中科院海洋生物资源可持续利用重点实验室（依托于南海海洋研究所）秦启伟研究员领导的团队，在海洋病毒编码的miRNA研究方面取得突破性进展。团队成员闫阳博士等首次鉴定并验证了一种新的海洋病毒——石斑鱼虹彩病毒能够编码功能性miRNA。研究成果已于近日发表在国际知名的综合类学术期刊《公共科学图书馆—综合》（Yan ,et al. 2011. Identification of a Novel Marine Fish Virus, Singapore Grouper Iridovirus-Encoded MicroRNAs Expressed in Grouper Cells by Solexa Sequencing. PloS ONE. 2011. 6(4): e19148）。
虹彩病毒（iridovirus）是一类广泛感染无脊椎和低等水生脊椎动物的大分子DNA病毒，对海洋野生动物种群和海水养殖动物造成极大威胁。石斑鱼虹彩病毒（Singapore grouper iridovirus, SGIV）是该团队从海水养殖石斑鱼体中分离出的一种新的高致病性虹彩病毒，是中国和东南亚海水养殖鱼类最重要的病毒性病原之一。然而，目前对SGIV感染致病的分子机理尚不十分清楚，缺乏有效的防治手段。
病毒编码的miRNA研究方兴未艾，因其长度较短、没有免疫原性等特点而成为治疗疾病的新型靶点。团队为揭示SGIV在其感染过程中是否编码miRNA，将感染了SGIV不同时期的石斑鱼胚胎细胞混合后构建小RNA库，进行Illumina/Solexa RNA深度测序。通过一系列生物信息学分析，最终确定16条SGIV编码的miRNA序列，其中一条与2型单纯疱疹病毒编码的miR-H4-5p具有高度同源性，提示miRNA在远缘病毒种中具有一定的保守性。这16个新发现的miRNA散在分布于SGIV基因组中，其中三个定位于ORF057L。同时发现SGIV编码的miRNA在其3' 端或5' 端存在序列或长度的异质性，可能会影响其与靶基因的识别。荧光定量PCR及荧光素酶报告基因分析了这16个miRNA的表达水平和潜在的生物学活性，证实其中11个miRNA确实存在于SGIV感染的石斑鱼胚胎细胞中，并发挥生物学功能。
以前只有人类和哺乳动物编码miRNA的研究报道，其中报道较多的为疱疹病毒科的各成员。该研究首次在国际上证实了海洋病毒也可以编码功能性miRNA，将miRNA研究推向了一个新的层次。
该项研究受到国家杰出青年基金、国家重点基础研究计划（“973”计划）和中科院知识创新工程重要方向项目资助。(生物谷 Bioon.com)
doi:10.1371/journal.pone.0019148
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Identification of a Novel Marine Fish Virus, Singapore Grouper Iridovirus-Encoded MicroRNAs Expressed in Grouper Cells by Solexa Sequencing
Yang Yan1, Huachun Cui1, Songshan Jiang1, Youhua Huang2, Xiaohong Huang2, Shina Wei2, Weiyi Xu1, Qiwei Qin2
Background MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms. Methodology/Principal Findings To determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3鈥?and/or 5鈥?end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells.ConclusionsOur study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis.
