Institute of Molecular Biology1 and Infectology2, Friedrich-Loeffler Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany
Author for correspondence: Nikolaus Osterrieder. Fax +49 38351 7151. e-mail klaus.osterrieder{at}rie.bfav.de
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MD can be controlled by immunization, but MDV strains have a tendency towards increased virulence despite vaccination, and so-called virulent (v), very virulent (vv) and very virulent plus (vv+) strains have evolved with time (Benton & Cover, 1957 ; Schat et al., 1982
; Witter, 1983
, 1997
). The appearance of vv+ MDV-1 has led to the introduction in the United States of the CVI988/Rispens vaccine, which has been used in Europe since the 1970s (Rispens et al., 1972a
, b
; Witter, 1992
; Witter et al., 1995
). The cell culture of choice for propagation of MDV strains and production of vaccines, which include all three MDV serotypes, is chicken embryo cells (CEC) (Witter, 2001
). For isolation of virulent strains and virus isolates that are not cell culture-adapted, primary chicken kidney cells (CKC) or duck embryo cells (DEF) can be used (Biggs, 2001
). Permanent cell lines for MDV propagation have been described; however, long periods of virus adaptation to the cell culture system used were necessary. With the CHCC-OU2 cell line, a permanent cell line derived from CEC, 4 weeks of culture were needed before MDV-1-specific plaques were visible. The resulting CEC cell lines OU2.1 and OU2.2 contain a latent form of virulent MDV as long as the cells are not confluent. This latent virus is reactivated as soon as cell-to-cell contacts are formed (Abujoub & Coussens, 1995
, 1997
). CHCC-OU2 cells have also been used for production of cell lines containing vaccine strains of all three MDV serotypes. Recently, adaptation of MDV strains to the continuous Vero cell line after several rounds of passaging has been described (Jaikumar et al., 2001
).
In this communication, the generation of a permanent recombinant cell line is described, which supports efficient growth of both virulent and vaccine MDV strains. The cell line by itself was not tumorigenic and vaccine virus produced on this cell line was able to protect conventional chickens as efficiently as a standard vaccine produced on CEC.
Quail muscle QM7 cells (ATCC, CRL-1632) were grown on six-well plates and transfected with 10 µg of recombinant plasmid pcMgE, which contains the glycoprotein E (gE) open reading frame of the MDV-1 vaccine strain CVI988 under the control of the human cytomegalovirus immediate-early promoter (Brunovskis & Velicer, 1995 ; Schumacher et al., 2001
). Transfected cells were propagated in Dulbecco's minimal essential medium (DMEM) containing 10% foetal calf serum and 1 mg/ml G-418 (Gibco BRL). A cell clone in which virtually every cell expressed gE was identified by indirect immunofluorescence (IIF) and termed SOgE (Fig. 1A
). The presence of gE DNA in SOgE but not in QM7 cells was confirmed by PCR analysis (Fig. 1B
).
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Production of MD vaccines could be facilitated using a permanent cell line allowing propagation of MDV-1 vaccine strains. Therefore, CVI988 DNA was transfected into CEC, QM7 or SOgE cells. Six days after transfection, cells were harvested and 103 infected cells were co-seeded with 107 uninfected cells of the matching cell type. Five days post-infection (p.i.), infected cells were trypsinized and titrated on freshly prepared cells. Four days after titration, the numbers of virus plaques were determined by IIF. It could be shown that mean CVI988 titres on SOgE cells reached 1·8x106 p.f.u./107 cells whereas titres of only 5·2x102 p.f.u./107 cells were obtained on QM7 cells. The titres on SOgE cells were virtually identical to those on primary CEC. From the results of the plaque size determinations and the titration experiments, we concluded that propagation of MDV strains on SOgE cells was as effective as or even superior to propagation on primary CEC.
