scholarly journals In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells

2001 ◽  
Vol 82 (6) ◽  
pp. 1429-1438 ◽  
Author(s):  
M. Grazia Revello ◽  
Fausto Baldanti ◽  
Elena Percivalle ◽  
Antonella Sarasini ◽  
Luciana De-Giuli ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Polymorphonuclear Leukocytes ◽  
In Vitro Selection ◽  
Biological Properties ◽  
Lung Fibroblasts ◽  
Human Embryonic Lung ◽  
Infected Cells ◽  
Selection Of

Four human cytomegalovirus (HCMV) isolates from different clinical sources were extensively propagated in human embryonic lung fibroblasts (HELF). Plaque isolates from each of the four virus strains were evaluated for their ability to be transferred to polymorphonuclear leukocytes (PMNL) and to grow in endothelial cells (EC). While all four of the clinical strains were found to be both PMNL- and EC-tropic, variants were identified from each of the four strains that lacked both biological properties, while three of the four parental strains lost their transfer capacity before passage 50 in HELF. It was demonstrated that one of the four field isolates (VR6110) and its transfer-deficient variant were genetically related, but showed different curves of virus yield in HELF. In addition, neither the immediate-early (IE) mRNA nor the IE protein p72 were found to be transferred to PMNL before 72 h post-infection (late in infection) or in the presence of viral DNA replication inhibitors. These findings link EC and PMNL tropism and suggest that PMNL tropism is a late HCMV function.

scholarly journals Human Cytomegalovirus Replicates Abortively in Polymorphonuclear Leukocytes after Transfer from Infected Endothelial Cells via Transient Microfusion Events

2000 ◽  
Vol 74 (12) ◽  
pp. 5629-5638 ◽  
Author(s):  
Giuseppe Gerna ◽  
Elena Percivalle ◽  
Fausto Baldanti ◽  
Silvano Sozzani ◽  
Paolo Lanzarini ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Copy Number ◽  
Polymorphonuclear Leukocytes ◽  
Biologically Active ◽  
Lung Fibroblasts ◽  
Wild Type ◽  
Viral Dna ◽  
Infected Cells ◽  
Type Strains

ABSTRACT Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

scholarly journals Human Cytomegalovirus Replication and Infection-Induced Syncytia Formation in Labial, Foreskin, and Fetal Lung Fibroblasts

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2355
Author(s):  
Alexis Aguiar ◽  
Melissa Galinato ◽  
Maite’ Bradley Silva ◽  
Bryant Toth ◽  
Michael A. McVoy ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Anatomical Site ◽  
Cmv Infection ◽  
Viral Spread ◽  
Syncytia Formation

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

scholarly journals Binding of human and animal immunoglobulins to the IgG Fc receptor induced by human cytomegalovirus

2001 ◽  
Vol 82 (5) ◽  
pp. 1137-1145 ◽  
Author(s):  
Annika Antonsson ◽  
P. J. Hugo Johansson
Keyword(s):  
Human Cytomegalovirus ◽  
Binding Sites ◽  
Fc Receptor ◽  
Lung Fibroblasts ◽  
Affinity Constant ◽  
Human Embryonic Lung ◽  
Binding Properties ◽  
Human Igg ◽  
Infected Cells ◽  
Simplex Virus

Human cytomegalovirus (HCMV)-infected cells express a virus-encoded receptor that is able to bind the Fc part of IgG. Some basic binding properties of this Fc receptor (FcR) have been examined. The affinity constant (K a) for human IgG Fc fragment in its interaction with acetone-fixed, HCMV-infected human embryonic lung fibroblasts was estimated to be around 2×108 M−1 and the number of binding sites was estimated to be around 2×106 per cell. Of the human IgG, IgA, IgM and IgD classes, only IgG reacted with the receptor, and all four of the IgG subclasses were reactive. IgG from rabbit, hamster, cat, swine and horse exhibited binding to the HCMV FcR, in contrast to IgG from mouse, rat, guinea pig, dog, sheep, goat, cow and chicken. Immunoglobulins with and without HCMV IgG FcR-binding properties, like IgG from rabbit and mouse, can be of value in revealing the functional importance of the receptor. When the immunoglobulins were tested against herpes simplex virus type 1-induced FcR, both similarities and differences in immunoreactivity were seen relative to the HCMV FcR, which makes it unlikely that the binding sites for these two herpesvirus FcRs on the IgG molecule are identical.

