Journal of General Virology (2003), 84, 1431–1436
Short
Communication
DOI 10.1099/vir.0.19055-0
Rescue of human cytomegalovirus strain AD169
tropism for both leukocytes and human endothelial
cells
Giuseppe Gerna, Elena Percivalle, Antonella Sarasini, Fausto Baldanti,
Giulia Campanini and M. Grazia Revello
Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
Correspondence
Giuseppe Gerna
[email protected]
Received 20 December 2002
Accepted 12 February 2003
Endothelial cell-tropism- and leukocyte- (polymorphonuclear- and monocyte-) tropism
(leukotropism) are two important biological properties shared by all recent clinical isolates of human
cytomegalovirus (HCMV). These properties are lost during extensive propagation of HCMV isolates
in human fibroblasts, as shown by reference laboratory-adapted strains AD169 and Towne. Here
we show that strain AD169 may reacquire both properties in vitro, endothelial (both venous and
arterial) cell-tropism preceding leukotropism (predominantly involving monocytes). Restriction
fragment length polymorphism analysis and sequencing performed on the original virus inoculum
from human fibroblasts and serial passages on endothelial cells confirmed virus identity. Thus,
fundamental biological properties may be lost and reacquired in vitro according to the cell culture
system employed. The lack of a 15 kb DNA fragment in the strain AD169 genome does not prevent
the rescue of these biological functions, thus indicating that they are likely to be encoded by viral
genes located elsewhere.
The most widely used reference strain of human cytomegalovirus (HCMV) is AD169, originally recovered from
adenoid tissue (Rowe et al., 1956). AD169, as well other wellknown laboratory-adapted or attenuated HCMV strains
such as Towne (Plotkin et al., 1975) or Davis (Weller et al.,
1957), have been extensively passaged in human fibroblast
cell cultures. During propagation, a large DNA fragment
within a region referred to as ULb9 was lost in strain AD169
and to a slightly lesser extent, in strain Towne (Cha et al.,
1996), while another AD169 viral variant, the UK variant,
has shown to lack an additional 929 bp in genes UL42 and
UL43 (Dargan et al., 1997; Mocarski et al., 1997). Recently, it
has been reported that during propagation all laboratory
strains have lost some basic biological properties consistently shared by recent clinical HCMV isolates, such as
the ability to grow in endothelial cells (EC-tropism) and
to be transferred to peripheral blood leukocytes (leukocytetropism or leukotropism), either polymorphonuclear leukocytes (PMNL) or monocytes (Revello et al., 1998; Gerna
et al., 2000, 2002a). It has also been demonstrated that
clinical isolates recovered from different body sites lost both
properties following a high number of passages in fibroblasts
(Revello et al., 2001). However, both the laboratory strain
Towne (Gerna et al., 2002b) and some clinical isolates
lacking both EC-tropism and leukotropism after propagation in fibroblasts (unpublished data) reacquired both
properties following inoculation onto human umbilical vein
endothelial cells (HUVEC).
0001-9055 G 2003 SGM
In this report, we show that strain AD169, currently considered unable to replicate in HUVEC, can be adapted to
growth in both HUVEC and human umbilical artery
endothelial cells (HUAEC), thus reacquiring an original
biological property which, in the absence of the ULb9 DNA
fragment presumably present in the original virus isolate,
was associated to a peculiar type of leukotropism mostly
restricted to monocytes.
Strain AD169 was propagated in human embryonic lung
fibroblasts (HELF) developed in the laboratory and used at
passages 23–28. HUVEC were obtained by trypsin treatment
of umbilical cord veins and used at passages 2–5 as reported
(Revello et al., 1998). HUAEC were obtained similarly by trypsin treatment of arteries of the same umbilical cords and
used within the same range of passages. All EC preparations
were tested for HCMV DNA by nested PCR (Gerna et al.,
1998b) to exclude asymptomatic congenital HCMV infection.
