草蝦裸病毒基因體與包埋體特性之研究

Abstract

草蝦裸病毒為蝦類球體桿狀病毒症之病原，此疾病會造成蝦苗死亡且可能抑制蝦苗生長與降低存活率，進而影響水產養殖之產值。此病毒之命名與分類地位曾因型態觀察與部分基因體序列之演化樹分析而有幾度變動，因此在本研究中，我們透過基因體解序與完整的演化樹分析策略，進一步釐清草蝦裸病毒之分類地位。草蝦裸病毒為海洋病毒中，少數可產生包埋體構造之特殊病毒，為了瞭解此海洋病毒的包埋體特性，我們分析了組成包埋體的多角體蛋白質之化學特性，並與加州苜蓿夜蛾核多角體病毒之多角體蛋白質相比較。在研究策略上，我們利用次世代定序與傳統定序方法獲得其全長基因體序列，共 119,638 鹼基對，並預測有115個開放譯讀區。根據預測的假定開放譯讀區，進行演化樹分析，結果顯示草蝦裸病毒與非包埋體裸病毒歸類於同一分群，而不應歸類於具有包埋體之桿狀病毒。其多角體蛋白質，為形成包埋體構造中重要與主要蛋白質，無論是重組包埋體蛋白質或是野生型草蝦裸病毒之包埋體皆在酸性條件下易解離，此與桿狀病毒包埋體在鹼性條件下較易解離的特性不同。總之，依本病毒基因體的研究與演化樹分析，我們認為本病毒並不屬於桿狀病毒科而應歸類於裸病毒科，同時由於本病毒之特異性，應訂立新屬，稱為Gammanudivirus。

Penaeus monodon nudivirus (PmNV) is the causative agent of spherical baculovirosis in shrimp (Penaeus monodon). This disease causes significant mortalities at the larval stage and early postlarval (PL) stage and may suppress growth and reduce survival and production in aquaculture. The nomenclature and classification status of PmNV has been changed several times due to morphological observation and phylogenetic analysis of its partial genome sequence. In this study, we therefore completed the genome sequence and constructed phylogenetic trees to clarify PmNV’s taxonomic position. To better understand the characteristics of the occlusion bodies formed by this marine occluded virus, we also compared the chemical properties of the polyhedrin produced by PmNV and the baculovirus AcMNPV (Autographa californica nucleopolyhedrovirus). We used next generation sequencing and traditional PCR methods to obtain the complete PmNV genome sequence of 119,638 bp encoding 115 putative ORFs. Phylogenetic tree analysis showed that several PmNV genes and sequences clustered with the non-occluded nudiviruses and not with the baculoviruses. We also investigated the characteristics of PmNV polyhedrin, which is a functionally important protein and the major component of the viral OBs (occlusion bodies). We found that both recombinant PmNV polyhedrin and wild-type PmNV OBs were sensitive to acid conditions, but unlike the baculoviral OBs, they were not susceptible to alkali treatment. From the viral genome features and phylogenetic analysis we conclude that PmNV is not a baculovirus, and that it should be assigned to the proposed Nudiviridae family with the other nudiviruses, but into a distinct new genus (Gammanudivirus).