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Characterization of Virus Distribution in Rock Bream (Oplegnathus fasciatus; Temminck and Schlegel) Infected with Megalocytivirus
J. Comp. Path. 2009, Vol. 141, 63e69
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Characterization of Virus Distribution in Rock Bream (Oplegnathus fasciatus; Temminck and Schlegel) Infected with Megalocytivirus N.-S. Lee*, J. W. Do*, J. W. Park† and Y. C. Kim‡ * Pathology Division, National Fisheries Research & Development Institute, Busan, † Department of Biological Sciences, University of Ulsan and ‡ Southern Inland Fisheries Research Institute, Jin-hae, Kyungnam, South Korea
Summary The distribution of virus-infected cells in the organs of Rock Bream naturally infected with megalocytivirus is reported. Examination of sections of liver, spleen and kidney stained by haematoxylin and eosin (HE) and periodic acid Schiff (PAS) revealed the presence of swollen and degenerate cells having morphology consistent with leucocytes. Many of these cells were shown to contain viral DNA by in-situ hybridization (ISH). Cells containing viral DNA were also found in the connective tissue of other organs in which there was no prominent infiltrate of degenerate leucocytes. Viral DNA was also found in the cytoplasm of leucocytes in blood smears. Transmission electron microscopy (TEM) confirmed the presence of viral particles in the cells within tissue and free within blood. The tissue distribution of virus in this infection is suggested to reflect the infiltration of virusinfected leucocytes. Ó 2009 Elsevier Ltd. All rights reserved. Keywords: in-situ hybridization; leucocyte; megalocytivirus; Rock Bream iridovirus
Introduction Iridoviruses are large cytoplasmic DNA viruses with an icosahedral morphology (Williams, 1996) and the family Iridoviridae consists of five genera including Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus and Megalocytivirus (Chinchar et al., 2005). Iridoviral infections are one of the most prevalent viral diseases of cultured freshwater and marine fish species. White Sturgeon iridovirus (WSIV) (Hedrick et al., 1990), lymphocystis disease virus (LCDV) (Tidona and Darai, 1997), epizootic haematopoietic necrosis virus (EHNV) (Langdon et al., 1988), Orange-Spotted Grouper iridovirus (GIV) (Lu et al., 2005), Sea Bass iridovirus (SBIV) (Sudthongkong et al., 2002), infectious spleen and kidney necrosis iridovirus (ISKNV) (He et al., 2000) and Red Sea Bream iridovirus (RSIV) (Inouye et al., 1992) are among the best known iridoviral infections. According to the International Committee on Taxonomy of Viruses (ICTV),
fish iridoviruses are members of the genera Lymphocystivirus, Ranavirus or Megalocytivirus. Since 1998, mass mortality has been observed in cage-cultured Rock Bream off the southern coast of the Korean peninsula, and megalocytivirus has been shown to be the causative agent (Do et al., 2004, 2005). In 2005, an iridoviral epizootic occurred amongst Rock Bream cultured in the Tongyeong area of South Korea, and megalocytivirus was detected in 34% of the moribund fish (unpublished data). The pathogenesis of megalocytivirus infection in fish has been incompletely characterized. Accordingly, the aims of the present study were to describe the histopathological changes in the tissues of naturally infected fish and to determine the distribution of virus-infected cells within these tissues.
Materials and Methods Fish
Correspondence to: Y. C. Kim (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2009.03.008
During the summer of 2005 there were epizootics of megalocytivirus infection amongst Rock Bream Ó 2009 Elsevier Ltd. All rights reserved.
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cultured in the Tongyeong area of South Korea. The moribund fish developed severe anaemia and petechial haemorrhages in gill filaments and intestines, and they showed abnormal swimming behaviour. Viscera and blood samples were collected from these moribund fish (of approximate body length 12 cm) and frozen at 80 C. Samples of viscera were also fixed in 10% neutral buffered formalin. Samples from 15 fish with confirmed megalocytivirus infection were selected for the present study. Confirmation of infection was by polymerase chain reaction (PCR) with primers specific for the gene encoding Rock Bream iridovirus major capsid protein (RBIVMCP). The primer sequences were forward 50 CGTGATGGAGGGGATCTTAA-30 and reverse 50 -GAAAAACGAGGCCGATCATA-30 . Samples of spleen and kidney were used for this confirmatory testing. Histology and Transmission Electron Microscopy
Fixed tissue samples were processed routinely and embedded in paraffin wax. Sections (4e5 mm) were stained with haematoxylin and eosin (HE) or periodic acid-Schiff (PAS). For transmission electron microscopy (TEM), tissues and blood cells were fixed overnight at 4 C in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sections were stained with lead citrate and uranyl acetate. DNA Extraction and Preparation of Probes
Digoxigenin (DIG)-labelled probes for in-situ hybridization (ISH) were prepared by PCR using RBIV DNA extracted from infected Rock Bream collected in 2003 (Do et al., 2004) as a template. Viral DNA was extracted with the High PureÔ PCR Template Preparation Kit (Roche, Mannheim, Germany) and the AccuPrepÔ Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea), following the method described by Do et al. (2004). Primers for PCR were designed from nucleotide sequences in the GenBank database. Primer sequences for amplification of the gene encoding viral ATPase (GenBank accession number AY532614) were RBIV-AP forward 50 -GTAGTGATATCGGGCTCCGA-30 and RBIV-AP reverse 50 -CCGTTCTTGAACAGGTCCAT-30 . Primer sequences for amplification of the gene encoding the major capsid protein (MCP) were the same as those described above for confirmation of megalocytivirus infection. DIG-labelled probes were prepared by multiplex PCR using these two primer sets (Fig. 1). Briefly, a mixture was prepared in one PCR tube containing
Fig. 1. Digoxigenin (DIG) labelled PCR products. Product (c) was used in the present study. M, marker; a, ATPase; b, MCP; c, multiplex PCR of (a) and (b).
