Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain

Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations.     

Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel)

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.     

Partial susceptibility of the SSN-1 fish cell line to a crustacean virus: a defective replication study

This study evaluated the possible use of the fish SSN-1 cell line to investigate the development of Macrobrachium rosenbergii nodavirus (MrNV). Cells were incubated with viral particles and cytopathic effects were observed. De novo synthesis of viral capsid proteins was shown by immuno-fluorescence labelling and a sandwich ELISA test. Viral genomic replication was demonstrated by RT-PCR using primers specific to RNA-1 as well as by quantitative RT-PCR (RT-qPCR). Using electron microscopy, only a few empty particles were observed and attempts to isolate complete infectious particles or to re-infect healthy cells (second passage) were unsuccessful. As complete viral particles were rarely observed, it appeared that defaults in MrNV virogenesis might arise resulting in the formation of scarce and non-infectious particles. SSN-1 cells were found to be partially permissive to MrNV infection that induced cell lysis, but key elements for viral infection were lacking such as regulatory factors for gene replication or post-translational modifications.     

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli – expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.     

Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product.     

逆转录聚合酶链式反应(RT-PCR)检测5种养殖石斑鱼的神经坏死病毒

神经坏死病毒(Nervous necrosis virus)是导致多种海水鱼类神经性病害的致病原.发病及死亡的石斑鱼除了表现神经异常症状外,无明显的临床病症,体表及内脏组织也未发现明显病变及寄生虫感染.2003年4～8月,应用逆转录聚合酶链式反应(RT-PCR)技术从福建南部人工养殖的5种石斑鱼即紫石斑鱼(Epinephelus lanceolatus)、马拉巴石斑鱼(E. malabaricus)、青石斑鱼(E. awoara)、赤点石斑鱼(E. akaara)和云纹石斑鱼(E. moara)中检出5个神经坏死病毒分离株.检测了76份石斑鱼样品,这些石斑鱼NNV病毒的平均感染率约为90%.对这些病毒的RT-PCR产物421 bp核酸进行了测序和序列分析,其相同的序列超过99%.将这些序列与GenBank的石斑鱼(Epinephelus spp.)神经坏死病毒相关基因序列作比较,同源性在97%以上.对神经坏死病毒在石斑鱼体内的分布也进行了分析,在脑和眼组织的检出率最高,部分病鱼的肝、脾和肾组织也能检出病毒.结合流行病学特征,可确认神经坏死病毒为该传染病的主要致病原.RT-PCR方法是检测NNV等病原的一种理想的诊断方法.     

棕点石斑鱼Piscidin样肽基因的克隆及其成熟肽的原核表达

以简并引物通过RT-PCR从重要经济海水鱼类棕点石斑鱼Epinephelus fuscoguttatus白细胞中扩增出一条388bp具有完整编码框的cDNA序列。Blast分析初步显示,该序列属于鱼类抗菌肽Piscidin家族的一员,将其命名为棕点石斑鱼Piscidin样肽(GU592793),推导氨基酸序列具有界定鱼类Piscidin的以下共同特征:(1)预测的信号肽序列与其他Piscidin的同源性较高,在60%~95%之间;(2)推测的成熟肽N端6个氨基酸残基富含Phe、Ile及His;(3)成熟肽第8个和第13个氨基酸位点均为Gly。NJ进化树分析表明,该cDNA推导的氨基酸序列与赤点石斑鱼Epinephelusakaarapiscidin-like peptide(ACE78290)、斜带石斑鱼Epinephelus coioidesPiscidin-like peptide(ACE78291)以高达81%的支持率聚为一支。也表明本研究成功克隆到棕点石斑鱼首条PiscidincDNA序列。推测的成熟肽为两亲性阳离子肽,等电点12.48,Schiffer-Edmunson预测其二级结构呈α螺旋构型。将...     

Development and characterization of five novel cell lines from snubnose pompano,Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies

Five novel permanent cell lines have been established from gill, heart, kidney, eye and fin of snubnose pompano, Trachinotus blochii. They were designated as snubnose pompano gill (SPG), snubnose pompano heart (SPH), snubnose pompano kidney (SPK), snubnose pompano eye (SPE) and snubnose pompano fin (SPF), respectively. All these cell lines were characterized and cryopreserved successfully at different passage levels. Cell lines were passaged every alternate day; SPG, SPH, SPK, SPE and SPF cell lines attained passage levels of 68, 74, 82, 79 and 106, respectively, since the initiation of their development in 2019. The cell lines grew well in Leibovitz's 15 medium containing 15% foetal bovine serum at 28°C. Immunophenotyping of the cell lines revealed the presence of fibronectin and pancytokeratin. No mycoplasma contamination was found. The transfection study revealed the gene expression efficiency of these cell lines by expressing the green fluorescent protein (GFP). The authentication on origin of cell lines from T. blochii was confirmed by amplification of species-specific mitochondrial cytochrome oxidase I gene. The results showed the susceptibility of these cell lines to fish nodavirus (FNV) and tilapia lake virus (TiLV) and resistance to cyprinid herpesvirus 2 (CyHV-2). The FNV infection in the cell lines was confirmed by RT-PCR, Western blot, ELISA and immunocytochemistry, while TiLV infection was confirmed by RT-PCR assay. These results revealed that these cell lines are suitable for virological and foreign gene expression studies.     

Development and characterization of a new marine fish cell line from turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>)

A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n = 44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.     

鱼类诺达病毒及其所导致的疾病

In recent years, piscine nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish resulting in high mortality in aquaculture worldwide. Affected fish exhibit a range of neurological signs, such as erratic swimming behaviour with the associated microscopic lesions of necrosis and vacuolation of the central nervous tissues and retina. Numerous roundshaped, unenveloped and 25-30 nm in diameter virus particles were found in the cytoplasm of affected retinal and nerve cells. Nodaviruses have a bipartite genome of positivesense RNA,with RNA1 encoding the RNAdependent RNA polymerase and RNA2 encoding the capsid protein. Both RNA are capped, but not polyadenylated. The family Nodaviridae comprises two genera: Alphanodavirus and Betanodavirus, members of which primarily infect insects and fish, respectively. Therefore, betanodavirus is also named piscine nodavirus. At present, piscine nodaviruses are divided into four genotypes based on partial sequences of the coat protein gene. ELISA and RT-PCR amplification have been developed as specific diagnostic methods for the d etection of the virus. Antibodies to striped jack (Pseudocaranx dentex) nervous necrosis (SJNNV) were found in 65% of plasma samples collected from wild and domestic brood stocks of striped jack, suggesting that the virus is very prevalent. Viral antigens were detected in eggs, larvae, and ovaries of hatcheryreared and wild spawner fish, suggesting both horizontal and vertical modes of transmission of the virus. Selection of nodavirusfree spawners using ELISA for detection of antigens and RT-PCR techniques have successfully reduced incidences of the virus infections in juvenile sea bass (Dicentrarchus labrax),striped jack and barfin flounder (Verasper moseri). The SSN1 and GF cell lines have been successfully used in isolating piscinenodaviruses.Although there are many papers describing the molecular characteristics of betanodavirus, our knowledge of the genomic attributes of these viruses is still limited. Vaccination studies are being undertaken by a number of researchers and need to be fostered. In particular, the use of passive immunization of broodfish with homologous and heterologous, high titre antisera are worthy of investigation.