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Nov 25, 2015 - genotypes: SJNNV (striped jack nervous necrosis virus), RGNNV ..... of the betanodavirus isolated from a dead sevenband grouper (SGEhi13), ..... (Hamilton): a cell line susceptible to grouper nervous necrosis virus. (GNNV).
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Cell Culture Isolation of Piscine Nodavirus (Betanodavirus) in Fish-Rearing Seawater Shinnosuke Nishi,a Hirofumi Yamashita,b Yasuhiko Kawato,c Toshihiro Nakaia Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japana; Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Uwajima, Japanb; National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, Japanc

Piscine nodavirus (betanodavirus) is the causative agent of viral nervous necrosis (VNN) in a variety of cultured fish species, particularly marine fish. In the present study, we developed a sensitive method for cell culture isolation of the virus from seawater and applied the method to a spontaneous fish-rearing environment. The virus in seawater was concentrated by an iron-based flocculation method and subjected to isolation with E-11 cells. A real-time reverse transcriptase PCR (RT-PCR) assay was used to quantify the virus in water. After spiking into seawater was performed, a betanodavirus strain (redspotted grouper nervous necrosis virus [RGNNV] genotype) was effectively recovered in the E-11 cells at a detection limit of approximately 105 copies (equivalent to 102 50% tissue culture infective doses [TCID50])/liter seawater. In an experimental infection of juvenile sevenband grouper (Epinephelus septemfasciatus) with the virus, the virus was isolated from the drainage of a fish-rearing tank when the virus level in water was at least approximately 105 copies/liter. The application of this method to sevenband grouper-rearing floating net pens, where VNN prevailed, resulted in the successful isolation of the virus from seawater. No differences were found in the partial sequences of the coat protein gene (RNA2) between the clinical virus isolates of dead fish and the cell-cultured virus isolates from seawater, and the viruses were identified as RGNNV. The infection experiment showed that the virus isolates from seawater were virulent to sevenband grouper. These results showed direct evidence of the horizontal transmission of betanodavirus via rearing water in marine aquaculture.

M

arine aquaculture has been highly developed for the past few decades; however, various types of diseases have negatively impacted the practice. In particular, viral infections, some of which induced high mortality in both hatchery and culture facilities, have seriously damaged the aquaculture industry. One of such viral infections is viral nervous necrosis (VNN), or viral encephalopathy and retinopathy (VER), which is caused by piscine nodaviruses (genus Betanodavirus, family Nodaviridae). The disease was first described in 1990 in hatchery-reared Japanese parrotfish (Oplegnathus fasciatus) in Japan (1) and barramundi (Lates calcarifer) in Australia (2) and has since caused high mortality in a variety of hatchery-reared or cultured marine and freshwater fish species, particularly marine fish (3, 4, 5). Betanodaviruses are nonenveloped and icosahedral in shape (25 to 30 nm in diameter), and the genome consists of two positive-sense single-stranded RNAs. RNA1 (3.1 kb) encodes the replicase (110 kDa), and RNA2 (1.4 kb) encodes the coat protein (42 kDa); both lack poly(A) tails at their 3= ends (6, 7, 8). A subgenomic RNA, RNA3 (0.4 kb), is derived from RNA1 in infected cells (9) and encodes a protein with potent RNA silencing-suppression activity (10). Based on the RNA2 sequences, betanodaviruses are classified into four major genotypes: SJNNV (striped jack nervous necrosis virus), RGNNV (redspotted grouper nervous necrosis virus), TPNNV (tiger puffer nervous necrosis virus), and BFNNV (barfin flounder nervous necrosis virus) (11, 12). In VNN, two transmission modes of betanodaviruses are known. Vertical transmission of the virus from broodstocks to offspring has been well described in several fish species (13, 14, 15, 16). The disease has been controlled in hatchery-reared larvae and juveniles of some fish species by the elimination of virus-carrying broodstocks and the disinfection of fertilized eggs and rearing waters (17, 18, 19). However, some fish species, such as sevenband grouper (Epinephelus septemfasciatus) (20), European sea bass

