www.ksfp.org 한국어병학회지 제32권 제1호 (2019) pISSN 1226-0819, eISSN 2233-5412 J. Pathol., 32(1) : 001~008 http://dx.doi.org/10.7847/jfp.2019.32.1.001

Correlation of replication and index in rock bream infected rock bream Oplegnathus fasciatus

Myung-Hwa Jung and Sung-Ju Jung†

Department of Aqualife Medicine, Chonnam National University, 50 Daehak-ro, Yeosu, Jeonnam, 59626, Korea

Rock bream iridovirus (RBIV) is a member of the Megalocytivirus genus that causes severe mortality to rock bream (Oplegnathus fasciatus) with characteristic clinical signs of spleen enlargement. In this study, we assessed spleen size and RBIV copy number patterns in RBIV-infected rock bream to determine lethal and safe levels of virus copy number/spleen index that may define disease progress. We found that rock bream infected with RBIV (1.1 × 107 virus copy number/100 μl) and held at 29, 26, 23 or 20°C exhibited significantly higher levels of spleen size compared to 17°C. In dead condition (100% mortality at 20~29°C), the spleen index (spleen weight / fish weight × 100) and virus copy number were 3.00~5.38 and 106~108/μl, respectively. Conversely, in survived condition (0% mortality at 17°C), spleen index and virus copy number was as low as not-infected control (0.34~1.22/100~101/μl, respectively). These findings suggest that spleen index can be an indicator of disease severity of RBIV disease.

Key words: rock bream iridovirus, rock bream, virus replication, spleen index, spleen enlargement

Iridoviridae is a family of large double-stranded bream Oplegnathus fasciatus (Jung and Oh, 2000). DNA virus (120~300 nm) with an icosahedral mor- Various indices have been used to evaluate the con- phology (Williams, 1996). The family includes five dition or well-being of fish, including the relative con- genera: Iridovirus, , , Lym- dition factor (Le Cren, 1951), relative weight (Wege phocystivirus and Megalocytivirus. Megalocytivirus and Anderson, 1978), gut index (Jensen, 1980), RNA- cause disease in more than 50 fish species and cur- DNA ratios of liver and muscle (Bulow et al., 1981), rently threaten the industry, causing great visceral somatic index (Delahunty and de Vlaming, economic losses in Korea, Japan, China and Southeast 1980; Adams et al., 1982) and liver somatic index Asia (Inouye et al., 1992; Nakajima and Sorimachi, (Edwards et al., 1972; Tyler and Dunn, 1976; Valto- 1994; Chua et al., 1994; Matsuoka et al., 1996; Miyata nen, 1974; Heidinger and Crawford, 1977; Bulow et et al., 1997; Chou et al., 1998; Jung and Oh, 2000; al., 1978; Delahunty and de Vlaming, 1980; Allen and He et al., 2002). Rock bream iridovirus (RBIV), Wootton, 1982; Adams and McLean, 1985). Liver so- which belongs to the genus Megalocytivirus (Do et matic index is a useful biomarker to detect the envi- al., 2004; Song et al., 2008; Kurita and Nakajima, ronmental stressors, and it is one of the most sensitive 2012) remains an important health problem in rock growth indicators (Edwards et al., 1972; Valtonen, 1974; Tyler and Dunn, 1976; Heidinger and Craw- † Corresponding author: Sung-Ju Jung ford, 1977; Bulow et al., 1978; Allen