging, as the artificial and endogenous TCR compete for expression. Moreover, the use of expanded/reactivated anti-CMV T cells to target CMV peptide-expressing glioblastomas is discussed. This review highlights the most important findings and compares the benefits, disadvantages, and challenges of each strategy. Finally, we discuss how anti-CMV T cell therapies can be further improved to enhance treatment efficacy.
... The genome comprises two unique regions, unique long and unique short, flanked by repeated sequences [3][4][5]. Following the initial infection, CMV is not cleared from the host and establishes lifelong latent infection in undifferentiated CD34 + stem cells and CD33 + myeloid progenitor cells and the CD14 + monocytes and dendritic cells that they mature into [6,7]. CMV is also latent in other tissues, such as lung [8]. ...
Cytomegalovirus (CMV) is a human herpes virus that causes significant morbidity and mortality in immunosuppressed children. CMV primary infection causes a clinically mild disease in healthy children, usually in early childhood; the virus then utilises several mechanisms to establish host latency, which allows for periodic reactivation, particularly when the host is immunocompromised. It is this reactivation that is responsible for the significant morbidity and mortality in immunocompromised children. We review CMV infection in the primary immunodeficient host, including early identification of these infants by newborn screening to allow for CMV infection prevention strategies. Furthermore, clinical CMV is discussed in the context of children treated with secondary immunodeficiency, particularly paediatric cancer patients and children undergoing haematopoietic stem cell transplant (HSCT). Treatments for CMV are highlighted and include CMV immunotherapy.
... Most patients are latently infected with CMV. CMV pro-liferates in hematopoietic cells and spreads to various organs via the vascular endothelium under immunocompromised conditions (34). The diagnosis of CMV colitis is based on gastrointestinal symptoms, the presence of gastrointestinal erosions or ulcers during endoscopy, and tissue or serological proof of the virus (35). ...
Pneumocystis jirovecii pneumonia (PJP) and cytomegalovirus (CMV) colitis are opportunistic infections that occur during immunosuppressive treatments for ulcerative colitis (UC). The prognosis of PJP and CMV colitis is very poor. We herein report a rare case of a 74-year-old UC patient with PJP and CMV colitis that was successfully treated with intensive therapy. PJP progresses rapidly, so the timing and choice of treatment are critical. Furthermore, a literature review of similar cases suggested that prophylactic therapy for opportunistic infections might be important, especially in the elderly. This case will serve as a reference for successful treatment in future cases.
... n INTRODUCTION C ytomegalovirus (CMV), a human betaherpesvirus, has an estimated worldwide seroprevalence of 45% to 100% increasing with age [1]. Primary infection usually occurs asymptomatically in immunocompetent subjects, and the virus remains in its latent form in CD34 + Hematopoietic Stem Cells (HSCs) and in CD33 + myeloid progenitors [2,3]; however, only myeloid dendritic cells and monocytes can productively spread viral particles leading to CMV reactivation [4]. Immunosuppression is one of the major causes of reactivation, and immunocompromised subjects, such as solid-organ and HSC transplanted patients, can experience a more aggressive disease with hepatitis, severe pneumonia, central and peripheral nervous system manifestations, myelosuppression, and graft rejection [5][6][7]. ...
Cytomegalovirus (CMV) reactivation is a major cause of morbidity and mortality after organ or hemato-poietic stem cell transplantation (HSCT). Letermo-vir (LTV) is a novel antiviral agent approved for CMV prophylaxis after allogeneic transplantation. In this single-center real-world study, we evidenced efficacy and safety of LTV for CMV prophylaxis in allogeneic HSCT recipients. A total of 133 consecutive patients who underwent autologous or allogeneic HSCT were included in the study, and a subgroup of 13 allogeneic HSCT recipients received CMV prophylaxis with LTV 240 mg/daily from day +7 to +100 (allo-LTV cohort). All patients in the allo-LTV cohort were at moderate or high risk of reactivation based on donor/recipient serology status, and 62% also received haploidentical HSCT and cyclophosphamide which further increased CMV reactivation risk. CMV infection rate was also compared to that observed in allogeneic HSCT patients without CMV prophylaxis and autologous recipients who have the lowest reported CMV infection incidence and were used as a control cohort. In our ex-SUMMARY perience, patients receiving LTV showed a significant decline in CMV reactivation incidence to similar rates described in autologous HSCT recipients (7.7% of al-logeneic LTV-treated vs 68% of allogeneic recipients without prophylaxis vs 15% of autologous patients; p<0.0001). The only patient in the allo-LTV cohort with CMV reactivation was a 25-year-old female with a diagnosis of very high-risk acute lymphoblastic leuke-mia who received a haploidentical HSCT after ex vivo T cell depletion. CMV reactivation occurred beyond LTV course, at +187 days from transplantation. In addition, we confirmed efficacy and safety of valganciclovir 450 mg/daily as pre-emptive therapy or for treatment of CMV disease in allogeneic and autologous HSCT recipients who experienced CMV reactivation even after LTV prophylaxis. However, further clinical trials in larger populations and longer follow-up are required to confirm our preliminary results.
... Primary CMV infection is followed by lifelong latencycharacterized by the persistence of the viral genome with no production of new infectious viral particlesdue to the ability of the virus to evade the host's immune response. Although CMV has been detected in most tissues and body compartments, the main reservoir is constituted by monocytes and pre-monocytic cells [2] and it has been shown that the virus can reactivate from latency when these cells differentiate into macrophages and myeloid-derived dendritic cells [3,4]. Reactivation of latent infection constitutes one of the major sources of CMV exposure in immunocompromised patients, since this phenomenon is documented in up to 60% of SOT recipients [5,6]. ...
Introduction: Cytomegalovirus (CMV) infection after kidney transplantation (KT) has been implicated in the so-called “indirect effects” attributable to the viral ability to evade host’s immunity and trigger sustained inflammation. Whether CMV exposure contributes to the development of post-transplant atherosclerotic events (AEs) remains controversial.
Areas covered: This review (based on a PubMed/MEDLINE search from database inception to October 2019) summarizes the proposed mechanisms for the role of CMV in atherogenesis, including accelerated immunosenescence, endothelial injury and inflammatory milieu in the vessel wall. Sero-epidemiological evidence linking CMV exposure and cardiovascular disease in the general population is discussed. Finally, we performed a comprehensive review of observational studies investigating the impact of CMV infection on the occurrence of AE after KT, as well as the potential protective effect of antiviral prophylaxis.
Expert opinion: Reviewed studies provide biological plausibility and preliminary clinical evidence pointing to the pathogenic role of CMV in post-transplant atherogenesis. However, no definitive recommendations can be made regarding the use of antiviral prophylaxis to prevent post-transplant AE, since existing evidence is mainly founded on inadequately powered post hoc analyses. Well-designed observational studies should clarify the differential impact of prophylactic or preemptive approaches on the occurrence of CMV-associated post-transplant AE among KT recipients.
... HCMV latency is characterized by maintenance of the viral genome as an intranuclear episome (Bolovan-Fritts et al., 1999) without replication, but with the potential to reactivate to a productive infection. A wide range of cell types support productive infection (Ibanez et al., 1991;Sinzger et al., 2008), but latency appears to be restricted to primitive bone-marrow-resident CD34 + cells and CD33 + myeloid progenitor cells (Mendelson et al., 1996;Hahn et al., 1998;Reeves et al., 2005b), which retain the latent viral genome as they differentiate into peripheral blood CD14 + monocytes and myeloid dendritic cells (mDCs) (Taylor-Wiedeman et al., 1991Hahn et al., 1998;Khaiboullina et al., 2004;Reeves et al., 2005b). A recent study found that CD14 + monocytes expressing the surface marker B7-H4 were a predominant site of latency in peripheral blood of healthy donors (Zhu et al., 2018). ...
Human cytomegalovirus (HCMV) reactivation is a major infectious cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). HCMV is a ubiquitous beta-herpesvirus which asymptomatically infects immunocompetent individuals but establishes lifelong latency, with the potential to reactivate to a life-threatening productive infection when the host immune system is suppressed or compromised. Opportunistic HCMV reactivation is the most common viral complication following engraftment after HSCT and is associated with a marked increase in non-relapse mortality, which appears to be linked to complex effects on post-transplant immune recovery. This minireview explores the cellular sites of HCMV latency and reactivation in HSCT recipients and provides an overview of the risk factors for HCMV reactivation post-HSCT. The impact of HCMV in shaping post-transplant immune reconstitution and its relationship with patient outcomes such as relapse and graft-versus-host disease will be discussed. Finally, we survey current and emerging strategies to prevent and control HCMV reactivation in HSCT recipients, with recent developments including adoptive T cell therapies to accelerate HCMV-specific T cell reconstitution and new anti-HCMV drug therapy for HCMV reactivation after HSCT.
... 10-15% of the subclinically infected infants subsequently develop late-onset sequelae including sensorineural hearing loss, mental retardation and learning disabilities within 3 years (Leung et al., 2003). HCMV reactivation has largely focused on hematopoietic stem cells (Goodrum et al., 2002;Hahn et al., 1998a;Khaiboullina et al., 2004;Kondo et al., 1994;Mendelson et al., 1996;Soderberg-Naucler et al., 2001). Certainly late-onset neurodevelopmental disorders could be caused by virus reactivation from the myeloid reservoirs, persistent infection, or a new lytic infection. ...
