短報
Virucidal Activities of Common Disinfectants Against the Japanese Eel Endothelial Cells-Infecting Virus (JEECV)
2025 年 60 巻 4 号 p. 199-202
詳細
2025 年 60 巻 4 号 p. 199-202
In this study, we investigated the virucidal activities of common disinfectants against the Japanese eel endothelial cells-infecting virus (JEECV) in vitro using JEE-2, a Japanese eel vascular endothelial cell line. After treatment with different concentrations of each disinfectant, virus suspension was added to the cells in 96-well plates and incubated for 10 days at 25°C. No cytopathic effects were observed after 1-min treatment with ≥50% ethanol, ≥20 ppm sodium hypochlorite, ≥50 ppm povidone-iodine, ≥500 ppm cresol, and ≥1,000 ppm benzalkonium chloride. These results suggest that appropriate disinfection controls the spread of JEECV infection on eel farms.
Viral endothelial cell necrosis of eel (VECNE), a disease caused by the Japanese eel endothelial cells-infecting virus (JEECV), has posed a significant challenge to eel aquaculture in Japan since the 1980s (Egusa et al., 1989; Kusuda et al., 1989; Ono et al., 2007; Mizutani et al., 2011). JEECV is a 15-kbp, non-enveloped, circular double-stranded DNA virus with hexagonal particles and 75 nm diameter (Ono et al., 2007; Mizutani et al., 2011). This virus contains a papillomavirus/polyomavirus-like replicase gene and an adenovirus-like virion maturation protease (Mizutani et al., 2011; Welch et al., 2020). Owing to the chimeric structure of its genome, a new family, Adomaviridae, has been proposed for JEECV and other fish-infecting viruses (Welch et al., 2020). JEECV primarily infects vascular endothelial cells and induces cytopathic effects (CPEs), such as cell nucleus enlargement and necrosis (Ono et al., 2007; Mizutani et al., 2011; Umeda et al., 2025). Disease symptoms include intense congestion of the central venous sinus of the gill filaments, liver and kidney hemorrhages, fin and skin reddening, and even death in severe cases (Egusa et al., 1989; Inouye et al., 1994; Ono and Nagai, 1997).
Previous studies have explored ways to prevent viral infections and control disease mortality. Increasing the water temperature to 35°C and feed withdrawal reduce infected fish mortality (Tanaka et al., 2008; Umeda et al., 2024). However, as JEECV is less infectious (Umeda et al., 2024) but not inactivated at this temperature (Ono et al., 2007; Tanaka et al., 2008; Umeda et al., 2025), decreasing the temperature possibly increases the risk of infection due to viral transmission from carrier fish to uninfected fish. We previously reported that virus-contaminated water acts as an infection source (Umeda et al., 2024). This suggests that the virus spreads from one pond to another via contaminated hands, rubber boots, waders, scoop nets, and eel baskets during daily operations, such as the removal of dead fish from ponds. Therefore, viral disinfection is necessary to prevent horizontal infections on eel farms. However, to date, no study has investigated the specific effects of different disinfectants on JEECV. We recently established a new JEECV-susceptible Japanese eel vascular endothelial cell line, JEE-2 (Umeda et al., 2025). In this study, we used this cell line to assess the virucidal activities of common disinfectants against JEECV in vitro.
JEE-2 cells were routinely cultured with 3 mL of the Humedia-EB2 medium (Kurabo Industries Ltd.) supplemented with the EGM-2 Endothelial SingleQuots Kit (Lonza Bioscience) and 1× penicillin–streptomycin solution (100 U/mL of penicillin G and 100 μg/mL of streptomycin sulfate; Fujifilm Wako Pure Chemical Corporation) in 25-cm2 culture flasks at 25°C in a humidified atmosphere with 5% CO2. The medium was changed every 3–4 days. Confluent cells were washed with phosphate-buffered saline without calcium and magnesium (PBS, pH 7.4) and detached with Accutase (Innovative Cell Technologies, Inc.) for 5–10 min. For the viral CPE assay, the cells were seeded in a 96-well plate at a density of 1.0 × 104 cells/well in 200 μL and incubated for one week at 25°C and 5% CO2. The passage number of the cells used in the assays ranged from 30 to 55. Susceptibility of the cells to JEECV was confirmed by observing viral CPEs in the cells to which the untreated virus was added in each assay.
Virus cultureJEECV (isolate JEECV-SO2101) was isolated from a diseased eel in a previous study (Umeda et al., 2025). The virus was routinely cultured using JEE-2 cells in 25-cm2 flasks at 25°C and 5% CO2. The infected cells and culture supernatants were harvested via scraping and passaged to uninfected cells every seven days. Virus suspensions were stored at -80°C until use and titrated by adding to the cells in a 96-well plate after a 10-fold serial dilution. The plate was incubated at 25°C, and CPEs were observed. The median tissue culture infectious dose (TCID50) was determined 10 days post-infection (Spearman, 1908; Kärber, 1931).
