Susceptibilities of medaka (Oryzias latipes) cell lines to a betanodavirus
© Adachi et al; licensee BioMed Central Ltd. 2010
Received: 2 June 2010
Accepted: 12 July 2010
Published: 12 July 2010
Betanodaviruses, members of the family Nodaviridae, have bipartite, positive-sense RNA genomes and are the causal agents of viral nervous necrosis in many marine fish species. Recently, the viruses were shown to infect a few freshwater fish species including a model fish medaka (Oryzias latipes). Although virological study using cultured medaka cells would provide a lot of insight into virus-fish interactions in molecular aspects, no such cells have yet been tested for virus susceptibility.
We tested ten medaka cell lines for susceptibilities to redspotted grouper nervous necrosis virus (RGNNV). Although the viral coat protein was detected in all the cell lines inoculated, the levels of cytopathic effect development and viral propagation were quite different among the cell lines. Those levels were especially high in OLHNI-1 and OLHNI-2 cells, but were extremely low in OLME-104 cells. Some cell lines entered into antiviral state after RGNNV infections probably because of inducing an antiviral system. This is the first report to examine the susceptibilities of cultured medaka cells against a virus.
OLHNI-1 and OLHNI-2 cells are candidates of new standard cells for betanodavirus study because of their high susceptibilities to the virus and their several advantages as model fish cells.
Betanodaviruses, members of the family Nodaviridae, are small non-enveloped viruses with a genome composed of a bipartite single-stranded, positive-sense RNA [1, 2]. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA-dependent RNA polymerase and the smaller genomic segment, RNA2 (1.4 kb), encodes the coat protein (CP) . During viral RNA replication, a subgenomic RNA3 is produced, which encodes the RNA interference inhibitor protein B2 [3–5]. Betanodaviruses are classified basically into four genotypes based on the phylogenetic analysis of their genomic RNA2 sequences [6–8]. These genotypes are striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV) and redspotted grouper nervous necrosis virus (RGNNV). Recently, a betanodavirus isolate from turbot (Scophthalmus maximus) was suggested to belong to a fifth genotype .
Betanodaviruses are the causative agents of a highly destructive disease of marine fish designated viral nervous necrosis. The viruses have been isolated from a large number of marine fish species [10, 11]. Betanodaviruses propagate in various established cell lines derived from not only fish [2, 12] but also mammals . Recently, it was revealed that larvae of freshwater fish guppy (Poicelia reticulata)  and tilapia (Oreochromis niloticus)  were affected naturally by RGNNV. Some freshwater fish including medaka (Oryzias latipes) [16, 17] and zebrafish (Danio rerio)  are lethally susceptible to betanodaviruses under experimental conditions. Medaka has several experimental advantages as a model fish compared to other fish and higher vertebrates. For example, medaka is small, cost-effective, easy to breed in large numbers, and has a short life cycle. Furthermore, whole medaka genomic sequences are available and many experimental techniques for gene function analysis can be applied to medaka [19, 20]. However, one obstacle to study betanodavirus-medaka interactions in molecular aspects is the lack of cultured medaka cells which are susceptible to a betanodavirus. Therefore, in this study, we examined the susceptibilities of ten medaka cell lines derived from different strains and organs to RGNNV.
Virus infection and cytopathic effect (CPE) development
Medaka cell lines used in this study
Production of progeny virus
Betanodavirus propagation in the medaka cell lines
Inoculum a (TCID50)
Viral titer (TCID50/ml) b
4.4 × 104 ± 1.2 × 104
4.9 × 108 ± 7.0 × 107
1.8 × 109 ± 7.5 × 108
4.0 × 104 ± 0
4.0 × 108 ± 1.7 × 108
1.4 × 109 ± 4.0 × 108
3.3 × 103 ± 7.5 × 102
4.4 × 107 ± 1.2 × 107
2.6 × 108 ± 6.0 × 107
4.0 × 103 ± 0
2.9 × 108 ± 3.5 × 107
3.2 × 108 ± 8.5 × 107
4.8 × 103 ± 8.0 × 102
1.9 × 108 ± 1.0 × 107
2.9 × 108 ± 3.5 × 107
2.9 × 105 ± 3.5 × 104
2.1 × 107 ± 4.0 × 106
2.5 × 107 ± 1.5 × 107
3.6 × 107 ± 4.0 × 106
5.1 × 104 ± 1.9 × 104
4.1 × 106 ± 1.6 × 106
3.7 × 106 ± 1.9 × 106
1.9 × 106 ± 1.3 × 106
3.6 × 105 ± 4.0 × 104
5.6 × 106 ± 0
5.6 × 106 ± 0
7.8 × 106 ± 2.2 × 106
1.1 × 105 ± 6.9 × 104
4.8 × 106 ± 8.0 × 105
7.8 × 106 ± 2.2 × 106
2.1 × 107 ± 1.1 × 107
4.8 × 104 ± 8.0 × 103
4.8 × 104 ± 8.0 × 103
3.6 × 104 ± 4.0 × 103
3.6 × 104 ± 4.0 × 103
2.7 × 104 ± 1.3 × 104
1.4 × 108 ± 1.3 × 108
1.4 × 108 ± 4.0 × 107
We have demonstrated the susceptibilities of established medaka cells to the betanodavirus (RGNNV), the levels of which varied irrespective of the originated tissues. Medaka cell lines could be classified into three categories in terms of the infectivity and/or the productivity of RGNNV in the cells as follows: (1) cells are efficiently infected by virus, and give CPE and a high titer of progeny virus as is the cases for OLHNI-1, OLHNI-2, OLHE-131, OLKaga-e1, and OLHdrR-e3 cells, (2) cells are infected by virus though CPE and viral spread are tightly limited, which resulted in production of a low amount of progeny virus as is the cases for OLCAB-e3, OLCAB-e21, OLCAB-e31 and OLF-136 cells, (3) cells are hardly infected by virus as is the cases for OLME-104 cells. There would be two possible processes which determined the levels of the susceptibilities to RGNNV. One is the presence or absence of cellular factors required for RGNNV infection, such as cell-specific receptors. The other is the presence or absence of cellular factors which repress RGNNV infection. With regard to the former possibility, fibronectin 2 of zebrafish (Danio rerio) is the only cellular factor which has so far been identified for fish viruses. Zebrafish fibronectin 2 mediates infectious hematopoietic necrosis virus attachment and cell entry . Cell-surface sialic acid is involved in binding of RGNNV to SSN-1 cells and other cellular molecules are required along with sialic acid for RGNNV penetration into some human cell lines [22, 23]. However, such a cellular molecule essential for betanodavirus infection has not yet been identified. OLME-104 cells were severely less susceptible to RGNNV (Figure 1), suggesting the lacks of specific cellular factors for RGNNV. Meanwhile, OLHNI-1 and OLHNI-2 cells were highly susceptible to RGNNV (Figure 1), suggesting that these cell lines possess a positive cellular factor for betanodavirus infection. With respect to the latter possible mechanism, the culture supernatant of OLME-104 cells included an antiviral substance that protected some of the medaka cell lines from RGNNV-infection (authors' unpublished data). These results indicate that OLME-104 cells produce an interferon-like signal molecule irrespective of viral infection, which brings themselves into antiviral state. Furthermore, these data also indicate that some medaka cells used in this study are sensitive to such a defense signal molecule.