10 ml of 10 PCR buffer that contained 15 mM MgCl2 (TAKARA, Tokyo, Japan), 2 ml each of forward and reverse primers (10 pmol/ml), 1 ml of Taq polymerase (5 U/ml, TAKARA), 10 ml of PCRlabelled mixture or 10 dNTPs solution (for non-DIG-labelled probes) (Roche), 2 ml of either DNA template or distilled water (DW, as a negative control), and DW to a volume of 100 ml. After an initial denaturation for 5 min at 94 C, PCR was carried out for 35 cycles. Each cycle consisted of 30 s of denaturation at 94 C followed by 30 s of annealing at 57 C and 45 s of elongation at 72 C. Further elongation was carried out for 7 min at 72 C. In-Situ Hybridization
Serial sections from the paraffin wax embedded tissues were prepared on VECTABONDÔ slides (Vector Laboratories, Burlingame, USA) and were dried overnight at 55 C. Peripheral blood smears were prepared using samples collected from the caudal vein. Smears were made on VECTABONDÔ slides or clean glass slides and dried at room temperature (RT). Thereafter, slides were rinsed with DW after fixation with 10% neutral buffered formalin (2 h for ISH) or with formalinealcohol solution (5 min for PAS staining). For ISH, the paraffin wax was removed from sections by incubation in xylene (5 min) and then 100% ethanol (5 min). Slides were then air-dried in a fume cabinet for 5 min, incubated with 10 mg/ml proteinase K in Tris buffer (TB, 0.1 M, pH 8.0) for 30 min at 37 C in a humid chamber, and then washed
Megalocytivirus Infection of Rock Bream
in TB for 3 min. The slides were further dehydrated in 95% ethanol for 1 min, followed by 100% ethanol for 1 min, and then air-dried for 5 min in a fume cabinet. A hybridization mixture was prepared by adding 1 part hybridization buffer (50% formamide, 10% dextran sulphate, 4 SSC, 250 mg/ml yeast tRNA, 1 Denhart’s solution) to 1 part probe, with a final concentration of at least 50 ng/ml of probe. The target virus DNA was denatured by incubating the slides for 5 min at 95 C and rapidly cooling them. The slides were then hybridized overnight at 42 C, and the excess probe was subsequently removed with washing buffer (DIG Wash and Block Buffer SetÔ, Roche) at 40 C for 10 min. Tissue sections were then blocked in the blocking buffer for 30 min at RT prior to antibody binding. Specific probe binding within the tissue sections was visualized using a sheep anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche). The conjugated antibody was diluted 1 in 50 with blocking buffer and was incubated with the sections for 1 h at RT in a darkened humid chamber. The slides were washed three times with washing buffer over a period of 30 min and subsequently incubated with the detection buffer (DIG Wash and Block Buffer Set, Roche) for 2 min before the substrate was added. BCIP/NBT substrate was prepared as recommended by the manufacturer (Roche) and incubated with the sections for 1 h in a darkened humid chamber. Excess substrate was removed by three washes of 2 min each with 100 mM TriseHCl buffer (pH 9.5). The slides were air-dried and mounted under buffered glycerol for light microscopical examination. Bismarck brown Y counterstain was used in some cases.
Results The major findings affecting each of the tissues examined are reported below.
