Foamy viruses (FVs) comprise the only genus in the Spumaretrovirinae subfamily of the Retroviridae and have been isolated from many mammalian species including human, non-human primates (chimpanzee, baboon), bovine, equine and feline species [1, 4, 6, 15, 16, 25]. FVs have three hallmark retroviral genes, gag, pol and env. Meanwhile, there are at least two accessory genes, named tas and bet, located between env and the 3′ long terminal repeat (LTR) . However, different from ordinary retrovirus, such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), FVs have a separate pol mRNA, an infectious DNA genome and two promoters in their genome, the LTR and the internal promoter (IP) . Tas, the product of tas, can activate both the LTR and IP promoters in trans as the pivotal regulator of viral gene expression during FVs′ replication [5, 10]. All these special features of FVs set them apart from the other known retroviruses and make them attractive targets for study in order to understand more details about the FVs evolutionary advantages and to investigate their suitability as transfer vectors. Strain 3026 of bovine foamy virus (BFV3026) was isolated by our laboratory in 1995 .The strain shows typical features of FVs and has been investigated with the aim of constructing a gene transfer vector.
FV infection seems to lead to life-long latency in almost all hosts since these viruses are nonpathogenic in their natural hosts or in experimentally infected animals [7, 11, 12]. However, their infection can induce cellular syncytia formation only in susceptive cells in vitro, while in some other cells they cannot [17, 22]. Therefore, the traditional titration methods based on cytopathic effect (CPE) may have some limitations in titrating foamy viruses. Indicator cell lines have been developed for the rapid and efficient detection and quantification of viral infections. Since the viral transactivator, Tas, can activate the promoter of a reporter gene, the viral infection can be determined through the expression of the viral protein. This technique has been applied widely for the diagnosis of various viruses, such as HIV, herpes simplex virus (HSV), primate foamy virus (PFV) and feline foamy virus (FeFV) [19, 23]. To date the chloramphenicol acetyltransferase (CAT) gene, the β-galactosidase gene, the enhanced green fluorescent protein (EGFP) gene and the luciferase gene have been used as reporter genes.
In this study, we established the BFV indicator cell line-BFVL, in which the firefly luciferase gene was employed as a reporter gene, to detect the presence of infectious BFV based on activation of the LTR to express firefly luciferase. The BFVLTR-driven luciferase plasmid was transfected into baby hamster kidney cells (BHK21) and the stable transfected cells were screened and isolated. This cell line was determined to be stable, sensitive and specific in detecting and quantitating the active BFV infection.
A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection
- Received Date: 30 May 2011
- Accepted Date: 05 August 2011
Abstract: In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.