Abstract
Frog virus 3 (FV3) is a large DNA virus that is the prototypic member of the family Iridoviridae. To examine levels of FV3 gene expression we generated a polyclonal antibody against the FV3 protein 75L. Following a FV3 infection in fathead minnow (FHM) cells 75L was found in vesicles throughout the cytoplasm as early as 3 hours post-infection. While 75L expressed strongly in FHM cells, our findings revealed no 75L expression in mammalian cells lines despite evidence of a FV3 infection. One explanation for the lack of gene expression in mammalian cell lines may be inefficient codon usage. As a result, 75L was codon optimized and transfection of the codon optimized construct resulted in detectable expression in mammalian cells. Therefore, although FV3 can infect and replicate in mammalian cell lines, the virus may not express its full complement of genes due to inefficient codon usage in mammalian species.
Highlights
Iridoviridae family members are large, icosahedral, double-stranded DNA viruses that are unique among eukaryotic virus genomes because they are both circularly permuted and terminally redundant [1]
The Iridoviridae family of viruses is comprised of five genera that can infect a variety of invertebrates (Iridovirus, Chloriridovirus) and ectothermic vertebrates (Lymphocystivirus, Ranavirus, Megalocytivirus) [2]
Frog virus 3 (FV3) has not been isolated from fish, closely related viruses to FV3 including epizootic haematopoietic necrosis virus (EHNV) and Bohle virus (BIV) have both been previously isolated from a variety of fish species [4,5,6]
Summary
Iridoviridae family members are large, icosahedral, double-stranded DNA viruses that are unique among eukaryotic virus genomes because they are both circularly permuted and terminally redundant [1]. CO75L was cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen) and transfected into BGMK cells and twenty-four hours posttransfection cells were fixed and indirect IF was used to detect 75L (mouse anti-myc and goat anti-mouse FITC conjugated antibodies). The corresponding amino acids are shown on the bottom row and are the same for both the original and optimized sequence This data demonstrates that at least one FV3 gene does not produce detectable proteins in mammalian cell lines. Essential viral genes must express in mammalian cell lines since the virus is able to infect and successfully replicate in many cell lines, including rodent, human, and simian cell lines (Figure 2) [10,11]. This work has provided a means for further characterization of the function of 75L
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