Development and characterization of a sustainable chick cell line infected with Marek's disease virus

Marek's disease virus (MDV) is an oncogenic, highly infectious, cell-associated avian herpesvirus. Fully productive MDV infections are restricted to feather follicle epithelium of afflicted birds. In cell culture, MDV infection of primary chick and duck fibroblast cells is semi-productive. Passage of MDV and production of MDV vaccines are limited to these primary cell systems. The limited life span of primary avian cell cultures has hampered efforts to use positive selection in generation of recombinant MDV and has complicated studies of temporal gene regulation.; We have developed a sustainable cell culture system (MDV OU2) using the nononcogenic, immortalized CHCC-OU2 chick cell line, which supports MDV replication. Southern blot and PCR analyses demonstrated that these cell lines do harbor MDV. MDV pp38 and pp14 expression was detected in sparse and confluent MDV OU2 cells by western blot analysis and IIFA staining, but expression of MDV structural glycoproteins (gB and gI) were detected only in confluent MDV OU2 cells. We also demonstrate using RT-PCR that MDV latency associated transcripts (LATs) are expressed at higher levels in sparse MDV OU2 cells than confluent monolayers and LAT expression is down regulated in confluent cells. MDV ICP4 expression was inversely proportional to the level of MDV LAT expression. Presence of distinct plaques and expression of glycoproteins in confluent MDV OU2 cell monolayers is consistent with a cytolytic infection. Whereas the pattern of MDV LAT/ICP4 expression, and lack of glycoprotein expression in sparse MDV OU2 cells are consistent with a latent infection. Data presented in this dissertation suggest that MDV OU2 cells are latent when subconfluent and the virus is reactivated when cells become confluent.; MDV OU2 cells are capable of transferring MDV infection to CEF in vitro and can induce MD in vivo. PCR analysis and in vivo experiments also demonstrated that MDV genomes are stabilized in this cell culture system. Unlike MDV passaged in CEF cells, MDV OU2 cells are still oncogenic and can induce clinical symptoms of MD after more than two and a half years in active continuous culture.