65
virus particles within the cytoplasm of degenerate cells within the central vein (Fig. 5A). Spleen
Numerous enlarged and basophilic cells were noted within the spleen in HE- and PAS-stained sections. Most of these cells were shown by ISH to contain viral DNA (Fig. 2B) and were scattered throughout the splenic tissue. Some labelled cells were located in the centre of ellipsoids. In certain individuals, many ISH-positive cells were located in the centre of the splenic cord. TEM revealed the presence of cytoplasmic virus particles within many abnormal leucocytes (Fig. 5B). Kidney
Numerous enlarged and basophilic cells or cells with cloudy cytoplasm were observed in the renal interstitial tissue in HE- and PAS-stained sections. The morphology of these cells was similar to those found in the spleen and liver. The majority of these cells correlated with those shown to contain viral DNA by ISH (Fig. 2C). Heart
There was no evidence of inflammatory or degenerative change within the myocardium (Fig. 2D) of the fish examined. However, leucocytes within this tissue were shown to contain viral DNA by ISH. Intestine
No abnormal cells were detected within HE- and PAS-stained sections of the intestine; however, numerous cells within the lamina propria and submucosa contained viral DNA as demonstrated by ISH. Only sparse labelled cells were identified within the serosa (Fig. 3A). Gills
Liver
Numerous enlarged and degenerate cells were observed in the HE- and PAS-stained sections, but these cells did not stain positively by PAS. These enlarged and degenerate cells corresponded with those identified by ISH and were located around or within the portal veins, central veins and sinusoids. This localization suggested that the cells were infiltrating leucocytes. Additionally, cells expressing viral DNA were present around the pancreatic tissue that may normally be found within the liver of fish (Fig. 2A). Ultrastructural examination revealed the presence of
No histological abnormality was detected on examination of HE- and PAS-stained sections; however, numerous cells within the central connective tissue of the gill filaments and the gill arches were shown to contain viral DNA by ISH (Fig. 3B). Some positively labelled cells were also observed within the tips of secondary gill filaments and immediately beneath the epithelium. Brain
There was no histological abnormality of brain tissue but cells containing viral DNA were identified by ISH
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Fig. 2. Serial sections stained by PAS (left panel) and subject to ISH with Bismarck brown Y counterstaining (right panel). (A) Section of liver indicating the portal vein (p), central vein (c) and bile canaliculi (b). Arrows indicate the position of swollen and basophilic cells that are unlabelled by ISH. 200. (B) Section of spleen indicating the aorta (a). Arrows indicate the position of swollen cells that are unlabelled by ISH. 400. (C) Section of kidney indicating the position of the glomerulus (g), renal tubules (r) and aorta (a). Arrows indicate the position of swollen cells that are unlabelled by ISH. 200. (D) Section of heart indicating the position of ventricular epicardium (e), myocardium (m) and an arteriole (a). These tissues have normal histological structure, but individual cells positively labelled by ISH are clearly seen associated with all three regions. 200.
Megalocytivirus Infection of Rock Bream
67
Fig. 3. Sections of (A) intestine, (B) gill and (C) brain after ISH with Bismarck brown Y counterstain. Arrows indicate the position of cells containing viral DNA. 200.
within the dura mater and arachnoid membrane in the outer layer of the cerebrum (Fig. 3C). Blood
Two types of leucocyte were observed within the PASstained blood smears. The first was characterized by having PAS-positive cytoplasm and the second by having basophilic cytoplasm. The diameter of both cell types was within the range 5e10 mm (Fig. 4B). Cells of similar diameter were shown to contain viral DNA by ISH (Fig. 4A). TEM demonstrated the presence of viral particles within the plasma (Fig. 5C) and the cytoplasm of some leucocytes. These infected leucocytes also contained cytoplasmic Russell bodies, mitochondria and abundant ribosomes.
Discussion It has been previously suggested that systemic iridoviruses of fish are mesotheliotropic and therefore comparable with LCDV (Gibson-Kueh et al., 2003). However, the findings of previous investigations and the present study would suggest that the pathogenesis of megalocytivirus infection is distinct from that of infection by LCDV. Heavily infected moribund fish display abnormal swimming behaviour and have petechial haemorrhages of the skin and viscera. Swol-
len and degenerate cells with morphology consistent with leucocytes are observed in the liver, spleen, kidney and other organs (Inouye et al., 1992; He et al., 2000, 2002; Jung and Oh, 2000; Wang et al., 2003). The results of the present study have shown that these leucocytes contain megalocytivirus DNA and virions and that the majority of such infected leucocytes are found within haemopoietic tissue of the spleen, kidney and liver. The morphology of the virus-infected cells within the viscera was similar to that of leucocytes within blood vessels, consistent with these cells having migrated into these tissues from the circulation. The size and shape of the infected leucocytes within the blood suggest that these cells are monocytes. This interpretation is compatible with the observation that the activity of macrophages is impaired by iridovirus infection (Siwicki et al., 2001) and the suggestion by Chao et al. (2004) that the enlarged cells in iridovirus-infected fish are monocytes. However, as plasma cells may also migrate into connective tissue and stain by PAS, it may also be possible that some of the virusinfected leucocytes that were observed in the tissues of these fish may have been of this lineage. Monocytes and eosinophilic granulocytes are abundant in the peripheral blood of normal bream (Ikeda et al., 1986); however, in the virus-infected
Fig. 4. Blood smear. (A) The arrowed cells are leucocytes positive for viral DNA expression on ISH. (B) Some leucocytes also stain positively with PAS (arrows), but other leucocytes are PAS negative (arrowheads). 400.
