Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall, Edinburgh, UK1
Moredun Research Institute, Pentlands Science Park, Penicuik, UK2
Author for correspondence: James Stewart. Present address: Centre for Comparative Infectious Diseases, The University of Liverpool, Dept of Medical Microbiology and Genitourinary Medicine, Duncan Building, Daulby Street, Liverpool L69 3GA, UK. Fax +44 151 706 5805. e-mail j.p.stewart{at}liv.ac.uk
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Abstract |
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Main text |
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The detection of DNA in diseased animals and inapparently infected sheep has confirmed that OvHV-2 is the aetiological agent involved in SA-MCF (Baxter et al., 1993 ; Li et al., 1998
; Muller-Doblies et al., 1998
; Wiyono et al., 1994
). OvHV-2 DNA can be found in epithelial tissues and B cells from sheep (Baxter et al., 1997
). However, in SA-MCF-affected ruminants, virus DNA has only been observed in hyperplastic T cells (Baxter et al., 1993
; Bridgen & Reid, 1991
). These T cells can grow in culture into lines and will transmit disease back to cattle as well as to experimental animals such as rabbits and hamsters (Burrells & Reid, 1991
; Buxton et al., 1984
, 1988
). In turn, T cell lines can subsequently be isolated and passaged from these secondary hosts (Reid et al., 1983
).
AlHV-1 has been isolated, completely sequenced and will productively infect epithelial cell lines in culture (Ensser et al., 1997 ; Plowright et al., 1960
). In contrast, research on OvHV-2 has been hampered by the lack of reagents and a consistent failure to isolate the virus and define a productive cell culture system. The aim of this work was to ascertain the nature of OvHV-2 genomes in T cell lines and to define a productive culture system for OvHV-2. Here, we show that infected rabbit T cell lines support aspects of the virus productive cycle.
To identify cells supporting productively replicating OvHV-2, we analysed infected T cell lines by Gardella (in situ lysis) gel electrophoresis and Southern blotting exactly as described previously (Gardella et al., 1984 ; Usherwood et al., 1996
). This technique discriminates linear from covalently closed circular (CCC) viral genomes which are characteristic of cells supporting either productive or latent herpesvirus infection respectively. OvHV-2-infected T cell lines were derived from an affected cow (BJ/1035) or affected rabbits (BJ/2222, BJ/2223) as described previously (Buxton et al., 1985
). The control cell lines, S11 (Usherwood et al., 1996
) and BCP-1 (Boshoff et al., 1998
), are latently infected with the related MHV-68 and KSHV respectively. Equal numbers (2x106) of cells were loaded onto each lane of the gel. A Southern blot of the gel was probed simultaneously with 32P-labelled DNA fragments derived from the OvHV-2 ORF75, the MHV-68 gp150 gene (Stewart et al., 1996
) and the KSHV K13 gene. The results are shown in Fig. 1
. S11 (MHV-68-infected) and BCP-1 (KSHV-infected) cells contained both CCC and linear genomes but S11 cells contained a higher proportion of linear relative to circular genomes. Indeed, the amount of linear genomes in BCP-1 cells was on the borderline of detection (shown by asterisk). This corresponds to a relatively high level of spontaneous reactivation (up to 20% of cells) in S11 (Usherwood et al., 1996
), compared with BCP-1 where the level of spontaneous reactivation is only 12% of cells (Talbot et al., 1999
). An OvHV-2-infected bovine T cell line (BJ/1035) contained predominantly circular genomes and a barely detectable level of linear genomes (shown by asterisk). In contrast, two OvHV-2-infected rabbit T cell lines (BJ/2222 and BJ/2223) contained comparatively large amounts of linear genomes with only a relatively small amount of circular genome apparent. These results strongly suggested that the OvHV-2-infected bovine line that we studied contained predominantly latently infected cells, whereas the two rabbit lines contained latently infected cells but also a relatively large proportion of cells that might be undergoing productive replication.
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Acknowledgments |
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References |
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Usherwood, E. J., Roy, D. J., Ward, K., Surman, S. L., Dutia, B. M., Blackman, M. A., Stewart, J. P. & Woodland, D. L. (2000). Control of gammaherpesvirus latency by latent antigen-specific CD8+T cells. Journal of Experimental Medicine 192, 943-952.
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Received 4 July 2002;
accepted 28 August 2002.
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