T98G cells have been shown to support long-term human cytomegalovirus (HCMV) genome maintenance without infectious virus release. However, it remains unclear whether these viral genomes could be reactivated. To address this question, a recombinant HCMV (rHCMV) containing a GFP gene was used to infect T98G cells, and the infected cells absent of infectious virus production were designated T98G-LrV. Upon dibutyryl cAMP plus IBMX (cAMP/IBMX) treatment, a serial of phenomena were observed, including GFP signal increase, viral genome replication, lytic genes expression and infectious viruses release, indicating the reactivation of HCMV in T98G-LrV cells from a latent status. Mechanistically, HCMV reactivation in the T98G-LrV cells induced by cAMP/IBMX was associated with the PKA-CREB signaling pathway. These results demonstrate that HCMV was latent in T98G-LrV cells and could be reactivated. The T98G-LrV cells represent an effective model for investigating the mechanisms of HCMV reactivation from latency in the context of neural cells.
... The first studies that proposed a role of PML-NBs directly in the control of incoming viral genomes came from Gerd Maul's laboratory Ishov et al. 1997). Using immuno-FISH, the authors showed that PML-NBs were sites of viral DNA deposition in cells infected by adenovirus Ad5, polyomavirus SV40, HSV-1, or human cytomegalovirus (HCMV) [HCMV genomes have also been detected around the nucleolus in CD34þ bone marrow myeloid progenitor cells, but no significance to this particular localisation has been proposed (Khaiboullina et al. 2004)]. Rather than inducing negative regulation of viral genome activity, PML-NBs were found as the primary sites of viral DNA replication and/or transcription for all four viruses. ...
The nucleus is composed of multiple compartments and domains, which directly or indirectly influence many cellular processes including gene expression, RNA splicing and maturation, protein post-translational modifications, and chromosome segregation. Nuclear-replicating viruses, especially herpesviruses, have co-evolved with the cell, adopting strategies to counteract and eventually hijack this hostile environment for their own benefit. This allows them to persist in the host for the entire life of an individual and to ensure their maintenance in the target species. Herpesviruses establish latency in dividing or postmitotic cells from which they can efficiently reactivate after sometimes years of a seemingly dormant state. Therefore, herpesviruses circumvent the threat of permanent silencing by reactivating their dormant genomes just enough to escape extinction, but not too much to avoid life-threatening damage to the host. In addition, herpesviruses that establish latency in dividing cells must adopt strategies to maintain their genomes in the daughter cells to avoid extinction by dilution of their genomes following multiple cell divisions. From a biochemical point of view, reactivation and maintenance of viral genomes in dividing cells occur successfully because the viral genomes interact with the nuclear architecture in a way that allows the genomes to be transmitted faithfully and to benefit from the nuclear micro-environments that allow reactivation following specific stimuli. Therefore, spatial positioning of the viral genomes within the nucleus is likely to be essential for the success of the latent infection and, beyond that, for the maintenance of herpesviruses in their respective hosts.
... In the natural setting, it is estimated that a very small number of myeloid progenitor cells harbor latent HCMV (0.004 to 0.01%) of total bone marrow or mobilized mononuclear cells 66 and is more prominent in bone marrow CD34 1 cells, where the frequency ranges from 0.02% to 0.1%. 67 Thus, it may be difficult to identify coinfected cells in natural specimens, especially in normal PB. It is postulated that nondetectable HCMV reactivation and replication can lead to new infections in other cell types and in turn repopulate the latent viral genome in CD34 1 cells. ...
Key Points
HCMV latency modulates host CD34+ cells in favoring HIV-1 infection. Latent HCMV upregulates HIV entry coreceptors and downregulates HIV restriction factors in CD34+ cells.
... Concerning HCMV latent reservoirs, peripheral blood monocytes have been shown to be one of the most relevant cell populations harbouring latent HCMV in vivo; furthermore, HCMV latency sites have also been identified within hematopoietic stem cells in the bone marrow, particularly in undifferentiated cells of the myeloid lineage [19][20][21][22][23][24][25][26][27]. ...
Background
Human cytomegalovirus (HCMV) is an opportunistic pathogen leading to severe and even fatal diseases in ‘at-risk’ categories of individuals upon primary infection or the symptomatic reactivation of the endogenous virus. The mechanisms which make the virus able to reactivate from latency are still matter of intense study. However, the very low number of peripheral blood monocytes (an important latent virus reservoir) harbouring HCMV DNA makes it very difficult to obtain adequate viral quantities to use in such studies.Thus, the aim of the present study was to demonstrate the usefulness of human THP-1 monocytes, mostly employed as HCMV latent or lytic infection system, as a reactivation model. MethodsTHP-1 monocytes were infected with HCMV TB40E strain (latency model) at multiplicities of infection (MOI) of 0.5, 0.25 or 0.125. After infection, THP-1 aliquots were differentiated into macrophages (reactivation model). Infections were carried out for 30 h, 4, 6 and 7 days. Viral DNA evaluation was performed with viable and UV-inactivated virus by q-Real-Time PCR. RNA extracted from latency and reactivation models at 7 days post-infection (p.i.) was subjected to RT-PCR to analyse viral latency and lytic transcripts. To perform viral progeny analysis and titration, the culture medium from infected THP-1 latency and reactivation models (7 days p.i.) was used to infect human fibroblasts; it was also checked for the presence of exosomes.For viral progeny analysis experiments, the Towne strain was also used. ResultsOur results showed that, while comparable TB40E DNA amounts were present in both latent and reactivation models at 30 h p.i., gradually increased quantities of viral DNA were only evident in the latter model at 4, 6, 7 days p.i.. The completion of the lytic cycle upon reactivation was also proved by the presence of HCMV lytic transcripts and an infectious viral yield at 7 days p.i. Conclusions
Our data demonstrate the effectiveness of THP-1 cells as a “switch” model for studying the mechanisms that regulate HCMV reactivation from latency. This system is able to provide adequate quantities of cells harbouring latent/reactivated virus, thereby overcoming the intrinsic difficulties connected to the ex vivo system.
... where, in general, the majority of lytic cycle-associated genes are robustly repressed, resulting in a lack of infectious virus production 1 . In healthy seropositive individuals, one site of HCMV latency is in the cells of the myeloid lineage where latent genome is found in monocytes and their CD34 + haemopoietic progenitors [2][3][4][5][6] . Despite the fact that haemopoietic cell types derive from CD34 + progenitor cells, HCMV is only detectable in the myeloid cells of the peripheral blood and not lymphocytes 7 . ...
Scientific Reports 6 : Article number: 31205; 10.1038/srep31205 published online: 05 August 2016 ; updated: 26 September 2016 . The original version of this Article contained an error in the spelling of the author Immaculada Montanuy which was incorrectly given as Immaculada Sellart.
... Being present at densities of 20–80 cells/mm 2 in the outermost layers of the oral mucosa (Cruchley et al., 1989Cruchley et al., , 1994 Lasisi et al., 2013; Upadhyay et al., 2013; Omine et al., 2015), LC are among the first cell types to encounter CMV during entry, together with epithelial cells. Their differentiation from CD34 + hematopoietic progenitors (Iijima et al., 2007), a well-known site of CMV latency (Minton et al., 1994; Mendelson et al., 1996; Zhuravskaya et al., 1997; Slobedman and Mocarski, 1999; Khaiboullina et al., 2004), was also shown to be associated with viral genome maintenance (Reeves et al., 2005a,b; Reeves and Sinclair, 2010), and reactivation (Reeves et al., 2005a,b; Reeves and Sinclair, 2010; Huang et al., 2012). Although mucosal LC are conventionally considered immature, their actual maturation status is likely to be more heterogeneous, as the oral mucosa is normally colonized by a variety of microorganisms whose identity and pathogenicity depend on the health and hygiene of the host. ...
Oral mucosal Langerhans cells (LC) are likely to play important roles in host defense against infection by human cytomegalovirus (CMV). We previously showed that in vitro-differentiated immature LC (iLC) populations contain smaller amounts of infected cells but produce higher yields than mature LC (mLC) cultures, obtained by iLC stimulation with fetal bovine serum (FBS), CD40 ligand (CD40L) and lipopolysaccharide (LPS). Here we sought to determine if exposure to select stimuli can improve LC permissiveness to infection, if specific components of the mLC cocktail are responsible for lowering viral yields, if this is due to defects in progeny production or release, and if these restrictions are also effective against reactivated virus. None of the stimuli tested extended the proportion of infected cells to 100%, suggesting that the block to infection onset cannot be fully removed. While CD40L and FBS exerted positive effects on viral progeny production per cell, stimulation with LPS alone or in combination with CD40L was detrimental. Reductions in viral titers were not due to defects in progeny release, and the permissive or restrictive intracellular environment established upon exposure to each stimulus appeared to act in a somewhat similar way towards lytic and latent infections.
... where, in general, the majority of lytic cycle-associated genes are robustly repressed, resulting in a lack of infectious virus production 1 . In healthy seropositive individuals, one site of HCMV latency is in the cells of the myeloid lineage where latent genome is found in monocytes and their CD34 + haemopoietic progenitors [2][3][4][5][6] . Despite the fact that haemopoietic cell types derive from CD34 + progenitor cells, HCMV is only detectable in the myeloid cells of the peripheral blood and not lymphocytes 7 . ...
