Tunic morphology and viral surveillance in diseased Korean ascidians: soft tunic syndrome in the edible ascidian,Halocynthia roretzi (Drasche), in aquaculture

‘Soft tunic syndrome’ causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non‐diseased specimens using chinook salmon embryo (CHSE‐214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.     

Comparative sensitivity of five fish cell lines to wild type infectious haematopoietic necrosis virus from two Oregon sources

Abstract. Five fish cell lines (CHSE-214, STE-137, RTG-2, EPC and FHM) were compared for sensitivity to infectious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infectious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Richardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polycation, polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells.     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.     

Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV)

A new continuous cell line (GF-1) was established and characterized. The GF-1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF-1 cells are fibroblast-like, together with some epithelioid cells. Spontaneous transformation of GF-1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF-1 cells 3–5 days post-infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV-infected GF-1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV-infected cells after CPE appeared. The experimental results indicated that GF-1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.     

山东沿海部分地区真鲷虹彩病毒病初步调查与分析

2008年11月～2010年11月,采集山东海域大菱鲆、石鲽、鲈鱼各20批,按照世界动物卫生组织推荐的PCR检测方法对真鲷虹彩病毒病(Red Sea Bream Iridoviral Disease,RSIVD)进行初步调查.结果显示,共检出4例RSIVD感染样品.以真鲷虹彩病毒(Red Sea Bream Iridovirus,RSIV)和传染性脾肾坏死病毒(Infectious Spleen and Kidney Necrosis Virus,ISKNV)主要衣壳蛋白基因为基础,设计简并引物,PCR扩增本次检出阳性样品的RSIV/ISKNV MCP基因.将MCP基因PCR扩增产物测序,提交GenBank,并以MCP基因为基础,对被检出的阳性样品进行虹彩病毒属系统分类,绘制进化树.由进化树得出,4例阳性病毒株均属于虹彩病毒科细胞肿大病毒属.     

Inhibition of infectious pancreatic necrosis virus infectivity by extracts of rainbow trout, Salmo gairdneri Richardson, tissue

Abstract. Extracts of healthy rainbow trout liver, kidney, spleen and whole fry inhibited plaque production of infectious pancreatic necrosis virus (IPNV) in cell cultures. The mode of inhibition is not known, although it appears not to be manifest at the cellular level, as pre-treatment of the cell cultures with tissue extracts did not inhibit plaque production. Any effect on the virus itself was not permanent as the inhibition could be mitigated by treatment of virus/extract mixtures with 1,1,2-trichloro-1,1,2-trifluoroethane. The inhibition may be caused by prevention or reduction of virus attachment to the cell surface or, alternatively, the tissue extract may cause aggregation of the virus and thereby reduce the number of available infectious units. The inhibitory effect is also lost by dilution of the extract, reinforcing the claim that adequate dilutions of fish extracts prior to attempted virus isolation are necessary, especially for the detection of carrier fish.     

Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UK

Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14-15 to 19-21 degrees C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 degrees C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at -70 degrees C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2.     

牙鲆免疫相关组织特征及抗体阳性细胞的免疫组化定位

应用抗牙鲆免疫球蛋白单克隆抗体(2D8、1H1)对牙鲆外周血系统、肾、脾、肝、胰、肠道组织中抗体阳性细胞进行了定位观察,并对其组织学特征进行了描述。两株单抗均能在外周血滴片和组织切片中成功地检测到抗体阳性细胞。外周血系统中的抗体阳性细胞主要为淋巴细胞,没有发现抗体阳性的巨噬细胞;牙鲆头肾中没有肾单位,肾小管、肾小球等主要存在于后肾中,脾脏和肾脏都含有巨噬细胞、粒细胞、淋巴细胞等免疫相关细胞,抗体阳性细胞存在方式也极为相似,成簇或单独分布于黑色素巨噬细胞中心和血管周围;牙鲆的胰组织镶嵌在肝上,形成肝胰脏,也参与免疫应答,抗体阳性细胞单个存在,分布于肝组织中,胰组织中没有发现抗体阳性细胞;肠道抗体阳性细胞主要存在于固有层中,有成簇存在现象,在上皮层也可见到单个存在的抗体阳性细胞。     

Establishment and characterization of a heart-derived cell line from goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 10^5.0, 10^4.5, and 10^5.0 TCID₅₀/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.     

