Maintenance of human cytomegalovirus genome in human diploid fibroblast cells

Human embryo lung cells pretreated with a combination of human leukocyte interferon and acyclovir were infected with human cytomegalovirus (HCMV) and treated daily for 14 days with the same inhibitor combination. After removal of the inhibitors, the incubation temperature was maintained at either 37 or 40.5 degrees. Incubation of infected cells at 37 degrees after inhibitor removal resulted in progressive virus-specific cytopathology and eventually total destruction of the cell culture. However, HCMV-infected cells incubated at 40.5 degrees after inhibitor removal exhibited little or no virus-induced cytopathology and HCMV remained noninfectious for 8 days. After extended incubation at 40.5 degrees, infectious HCMV was readily detectable even though virus-specific cytopathology was not evident. Reducing the incubation temperature from 40.5 to 37 degrees resulted in stimulation of infectious virus replication and subsequent destruction of the infected cell culture monolayer within 10 days.     

Expression of the human cytomegalovirus genome in mouse cells and in human-mouse heterokaryons

Summary Mouse cells with an established human cytomegalovirus (HCMV) infection were fused with susceptible human embryonic fibroblast cells. CMV-specific early antigens could be demonstrated in the cytoplasm and cell-membrane of the heterokaryons. Treatment with 5-iodo-2-deoxyuridine (IUdR) of the heterokaryons or of the latently infected mouse cells, prior to their fusion with human cells, could induce the appearance of immunofluorescent elements, characterised as late antigens, and of infectious virus. Our data show that the mouse cells, in the latent stage of infection, contain the whole virus genome and that the replication of the virus is controlled by a genetic mechanism of the host cells both in virus-harbouring mouse cells and in heterokaryons.With 2 Figures     

Quantification of human cytomegalovirus using bronchoalveolar lavage cells in pulmonary complications associated with hematologic neoplasia

Human cytomegalovirus (CMV) has been recognized as a frequent pathogen involved in interstitial pneumonia (IP), and CMV-IP is a severe and life-threatening complication in the immunocompromised patients. The use of real-time PCR in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting a wide variety of templates including viruses. Therefore, we developed a rapid quantification system of CMV using a LightCycler in order to clarify the possible role of CMV reactivation in patients with hematologic neoplasia showing pulmonary complications. Sixty-nine bronchoalveolar lavage fluid (BALF) specimens were obtained from consecutively treated patients showing interstitial shadow including 20 patients with hematologic neoplasia. First, we determined the viral burden in BAL cells from healthy volunteers, idiopathic interstitial pneumonia (IIP) and sarcoidosis. CMV copy numbers in samples obtained from healthy volunteers, IIP and sarcoidosis, were less than 10(2) copies per 1 microg of DNA, whether or not BAL cells were composed of high percentage of lymphocytes. Among 20 patients with hematologic neoplasia analyzed, two specimens obtained from leukemia patients with pulmonary alveolar proteinosis, two from GvHD, one with CMV interstitial pneumonia and one with Hodgkin's disease had high level of CMV viral DNA. Our results suggest that measurement of CMV genomes in BAL cells using real-time PCR may be useful not only to understand the involvement of CMV in systematic respiratory tract disease but also in management of the care of respiratory complications in hematologic neoplasia.     

Cultured human brain capillary endothelial cells are permissive for infection by human cytomegalovirus

Human cytomegalovirus (HCMV) infection is associated with a variety of systemic and neurologic diseases. In vitro HCMV growth is usually studied in fibroblasts, while in vivo HCMV growth is frequently observed in a wide variety of cell types including glia, neurons, and human brain capillary endothelial (HBCE) cells. To examine the biology of HCMV in HBCE cells, we have established a procedure for isolating these cells from human brain temporal lobectomies. Greater than 99.0% of these cultured cells were identified as HBCE cells on the basis of positive staining for factor VIII-related antigen-Von Willebrand's factor (F VIII) and Ulex Europaeus agglutinin I (UEA I). HCMV antigens were detected by immunocytochemistry in HBCE cells after infection with strain AD 169. Intracellular virions were observed in infected cells by electron microscopy and infectious virus was released from HBCE cells. In addition, infected cells were confirmed as endothelial cells by double staining with antibodies to F VIII and HCMV.     

