Genes diferencialmente expressos em camarões de cultivo Litopennaeus vannamei infectados pelo vírus da Síndrome da Mancha Branca e genotipagem de isolados geográficos brasileiros do vírus
Genes diferencialmente expressos em camarões de cultivo Litopennaeus vannamei infectados pelo vírus da Síndrome da Mancha Branca e genotipagem de isolados geográficos brasileiros do vírus
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发表时间：
2012-10
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通讯作者：
I. Müller
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作者：
I. Müller
白色斑点综合征病毒（White Spot Syndrome Virus，WSSV）是一种危害多种甲壳动物的高致死率病害，给对虾养殖区造成了巨大的经济损失。本研究的目的是标准化的真实的时间PCR检测方法WSSV，基因型的地理分离的病毒和分离差异表达的基因在养殖对虾感染WSSV。对巢式PCR阳性和阴性标本进行真实的时间PCR分析。对样本进行病毒载量测定，部分巢式PCR阴性样本经真实的时间PCR检测为阳性，病毒载量较低。在从圣卡塔里纳和巴伊亚采集的虾分离的病毒中，比较了三个小卫星区域的模式、缺失和转座酶的存在。所有的圣卡塔里纳样本都有相同的模式的小型卫星分析。巴伊亚州的样品显示出不同的基因座模式，表明在巴西有两种不同的WSSV基因型。巴西分离株在ORF 23/24中有10，780 bp缺失，并且没有转座酶序列。分析的三颗小卫星的重复数模式在巴西分离株和来自美国国家的分离株之间存在差异。然而，当仅分析ORF 94时，巴西南部、墨西哥、尼加拉瓜、洪都拉斯和巴拿马都有超过9个重复，而巴西东北部和美国样品的重复少于9个。这种重复序列的相似性可能与毒力的相似性有关。我们的研究结果表明，在巴伊亚和圣卡塔里纳州发现的WSSV来自不同的污染源，或者病毒在一段时间内发生了突变。从感染和未感染WSSV的对虾鳃中提取mRNA，进行逆转录和抑制性消减杂交（SSH）。文库1（诱导基因）有300个克隆，形成36个重叠群和36个单重体。其中69条（88.4%）与GeneBank中的序列相似。文库2（阻遏基因）有403个克隆，形成23个重叠群和36个单重群，40个序列（75.5%）与已知序列相似。这些基因根据它们的功能分组。在诱导基因中，有涉及ATP合成、细胞骨架、细胞粘附、代谢和分子转运的基因。在受抑制的基因中，有参与抗菌防御、信号转导和核酸生物合成的基因。WSSV感染似乎改变了几种代谢途径。
White Spot Syndrome Virus (WSSV) is a disease that affects several species of crustaceans, causing high mortality rates, which has been causing great economical losses in shrimp farming regions. The aims of this study were to standardize the Real Time PCR methodology for WSSV detection, to genotype geographical isolates of the virus and to isolate differentially expressed genes in farmed shrimps infected with WSSV. Samples positives and negatives by nested PCR were analyzed by Real Time PCR. The viral load of the samples was determined and some samples negative by nested PCR were positive by Real Time PCR, with low viral load. The pattern of three minisatellite regions, the presence of a deletion and a transposase were compared in virus isolated from shrimp collected in Santa Catarina and Bahia. All Santa Catarina samples had the same pattern for the minisatellites analyzed. Bahia samples showed a different pattern for the loci, indicating that there are two different WSSV genotypes in Brazil. Brazilian isolates have a 10,780 bp deletion in ORF 23/24 and do not have a transposase sequence. The repeat number pattern for the three minisatellite analyzed was different between the Brazilian isolates and isolates from America’s countries. However, when analyzing only ORF 94, South Brazil, Mexico, Nicaragua, Honduras and Panama have all more than nine repeats, while Northeast Brazil and USA samples have less than nine repeats. This similarity of repeats could be related to a similarity in virulence. Our results show that WSSV found in Bahia and Santa Catarina States have originated from different sources of contamination or the virus have undergone mutations during time. mRNA was extracted from gills of shrimps infected and non-infected with WSSV, reverse transcribed and a suppression subtractive hybridization (SSH) was performed. Library 1 (induced genes) has 300 clones, which formed 36 contigs and 36 singlets. Of these sequences, 69 (88,4%) are similar to sequences in GeneBank. Library 2 (repressed genes), has 403 clones, which formed 23 contigs and 36 singlets, and 40 sequences (75,5%) are similar to known sequences. These genes were organized in groups, according to their functions. Among the induced genes there are genes involved in ATP synthesis, cytoskeleton, cell adhesion, metabolism and molecule transport. Among the repressed genes, there are genes involved in antibacterial defence, signal transduction and nucleic acids biosynthesis. WSSV infection seems to alter several metabolic routes.