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Fig. 5. Ultrastructural examination of cells from RBIV infected Rock Bream. (A) Liver showing free virus particles within a sinusoid (arrowheads). The positions of a hepatocyte (H), reticulin fibre (R), the space of Disse (arrow) and an endothelial cell (E) are indicated. (B) Spleen showing a single swollen and degenerate leucocyte containing cytoplasmic virus particles (arrowheads). (C) Blood smear showing free virus particles (arrowheads). The position of adjacent leucocytes (L) and erythrocytes (R) is indicated.
fish the number of abnormal basophilic leucocytes was increased (data not shown). These basophilic leucocytes did not appear to be consistently labelled by ISH. The origin of the basophilic leucocytes has not been determined but it is likely that they originate from the thymus and/or kidney (Iwama, 1996). Iridovirus infection of monocytes or plasma cells might result in the dysfunction of haemopoietic organs (spleen and kidney) and induce the production of abnormal cells such as the enlarged basophilic cells observed in these fish. The results of the present study have shown that RBIV virions are present in the blood and that viral DNA and complete virions are contained within the cytoplasm of circulating leucocytes. Furthermore, many ISH-positive cells were observed within the connective tissue of various organs. These findings suggest that virus-infected cells migrate into the connective tissues of various organs from the blood. Jung et al. (1997) named these enlarged cells in iridovirusinfected fish as ‘inclusion body bearing cells’ (IBCs), however we have shown that not all of these enlarged cells contain virus DNA. Moreover, some cells positively labelled by ISH were of normal size and staining characteristics, suggesting that small cells can also contain megalocytivirus DNA. Rock Bream are highly susceptible to megalocytivirus infection. It is therefore of note that there is no significant difference in the number of tissue cells containing megalocytivirus DNA when infected Rock Bream are compared with species that are less sensitive to iridovirus, such as Flounder and Sea Bass (data not shown). This would suggest that megalocytivirus can infect different fish species with equally
efficacy, but induce different levels of pathology such as dysfunction of haemopoietic organs and lesions of blood vessels and connective tissues. The precise classification of granular leucocytes and lymphoid cells in fish has been poorly described to date, however the morphological and biochemical characteristics of lymphoid cells in fish are similar to those of higher vertebrates (Ainsworth, 1992). Therefore, the application of markers for myeloid or lymphoid cells that have been used to define these populations in higher vertebrates would be very useful for future studies aimed at defining the nature of the fish leucocytes that are susceptible cells to megalocytivirus infection.
Acknowledgment This work was supported by the Korea Research Foundation Grant KRF-2004-037-F00018.
References Ainsworth AJ (1992) Fish granulocytes: morphology, distribution, and function. Annual Review of Fish Diseases, 2, 123e148. Chao CB, Chen CY, Lai YY, Lin CS, Huang HT (2004) Histological, ultrastructural, and in situ hybridization study on enlarged cells in grouper Epinephelus hybrid infected by grouper iridovirus in Taiwan (TGIV). Diseases of Aquatic Organisms, 58, 127e142. Chinchar VG, Essbauer S, He JG, Hyatt A, Miyazaki T et al. (2005) In: Iridoviridae. Virus Taxonomy, VIIIth Report of the International Committee on Taxonomy of Viruses, CM Fauqet, MA Mayo, J Maniloff, U Desselberger and LA Ball, Eds, Academic Press, London, pp. 163e175.
Megalocytivirus Infection of Rock Bream
Do JW, Cha SJ, Kim JS, An EJ, Lee NS et al. (2005) Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders, Paralichthys olivaceus, in Korea. Diseases of Aquatic Organisms, 64, 193e200. Do JW, Moon CH, Kim HJ, Ko MS, Kim SB et al. (2004) Complete genomic DNA sequence of Rock Bream iridovirus. Virology, 325, 351e363. Gibson-Kueh S, Netto P, Ngoh-Lim GH, Chang SF, Ho LL et al. (2003) The pathology of systemic iridoviral disease in Fish. Journal of Comparative Pathology, 129, 111e119. He JG, Wang SP, Zeng K, Huang ZJ, Chan SM (2000) Systemic disease caused by an iridovirus-like agent in cultured mandarin fish, Siniperca chuatsi (Basilewsky) in China. Journal of Fish Diseases, 23, 219e222. He JG, Zeng K, Weng SP, Chan SM (2002) Experimental transmission, pathogenicity and physicalechemical properties of infectious spleen and kidney necrosis virus (ISKNV). Aquaculture, 204, 11e24. Hedrick RP, Groff JM, Mcdowell T, Wingfield WH (1990) An iridovirus infection of the integument of the white sturgeon, Acipenser transmontanus. Diseases of Aquatic Organisms, 8, 29e44. Inouye K, Yamano K, Maeno Y, Nakajima K, Matsuoka M et al. (1992) Iridovirus infection of cultured Red Sea Bream, Pagrus major. Fish Pathology, 27, 19e27. Ikeda I, Osaki H, Sesaki K (1986) Blood Atlas of Fishes. Midori Shobo Co., Japan, pp. 244e259. Iwama G (1996) The Immune System. Organism, Pathogen, and Environment. Academic Press Inc., California, pp. 1e62. Jung SJ, Oh MJ (2000) Iridovirus-like infection associated with high mortalities of striped Beakperch, Oplegnathus fasciatus (Temminck and Schlegel), in southern coastal areas of the Korean peninsula. Journal of Fish Diseases, 23, 223e226.
