Evidence that Marek's disease virus exists in a latent state in a sustainable fibroblast cell line

Abstract

Previously, we reported the development of two fibroblastic cell lines (MDV OU2.1 and OU2.2) infected with Marek's disease virus (MDV). The two cell lines, in nonconfluent continuous cultures, displayed characteristics consistent with MDV existing in a latent state. However, presence of distinct plaques in confluent cell monolayers and the ability to transfer cytolytic infection to susceptible birds and primary chick embryo fibroblasts, suggest that, if latent, the virus is easily reactivated from MDV OU2 cell lines. In this report, we present evidence which supports the hypothesis that MDV genomes in MDV OU2 cells are latent. PCR analyses and in vivo experiments demonstrate that CHCC-OU2 cells stabilize MDV so that serial in vitro passage does not attenuate the virus. Following two years of active culture, MDV genomes in MDV OU2 cells are still oncogenic, similar to that seen in MDV-lymphoblastoid cell lines. Expansion of the 132-bp repeat within MDV BamHI fragments H and D, typical of highly passaged serotype 1 MDV, has not been observed beyond two copies in MDV OU2 cells. Indirect immunofluorescence assays clearly demonstrate that MDV OU2 cells do not express glycoproteins B and I when subconfluent. However, upon reaching confluence these proteins are expressed in readily detectable amounts. Using RT-PCR we demonstrate that glycoproteins E and D are highly expressed in confluent MDV OU2 cells but absent from subconfluent cells, and MDV latency-associated transcripts (LATs), which are antisense to ICP4 transcripts and have been associated with latent MDV infection, are expressed in subconfluent MDV OU2 cells. Coincident with an increase in ICP4 expression, MDV LAT expression is down regulated when MDV OU2 cells become confluent.