RESUMO
White spot syndrome, which is caused by white spot syndrome virus (WSSV), is a highly contagious disease of penaeid shrimp. However, there is currently incomplete understanding of the infection mechanism and pathogenesis of WSSV. In this study, a novel gene of a previously uncharacterized WSSV response protein (LvWRP) in Litopenaeus vannamei was identified and characterized. The LvWRP gene has an open reading frame (ORF) of 879 bp encoding a putative protein of 292 amino acids. Sequence analysis revealed that LvWRP shared 24.9% identity with an uncharacterized protein of Penaeus monodon nudivirus. Real-time qPCR analysis showed that LvWRP was ubiquitously expressed in shrimp tissues, with transcript levels induced in hemocytes upon immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and WSSV. In addition, RNA interference-mediated knockdown of LvWRP followed by WSSV challenge revealed significant decrease in the transcript levels of WSSV IE1 and VP28 genes coupled with a reduction in WSSV copies in shrimp hemocytes. Moreover, depletion of LvWRP followed by WSSV challenge significantly increased the transcript levels of Vago4 and Vago5 as well as increased the phosphorylation of STAT, while hemocytes apoptosis in terms of caspase 3/7 activity was decreased. These results suggest that LvWRP is important for WSSV replication in shrimp, and therefore one of the vital host factors in WSSV infection.
Assuntos
Proteínas de Artrópodes/imunologia , Hemócitos/imunologia , Penaeidae/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , Perfilação da Expressão Gênica/métodos , Hemócitos/metabolismo , Hemócitos/virologia , Penaeidae/genética , Penaeidae/virologia , Interferência de RNA , Análise de Sequência de DNA/métodos , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genética , Vibrio parahaemolyticus/imunologia , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Although vertebrate immunity has been well studied for the past decades, invertebrate immunity was much less explored. One possible reason was that in vitro culture system was not well established. In this study, Litopenaeus vannamei was applied as an invertebrate study model. Primary culture conditions for L. vannamei hemocytes were optimized to get relatively quiescent state cells. LPS was used as an immune stimulator and the responses of primary cultured hemocytes were transcriptomically analyzed. Our results showed that around 1,600 genes were upregulated and 800 genes were downregulated from LPS treated hemocytes. The altered genes could be classified into three categories: upregulated, downregulated, upregulated and then downregulated. Further qPCR validation showed that ubiquitin, ubiquitin-conjugating enzyme E2 C, ubiquitin-conjugating enzyme H1 and ubiquitin-conjugating enzyme H5b in ubiquitin-proteasome pathway were upregulated, cytochrome c oxidase 1, NADH dehydrogenase 1, Inosine-5'-monophosphate dehydrogenase 1b and phospholipid-transporting ATPase IA in mitochondria oxidation phosphorylation were downregulated. Our results showed that L. vannamei hemocyte inflammation responses share a lot of similarities with mammalian macrophage inflammation responses.