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(Dicentrarchus labrax) (21, 22), Atlantic halibut (Hippoglossus hippoglossus) (23), and Atlantic cod (Gadus morhua) (24), remain susceptible to the virus (RGNNV or BFNNV) even at the growout stages during net pen culture in the open sea. Although this type of infection is seemingly caused by the waterborne transmission of the virus, the nature of the transmission mode of the virus in the open sea remains unclear, mainly due to a lack of reliable methods for isolating infectious betanodaviruses from seawater. The number of pathogenic viruses in environmental waters is generally low. Thus, a method for concentrating viruses in water is indispensable for their isolation in cell culture. Previously reported methods for the concentration of fish-pathogenic viruses in seawater were somewhat time-consuming or complex, with low recovery rates (25, 26, 27, 28). However, a simple and rapid method for the concentration of ocean virus on the basis of iron flocculation was recently reported (29). The present report describes the successful cell culture isolation of betanodavirus from seawater utilizing an iron-based flocculation method and the dynamics of the virus in seawater. MATERIALS AND METHODS Fish. Sevenband grouper, which were produced and reared under specific-pathogen-free conditions at a facility of the Fishery Research Center,

Received 25 November 2015 Accepted 11 February 2016 Accepted manuscript posted online 19 February 2016 Citation Nishi S, Yamashita H, Kawato Y, Nakai T. 2016. Cell culture isolation of piscine nodavirus (betanodavirus) in fish-rearing seawater. Appl Environ Microbiol 82:2537–2544. doi:10.1128/AEM.03834-15. Editor: H. Goodrich-Blair, University of Wisconsin—Madison Address correspondence to Toshihiro Nakai, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Ehime Research Institute of Agriculture (ERIA), were used in this study. Prior to the experiments, fish (n ⫽ 20) were randomly sampled from stocks and the brains were examined for betanodavirus by PCR according to a previously described method (30). No specific PCR products were detected in any fish examined. Virus strain and propagation. A betanodavirus SGEhi00 strain, which had been isolated from a diseased sevenband grouper in Japan and identified as RGNNV (31), was used in this study. The virus was propagated in E-11 cells cultured at 25°C in Leibovitz’s L-15 medium (Life Technologies, USA) supplemented with 5% fetal bovine serum (FBS), as previously described (32). Briefly, monolayer E-11 cell cultures in a 25cm2 flask were washed twice with Hanks’ balanced salt solution (HBSS; Life Technologies) and inoculated with SGEhi00 at a dose of 106.5 50% tissue culture infective doses (TCID50)/flask. After a 3-day incubation with L-15 medium supplemented with 5% FBS at 30°C, the culture supernatant was collected, filtered through a 0.2-␮m-pore-size membrane filter (Advantec; Toyo Roshi Kaisha, Ltd., Japan), and stored at ⫺80°C until use. The virus stock was titrated using 96-well plates (Asahi Glass Co., Ltd., Japan) seeded with E-11 cells, and the virus infective titer was estimated according to the Reed and Muench method (33). Purification of virus. The crude virus suspension (30 ml) was mixed with 10 ml of polyethylene glycol (PEG) solution (40% polyethylene glycol 6000, 2 M NaCl), followed by incubation on ice for 1 h. After centrifugation (25,000 ⫻ g, 10 min, 4°C), the sediment was resuspended in 1 ml of 10 mM (pH 7.0) phosphate-buffered saline (PBS). Solid CsCl was added to the virus suspension to bring the density to approximately 0.36 g/ml, and the suspension was centrifuged (150,000 ⫻ g, 18 h, 4°C) with a Hitachi CS100GS rotor (Hitachi, Ltd., Japan). The virus fraction was dialyzed against HBSS for 6 h (4°C), titrated as described above, and stored at ⫺80°C until use. Real-time PCR (qPCR) assay. Total RNA was extracted from the purified SGEhi00 strain (108.5 TCID50/ml) or seawater samples for virus by using ISOGEN (Nippon Gene Co., Ltd., Japan) according to the directions of the manufacturer. RNA was used to quantify the copy number of virus genome by quantitative PCR (qPCR). Using a previously described method (34), qPCR analysis of the viral gene was performed using a PrimeScript RT-PCR kit (Perfect Real Time) (TaKaRa Bio, Inc., Japan), forward primer qR2TF (5=-CTTCCTGCCTGATCCAACTG-3=), reverse primer qR2TR (5=-GTTCTGCTTTCCCACCATTTG-3=), and probe R2probe2 (6-carboxyfluorescein [FAM]-CAACGACTGCACCACGAGTTG-black hole quencher 1 [BHQ1]). This was expected to generate a PCR product of 93 bp in base positions 378 to 470 (428 to 448 for the probe) from the start codon of RNA2 (34). The reverse transcription conditions were as follows: 20 min at 50°C and 5 min at 95°C, followed by 45 PCR amplification cycles of 15 s at 95°C and 45 s at 60°C. Concentration of virus in seawater. A previously reported iron-based flocculation method (29) was used to concentrate the virus in seawater, with slight modifications. An FeCl3 solution was added to 500 ml of seawater to prepare a final concentration of 1 mg Fe/liter. The seawater was gently (200 rpm) agitated at room temperature for 1 h using a magnetic stirrer, and the resulting Fe-virus flocculate was trapped on a 0.8-␮mpore-size polycarbonate filter (Advantec) under conditions of reduced pressure. Quantification of virus genome in seawater. The flocculate-trapped filter was transferred to a small tube, to which 100 ␮l of HBSS and 2.5 ml of chloroform were added to lyse the filter. After centrifugation at 5,000 ⫻ g for 20 s (4°C), total RNA was extracted from the aqueous and sediment layers by using ISOGEN. RNA was dissolved in 50 ␮l of RNase-free water and diluted 10 times with the same water, and 2 ␮l of RNA was used to quantify the copy number of virus genomes by qPCR, as described above. Cell culture isolation of virus in seawater. Virus isolation was performed according to a method reported previously (29) with slight modifications. The filter-trapped Fe-virus flocculate was transferred to a small tube, to which 1 ml of oxalate-EDTA buffer (0.25 M oxalic acid, 0.2 M Mg2EDTA, pH 6) was added to release the virus. After gentle shaking at