Inflammatory cells in transplanted kidneys are infected by human cytomegalovirus.

To determine which cells in kidney grafts are infected with human cytomegalovirus (HCMV) before and after transplantation, kidney specimens were studied by in situ hybridization with 35S-labeled DNA probes representing HCMV immediate-early and late genes. Pretransplantation biopsies and serial posttransplantation biopsies were obtained from 7 renal grafts. All of the transplant recipients were HCMV-seronegative at the time of transplantation and all developed primary HCMV infections. HCMV nucleic acids were not detected in biopsies taken from the healthy donor kidneys before transplantation. However, biopsies taken at various intervals after transplantation showed abundant hybridization with HCMV immediate-early and late gene probes. Virtually all of the hybridizing cells were mononuclear inflammatory cells in the interstitial spaces of the kidney. Occasional hybridization was seen with renal tubular or glomerular cells. No cytomegalic cells were seen. Biopsy specimens taken after systemic anti-HCMV chemotherapy with phosphonoformate showed no uniform reduction in HCMV gene expression. These studies demonstrate that the principal HCMV-infected cells in kidneys of renal transplant patients with primary HCMV infections are infiltrating inflammatory cells.     

Tetracycline-mediated regulation of gene expression within the human cytomegalovirus genome.

To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sites which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547-5551). Two modifications of PhCMV*-1 were also studied: P1129, in which the tet operator sites were reduced from seven to one; and P1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P1125 exhibited modest differential regulation but were strongly activated by viral infection. P1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late times of infection, whereas P1125 displayed nearly 100-fold regulation. Regulation of P1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes.     

A polymorphic region of the human cytomegalovirus genome encoding putative glycoproteins

Summary A region of the human cytomegalovirus genome (corresponding to nt 17973-19945 of strain AD169) was analyzed by restriction fragment length polymorphism. Rsa I digestion of the region showed 20 distinct patterns in agarose gel electrophoresis.     

Persistence and replication of the human cytomegalovirus genome in lymphoblastoid cells of B and T origin.

Human lymphoblastoid cells infected in vitro with human cytomegalovirus (CMV) were examined by DNA-DNA reassociation kinetics at various times after virus infection. The results indicate that CMV DNA replicates in two B lymphoblastoid cell lines (Raji and Simpson) and one T-cell line (Molt-4). Multiple copies of the entire CMV genome were found in each cell line. After an initial round of CMV DNA replication, the number of CMV DNA copies per cell gradually decreased with time in Raji and Molt-4 cells. In contrast, Simpson cells maintained a fairly constant number of CMV DNA copies throughout the 15-day experiments.     

Rabbit kidney cells abortively infected with human cytomegalovirus are arrested in mitotic phase

Summary In rabbit kidney epithelial cells (RK₁₃) abortively infected with human cytomegalovirus (HCMV), DNA synthesis at 1 or 2 days post-infection was enhanced 4 to 5 fold, compared to mock-infected cells. DNA analysis by isopycnic centrifugation revealed that the DNA newly synthesized in the virus infected RK₁₃ cells was of cellular origin. HCMV infection also caused a marked increase in the mitotic activity of RK₁₃ cells. When semi-confluent RK₁₃ cells were infected more than 20 per cent of cells demonstrated mitosis at 72 hours post-infection although the rate of cell growth was considerably reduced compared to that of uninfected cells. The most frequent chromosomal change observed was fragmentation although other aberrations, gap, break, deletion etc. occurred also.Two immediate-early viral polypeptides with apparent molecular weights 72,000 (72K) and 76,000 (76K) daltons were produced in both RK₁₃ cells and human embryonic lung cells (HEL) by 3 hours post-infection. Synthesis of the 76K polypeptide was greater than that of the 72K polypeptide in non-permissive RK₁₃ cells whereas the reverse occurred in permissive HEL cells. Furthermore, of three early polypeptides which were expressed in productively infected HEL cells two, 88K and 80K, were not detected in abortively infected RK₁₃ cells.These results suggest that the arrest in mitosis of the abortively infected RK₁₃ cells and the subsequent chromosomal changes are associated with the altered expression of immediate-early or early virus functions in these cells.Following HCMV infection permissive HEL cells undergo a characteristic sequence of morphological changes (3) of which cell rounding and contraction are observed in the early stages and could be related to either the action of cellular contractile proteins or changes in the plasma membrane permeability and a concomitant Ca⁺⁺ influx (2). Relaxation and cell enlargement are other changes considered to be initiated by early virus functions although they occur after the commencement of virus DNA synthesis. Thus, the expression of immediate-early and early virus functions has an important role in controlling the sequential morphological changes in infected cells and may also be important in determining whether infection with HCMV will be productive or abortive.With 4 Figures     