The laboratory-adapted HCMV strain AD169, originally
obtained from ATCC and propagated in HELF, was used
for inoculation of HUVEC and HUAEC and was shown
to lack both EC-tropism and leukotropism. In addition,
a low-passage strain (VR1814) was used routinely as a
reference HUVEC-tropic and leukotropic (both PMNLand monocyte-tropic) HCMV strain (Revello et al., 2001).
Cell-free strain AD169 was inoculated onto confluent
HUVEC monolayers grown in 24-well plates. Virus
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G. Gerna and others
inoculation was followed by plate centrifugation for 45 min
at 600 g. After a 7 day incubation at 37 ˚C in a 5 % CO2
atmosphere, infected HUVEC monolayers were trypsinized
and mixed at a ratio of 1 : 2 with uninfected HUVEC. This
procedure was repeated weekly. Virus growth in HUVEC
was checked 7 days post-inoculation (p.i.), at each passage,
by immunofluorescence using monoclonal antibodies to
the major immediate-early protein p72 (Gerna et al., 1990)
and gB (kindly provided by L. Pereira, UCSF, CA, USA).
The degree of infection was determined subjectively by
light microscopy following counterstaining with 0?0005 %
Evans’ blue.
Assays for PMNL- and monocyte-tropism were performed
as reported (Gerna et al., 2002b). Briefly, PMNL preparations from healthy blood donors were first cocultured with
infected HUVEC (Revello et al., 1998), and then placed in
the upper compartment of a cell culture device separated by
a transwell filter (5 mm pore size, Costar) from the lower
compartment containing 1028 M N-formyl-Met-Leu-PheAla (FMLP; Sigma). In these conditions, PMNL are attracted
to the lower compartment, 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 suspensions were purified from the
monocyte fraction through a Percoll gradient. Procedures
for coculture and purification of cocultured monocytes were
the same as for PMNL, except for a higher concentration of
FMLP (1027). Purified PMNL and monocyte suspensions
were then tested for the presence of HCMV pp65 in cytospin
preparations, according to a procedure developed for monitoring of HCMV antigenaemia in immunocompromised
patients (Gerna et al., 1992, 1998a).
Restriction fragment length polymorphism (RFLP) analysis
was performed by PCR amplification of the HCMV genomic
regions UL54, UL55, UL123 and ULb9 (Revello et al., 2001),
and then cleaving PCR products with two to four of the
following endonucleases: HaeIII, MspI, HinP1I, AluI and
BstUI (New England Biolabs). RFLP patterns were compared by agarose gel electrophoresis.
Seven days after inoculation of cell-free strain AD169 virus
preparation (titrated on microtitre plates of HELF 96 h
p.i. following immunostaining with a monoclonal antibody
to a late HCMV antigen) at an m.o.i. of 5 onto confluent
monolayers of HUVEC, followed by centrifugation, immunostaining of the cell monolayer revealed the presence of a
considerable number of discrete plaques (Fig. 1A), initially
consisting of a central infected cell, stained for both p72
(nuclear) and gB (cytoplasmic), often surrounded by a
variable number of contiguous cells showing nuclei stained
for p72 only. Upon subsequent weekly passages, the number
of HUVEC stained for both p72 and gB increased
progressively, reaching about 1 % at passage 2, 10 % at
passage 3 and 50 % at passage 4, while cytopathic effect
increased in parallel from 1+ at passage 2 to 3+ at passage
4. Tests for leukotropism, performed for both PMNL and
monocytes, were negative.
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Subsequently, between passages 5 and 10, the weekly
passages on HUVEC were associated with a progressive
extension of the infection to about 100 % of the cell monolayer (Fig. 1A–F), while tests for leukotropism remained
negative, thus documenting the same dissociation between
HUVEC-tropism and leukotropism already observed during
adaptation to growth in HUVEC of the Towne strain (Gerna
et al., 2002b). Afterwards, following coculture, a small
number of leukocytes started becoming positive for pp65.