and Wootton, Tel: +82-61-659-7175; Fax: +82-61-659-7179 E-mail: [email protected] 1982; Adams and McLean, 1985). Liver is the meta- 2 Myung-Hwa Jung and Sung-Ju Jung bolic organ, and fish store energy in the liver during firm presence of abnormally enlarged cells in Giemsa- periods of high energy intake. Most of this stored en- stained stamp-smear of the spleen. Basophilic enlarged ergy is in the form of glycogen (Busacker et al., cells by Giemsa stain are compactly packed with virus 1990). Therefore, the relative size of the liver should particles in the cytoplasm. Hence, we hypothesised be correlated to the nutritional state of the fish as that splenomegaly of rock bream can be an indicator well as the growth rate. of severity of RBIV disease. Other organ indexes except liver have not been In the present study, rock bream were artificially commonly used as biomarkers for the evaluation of infected with RBIV, and RBIV copy number and fish health. Spleen is one of the major filtering organs spleen size were measured. Furthermore, lethal and in the vascular system, removing effete blood cells safe levels of RBIV copy number and spleen size and foreign agents. In humans, vertebrate and fish, (index) were estimated. the spleen is the major site of pathogen growth and disease pathology (Chinabut et al., 1990; Toranzo et Materials and Methods al., 1991; Inouye et al., 1992; Santos et al., 1992; Fryer and Mauel, 1997; Jung and Oh, 2000; Kim et Experimental infection al., 2005; Kim et al., 2009; Jeffery et al., 2010). The RBIV was obtained from RBIV-infected rock Hence, the spleen is frequently used in the diagnosis bream in 2010 (Jung et al., 2014). Virus amount was of damage caused by pathogen infection. Studies on quantified by quantitative real-time polymerase chain fish disease such as Flexibacter psychrophilus (Santos reaction (qRT-PCR). The RBIV major capsid protein et al., 1992), Pasteurella piscicida (Toranzo et al., (MCP) gene copies of the original virus in the super- 1991), Piscirickettsia salmonis (Fryer and Mauel, natant preparations were 7.5 × 107/100 μl MCP gene 1997), Francesella sp. (Jeffery et al., 2010), Micro- copies, and that was suspended in phosphate-buffered bacteriosis (Chinabut et al., 1990), Cyprinid herpesvi- saline (PBS) to 1.1 × 107/100 μl as previously de- rus 2 (Jeffery et al., 2007), Viral haemorrhagic septi- scribed (Jung et al., 2014). caemia (Kim et al., 2009), Trypanoplasma salmosi- The RBIV-free rock bream were reared by the tica (Li et al., 2013) and Haemogregarina sachai Fisheries Science Institute at Chonnam National (Kirmse, 1980) have demonstrated a clear clinical University. The experimental design was identical to sign of spleen enlargement (splenomegaly). Nearly all that explained previously (Jung et al., 2015). Rock deaths by RBIV are accompanied by enlargement of bream (10.8 ± 1.5 cm, 25.1 ± 3.1 g) were used for spleen (Jung and Oh, 2000; Zhang et al., 2012). The evaluation of spleen weight at different water temper- simplest diagnostic method of RBIV disease is to con- atures (29, 26, 23, 20 and 17°C) (Table 1). For viral