Manipulation of the human genome in human embryonic stem cells

Human embryonic stem cells (HESCs) have a tremendous clinical and scientific importance since they may serve as a cell source for transplantation and as a system for the study of human development and disease. The genetic engineering of HESCs has become instrumental in achieving these goals. Here we discuss various methodologies to genetically manipulate HESCs and propose a variety of applications of the modified cells in basic and applied research.     

Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts.

In heart transplantation, long-term engraftment success is severely limited by the rapid development of obliterative disease of the coronary arteries. Data from various groups have been suggestive of a pathogenetic role of herpesviruses, particularly human cytomegalovirus, in accelerated allograft coronary artery disease; however, results are not yet conclusive. This study examines the hypothesis that human cytomegalovirus infection of allograft tissues is related pathogenetically and directly to accelerated coronary artery disease. Using in situ DNA hybridization and polymerase chain reaction, we examined particular coronary artery segments from 41 human heart allografts (ranging from 4 days to greater than 4 years after transplantation; mean, 457 days) and 22 donor age- and gender-comparable, coronary site-matched trauma victims for presence of human cytomegalovirus DNA. Human cytomegalovirus genome was detected in 8 of 41 (19.5%) allografts and in 1 of 22 (4.5%) control hearts. This difference in positivity was not statistically significant (P = 0.10). In the human cytomegalovirus-positive hearts, viral genome was localized to perivascular myocardium or coronary artery media or adventitia. Human cytomegalovirus genome was not detected in arterial intima of any allograft or control heart, although human cytomegalovirus genome was readily identified within intima of small pulmonary arteries from lung tissue with human cytomegalovirus pneumonitis. By statistical analyses, the presence of human cytomegalovirus genome was not associated with the nature or digitized extent of transplant arteriopathy, evidence of rejection, allograft recipient or donor serological data suggestive of human cytomegalovirus infection, duration of allograft implantation, or causes of death or retransplantation. Thus, our data indicate a low frequency of detectable human cytomegalovirus genome in accelerated coronary artery disease and do not support a direct role for human cytomegalovirus in vascular wall infection or in the development of accelerated coronary artery disease.     

Early interactions between human cytomegalovirus and cells

Summary Adsorption of cytomegalovirus (CMV) to human fibroblasts was not inhibited by preincubation with other herpes viruses (HSV-1, HSV-2, VZV). Transport of virus to the nucleus was studied using virus labelled with³H-thymidine. Radioactivity was found in the nucleus 20 minutes after virus had been added to the cells. In order to assess the expression of the virus genome, synthesis of early (EA) and late (LA) antigens was studied. Assayed on permissive human lung fibroblasts, a plaque purified virus contained more EA inducing than both EA and LA inducing viral units. Since this was a consistent finding with virions of all densities, it seems to be an effect of viral dose rather than of virion density. Alternatively, LA-defectiveness is a virion property which does not vary with virion density.With 3 Figures     

Latent infection of human ovarian teratocarcinoma cells with human cytomegalovirus

Summary Persistent infection with human cytomegalovirus (HCMV) can be established in cultures of human ovarian teratocarcinoma (PA₁) cells, and maintained for more than 200 days. Infected cultures maintained at 34°C (PA₁CMV₃₄) and 37°C (PA₁CMV₃₇) entered crisis and subsequently displayed massive cytopathic effects (CPE), whereas infected cultures maintained at 32°C (PA₁CMV₃₂) and 39°C (PA₁CMV₃₉) continued to release small amounts of infectious virus until 240 or 151 days post-infection (p. i.) respectively. PA₁CMV₃₂ cultures shifted to 37°C at 258 days p.i. resumed synthesis of infectious virus which resulted in cell destruction, indicating that latent infection with HCMV was maintained in PA₁ cells at 32°C. In contrast, PA₁CMV₃₉ cells did not produce infectious virus even when cultured at 37°C for more than 100 days after the temperature shift.With 1 Figure     

Infection of human amnion cells with cytomegalovirus.