JEECV inactivationVirus suspensions were treated with different concentrations of each disinfectant for different periods. The following disinfectants were used: Ethanol (Fujifilm Wako Pure Chemical Corporation), sodium hypochlorite (Aron Clean; Toagosei Co., Ltd.), povidone-iodine (Isodine; DS Pharma Animal Health Co., Ltd.), cresol (Saponated cresol solution; Taiyo Pharmaceutical Co., Ltd.), and benzalkonium chloride (Osvan S; Nihon Pharmaceutical Co., Ltd.) (Table 1).
| Group | Product name | Ingredient | Concentration for normal use |
|---|---|---|---|
| Alcohols | Ethanol | Ethanol (99.5%) | 76.9–81.4% (v/v) |
| Halogens | Aron Clean | Sodium hypochlorite (12%) | 100–200 ppm |
| Isodine | Povidone-iodine (10%) | 50 ppm | |
| Phenols | Saponated cresol solution | Cresol (42–52% [v/v]) | 1:33 to 1:100 |
| Cationic surfactants | Osvan S | Benzalkonium chloride (10% [w/v]) | 1:100 to 1:1,000 |
Ethanol was diluted in PBS to achieve 10–70% (v/v) concentration. One volume of the virus suspension (106.0 TCID50/mL) was mixed with nine volumes of diluted ethanol. The mixture was incubated for 1, 5, 15, and 30 min at room temperature (20–25°C) and diluted 10-fold with fresh medium to attenuate the impact of ethanol on the JEE-2 cells. Because of the preparation method, the concentration of the disinfectant at the reaction time was 9/10 of the concentration of the disinfectant solution used. The same applies to the following procedures.
Sodium hypochlorite was diluted to 2–50 ppm (1:60,000 to 1:2,400 dilution of the original solution). The available chlorine in the original solution was measured using a free chlorine handheld colorimeter (Hanna Instruments, Inc.). The virus suspension (106.0 TCID50/mL) was mixed with the diluted solution at a 1:9 ratio and incubated for 1, 5, 15, and 30 min. After each period, 50 μL of the mixture was neutralized with 5 μL of 0.1 M sodium thiosulfate solution and immediately diluted 10-fold with fresh medium.
Povidone-iodine was diluted to 2–100 ppm (1:50,000 to 1:1,000 dilution of the original solution). The available iodine in the original solution was measured using an iodine handheld colorimeter (Hanna Instruments, Inc.). The virus suspension (106.0 TCID50/mL) was mixed with the diluted solution at a 1:9 ratio and processed in the same manner as that described for sodium hypochlorite.
Cresol was diluted to 31.25–500 ppm (1:16,000 to 1:1,000 dilution of the original solution). The virus suspension (106.0 TCID50/mL) was mixed with the diluted solution at a 1:9 ratio. The mixture was incubated for 1, 5, 15, and 30 min and diluted 10-fold with fresh medium.
Benzalkonium chloride was diluted to 62.5–1,000 ppm (1:1,600 to 1:100 dilutions of the original solution). The virus suspension (106.75 TCID50/mL) was mixed with the diluted solution at a 1:9 ratio. The mixture was incubated for 1, 5, 15, and 30 min and diluted 1,000-fold with fresh medium to attenuate the cytotoxic effects of the disinfectant.
CPE inhibition assayFor the CPE inhibition assay, the medium in each well of a 96-well plate containing confluent JEE-2 cells was aspirated and replaced with 100 μL of fresh medium. Subsequently, 100 μL of the treated virus suspension was added to four replicated wells. PBS without disinfectants was used as a negative control. Finally, number of wells with visible CPEs was counted 10 days post-infection. Since the final concentration of the virus added was 101.75 TCID50 per well for benzalkonium chloride and 103.0 TCID50 per well for the other disinfectants, the absence of CPE in the well indicates that the treatment inactivated the corresponding amount of the virus.