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4°C for 2 h, the water content was transferred into a cellulose tube (Viskase Companies, Inc., USA), dialyzed against HBSS for 6 h, and filtered through a 0.2-␮m-pore-size membrane filter (Advantec). The virus filtrate (500 ␮l) was inoculated into an E-11 cell monolayer culture using L-15 medium supplemented with 5% FBS and antibiotics (100 units/ml penicillin and 100 ␮g/ml streptomycin) in a 25-cm2 flask (Primaria; Corning Incorporated-Life Sciences, USA). The flask was incubated at 25°C for 10 days to observe cytopathic effects (CPE). Blind passages were conducted for flasks showing no CPE. For virus identification, a reverse transcriptase PCR (RT-PCR) was performed using a PrimeScript RT-PCR kit (TaKaRa Bio, Inc.) according to the directions of the manufacturer. The following primers were used for the PCR amplification of the RNA2 gene of RGNNV: RGRp (5=-CCGGATGACCCGGTTAGTTT-3=) and qR2TF (5=-CTTCCTGCCTGATCCAACTG-3=) (34). This was expected to generate a PCR product of 659 bp (base positions 378 to 1036), including a variable region (T4) of RNA2 (11, 30). The PCR products were examined using 2% agarose gel electrophoresis. Validation of virus detection method for seawater. An FeCl3 solution was added to 500 ml of artificial seawater (Marine Art SF-1; Tomita Pharmaceutical Co., Ltd., Japan) (salinity, 36 ppt) to prepare a final concentration of 1 mg Fe/liter. The purified virus (strain SGEhi00) was spiked at different doses ranging from 101.8 to 109.8 copies/500 ml. Three samples were used for each dose. The seawater was gently (200 rpm) agitated at room temperature for 1 h using a magnetic stirrer, and the resulting Fevirus flocculate was trapped on a 0.8-␮m-pore-size polycarbonate filter (Advantec) under conditions of reduced pressure. Two flocculatetrapped filters were prepared in every experiment. The virus quantification (qPCR) and isolation (E-11 cells) were performed using each one of the filters, as described above. Detection of virus in fish-rearing seawater in a laboratory setting. Juvenile sevenband grouper (initial average body weight, 30.2 g) were used in this experiment. Twenty-five fish, as virus donors, were intramuscularly injected with cell-cultured strain SGEhi00 (103.5 TCID50/fish) and placed in a 54-liter plastic tank. The drainage from the tank was introduced into another 54-liter tank, where 25 virus-free fish acting as virus recipients were kept. As a negative control, in a second set of tanks, 25 fish received HBSS injections (donors), and 25 virus-free fish (recipients) were placed in separate 54-liter tanks. UV-treated seawater (25°C ⫾ 1°C) was introduced into the donor fish tanks at a flow rate of 120 liters/h. The fish were fed daily with commercial pellets (Nisshin Marubeni Feed Co., Ltd.) and observed daily to monitor their health conditions throughout an experimental period of 24 days. During this period, the drainage seawater from the donor fish tank in the experiment set was sampled at 1 to 9, 15, 17, 19, 22, and 24 days postinjection (dpi). In the control set, the seawater from the mock-infected fish tank was sampled only at 0 and 24 dpi. The sampled seawater (1 liter) was filtered through an 8.0-␮m-pore-size polycarbonate filter (Advantec) and subjected to the iron-based flocculation and qPCR assay. Additionally, 1 liter of seawater was similarly filtered and subjected to the iron-based flocculation. The flocculate was resuspended in oxalate-EDTA buffer and subjected to cell culture isolation using L-15 medium. Fish that died during the experimental period and survivors at 24 dpi were subjected to virus titrations with E-11 cells, followed by an indirect fluorescent antibody test using a monoclonal antibody according to a method described previously (31). Detection of virus in fish-rearing seawater in a field setting. The experiment was performed at ERIA in 2013. A total of 115,000 juvenile sevenband grouper (initial body weights; 15 to 25 g), which were all injected with a commercial vaccine for VNN (OceanTect VNN; Nisseiken Co., Ltd., Japan) 3 weeks before being transferred to rearing pens, were put in 16 net pens (each measuring 5 by 5 by 3 m) from 17 September to 7 November. Seawater was collected 6 times near the pens before introduction (5 August) and after introduction (25 September, 30 October, 10 November, 27 November, and 20 December) of the fish into the net pens. During the experiment, the water salinity and temperature around the net pens ranged from 34 to 35 ppt and from 16.2°C to 24.7°C, respectively.

