TREATED WITH RED SEA BREAM IRIDOVIRUS ( RSIV ; family Iridoviridae , genus Megalocytivirus ) IN COMBINATIONS WITH INTERFERONS AND SPLENIC SUBSTANCES

The genus Megalocytivirus in the family of Iridoviridae encompasses isolate of red sea bream iridovirus (RSIV). In the present study, grunt fin (GF) cells were treated with red sea bream iridovirus (RSIV) in combinations with interferons (IFNs) and splenic substances. The viral titer in the combination with primary splenic substance was higher than the other combinations of 10-1 and 10-2 diluted splenic substances, and the positive control. The viral titer was not decreased by all combinations with recombinant murine interferon-α (rMuIFN-α), recombinant murine IFN-β (rMuIFN-β), and recombinant feline interferon-ω (rFeIFN-ω). Electron microscopy revealed inclusion body bearing cells (IBCs) and enlarged cells allowing virus propagation within the intracytoplasmic virus assembly site (VAS). Most were enlarged cells. These enlarged cells were divided into three cell types. Cells of Type II, which contained many mature virions within the VAS, were numerous in number in all treated cells. Cells of Type I allowing assembly of few virions and cells of Type III containing many immature viral particles were rather fewer in number. Their percentage was almost the same in all combinations with the splenic substances and IFNs. These results determined in in vitro treatment with IFNs did not prevent viral replication of RSIV, as well as the splenic substances which were derived from the RSIV-infected spleen of red sea bream did not contain any factors to disturb RSIV replication.

In our previous study (Mahardika et al., 2004), we performed artificial infection in juvenile humpback grouper with grouper sleepy disease iridovirus (GSDIV).As a result, the mortality was lower in the groups injected with the primary filtrate than in groups receiving the 10 -4 diluted inoculum.Electron microscopy revealed the presence of many unusual inclusion body-bearing cells (IBCs) containing abnormal inclusion body with a small number of deformed virions in the former groups.This impaired virus assembly and appeared to prevent mortality in the challenged fish and was assumed to be due to an interferon-like effect of a previously unknown substance that was passed with a filtrate from the donor fish.

Cell Culture
GF (grunt fin) cells were used in this study.GF cells were grown in 25 cm 2 cell culture flasks at 25 o C to confluence in Eagle's minimum essential medium (MEM: Nissui, Japan) supplemented with fetal bovine serum (FBS: Sigma, USA), L-glutamine, and buffered with NaHCO to pH 7.2-7.4.For routine passage and virus inoculation, 10% and 2% FBS was added to the medium (MEM-10 and 2), respectively.

Preparation of Viral Inoculum and Splenic Substances
Semi-purified virus was prepared from the spleens of the RSIV-infected red sea bream from a mariculture farm in Mie Prefecture, Japan, likewise our previous preparation of semipurified virus (Mahardika & Miyazaki, 2009) but with some modifications.The procedures were as follows: A total of 0.6 g of swollen spleens from the diseased fish was homogenized in 6 mL of MEM-2 containing antibiotics (penicillin 50 IU mL -1 and streptomycin 50 μg mL -1 ) in a glass homogenizer.The obtained homogenate was confirmed to be RSIV-positive by PCR assay described by Kurita et al. (1998).The homogenate was centrifuged (CT4D Himac, Hitachi, Japan) at 1,200 x g for 15 min.at room temperature, and the supernatant was filtered (450 nm).The obtained filtrate was diluted to be a final volume of 12 mL and ultracentrifuged (L-70 Ultracentrifuge, Beckman, USA) at 100,000 x g for 10 min.at 4 o C. The supernatant was collected, filtered (450 nm) and used as the primary splenic substance.This substance was diluted 10 and 100 times (10 -1 and 10 -2 dilution) with MEM-2.
The sediment was resuspended in 12 mL of MEM-2 containing antibiotics and ultracentrifuged at 100,000 x g for 10 min.at 4 o C.Then, the recovered sediment was resuspended in 5 mL of MEM-2 containing antibiotics and filtered (450 nm).The obtained semipurified virus was used for an inoculum although, as the result, the virus concentration was decreased in number by repeated ultracentrifugation.

