Because of the exquisite quality of flesh, grouper is one of the aquaculture species with high economic value. In past decades, the grouper industry has developed rapidly in many Asian countries. In Taiwan alone, the production volume of grouper was about 9,300 mt in 2006, reaching a total value of NT$ 1.73 billion, approximate US$60 million
. However, the sustainable development of grouper industry has been threatened by infection caused by nervous necrosis virus (NNV), which could cause mass mortality of up to 100% in the fish at larval and juvenile stages
NNV, also known as viral encephalopathy and retinopathy (VER), belongs to the genus Betanodavirus of the family Nodaviridae. NNV virion is non-enveloped, spherical-shaped particle with a diameter of 25–34 nm
[2, 6, 7]. It consists of two single-stranded, positive-sense RNA genomes: RNA1 (3.1kb) encodes the viral RNA-dependent RNA polymerase (RdRp) and RNA2 encodes the viral capsid protein
. In addition, a subgenomic RNA3 from RNA1 contains two open reading frames that encode protein B1 (111 amino acids) and B2 (75 amino acids), which have been shown to be an anti-necrosis and a suppressor of host siRNA silencing factor, respectively
[9, 10]. Protein B2 might also function as necrotic death factor
. The first NNV outbreak was reported in Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel) in 1990
, and has been since widely reported in various teleost species, including grouper. In grouper, the infected fish exhibit signs of uncoordinated darting, spiral swimming, abnormalities of swimming bladder control, and vacuolation in brain and retina
[2, 4, 12–16].
NNV infection remains as now a major threat to the grouper industry, despite extensive efforts have been invested to develop therapeutic and prophylactic regiments against the viral infection. Extended understanding of the molecular mechanism of the viral pathogenicity can facilitate the development of antiviral strategy, yet such knowledge is currently inadequate. For examples, the infection route of NNV in the host is not fully illustrated, and the cellular response of the host toward NNV remains largely in puzzle. NNV is known to cause apoptotic death of infected cells. Recent studies have further indicated a connection between the viral-induced endoplasmic reticulum (ER) stress and the apoptotic death
[17–19]. Many studies have demonstrated that virus-induced ER stress could determine the fate of infected cell, survival or death
[20, 21]. Cell which does not overcome the ER stress will succumb to apoptosis ultimately. In the stressed cell, the ER chaperone immunoglobulin heavy-chain binding protein (BiP), also known as glucose-regulated protein 78 (GRP78), functions as a master control to relieve the stress by initiating the unfolding protein response (UPR) pathway. BiP activates the three mediators of UPR: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring 1 (IRE1). Activation of PERK could phosphrylate eIF-2α, resulting in attenuation of protein synthesis. On the other hand, ATF6 and IRE1 function at the later stage of UPR as compared to PERK. After being translocated into nucleus, the activated ATF6 stimulates the expression of several chaperones that are capable of refolding misfolded proteins. ATF6 also stimulates the expression of the X-box-binding protein (XBP1) mRNA, which would produce a transcription factor once one of its intron is removed by IRE1. The product of the spliced XBP1 mRNA stimulates the expression of proteins that could facilitate the degradation of misfolded proteins
. All together, these mediators alleviate ER stress by assisting protein folding, reducing translocation of newly synthesized proteins into ER, and facilitating protein degradation in ER lumen. Currently, the understanding of NNV-induced ER stress response is insufficient and requires further investigation.
One hurdle to the study of the molecular mechanism of NNV pathogenicity in grouper cells is the lack of sufficient genomic information of the host. The recent advance in the next generation sequencing (NGS) technology has exerted profound impact on the biological science research. The technique has been applied in decoding the genomes of several non-model organisms by transcriptome sequencing, providing valuable information in the understanding of gene function, cell responses and evolution. Transcriptome sequencing is cost effective compared to traditional Sanger sequencing method, and is a suitable alternative to whole genome sequencing as it can provide information of transcribed portion of genes at a lower cost
In this study, grouper kidney cell (GK cell) was used to establish a transcriptome library by using the NGS technology. A total of 66,584 unigenes were obtained by SOAP de novo transcriptome assembly software from 51 million reads of raw mRNA sequencing data. By matching the assembled unigenes with various databases, we have characterized these unigenes into different functional categories and pathways. The analysis resulted in the identification of 25 unigenes that are relevant to ER stress-associated genes, including BiP, PERK, ATF6, IRE1, and XBP-1. We further found that BiP could interact with the NNV capsid protein in the infected cells, suggesting a role of these two proteins in the ER stress response after viral infection. These data will benefit future study of NNV pathogenicity and development of therapeutic or prophylactic treatment against NNV.