Finally, between passages 16 and 20, coculture of strain
AD169-infected HUVEC with either PMNL or monocytes
yielded a high number of pp65-positive leukocytes, predominantly of the monocyte subpopulation (Fig. 1K–L).
Compared to monocytes, the relative proportion of pp65positive PMNL ranged between 1?0 and 10?0 % (Table 1).
At this time, strain AD169 has reached passage 70 on
HUVEC, where it continues growing efficiently.
The growth kinetics of AD169 in HUAEC was not substantially different from that reported for HUVEC (Fig. 1G–J).
In fact, the rate of adaptation to growth, the chronological
dissociation between appearance of EC-tropism and leukotropism, and the difference in tropism for the two leukocyte
subpopulations, were comparable.
The genetic identity of strain AD169 at passages HELF/35,
HUVEC/70 and HUAEC/28 was verified by RFLP analysis of
multiple genome regions amplified by PCR. The identity of
the three virus preparations was documented in 100 % (16/
16 combinations) of RFLP profiles of amplified regions,
while strains Toledo and Towne as well as a number of
clinical isolates (not reported) were readily differentiated
from strain AD169 and from each other. Representative
RFLP profiles are shown in Fig. 2. In addition, gB (nt
80772–83492) and the DNA polymerase region encompassing all functional domains (nt 77619–79633) were
sequenced in the same three strain AD169 preparations,
showing no nucleotide variations.
In this study, we report the adaptation to growth in both
HUVEC and HUAEC of the laboratory strain AD169.
Rescue of EC-tropism was followed by rescue of leukotropism, which was mostly restricted to monocytes. EC-tropism
can be defined as the property of a virus strain to infect and
propagate extensively in EC. Thus, EC-tropism involves
virus adsorption, penetration, transport through the cytoplasm and then viral DNA access to the nucleus to trigger
immediate-early transcriptional processes. AD169 has been
repeatedly reported not to grow in HUVEC (MacCormac &
Grundy, 1999; Sinzger et al., 2000) and the underlying
mechanism has been attributed to the impairment or
inefficiency of translocation of the viral genome into the
nucleus of infected cells (Slobbe van Drunen et al., 1998;
Sinzger et al., 2000; Bolovan-Fritts & Wiedeman, 2002).
However, strain AD169 may infect and replicate to some
extent in aortic endothelial cells (Fish et al., 1998; BolovanFritts & Wiedeman, 2001), where virus infection does not
spread to contiguous cells, resulting in a nonlytic persistent
infection (Fish et al., 1998). Opposite results have been
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Journal of General Virology 84
Rescue of HCMV AD169 EC- and leukotropism
Fig. 1. Adaptation to growth in EC of a cell-free HCMV AD169 preparation. (A–F) Adaptation to HUVEC. (A) Passage 1, rare
foci of virus infection. (B–D) Passages 2 to 4 showing a progressive increase in the number of infected cells. (E, F)
Generalized spreading of the infection to the entire cell monolayer at passage 8. (A, F) 6006. (B–E) 1506. (G–J) Adaptation
to HUAEC. (G) Passage 1 (6006). (H) Passage 4 (1506). (I, J) Passage 7 at a lower (1506) and higher (6006)
magnification, respectively. (K, L). In vitro generated pp65-positive (K) monocytes and (L) polymorphonuclear leukocytes
following coculture with HUVEC infected with AD169. The relative proportion of positive cells of the two leukocyte
subpopulations can be seen. 6006. (A–J) Indirect immunofluorescent staining at 7 days p.i. using a pool of monoclonal
antibodies to the immediate-early protein p72 and gB. (K, L) Indirect immunofluorescent staining with a pool of pp65-specific
monoclonal antibodies (Gerna et al., 1992).
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G. Gerna and others
Table 1. Sequential reversion to EC- (both HUVEC- and HUAEC-) tropism and leukotropism of an AD169 virus preparation
lacking both biological properties
N, negative; L, low (1–50 per 16105 cells) number of pp65-positive leukocytes; M, medium (51–500) number; H, high (>500) number;
HUVEC, human umbilical vein endothelial cells; HUAEC, human umbilical artery endothelial cells; PMNL, polymorphonuclear leukocytes;
HELF, human embryonic lung fibroblast cell strain established in the laboratory in 1980.