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Jung SJ, Miyazaki T, Miyata M, Danayadol Y, Tanaka S (1997) Pathogenicity of iridovirus from Japan and Thailand for the Red Sea Bream Pagrus major. Fisheries Science, 63, 735e740. Langdon JS, Humphrey JD, Williams LM (1988) Outbreaks of an EHNV-like iridovirus in cultured rainbow trout, Salmo gairdneri, in Australia. Journal of Fish Diseases, 11, 93e96. Lu L, Zhou SY, Chen C, Weng SP, Chan SM et al. (2005) Complete genome sequence analysis of an iridovirus isolated from the Orange-Spotted Grouper, Epinephelus coioides. Virology, 339, 81e100. Siwicki A, Pozet F, Morand M, Terech-Majewska E, Bernard D (2001) Pathogenesis of iridovirus: in vitro influence on macrophage activity and cytokine-like protein production in fish. Acta Veterinaria Brno, 70, 451e456. Sudthongkong C, Miyata M, Miyazaki T (2002) Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries. Archives of Virology, 147, 2089e2109. Tidona CA, Darai G (1997) The complete DNA sequence of lymphocystis disease virus. Virology, 230, 207e216. Wang CS, Shin HH, Ku CC, Chen SN (2003) Studies on epizootic iridovirus infection among Red sea Bream, Pagrus major (Temminck and Schlegal), cultured in Taiwan. Journal of Fish Diseases, 26, 127e133. Williams T (1996) The iridoviruses. Advances in Virus Research, 46, 345e412.
August 25th, 2008 ½ Received, Accepted, March 29th, 2009
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
Characterization of Virus Distribution in Rock Bream (Oplegnathus fasciatus; Temminck and Schlegel) Infected with Megalocytivirus N.-S. Lee*, J. W. Do*, J. W. Park† and Y. C. Kim‡ * Pathology Division, National Fisheries Research & Development Institute, Busan, † Department of Biological Sciences, University of Ulsan and ‡ Southern Inland Fisheries Research Institute, Jin-hae, Kyungnam, South Korea
Summary The distribution of virus-infected cells in the organs of Rock Bream naturally infected with megalocytivirus is reported. Examination of sections of liver, spleen and kidney stained by haematoxylin and eosin (HE) and periodic acid Schiff (PAS) revealed the presence of swollen and degenerate cells having morphology consistent with leucocytes. Many of these cells were shown to contain viral DNA by in-situ hybridization (ISH). Cells containing viral DNA were also found in the connective tissue of other organs in which there was no prominent infiltrate of degenerate leucocytes. Viral DNA was also found in the cytoplasm of leucocytes in blood smears. Transmission electron microscopy (TEM) confirmed the presence of viral particles in the cells within tissue and free within blood. The tissue distribution of virus in this infection is suggested to reflect the infiltration of virusinfected leucocytes. Ó 2009 Elsevier Ltd. All rights reserved. Keywords: in-situ hybridization; leucocyte; megalocytivirus; Rock Bream iridovirus
Introduction Iridoviruses are large cytoplasmic DNA viruses with an icosahedral morphology (Williams, 1996) and the family Iridoviridae consists of five genera including Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus and Megalocytivirus (Chinchar et al., 2005). Iridoviral infections are one of the most prevalent viral diseases of cultured freshwater and marine fish species. White Sturgeon iridovirus (WSIV) (Hedrick et al., 1990), lymphocystis disease virus (LCDV) (Tidona and Darai, 1997), epizootic haematopoietic necrosis virus (EHNV) (Langdon et al., 1988), Orange-Spotted Grouper iridovirus (GIV) (Lu et al., 2005), Sea Bass iridovirus (SBIV) (Sudthongkong et al., 2002), infectious spleen and kidney necrosis iridovirus (ISKNV) (He et al., 2000) and Red Sea Bream iridovirus (RSIV) (Inouye et al., 1992) are among the best known iridoviral infections. According to the International Committee on Taxonomy of Viruses (ICTV),
fish iridoviruses are members of the genera Lymphocystivirus, Ranavirus or Megalocytivirus. Since 1998, mass mortality has been observed in cage-cultured Rock Bream off the southern coast of the Korean peninsula, and megalocytivirus has been shown to be the causative agent (Do et al., 2004, 2005). In 2005, an iridoviral epizootic occurred amongst Rock Bream cultured in the Tongyeong area of South Korea, and megalocytivirus was detected in 34% of the moribund fish (unpublished data). The pathogenesis of megalocytivirus infection in fish has been incompletely characterized. Accordingly, the aims of the present study were to describe the histopathological changes in the tissues of naturally infected fish and to determine the distribution of virus-infected cells within these tissues.