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TABLE 1 Recovery of RGNNV from experimentally virus-spiked seawater by qPCR and cell culture isolationa qPCR result (no. of copies/500 ml) No. of spiked virus (no. of copies/500 ml) 109.8 108.8 107.8 106.8 105.8 104.8 103.8 102.8 101.8 a

Sample no. 1

2

3

Average

Isolation result

109.8 108.0 107.5 106.9 105.9 105.1 104.0 103.4 ns

109.9 108.5 107.7 106.8 105.9 105.2 105.1 103.4 ns

109.8 108.6 107.7 106.7 106.1 105.4 104.3 103.4 ns

109.8 108.5 107.7 106.8 106.0 105.3 104.7 103.4 ns

ND ND ⫹ ⫹ ⫹ ⫹ ⫺ ND ND

ND, no data; ns, no signal.

FIG 1 The relationship of the spiked number and the recovery number of the The 1-liter samples of seawater were filtered serially through 8.0-␮m-, 0.8-␮m-, and 0.2-␮m-pore-size polycarbonate filters (Advantec) and subjected to iron-based flocculation, qPCR assay, and cell-culture isolation, as described above. Throughout the experimental period, randomly sampled dead fish were examined using the RT-PCR test. Infection experiment. Virus isolates (n ⫽ 3) from seawater in the field setting were cultured in E-11 cells at 25°C for 3 days. The cultured viruses were intramuscularly injected in sevenband grouper (average body weight, 103 g) at a dose of 105.5 TCID50/fish (each group; n ⫽ 15). The fish were observed in tanks at 25°C ⫾ 1°C water temperature for 12 days. Dead and surviving fish were subjected to virus isolation with E-11 cells as described above. Sequence analysis of the virus detected or isolated from seawater. Total RNAs were extracted from the Fe-flocculated filter samples and virus isolates, which were prepared from seawaters during the net pen culture of sevenband grouper mentioned above. The RT-PCR analysis was performed as described above, and the PCR products were examined by 2% agarose gel electrophoresis and purified using a QIAquick gel extraction kit (Qiagen). Sequencing analyses were performed at the Natural Science Center for Basic Research and Development at Hiroshima University using an ABI Prism 3130xl analyzer (Life Technologies). Nucleotide sequence accession numbers. Partial genome sequences of the betanodavirus isolated from a dead sevenband grouper (SGEhi13), the betanodaviruses isolated from seawater (SWiso10-30, SWiso11-10, and SWiso11-27), and two betanodavirus RNA extracts from seawater (SWpcr10-30 and SWpcr11-10) were submitted to the DNA Data Bank of Japan (DDBJ) under accession numbers LC095623 (SGEhi13), LC095624 (SWiso10-30), LC095625 (SWiso11-10), LC095626 (SWiso11-27), LC095627 (SWpcr10-30), and LC095628 (SWpcr11-10).