% HCMV-infected
Virus preparation
AD169 HELF/35 (cell-free)
Leukotropism
Sequential passages
HUVEC
HUAEC
HUVEC
HUAEC
PMNL
Monocytes
1
2
3
4
10
15
30
70
0?1
1?0
10?0
50?0
100?0
100?0
100?0
100?0
0?01
0?1
1?0
20?0
80?0
100?0
100?0
Not determined
±
+
2+
3+
4+
4+
4+
4+
2
2
±
+
4+
4+
4+
Not determined
N
N
N
N
N
N–L
L
L
N
N
N
N
N
M
H
H
reported by others showing that arterial EC may undergo a
lytic infection if an EC-adapted HCMV isolate is tested
(Kahl et al., 2000). Our results show that even strain AD169
Fig. 2. RFLP analysis of: (A) two HCMV DNA polymerase
fragments, POL 1-2 (nt 77619–78544) and POL 3-4 (nt
78632–79633), digested with BstUI, and HaeIII and HinP1I
endonucleases, respectively; and (B) gB (nt 81873–82174)
digested with AluI and MspI. RFLP profiles are identical for
AD169 grown in (1) fibroblasts (HELF/35), (2) HUVEC (HUV/
70) and (3) HUAEC (HUA/28), while (4) Toledo and (5)
Towne are differentiated from AD169 in 4/5 (except for gB
digested with MspI) and 5/5 combinations, respectively.
1434
CPE
is cytopathogenic for both venous and arterial EC if it has
been adapted to growth in this cell system. These results may
be explained by hypothesizing that adaptation to growth in
EC occurs through the in vitro selection of an endotheliotropic viral variant which is present in a minor proportion
in the viral population prior to inoculation onto EC, and
is rescued during the adaptation process. An alternative
hypothesis is reversion of mutations acquired during HELF
propagation.
We have previously shown that the most efficient way to
adapt a HCMV clinical isolate to growth in EC is to recover
the virus directly from blood or, alternatively, to select the
endotheliotropic variant present in a virus strain recovered
from another body site by means of PMNL (Gerna et al.,
2002c). However, even in the presence of a virus strain not
able to grow in EC, a small number of EC are infected upon
inoculation of cell-free virus and become permissive for
virus replication with production of new viral progeny. The
latter cannot spread from parental infected cells to contiguous uninfected cells, thus preventing plaque formation
(our unpublished observations). Therefore, EC-tropism
seems to be determined by the ability to spread from cell to
cell rather than to infect EC. In this respect, cell-to-cell
spread during propagation of the infection in cell culture,
and transfer of virus and virus products from infected EC to
uninfected leukocytes, appear to be similar events, both
requiring microfusion of the cell membranes of the infected
and the contiguous uninfected cell and thus, enabling virus
transmission (Gerna et al., 2000). This process may be
mediated by an-as-yet unidentified virus gene product
directly or indirectly intervening in the cell fusion process.
The same susceptibility to growth in both HUVEC and
HUAEC has been recently reported by our group for
another laboratory strain, Towne (Gerna et al., 2002b).
However, in both cases, we have observed a dissociation
between rescue of EC-tropism and leukotropism. In fact,
leukotropism was reacquired following a number of
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Journal of General Virology 84
Rescue of HCMV AD169 EC- and leukotropism
passages after adaptation to growth in EC, thus suggesting
that different viral gene functions might be involved in the
expression of the two biological properties.
In addition, unlike leukotropism of either EC-adapted strain
Towne or recent clinical isolates, which consistently involves
both PMNL and monocytes to about the same extent (Gerna
et al., 2002b), leukotropism of EC-adapted AD169 was
predominantly restricted to monocytes. The recent identification of a potent granulocyte-attractant a (CXC) chemokine encoded by UL146 in the ULb9 genome fragment
missing in strain AD169 may reasonably explain why pp65positive monocytes are preferentially detected during coculture with strain AD169-infected EC (Penfold et al., 1999).
