Replication of varicella zoster virus in Raji cells

331

Virus Research, 4 (1986) 337-342

Elsevier VRR 00247

Replication of varicella zoster virus in Raji cells R. Cauda 1,2, S. Chatterjee 2, A.B. Tilden 3,4, C.E. Grossi * and R.J. Whitley 2 ’ Department of Pathology, ’ Pediatrics, and ’ Surgery and ’ The Veterans Administration Medical Center, The University of Alabamaat Birmingham, Birmingham, AL 35294, U.S.A. (Accepted 14 January 1986)

Summary This report provides evidence for the replication of varicella zoster virus (VZV) in Raji cells. Infection was achieved by co-cultivation of Raji cells with VZV-infected human fibroblasts. Replication of VZV, as assessed by immunofluorescence using monoclonal antibodies against VZV-glycoproteins, ranged from 18 to 24% of the cells. Electron microscopy detected complete virions within the membrane-bound cytoplasmic vesicles and free viral particles in the nuclear matrix as late as 12 days post-infection. Western blot analysis of infected Raji cells demonstrated VZV-specific glycoproteins. The availability of a VZV-susceptible cell line growing in suspension culture provides a useful model for future studies. replication of varicella zoster virus, infection of Raji cells, monoclonal VZV glycoproteins, VZV-specific glycoproteins

antibodies,

Varicella zoster virus (VZV) propagates in cell culture monolayers primarily of human or simian origin, as well as other continuous cell lines. In susceptible cell cultures VZV causes a characteristically focal cytopathic effect (CPE) and spreads from the infected cell to the neighboring cells. Few infectious particles become free under standard conditions. Therefore, it is difficult to obtain routinely large quantities of infectious cell-free VZV. Furthermore, freeze-thawing and trypsin treatment of sonicates substantially reduces the amount of recoverable virus (Schmidt and Lennette, 1976; Dumas et al., 1980). In infected cell cultures at least sixteen VZV-specific polypeptides have been recognized by heterologous antisera obtained by immunization of guinea pigs with VZV or by serum from humans with herpes zoster (Asano and Takahashi, 1980; 016%1702/86/$03.50

0 1986

Elsevier Science Publishers B.V. (Biomedical Division)

338 Grose, 1981; Grose and Friedrichs, 1982). Three of these, gp 62, gp 98 and gp 118, are the major glycoproteins of the virus envelope. Newly developed murine monoclonal antibodies have localized VZV-specific glycoproteins to sites of virus replication using electron microscopy (Achong and Meurisse, 1968; Grose et al., 1983; Weigle and Grose, 1983). The possibility of infecting Raji cells, a lymphoblastoid cell line that carries the EBV genome but does not produce virions or electron microscopic evidence of infection with VZV (Levanton-Kriss et al., 1979; Iltis et al., 1982) as well as such other herpesviruses as cytomegalovirus and herpes simplex (St. Jeor and Weisser, 1977; Henle et al., 1969) has been reported. However, detection of viral antigens and mature virions has not been provided. In this study, we present evidence for replication of VZV in Raji cells by electron microscopy, immunofluorescence assays, and Western blot analysis. Confluent monolayers of human foreskin fibroblast (HFS) cells, grown in Eagle’s minimal essential medium supplemented with 10% heat inactivated fetal calf serum (FCS) and gentamicin, were inoculated with a clinical isolate of VZV at a MO1 of 10. When a 90% cytopathic effect was achieved, medium was removed and 5 X 10” Raji cells per ml grown in RPM1 1640, supplemented with 10% heat inactivated FCS and gentamicin, were overlaid on the HFS cells. After a 2 h incubation at 37°C non-adherent Raji cells and medium were removed and fresh medium (RPM1 1640 and gentamicin with 10% FCS) was added. Contact of Raji cells with VZV infected HFS continued for an additional 24 h and non-adherent cells were subsequently removed. This resulting suspension of non-adherent cells was incubated for 3 h at

Fig. 1. lmmunofluorescence pattern of VZV-infected RaJi cells after fixation with cold acetone. (A) Stained with monoclonal antibodies against a VZV glycoprotein complex. (B) Stained with a polyspecific convalescent serum.

