Propagation of Rat Parvovirus in Thymic Lymphoma Cell Line C58(NT)D and Subsequent Appearance of a Resistant Cell Clone after Lytic Infection

Abstract

Rat parvovirus (RPV) is nonpathogenic in rats but causes persistent lymphocytotropic infection. We found that RPV was propagated in rat thymic lymphoma cell line C58(NT)D and induced apoptosis. Interestingly, a resistant subclone, C58(NT)D/R, from surviving cells after lytic infection had differentiated phenotypic modifications, such as increased cell adherence, resistance to apoptosis, and suppressed tumorigenicity.Recent molecular studies on parvoviral pathogenicity suggest that the viral nonstructural (NS) protein in which coded genes are highly homologous among parvoviruses correlates with cytotoxicity (2,8,17). The productive and cytotoxic activity of the NS protein is modulated by cellular factors that may vary with the host cell type, particularly in oncogene-transformed cells (2, 13). We have shown that a transcriptional coactivator, CREB binding protein, is required for NS1-mediated viral and cellular transcription in parvovirus-infected cells, resulting in cell proliferation and differentiation to achieve its lytic cycle (16).Newly recognized rodent parvoviruses, also called orphan parvoviruses, are widespread among laboratory mice and rats (21,23,24), and viruses isolated have been classified as mouse parvovirus and rat parvovirus (RPV) (1, 5, 6). Although mouse parvovirus grows well in a murine T-cell clone (L3), causing a cytopathic effect (CPE) (10), effective in vitro RPV propagation has not been established. We found that RPV involved lytic infection in the rat thymic lymphoma cell line C58(NT)D and developed in vitro propagation for RPV. Interestingly, virus-resistant cell clones isolated from subcultures of surviving cells acquired differentiated phenotypes such as reduced tumorigenicity and sensitivity to apoptosis. Viral propagation in cell lines. The RPV strain was isolated from rats infected spontaneously at our facility (23) and passaged three times in specific-pathogen-free newborn SpragueDawley rats. The virus stock was prepared from infected spleens at 7 days postinoculation (p.i.) We initially inoculated a supernatant of 5% infected spleen homogenate into cell lines and primary cell cultures of rats and hamsters to find cells permitting virus propagation. C6 (rat glioma), BRL-3A (Buffalo rat liver), RBL-2H3 (rat basophilic leukemia), BHK-21 (Syrian hamster kidney), and Y3-Ag1.2.3 (rat myeloma) cells were obtained from the Riken Cell Bank, Tsukuba, Japan, and C58(NT)D (rat thymic lymphoma) cells were purchased from the American Type Culture Collection, Manassas, Va. Infected cells were observed for appearance of the viral CPE and examined for presence of the viral antigen by immunofluorescent antibody (IFA) and hemagglutination (HA) ability assays. The isolated virus was propagated only in C58(NT)D cells, not in other cells tested (Table 1). Parvoviral DNA was also detected only in infected C58(NT)D cells (data not shown). In contrast, prototype RV-13 (7) was propagated, with titers in C58(NT)D cells lower than those in other cells. Propagation of the isolate in C58(NT)D cells and a dif...

Lurking in the Shadows: Emerging Rodent Infectious Diseases

“…RPV-1 was amplified in vivo by inoculating Fisher rat leukemia cells into "ROPV-seropositive" rats, followed by in vitro isolation in 324K cells (Ball-Goodrich et al 1998). There have been reports of an additional isolate (RPV/UT) in Japan, but researchers have not yet decoded its complete genome sequence, so its relatedness to the other rat parvoviruses is unknown (Iseki et al 2005;Ueno et al 1996Ueno et al , 1997Ueno et al , 2001.…”

Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species

“…In particular, depending on cell type and growth conditions, H-1PV is able to induce apoptosis (35,42,46), necrosis (41), or cathepsin B-dependent cell death (16). It is presently unclear why some cells differ in the way they are killed by parvovirus and whether a common trigger initiates these various death processes.…”

Retargeting of Rat Parvovirus H-1PV to Cancer Cells through Genetic Engineering of the Viral Capsid

“…This study focuses on rat H-1PV, which infects and kills human tumor cell lines of various origins (e.g., of brain [23], pancreas [4,14], blood [3], colon [38], cervix [20], and breast [66,67]) and which is currently under evaluation in a phase I/IIa clinical trial for the treatment of patients with recurrent glioblastoma multiforme (57). H-1PV has the ability to induce different cell death pathways in cancer cells, including necrosis (53), apoptosis (28,46,54,65), and lysosome-dependent cell death (16), while sparing nontransformed cells. Recently, we have reported the capacity of the virus to induce oxidative stress in cancer cells leading to DNA damage, cell cycle arrest, and apoptosis.…”