Table 1 Experimental details of artificial infection Infection dose/fish Days Mortality Group Sampling point (virus copy number) observed (%) 29°C 1.1 × 107/fish 10 6, 7, 8, 9 and 10 dpi1) 100 26°C 1.1 × 107/fish 14 8, 9, 10, 11, 12 and 14 dpi 100 23°C 1.1 × 107/fish 15 13, 14 and 15 dpi 100 20°C 1.1 × 107/fish 26 19, 20, 21, 22, 23, 24 and 26 dpi 100 17°C 1.1 × 107/fish 150 100 and 150 dpi 0 1)dpi: days post infection Correlation of virus replication and spleen index in rock bream iridovirus infected rock bream Oplegnathus fasciatus 3 infection, 15 fish was intraperitoneally (i.p.) injected 79 to 140 mg (average 110.3 mg) (Fig. 1B), and with 100 μl/fish containing 1.1 × 107 MCP gene copy, spleen index was in the range of 3.06 to 5.38 (average while the control fish was i.p. injected with the PBS 3.65) and was regarded as ‘lethal’ (Fig. 1C). Fig. 1D (100 μl/fish) and then maintained in the aquaria con- shows cumulative mortality of fish sampled for this taining 30 L of UV-treated seawater. To determine study (previously published by Jung et al., 2015). the RBIV replication pattern, spleen was collected from dead fish at 29, 26, 23 and 20°C and surviving Survived condition (RBIV infection at 17oC) fish at 17°C. Samples were stored at -80°C after being At 17°C, all the fish survived with no clinical flash frozen in liquid nitrogen. Table 1 summarizes signs. The five sampled fish at 17°C did not have experimental conditions. enlarged (range of 17 to 49 mg) and had low virus copy numbers (average 3.0 × 101/μl) at 100 dpi Determination of RBIV copy number in the (Fig. 1A). The remaining survivors (10 fish) were spleen and spleen index placed in increased water temperature to 26°C; at 100 Genomic DNA was extracted from the whole spleen dpi, the fish did not die and had low virus copy num- tissues (20~150 mg) of the sampled fish using an bers (average 5.6 × 101/μl) at 150 dpi. All the 15 AccuPrep®Genomic DNA extraction kit (Bioneer, surviving fish showed a spleen weight of below 50 Korea) according to the manufacturer’s instructions. mg against RBIV infection during the experimental Quantification of RBIV copy numbers were deter- period (Fig. 1B). The spleen index in survivors was mined by qRT-PCR using Exicycler 96 Real-Time in the range of 0.53 to 1.44 (average 0.84) (Fig. 1C). Quantitative Thermal Block (Bioneer) with RBIV MCP gene specific primer set (F 5’ tgcacaatctagttgagg Control aggtg 3’ and R 5’ aggcgttccaaaagtcaagg 3’) according PBS-injected fish maintained at 29, 26, 23 and to the standard curve and method described pre- 20°C had a 100% survival rate. The fish sampled in viously (Jung et al., 2014). The virus copy number all groups were distributed in the below detection lim- was expressed as viral DNA copies 1 μl of DNA of it level of RBIV MCP copy number (1.0 × 101/μl) 100 μl of total DNA from a whole spleen. The de- (Fig. 1A). Spleen weight of all control fish was under tection limit level of RBIV MCP copy number was 50 mg in the experimental period (Fig. 1B). The 1.0 × 101/µl. The spleen indexes were defined by the spleen index was in the range of 0.58 to 1.50 (average following formula: 1.13) and was regarded as ‘safe’ (Fig. 1C). Spleen index = (Spleen weight (g)/ Fish weight (g)) × 100. Discussion

