Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy1
Author for correspondence: Giuseppe Gerna. Fax +39 0382 502599. e-mail g.gerna{at}smatteo.pv.it
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Abstract |
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Introduction |
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Recently the Towne strain, like other high-passage strains such as AD169 or Davis, has been shown to lack two biological properties which have been found to be shared by all of the recent clinical HCMV isolates thus far tested (more than 100 strains), i.e. endothelial cell (EC) tropism and leukotropism (Revello et al., 1998 , 2001
; Gerna et al., 2000
, 2002a
). Endothelial cell tropism is the ability of an HCMV strain to infect and to grow in endothelial cells and, in particular, in human umbilical vein endothelial cells (HUVEC), while leukotropism is the ability of an HCMV strain to enter peripheral blood leukocytes and to replicate either abortively, as in polymorphonuclear leukocytes (PMNL) or productively, as in monocytes/macrophages, according to several reports (Lathey & Spector, 1991
; Ibanez et al., 1991
; Taylor-Wiedeman et al., 1991
, 1994
; Söderberg et al., 1993
; Söderberg-Nauclér et al., 1997
, 2001
; Fish et al., 1995
, 1996
; Gerna et al., 2000
). In addition, we have shown that all clinical HCMV isolates lose both biological properties after extensive propagation in human fibroblast cell cultures, thus documenting that modifications of biological properties of laboratory-adapted HCMV strains had occurred during propagation in cell cultures (Revello et al., 2001
). Loss of endothelial cell tropism and loss of leukotropism have been proposed as potential in vitro correlates of a true in vivo attenuation in terms of pathogenicity (Gerna et al., 2002a
).
Reversion of high-passage (laboratory-adapted) HCMV strains to endothelial cell tropism and leukotropism has never been fully investigated. In the present report, we describe the adaptation to growth in HUVEC of two different preparations of the Towne strain, which reacquired leukotropism more than 20 passages after initial adaptation to growth in HUVEC. Thus, endothelial cell tropism and leukotropism, which have been so far found to be apparently associated with one another, are two distinct biological properties.
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Methods |
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Virus strain.
The HCMV isolate VR6110, originally recovered from the blood of a patient with AIDS, was previously adapted to growth in HUVEC and was initially used as a reference HUVEC-tropic and PMNL-tropic low-passage strain (Revello et al., 1998 ). Subsequently, a new low-passage HCMV strain, originally recovered from cervical secretions of an immunocompetent pregnant woman (VR1814), was used routinely as a reference HUVEC-tropic PMNL-tropic HCMV strain (Revello et al., 2001
). The three laboratory-adapted HCMV strains Towne (originally obtained from E. Gönczöl, Wistar Institute, PA, USA), AD169 and Davis (both obtained from ATCC) were used as reference high-passage HCMV strains grown in human fibroblasts and lacking both HUVEC tropism and PMNL tropism (Revello et al., 1998
, 2001
). Two preparations of the Towne strain were used for adaptation of the virus to growth in HUVEC, both at passages between 129 and 134 in human fibroblasts: one obtained from E. Gönczöl (Wistar) on MRC-5, and the other one from RIT (Genval, Belgium). The former was inoculated onto HUVEC as cell-associated virus, the latter as cell-free virus. Finally, the Toledo strain (currently referred to as the reference wild-type or low-passage strain), also kindly provided by E. Gönczöl (Wistar), and shown recently to lack both HUVEC tropism and PMNL tropism (Gerna et al., 2002a
), was tested as a reference strain.
Assay for HUVEC tropism.
Two protocols were used to test for endothelial cell tropism (Gerna et al., 2002b ). The first was relevant to transmission of HELF-associated virus to HUVEC (Revello et al., 2001
). HELF were infected with Towne Wistar (Towne-W) at an m.o.i. of 15. Following a 7 day incubation at 37 °C, infected HELF showing 100% CPE were trypsinized and inoculated at a ratio of 1:3 onto confluent monolayers of uninfected HUVEC grown in 24-well plates. After a further 7 days of incubation, infected HUVEC were trypsinized and mixed at a ratio of 1:2 with uninfected HUVEC. This procedure was repeated weekly until passage 6, when cells were sonicated and cell-free virus propagated. Virus growth in HUVEC was checked 7 days p.i., at each passage, by immunofluorescence using monoclonal antibodies to the major immediate-early (IE) protein p72 or glycoprotein B (gB) (Gerna et al., 1990
). Monoclonal antibodies to gB were kindly provided by Lenore Pereira (UCSF, CA, USA). The second protocol was relevant to infection of HUVEC with cell-free Towne RIT (Towne-RIT) virus preparation grown in HELF at an m.o.i. of 5. Weekly passages of infected HUVEC followed the same protocol. Virus growth was checked 7 days p.i. by immunofluorescence using monoclonal antibodies as reported above. The degree of infection was determined subjectively by light microscopy following counterstaining with 0·0005% Evans blue.
