Establishment and characterization of transformed goat primary cells by expression of simian virus 40 large T antigen for orf virus propagations

Abstract

Due to the limited host range of orf virus (ORFV), primary cells derived from its natural hosts, such as goats and sheep, are recommended for isolation and propagation of wild type ORFV. This situation limits the option for the study of virus-host interaction during ORFV infection since primary cells only support a few numbers of passages. SV40 T antigen is a viral oncoprotein that can abrogate replicative senescence, leading to an extended life span of cells. In this study, the transformation of two goat primary cells, fibroblast (FB) and testis (GT) cells, were achieved by stably expressing SV40 T antigen using the lentiviral technique. The presence of the gene encoding SV40 T antigen was validated by polymerase chain reaction (PCR) and western blot analyses. As evidenced by immunofluorescent microscopy, the two types of cells expressing SV40 T antigen (namely, FBT and GTT) were purified to homogeneity. Moreover, faster growth kinetics and a lower serum dependency were noticed in FBT and GTT, as compared with their counterpart parental cells. FBT and GTT remain permissive and can form plaque of ORFV, despite with different profiles; generally speaking, with SV40 T expression, ORFV forms plaques with smaller size and distinct margin. Most importantly, the prolonged life span of goat FBT and GTT serves as an ideal cell culture resource for ORFV isolation from the field, studies of ORFV pathogenesis and efficient vaccine development.

Immortalizing Cells for Human Consumption

“…The earliest method for immortalizing cell lines via inactivation or bypass of the p53/p16/Rb stress response has been transformation with viral genes. A notable example is the simian virus 40 (SV40) large T-antigen (TAg), which binds to and inactivates p53/Rb as well as other tumor suppressor factors in a number of species and organ types [ 28 , 29 , 30 ]. In the wild, SV40 is thought to infect senescent kidney epithelial cells in Rhesus macaques; here, TAg reactivates the host cell in order to promote replication of the SV40 virion [ 31 ].…”

The orf virus (ORFV) protein OV20.0 interacts with the microprocessor complex subunit DGCR8 to regulate miRNA biogenesis and ORFV infection

“…Human embryonic kidney 293T cell (293T), DGCR8 knockdown (KD) 293T cell, human lung adenocarcinoma A549 cell, immortalized goat testis cell (GTT) [21] and Madin‐Darby Canine Kidney cells (MDCK) were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS (Gibco BRL, Life Technologies Corporation, Carlsbad, CA, USA) and 1% of penicillin‐streptomycin. Cells were maintained at 37 °C with 5% CO 2 .…”

“…Recombinant ORFV expressing eGFP (ORFV‐eGFP) was described in the previous study [7]. In this study, cells were infected with ORFV‐eGFP at the indicated multiplicity of infection (MOI) for 1 h, and were subsequently cultured in DMEM supplemented with 2.5% FBS for 24 or 48 h. Viral yield was determined by plaque assay in GTT cells [21].…”

“…The filters were blocked in 5% skimmed milk, followed by immunoblotting with specific antibodies against target proteins. The dilutions of antibodies were as follows: FLAG antibody (1 : 2000) (F7425; Sigma‐Aldrich), PKR antibody (1 : 1000) (ab32052; Abcam, Cambridge, UK), DGCR8 antibody (1 : 1000) (GTX130061; Genetex), actin antibody (1 : 5000) (NB600‐501; Novus Biologicals, Centennial, CO, USA), tubulin antibody (1 : 5000) (NB100‐690; Novus Biologicals), eGFP antibody (1 : 2000) (YH80005; Yao‐Hong Biotechnology, Taiwan), hemagglutinin (HA) antibody (1 : 2000) (YH80008; Yao‐Hong Biotechnology, Taiwan), c‐myc antibody (1 : 1000) (YH80009; Yao‐Hong Biotechnology, Taiwan), NS1 antibody (1 : 2000) (GTX125990; Genetex) and F1L antibody (1 : 2000) [21]. Secondary antibodies conjugated with horseradish peroxidase (HRP) were diluted in 5% skimmed milk and incubated with membranes for 1 h at room temperature.…”

K160 in the RNA‐binding domain of the orf virus virulence factor OV20.0 is critical for its functions in counteracting host antiviral defense

“…Transferred membranes were blocked in 5% skimmed milk, followed by immunoblotting with specific antibodies against target proteins. In particular, the antibodies (and corresponding dilutions) were the following: FLAG antibody (1 : 2000) (F7425; Sigma-Aldrich), PKR antibody (1 : 1000) (ab32052; Abcam, Cambridge, UK), phosphor-PKR (PKR-p) antibody (1 : 1000) (ab32036; Abcam), ADAR1 antibody (1 : 500) (sc-73408; Santa Cruz Biotechnology), actin antibody (1 : 5000) (NB600-501; Novus Biologicals, Centennial, CO, USA), actin of N. benthamiana antibody (1 : 5000) [32], EGFP antibody (1 : 2000) (YH80005; Yao-Hong Biotechnology, New Taipei City, Taiwan), and F1L antibody (1 : 2000) [33]. Secondary antibody-conjugated horseradish peroxidase (HRP) was diluted in 5% skin milk and incubated with membranes for 1 h at room temperature.…”