The successful establishment and maintenance of human cytomegalovirus (HCMV) latency is dependent on the expression of a subset of viral genes. Whilst the exact spectrum and functions of these genes are far from clear, inroads have been made for protein-coding genes. In contrast, little is known about the expression of non-coding RNAs. Here we show that HCMV encoded miRNAs are expressed de novo during latent infection of primary myeloid cells. Furthermore, we demonstrate that miR-UL148D, one of the most highly expressed viral miRNAs during latent infection, directly targets the cellular receptor ACVR1B of the activin signalling axis. Consistent with this, we observed upregulation of ACVR1B expression during latent infection with a miR-UL148D deletion virus (ΔmiR-UL148D). Importantly, we observed that monocytes latently infected with ΔmiR-UL148D are more responsive to activin A stimulation, as demonstrated by their increased secretion of IL-6. Collectively, our data indicates miR-UL148D inhibits ACVR1B expression in latently infected cells to limit proinflammatory cytokine secretion, perhaps as an immune evasion strategy or to postpone cytokine-induced reactivation until conditions are more favourable. This is the first demonstration of an HCMV miRNA function during latency in primary myeloid cells, implicating that small RNA species may contribute significantly to latent infection.
... Herpesviral latency is defined as a stage in which the viral genome exists as an episome within the nucleoplasm in the absence of lytic gene expression and production of infectious virus [1]. It is now generally believed that human cytomegalovirus (HCMV) is able to establish latency in hematopoietic progenitor cells of the myeloid lineage and differentiation of these cells to a mature phenotype is linked to viral reactivation [2][3][4][5][6][7]. The ability of HCMV to establish lifelong infection in humans and to reactivate from latency in immunocompromised individuals leading to devastating disease underscores the importance of understanding the mechanisms that regulate latency and reactivation. ...
Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.
... From these peripheral sites, CMV is thought to reach the circulation and hence the bone marrow, where latency is established in myeloid cells, including CD34 ϩ hematopoietic progenitors (1)(2)(3)(4)(5). Reactivation from latency in the myeloid progeny of these cells is then believed to be the source of newly produced viral particles, which upon amplification in oral epithelial cells are released in the saliva and contribute to CMV horizontal transmission in vivo (6)(7)(8). ...
Unlabelled:
Acquisition of human cytomegalovirus (CMV) usually occurs by contact between contaminated bodily fluids, such as urine and saliva, and host mucosal cells. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of the oral mucosae, where they not only provide a first line of defense against CMV but can easily be targeted by orally administered vaccines, while their bone marrow resident progenitors are important sites of virus latency. In this work, we tracked the progress of infection in CD34(+) progenitor cells, immature LC (iLC), and mature LC (mLC) exposed to the clinical-like strain TB40-BAC4 or to the vaccine strain AD169varATCC, prior to their long-term maintenance under either immature or mature conditions. We show that the genomes of both strains are efficiently maintained in CD34(+) cells during their differentiation into iLC, although this requires the presence of larger amounts of input AD169varATCC DNA. Lipopolysaccharide- and CD40 ligand-induced maturation of iLC derived from latently infected progenitors was not associated with robust viral genome replication and progeny production, while maturation of directly infected iLC increased and prolonged expression of the viral immediate early proteins. While effective replication of viral genomes from both strains occurred only in mLC, both iLC and mLC produced viral progeny, suggesting that both types of LC may contribute to CMV horizontal transmission in vivo.
Importance:
Human CMV is usually acquired via the oral and nasal mucosae. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of these tissues. Understanding how CMV interacts with LC and their hematopoietic progenitors is thus essential to develop innovative means of defense against this virus. Here we show that the genomes of a virulent and an attenuated strain of CMV are maintained in hematopoietic progenitor cells during their differentiation into immature LC and that maturation of these cells by exposure to lipopolysaccharide and CD40 ligand is not sufficient to trigger virus reactivation. While the extents of viral protein expression and genome replication were broadest in directly infected mature LC populations, similar amounts of viral progeny were detected in the supernatants of immature and mature LC, suggesting that these immune cells of the oral mucosa are likely to be important for CMV transmission within the human population.
... Consistent with other members of the Herpesviridae family, HCMV establishes latency following primary infection. Although the molecular aspects of latency and reactivation have not been fully elucidated, latency is maintained in cells of the myeloid lineage, with the viral DNA being present in an extrachromosomal circular form 7 . In vitro, reactivation and triggering of the immediate early promoter is accompanied by chromatin remodeling and activation of histone deacetylase 8 . ...
Human cytomegalovirus remains the leading infectious complication following solid organ transplantation and leads to a range of direct and indirect effects that contribute to patient morbidity. The primary objective of this review is to provide an up-to-date evaluation of the evidence base for the indirect effects of human cytomegalovirus in solid organ transplant recipients. We shall review data from the rat cytomegalovirus model of transplantation and also data from individual studies in human transplantation and controlled and non-con-trolled antiviral intervention trials to argue that the burden of evidence supports a central role for human cytomegalovirus in the indirect effects. In addition, we will review, where data is available, the likely biological mechanisms that underlie the indirect effects after trans-plantation.
... As a latent reservoir of HCMV, hematopoietic progenitor cells of the myeloid lineage are discussed (Sindre et al., 1996;Mendelson et al., 1996;Khaiboullina et al., 2004). Induction of terminal differentiation of these cells results in HCMV reactivation associated with lytic replication and virus production (Taylor-Wiedeman et al., 1994;Soderberg-Naucler et al., 1997). ...
... Quite interestingly, mLC not only support lytic infection, but also viral reactivation from latency [96,97]. In latently-infected CD34 + cells, viral genomes are maintained as nuclear episomes in the absence of lytic gene expression [98][99][100], and the transcriptionally silent MIEP is associated with deacetylated H4 histones and with the transcriptional repressor heterochromatin protein 1 [96,97,101]. CD34 + cell differentiation and maturation into mLC stimulates the acetylation of H4 histones and the release of heterochromatin protein 1 from the MIEP, enabling expression of the IE1/IE2 proteins and, ultimately, the release of viral progeny [96,97,101]. ...
Human CMV infections are a serious source of morbidity and mortality for immunocompromised patients and for the developing fetus. Because of this, the development of new strategies to prevent CMV acquisition and transmission is a top priority. Myeloid dendritic cells (DC) residing in the oral and nasal mucosae are among the first immune cells to encounter CMV during entry and greatly contribute to virus dissemination, reactivation from latency, and horizontal spread. Albeit affected by the immunoevasive tactics of CMV, mucosal DC remain potent inducers of cellular and humoral immune responses against this virus. Their natural functions could thus be exploited to generate long-lasting protective immunity against CMV by vaccination via the oronasal mucosae. Although related, epithelial Langerhans-type DC and dermal monocyte-derived DC interact with CMV in dramatically different ways. Whereas immature monocyte-derived DC are fully permissive to infection, for instance, immature Langerhans-type DC are completely resistant. Understanding these differences is essential to design innovative vaccines and new antiviral compounds to protect these cells from CMV infection in vivo. Copyright © 2014 John Wiley & Sons, Ltd.
... 40 Donors are tested for a specific number of temporarily known pathogens. 41 Bone marrow can contain viruses with a long-term viral latency, such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human parvovirus B19, 42,43 as well as the human T-cell leukemia virus 1. 44 The risk of transmission of Creutzfeldt-Jakob disease (CJD) iscurrently considered low because none of the 469 cases of iatrogenic CJD that have been reported worldwide involved bone allograft. 45 A further study detected JC virus DNA by quantitative polymerase chain reaction in human bone marrow samples to serve as a reservoir for a progressive multifocal leukoencephalopathy. 46 Viruses within a virus family theoretically are able to develop different resistances to chemicals, and the virus types differ in their thermostability. ...
... CMV infects both HSPCs and stromal cells, so that the entire haematopoietic system is a target for CMV dissemination and latency [139]. As a result, myelosuppression can result from both direct inhibition of HSPC growth and indirect impairment of the marrow stromal cell support for HSPC commitment to myeloid lineage development [139][140][141][142][143]. Although the primary site of HBV pathogenic impact occurs in the liver, involvement of bone marrow hematopoietic cells and stromal cells as well as peripheral blood granulocytes has been observed during HBV infection [144][145][146]. ...
Neutropenia frequently occurs in patients with Human immunodeficiency virus (HIV) infection. Causes for neutropenia during HIV infection are multifactoral, including the viral toxicity to hematopoietic tissue, the use of myelotoxic agents for treatment, complication with secondary infections and malignancies, as well as the patient's association with confounding factors which impair myelopoiesis. An increased prevalence and severity of neutropenia is commonly seen in advanced stages of HIV disease. Decline of neutrophil phagocytic defense in combination with the failure of adaptive immunity renders the host highly susceptible to developing fatal secondary infections. Neutropenia and myelosuppression also restrict the use of many antimicrobial agents for treatment of infections caused by HIV and opportunistic pathogens. In recent years, HIV infection has increasingly become a chronic disease because of progress in antiretroviral therapy (ART). Prevention and treatment of severe neutropenia becomes critical for improving the survival of HIV-infected patients.
... 26 One reason for this discrepancy may be because extremely small amount of DNA can be detected by traditional PCR techniques that were utilized such as nested-PCR utilized in the previous study 1 in which 35-50 cycles of amplification followed by a second round of amplification, and HCMV DNA from cells other than the tumor cells were detected. Glioblastoma is often accompanied with reactive gliosis at the edge of the tumor tissue, and microglial cells that originate from bone marrow myeloid cells, which may be persistently infected by HCMV, 27 could have important roles in the development of gliosis. 28 Thus, we consider that these positive PCR results different from ours may be because of relatively smaller content of reactive glial cells in our samples. ...