SOgE cells were derived from a chemically induced quail tumour, which raised the remote possibility that it may cause neoplasia after systemic application of whole cell preparations. To address this important issue, 12 conventional White Leghorn chickens (Lohmann Tierzucht) were inoculated at 1 day old with either SOgE cells (six chickens) or parental QM7 cells (six chickens). Each individual chicken simultaneously received 106 cells by the intramuscular and 106 cells by the intra-abdominal route. The fate of the inoculated chickens was followed for 12 weeks, after which post-mortem examination was performed. None of the chickens exhibited any clinical sign during the course of the experiment. All chickens appeared in a good nutritional state and without any sign of tumour formation at the post-mortem examination. From these results, we concluded that SOgE and parental QM7 cells do not cause tumours in chickens, even when approximately 1000-fold more cells compared with a vaccine dose are administered.
The protective capacity of the vaccine strain CVI988 propagated on SOgE cells compared with that produced on CEC was analysed by injecting 106 SOgE (seven chickens) or QM7 cells (six chickens), or 103 p.f.u. CVI988 produced on either SOgE cells (eight chickens) or CEC (eight chickens) into 1-day-old chickens. Vaccine virus was produced after transfection of CVI988 DNA isolated from CEC into SOgE cells or CEC. Transfected cells were co-seeded with fresh uninfected cells and vaccine virus was harvested on day 5 p.i. Immunized birds were challenge-infected with hypervirulent MDV-1 strain EU1 on day 12 after immunization. Strain EU1 was isolated from a vaccinated flock in Italy exhibiting lethal MD in 1992 (F. Fehler and others, unpublished). The results of the experiment are summarized in Fig. 3(A). In mock-immunized animals (QM7 cells), chickens showed signs of MD starting at day 7 p.i., and one bird died on each of days 9 and 10. By day 37 p.i., all birds had died as a consequence of the infection. In SOgE-immunized animals, three birds survived until termination of the experiment on day 65 p.i. The three surviving birds, however, exhibited MD as evidenced by a gross pathology examination and detection of tumours in inner organs. In stark contrast, chickens immunized with CVI988 produced on either SOgE cells or CEC did not suffer from MD until the termination of the experiment, but in one bird in each of these two groups, signs of MD were identified by gross pathology. Serological anti-MDV-1 responses in immunized and challenged birds were followed by an MDV-specific ELISA (Fig. 3B
). It could be demonstrated that protected birds exhibited antibody titres that gradually rose with time after challenge infection, whereas chickens inoculated with SOgE or QM7 cells did not develop high antibody titres (Fig. 3B
). We concluded that the CVI988 vaccine produced on permanent SOgE cells provided as good a protection as CVI988 produced conventionally on CEC. Protection against MD was coincident with gradually rising antibody titres, suggesting that a permanent boost of the chicken immune system by either lytically replicating vaccine virus or low level replication of EU1 is a prerequisite for protection against MD.
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The plaque sizes and virus titres obtained on SOgE cells exceeded that of the parental QM7 cells by 1000-fold. These results reflect the crucial function of MDV-1 gE in cell-to-cell spread, and indicate that the presence of large amounts of gE, which may not be complexed with gI as is the case in MDV-1-infected CEC, serves as a transporter' of MDV-1 infectivity from an infected to an uninfected cell. It is important to note that MDV-1 devoid of gE is unable to spread in CEC or QM7 cells (Schumacher et al., 2001 ). Future work will concentrate on the mechanism of MDV-1 cell-to-cell spread and the functions of the individual membrane and tegument proteins involved in this process. Cell-to-cell spread appears to be regulated and performed differently in MDV-1 and possibly varicella zoster virus when compared with other Alphaherpesvirinae, because all four proteins forming the membrane protein heterodimers gE/gI and gM/UL49.5 are essential in the case of MDV-1 (Schumacher et al., 2001
, Tischer et al., 2002
). In addition, the tegument component VP22, which may interact with either the gE and/or gM cytoplasmic tails, is also essential for MDV-1 (Dorange et al., 2002
; Mettenleiter, 2002
).
In summary, the permanent SOgE cell line exhibits unique features inasmuch as it provides the possibility for use as a permanent production system for MDV replication and may be a useful tool for experiments leading to the elucidation of the mechanisms underlying cell-to-cell spread in strictly cell-associated Alphaherpesvirinae.
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Received 31 January 2002;
accepted 15 March 2002.