Materials and Methods Fish
Correspondence to: Y. C. Kim (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2009.03.008
During the summer of 2005 there were epizootics of megalocytivirus infection amongst Rock Bream Ó 2009 Elsevier Ltd. All rights reserved.
64
N.-S. Lee et al.
cultured in the Tongyeong area of South Korea. The moribund fish developed severe anaemia and petechial haemorrhages in gill filaments and intestines, and they showed abnormal swimming behaviour. Viscera and blood samples were collected from these moribund fish (of approximate body length 12 cm) and frozen at 80 C. Samples of viscera were also fixed in 10% neutral buffered formalin. Samples from 15 fish with confirmed megalocytivirus infection were selected for the present study. Confirmation of infection was by polymerase chain reaction (PCR) with primers specific for the gene encoding Rock Bream iridovirus major capsid protein (RBIVMCP). The primer sequences were forward 50 CGTGATGGAGGGGATCTTAA-30 and reverse 50 -GAAAAACGAGGCCGATCATA-30 . Samples of spleen and kidney were used for this confirmatory testing. Histology and Transmission Electron Microscopy
Fixed tissue samples were processed routinely and embedded in paraffin wax. Sections (4e5 mm) were stained with haematoxylin and eosin (HE) or periodic acid-Schiff (PAS). For transmission electron microscopy (TEM), tissues and blood cells were fixed overnight at 4 C in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sections were stained with lead citrate and uranyl acetate. DNA Extraction and Preparation of Probes
Digoxigenin (DIG)-labelled probes for in-situ hybridization (ISH) were prepared by PCR using RBIV DNA extracted from infected Rock Bream collected in 2003 (Do et al., 2004) as a template. Viral DNA was extracted with the High PureÔ PCR Template Preparation Kit (Roche, Mannheim, Germany) and the AccuPrepÔ Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea), following the method described by Do et al. (2004). Primers for PCR were designed from nucleotide sequences in the GenBank database. Primer sequences for amplification of the gene encoding viral ATPase (GenBank accession number AY532614) were RBIV-AP forward 50 -GTAGTGATATCGGGCTCCGA-30 and RBIV-AP reverse 50 -CCGTTCTTGAACAGGTCCAT-30 . Primer sequences for amplification of the gene encoding the major capsid protein (MCP) were the same as those described above for confirmation of megalocytivirus infection. DIG-labelled probes were prepared by multiplex PCR using these two primer sets (Fig. 1). Briefly, a mixture was prepared in one PCR tube containing
Fig. 1. Digoxigenin (DIG) labelled PCR products. Product (c) was used in the present study. M, marker; a, ATPase; b, MCP; c, multiplex PCR of (a) and (b).
10 ml of 10 PCR buffer that contained 15 mM MgCl2 (TAKARA, Tokyo, Japan), 2 ml each of forward and reverse primers (10 pmol/ml), 1 ml of Taq polymerase (5 U/ml, TAKARA), 10 ml of PCRlabelled mixture or 10 dNTPs solution (for non-DIG-labelled probes) (Roche), 2 ml of either DNA template or distilled water (DW, as a negative control), and DW to a volume of 100 ml. After an initial denaturation for 5 min at 94 C, PCR was carried out for 35 cycles. Each cycle consisted of 30 s of denaturation at 94 C followed by 30 s of annealing at 57 C and 45 s of elongation at 72 C. Further elongation was carried out for 7 min at 72 C. In-Situ Hybridization
Serial sections from the paraffin wax embedded tissues were prepared on VECTABONDÔ slides (Vector Laboratories, Burlingame, USA) and were dried overnight at 55 C. Peripheral blood smears were prepared using samples collected from the caudal vein. Smears were made on VECTABONDÔ slides or clean glass slides and dried at room temperature (RT). Thereafter, slides were rinsed with DW after fixation with 10% neutral buffered formalin (2 h for ISH) or with formalinealcohol solution (5 min for PAS staining). For ISH, the paraffin wax was removed from sections by incubation in xylene (5 min) and then 100% ethanol (5 min). Slides were then air-dried in a fume cabinet for 5 min, incubated with 10 mg/ml proteinase K in Tris buffer (TB, 0.1 M, pH 8.0) for 30 min at 37 C in a humid chamber, and then washed
Megalocytivirus Infection of Rock Bream
