Complete genome sequence and pathogenic analysis of a new betanodavirus isolated from shellfish

We determined the complete genomic RNA sequence of a new type of betanodavirus Korea shellfish nervous necrosis virus (KSNNV) isolated from shellfish. Compared with other isolates representing four genotypes of betanodaviruses, the identity of the whole nucleotide sequence of the virus was in the range of 76%–83% with the presence of specific genetic motifs and formed a separate new branch in the phylogenetic analysis. In pathogenic analysis by immersion method, KSNNV‐KOR1 shows 100% cumulative mortality like SFRG10/2012BGGa1 (RGNNV) in newly hatched sevenband grouper and mandarin fish, which is clearly different from those found in negative control groups. There were no significant differences in increasing rates of mortality and viral intra‐tissue concentration of larval fishes infected with KSNNV‐KOR1 at both 20 and 25°C water temperature. Histopathological examination of each fish species in the moribund stage revealed the presence of clear vacuoles in both brain and retinal tissues similar to typical histopathology features of RGNNV. In the present study, we first report a new betanodavirus from shellfish as the aetiological agent of viral nervous necrosis disease in fish with complete genomic nucleotide sequence and pathogenic analysis.     

Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty‐eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000–2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs.     

Occurrence and molecular characterization of betanodaviruses in fish and invertebrates of the Greek territorial waters

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter‐RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.     

Identification of B-cell epitopes on the betanodavirus capsid protein

The pepscan procedure was used to identify betanodavirus B-cell epitopes recognized by neutralizing mouse monoclonal antibodies (MAbs) and serum samples obtained from sea bass, Dicentrarchus labrax, naturally infected with betanodavirus. Pepscan was performed with a panel of thirty-four 12-mer synthetic peptides that mimicked the entire betanodavirus capsid protein. Sea bass serum samples reacted strongly with three regions of the capsid protein comprising amino acid residues 1-32, 91-162 and 181-212. The latter region was also recognized by neutralizing MAbs and coincided with a region of high antigenic propensity identified by an antigen prediction algorithm. These data suggest that a region of the betanodavirus capsid protein spanning amino acid residues 181-212 may represent a neutralization domain that could potentially be used to inform the development of nodavirus vaccines and immunodiagnostic reagents.     

蓝太阳鱼生长激素全长cDNA的克隆与序列分析

The full length cDNA encoding growth hormone of a freshwater fish, Lepomis cyanellus, (LcGH) was cloned from pituitary RNA with RT-PCR, 3‘ and 5‘ RACE (rapid amplification of cDNA ends). The LcGH cDNA (Genbank No. AY530822), about 989nt (nucleotide) long, consisted of a open reading frame with 615nt long, 5‘and 3‘untranslated regions with 93nt and 224nt long respectively, and a 57nt poly (A) tail. The DNA sequence analysis showed that there are typical Kozak sequence and polyadenylation signal. The pregrowth hormone peptide of 204aa deduced from LcGH cDNA included a putative signal peptide (17aa) locating in its Nterminal. There exist a Asn-Cys-Thr glycosylation site at amino acid 201, and 4 cysteine residues (No. 69, 177, 194, 202) that are essential to construct two S-S bonds in this pregrowth hormone peptide. Homological comparision among LcGH and other species growth hormones showed that There is high homology (more than 85%) between growth hormone of Lepomis cyanellus and that of most perciformes fish, but low homology (less than 70%) in comparison with other species such as Siluriformes and Cypriniformes fish.     

Detection of betanodavirus in juvenile barramundi, Lates calcarifer (Bloch), by antigen capture ELISA

Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries.     