Table 2 presents the test results for each disinfectant. The minimum concentration necessary for complete CPE inhibition after 1-min treatment was 50% for ethanol, 20 ppm (1:6,000 dilution of the original solution) for sodium hypochlorite, 50 ppm (1:2,000 dilution) for povidone-iodine, 500 ppm (1:1,000 dilution) for cresol, and 500 ppm (1:200 dilution) for benzalkonium chloride. Although ethanol is less effective against some non-enveloped viruses (Kampf, 2018), JEECV was inactivated by the 50% ethanol solution, and the 30% ethanol solution also inhibited the CPEs after ≥5 min of treatment in this study. This is possibly because the viral surface proteins associated with JEECV infectivity are susceptible to denaturation by ethanol. These results suggest that disinfectant ethanol is useful for hand and equipment disinfection to protect against JEECV infection. Previous studies have detected the JEECV genomic DNA in mature eels in spawning areas and elvers in rivers, although no clear evidence of its vertical transmission has been reported to date (Okazaki et al., 2016; Okazaki-Terashima et al., 2016). With the advancement in the larval production techniques for Japanese eels (Tanaka, 2015), povidone-iodine can possibly be used to disinfect eel eggs to prevent vertical infection by JEECV. However, povidone-iodine should be used with caution because egg disinfection with 25 ppm povidone-iodine negatively impacts the hatching success and larval survival of the European eel, Anguilla anguilla (Jéhannet et al., 2024). For halogen disinfectants, such as sodium hypochlorite and povidone-iodine, effective concentrations may vary depending on the virus suspension preparation method as the disinfectants are rapidly inactivated in the presence of organic matter (Inouye et al., 1990b). On farms, adequate concentrations of these disinfectants should be maintained via frequent solution replacement. As the minimum effective concentrations determined in this study were equivalent to or below the normal concentration range, the tested halogen disinfectants are useful for the disinfection of rubber boots and scoop nets against JEECV during daily aquaculture operations. Notably, minimum effective concentration of cresol was lower for JEECV than for other non-enveloped fish viruses, such as the infectious pancreatic necrosis virus (Inouye et al., 1990b), and dropped to 62.5 ppm at ≥5 min treatment durations. Therefore, long treatment durations are recommended when cresol is used for JEECV disinfection. Benzalkonium chloride inhibited the CPEs of JEECV but was toxic to the JEE-2 cells at high concentrations, consistent with previous reports in other fish cell lines (Inouye et al., 1990a, 1991). Although lecithin and Tween 80 have been shown to neutralize the cytotoxicity of benzalkonium chloride in previous studies (Sozzi et al., 2019), they did not attenuate its toxicity in the cell line used in this study (data not shown). Nevertheless, benzalkonium chloride can be used for JEECV disinfection due to its ability to inactivate JEECV at non-cytotoxic concentrations.
| Treatment time (min) | Ethanol (%) | ||||||
| 0 | 10 | 30 | 50 | 70 | |||
| 1 | 4*1 | 4 | 3 | 0 | 0 | ||
| 5 | 4 | 4 | 0 | 0 | 0 | ||
| 15 | 4 | 4 | 0 | 0 | 0 | ||
| 30 | 4 | 4 | 0 | 0 | 0 | ||
| Sodium hypochlorite (ppm) | |||||||
| 0 | 2 | 5 | 10 | 20 | 50 | ||
| 1 | 4 | 4 | 4 | 3 | 0 | 0 | |
| 5 | 4 | 4 | 3 | 0 | 0 | 0 | |
| 15 | 4 | 4 | 0 | 0 | 0 | 0 | |
| 30 | 4 | 4 | 0 | 0 | 0 | 0 | |
| Povidone-iodine (ppm) | |||||||
| 0 | 2 | 5 | 10 | 20 | 50 | 100 | |
| 1 | 4 | 4 | 4 | 4 | 4 | 0 | 0 |
| 5 | 4 | 4 | 4 | 4 | 4 | 0 | 0 |
| 15 | 4 | 4 | 4 | 4 | 4 | 0 | 0 |
| 30 | 4 | 4 | 4 | 4 | 4 | 0 | 0 |
| Cresol (ppm) | |||||||
| 0 | 31.25 | 62.5 | 125 | 250 | 500 | ||
| 1 | 4 | 4 | 4 | 3 | 1 | 0 | |
| 5 | 4 | 1 | 0 | 0 | 0 | 0 | |
| 15 | 4 | 0 | 0 | 0 | 0 | 0 | |
| 30 | 4 | 0 | 0 | 0 | 0 | 0 | |
| Benzalkonium chloride (ppm) | |||||||
| 0 | 62.5 | 125 | 250 | 500 | 1,000 | ||
| 1 | 4 | 4 | 4 | 3 | 0 | CTE*2 | |
| 5 | 4 | 4 | 3 | 0 | 0 | CTE | |
| 15 | 4 | 4 | 0 | 0 | 0 | CTE | |
| 30 | 4 | 4 | 0 | 0 | 0 | CTE | |
This study demonstrated the JEECV inactivation efficiency of five common disinfectants. Although we only examined one available isolate of JEECV, and more isolates should be collected and investigated for the sensitivity to disinfectants in future studies, our results suggest that JEECV is not highly resistant to common disinfectants at normal concentrations indicated in the manufacturers’ instructions. Since size selection and transfer between ponds are frequent in eel aquaculture (Miyoshi, 1977), the virus can easily spread in eel farms via contaminated tools. With careful consideration of their potential negative effects on fish, farm workers, and the environment, the appropriate use of disinfectants can prevent viral infection and transmission in eel aquaculture.
This study was a part of the “Regulatory research projects for food safety, animal health and plant protection (JPJ008617.19190702)” funded by the Ministry of Agriculture, Forestry and Fisheries of Japan and supported by the JSPS KAKENHI Grant-in-Aid for Young Scientists (JP22K14953). We would like to thank Editage (www.editage.jp) for English language editing.