in TB for 3 min. The slides were further dehydrated in 95% ethanol for 1 min, followed by 100% ethanol for 1 min, and then air-dried for 5 min in a fume cabinet. A hybridization mixture was prepared by adding 1 part hybridization buffer (50% formamide, 10% dextran sulphate, 4 SSC, 250 mg/ml yeast tRNA, 1 Denhart’s solution) to 1 part probe, with a final concentration of at least 50 ng/ml of probe. The target virus DNA was denatured by incubating the slides for 5 min at 95 C and rapidly cooling them. The slides were then hybridized overnight at 42 C, and the excess probe was subsequently removed with washing buffer (DIG Wash and Block Buffer SetÔ, Roche) at 40 C for 10 min. Tissue sections were then blocked in the blocking buffer for 30 min at RT prior to antibody binding. Specific probe binding within the tissue sections was visualized using a sheep anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche). The conjugated antibody was diluted 1 in 50 with blocking buffer and was incubated with the sections for 1 h at RT in a darkened humid chamber. The slides were washed three times with washing buffer over a period of 30 min and subsequently incubated with the detection buffer (DIG Wash and Block Buffer Set, Roche) for 2 min before the substrate was added. BCIP/NBT substrate was prepared as recommended by the manufacturer (Roche) and incubated with the sections for 1 h in a darkened humid chamber. Excess substrate was removed by three washes of 2 min each with 100 mM TriseHCl buffer (pH 9.5). The slides were air-dried and mounted under buffered glycerol for light microscopical examination. Bismarck brown Y counterstain was used in some cases.
Results The major findings affecting each of the tissues examined are reported below.
65
virus particles within the cytoplasm of degenerate cells within the central vein (Fig. 5A). Spleen
Numerous enlarged and basophilic cells were noted within the spleen in HE- and PAS-stained sections. Most of these cells were shown by ISH to contain viral DNA (Fig. 2B) and were scattered throughout the splenic tissue. Some labelled cells were located in the centre of ellipsoids. In certain individuals, many ISH-positive cells were located in the centre of the splenic cord. TEM revealed the presence of cytoplasmic virus particles within many abnormal leucocytes (Fig. 5B). Kidney
Numerous enlarged and basophilic cells or cells with cloudy cytoplasm were observed in the renal interstitial tissue in HE- and PAS-stained sections. The morphology of these cells was similar to those found in the spleen and liver. The majority of these cells correlated with those shown to contain viral DNA by ISH (Fig. 2C). Heart
There was no evidence of inflammatory or degenerative change within the myocardium (Fig. 2D) of the fish examined. However, leucocytes within this tissue were shown to contain viral DNA by ISH. Intestine
No abnormal cells were detected within HE- and PAS-stained sections of the intestine; however, numerous cells within the lamina propria and submucosa contained viral DNA as demonstrated by ISH. Only sparse labelled cells were identified within the serosa (Fig. 3A). Gills
Liver
Numerous enlarged and degenerate cells were observed in the HE- and PAS-stained sections, but these cells did not stain positively by PAS. These enlarged and degenerate cells corresponded with those identified by ISH and were located around or within the portal veins, central veins and sinusoids. This localization suggested that the cells were infiltrating leucocytes. Additionally, cells expressing viral DNA were present around the pancreatic tissue that may normally be found within the liver of fish (Fig. 2A). Ultrastructural examination revealed the presence of
No histological abnormality was detected on examination of HE- and PAS-stained sections; however, numerous cells within the central connective tissue of the gill filaments and the gill arches were shown to contain viral DNA by ISH (Fig. 3B). Some positively labelled cells were also observed within the tips of secondary gill filaments and immediately beneath the epithelium. Brain
There was no histological abnormality of brain tissue but cells containing viral DNA were identified by ISH
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Fig. 2. Serial sections stained by PAS (left panel) and subject to ISH with Bismarck brown Y counterstaining (right panel). (A) Section of liver indicating the portal vein (p), central vein (c) and bile canaliculi (b). Arrows indicate the position of swollen and basophilic cells that are unlabelled by ISH. 200. (B) Section of spleen indicating the aorta (a). Arrows indicate the position of swollen cells that are unlabelled by ISH. 400. (C) Section of kidney indicating the position of the glomerulus (g), renal tubules (r) and aorta (a). Arrows indicate the position of swollen cells that are unlabelled by ISH. 200. (D) Section of heart indicating the position of ventricular epicardium (e), myocardium (m) and an arteriole (a). These tissues have normal histological structure, but individual cells positively labelled by ISH are clearly seen associated with all three regions. 200.
