Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 02 Dec 2018 21:11:57 Journal of General Virology (2001), 82, 1429–1438. Printed in Great Britain ................................................................................................................................................................................................................................................................................... In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells M. Grazia Revello, Fausto Baldanti, Elena Percivalle, Antonella Sarasini, Luciana De-Giuli, Emilia Genini, Daniele Lilleri, Nazarena Labo  and Giuseppe Gerna Servizio di Virologia, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, 27100 Pavia, Italy Four human cytomegalovirus (HCMV) isolates from different clinical sources were extensively propagated in human embryonic lung fibroblasts (HELF). Plaque isolates from each of the four virus strains were evaluated for their ability to be transferred to polymorphonuclear leukocytes (PMNL) and to grow in endothelial cells (EC). While all four of the clinical strains were found to be both PMNL- and EC-tropic, variants were identified from each of the four strains that lacked both biological properties, while three of the four parental strains lost their transfer capacity before passage 50 in HELF. It was demonstrated that one of the four field isolates (VR6110) and its transfer-deficient variant were genetically related, but showed different curves of virus yield in HELF. In addition, neither the immediate-early (IE) mRNA nor the IE protein p72 were found to be transferred to PMNL before 72 h post-infection (late in infection) or in the presence of viral DNA replication inhibitors. These findings link EC and PMNL tropism and suggest that PMNL tropism is a late HCMV function. Introduction The presence of human cytomegalovirus (HCMV) in blood is the hallmark of a disseminated infection in immuno- compromised patients (Van der Bij et al., 1988; Van den Berg et al., 1991 ; Gerna et al., 1991, 1994, 1998 a, 1999) and a recent primary infection in immunocompetent patients (Revello et al., 1998 a). During in vivo infection, HCMV interacts with a variety of leukocyte subsets, yet polymorphonuclear leuko- cytes (PMNL) represent the major cell subpopulation carrying infectious virus and virus products (Gerna et al., 1992 a ; Grefte et al., 1994). HCMV-infected endothelial cells (EC) secrete a series of virus-induced α (CXC) chemokines, such as IL-8 and Groα, which recruit PMNL (Grundy et al., 1998) and a novel HCMV-encoded α chemokine (the UL146 gene product) similar to IL-8 has been reported recently (Penfold et al., 1999). This virus-encoded chemokine, designated vCXC-1, along with virus-induced α chemokines, may play a major role in the active recruitment of PMNL. Although PMNL carry infectious virus, they do not seem to be productively infected in vivo (Gerna et al., 1992 a, Author for correspondence : Giuseppe Gerna. Fax 39 0382 502599. e-mail g.gernasmatteo.pv.it 2000 ; Grefte et al., 1994). However, results obtained using an in vitro model for the study of the PMNL–ECHELF (human embryonic lung fibroblasts) interaction suggest that PMNL may contribute to the haematogenous dissemination of HCMV in immunocompromised patients (Grundy et al., 1998 ; Revello et al., 1998 b). In the present study, we have plaque-purified isolates that do not have the capacity of the parental strains to be transferred to PMNL (PMNL tropism) or to grow in EC (EC tropism). These virus variants, referred to as transfer-deficient variants, are genetically related to the parental strains and behave in a way similar to laboratory-adapted HCMV strains in terms of PMNL and EC tropism. Transfer-deficient variants are useful tools for studying HCMV–PMNL interactions and may help to identify the viral gene(s) that is responsible for PMNL and EC tropism. Methods  Cell cultures and virus strains. HELF derived from a cell strain originally isolated in our laboratory were used at passages 23–28. Human umbilical vein EC (HUVEC) obtained by trypsin treatment of umbilical cord veins were used at passages 2–6. All primary HUVEC preparations were tested for the presence of the HCMV genome by nested PCR, as 0001-7566  2001 SGM BECJ