RESULTS

Efficacy of the iron-based flocculation for qPCR assay and virus isolation. By spiking RGNNV (strain SGEhi00) at different doses (101.8 to 109.8 copies/500 ml) into artificial seawater, the method combining iron-based flocculation and qPCR was shown to be effective in revealing a linear relationship (R2 ⫽ 0.9905) between the spiked number and the recovery number of the virus, when virus was spiked at a dose of 102.8 copies/500 ml or higher (Table 1 and Fig. 1). The ratio of the recovery number to the spiked number was a little higher at higher dilutions. We determined that the quantitative detection limit of the iron-based flocculation method followed by qPCR was approximately 103 copies/500 ml. In cell culture isolation, betanodavirus-characteristic CPE appeared when seawater was spiked with the virus at doses of at least 104.8

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RGNNV genome determined by the iron-based flocculation method and qPCR. The error bars indicate standard deviations of the means (n ⫽ 3).

copies/500 ml followed by inoculation of the Fe flocculates into E-11 cell cultures (Table 1). Dynamics of the virus from fish-rearing seawater in a laboratory setting. Donor fish injected with RGNNV (strain SGEhi00) showed abnormal swimming behaviors at 4 dpi and died between 5 and 10 dpi. The cumulative mortality rate reached 44%; however, a few fish displaying abnormal swimming behavior were observed in the tank even at the termination of the experiment (24 dpi). RGNNV was first detected at 2 dpi (104.1 copies/liter) from the drainage of the donor fish, and the levels increased with fish mortality. The highest level of the virus was 108.6 copies/liter at 9 dpi. After the donor fish deaths ceased, the virus level in the water decreased and became undetectable (lower than 103 copies/liter) at 24 dpi. One week after the last death in the donor group, mortality was observed in the recipient group, resulting in an 8% cumulative mortality rate (Table 2 and Fig. 2). RGNNV was reisolated from 76% and 100% of the brains of the donor and recipient surviving fish, respectively. Virus titers in the dead fish of the donor and recipient groups were 109.5 and 1010.0 TCID50/g on

TABLE 2 Cell culture isolation and qPCR detection of RGNNV from fish-rearing seawater in laboratory setting Day postinjection (dpi)

Cell culture isolation result

qPCR result (no. of copies/liter)

1 2 3 4 5 6 7 8 9 15 17 19 22 24

⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺

—a 104.1 104.4 103.3 104.3 106.8 106.7 104.8 108.6 106.0 106.1 104.3 104.9 —

a

—, under detection limit of 103 copies/liter.

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FIG 2 RGNNV detection from the fish-rearing seawater and cumulative mortality of fish in experimental infection. Sevenband grouper were injected with RGNNV (donor). Drainage (1 liter) from the donor fish tank was subjected to iron-based flocculation and qPCR analysis at 1 to 9, 15, 17, 19, 22, and 24 days postinfection. The drainage was introduced into recipient fish tank at a flow rate of 120 liters/h. Asterisks indicate the seawater samples from which the virus was isolated.