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Fig. 3. Sections of (A) intestine, (B) gill and (C) brain after ISH with Bismarck brown Y counterstain. Arrows indicate the position of cells containing viral DNA. 200.
within the dura mater and arachnoid membrane in the outer layer of the cerebrum (Fig. 3C). Blood
Two types of leucocyte were observed within the PASstained blood smears. The first was characterized by having PAS-positive cytoplasm and the second by having basophilic cytoplasm. The diameter of both cell types was within the range 5e10 mm (Fig. 4B). Cells of similar diameter were shown to contain viral DNA by ISH (Fig. 4A). TEM demonstrated the presence of viral particles within the plasma (Fig. 5C) and the cytoplasm of some leucocytes. These infected leucocytes also contained cytoplasmic Russell bodies, mitochondria and abundant ribosomes.
Discussion It has been previously suggested that systemic iridoviruses of fish are mesotheliotropic and therefore comparable with LCDV (Gibson-Kueh et al., 2003). However, the findings of previous investigations and the present study would suggest that the pathogenesis of megalocytivirus infection is distinct from that of infection by LCDV. Heavily infected moribund fish display abnormal swimming behaviour and have petechial haemorrhages of the skin and viscera. Swol-
len and degenerate cells with morphology consistent with leucocytes are observed in the liver, spleen, kidney and other organs (Inouye et al., 1992; He et al., 2000, 2002; Jung and Oh, 2000; Wang et al., 2003). The results of the present study have shown that these leucocytes contain megalocytivirus DNA and virions and that the majority of such infected leucocytes are found within haemopoietic tissue of the spleen, kidney and liver. The morphology of the virus-infected cells within the viscera was similar to that of leucocytes within blood vessels, consistent with these cells having migrated into these tissues from the circulation. The size and shape of the infected leucocytes within the blood suggest that these cells are monocytes. This interpretation is compatible with the observation that the activity of macrophages is impaired by iridovirus infection (Siwicki et al., 2001) and the suggestion by Chao et al. (2004) that the enlarged cells in iridovirus-infected fish are monocytes. However, as plasma cells may also migrate into connective tissue and stain by PAS, it may also be possible that some of the virusinfected leucocytes that were observed in the tissues of these fish may have been of this lineage. Monocytes and eosinophilic granulocytes are abundant in the peripheral blood of normal bream (Ikeda et al., 1986); however, in the virus-infected
Fig. 4. Blood smear. (A) The arrowed cells are leucocytes positive for viral DNA expression on ISH. (B) Some leucocytes also stain positively with PAS (arrows), but other leucocytes are PAS negative (arrowheads). 400.
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Fig. 5. Ultrastructural examination of cells from RBIV infected Rock Bream. (A) Liver showing free virus particles within a sinusoid (arrowheads). The positions of a hepatocyte (H), reticulin fibre (R), the space of Disse (arrow) and an endothelial cell (E) are indicated. (B) Spleen showing a single swollen and degenerate leucocyte containing cytoplasmic virus particles (arrowheads). (C) Blood smear showing free virus particles (arrowheads). The position of adjacent leucocytes (L) and erythrocytes (R) is indicated.
fish the number of abnormal basophilic leucocytes was increased (data not shown). These basophilic leucocytes did not appear to be consistently labelled by ISH. The origin of the basophilic leucocytes has not been determined but it is likely that they originate from the thymus and/or kidney (Iwama, 1996). Iridovirus infection of monocytes or plasma cells might result in the dysfunction of haemopoietic organs (spleen and kidney) and induce the production of abnormal cells such as the enlarged basophilic cells observed in these fish. The results of the present study have shown that RBIV virions are present in the blood and that viral DNA and complete virions are contained within the cytoplasm of circulating leucocytes. Furthermore, many ISH-positive cells were observed within the connective tissue of various organs. These findings suggest that virus-infected cells migrate into the connective tissues of various organs from the blood. Jung et al. (1997) named these enlarged cells in iridovirusinfected fish as ‘inclusion body bearing cells’ (IBCs), however we have shown that not all of these enlarged cells contain virus DNA. Moreover, some cells positively labelled by ISH were of normal size and staining characteristics, suggesting that small cells can also contain megalocytivirus DNA. Rock Bream are highly susceptible to megalocytivirus infection. It is therefore of note that there is no significant difference in the number of tissue cells containing megalocytivirus DNA when infected Rock Bream are compared with species that are less sensitive to iridovirus, such as Flounder and Sea Bass (data not shown). This would suggest that megalocytivirus can infect different fish species with equally
efficacy, but induce different levels of pathology such as dysfunction of haemopoietic organs and lesions of blood vessels and connective tissues. The precise classification of granular leucocytes and lymphoid cells in fish has been poorly described to date, however the morphological and biochemical characteristics of lymphoid cells in fish are similar to those of higher vertebrates (Ainsworth, 1992). Therefore, the application of markers for myeloid or lymphoid cells that have been used to define these populations in higher vertebrates would be very useful for future studies aimed at defining the nature of the fish leucocytes that are susceptible cells to megalocytivirus infection.
Acknowledgment This work was supported by the Korea Research Foundation Grant KRF-2004-037-F00018.
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August 25th, 2008 ½ Received, Accepted, March 29th, 2009