Treatments
Two treatments were performed.The first was treatment with the splenic substances using five tubes: 0.1 mL of the semi-purified virus inoculum mixed into each of the four tubes containing 0.2 mL of different dilutions of the splenic substances (primary, 10 -1 , 10 -2 dilution) and MEM-2 as positive control, respectively.In the fifth tube, the same volume of the primary splenic substances and MEM-2 were mixed as negative control.These five mixtures were kept at room temperature for 15 min., and inoculated into monolayer of GF cells for 1 hour at 25 o C, followed by adding the MEM-2 to a final volume of 5 mL.This treatment was repeated two times.
Indonesian Aquaculture Journal Vol.5 No.1, 2010 The second was treatment with IFNs and set in two trials.First trial consisted of five tubes: 0.1 mL of the semi-purified virus inoculum was mixed into each of the five tubes, which contained 0.1 mL of different concentrations of rMuIFN-α (10 4 and 10 3 units mL -1 ), rMuIFN-β (10 4 and 10 3 units mL -1 ) and MEM as control, respectively.The second trial consisting of three tubes: 0.1 mL of the semi-purified virus inoculum was mixed into each of the three tubes which contained 0.1 mL of rMuIFN-β (10 5 units mL -1 ), rFeIFN-ω (10 6 units mL -1 ) and MEM as control, respectively.This second trial was repeated two times.All mixtures were kept at room temperature for 15 min., and inoculated into monolayer of GF cells for 1 hour at 25 o C, followed by adding the MEM-2 to reach 5 mL.The final concentration of both rMuIFN-α and rMuIFN-β was 2 x 10 2 and 2 x 10 1 units mL -1 in the first trial.In the second trial, the final concentration was 2 x 10 3 units mL -1 of rMuIFN-β and 2 x 10 4 units mL -1 of rFeIFN-ω.
Monolayer of GF cells were infected with semi-purified virus inoculum at a multiplicity of infection (MOI) of approximately 0.5-1.Infected cells were incubated at 25 o C. The rounded and enlarged cells were harvested by shaking the culture medium in order to separate these cells from the monolayer at 6 days post infection (p.i.).The recovered cells were used for virus titration and electron microscopy (EM).

Virus Titration
Viral titration in RSIV-infected GF cells was conducted using the TCID 50 method with GF cells grown in 96-well tissue culture plates.This assay was analyzed in duplicate.

Electron Microscopy
For transmission electron microscopy, a 3.5 mL of harvested medium containing RSIV-infected GF cells were centrifuged at 1,200 x g for 10 min.The obtained pellets were fixed with 70% Karnovsky's solution (1.6 g paraformaldehyde in 20 mL distilled water, 2 mL of 70% glutaraldehyde, 35 mL of 0.2 M phosphate-buffered saline, and 13 mL distilled water), post-fixed in 1% OsO 4 and processed for electron microscopy, as previously described by Mahardika et al. (2008).Rounded and enlarged cells contained within 800 mm 2 of the semi-thin section per sample were examined.These cells were counted at an instrument magnification of 5,000X to 10,000X.The data were expressed as percentage per cells.All of obtained data were evaluated using fisher's exact test.

Electron Microscopic Features of GF Cells Treated with RSIV and Splenic Substances
Electron microscopic examination of GF cells, which were treated with mixture of semipurified virus and different dilution of the splenic substances revealed IBCs and necrotic cells allowing virus propagation within the intracytoplasmic virus assembly site (VAS) at 6 d p.i. IBCs were observed a few in the number (2.1%-5.7%)(Table 1).Some small cells were also observed forming the intracytoplasmic VAS containing a small number of virus particles (10.8%-12.7%).Most were enlarged cells (83.0%-86.6%)and had fragmented nuclei whose components were assimilated with the cytoplasm, resulting in the disappearance of the nuclei.These cells developed intracytoplasmic VAS within which many virions were propagated.These enlarged cells were divided into three cell types.Cell type-I allowed development of the VAS, which showed the assembly of a few number of virions (less than 20).Cell type-II had assembled many mature virions (more than 20) within the VAS.Cell type-III contained many immature viral particles (more than 20) within the VAS.In the GF cells treated with the mixture of semi-purified virus inoculum and different dilution of the splenic substances (primary, 10 -1 and 10 -2 ), the percentages of cell type-I and cell type-III were 11.6%-18.8%and 2.9%-10.6%,respectively, which were lesser than cell type-II (75.8%-85.5%).The percentages of these cell types were similar (P>0.05) to the positive control (Table 2, Figure 3 & 4).

Electron Microscopic Features of GF Cells Treated with RSIV and IFNs
Electron microscopic examination of the GF cells treated with mixture of semi-purified virus and various IFNs focused to the enlarged cells.In combination with rMuIFN-α and rMuIFN- β (2 x 10 2 and 2 10 1 units mL -1 ), the percentages of cell type-I and cell type-III were lesser (13.5%-14.9%and 3.4%-4.4%)than cell type-II (81.0%-82.7%),which was almost same (P>0.05)as the control (Table 3, Figure 5).Some of cell type-I in combination with 2 x 10 2 units mL -1 of rMuIFN-α contained tubule structures among immature viral particles within the VAS.