暗纹东方鲀芳香化酶基因的结构及表达分析

性腺型芳香化酶基因是一种调节雌雄激素平衡的基因。为初步探究暗纹东方鲀性腺型芳香化酶基因在性腺发育和性别分化过程中的作用,通过RACE及荧光定量PCR技术,成功克隆出暗纹东方鲀性腺型芳香化酶基因的cDNA全长序列,共1786 bp,编码514个氨基酸,与红鳍东方鲀及星点东方鲀同源性最高。氨基酸序列分析显示,该基因编码的蛋白是一种稳定的亲水性蛋白,但不存在信号肽序列;性腺型芳香化酶含有38个磷酸化位点、4个N-糖基化位点、2个跨膜保守结构域;其氨基酸序列在哺乳动物和鱼类中均十分保守,且含有跨膜区、Ⅰ-螺旋区、Ozol′s肽区、芳香化酶特异性保守区和亚铁血红素结合区等功能保守区。表达分析显示,暗纹东方鲀性腺型芳香化酶基因主要在肌肉和卵巢中表达,其次在其他组织中也有少量表达,而且在幼鱼不同发育期卵巢中的表达量呈现出逐渐升高再降低的表达趋势,在精巢中则基本不表达。研究结果表明,性腺型芳香化酶基因很可能参与了暗纹东方鲀雌鱼性腺分化后卵巢的发育、雌性特征的维持和其他组织的发育等过程。     

Characterization of a γ‐aminobutyrate type A receptor‐associated protein gene,which is involved in the response of Portunus trituberculatus to CO2‐induced ocean acidification

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in membrane trafficking and fusion events involving the γ‐aminobutyrate type A receptor, autophagy and apoptosis. In this study, the gene encoding GABARAP was cloned from swimming crab Portunus trituberculatus (PtGABARAP) based on the expression sequence tag (EST). The full‐length cDNA of 664 bp includes a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 223 bp with a poly(A) tail, and an open reading frame (ORF) of 354 bp encoding a polypeptide of 117 amino acids with a predicted molecular weight of 13.96 kDa. The deduced amino acid sequence shares high similarity (93%–100%) with GABARAPs from other species and includes a conserved Atg8 domain. In a phylogenetic analysis PtGABARAP clustered with GABARAPs from other species, and more widely with other GABARAP family proteins. The impact of elevated ocean acidification (OA) on P. trituberculatus behaviours was investigated, and real‐time RT‐PCR revealed that PtGABARAP expression was up‐regulated after OA exposure. Ocean acidification also caused crabs anxiety‐like behaviours, like the shoal average speed increase, preference for dark environment (scototaxis) and fast exploration. The results indicated that GABARAP might be involved in the interactions of GABA_A receptors and elevated‐CO₂ seawater.     

锯缘青蟹蜕皮抑制激素cDNA的分子克隆及其表达分析

根据近缘种类同源序列设计简并引物,采用逆转录聚合酶链式反应(RT—PCR)技术,首次扩增获得锯缘青蟹(Scylla serrata)眼柄组织中编码蜕皮抑制激素(MIH)成熟肽全长cDNA序列及部分信号肽序列。将该序列克隆到pUCm—T载体上进行序列测定。结果表明,编码锯缘青蟹MIH成熟肽的cDNA由234个碱基组成,由此推测MIH成熟肽含78个氨基酸残基。该序列不仅与其它短尾类甲壳动物的MIH氨基酸序列具有高度的同源性(79％～9l％),还与该激素同一家族的性腺抑制激素、大颚器抑制激素的氨基酸序列具有较高的相似性。RNA斑点杂交结果显示,MIH基因在眼柄神经节及脑组织中均有表达,而在肌肉、中肠腺中不表达。     

The responsive expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis against pH stress

Caspases are a family of proteases, which play an important role in apoptosis. To evaluate the relationship between apoptosis and pH stress in crustaceans, a caspase gene (FcCasp) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length of FcCasp was 1329 bp with a 972 bp ORF, encoding a polypeptide of 323 amino acids with a calculated molecular weight and pI of 36.0 kDa and 6.27 respectively. The deduced amino acid sequence of FcCasp contained a potential active site (QACRG pentapeptide) conserved in most caspases and two profile hits (p20 and p10 domain profile). Comparison of amino acid sequences revealed that FcCasp had an overall similarity of 76–83% with other penaeid shrimp caspases. The amino acid sequence of recombinant FcCasp protein expressed in Escherichia coli was identified by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometer analysis. High‐level expression of FcCasp in six different tissues was detected by real‐time polymerase chain reaction after exposure to pH stress for 96 and 148 h. TUNEL analysis indicated that apoptosis began to appear in F. chinensis hepatopancreas exposed to extreme pH for 12 h. The amount of apoptosis seems positively correlated with the length of exposure to the pH stressor. The results suggested that FcCasp was involved in the response to environmental pH stress.     