Recent reports have indicated human cytomegalovirus (HCMV) to be associated with human glioblastoma carcinogenesis. In established examples of viral carcinogenesis, viral DNA and one or more of its products have been detected in most tumor cells of biopsies in the majority of cases. To test whether HCMV is associated with human glioblastoma based on this criterion, we measured the number of viral DNA molecules per cell in both frozen and paraffin-embedded tumor biopsies from 58 patients using real-time quantitative PCR (QPCR). Immunohistochemical and fluorescence in situ hybridization (FISH) to detect HCMV proteins and genome was performed in 10 cases using formalin-fixed paraffin-embedded glioblastoma tissues. Southern blotting using DNA extracted from 4 glioblastoma cell lines together with immunoblotting using the 4 cell lines and 5 glioblastoma tissue samples were also performed. We further confirmed the immunoblot bands using liquid chromatography-tandem mass spectrometry assay.
As a result, HCMV DNA was not detected in the tumor cells from any of the glioblastoma cases by QPCR detecting two different HCMV genes, in clear contrast to samples from patients with HCMV infection. Southern blotting and immunoblotting of cell lines and FISH using paraffin-sections were all negative. However, immunoblotting and immunohistochemistry using tissue samples were partly positive, but HCMV proteins were not detected by proteomic analysis, suggesting false-positivity of the analyses. Since our QPCR analysis could detect 10 copies of HCMV DNA mixed with DNA extracted from 104 HCMV-negative cells, we conclude that HCMV is not persistent, at least in the tumor cells, of developed human glioblastoma.
... Infection is normally asymptomatic; however, the virus can produce severe disease in immunocompromised individuals. Interestingly, CMV latently infects BM cells, particularly those of the myeloid lineage, and these cells may serve as a reservoir for the virus [19][20][21]. The effects of CMV on the BM and haematopoiesis during infection in vivo, however, require further analysis. ...
Cytomegalovirus latently infects myeloid cells; however, the acute effects of the virus on this cell subset are poorly characterised. We demonstrate that systemic cytomegalovirus infection induced rapid activation of monocytes in the bone marrow, characterised by upregulation of CD69, CD11c, Ly6C and M-CSF receptor. Activated bone marrow monocytes were more sensitive to M-CSF and less sensitive to GM-CSF in vitro, resulting in the generation of more macrophages and fewer dendritic cells, respectively. Monocyte activation was also observed in the periphery and resulted in significant accumulation of monocytes in the spleen. MyD88 expression was required within the haematopoietic compartment to initiate monocyte activation and recruitment. However, monocytes lacking MyD88 were activated and recruited in the presence of MyD88-sufficient cells in mixed bone marrow chimeras, indicating that once initiated, the process was MyD88-independent. Interestingly, we found that monocyte activation occurred in the absence of the common inflammatory cytokines, namely type I interferons (IFNs), IL-6, TNF-α, and IL-1 as well as the NLRP3 inflammasome adaptor protein, ASC. We also excluded a role for the chemokine-like protein MCK-2 (m131/129) expressed by MCMV. Taken together, these results challenge the notion that a single inflammatory cytokine mediates activation and recruitment of monocytes in response to infection. This article is protected by copyright. All rights reserved.
... These data are consistent with the view that HCMV latency is established in early progenitor cells in the bone marrow, the viral genome then persists in the myeloid/monocyte compartment in the peripheral blood without detectable viremia, and finally, terminal differentiation of latent monocytes to a macrophage or DC phenotype instigates a chain of events leading to reactivation of the virus in the periphery due to a more favorable transcriptional environment for MIEP activity and that this is likely augmented by inflammatory signaling in vivo (1,46). The paucity of circulating myeloid DCs in the peripheral blood compartment has led many investigators to use in vitro differentiation of early myeloid progenitors to model HCMV reactivation (4,5,(20)(21)(22)(47)(48)(49). However, we wished to determine whether the robust link between HCMV reactivation and experimental differentiation of monocytes to DCs ex vivo was recapitulated in differentiated circulating myeloid DCs in vivo: in that case, direct isolation of the DC compartment directly from peripheral blood of healthy seropositive donors should yield genome-positive cells that can support HCMV reactivation without further ex vivo differentiation. ...
Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong
infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection
and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34+ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous
analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating
blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived
DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly,
are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes
with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory
stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals,
thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.
... Contributing to the success of this pathogen, HCMV establishes latent infections allowing for persistence in the face of robust antiviral immune responses and thus maintains a lifelong presence in its host (1,3). HCMV establishes latency in undifferentiated cells of the myeloid lineage (4)(5)(6)(7)(8)(9). Because viral DNA, but no evidence of productive replication, has been detected in peripheral blood monocytes and in the CD34 ϩ hematopoietic progenitor cells (HPCs) from which they are derived (7,10), it is thought that a CD34 ϩ HPC represents at least one in vivo latent reservoir (4,7). ...
Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections
in undifferentiated cells of the myeloid lineage, such as CD34+ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular
intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels
to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm.
Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34+ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent
infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34+ cells. However, in contrast to what occurs in primary CD34+ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC
inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3
cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections
differ in important parameters from those in primary CD34+ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered
independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed.
... However the cis acting element required for maintenance of the viral genome is unknown. Lifelong HCMV latency is established in myeloid lineage, from bone marrow-derived CD34 (+) progenitors through peripheral blood to CD14 (+) monocytes[26][27][28][29][30][31][32][33]. Latently infected cells contain HCMV DNA without supporting lytic replication although virus can be reactivated and recovered through differentiation[34][35][36][37][38][39][40]. ...
The parameters involved in human cytomegalovirus (HCMV) latent infection in CD14 (+) and CD34 (+) cells remain poorly identified. Using next generation sequencing we deduced the transcriptome of HCMV latently infected CD14 (+) and CD34 (+) cells in experimental as well as natural latency settings. The gene expression profile from natural infection in HCMV seropositive donors closely matched experimental latency models, and included two long non-coding RNAs (lncRNAs), RNA4.9 and RNA2.7 as well as the mRNAs encoding replication factors UL84 and UL44. Chromatin immunoprecipitation assays on experimentally infected CD14 (+) monocytes followed by next generation sequencing (ChIP-Seq) were employed to demonstrate both UL84 and UL44 proteins interacted with the latent viral genome and overlapped at 5 of the 8 loci identified. RNA4.9 interacts with components of the polycomb repression complex (PRC) as well as with the MIE promoter region where the enrichment of the repressive H3K27me3 mark suggests that this lncRNA represses transcription. Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE), which identifies nucleosome-depleted viral DNA, was used to confirm that latent mRNAs were associated with actively transcribed, FAIRE analysis also showed that the terminal repeat (TR) region of the latent viral genome is depleted of nucleosomes suggesting that this region may contain an element mediating viral genome maintenance. ChIP assays show that the viral TR region interacts with factors associated with the pre replication complex and a plasmid subclone containing the HCMV TR element persisted in latently infected CD14 (+) monocytes, strongly suggesting that the TR region mediates viral chromosome maintenance.
... [36][37][38] HCMV DNA follows the myeloid differentiation pathway. 39 Latent HCMV can be reactivated in myeloid DCs from HCMV-seropositive donors after ex vivo differentiation to mature DCs 40 or in macrophages produced by allogeneic stimulation of peripheral blood mononuclear cells (PBMCs). 41 The inflammatory cytokines IFN-c and TNF-a produced by allostimulated T cells are important for the reactivation of latent virus and the growth of HCMV in differentiated macrophages. ...
Human cytomegalovirus (HCMV), a member of the herpesvirus family, establishes life-long persistence and latency after primary infection and can be reactivated later in life. In immunosuppressed patients, it is an important pathogen that can cause severe disease. HCMV is also thought to play a causative role in inflammatory diseases and cancer. The virus can infect different immune cells, including dendritic cells (DCs) and can take advantage of host immune functions to avoid immune recognition. These characteristics have sparked major interest in understanding HCMV and its interaction with immune cells and their relevance to disease pathogenesis. In this review, we focus on the complex host-pathogen relationship between HCMV and DCs, including the persistence of the virus in these cells, their function in the immune response to HCMV infection and the potential clinical consequences of HCMV infection in DCs.
... After treatment with phorbol esters, THP-1 cells differentiate into macrophage-like cells, which mimic native monocyte-derived macrophages (Beisser et al., 2001;Maeß et al., 2010;Turtinen & Seufzer, 1994;Weinshenker et al., 1988). Substantial evidence has demonstrated that non-differentiated THP-1 cells do not support HCMV lytic infection unless they are differentiated by the addition of phorbol 12-myristate 13-acetate (PMA) or 12-O-tetradecanoylphorbol-13-acetate (Khaiboullina et al., 2004;Turtinen & Seufzer, 1994;Weinshenker et al., 1988). It has been suggested that immediate-early (IE) proteins IE1 and IE2, pp65 and pp150 are undetectable in undifferentiated THP-1 cells and that HCMV DNA polymerase UL54 and MIE protein levels are higher in differentiated than in undifferentiated THP-1 cells (Ioudinkova et al., 2006;Lee et al., 1999;Turtinen & Seufzer, 1994;Yee et al., 2007). ...