FIG 3 Daily mortality of sevenband grouper in floating net pens. A total of

average, respectively. In the surviving fish, the mean virus titers in the donor fish were slightly lower than those in the recipient fish, regardless of behavioral conditions (Table 3). In the negative-control group, no fish abnormalities were observed. Furthermore, the virus genome was not detected in water, and the virus could not be reisolated in the surviving fish. Dynamics of the virus from seawater around VNN-prevailing net pens in a field setting. Three weeks after the introduction of the first batch of juvenile sevenband grouper into the net pens, mortality began in early October and continued until late December 2013 (Fig. 3). During this period, the total number of dead fish was approximately 7,800 (6.8% cumulative mortality). The RTPCR tests performed on randomly sampled dead fish demonstrated that the mortality was due to VNN (RGNNV infection). The virus genome was detected from the seawaters examined on 30 October, 10 November, and 20 December at 104.2, 105.6, and 103.7 copies/liter, respectively, while the isolations of the virus with the E-11 cells were positive in the 30 October and 10 November seawaters and in the qPCR-negative seawater of 27 November (Table 4 and Fig. 3). Pathogenicity of the virus isolates from seawater in sevenband grouper. Three virus isolates from seawater, SWiso10-30, SWiso11-10, and SWiso11-27, which were isolated on 30 October,

10 November, and 27 November, respectively, were examined. The fish that were injected with the virus isolates from seawater died between 5 and 9 dpi. These fish showed abnormal swimming behaviors characteristic of VNN and had a cumulative mortality rate ranging from 26.7% to 33.3%. The inoculated virus was reisolated in the E-11 cells from all of dead fish and from around half of the surviving fish (Table 5). In the control fish, swimming abnormalities were not observed, and virus isolation was negative for the survivors. Sequence analysis of the virus obtained in a field setting. Two RNA samples extracted from seawaters on 30 October (SWpcr1030) and 10 November (SWpcr11-10), three RNA samples of the virus isolates from seawaters (SWiso10-30, SWiso11-10, and SWiso11-27), and one RNA sample of the virus isolate (SGEhi13) from a dead sevenband grouper collected on 31 October, when VNN prevailed, were subjected to sequencing for the T4 region of RNA2. All sequences were identical, except for the sequence of the RNA from SWpcr10-30, which had 3 substituted bases (Fig. 4). These sequences had 98% or higher similarity to those of SGWak97 (a representative strain of the RGNNV genotype) (32) or SGEhi00 (31), which was used in this study as RGNNV.

115,000 fish were placed in 16 net pens from 17 September to 7 November. Arrowheads indicate the dates when seawater samples were collected.

TABLE 3 Virus titers in the brain of sevenband groupers challenged with RGNNVa Virus isolation from the brain Group

Grouper category

No. of fish examined

Condition of fish at 24 dpi

No. of fish with positive result

Avg (range) TCID50/g

Control

Donor Recipient

25 25

Normal Normal

0 0

⬍103.1 (⬍103.1) ⬍103.1 (⬍103.1)

Experiment

Donor

5 9 11 8 15 2

Normal Abnormal Dead (5–10 dpi) Normal Abnormal Dead (17–19 dpi)

2 6 11 8 15 2

104.5 (⬍103.1–104.9) 104.8 (⬍103.1–105.4) 109.5 (108.1–1010.3) 105.8 (104.9–106.8) 106.2 (104.3–107.6) 1010.0 (109.9–1010.0)

Recipient

a

Fish were intramuscularly injected with RGNNV and observed at 25°C for 24 days. dpi, days postinjection; Abnormal, abnormal swimming behavior.

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TABLE 4 Cell culture isolation and qPCR detection of RGNNV from seawater around fish-rearing net pens in field setting Sampling date in 2013

Cell culture isolation result

qPCR result (no. of copies/liter)

6 August 25 September 30 October 10 November 27 November 20 December

⫺ ⫺ ⫹ ⫹ ⫹ ⫺

—a — 104.2 105.6 — 103.7

a

—, under detection limit (103 copies/liter).

