Department of Medical Virology1 and Department of Medicine3, University of Tü bingen, Calwerstraße 7/6, D-72076 Tübingen, Germany
Department of Pathology, Ohio State University, Columbus, USA 2
Author for correspondence: Christian Sinzger.Fax +49 7071 295790.e-mail christian.sinzger{at}med.uni-tuebingen.de
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Abstract |
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Introduction |
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A number of clinical and experimental observations support the hypothesis that interstrain differences in the virulence of HCMV variants may contribute to differences in the clinical course of infections. Vaccination studies in healthy human volunteers have demonstrated that fibroblast-adapted HCMV strains lack virulence as compared with recent isolates (Elek & Stern, 1974 ; Quinnan et al., 1984
). Similar interstrain differences have been reported in SCID mice carrying human thymus/liver transplants (Brown et al., 1995
). In vitro studies have revealed that apathogenic laboratory strains fail to propagate in endothelial cells or macrophages, while recent HCMV isolates can efficiently infect these cells (Waldman et al., 1989
; Ibanez et al., 1991
; Lathey & Spector, 1991
; Minton et al., 1994
; Sinzger et al., 1997
). Immunohistochemical analyses have demonstrated that tissue macrophages and endothelial cells are in vivo targets of HCMV in infected organs (Wiley & Nelson, 1988
; Roberts et al., 1989
; Sinzger et al., 1993
, 1995
, 1996
; Ng Bautista & Sedmak, 1995
). Transmission of virus from endothelial cells to blood cells in cell culture models has further underscored the potential role of infected endothelial cells for the dissemination of virus (Waldman et al. , 1995
; Grundy et al., 1998
). Clinical observations suggest that the appearance of circulating infected endothelial cells in the peripheral blood was associated with increased risk of symptomatic disease (Grefte et al., 1993
; Percivalle et al., 1993
). Loss of endothelial cell tropism of a clinical HCMV isolate during adaptation to fibroblast culture has been reported (Waldman et al., 1991
). Based on these findings it was hypothesized that the potential of HCMV strains to infect endothelial cells might influence the clinical course of infections (Grefte et al., 1995
). However to date, phenotypic analyses have only compared single clinical isolates with laboratory strains. Thus, the degree of variability in natural tropism among clinical isolates, as well as the nature of the determinants of tropism, remain unknown.
Recently large-scale genomic differences between fibroblast-adapted strains and low-passage HCMV isolates have been described, which might account for the phenotypic interstrain differences that had been observed in the hu-SCID mouse model (Cha et al., 1996 ). With regard to interstrain differences in endothelial cell tropism, this hypothesis has yet to be tested.
This study is focused on the endothelial cell tropism of HCMV isolates. We investigated 30 recent isolates to quantify the distribution of this phenotype in wild-type HCMV strains. The stability of the endotheliotropic phenotype during differential propagation in fibroblasts or endothelial cells was tested. Differentially adapted isolate pairs were compared on a large-scale genomic level, and transfection experiments revealed that the endothelial cell tropism is determined by genomic variants of HCMV strains.
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Methods |
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Clinical HCMV strains were isolated from throat swabs, urine, bronchoalveolar lavage or blood of immunocompromised patients. Underlying conditions of those patients included allogeneic bone marrow transplantation, liver transplantation, human immunodeficiency virus infection, autoimmune disease and congenital HCMV infection. All patients had HCMV-DNAemia, indicating systemic HCMV infection. Recent clinical HCMV isolates were initially propagated on HFF until about 10% of cells showed cytopathic effect (CPE). Infected cultures were subsequently trypsinized and stored at -80 °C until analysis in focus expansion (FE) assays. At that time no infectivity was detectable in the supernatant of isolate cultures, implying that virus remained strictly cell-associated. To differentially adapt recent isolates to endothelial cells or fibroblasts, isolate cultures at a CPE of 10% were treated with 2x10-6 M mitomycin C to induce terminal differentiation (Niggli et al., 1989 ) and abolish mitotic activity. After this treatment aliquots of the isolate cultures were cocultured both with HUVEC and HFF. Mitomycin C treatment prevented outgrowth of fibroblasts in HUVEC cultures and allowed for the adaptation of cell-associated recent isolates to HUVECs. For adaptation of isolates to fibroblasts, cell- associated isolate cultures were subcultured in HFF until infectivity became detectable in the supernatant of those cultures by 50% tissue culture infectious dose (TCID50) assays. Cell-culture- adapted strains were propagated by infection of subconfluent HFF monolayers at an m.o.i. of 0·1 and harvest of virus progeny at day 7 after infection. Infectious supernatants were either used for further propagation of the virus or for experiments, or were stored at -80 °C. The unique nature of clinical isolates was confirmed by restriction fragment length analysis (RFLA) in the a- sequence of the viral genome (Zaia et al., 1990
; Weber et al., 1993
), or by RFLA of the complete viral genome when results from the a-sequence were equivocal. All isolates were different from laboratory strains and from each other.