Assays for PMNL and monocyte tropism.
Concentrated PMNL preparations from healthy blood donors were cocultured for 3 h with infected HUVEC 96168 h p.i. (Revello et al., 1998 ). Following coculture, to separate PMNL from infected HUVEC detached from the growth surface, cell suspensions were placed for 3 h at 37 °C in the upper compartment of a cell culture device separated by a transwell filter (5 µm pore size, Costar) from the lower compartment containing 10-8 M N-formyl-Met-Leu-Phe-Ala (FMLP, Sigma) according to a reported procedure (Revello et al., 1998
; Gerna et al., 1998a
). In these conditions, PMNL migrate to the lower compartment, thus separating from contaminating HUVEC and reaching a level of purity comparable to that of fluorescence-activated cell sorting (Revello et al., 1998
). In parallel, Ficoll-prepared peripheral blood mononuclear cell (PBMC) suspensions were separated in the monocyte subpopulation by purification through a Percoll gradient. Following overnight coculture according to the above protocol, monocytes were separated from infected HUVEC by the same procedure used for PMNL, except for a higher concentration of FMLP (10-7) in the lower compartment of the transwell device. PMNL and monocyte suspensions were then tested for presence of pp65 in cytospin preparations of 2x105 cells, according to a procedure originally reported for monitoring of HCMV antigenaemia in blood of immunocompromised patients (Gerna et al., 1992
) and more recently standardized (Gerna et al., 1998a
). Cytospin preparations of either PMNL or monocytes were examined for pp65 antigen following immunofluorescent staining with a pool of three monoclonal antibodies reactive to different epitopes of HCMV pp65 (Gerna et al., 1992
). In addition, monocyte preparations were examined for p72 and gB.
Restriction fragment length polymorphism (RFLP) analysis.
Genomic regions UL54, UL97 and UL123 of the HCMV strains examined were amplified by PCR using the pairs of primers previously reported (Revello et al., 2001 ). PCR products were then cleaved using two to four of the following endonucleases: HaeIII, MspI, HinP1I, AluI and BstUI (New England Biolabs). RFLP patterns were compared by agarose gel electrophoresis.
Southern blot analysis.
The genomes of Towne-W variants and Toledo were digested with EcoRI, HindIII and BamHI, blotted onto nylon membranes (Boehringer Mannheim) and hybridized using a set of cosmid probes from the VR6110 genome library spanning almost (9498%) the entire virus genome. The VR6110 cosmid library was constructed as previously reported (Revello et al., 2001 ).
Growth of Towne strain in human umbilical artery endothelial cells (HUAEC).
HUAEC were prepared from the same umbilical cords used for HUVEC preparations. However, the cell yield for HUAEC was markedly lower compared to HUVEC. Cells could be propagated and used within 45 passages. Virus growth in HUAEC was monitored, as reported for HUVEC, by looking for CPE and immunostaining cell cultures with monoclonal antibodies to both p72 and gB.
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Results |
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When using the cell-free Towne-RIT virus preparation, the number of infected HUVEC, which was very low upon first passage, rapidly increased till reaching about 30% and 50% of the cell monolayer at passage 4 and 5, respectively. The great majority of HUVEC were infected after passage 10. However, until at least passage 20, no PMNL nor monocyte was found to be pp65-positive after coculture, thus documenting a striking dissociation between EC tropism and leukotropism (Table 1). The rapid process of adaptation of Towne-RIT to growth in HUVEC during the very first passages is documented in Fig. 1
, where a large number of HUVEC are already productively infected in panel (F) corresponding to passage 8.
Starting at passage 25, coculture of Towne-RIT-infected HUVEC with either PMNL or monocytes yielded a high number of pp65-positive cells of either leukocyte subpopulation (Fig. 2), while the relative number of positive cells of the two subpopulations was comparable, suggesting that the efficiency of transmission to PMNL and monocyte was similar. In addition, following overnight coculture with infected HUVEC, monocytes were positive for IE protein p72 in the nucleus, while showing granules of gB in the cytoplasm.
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Discussion |
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On the other hand, all viral variants selected in vitro after extensive propagation in HELF and shown to be unable to infect leukocytes were also shown to be unable to grow in HUVEC (Revello et al., 2001 ). According to these findings, laboratory-adapted HCMV strains such as Towne were considered to lack both biological properties, EC tropism and leukotropism.
