Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii

A disease of Macrobrachium rosenbergii, the giant freshwater prawn, farmed in China was recently recorded in Zhejiang, Jiangsu, Shanghai, Guangxi and Guangdong provinces. The clinical sign of the disease, which develops in post-larvae (PL), is a whitish appearance of the muscles, particularly noticeable in the abdomen. Mortalities may reach 100% in some hatcheries. Investigations by transmission electron microscopy after negative staining of diseased PL homogenates showed the presence of two types of viral particles: one, unenveloped, icosahedral in shape, 26-27 nm in diameter, the second, much smaller, about 14-16 nm in diameter, designated extra small virus particle (XSV). The large virus has a genome with two pieces of ssRNA (RNA-1 and RNA-2), of 3 and 1.2 kb, respectively. Hybridization tests confirmed that this large virus is closely related to M. rosenbergii nodavirus (MrNV) which was isolated from diseased prawns in a hatchery in the French West Indies. Its very small size and hypothesized biochemical and biological characteristics suggest XSV is a new type of crustacean virus. As XSV has always been found associated with the larger virus (nodavirus) and is located in muscle and connective cells of diseased animals, it could be an autonomous virus, a helper-type virus or a satellite-like virus.     

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.     

我国一株新型黄头病毒的分子流行病学

为查明黄头病毒(Yellow Head Virus,YHV)在我国的存在和变异情况,本研究采用世界动物卫生组织(OIE)《水生动物诊断手册》中YHV套式RT-PCR检测方法对2012-2014年采集的299份样品进行了YHV监测,并对部分YHV阳性样品基因进行了克隆测序及系统发育分析.流行病学调查显示,299份样品中YHV的阳性率为11％,我国养殖的中国明对虾、凡纳滨对虾、日本囊对虾以及罗氏沼虾的部分样品中均检出了YHV,中国明对虾和罗氏沼虾是本次调查中新发现的YHV自然宿主,而且YHV在中国明对虾中的检出率最高.对6份较强阳性样品YHV基因组ORF1b内1002 bp的分型片段进行克隆测序和序列分析,序列比对结果显示,阳性样品的YHV与国外报道的YHV的6个基因型相似度为81.0％-90.5％;系统发育分析显示,6份阳性样品归于同一分支,但与已知YHV的6个基因型均不在同一分支内,其与YHV基因1型(YHV-1)亲缘关系较近.对阳性样品YHV基因组ORF3内编码gp116蛋白的一段序列进行克隆测序,得出其序列长度为509 bp,与YHV-1a的545 bp、YHV-1b的383 bp和YHV-2(即鳃联病毒GAV)的476 bp均不同;依据该片段构建的系统发育树显示,6份阳性样品归于同一分支,与YHV已知的6个基因型不同,与YHV-1亲缘关系较近,且与YHV-1a相似度大于YHV-1b.对其中两份样品的ORF2序列进行比对显示,两份样品序列相似性为99.8％,蛋白序列完全相同,与YHV-1的序列相似度为85.9％,与YHV-2相似性为80.9％.样品调查结果对增补YHV的宿主范围有重要意义;YHV核酸检测和序列比对结果表明,感染我国养殖对虾的YHV为一种新的致病株型.     

White tail disease of the giant freshwater prawn, Macrobrachium rosenbergii: separation of the associated virions and characterization of MrNV as a new type of nodavirus

White tail disease of the farmed freshwater prawn, Macrobrachium rosenbergii, is the cause of mortalities in the French West Indies, China and India. Two different sized particles, both developing in the cytoplasm of target cells, are found associated with diseased animals. These two viruses were separated, purified and subsequently characterized. The larger one, called MrNV, is icosahedral in shape and 27 nm in diameter. Its genome is composed of two fragments of linear single-stranded RNA (ss-RNA), of 2.9 and 1.3 kb, respectively and its capsids exhibited a single polypeptide of 43 kDa. These characteristics and the partial sequence of a cloned fragment of RNA-1 suggest this agent is a member of the family Nodaviridae, but with differences from both the genera Alphanodavirus and Betanodavirus. The smaller virus, named XSV, is icosahedral in shape, 15 nm in diameter, possesses a linear ss-RNA genome of about 0.9 kb, and its capsid exhibits two polypeptides of 16 and 17 kDa, respectively. The relationships between these two viruses remain unknown.     