For supernatant-mediated infection of cell cultures, media were removed and replaced by fresh MEM containing glutamine (2·4 mM), gentamicin (100 µg/ml) and foetal calf serum (5%) 60 min prior to infection. Cells were incubated with infectious HCMV at an m.o.i. of 0·1 at 37 °C for 60 min in a low volume of MEM with occasional rocking. Appropriate media were then added and infected cultures were maintained at 37 °C in a 5% CO2 atmosphere. Virus stocks were harvested 6 days after infection by removal of cell debris from infected fibroblast supernatant. If necessary, virus stocks were concentrated by ultracentrifugation at 80000 g for 70 min. Fractioning of virus particles was done by ultracentrifugation on glycerol tartrate gradients (Irmiere & Gibson, 1983 ). Virus preparations were stored at -80 °C until used for experiments.
Analysis of virus replication.
The titre of infectious virus in preparations of cell-culture- adapted virus was determined by end-point titration to determine the TCID50 assay. Briefly, tenfold dilution series of infectious supernatants were incubated with HFF or HUVEC in 96-well plates in quadruplicate. After 24 h of incubation at 37 °C in a 5% CO2 atmosphere HCMV immediate early antigen was detected by indirect immunoperoxidase staining, employing MAb E13 (Biosoft) as a primary antibody, peroxidase-labelled rabbit anti-mouse Ig serum (Dako) as a secondary antibody, and aminoethylcarbazole as a chromagen. TCID 50 values of supernatants were calculated by the method of Spearman and Kaerber (Mahy & Kangro, 1996 ).
The replication of cell-associated isolates in a given cell culture was quantified by FE assays as previously described (Fig. 1 ) (Sinzger et al., 1997
). Briefly, frozen infected cells were thawed, washed and cocultured in 96-well plates together with either uninfected HFF or uninfected HUVEC. To quantify HCMV replication in endothelial cell cultures (FEHUVEC), 2x104 uninfected HUVEC per well were cocultured with serial dilutions of infected fibroblasts (104 to 100) for 5 days at 37 °C with 5% CO2 in endothelial cell medium. To determine HCMV replication in fibroblast cultures (FEHFF), 2x104 uninfected HFF per well were cocultured in the same way, using fibroblast medium. After 5 days of cocultivation, cells were fixed with cold methanol and HCMV immediate early antigen was detected by indirect immunoperoxidase staining, as described above. All tests were done in quadruplicate. Stained slides were read with an Axiovert 135 microscope (Zeiss). Infectious foci were defined as clusters of three or more antigen-positive cells. The number of infected cells in the largest focus in each of the four parallel tests was counted. The highest and the lowest counts were eliminated and the mean values of the remaining two counts were defined as the FE value of the respective isolate.
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Restriction enzyme digests.
Viral DNA was digested with either EcoRI, BamHI, XbaI or HindIII. Digestion mixtures (2040 µl), each containing 14 µg of viral DNA, digestion buffer, H2O and 1040 IU of the enzyme, were incubated for 12 h at 37 °C. Digestion was stopped by adding 1/10 vol. of loading buffer containing 20% Ficoll 400, 0·1 M Na2EDTA (pH 8·0), 1% SDS, 0·25% bromophenol blue and 0·25% xylene cyanol.
Agarose gel electrophoresis and Southern blot analysis.
Viral DNA fragments were run on 1% agarose gel for 1248 h at 40 V, stained with ethidium bromide, and visualized by transillumination with UV light. Gels were soaked in 1 M KOH denaturation solution for 30 min and then incubated in 0·5 M TrisHCl (pH 7·5) neutralization solution. DNA fragments were transferred by capillary action to a positively charged nylon membrane for 312 h. DNA was cross-linked to the nylon membrane by UV-cross-linking. Membranes were prehybridized for 30 min at 42 °C. The denatured DNA probe was added to the hybridization buffer and allowed to hybridize at 42 °C overnight. DNA probes were labelled with digoxigenin (DIG)dUTP according to the manufacturer's protocol (Boehringer Mannheim). Hybridized DIG-labelled probes were detected with an anti-DIG antibody conjugated to alkaline phosphatase and developed as recommended by the manufacturer.