KK-42对日本沼虾表皮蛋白基因MnCP-1表达的上调效应

为探讨KK-42显著缩短日本沼虾(Macrobrachium nipponense)蜕皮周期的可能机制,本研究采用RACE技术首次从头胸甲中获得了一个含几丁质结合-4(chitin_bind_4)结构域的表皮蛋白(cuticle proteins,CPs)c DNA基因全长,命名为Mn CP-1。该序列全长604 bp,可编码122个氨基酸。氨基酸序列比对显示,Mn CP-1与普通黄道蟹(Cancer pagurus,P81576.1)相似性最高(61%)。系统进化分析发现,Mn CP-1与地中海实蝇(Ceratitis capitata)聚类为一支。Real-time PCR结果显示,头胸甲中Mn CP-1在蜕皮前期晚期(D3-4期)和蜕皮后期(A期)的表达量较高,而在蜕皮间期(C期)和蜕皮前期早期(D0-2期)的表达量较低。KK-42处理能显著上调C期和D0-2期Mn CP-1的表达,在处理后6、24和48 h,其mRNA表达水平较对照组高3倍以上。结果表明,头胸甲上皮细胞Mn CP-1的表达与蜕皮周期有关,KK-42可显著上调C期和D0-2期Mn CP-1的表达。     

Cloning,expression and sequence analysis of Macrobrachium rosenbergii nodavirus genes: Indian isolate

RNA‐dependent RNA polymerase (RdRp), B2 and capsid genes of Macrobrachium rosenbergii nodavirus (MrNV) of Indian isolate were polymerase chain reaction amplified, cloned and sequenced. Expression of the MrNV fusion recombinant proteins of RdRp (44.5 kDa), B2 (32.2 kDa) and capsid (58.4 kDa) was confirmed by Western blot analysis using anti‐His mouse monoclonal antibodies. Polyclonal antibodies specific to purified recombinant MrNV capsid protein showed specificity against the capsid protein by Western blot. The protein sequence analysis of the partial RdRp gene of MrNV revealed the signature sequence along with the conserved core residues of the catalytic domain and indicated the presence of active sites, metal ion‐binding site and nucleic acid‐binding site residues. The Indian isolate of MrNV showed high RdRp and capsid gene sequence homology with the other MrNV geographical isolates. However, the Belize isolate was found to be the most distinct among the different geographical prawn nodavirus isolates due to the host specificity. Secondary structure prediction analysis of the MrNV capsid predicted it to be a DNA‐binding protein consisting of α helix (22.91%), extended strand (24.80%), β turn (5.39%) and random coil (46.90%) regions.     

Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (SJNNV, RGNNV, TPNNV and BFNNV) and it is suggested that host specificity is different among these genotypes. In the present study, a betanodavirus [sevenband grouper nervous necrosis virus (SGNNV)] belonging to the redspotted grouper nervous necrosis virus (RGNNV) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of several marine fish species. When challenged with the virus by a bath method (10(5.1) TCID50 mL(-1)), sevenband grouper, Epinephelus septemfasciatus, Japanese flounder, Paralichthys olivaceus, and tiger puffer, Takifugu rubripes, displayed behavioural abnormalities and mortalities with distinct histopathological signs of viral nervous necrosis and heavily immunostained cells were observed in the central nervous tissues and retina. Bath-challenged rock fish, Sebastiscus marmoratus, and a hybrid of sevenband grouper and kelp grouper, E. moara, did not display any behavioural abnormality or mortality during the experimental period, although many fish showed slight signs of viral infection in nerve cells. Kelp grouper and red sea bream, Pagrus major, showed no behavioural abnormality, mortality or immunohistopathological changes after the virus challenge. These results are, in part, consistent with the natural host range of RGNNV, indicating the complexity in the host specificity of betanodaviruses.     

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.     

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.