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
g.gerna{at}smatteo.pv.it


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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.


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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 well-known 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 ULb' 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 (leukocyte-tropism 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).

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 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 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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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) 600x. (B–E) 150x. (G–J) Adaptation to HUAEC. (G) Passage 1 (600x). (H) Passage 4 (150x). (I, J) Passage 7 at a lower (150x) and higher (600x) 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. 600x. (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).

 
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 pp65-positive 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.


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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 1x105 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.

 
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.



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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.

 
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; Bolovan-Fritts & 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 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 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 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 {alpha} (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.