The OLCAB-e21, OLCAB-e31, and OLF-136 cells infected by RGNNV produced sufficient amounts of infectious virus particles in the culture supernatant (Table 2) though the levels of CPE and viral spread were tightly limited (Figures 2 and 3). These characteristics suggest that the cells entered into antiviral states after viral infections. Similar to mammalian Mx proteins, fish Mx proteins also possess type I interferon (IFN)-inducible antiviral activity in vitro and in vivo [24–27]. Grouper (Epinephelus coioides) Mx proteins inhibited the propagation of RGNNV in the grouper brain cells . In addition, the BB cell line derived from the brain of barramundi (Lates calcarifer) was infected persistently with RGNNV and this viral persistence in BB cells was well correlated with the expression of Mx gene [29, 30]. Thus, an IFN-like substance might be produced in the culture supernatant of the RGNNV-infected OLCAB-e21, OLCAB-e31, and OLF-136 cells, which induces the cells into antiviral states. However, Mx gene expression was detected by RT-PCR in OLCAB-e31 cells inoculated with RGNNV, not in inoculated OLCAB-e21 or OLF-136 cells (authors' unpublished data). These results suggest that a defense machinery other than the IFN system works in OLCAB-e21 and OLF-136 cells. Taken together, a few kinds of defense systems could function to protect the medaka cells from RGNNV infection.
E-11 cells  cloned from SSN-1 cells are infected latently with snakehead retrovirus (SnRV) . SnRV regulates positively  or negatively  the infections of fish cells with betanodaviruses. In our experiments, SnRV was detected by RT-PCR in all of the medaka cells inoculated with RGNNV prepared from infected E-11 cells. However, there was no correlation between susceptibilities of medaka cells to RGNNV and the levels of RT-PCR signals for SnRV (authors' unpublished data).
In this report, we examined the susceptibility of various medaka cell lines to RGNNV, and found that RGNNV can infect and propagate in many kinds of established medaka cells. Studies on host-betanodavirus interactions using these medaka cell lines would lead to the identification of host factors essential for betanodavirus infections. Especially, OLHNI-1 and OLHNI-2 cells would be suitable for such studies in molecular aspects.
Cells and viruses
The three medaka cell lines, OLHE-131, OLF-136, and OLME-104 (Table 1), were purchased from RIKEN BRC Cell Bank (Tsukuba, Japan). The other seven medaka cell lines (Table 1)  were provided from H. Mitani. All the medaka cells were cultured at 30°C in Leibovitz's L-15 medium (L-15) (Invitrogen, Carlsbad, CA, USA) containing 15% fetal bovine serum (FBS). E-11 cells  were cultured in L-15 medium supplemented with 5% FBS. The betanodavirus used in this study was RGNNV (SGWak97 strain) . Virus was prepared from the inoculated E-11 cells when more than 90% of the inoculated cells showed CPE. Viral titers were determined based on TCID50  using E-11 cells.
Viral inoculation and multiplication assay
Medaka cells were seeded in 24-well plates and were inoculated with RGNNV at 30°C for 1 h. For each cell line, 1.0-1.5 × 105 cells were inoculated with 103, 105, or 106 TCID50 of virus. The cells were washed to remove unbound viral particles and were further cultured at the same temperature. The culture supernatant was recovered periodically and its viral titer was determined by the TCID50 assay as described above.
Indirect immunofluorescence assay was performed using inoculated medaka cells as described previously . Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by treatment with 0.1% NP-40 in PBS. The cells then were treated with a 1:1000 dilution of anti-RGNNV CP antiserum, followed by the treatment with a 1:2000 dilution of Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), and were observed under the fluorescence microscope (ORCA-1394 and AQUA-Lite version 1.10 systems; Hamamatsu photonics K. K., Hamamatsu, Japan).
We would like to thank Dr. H. Mitani, The University of Tokyo, for providing the medaka cell lines. We also would like to express our thanks to Y. Ninomiya, Medaka Honpo Co., Ltd., for technical assistance of rearing medaka. This work was supported in part by a grant-in-aid for the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) and by a grant-in-aid for Scientific Research (20380111) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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