Transfection of fibroblasts with viral DNA.
For transfection assays genomic viral DNA was phenol-extracted from late-stage infected fibroblasts. To minimize shearing of genomic DNA, the aqueous DNA-containing phase was overlaid with 2 vols of isopropanol, and DNA was spooled at the interface onto a Pasteur pipette that had been heated at the tip to form a hook. DNA was air- dried and redissolved in TE buffer overnight. Fibroblasts were seeded into 6-well culture plates at a density of 200000 cells per well 24 h prior to transfection. For transfection 1 µg of viral DNA was introduced into fibroblasts using Superfect reagent (Qiagen) following the manufacturer's instructions. Infectious virus resulting from transfection assays was propagated in HFF and analysed by RFLA of complete genomic DNA. Pretransfection strains always resembled posttransfection strains with regard to RFLA of genomic DNA.
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Results |
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This study was performed (i) to test whether clinical HCMV isolates from patients with disseminated HCMV infection generally display an endotheliotropic phenotype, (ii) to analyse the time-course of cell culture adaptation, (iii) to analyse whether endothelial-cell-adapted and fibroblast-adapted strains of the same isolate differ in their genome, and (iv) to test whether the purified viral DNA of endotheliotropic HCMV variants is sufficient to reconstitute the endotheliotropic phenotype.
Definition of the endotheliotropic phenotype
All cytomegalovirus strains included in our analysis could infect endothelial cells to some degree. However, the attempt to propagate a certain strain continuously in endothelial cells sharply distinguished two groups of viruses. Nonendotheliotropic viruses like AD169, Towne and one clinical isolate, TB27/02, failed to increase the fraction of initially infected cells in the endothelial cell culture. Thus, they were unable to disseminate, and continuous passaging of inoculated cultures eventually resulted in complete loss of infectious virus. Endotheliotropic strains, in contrast, increased the number of infected cells by formation of infectious foci. These viruses were able to disseminate in endothelial monolayers, subsequently reaching 100% CPE. Endotheliotropic cytomegalovirus strains could be continuously passaged in endothelial cells yielding progressively higher titres of infectious virus. To allow for quantitative analysis of endothelial tropism of HCMV isolates we have developed an FE assay which is based on the coculture of few late-stage infected fibroblasts in a monolayer of uninfected EC as described previously (Sinzger et al., 1997 ) (Fig. 1
). This assay allows for the quantification of cell-associated focal dissemination of low-passage HCMV isolates in EC cultures prior to cell-culture-adaptation of those isolates. The number of immediate early antigen-positive (IE-positive) cells per focus at day 5 after coculture reflects the extent of virus replication and dissemination in the culture system (FEHUVEC ). The number of IE-positive cells after 5 days of culture was directly proportional to the number of late-stage infected cells after 8 days of culture. Therefore, to improve efficiency we chose to focus on the detection of viral immediate early antigen. We initially compared focus formation properties of three endotheliotropic isolates and the nonendotheliotropic strain AD169 in HUVEC, human aortic endothelial cells, and microvascular endothelial cells with essentially identical results regardless of endothelial cell type. Thus, because of ease of accessibility we used HUVEC for all subsequent analysis of endothelial cell tropism of clinical HCMV isolates. In summary, the ability of cytomegalovirus strains to disseminate in endothelial cell culture was indicated by their potential to form foci of
4 infected cells in a HUVEC monolayer within 5 days, starting from single late-stage infected cells.
Endothelial cell cytopathogenicity is a common but variable property of clinical HCMV isolates
In the phenotypic analysis we included 30 recent clinical isolates as well as the nonpathogenic laboratory strains AD169 and Towne. All clinical isolates used in this study originated from patients with confirmed disseminated HCMV infection. The assays were performed identically with isolates and laboratory strains. Neither laboratory strain was able to form infectious foci (>>3 cells) in endothelial monolayers, although in fibroblast culture they replicated and disseminated rapidly, forming comet-shaped foci (Fig. 1). In contrast, 29 out of 30 recent HCMV isolates generated cytopathic foci in endothelial monolayers (Figs 1
and 2
). This indicated that endothelial tropism actually distinguishes most recent isolates from long-term fibroblast-adapted strains. However, clinical isolates were not uniform regarding the extent of FE, but rather displayed up to tenfold variability in FEHUVEC values. The distribution of FEHUVEC values in the 30 tested HCMV isolates with a mean value of 12·6 cells per focus and a SD of 9·0 demonstrated a continuous spectrum of endothelial tropism rather than a rigid distinction between endotheliotropic and nonendotheliotropic strains. These findings suggest that endothelial cell tropism is a complex phenotype which is determined polygenically rather than by a single gene.