Development of a multiplex RT-PCR assay for simultaneous detection of the major viruses that affect rainbow trout (Oncorhynchus mykiss)

The major viral diseases that affect rainbow trout (Oncorhynchus mykiss) are viral haemorrhagic septicaemia, infectious haematopoietic necrosis, infectious pancreatic necrosis and sleeping disease. In the presented study, we developed a multiplex RT-PCR (mRT-PCR) assay for the simultaneous detection of these four rainbow trout viruses in a single assay. The choice of primers was carried out based on the expected size of the fragments, the temperature and time required for the amplification, and the specificity for the target sequence. Firstly, the method was optimised using reference strains of viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and sleeping disease virus (SDV) cultivated with permissive cell culture lines; subsequently, the method was used for the identification of these viral infections in rainbow trout samples. Twenty-two samples of rainbow trout, clinically suspected of having viruses, were analysed by the developed method to detect the presence of the four viruses, by directly analysing the animal tissues. The mRT-PCR method was able to efficiently detect the viral RNA in infected cell culture supernatants and in tissue samples, highlighting the presence of single infections as well as co-infections in rainbow trout samples. VHSV/SDV and IHNV/SDV co-infections were demonstrated for the first time in rainbow trout. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Studies of the ultrastructure and morphogenesis of fish pathogenic viruses grown in cell culture

A birnavirus (infectious pancreatic necrosis virus, IPNV), three rhabdoviruses (viral haemorrhagic septicaemia virus, VHSV; infectious haematopoietic necrosis virus, IHNV; and spring viraemia of carp virus, SVCV) and an iridovirus (isolate from a sheatfish) were investigated with regard to their morphogenetic interactions with cells in culture. In cells infected with birnavirus, a granular viromatrix, single virions randomly distributed in the cytoplasm, viral particles aggregated in pseudocrystals and cytoplasmic tubuli similar in diameter to that of the virus were found. Rhabdoviruses entered the cells by viropexis and replicated within the cytoplasm. Maturation occurred predominantly at the cell membrane and sporadically at membranes of the Golgi cisternae. Inclusion bodies were found partially consisting of viral nucleocapsids. After budding, new virions were found adsorbed to the cell membrane. Viral haemorrhagic septicaemia virus, known to exhibit an atypical shape because of preparative procedures, could be identified by immunostaining using two monoclonal antibodies directed against G- and N-proteins and colloidal gold. Iridoviruses entered the cells by viropexis. Viral particles were found in coated vesicles. Subsequently, vesicles without a clathrin coat were detected. Replication occurred within prominent cytoplasmic inclusion bodies. Isometric viral nucleocapsids were transported in an unknown manner to the cell membrane and matured by budding.     

Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout,Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested‐PCR study

This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E‐stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin‐fixed, paraffin‐embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.     

抗迟缓爱德华菌单克隆抗体的应用

检测了抗迟缓爱德华菌单抗对牙鲆的保护性。从１０株自制单抗中筛选出一株对牙鲆具有较强保护性的单抗３Ｆ７。与仅以该菌感染的对照组相比,该抗体可显著提高牙鲆感染迟缓爱德华菌后的存活率。在以每条鱼０．１ｍＬ（１０＾９ＣＦＵ）菌量做攻击保护时,其保护率可达８０％。另外,利用所制单克隆抗体以免疫组织组织化学ＳＡＢＣ法检查了迟缓爱德华菌经腹腔感染牙鲆鱼后细菌的侵染途径,结果显示牙鲆对该菌易感的器官为肝、肾、脾等     

Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

Atlantic cod, Gadus morhua , averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2^−ΔΔCt method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.     

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV‐3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin‐4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.     

军曹鱼淋巴囊肿的病理学研究

应用病理组织学和电镜方法,对患疑似淋巴囊肿病的军曹鱼(Rachycentron canadum)的各器官进行观察.结果表明,患病军曹鱼的皮肤囊肿组织由一些淋巴囊肿细胞集合体组成,这些囊肿细胞排列紧密,直径为10～150 μm,细胞呈圆形、锥形不规则状;细胞外有一层厚的囊膜;细胞质内散布有大量的嗜碱性包涵体,且多数集中在细胞的边缘部分;电镜观察到囊肿细胞质中有大量二十面体的病毒粒子,病毒颗粒直径220 nm.据此确认该病为病毒性淋巴囊肿病.其他器官主要组织病理学变化有:在心脏、肝脏、脾脏和头肾中也存在囊肿细胞,心肌纤维水肿;肾间质淋巴细胞增生,巨噬中心出现,肾小管上皮细胞变性和坏死;脾淋巴细胞增生,脾髓质出血;肝脂肪变性;鳃上皮肿胀.根据观察结果可以认为,该病毒不仅损伤鱼的皮肤,致使病鱼外观异样而严重影响其商品价值,而且对鱼的内脏和免疫器官也造成严重的致命损伤.