Furthermore, the high number of pp65-positive monocytes
suggests that some other viral gene should be responsible
for producing an hypothetical viral monocyte attracting
chemokine.
Molecular monitoring of the strain AD169 genome indicated that no other strain contamination occurred during
EC adaptation. In particular, RFLP analysis together with
sequencing of POL (UL54) and gB (UL55) genes has shown
the identity of strain AD169 grown in fibroblasts and the
virus grown in both venous and arterial EC. This supports
the hypothesis that only minor mutations are likely to be
responsible for the loss of EC-tropism and leukotropism
during propagation in fibroblasts.
In conclusion, if leukotropism and EC-tropism may be
considered in vitro correlates of in vivo pathogenicity (Gerna
et al., 2002a), then the reversion to pathogenicity of strain
AD169 may be considered a warning against administration
of attenuated strains to humans as a vaccine, as already
suggested for the Towne strain (Gerna et al., 2002b).
Gerna, G., Revello, M. G., Percivalle, E. & Morini, F. (1992).
Comparison of different immunostaining techniques and monoclonal antibodies to the lower matrix phosphoprotein (pp65) for
optimal quantitation of human cytomegalovirus antigenemia. J Clin
Microbiol 30, 1232–1237.
Gerna, G., Percivalle, E., Torsellini, M. & Revello, M. G. (1998a).
Standardization of the human cytomegalovirus antigenemia assay by
means of in vitro-generated pp65-positive peripheral blood polymorphonuclear leukocytes. J Clin Microbiol 36, 3585–3589.
Gerna, G., Zavattoni, M., Baldanti, F., Sarasini, A., Chezzi, L.,
Grossi, P. & Revello, M. G. (1998b). Human cytomegalovirus
(HCMV) leukoDNAemia correlates more closely with clinical
symptoms than antigenemia and viremia in heart and heart-lung
transplant recipients with primary HCMV infection. Transplantation
65, 1378–1385.
Gerna, G., Percivalle, E., Baldanti, F., Sozzani, S., Lanzarini, P.,
Genini, E., Lilleri, D. & Revello, M. G. (2000). Human cytomegalo-
virus replicates abortively in polymorphonuclear leukocytes after
transfer from infected endothelial cells via transient microfusion
events. J Virol 74, 5629–5638.
Gerna, G., Percivalle, E., Baldanti, F. & Revello, M. G. (2002a).
Lack of transmission to polymorphonuclear leukocytes and human
umbilical vein endothelial cells as a marker of attenuation of human
cytomegalovirus. J Med Virol 66, 335–339.
Gerna, G., Percivalle, E., Sarasini, A., Baldanti, F. & Revello, M. G.
(2002b). The attenuated Towne strain of human cytomegalovirus
may revert to both endothelial cell tropism and leuko- (neutrophiland monocyte-) tropism in vitro. J Gen Virol 83, 1993–2000.
Gerna, G., Percivalle, E., Sarasini, A. & Revello, M. G. (2002c).
Human cytomegalovirus and human umbilical vein endothelial cells:
restriction of primary isolation to blood samples and susceptibilities
of clinical isolates from other sources to adaptation. J Clin Microbiol
40, 233–238.
Kahl, M., Siegel-Axel, D., Stenglein, S., Jahn, G. & Sinzger, C.
(2000). Efficient lytic infection of human arterial endothelial cells by
human cytomegalovirus strains. J Virol 74, 7628–7635.
MacCormac, L. P. & Grundy, J. E. (1999). Two clinical isolates and
the Toledo strain of cytomegalovirus contain endothelial cell tropic
variants that are not present in the AD169, Towne or Davis strains.
J Med Virol 57, 298–307.
Mocarski, E. S., Prichard, M. N., Tan, C. S. & Brown, J. M. (1997).
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