Unexpectedly, the results of the present study indicate that two different preparations of the Towne strain, both following extensive propagation in HELF, were able to grow readily in HUVEC after a few passages, thus suggesting that EC-tropic viral variants are still represented in the Towne strain preparations after more than 100 passages in human fibroblasts. The apparent identity at the molecular level (as shown by both RFLP and Southern blot analysis) of the Towne strain extensively propagated in human fibroblasts and shown to have lost leukotropism and the Towne strain grown in HUVEC and shown to have reacquired leukotropism, indicated that only minor genomic mutations should be responsible for the reversion of a laboratory attenuated virus to biological properties commonly shared by recent clinical isolates. To explain this reversion, the hypothesis of selection in vitro of viral variants previously represented in a minor proportion of the population appears more plausible than that of the sudden emergence of a new mutation rapidly representing the majority of the viral population.
However, the result which was most unexpected in this study was the dissociation (observed for the first time) of EC tropism and leukotropism. In fact, the two properties were found to be clearly distinct during the reversion process, with leukotropism being reacquired a number of passages after adaptation of Towne to growth in HUVEC. This dissociation was observed during adaptation of both Towne preparations, suggesting that different viral gene functions might be involved in the expression of the two biological properties. However, adaptation to growth in HUVEC appears to precede leukotropism, thus suggesting a link between the two properties.
It is well known that, when administered to humans, the Towne vaccine has never been found to be pathogenic nor to undergo latency (Plotkin et al., 1976 , 1989
; Quinnan et al., 1984
). However, if leukotropism and EC tropism, as recently suggested (Gerna et al., 2002a
), are in vitro correlates of in vivo pathogenicity, data herein reported do not seem to exclude the risk of reversion of the Towne strain to pathogenicity when administered as a vaccine to humans. In fact, replication in EC during primary infection of the immunocompetent or during both primary and reactivated infections of the immunocompromised host may be one of the first steps preceding virus dissemination in vivo through interaction of EC with circulating leukocytes, both PMNL and monocytes (Grundy et al., 1998
; Revello et al., 1998
; Gerna et al., 2000
).
Recently the Toledo strain, which is currently considered the prototype of wild-type HCMV strains, has been unexpectedly reported to lack both leukotropism and EC tropism in three different virus preparations from three different sources (Gerna et al., 2002a ), in contrast to the large number of recent clinical isolates tested and sharing consistently both properties (Revello et al., 2001
). However, low-passage Toledo has been actually shown to be pathogenic when inoculated in seronegative immunocompetent individuals (Quinnan et al., 1984
; Plotkin et al., 1989
). Whether Toledo strain lacking leukotropism and EC tropism is a true low-passage virus is uncertain at this time. Furthermore, results consistently obtained with the other virus strains representing true recent clinical isolates suggest that it is likely that Toledo strain preparations now circulating within laboratories most likely are no longer low-passage viruses. On this basis, we were interested in analysing at a molecular level the Toledo strain which has recently been reported to be adapted to growth in HUVEC (Grundy et al., 1998
). We used both RFLP and Southern blot analysis to demonstrate that the Toledo strain grown in HUVEC was not the true Toledo strain, but instead was likely to be an entirely different HCMV strain, a new virus strain resulting from recombination of Toledo with a new HCMV strain (F. Baldanti, M. G. Revello, E. Percivalle, N. Labò & G. Gerna, unpublished). Thus, these data further confirm that Toledo strain is not per se able to grow in HUVEC, as shown in our laboratory by consistently unsuccessful adaptation attempts.
In conclusion, we believe that leukotropism and EC tropism, which are lost following extensive propagation in HELF of laboratory-adapted HCMV strains, such as the Towne vaccine strain, may be reacquired if EC-tropic viral variants are still represented in HELF-derived viral populations. However, reversion to the ability to grow in HUVEC is not simultaneously accompanied by reversion to leukotropism, which is regained a series of passages afterwards. These findings suggest that a vaccine strain such as Towne may reacquire biological markers that are typical of wild-type HCMV strains.
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Acknowledgments |
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The authors thank Linda D'Arrigo for revision of the English, and the Personnel of the Obstetrics and Gynecology ward of the Clinic Città di Pavia', together with Nazarena Labò and Daniele Lilleri, for providing umbilical cords. In addition, the authors are indebted to the entire technical personnel of Servizio di Virologia, and to Daniela Sartori for typing the manuscript.
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References |
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Received 15 February 2002;
accepted 19 April 2002.