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Immediately after isolation from patient specimens in fibroblast culture, HCMV isolates were passaged weekly by subculturing of the infected cell cultures. Aliquots of the culture supernatant were assayed for infectious virus. Aliquots of infected cells were tested by FE assays for fibroblast tropism (FEHFF) and endothelial cell tropism (FEHUVEC). Cell culture adaptation in this context was defined as the appearance of detectable infectivity in the supernatant of infected fibroblast cultures. Fibroblast culture adaptation was always associated with a sudden increase of the FEHFF value (Fig. 3). At that time foci became significantly larger than before and developed a comet shape. Fibroblast culture adaptation of those isolates occurred between passage 5 and passage 12. While these changes occurred in FEHFF assays, FEHUVEC values of the same isolate passages remained constant (Fig. 3
). This indicated that endothelial tropism is a relatively stable property under these conditions (at least through passage 12).
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Differences in endothelial cell cytopathogenicity are associated with genomic variations
We then sought to determine whether these phenotypic changes, which occurred during differential selection of a given HCMV isolate in fibroblasts or EC, were accompanied by alterations at the DNA level. Differentially adapted HCMV isolate pairs were subjected to a comparative analysis of the genome by RFLA. As such isolate pairs originate from a common parental HCMV isolate, they do not display a priori genetic polymorphism. Therefore, genomic differences between members of these pairs are likely linked to the phenotypic differences resulting from differential propagation. We analysed two isolate pairs generated by differential long-term selection as mentioned above (VHL/E and VHL/F; TB40/E and TB40/F). Genomic DNA of each strain was digested with restriction enzymes BamHI, EcoRI, HindIII or XbaI. The resulting digestion profiles were visualized by electrophoresis on 0·6% agarose gels for RFLA. In addition, it would have been of great interest to compare TB40/E and TB40/F with the DNA of the original TB40 isolate. However, the strict cell association of the recent isolate TB40 and the limited CPE at passage 5 when both strains were separated made it impossible to obtain viral DNA without excess cellular DNA background. Therefore, only the long-term adapted strains could be analysed. The RFLA pattern of VHL/E closely resembled the pattern of VHL/F, indicating that both variants originated from the same isolate. Digests of VHL/E and VHL/F genomes with BamHI, EcoRI and XbaI yielded at least one differing fragment whereas no difference was detected in the HindIII digest (Fig. 4). The isolate pair TB40/E and TB40/F displayed a greater number of differing fragments in RFLA than the VHL pair did. With each enzyme at least four different fragments were detected in a viral genome RFLA (Fig. 4
). However, TB40 variants more closely resembled each other than any unrelated isolate (data not shown), indicating that TB40 viruses also originated from a common parental isolate. At this point two assumptions were deduced from our phenotypic and genotypic comparisons of HCMV strains: (i) genomic differences between endotheliotropic and nonendotheliotropic HCMV strains might account for the phenotypic differences, and (ii) the continuous distribution of the endotheliotropic phenotype in a population of HCMV strains may indicate a polygenic nature of this phenotype. This is further supported by the finding that fragment differences of the VHL pair are not identical with fragment differences of the TB40 pair.
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Discussion |
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A clear definition of the endotheliotropic phenotype was prerequisite to this analysis. In this study, endothelial cell tropism was defined as the ability of an HCMV isolate or strain to disseminate within endothelial cell culture. Endotheliotropic HCMV variants were sharply distinguished from nonendotheliotropic HCMV by their characteristics during long-term propagation in endothelial cells. With nonendotheliotropic variants the fraction of infected cells decreased over time, eventually resulting in complete loss of virus. In contrast, with endotheliotropic variants the number of infected cells increased over time, finally resulting in 100% CPE. The expression of endothelial cell tropism was then quantified by an FE assay. This readout system had practical advantages: it could be applied to cell-associated HCMV isolates directly after primary isolation; the phenotype was consistent not only in HUVEC cultures but also in aortic endothelial cells and in microvascular endothelial cells (data not shown); mixed phenotypes in a heterogeneous virus preparation could be discriminated even if the endotheliotropic virus was the minority.