DISCUSSION

Various types of real-time reverse transcriptase PCR (RT-qPCR) methods for the quantitative detection of betanodaviruses have been reported, as summarized by Hick and Whittington (34). In the present study, to quantify RGNNV in seawater, we used the method optimized in that paper (34), which is particularly specific to RGNNV among the 3 other genotypes of betanodaviruses (SJNNV, TPNNV, and BFNNV). In our RT-qPCR analysis of RGNNV, 1 TCID50 unit corresponded to approximately 1,900 copies. This TCID50 estimation and the RGNNV genome copy number nearly matched those reported for BFNNV (35) or RGNNV, SJNNV, and BFNNV (36). Nerland et al. (35) developed an RT-qPCR method to detect AHNV (Atlantic halibut nodavirus; BFNNV) from seawater without any previous treatment, i.e., without concentration of the virus, and the detection limit of the method was approximately 104 copies/ml (equivalent to 106 or 107 copies/500 ml). In the present study, we combined iron-based flocculation and RT-qPCR to detect RGNNV at up to 102.8 copies/500 ml (equivalent to 1.2 copies/ ml) from RGNNV-spiked seawater. Therefore, our method, which is applicable to at least 500-ml volumes of seawater, is effective for detecting smaller amounts of RGNNV in seawater. Additionally, the present method of direct extraction of RNA from iron flocculate-trapped filters is relatively simple and does not require other cumbersome steps (27, 29). The only disadvantage of this method was that the RNA extracted from the iron-virus flocculate filter was slightly brown-colored, which affected the genome copy determination in RT-qPCR. However, this was solved by diluting the RNA solution 10-fold. Since the first successful isolation of a betanodavirus

TABLE 5 Pathogenicity of the RGNNV isolates from natural seawater in sevenband grouper

Inoculated virus isolate

% mortality (no. of dead fish/no. of fish examined)

SWiso10-30a SWiso11-10b SWiso11-27c Control

26.7 (4/15) 26.7 (4/15) 33.3 (5/15) 0 (0/15)

a b c

Isolate from seawater collected on 30 October. Isolate from seawater collected on 10 November. Isolate from seawater collected on 27 November.

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% reisolation (no. of fish with positive result/no. of fish examined) of the virus from: Dead fish 100 (4/4) 100 (4/4) 100 (5/5)

Surviving fish 54.5 (6/11) 54.5 (6/11) 50.0 (5/10) 0 (0/15)

FIG 4 Multiple alignment of nucleotide sequences of the T4 region of RGNNVs. SGWak97, a representative strain of RGNNV; SGEhi00, an RGNNV strain used in the present in vitro and in vivo experiments; SGEhi13, an isolate from the present dead fish; SWiso10-30, SWiso11-10, and SWiso11-27, 3 isolates collected from the present seawater on 30 October, 10 November, and 27 November, respectively; SWpcr10-30 and SWpcr11-10, two RNA extracts collected from the present seawater on 30 October and 10 November, respectively.

(RGNNV) with the SSN-1 cell line (37), several cell lines for the isolation and propagation of betanodaviruses have been reported (32, 38, 39, 40). Among them, the E-11 cell line (32) derived from the SSN-1 cell line has proven to be highly permissive to all genotypic variants of betanodavirus. Furthermore, the virus productivity in E-11 cells is high, with titers reaching 1011 TCID50/ml after 96 h of incubation at 30°C (41). In the present study, RGNNV was easily isolated from the iron-virus flocculate filter by E-11 cells. However, we failed to quantify the virus titers in seawater using 96-well plates due to unidentified cytotoxic effects of