Human cytomegalovirus (HCMV) is a large DNA virus belonging to the subfamily Betaherpesvirinae. Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV. However, following terminal differentiation of these cells, virus is reactivated, and in an immunocompromised host acute infection can occur. It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections. Cyclophilin A (CyPA) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses, including human immunodeficiency virus, hepatitis C virus, murine cytomegalovirus, influenza A virus and vaccinia virus. This study investigated the role of CyPA during HCMV infection. CyPA expression was silenced in human foreskin fibroblast (HF) and THP-1 cells using small interfering RNA (siRNA) technology, or the cells were treated with cyclosporin A (CsA) to inhibit CyPA activity. Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early (IE) proteins, decreased viral DNA loads and reduced titres. Furthermore, silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state. Interestingly, it was observed that treatment of THP-1 cells with CsA prevented the cells from establishing a fully latent infection. In summary, these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells, and is a major component in virus reactivation from infected THP-1 cells.
Human cytomegalovirus (HCMV) is an important opportunistic pathogen that is the most
significant viral cause of congenital birth abnormalities and is responsible for a high morbidity and
death rate in immunocompromised patients. HCMV's rising severity and the limitations of current
vaccines in preventing infection highlight the urgent need for effective antiviral drugs. This study
aimed to identify small compounds using extensive & appropriate in-silico drug design techniques,
including molecular docking, Post-docking MM-GBSA, ADMET, MD simulation, PCA, and
DCCM were employed in this study capable of binding to the viral protease and inhibiting its
activity, thereby preventing proteolytic processing during capsid maturation. 3516 compounds
from life chemical were used in molecular docking, and the top four compounds having high
binding affinity and promising ADMET properties with life chemicals ID: F3407-0101 (CID:
49667672), F6559-3323 (CID: 121022124), F6456-1266 (CID: 71810903), and F3411-7969
(CID: 50785034). MD simulation was also used to assess the stability of the protein-ligand
complex structure. Finally, after MD simulation, principal component analysis (PCA), and
dynamic cross-correlation matrix (DCCM) analysis were performed using trajectories, and we can
suggest the best drug candidate which is CID: 50785034 (N-(3-fluoro-4-methylphenyl)-2-({7-oxo-
8-[(oxolan-2-yl) methyl]-5-thia-1,8,10,11-tetraazatricyclo [7.3.0.0^ {2,6}] dodeca-2(6),3,9,11-
tetraen-12-yl}sulfanyl)acetamide), another two compounds CID: 121022124, and CID: 71810903
which comes after CID: 50785034. All three compounds may have the potential to be developed
as a therapy option for HCMV infection.
Intractable diarrhoea in infancy is defined as diarrhoea persisting for more than 2 weeks in infants up to 3 months of age and requiring parenteral nutrition. It is often a diagnostic and therapeutic challenge for paediatricians. The following case report suggests that, once the most common diagnoses for this age group have been excluded, a cause of persistent diarrhoea that is usually overlooked and neglected such as cytomegalovirus enterocolitis should be always kept in mind.
Cytomegalovirus (CMV) may no longer be the menace that plagues stem cell transplant outcomes, owing to marked improvements in transplantation techniques and methods of prophylaxis. However, it still remains a common and morbid problem for recipients of stem cell transplant and patients with certain hematologic malignancies. This article discusses the epidemiology and risk factors of CMV infection and disease, associated morbidity and mortality, diagnosis, and clinical features of clinical syndromes associated with CMV and the principles of management of CMV, with special attention to resistant CMV as it pertains to infectious disease specialists.
Frailty is a common geriatric syndrome that has been variously characterized as a wasting state of decreased physiologic reserve, loss of physiologic complexity, and accumulation of deficits, and is an independent risk factor for adverse outcomes in older adults. A physiologic phenomenon that has been repeatedly observed in frail older individuals is a generalized inflammatory state, beyond age-related changes. Human cytomegalovirus (HCMV) is a prevalent herpesvirus that has come to be best known by the opportunistic diseases it causes in immunocompromised or immunosuppressed individuals. However, little is known regarding the long-term clinical effects of persistent HCMV infection in immunocompetent adults. We discuss here the epidemiologic associations between persistent HCMV infection and frailty in older adults and the limitations in such findings. We raise questions regarding the potential role of HCMV infection in the development of frailty. To accomplish this, we examine the biology of HCMV infection and immunologic mechanisms of pathogenesis. We discuss the immune responses observed in HCMV infection and consider the potential pathogenic effects that such responses could have in the long term. We consider why HCMV infection might contribute to the development of frailty in some individuals but not others, and raise further questions regarding the possible role of HCMV infection in the pathogenesis of frailty in older adults and attempt to answer them.
Human cytomegalovirus (HCMV) is universally distributed among humans without any adverse effects; however, it induces severe diseases in immunocompromised patients such as organ transplant recipients and AIDS patients. To manage these immunocompromised patients, an easy clinical examination for the monitoring of disease risk is required. In this study, we modified the interferon-γ (IFN-γ) release test (QuantiFERON®-CMV) using HCMV immediate early-1 (IE-1) or pp65 whole proteins, or UV-inactivated HCMV particles as an antigen. The response of heparinized peripheral blood from healthy volunteers to the pp65 protein showed an obvious dose-dependent sigmoid curve, although no correlation was observed between results of this assay and an ELISPOT assay. The addition of pp65 to the blood samples at a final concentration of 1×10³ to 1×10⁵ pg/ml was found to be optimum. Using this assay, we observed a significant enhancement in cellular immunity in volunteers after the daily ingestion of yogurt for 8 weeks, which suggested a novel application of the assay in addition to monitoring HCMV infection risk. IFN-γ secretion from peripheral blood cells on HCMV-antigen stimulation differed significantly between individuals; therefore, the assay could not be normalized. Nevertheless, it was found to be particularly useful for observing fluctuations in cellular immune activity on an individual level.
Promyelocytic leukemia (PML) nuclear bodies (PML-NBs), also referred to as nuclear domain 10 (ND10), have emerged as a cellular subnuclear structure that mediates an intrinsic defense against viral infections via chromatin-based mechanisms. This structure is defined as nuclear protein accumulations consisting of >100 different factors including the major components PML, hDaxx, Sp100, and SUMO. After infection with human cytomegalovirus (HCMV), which is a herpesvirus of major clinical relevance, PML-NBs are able to induce the silencing of viral immediate-early (IE) gene expression. The tegument protein pp71 of HCMV efficiently antagonizes this PML-NB-based repression by inducing the proteasomal degradation of hDaxx thus facilitating the initiation of IE gene expression. Subsequently, the newly synthesized IE1 protein of HCMV disrupts PML-NBs via a deSUMOylation of PML thereby activating lytic replication. By infection of PML as well as hDaxx knockdown cells, we show that both factors contribute to the deposition of repressive chromatin at the major immediate-early promoter of HCMV thus further confirming the epigenetic silencing mechanism. PML-NB proteins were hypothesized to play a role for both lytic replication as well as for the establishment of latency. However, recent evidence using the latency model of THP1 monocytic cells suggested that PML, Sp100, and hDaxx primarily act as cellular restriction factors that affect the efficacy of differentiation-induced reactivation of HCMV from latency.
Human cytomegalovirus (HCMV), a member of the herpesvirus family, can cause significant morbidity and mortality in immune compromised patients resulting from either primary lytic infection or reactivation from latency. Latent infection is associated with a restricted viral transcription programme compared to lytic infection which consists of defined protein coding RNAs but also includes a number of virally encoded microRNAs (miRNAs). One of these, miR-UL112-1, is known to target the major lytic IE72 transcript but, to date, a functional role for miR-UL112-1 during latent infection has not been shown. To address this, we have analysed latent infection in myeloid cells using a virus in which the target site for miR-UL112-1 in the 3' untranslated region of IE72 was removed such that any IE72 RNA present during latent infection would no longer be subject to regulation by miR-UL112-1 through the RNAi pathway. Our data show that removal of the miR-UL112-1 target site in IE72 results in increased levels of IE72 RNA in experimentally latent primary monocytes. Furthermore, this resulted in induction of IE expression detectable by IE-specific cytotoxic T cells (CTLs); no such CTL recognition of monocytes latently infected with wild-type virus was observed. We also recapitulated these findings in the more tractable THP-1 cell line model of latency. These observations argue that an important role for miR-UL112-1 during latency is to ensure tight control of lytic viral IE gene expression thereby preventing recognition of latently infected cells by the host's potent pre-existing anti-viral CTL response.
After a brief introduction to modern bone substitute materials, this review article compares the properties of autologous and allogeneic bone substitute materials. Autologous bone substitute material remains the gold standard, yet the disadvantages of autologous material, including higher donor site morbidity, limited availability, and prolonged operation time, are not found when using allogeneic material. This is why allografts have become popular in preprosthetic surgery in the last decade. Recent literature with a low level of evidence underlines an implant survival rate of over 90% and a postulated new bone formation of up to 30% after a 6-month healing period. The current chemical and physical treatment methods for allogeneic bone substitute material, such as freeze-drying and gamma irradiation, are presented. The infectivity and antigenicity are discussed based on the postoperative infection rate after allograft augmentation, which is said to be 5%. Allogeneic bone replacement materials only seem to be an alternative in smaller bone defects, but their biologic safety regarding antigenicity and infectivity, as well as their long-term resorption rate, should be discussed further. Augmentation with allografts remains an individual decision that should be weighed thoroughly.