From the analysis of endothelial cell tropism of 30 clinical HCMV isolates, two conclusions can be drawn. These studies confirm previous anecdotal reports of natural endothelial cell cytopathogenicity of fresh clinical HCMV isolates. The vast majority of isolates was clearly distinguished from fibroblast-adapted HCMV strains by the ability to form infectious foci in endothelial cell culture. However, clinical isolates were not uniform with regard to the degree of endothelial cell tropism. This indicates that in vivo, strain variation in endothelial cell tropism might also exist and may influence the virulence of the respective strain.
Based on our findings of phenotypic alterations during continuous passaging of HCMV isolates in fibroblast culture, four sequential degrees of cell culture adaptation can be discriminated. (i) The hallmark of all recent HCMV isolates was their strict cell association during the initial fibroblast passages. Most recent isolates displayed endothelial cell tropism. (ii) Cell-culture-adapted HCMV strains have lost their strict cell association but have still retained their natural endothelial cell tropism. (iii) The hallmark of fibroblast- adapted HCMV strains was the loss of endothelial cell tropism. Endothelial cell-adapted strains retained the endotheliotropic phenotype. (iv) Plaque-purified fibroblast-adapted HCMV strains had a stable nonendotheliotropic phenotype. Similarly, plaque purification of endothelial-cell-adapted strains resulted in a stable endotheliotropic phenotype.
Regarding the underlying mechanism of cell culture adaptation, we propose three different hypotheses. (i) Propagation of HCMV in various cell types might alter the composition of the virion, resulting in cell tropism alterations independent of the viral genome. (ii) Loss of endothelial cell tropism might be caused by genomic de novo mutations occurring during extented propagation in fibroblasts. (iii) Loss of endothelial cell tropism might result from the selection of preexisting nonendotheliotropic variants in a genomically heterogeneous virus isolate.
The data obtained in this analysis are consistent with the assumption that cell culture adaptation is a result of selection of preexisting variants. Transfection of pure viral DNA into fibroblasts was sufficient to reconstitute the endotheliotropic or nonendotheliotropic phenotype of those HCMV variants from which the DNA was prepared. This clearly demonstrates that the endotheliotropic phenotype of HCMV is determined by the viral genome, independent of the cell type in which the virus is assembled. This is an expected finding, as there are many reports about genomic determination of cell tropism with other viruses (Sharma et al., 1976 ; Almond et al., 1984
; Christodoulou et al., 1990
; Equestre et al., 1991
; Stokes et al., 1993
; Willcocks et al., 1994
; Funkhouser et al., 1994
; Graff et al., 1994
; Georgescu et al., 1995
; McGoldrick et al., 1995
; Harrowe & Cheng Mayer, 1995
; Lukashov & Goudsmit, 1995
; Schr öder et al., 1997
; Schmidt et al., 1997
). However, it has not been shown for HCMV before. Two other findings further support the importance of genomic variants for expression of the endotheliotropic phenotype. (i) Strain pairs generated from a single isolate by collateral propagation in fibroblasts and EC displayed large-scale genomic differences. (ii) Coinfection of EC with two nonendotheliotropic HCMV strains yielded endotheliotropic recombinant HCMV variants. Variants from independent experiments resembled each other in their restriction fragment pattern, which was a combination of both parental strains. This strongly indicates that recombination of parental strains occurred during coinfection, and that a certain genome combination could restore the endotheliotropic phenotype in the recombinant virus.
Finally, the different stability of the endotheliotropic phenotype in the crude TB40 isolate versus the plaque-purified HCMV variant TB40/E favours the assumption of preexisting variants in the TB40 isolate, which was recently reported (MacCormac & Grundy, 1999 ). If the loss of endothelial cell tropism was due to de novo mutations occurring in cell culture, this would also have applied to long-term culturing of the plaque-purified virus. However, the endotheliotropic phenotype of plaque-purified TB40/E was stable through 40 passages in fibroblasts, while the endothelial cell tropism of the recent TB40 isolate was lost after 20 passages. Though no formal proof, this favours the existence of preexisting variants in the clinical HCMV isolate, which are lost after plaque purification.
In conclusion, our data show that phenotypic alterations during long- term cell culture of HCMV isolates are associated with genomic variations, that the viral genome is sufficient to reconstitute the phenotype, and that these genomic variations most likely result from selection of preexisting variants. We are aware that these experiments have not yet mapped the gene regions determining the endotheliotropic phenotype, but they provide the rationale for future attempts to define HCMV genes involved in endothelial cell cytopathogenicity.
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Acknowledgments |
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References |
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Received 19 April 1999;
accepted 16 July 1999.