Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
Correspondence
Giuseppe Gerna
g.gerna{at}smatteo.pv.it
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ABSTRACT |
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MAIN TEXT |
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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 ULb' 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 2328. HUVEC were obtained by trypsin treatment of umbilical cord veins and used at passages 25 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 PMNL- and monocyte-tropic) HCMV strain (Revello et al., 2001).
Cell-free strain AD169 was inoculated onto confluent HUVEC monolayers grown in 24-well plates. Virus 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 µm pore size, Costar) from the lower compartment containing 10-8 M N-formyl-Met-Leu-Phe-Ala (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 (10-7). 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 ULb' (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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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 8077283492) and the DNA polymerase region encompassing all functional domains (nt 7761979633) were sequenced in the same three strain AD169 preparations, showing no nucleotide variations.
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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 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
(CXC) chemokine encoded by UL146 in the ULb' genome fragment missing in strain AD169 may reasonably explain why pp65-positive 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
).
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Received 20 December 2002;
accepted 12 February 2003.