Human cytomegalovirus (HCMV) can establish latent infection in hematopoietic progenitor cells (HPCs) or CD14 (+) monocytes. While circularized viral genomes are observed during latency, how viral genomes persist or which viral factors contribute to genome maintenance and/or replication is unclear. Previously, we identified a HCMV cis-acting viral maintenance element (TR element) and showed that HCMV IE1 exon 4 mRNA is expressed in latently infected HPCs. We now show that a smaller IE1 pro-tein species (IE1x4) is expressed in latently infected HPCs. IE1x4 protein expression is required for viral genome persistence and maintenance and replica-tion of a TR element containing plasmid (pTR). Both IE1x4 and the cellular transcription factor Sp1 interact with the TR, and inhibition of Sp1 binding abrogates pTR amplification. Further, IE1x4 interacts with Topoisomerase IIb (TOPOIIb), whose activity is required for pTR amplification. These results identify a HCMV latency-specific factor that promotes viral chromosome maintenance and replication.
MicroRNAs (miRNAs) play important roles in regulating gene expression of plants, animals and viruses. Comprehensive characterization of host and viral miRNA will help uncover the molecular mechanisms that underlie the progression of human cytomegalovirus (HCMV) latent infection. To investigate the miRNA expression profile of HCMV and host cells during latent infection, we performed deep-sequencing analysis of the small RNAs isolated from HCMV-infected and mock-infected human monocytic leukemia cell line, THP-1.
We established a HCMV latent infection cell model using the THP-1 cells. High-throughput sequencing technology was used to sequence small RNA libraries of the HCMV-infected and mock-infected THP-1 and to investigate their small RNA transcriptomes. We found eight miRNAs including miR-US25-1, miR-US25-2-5p and miR-UL112 that were expressed by HCMV during latent infection. The expressions of the host miRNAs were also affected by HCMV latent infection. At least 49 cellular miRNAs were differentially expressed: 39 were up-regulated and 10 were down-regulated upon HCMV latent infection. The expression of the human miRNA hsa-miR-124-3p was significantly up-regulated in the HCMV latent infection library. In addition, we found 14 cellular novel miRNAs in the HCMV-infected and mock-infected THP-1 libraries. Functional annotation of the target genes of the differentially expressed miRNAs suggested that the majority of the genes are involved in melanogenesis, pathways in cancer, endocytosis and wnt signaling pathway.
The small RNA transcriptomes obtained in this study demonstrate the usefulness of the deep-sequencing combined with bioinformatics approach in understanding of the expression and function of host and viral small RNAs in HCMV latent infection. This approach can also be applied to the study of other kinds of viruses.
Frailty is a common geriatric syndrome that has been variously characterized as a wasting state of decreased physiologic reserve,
loss of physiologic complexity, and accumulation of deficits [11, 58, 83], and is an independent risk factor for poor outcomes
in older adults [7, 30, 113]. A physiologic phenomenon that has been consistently observed in frail older individuals is a
generalized inflammatory state, beyond age-related changes [26, 56, 87, 106]. Frail older adults have higher levels of systemic
inflammatory markers, including interleukin-6 and C-reactive protein, than older adults who are not frail, even when chronic
diseases are excluded [106]. The chronic activation of inflammatory pathways is known to influence skeletal muscle mass decline,
the anemia of chronic disease, hypothalamic-pituitary-adrenal axis (HPA axis) activity, cognition, and a number of chronic
disease states, and likely plays an important role in the pathogenesis of frailty through its effects on these multiple physiologic
systems [28].
Prophylaxis with vaccines is of great importance in geriatrics as, apart from specific protection, it reduces the incidence
of potentially fatal infectious complications and exacerbations of existing medical conditions. The level of postvaccination
protection strongly depends on immune system and therefore markers of its condition may be used to predict the efficiency
of vaccination. From the practical point of view, a link between some clinical features of the health status and condition
of immune system are desirable as they allow to find the patients who may need additional care necessary to avoid possible
complications, in case the vaccination did not protect them against the infection. This chapter reviews immune phenomena associated
with anti-influenza vaccination. Humoral and cellular markers of the immunization efficiency are discussed in respect of health
status of the elderly.
The bone marrow plays a unique role within the immune system. We compared the phenotype and function of virus-specific CD8(+) T cells from matched samples of human peripheral blood and bone marrow. Analysis of virus-specific memory CD8(+) T cells showed widely divergent partition of antigen-specific populations between blood and bone marrow. T cells specific for Epstein-Barr virus (EBV) lytic antigens were enriched 3-fold in marrow compared with blood, whereas the response to EBV latent epitopes was equivalent between the 2 compartments. No difference in EBV viral load or expression of the EBV lytic protein was observed between blood and bone marrow. In direct contrast, although cytomegalo-virus (CMV)-specific T cells were the largest virus-specific population within peripheral blood, they were reduced by 60% within marrow. Bone marrow T cells were found to exhibit a unique CCR5(+)CXCR6(+)CXCR3(-) homing phenotype which has not been observed on T cells from other secondary lymphoid organs or peripheral organs. Expression of CCR5 and CXCR6 was higher on EBV-specific T cells within peripheral blood compared with CMV-specific populations. These observations identify a novel bone marrow homing phenotype for CD8(+) memory T cells, which necessitates a reevaluation of the magnitude of antigen-specific populations within the lymphoid system.
Because of the central role of the transcription factor nuclear factor (NF)-κB in cell survival and proliferation in many kinds of cancer cells, NF-κB inhibitors may have a potential role in cancer therapy. Currently, many NF-κB inhibitors are used for immunosuppression to treat hematologic malignancy patients after stem cell transplantation (SCT). Human cytomegalovirus (HCMV) infection is one of the most common complications following SCT. Some workers have reported that HCMV infection has a close relationship to NF-κB activation; however, the specific effects of NF-κB inhibitors, such as pyrrolidinedithiocarbamate (PDTC), on infection with and activation of CMV in mesenchymal stem cells (MSCs) remain unknown. In our study, we isolated MSCs from the bone marrows of healthy human donors for infection with 1 tcid(50) of HCMV with or without 1 μmol/L PDTC. After 48 hours of culture in dmem supplemented with 10% (volume per volume) fetal calf serum, we tested MSCs using reverse transcription-polymerase chain reaction (RT-PCR) assays to detect messenger RNA (mRNA) expression of HCMV immediate early (IE) gene and the GAPDH gene. Flow cytometry was used to detect HCMV pp65 antigen-positive cells and transmission electron microscopy (TEM) for intra cellular HCMV particles. We observed that the shape of the MSCs changed in response to infection by 1 TCID(50) of HCMV. MSCs infected by 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC changed their shapes more profoundly; almost all cells went from a thin elongated profile to a round, thick ball. In contrast, the shape of cells treated with PDTC alone or the HCMV mock-infected elements did not change. The RT-PCR assay showed that there was a bright band corresponding to HCMV IE mRNA in MSCs infected with 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC, as compared with cells infected by only 1 TCID(50) of HCMV. The HCMV mock-infected MSCs did not express HCMV IE mRNA. Using flow cytometry, we detected more HCMV pp65 antigen-positive cells among MSCs infected with 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC. HCMV particles were observed by TEM in the nucleus and cytoplasm of MSCs infected with HCMV. There were more HCMV particles in cells infected by HCMV in combination with PDTC. In conclusion, NF-κB activation may affect HCMV infection efficiency of MSCs. An NF-κB inhibitor increased the infection by activation of HCMV in MSCs, thus we should pay close attention to HCMV infection when we prescribe an NF-κB inhibitor in clinical settings.
Clinical experience and laboratory data suggest that human cytomegalovirus (HCMV) is present in peripheral blood of seropositive individuals in a latent or persistent state and can be transmitted via blood products and be reactivated in seropositive imunocompromised patients. The pathophysiology of HCMV latency and the nature of HCMV interaction with hematopoietic cells remains unknown. In this study, we investigated the infection of bone marrow (BM) progenitor cells and their progeny as a model of HCMV latency. A clinical isolate and the recombinant laboratory strain Towne/lox containing the Escherichia coli β galactosidase (β-gal) gene regulated by immediately early (IE) HCMV promoter were used to infect highly purified CD34+ cells. Although the infection of these cells with a clinical isolate was associated with an inhibition of proliferation by 59%, an expansion of progeny derived from these cells was possible. Polymerase chain reaction analysis and staining for β-gal have shown that HCMV persisted in infected BM CD34+ cells and their progeny for up to 4 weeks. However, IE and late gene products (mRNA and protein) were detected only late in the course of infection and their expression correlated with terminal macrophage differentiation of the CD34+-derived progeny. Although early infection of CD34+ progenitor cells was not productive (as shown by the plaque assay), infectious virus could be recovered from the terminally differentiated cultures. BM progenitor cells may serve as a reservoir of the latent virus with limited transcription. Proliferation and monocytic maturation of infected progenitors may lead to the numerical expansion of HCMV-infected cells, which serve as a source of HCMV dissemination and reactivation.
The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.
The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.
Monocytes are one of the predominant cell types in the peripheral blood that are infected by human cytomegalovirus (HCMV). Although virus can be detected in these cells in vivo, HCMV replication in cultured monocytes has been unsuccessful. In this study, we demonstrate efficient HCMV replication in cultured monocytes. HCMV permissiveness in these cells was dependent on nonadherent cell-induced stimulation of the monocyte, with subsequent morphological differentiation into macrophages. Approximately 40% of the cells infected by virus were detected by immunofluorescent staining with both immediate-early and late antibodies. In addition, viral plaque assays demonstrated significant productive infection of macrophages. These observations are consistent with the suggestion that the monocyte/macrophage serves as a source of viral amplification and dissemination.