Human cytomegalovirus (CMV), known to replicate in vitro in human fibroblastic cells, was found to replicate in epithelial human amnion (HA) cells. Large syncytia formed in these cells after infection with CMV; inclusion bodies were observed in the nuclei, and CMV antigens were demonstrated in both the cytoplasm and the nucleus by indirect immunofluorescence techniques. The synthesis of virus DNA was also detected, and the production of infectious virus was followed. The titers were lower (from 10(4) to 6 X 10(5) using different isolates of CMV) than those obtained in human embryo fibroblast (HEF) cells, and the replication cycle was slower in HA than in HEF cells.     

Replication of cytomegalovirus in human thymic epithelial cells

Cytomegalovirus (CMV) has often been cited as a cause of immune suppression in children, yet little is known of the mechanisms through which this agent might affect immune function. We have succeeded in using CMV to productively infect cultured human fetal and infantile thymic epithelial (TE) cells. Morphological changes were apparent by 2–4 days after viral inoculation. CMV-related early antigen (EA) and late antigen (LA) were detected by immunofluorescence after 8 days, and progeny infectious CMV was recovered from culture media after 12–17 days. TE cells that reacted with monoclonal antibodies specific for keratin and for GQ ganglioside were predominant throughout the culture period. In contrast, infection by CMV resulted in a significant decrease in numbers of cells reactive with monoclonal antibodies specific for mesoderm-derived components. Inoculation of TE cells with CMV also caused a diminution in levels of detectable interleukin-1 (IL-1)-related antigen by 17 days after infection.     

Clinical utility of bronchoalveolar lavage cytomegalovirus viral loads in the diagnosis of cytomegalovirus pneumonitis in infants

Cytomegalovirus (CMV) pneumonitis is a significant cause of morbidity and mortality of children in Africa. The current practice for diagnosing CMV pneumonitis in this setting is based on interpretation of clinical, laboratory, and radiological findings. There is a need for a sensitive and specific laboratory test to objectively distinguish between patients with CMV pneumonitis and those with CMV infection, and non‐CMV pneumonia. In this study, we compared plasma and non‐bronchoscopic bronchoalveolar lavage (NBBAL) CMV viral loads in patients with CMV pneumonitis and those with CMV infection and non‐CMV pneumonia. Receiver operator characteristic curve analysis was used to establish a threshold and assess utility of viral loads in the diagnosis of CMV pneumonitis. We assessed the urea dilution method, and expression of viral loads relative to the total amount of extracted nucleic acids in correcting for NBBAL dilution. CMV quantification in NBBAL specimens was more predictive of CMV pneumonitis than blood CMV quantification. The threshold of 4.03 log IU/ml in NBBAL specimens has good predictive value and can be used to guide management of infants with suspected CMV pneumonitis. Adjusting for dilution of NBBAL specimens by using the urea dilution method or by expressing the viral load relative to the total nucleic acids extracted did not provide additional analytical benefits. J. Med. Virol. 89:1080–1087, 2017 . © 2016 Wiley Periodicals, Inc.

     

Intracellular forms of the parental human cytomegalovirus genome at early stages of the infective process.

Intracellular forms of human cytomegalovirus (HCMV) DNA molecules isolated from infected cells were examined by electron microscopy before and after the onset of viral DNA synthesis. In cells harvested before showing replicative forms of viral DNA, circular and concatemeric molecules were observed in addition to linear double-stranded molecules. The observation of circular, unbranched, unit-size molecules suggests that HCMV DNA has a repetitive sequence that is located at or near the termini and is exposed by exonuclease digestion within infected cells, and that single-stranded regions can complement each other to form circles. Linear molecules (unit size or smaller) and concatemers with replicative “eye” loops or forks were observed after DNA replication could be assumed to have begun. Viral DNA molecules with terminal loops were also observed. These structures indicate that an inverted repetition of the sequence may be present within the terminal region. The functions of these molecules and of circular molecules are unknown.