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the inoculum; instead, we used 25-cm2 flasks. Further modifications would be required for the quantitative isolation of the virus. As shown by the experiments performed with RGNNV and juvenile sevenband grouper, there was a clear correlation between disease progression in the virus-infected fish (donor) and the virus concentrations in the rearing water. The virus was first detected at 104 copies (equivalent to approximately 101 TCID50)/liter 3 days before fish death was observed. The virus copy number increased rapidly as fish mortality increased, reaching 108 copies (105 TCID50)/liter when fish deaths ceased. Subsequently, the copy number decreased despite the surviving fish exhibiting abnormal behavior. The virus was effectively isolated from the tank drainage, in which the virus level was at least approximately 105 copies/ liter. Because the virus titers in the brains of the dead fish were very high (108 –10 TCID50/g) compared with those in the brains of the surviving fish (104 –5 TCID50/g; Table 3), it seems that heavy virus shedding into the water occurred during rigorous proliferating processes, which led to overt infection. Moreover, we confirmed the transmission of the virus in the recipient fish exposed to the virus-contaminated water. More than 2 weeks elapsed until the initial occurrence of recipient fish death. Although the mortality rate was relatively low (8%), all surviving recipient fish carried high titers of the virus in the brain (104 –7 TCID50/g) (Table 3). In general, fish at younger stages are more susceptible to betanodaviruses (3, 42) and experimental infections by immersion (or bath) methods result in higher mortality at larval stages (43, 44, 45) but lower mortality in juveniles or older fish (43, 46, 47), regardless of the virus genotype. This age resistance has not helped elucidation of the infection mechanisms of VNN in fish at growout stages, although a few reports have described infections in juveniles performed by immersion or cohabitation challenges (48, 49, 50, 51). The present infection system using virus-contaminated water from infected fish seemed to mimic the naturally occurring infection mode and thus could be useful as an infection model for dynamic studies on the horizontal transmission of betanodaviruses. In this study, we succeeded in detecting RGNNV in seawater near floating net pens where VNN prevailed in cultured juvenile sevenband grouper. Compared with the results of a study in which AHNV was detected at 107.3 copies/ml (equivalent to 104.2 TCID50/ml) in the seawater of floating bags at VNN-prevailing facilities for Atlantic halibut larvae (35), the virus level detected in our field study was fairly low (ca. 10 to 100 copies/ml). This may have been due to differences in the rearing systems or mortality rates. The floating bag system in marine lagoons (40,000 larvae/ bag for 100-mg fish or 5,000 larvae/bag for 400-mg fish) for Atlantic halibut larvae cultures likely had a higher mortality rate than the net pen system for sevenband grouper grow-out cultures (6.8%). Incidentally, based on our experience with VNN in net pen-cultured sevenband grouper, when hatchery-produced juvenile sevenband grouper reared under virus-free conditions are transferred to an infectious open sea location, heavy mortality or the almost complete destruction of the fish population occurs within 2 weeks after transportation. Therefore, the present low mortality rate may be attributable to a vaccine effect. Our continuous field surveys on the vaccine effect in the same facility confirmed a low mortality rate of vaccinated sevenband grouper, i.e., less than 5% in 2014 and 2015. We succeeded in isolating RGNNV from fish-rearing water in the open sea containing very sparse virus numbers, i.e., 104 copies/

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liter or fewer, at levels that are near practical threshold values. Because of the complete sequence similarity (T4 region in RNA2) between clinical (dead fish) and environmental (seawater) virus isolates and the confirmation of their pathogenicity in sevenband grouper, we demonstrated that the present method is effective for determining the presence of infectious virions in fish-rearing environmental water. As mentioned earlier, the dynamics and natural infection mechanisms of piscine nodavirus, particularly at grow-out stages during net pen culture in the open sea, have been poorly elucidated. The present results provide direct evidence for the horizontal (waterborne) transmission of the virus via rearing waters in the open sea. RT-PCR or nested PCR analyses of betanodaviruses from fish suggested that possible sources of transmission of betanodaviruses to hatcheries and farms stem from a variety of subclinically or persistently infected cultured and wild fish (5). However, there has been no direct evidence for this hypothesis. Molecular analyses have indicated the presence of different betanodaviruses with high numbers of sequence variations or reassortant viruses in populations of wild fish compared with those isolated from diseased fish, which have relatively limited sequence variations (52). The virulent forms of these viruses may have originated from less-susceptible wild fish and subsequently shed in water, or various genetic variants may have been present as virions in water and only virulent forms infected susceptible cultured fish. In the present study, we found 3 nucleotide substitutions (T4 region) in one RNA extract from seawater (SWpcr10-30); this supports the possibility of the latter idea. Using the present method, we tried to detect and isolate betanodaviruses from the rearing tank water of a betanodavirus-positive (RT-PCR) wild-fish population but failed to do so (data not shown). More-sophisticated techniques may be required and will be explored in future studies. Currently, 40 or more marine and freshwater fish species are known to be betanodavirus-susceptible hosts (4, 5). Considering the frequent worldwide occurrence of the disease, particularly in aquaculture farms of marine fish, the ability to detect the presence of infectious pathogens is indispensable in establishing measures to control the disease. We expect that the present method will be useful for the future researches on VNN and betanodaviruses. ACKNOWLEDGMENT This study was supported in part by a special grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.

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