Immunomodulatory effect of Cynodon dactylon against white tail disease of giant freshwater prawn,Macrobrachium rosenbergii (de Man, 1879)

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important and extensively cultured crustacean worldwide. The viral pathogens, Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) are responsible for causing severe mortalities in the hatchery and nursery phases. This study investigates the protection of postlarvae of freshwater against white tail disease (WTD) using plant extract derived from Cyanodon dactylon and the modulation of the prawn non‐specific immunity. To determine the immunomodulatory effect of C. dactylon extract, the prawn was injected with plant extract and various immunological parameters were estimated. The immunological parameters such as proPO, SOD, THC and clotting time were found to be significantly higher in the plant extract‐injected prawn when compared with control groups. The results of real time PCR analysis revealed up regulation on the expression proPO, SOD and lysozyme genes in MrNV and XSV challenged prawn postlarvae treated with C. dactylon extract. Infectivity experiment showed high relative per cent survival in MrNV and XSV‐challenged prawn postlarvae treated with C. dactylon extract. These results strongly indicate that the administration of C. dactylon plant extract enhances immunity of the prawn. Based on the results, this study recommends that the immersion of postlarvae in C. dactylon plant extract is a potential prophylactic agent against WTD.     

Effect of chemical and physical treatments on the inactivation of Macrobrachium rosenbergii nodavirus and extra small virus

White tail disease (WTD) is found to cause immense economic losses in hatcheries, with mortalities often reaching 100% within 4 or 5 days. The pathogenic agents have been identified as Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which are 27 and 15 nm in diameter respectively. The effects of some chemical disinfectants hydrogen ions (pH), heat and ultraviolet (UV) irradiation on the inactivation of MrNV and XSV were investigated. The viral inoculum exposed to UV irradiation for a period of 5 min and more was totally inactivated and failed to cause mortality in postlarvae of prawn. The viruses were totally inactivated by this high pH (8.5, 9 and 10). The viral suspension treated with sodium hypochloride, formalin, Benzalkonium chloride and Benzethonium chloride at the concentration of 200 ppm caused 100% mortality in postlarvae of prawn. Iodine was found to be effective to inactivate MrNV and XSV at the concentration of 100 ppm or more, whereas the viral suspension treated with iodine at the concentration of 50 ppm or less caused mortality in postlarvae. The infected postlarvae in treated and positive control groups showed positive by RT‐PCR for these viruses.     

患铁虾综合征罗氏沼虾眼柄转录组研究

为探索罗氏沼虾(Macrobrachium rosenbergii)铁虾综合征(IPS)的分子机制,采用高通量测序平台(Illumina Hiseq-2500)分别对患IPS罗氏沼虾(IPS虾)和正常罗氏沼虾开展转录组测序,进行生物信息学分析。结果显示,高通量测序共获得56.42 G高质量数据,拼接后得到221 901条单基因序列(unigene),长度范围为201~30 985 bp,平均长度为1572 bp,N50长度为2867 bp,N90长度为646 bp。将单基因序列分别在Nr、Nt、Swissprot、KEGG、KOG、GO、PFAM数据库进行序列比对及功能注释,103 570条得到注释,其中,GO数据库注释到的单基因序列最多。差异表达分析显示,2003个基因在IPS虾眼柄中差异表达,包括1209个上调基因和794个下调基因,516个基因被注释到242条KEGG通路中,翻译、信号转导和免疫系统富集的差异基因数目最多。催乳素、雌激素、胰岛素、促性腺激素释放激素、胰高血糖素、催产素、谷氨酸能突触、血清素能突触等与生殖调控相关激素的代谢过程在IPS虾与正常虾眼柄之间存在差异。此外,一些已被证明在免疫反应中起重要作用的基因在IPS虾眼柄中显著上调,如血管内皮生长因子受体1、丝氨酸蛋白酶抑制剂6、C型凝集素、芳基硫酸酯酶B、酚氧化酶原激活酶2a、组织蛋白酶B、组织蛋白酶L、甲壳类抗菌肽4等。同时,注释到溶酶体、吞噬体、抗原处理与呈递、细胞凋亡、内吞作用等多条与免疫相关的途径,支持近期研究得出的罗氏沼虾IPS与病原感染相关的结论。本研究为解析罗氏沼虾IPS的成因和分子机制提供了数据支撑。     

Effect of brewer’s yeast on immune response of giant freshwater prawn, Macrobrachium rosenbergii, and its resistance to white muscle disease

The present study was conducted to find the effect of dietary brewer’s yeast on growth, survival, immune response, and resistance to white muscle disease in giant freshwater prawn, Macrobrachium rosenbergii. The brewer’s yeast was supplemented at graded levels 0, 5, 10, and 20?g per kg diet, and the experiment was conducted for 75?days. After the feeding trials, growth, survival, immune parameters like prophenoloxidase activity, respiratory burst, and total hemocyte count were assessed. The growth, specific growth rate, and survival were not found significantly different among the treatment and control groups “prophenoloxidase activity and respiratory burst” were found significantly different (p?<?0.05) among the treatment and control groups. The total hemocyte count was significantly different (p?<?0.05) among control and treatment groups. Cumulative percent survival was higher in M rosenbergii fed 1?% brewer’s yeast-supplemented diet when they were challenged with white muscle disease virus (MrNV (Macrobrachium rosenbergii nodavirus) and XSV (extra small virus)). The present work revealed that the incorporation of brewer’s yeast in the diet is effective in enhancing immune response and controlling the white muscle disease in M rosenbergii.     

PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.     

中华锯齿米虾卵黄蛋白原部分cDNA序列的克隆

卵黄蛋白原(Vitellogenin,Vg)是广泛地存在于卵生脊椎和无脊椎动物体内的雌性特异性蛋白,是卵黄蛋白(vitellin,Vn)的前体,本文通过同源克隆的方法从成熟中华锯齿米虾的卵巢中克隆到了一条长474 bp的Vg cDNA序列。用DNAMAN中的Blast功能将此片段与罗氏沼虾(Macrobrachium rosenbergii)、高背长额虾(Pandalus hypsinotus)的Vg部分序列进行比对,结果发现中华锯齿米虾与罗氏沼虾和高背长额虾的同一性分别为64%与62%。此片段为中华锯齿米虾Vg cDNA全长序列的克隆及其表达奠定了基础。     

罗氏沼虾白斑综合征病毒囊膜蛋白VP28基因的克隆及分析

根据已公布的罗氏沼虾白斑综合征病毒(WSSV)囊膜蛋白VP28基因序列设计一对特异性引物,从疑似患白斑病毒病的罗氏沼虾(Macrobrachium rosenbergii)中提取总DNA,并以此为模板,经PCR扩增、克隆并测序后将该片段通过GenBank比对,证实为WSSV的VP28基因;与20个已公布的WSSV VP28进行同源性比较,结果显示:从中国对虾、斑节对虾、南美白对虾、日本对虾、波纹龙虾提取的病毒株聚为一类,印度对虾WSSV VP28为另一类,罗氏沼虾WSSV VP28又单独为一类。根据测序结果推测VP28蛋白的二级结构在氨基酸的7～29区间可能为跨膜螺旋区,且该区域高度保守。     

日本沼虾cytMnSOD和mtMnSOD基因全长cDNA克隆及表达

利用RACE技术从日本沼虾肝胰腺中克隆了cytMnSOD和mtMnSOD基因cDNA全长序列。cytMnSOD基因cDNA全长1 233 bp,开放阅读框为858 bp,编码286个氨基酸,N端含有60个氨基酸残基组成的延伸区;mtMnSOD基因cDNA全长1 113 bp,开放阅读框为654 bp,编码218个氨基酸,N端含有20个氨基酸残基组成的信号肽;cytMnSOD和mtMnSOD预测蛋白分子量及等电点分别为31.33、24.05 ku和5.62、7.12。日本沼虾cytMnSOD推导的氨基酸序列与其mtMnSOD的相似性为40%,二者均含有MnSOD的特征肽段(DVWEHAYY)、4个Mn2+结合位点和2个N-糖基化位点。Real-time PCR结果表明,cytMnSOD和mtMnSOD在日本沼虾肝胰腺、肌肉、血细胞、大颚器官、卵巢和鳃等组织均有表达,其中肝胰腺表达量最高;肝胰腺cytMnSOD和mtMnSOD基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低。嗜水气单胞菌刺激后3 h,肝胰腺cytMnSOD和mtMnSOD的表达量显著增加,推测MnSOD是参与机体免疫防御反应的一种重要分子。     

Indian isolates of white spot syndrome virus exhibit variations in their pathogenicity and genomic tandem repeats

To detect genomic variation of white spot syndrome virus (WSSV) isolates from different geographical regions of India, the variable number of the tandem repeat (VNTR) region of the ORF 94 (Thailand WSSV isolate – GeneBank Accession No. AF369029 ) was analysed using five specific sets of primers. Analysis of 70 WSSV‐positive samples showed the presence of 14 different genotypes of WSSV with VNTRs ranging from 2 to 16 tandem repeats with the majority (85.47%) having 6–12 tandem repeats. Occurrence of different genotypes of WSSV was found to be neither correlated to any specific geographical region nor to the different growth stage of the tiger shrimp, Penaeus monodon. Pathogenicity studies conducted with 25 isolates of WSSV revealed the presence of virulent and avirulent strains of WSSV in Indian shrimp farms. However, an unambiguous link could not be established between the different genotypes and their virulence.