Saffold virus type 3 (SAFV-3) persists in HeLa cells

Abstract

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler's murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.

Figure 1. Growth kinetics of SAFV-3 and TMEV-DA on HeLa-N and HeLa-R cells. A: Growth kinetics of SAFV-3.

Solid and broken lines indicate the growth curves of SAFV-3 on HeLa-N and HeLa-R cells, respectively. The viruses (as a mixture of cell-free and cell-associated viruses) were harvested at several time points indicated and assayed for titers by a standard plaque assay on HeLa-N cells. Titers shown are the means ± S.D. in three independent experiments. B: Growth kinetics of TMEV-DA. Solid and broken lines indicate the growth curves of TMEV-DA on HeLa-N and HeLa-R cells, respectively. The viruses (as a mixture of cell-free and cell-associated viruses) were harvested at several time points indicated and assayed for titers by a standard plaque assay on BHK-21 cells. Titers shown are the means ± S.D. in three independent experiments.

Figure 2. The STR analysis to confirm the identity of HeLa cells.

Left and right panels show the data on HeLa-N and HeLa-R cells, respectively. Graphs were images generated by GeneMapper ver. 3.5 (Applied Biosystems). The allele data were presented as a table on the right side of each image. Locus indicates the name of gene analyzed. It is indicated that the gene presented by one datum was homo and the gene presented by two data was hetero.

Figure 3. The detection of virus antigen in PSAF/HeLa-R cells by Western blotting.

The anti-SAFV-3 antiserum detected the viral antigen of about 28∼30 kDa (arrow) in the lysates of PSAF/HeLa-R cells cultured for 30 days (5 passages) (lane 2) and HeLa-N cells infected with SAFV-3 (18 hours p.i.) used for a positive control (lane 3). The band of viral antigen was not detected in the lysate of HeLa-R cells used for a negative control (lane 1).

Figure 4. Effects of culture with FCS on PSAF/HeLa-R cells.

A: Representative microphotographs of the CPE on PSAF/HeLa-R cells Left panel shows the PSAF/HeLa-R cells maintained in MEM with 10% CS (62 days p.i.). Right panel shows the PSAF/HeLa-R cells maintained in DMEM with 10% FCS for 12 days (from 50 days to 62 days p.i.). CPE on the PSAF/HeLa-R cells significantly increased by the culture with FCS for 12 days. Magnification: ×100. B: Representative microphotographs of the immunocytochemistry Left 6 panels show the PSAF/HeLa-R cells maintained in MEM with 10% CS. Right 2 panels show the PSAF/HeLa-R cells maintained in DMEM with 10% FCS. Upper and lower panels show Nomarski and fluorescent images, respectively. Virus antigen was detected with anti-SAFV-3 antiserum pre-absorbed by the homogenates of HeLa-R cells and Alexa Fluor 594-conjugated anti-rabbit IgG antibody. Viral antigen positive cells were shown in a part of PSAF/HeLa-R cells cultured with CS. After cultivation with FCS for 12 days, however, viral antigen positive cells clearly increased. Magnification: x400.

Figure 5. Immunofluorescent detection of the virus binding.

The photos show the virus binding to the cell surface molecule(s) of each cell line. The cells fixed by 10% formalin were incubated with virus (MOI of 100). Then the viruses binding to the cell surface molecule(s) were detected by anti-SAFV-3 antiserum pre-absorbed by the homogenates of HeLa-R cells and Alexa Fluor 594-conjugated anti-rabbit IgG antibody. Left panels: HeLa-N cells, Right panels: HeLa-R cells. Upper and lower panels show Nomarski and fluorescent images, respectively. The viruses binding to cell surface of HeLa-R cells was significantly few, suggesting that the expression of receptor for SAFV infection is low in HeLa-R cells. Magnification: ×400.