In vitro selection of DNA aptamers against coxsackievirus B3 infected cells

2005 ◽  
Vol 2005 (Fall) ◽  
Author(s):  
Andreas Zerressen-Harte ◽  
Volker A. Erdmann
Keyword(s):  
In Vitro Selection ◽  
Coxsackievirus B3 ◽  
Dna Aptamers ◽  
Infected Cells ◽  
Selection Of

scholarly journals The attenuated Towne strain of human cytomegalovirus may revert to both endothelial cell tropism and leuko- (neutrophil- and monocyte-) tropism in vitro

2002 ◽  
Vol 83 (8) ◽  
pp. 1993-2000 ◽  
Author(s):  
Giuseppe Gerna ◽  
Elena Percivalle ◽  
Antonella Sarasini ◽  
Fausto Baldanti ◽  
M. Grazia Revello
Keyword(s):  
Endothelial Cell ◽  
Cell Cultures ◽  
Human Cytomegalovirus ◽  
Biological Properties ◽  
Clinical Isolates ◽  
Viral Gene ◽  
Cell Tropism ◽  
Human Embryonic Lung ◽  
Healthy Human

The Towne strain of human cytomegalovirus (HCMV), originally recovered from the urine of a congenitally infected newborn, was attenuated through 125 passages in human embryonic lung fibroblast cell cultures. Although reliable markers of attenuation were not identified, the virus was shown to be attenuated by inoculation of both healthy human volunteers and immunocompromised patients. More recently, Towne (like other laboratory-adapted strains) was shown not to have two biological properties typical of recent clinical isolates: endothelial cell tropism and polymorphonuclear leukocyte tropism. These markers of attenuation are lost by all clinical isolates on extensive propagation in cell cultures and are apparently associated with one another. Here, we show that Towne may reacquire both endothelial cell tropism and leuko- (polymorphonuclear- and monocyte-) tropism on adaptation to growth in endothelial cell cultures. However, reversion to endothelial cell tropism is dissociated from reversion to leukotropism, since the latter was reacquired 10–20 passages later. Thus, these two biological properties, which were considered to be encoded by the same viral gene(s), appear to be distinct. Both restriction fragment length polymorphism and Southern blot analysis demonstrated the identity of the attenuated and endothelial cell tropic variants of Towne, thus suggesting that only minor variations (mutations) of the viral genome may be responsible for loss or reacquisition of the two biological properties. Viral genes involved in endothelial cell tropism and leukotropism remain to be identified. However, reversion of attenuated strains to pathogenicity in vivo cannot be excluded a priori.

scholarly journals Efficient Lytic Infection of Human Arterial Endothelial Cells by Human Cytomegalovirus Strains

2000 ◽  
Vol 74 (16) ◽  
pp. 7628-7635 ◽  
Author(s):  
M. Kahl ◽  
D. Siegel-Axel ◽  
S. Stenglein ◽  
G. ◽  
Jahn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Umbilical Vein ◽  
Lytic Infection ◽  
Vascular Bed ◽  
Complete Lysis ◽  
Infected Cells ◽  
Arterial Endothelial Cells

ABSTRACT Endothelial cells (EC) are common targets of permissive infection by human cytomegalovirus (HCMV) in vivo during acute disease. However, studies of HCMV-EC interactions in vitro have generated discordant results. While lytic infection of cultured venous EC has been well established, Fish et al. (K. N. Fish, C. Soderberg Naucler, L. K. Mills, S. Stenglein, and J. A. Nelson, J. Virol. 72:5661–5668) have reported noncytopathic persistence of the virus in cultured aortic EC. We propose that interstrain differences in viral host cell tropism rather than the vascular bed of origin of infected EC might account for these discrepancies. In the present investigation we compared the responses of EC derived from human adult iliac artery, placental microvasculature, and umbilical vein to infection with various HCMV strains. Regardless of the vascular bed of origin, infection with EC-propagated HCMV strains induced 100% efficient cytopathic change progressing to complete lysis of inoculated monolayers. While fibroblast-propagated strains persisted at low titer in infected arterial EC cultures, they were also cytolytic for individual infected cells. The finding of cytopathic lytic infection of arterial EC by HCMV implicates a mechanism of vascular injury in the pathogenesis of HCMV infection.

scholarly journals Human Cytomegalovirus Infection of Endothelial Cells Triggers Platelet Adhesion and Aggregation

2005 ◽  
Vol 79 (4) ◽  
pp. 2211-2220 ◽  
Author(s):  
Afsar Rahbar ◽  
Cecilia Söderberg-Nauclér
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Atherosclerotic Disease ◽  
Viral Gene ◽  
Platelet Adherence ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 ± 15 [mean ± standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, α5β3-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% ± 5%, 74% ± 5%, or 18% ± 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.

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