339 37’C and subsequently treated with rabbit anti-human fibronectin antisera (Collaborative Research, Inc., Waltham, MA) at a 1: 2 dilution and guinea pig complement to remove HFS cells. Non viable cells were removed by Ficoll-Hypaque gradient cent~fugation. The final cell preparation (approximately 2 X lo6 per ml),

Fig. 2. Electron microscopic observation of VZV-infected Raji cells. Electron microscopy has been carried out essentially as described by Compans et al. (1966). Distinct viral particles (arrows) can be observed at 1 (A, B) and 12 (C) days post-infection. C = cytoplasm and N = nucleus. Ail magnifications are at X 15925.

340 containing more than 99% Raji cells, was cultured in RPM1 1640 supplemented with 10% heat-inactivated FCS and gentamicin at 37°C. This time point was designated as day 1 of the infection. The viability of the cells as assessed by trypan blue exclusion-test was determined as greater than 95%. The virtual absence of HFS cells was confirmed by immunofluorescence staining with anti-fibronectin antisera. The extent of VZV infection was determined by immunofluorescence studies using fluorescein-conjugated monoclonal antibodies (Ortho Diagnostic Systems, Inc., NJ) against major VZV glycoproteins. Infection ranged from 18 to 24% of the cells with a mean value of 20% 1 and 3 days after infection. Brightly fluorescing cells could be observed following incubation with monoclonal antibodies as well as convalescent serum from a patient with herpes zoster (Fig. lA, B). A higher percentage of positive cells (70-80%) was observed when the latter reagent was used. Uninfected Raji cells stained with anti-VZV monoclonal antibodies were negative as were the VZV-infected Raji cells stained with monoclonal antibodies against antiHSV glycoprotein B (J. Koga, S. Chatterjee, and R. Whitley, manuscript in prep.) or against 66 kDa glycoprotein of CMV (kindly provided by Dr. L. Pereira, California Department of Health Services, Berkeley, CA). In two different experiments, viral glycoproteins were expressed for 12 days. A progressive decrease in percentage of infected cells was observed from day 1 to 12, and only 0.1% of the Raji cells still expressed VZV-specific glycoproteins on day 12 of infection (data not shown).

1

2

,gP98/92

Fig. 3. Demonstration technique. The detailed Raji cells.

of virus-specific glycoproteins in VZV-infected Raji cells by Western blot procedure is described in the text. Lane 1, uninfected and lane 2. VZV-infected

341 In agreement with the immunofluorescence studies, electron microscopy detected the presence of both mature and immature viral particles either within vesicles in the cytoplasm or in the nucleus (Fig. 2). The presence of mature viral particles was shown on days 1 (Fig. 2A, B), 3 and 12 (Fig. 2C) post-infection, confirming the immunofluorescence data. To demonstrate the presence of VZV-specific glycoproteins in Raji cells, virus-infected and uninfected cell lysates were fractionated on SDS-polyacrylamide slab gel as described by Chatterjee et al. (1982) and processed for immunoblotting as described previously (Towbin et al., 1979; Chatterjee et al., 1985). Western blot analysis of infected Raji cells revealed the presence of a VZV-specific glycoprotein complex, gp98/92 (Fig. 3). In preliminary experiments, the VZV-infection was transferred from Raji cells on day 1 and 3 of infection to a monolayer of HFS cells as evidenced by focal CPE at the sites of Raji cell attachment. Raji cells infected with VZV provide an advantage over the widely employed cell culture in monolayers. The lymphoblastoid Raji cells grow continuously in cell culture and no enzymatic or mechanical procedures are needed which could affect the expression of viral glycoproteins. Although earlier studies have suggested the possibility to infect Raji cells with VZV, proof of VZV-infection was absent (Levanton-Kriss et al., 1979; Iltis et al., 1982). Our findings provide the first demonstration for infection of Raji cells by VZV using murine monoclonal antibodies directed against VZV specific glycoproteins. The replicative nature of the infection was confirmed by electron microscopy and Western blot analysis. Detection of enveloped viral particles on days 1 and 12 of infection either within vesicles or free in the cytoplasm of Raji cells rules out the possibility of non-specific binding of the virus to Raji cells. Since Raji cells do not produce Epstein-Barr virus, the electron microscopic findings are specific for VZV with the supporting studies. This is also confirmed by experiments in which infection was transferred from infected Raji cells to uninfected fibroblasts by cell to cell contact, 1 or 3 days following the infection of Raji cells. These results provide a useful model for diagnostic analysis and virological studies of human VZV infections.

Acknowledgements The authors wish to thank Ronald Acoff for excellent technical assistance and Ms. Jane Hamner for expert editorial work. R. Cauda was a recipient of NIH grant S07-RR-05349 and of a grant from Minister0 degli Affari Esteri, Rome, Italy.

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