Clinical experience and laboratory data suggest that human cytomegalovirus (HCMV) is present in peripheral blood of seropositive individuals in a latent or persistent state and can be transmitted via blood products and be reactivated in seropositive immunocompromised patients. The pathophysiology of HCMV latency and the nature of HCMV interaction with hematopoietic cells remains unknown. In this study, we investigated the infection of bone marrow (BM) progenitor cells and their progeny as a model of HCMV latency. A clinical isolate and the recombinant laboratory strain Towne/lox containing the Escherichia coli beta galactosidase (beta-gal) gene regulated by immediately early (IE) HCMV promoter were used to infect highly purified CD34+ cells. Although the infection of these cells with a clinical isolate was associated with an inhibition of proliferation by 59%, an expansion of progeny derived from these cells was possible. Polymerase chain reaction analysis and staining for beta-gal have shown that HCMV persisted in infected BM CD34+ cells and their progeny for up to 4 weeks. However, IE and late gene products (mRNA and protein) were detected only late in the course of infection and their expression correlated with terminal macrophage differentiation of the CD34+-derived progeny. Although early infection of CD34+ progenitor cells was not productive (as shown by the plaque assay), infectious virus could be recovered from the terminally differentiated cultures. BM progenitor cells may serve as a reservoir of the latent virus with limited transcription. Proliferation and monocytic maturation of infected progenitors may lead to the numerical expansion of HCMV-infected cells, which serve as a source of HCMV dissemination and reactivation.
Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of viral DNA was investigated by PCR-driven in situ hybridization (PCR-ISH), and the number of viral genomes per cell was estimated by quantitative competitive PCR during both experimental and natural latent infection. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in >90% of cells at a copy number of 1 to 8 viral genomes per cell. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow from seropositive donors, at a copy number of 2 to 13 genomes per infected cell. When evaluated by reverse transcription-PCR-ISH, only a small proportion of experimentally infected cells (approximately 2%) had detectable latent transcripts. This investigation identifies the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode currently identified latent transcripts.
Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34(+) cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42(+) MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the beta-galactosidase (beta-gal) gene. Using chromogenic beta-gal substrates, HCMV was detected during MK differentiation of infected CD34(+) cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV. (Blood. 2000;95:487-493)
The in vitro susceptibility of human peripheral lymhpocytes and lymphoblastoid (F265) cells to infection by human cytomegalovirus was examined. Infection of these cell types with cytomegalovirus resulted in a persistent type of infection rather than the typical growth curve observed with permissive fibroblastic cells. When infection of peripheral lymphocytes was associated with a blastogenic response, the virus persisted for a longer time and at a higher titer than in cells in which a blastogenic response did not occur. Autoradiographic studies and infectious-center assays indicated that only a small number of cells, resembling lymphocytes, were involved in virus persistence. Whether or not the persistence of the virus indicates release of input virus or synthesis or new virus was not determined.
We have used the nested polymerase chain reaction (PCR) combined with fluorescence-activated cell sorting to define sites of latency of human cytomegalovirus (HCMV) in the peripheral blood of healthy subjects. Peripheral blood mononuclear (PBM) cells were separated into T cell or non-T cell populations and monocytes, and were then analysed by PCR for the presence of HCMV DNA. In five of six seropositive subjects, HCMV was found predominantly in the non-T cell population. Further analysis suggested that the virus was present in adherent cells and CD14+ cells. In three of nine seronegative subjects we could demonstrate HCMV DNA, which we do not believe was due to contamination, reproducibly by PCR. In one of these seronegative subjects, HCMV DNA was present predominantly in the non-T cell fraction of PBM cells. No HCMV DNA was detectable in the remaining six seronegative subjects. We conclude that, within the PBM cells of normal asymptomatic seropositive and some seronegative subjects, HCMV is present predominantly in the monocyte fraction. In addition, the detection of HCMV sequences in seronegative subjects may indicate that infection with HCMV is more widespread than conventional seroepidemiology suggests.
The in vivo interaction of human cytomegalovirus (HCMV) with leukocytes from a group of immunosuppressed patients was studied
using specific subgenomic DNA probes and in situ cyto-hybridization. Study subjects had no recognized disease due to HCMV
or had HCMV retinitis or colitis. Viral nucleic acid was detected in lymphocytes, monocytes, and polymorphonuclear leukocytes
(PMNL). PMNL were consistently hybridization-positive for HCMV RNA and DNA in patients with HCMV viremia. Monocytes were occasionally
positive by hybridization and lymphocytes were rarely positive. These findings demonstrate that the HCMV genome is expressed
in PMNL of viremic patients and that PMNL may play an important role in virus dissemination.
Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants, with at least
two-thirds of these patients having CMV infection 1–4 months after transplantation. Latently infected allografts are the major
exogenous source of CMV infection in transplant recipients, although leukocyte-containing blood products can also transmit
the virus. Three patterns of CMV infection are recognized: primary infection, reactivation infection, and superinfection.
Primary infection has the greatest clinical impact. The clinical effects of CMV infection include infectious disease syndromes
such as pneumonia and chorioretinitis; an immunosuppressed state that predisposes to potentially lethal opportunistic infection;
and the initiation of a process that can result in allograft injury. Progress has been made in controlling CMV infection;
hyperimmune anti-CMV globulin and certain antiviral drugs appear promising for prophylaxis, and the combination of hyperimmunoglobulin
and ganciclovir appears promising for therapy.
Human cytomegalovirus (HCMV) infection has previously been associated with the production of immunosuppression. The mechanism by which any such immunosuppressive effect might be mediated is unclear but previous work has implicated an effect of the virus on monocytes. We have attempted to characterize the immunosuppressive activity produced by in vitro infection of normal monocytes with HCMV strain AD169. We first examined the ability of HCMV AD169 and recent clinical isolates to infect normal peripheral blood mononuclear cells in vitro. We have found by immunofluorescence analysis that only a very limited number of peripheral blood mononuclear cells (0.2 to 0.5%) showed evidence of virus infection as demonstrated by expression of the major immediate early protein. We found that the inhibitory activity of supernatants of monocytes exposed to HCMV which suppressed mitogen-driven T cell responsiveness was associated with a protein of about 95K. Experiments to investigate the mechanism of action of this inhibitor suggested the possibility of mycoplasma contamination and we were subsequently able to isolate Mycoplasma hyorhinis from our AD169 virus stock. When a series of low passage clinical isolates of HCMV were examined for their ability to cause immunosuppression, there was a direct correlation between suppression and the presence of contaminating mycoplasmas. Using mycoplasma-free isolates of HCMV we could demonstrate no immunosuppressive effect on mitogen-mediated T cell proliferation of both unseparated human peripheral blood lymphocytes and nylon wool non-adherent T cells; these virus isolates also did not suppress accessory cell function or interleukin 1 production by monocytes infected in vitro. We conclude that the previously reported immunosuppressive effects of HCMV in vitro may be attributable to the presence of mycoplasmas and are unlikely to be due to expression of HCMV in monocytes. We suggest that mycoplasma contamination of isolates of HCMV may be a more extensive problem than is currently recognized.
Cytomegalovirus infection isoneof a long list of latent human infections that, although normally controlled by the cellular
immune response, is activated after human immunodeficiency virus infection takes its toll on the T4 lymphocytes. Expression
of this infection isunique, its most prominent manifestations being a progressive retinitis, a colitis, an encephalitis, and
perhaps a pneumonia. The diagnosis and treatment of these conditions remain difficult and controversial clinical problems.
For many years Dr. W. Lawrence Drew has studied infections caused by cytomegalovirus. We have asked Dr. Drew to give his impressions,
based on the most current data available, of several aspects of this infection: When is cytomegalovirus a pathogen in the
patient with AIDS? How is the diagnosis of cytomegalovirus infection established? What agents are available to treat the infection?
In this investigation, we studied the ability of human cytomegalovirus to infect peripheral blood mononuclear cells. With monoclonal antibody technology, we demonstrated that cytomegalovirus could infect human lymphocytes of T- and B-cell lineage, natural killer cells, and monocytes. Furthermore, virus expression was limited to the synthesis of immediate-early cytomegalovirus polypeptides. These peripheral blood mononuclear cells did not produce infectious virus, nor were mature virions visualized by electron microscopy. This abortive infection of mononuclear cells was most convincingly shown with stocks of cytomegalovirus that had been recently isolated from infected patients and passaged minimally in fibroblasts. This argues for an increased lymphotropic effect of some isolates of cytomegalovirus, compared to strains of virus that are extensively adapted to growth in fibroblasts. Furthermore, immunocompetent cells that were shown to be abortively infected with cytomegalovirus lost selected differentiated functions.