Table 1. Electron microscopic cell count of different types of cells with virus particles derived from RSIV-infected GF cells in combination with splenic substances in vitro
IBCs were included in the enlarged cells with virus particles.I: First examination, II: Second examination In c ombination with 10 -2 dilution of splenic substanc e In c ombination with MEM-2 (positiv e control)

Samples Different t ypes of cells w it h virus part ic les ( % )
In c ombination with primary splenic substanc e In c ombination with 10

Samples
In c ombination with primary splenic substanc e In c ombination with 10 -1 dilution of splenic substanc e In c ombination with 10 -2 dilution of splenic substanc e In c ombination with MEM-2 as positiv e c ontrol   In the present study, inoculated RSIV infected GF cells and produced cellular enlargement as a definite cytopathic effect in all combinations with the splenic substances and IFNs.The viral titer in combination with primary splenic substances was higher than the other combinations of 10 -1 and 10 -2 diluted splenic substances, and positive control.The viral titer was not decreased by all combinations with rMuIFN-α, rMuIFN-β, and rFeIFN-ω.These results determined in vitro treatment with INFs did not prevent viral replication of RSIV, as well as the splenic substances which were derived from the RSIV-infected spleen of red sea bream, did not contain any factors that could disturb RSIV replication.In EM examination, IBCs seen in the GF cells that were infected with RSIV in combination with different dilutions of the splenic substances were same to those seen in the previous study (Mahardika & Miyazaki, 2009) as well as megalocytivirus-infected cells, in vivo (Jung et al., 1997;Sudthongkong et al., 2002a;2002b;Mahardika et al., 2004;2008;Miyazaki, 2007).Enlarged cells were also derived from RSIV-infected GF cells and allowed assembly of viral particles in the intracytoplasmic VAS.Cells of type II, which contained many mature virions within the VAS, were numerous in number in all treated cells.Cells of type I allowing assembly of a few virions and cells of type III containing many immature viral particles were rather small in the number.Their percentages were almost the same in all combinations with the splenic substances and IFNs.IFN-α and IFN-β are known to be potential as endogenous antiviral cytokines that suppress the replication of viruses (Pitha et al., 1979;Härle et al., 2002;Sainz & Harford, 2002;Sainz et al., 2004).rFeIFN-ω has clinical anti-viral activities (Yamamoto et al., 1990;Uchino, 1997;Ishiwata et al., 1998;Miyazaki et al., 2000).In the present study, there were cells with the formation of many immature viral particles within the VAS as cell type III, and cells with slight virion assembly as cell type I.However, because these cells were small in number, they did not appear to be resulted in prevention of viral replication.These results indicate that the in vitro treatment with IFNs (rMuIFN-α, rMuIFN-β, and rFeIFN-ω) did not affect on the RSIV propagation in GF cells.Our in vivo study (Mahardika et al., 2004) revealed that the mortality rate in the fish injected with the primary filtrate of the spleen of GSDIV-infected grouper were lower than the fish received the 10 -4 diluted inoculum.Results of our study in vivo and in vitro resembled results of studies by Sainz & Halford (2002) who revealed that T-cell derived gamma interferon (IFN-γ) inhibited the replication of herpes simplex virus type 1 (HSV-1; genus Simplexvirus, family Herpesviridae) in vivo, while in vitro, HSV-1 was highly resistant to the antiviral activity of IFNα/β or IFN-γ.Ellis (2001) reported that the cells after in vivo infection produced interferon to induce anti-viral defenses in neighboring cells, which able to lyse virally infected cells and reduce the rate of multiplication of virus within them.
It was concluded that the examined IFNs did not have antiviral efficacy to megalocytivirus in vitro.Further in vivo studies using IFNs will be needed to reveal real antiviral effects of IFNs on RSIV.

Figure 3
Figure 3. (A-B) Electron micrographs of IBC and derived from RSIV-infected GF cells in combination with splenic substances at 6 d p.i. (A) Mature IBC in the combination with primary splenic substance has a large inclusion body with a VAS containing a mass of coarse granules (c) and multiple virus particles (scale bar = 2,900 nm).(B) Enlarged cells in combination with 10 -2 dilution of splenic substance.One elarged cell (upper) has a disappearance of the nucleus and development of an intracytoplasmic VAS in which many virus particles are present.Another is small cell.This cell forms an intracytoplasmic VAS containing a small number of virus particles (scale bar = 2,400 nm).f: mass of fine granules; n: nucleus of host cell; VAS: virus assembly site

Table 2 .
Electron microscopic cell count of different types of enlarged cells with virus particles derived from RSIV-infected GF cells in combination with splenic substances in vitro

Table 3 .
Electron microscopic cell count of different types of enlarged cells with particles derived from RSIV-infected GF cells in combination with interferons (rMuIFN-α and rMuIFN-β) in vitro

Table 4 .
Electron microscopic cell count of different types of enlarged cells with virus particles derived from RSIV-infected GF cells in combination with interferons (rMuIFN-β and rFeIFN-ω) in vitroIBCs were included in the enlarged cells with virus particles.I: First examination, II: Second examination