Results Spleen acts as an important immune responsive or- gan against a variety of pathogens. It is crucial for RBIV replication and spleen size at different the capture and destruction of pathogens and the for- infection condition mation of adaptive immunity (Mebius and Kraal, Dead condition (RBIV infection at 29, 26, 23 and 2005). Spleen size of the fish is considered as a sim- 20°C). The virus copy number from 60 dead fish ple measurable immune parameter with a potential ranged from 1.11 × 107 to 9.56 × 108/μl and was re- role in immune response against pathogens (Skarstein garded as ‘lethal’ virus copy number (Fig. 1A). The et al., 2001; Taskinen and Korter, 2002; Korter and spleen weight from all of the dead fish ranged from Taskinen, 2004; Lefebvre et al., 2004), and European 4 Myung-Hwa Jung and Sung-Ju Jung

A B

C D

Fig. 1. Absolute major capsid protein (MCP) gene copies (A), spleen weight (B) and spleen index (C) of rock bream intraperitoneally injected with 1.1 × 107 MCP gene copy/fish at different water temperatures (29, 26, 23, 20 and 17°C). Surviving fish at 17°C were sampled at 100 (△) and 150 (▲) days post infection. The cumulative mortality (D) of rock bream previously published (Jung et al. 2015) is shown for reference. Virus copy number, 1.0 × 101/µl, in the PBS injected control group (A) was regarded as negative. starlings (Sturnus vulgaris) with larger spleens have 108/μl (109~1010/100 μl/whole spleen weight 79~140 been shown to mount stronger immune responses mg), and the lethal ratio of the spleen index due to (Ardia, 2005). However, RBIV-infected rock bream RBIV infection was 3.06 to 5.38. A similar virus copy are characterised by enlargement of spleen (Jung and range and spleen index of dead rock bream against Oh, 2000), and spleen size may reflect disease status megalocytivirus was observed in the range of 2.03 × after virus infection. 107/mg and 4.85 ± 1.06, respectively (Jin et al. 2011). First, absolute MCP gene copy and spleen weight In addition, control and surviving fish had very low were evaluated from the dead fish obtained from the virus copy at below 101/μl (detection limit level, be- RBIV-injected group and maintained higher than low 103/100 μl/whole spleen weight 20~47 mg) and 20°C. A threshold (dangerous limit) was then set up a similar range of spleen index (0.59~1.50 in the con- for the virus copy number and spleen index indicating trol group and 0.34~1.22 in the surviving group). possible virus copy number and spleen index, which These levels were regarded as safe. A contrasting ob- causes fish death. The virus copy range was 107 to servation has been reported by Hadidi et al., (2007) Correlation of virus replication and spleen index in rock bream iridovirus infected rock bream Oplegnathus fasciatus 5 that enlarged spleen (approximately 65 mg) and high reported for malaria-infected mouse (i.e. enlarged index (1.4) was observed in an Flavobacterium psy- spleen reduced to its normal size at several weeks chrophilum-resistant rainbow trout Oncorhynchus my- after primary malaria infection in the mouse) (Steven- kiss group, while reduced spleen weight (approxim- son and Kraal, 1989; Weiss, 1989; Achtman, 2003). ately 30 mg) with low index (0.7) was observed in Use of the size of immune system organs as an a susceptible group. Hence, spleen size and RBIV index of investment in the immune system is a com- copy number in this study suggest that the spleen size mon approach, and the spleen is of particular interest. (spleen enlargement due to virus infection) is not cor- The spleen is a relatively small but critical organ that related with RBIV disease resistance but severity of is involved in the production of lymphocytes that are the disease. used to fight against infections (John, 1994). Use of This was evident from our supplementary data that the size of the spleen as a proxy measure of immuno- previously published in shown the reference (Jung et logical activity has been widespread, particularly in al., 2017). In fixed water temperatures of higher than birds (Møller, 1997; Møller et al., 1998; Shutler et 23°C, rock bream mortality was extreme at 100% al., 1999), mammals (Cowan et al., 2009; Schulte- (Jung et al., 2014; Jung et al., 2015; Jung et al., Hostedde and Elsasser, 2011) and fish (Korter and 2016). For this reason, water temperature shifting Taskinen, 2004; Lefebvre et al., 2004; Skarstein et from 23°C to 17°C was tried in order to obtain survi- al., 2001; Taskinen and Korter, 2002), under the as- vors and detail the RBIV replication effect on the rock sumption that a larger spleen produces and stores bream spleen index and evaluate the spleen index for more lymphocytes than a smaller spleen (Nunn, 2002); a recovery stage from RBIV infection (Jung et al., hence, it can be induced disease resistance. Although 2017). Rock bream infected with RBIV and held for RBIV-infected rock bream are characterised by en- 7, 4 and 2 days at 23°C before the water temperature largement of spleen, it may not be due to the incre- was reduced to 17°C had mortality rates of 28% ment of lymphocytes but expansion of virus-infected (group A1), 0% (group A2) and 0% (group A3), re- cell size or number; it can be concluded that rock spectively (Jung et al., 2017). In groups A1, A2 and bream spleen enlargement is not related to disease re- A3, the acute stage of RBIV infection was from 7 sistance and that spleen size reflects RBIV disease to 22 dpi; when virus replication reached peak at 20 status. to 22 dpi (average range of 105~107/μl), spleen weight This study clearly showed spleen size (index) has and spleen index reached their highest (average range a strong positive relationship with mortality and virus of 108~125 mg and 2.97~3.76, respectively) (Supple- copy number, and suggests the possibility of using mentary Fig. 1). Furthermore, no mortality occurred spleen index as an indicator for the severity of RBIV from 30 to 100 dpi, and these fish showed no clinical disease. signs of RBIV. This time period was regarded as a recovery stage from infection. It was evident that Acknowledgement gradual decrease of virus copy numbers (average 107 This research was a part of the project titled ‘Fish reduced to 101/μl) accompanied with gradual reduc- Vaccine Research Center’, funded by the Ministry of tions of spleen weight and spleen index (average 94 Oceans and Fisheries, Korea to 27 mg and 2.79 to 0.84, respectively) (Supplemen- tary Fig. 1). This indicates that the spleen size of References RBIV-infected rock bream was positively co-related with virus replication. A similar observation has been Achtman, A.H.: The B cell response to Plasmodium cha- 6 Myung-Hwa Jung and Sung-Ju Jung

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