The identity of cells responsible for transmission of human cytomegalovirus (HCMV) in blood products or bone marrow transplants is unknown. We have tested the capacity of HCMV to in vitro infect human peripheral blood mononuclear cells (PBMC) from healthy donors and found that certain PBMC are permissive to HCMV infection. In vitro-infected viable cells were double stained for surface expression of different HMCV proteins and for cell-type-specific antigens to allow the identification of sensitive cells. All analysis were performed on viable cells, using HCMV-specific monoclonal antibodies and automated flow cytofluorimetry. PBMC were infected either with the laboratory-adapted HCMV strain AD169 or with a virus isolate obtained from a viremic patient. Up to 25% of all PBMC could express the major immediate-early antigen as well as the pp65 antigen, known at the lower matrix protein. Infected cells were mainly CD14+ monocytes, but also a small population of large CD8+ cells were susceptible to HCMV infection. CD19+ B lymphocytes were resistant to HCMV infection. Different populations of infected cells were enriched by using Dynabeads coated with cell-type-specific antibodies, and the presence of infectious virus was demonstrated by incubating the selected and sonicated cell material on human fibroblasts. Only material from infected monocytes and from CD3+ CD8+ cells gave rise to HCMV-specific plaques. The presence of HCMV mRNA as a sign of active viral transcription of the major immediate-early and late pp150 genes in infected cells was demonstrated by using nested reversed polymerase chain reaction. A common denominator was found for all cells that could be infected with HCMV. The CD13 antigen, a 130- to 150-kDa integral membrane protein identical to the enzyme aminopeptidase N, was expressed on all HCMV-permissive cells.
Peripheral blood samples from 313 normal donors were tested for prior human cytomegalovirus (HCMV) infection: 37%, 0.9%, and
43% of the samples were positive by antibody detection, DNA hybridization, and RNA hybridization assays, respectively. An
early mRNA, which is transcribed from a HindIII-b fragment of the CMV genome and detected with an antisense RNA probe, can be detected more frequently than antibody and
CMV DNA. The early CMV mRNA transcripts can be detected in the peripheral white blood cells in 44% of HCMV-seronegative blood
donors. Blood samples that were CMV RNA positive but antibody negative comprised 27% of the tested samples. Whether CMV RNA
in donor blood indicates that CMV can be transmitted via blood transfusion must be determined by further studies.
We have investigated the interaction of human cytomegalovirus (CMV) with cultured primary granulocyte-macrophage progenitors, a suspected natural site of viral latency, and have established conditions for latent infection and reactivation in this cell population. Progenitor cells from human fetal liver or bone marrow maintained a CD14+, CD15+, CD33+ cell surface phenotype during propagation in suspension culture. Exposure to human CMV did not reduce growth or alter the phenotype of these cells during a 4-week culture period. Viral replication was not detectable in these cells, although viral DNA, as measured by PCR analysis, persisted in a high proportion of cultured cells in the absence of delayed early (beta) gene expression. Viral gene expression was restricted such that only ie1 region transcripts were detected by PCR analysis of cDNA, and these transcripts were estimated to be present in no less than 2-5% of latently infected cells. Most of these transcripts remained unspliced, a result that strikingly contrasts with the splicing pattern normally seen during viral replication in permissive cells. Latent virus reactivated after prolonged, 16- to 21-day cocultivation of infected granulocyte-macrophage progenitors with permissive cells, results that support a role for the myelomonocytic cell population as a biological reservoir of latent human CMV and suggest that these cells may be the source of CMV DNA PCR-positive monocytes found in the peripheral blood of healthy carriers.
Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.
Polymorphonuclear leukocytes (PMNL) have been shown to harbour human cytomegalovirus (HCMV) in viraemic patients, but to date PMNL of asymptomatic healthy subjects have not been examined directly to determine whether this is a normal site of HCMV persistence. Using the polymerase chain reaction (PCR), paired DNA samples prepared from adherent peripheral blood mononuclear cells (PBMC), which are known to be a site of persistence of HCMV, and PMNL of 10 healthy adults were analysed. All of seven individuals who were HCMV seropositive, and one of three who were seronegative gave a reproducible signal for HCMV DNA in their adherent PBMC, whereas none of the paired PMNL DNA samples gave a positive result. The remaining two seronegative subjects showed no HCMV DNA in either the PBMC or PMNL samples. In every case where PCR for HCMV was negative, PCR amplification of a control human gene was used to show there was no inability to amplify the DNA. We conclude that within the leukocyte population of normal asymptomatic HCMV carriers, PMNL do not appear to harbour persistent HCMV whereas adherent PBMC in the same subjects are a site of persistence.
The cellular sites and mechanisms of human cytomegalovirus (HCMV) latency are still poorly defined. Although evidence suggests that peripheral blood monocytes are one site of latency in the healthy carrier, it is unlikely that monocytes represent a site of primary HCMV infection. Consequently, we have analysed CD34+ bone marrow progenitors, precursors of monocytes, to determine whether they are a site of HCMV carriage in normal virus carriers. For the first time, we demonstrate the presence of endogenous HCMV within bone marrow progenitors in the absence of HCMV lytic gene expression. These findings are consistent with previous evidence showing that the permissiveness of myeloid cells for HCMV is critically dependent on the differentiation state of the cell.
Reactivation of human cytomegalovirus (HCMV) results in severe disease in AIDS patients and immunocompromised patients receiving blood transfusions or organ or bone marrow grafts. Although the site of HCMV latency is unknown, blood cells have been implicated as a viral reservoir. In this study, we demonstrate HCMV reactivation in vitro from seven consecutive healthy donors through allogeneic stimulation of peripheral blood mononuclear cells (PBMCs). HCMV replication was detected at 17 days poststimulation, and virus was recovered after long-term culture from a macrophage expressing dendritic cell markers. Thus, these observations demonstrate that PBMCs harbor latent HCMV, which reactivates in a myeloid lineage cell upon allogeneic stimulation.
Human embryonal fibroblasts (HEF) are fully permissive for infection by human cytomegalovirus (HCMV) strain AD169, whereas human umbilical vein endothelial cells (HUVEC) seem to form an almost complete barrier to infection with this virus. To investigate this difference in permissiveness, HCMV infection of both cell types was studied using in situ hybridisation (ISH) as well as immunocytochemistry to detect viral DNA and viral proteins. At 2 h post-infection (p.i.), viral DNA was detected dispersed throughout the cytoplasm in both HEF and HUVEC, indicating that HCMV enters all cells of both cell types. At 4 h p.i., the viral DNA was found in the nucleus in HEF, and at the same time expression of immediate early (IE) antigen was found. In contrast, in HUVEC the expression of the IE proteins occurred in a limited number of cells at 8 h p.i., while in most HUVEC an accumulation of viral DNA around the nuclei was observed at this time point. In HUVEC, the nuclear localisation of viral DNA was detected 16 h p.i. in a minority of cells, indicating that transport of HCMV DNA into the nucleus is considerably slower in HUVEC than in HEF. Furthermore, the number of HUVEC containing HCMV DNA decreased about six-fold between 8 and 48 h p.i., indicating that HCMV DNA is either transported into the nucleus or eliminated. Apparently, the lower permissiveness of HUVEC for the HCMV strain AD169 relative to HEF is due to inefficient transport of HCMV DNA into the nuclei of infected HUVEC.
For a number of years it has been well established that human cytomegalovirus (HCMV) can be transmitted by the cellular components of blood. HCMV is also associated with a number of hematologic disorders. Although HCMV was thought to be present in blood cells in a latent or persistent form, it was not known how the virus was maintained and which cells were the carriers of HCMV. In addition to peripheral blood cells, there has been clinical evidence that HCMV may be associated with specific disorders of the hematopoietic system. Recently, a number of advances in cell and molecular biology have helped to develop a better understanding of the relationship between HCMV and the hematopoietic system. The application of the polymerase chain reaction (PCR) to the study of HCMV infection has revealed that the virus was present in mononuclear cells with only limited transcription of its genome. Studies conducted in our laboratory have demonstrated that both CD34+ progenitor cells and monocytes could be infected with HCMV and virus recovered when the cells were allowed to terminally differentiate. Subsequently, these results have been confirmed in vivo: HCMV DNA and limited RNA transcripts could be detected in in vivo infected hematopoietic progenitor cells and HCMV has been rescued from macrophages derived through in vitro differentiation of monocytes from normal seropositive blood donors. Although our understanding of the relationship between HCMV and the hematopoietic system has been advanced, the mechanisms by which the virus can be maintained in a latent state and how it is reactivated is still unclear. Furthermore, it remains to be determined what HCMV-mediated effect is responsible for the inhibition of hematopoiesis following an in vitro infection and its significance in vivo.
To more precisely determine the nature of chromosome 3 rearrangements in small cell lung carcinomas (SCLCs), we have applied molecular cytogenetic technologies to a newly characterized SCLC tumor and five SCLC cell lines. Fluorescent in situ hybridization, chromosome microdissection, and, on the previously uncharacterized tumor, spectral karyotyping was utilized to determine chromosome 3 rearrangements. In all cases, our studies were performed on previously G-banded chromosomes in a sequential manner to facilitate a direct comparison. A consistent breakpoint on the long arm of chromosome 3 at band 3q13.2 was identified in all six tumors. This breakpoint was commonly the result of complex chromosomal rearrangements. Loss of the entire short arm of a chromosome 3 was noted in all six tumor cultures. Two of these cell lines had two sublines, one of which contained a 3q13.2 rearrangement and the other of which contained a chromosome rearrangement that resulted in loss of a chromosome 3 short arm. This consistent rearrangement at chromosome band 3q13.2, as demonstrated by molecular cytogenetic methods, may indicate the location of a gene important in the tumorigenesis of SCLC.
Cytomegalovirus infects human lymphocytes and monocytes: virus expression is limited to immediate-early gene products
- G P Rice
- R D Schrier
- M B Oldstone
Rice, G.P., Schrier, R.D. & Oldstone, M.B. (1984) Cytomegalovirus
infects human lymphocytes and monocytes: virus expression is
limited to immediate-early gene products. Proceedings of the
National Academy of Sciences United States of America, 81, 6134-6138.