Abstract
Groupers (Epinephelus spp.) are important aquaculture fish species in Taiwan, but they are highly susceptible to nervous necrosis virus (NNV) and iridovirus which often cause significant economic losses to grouper aquaculture. NNV, a piscine nodavirus, has caused serious viral nervous necrosis and viral encephalopathy and retinopathy in hatchery-reared larvae and juveniles of a wide range of teleost species worldwide in the last two decades. Although the mortality of NNV-infected larvae is nearly 100%, there are still some larvae that survive this catastrophe. To comprehensively understand the variations of these survivors at the molecular level, we collected orange-spotted grouper (Epinephelus coioides) larvae that survived an NNV outbreak in an indoor hatchery in southern Taiwan to study differential gene expression. Survived larvae with high, medium and low levels of detected NNV were compared with morbid larvae using a 9600-clone-containing grouper larva cDNA microarray, and differential gene expression was further confirmed by a quantitative real-time polymerase chain reaction. Significant gene expression variation exists in survived larvae. The following genes were confirmed that had relative 2-folde higher expression in survived larvae: adenylate kinase 1-2, myosin binding protein H-like, myosin light chain 2, myosin light chain 3, tropomyosin, fast/white muscle troponin T embryonic isoform, and two types of parvalbumin genes. Four genes were confirmed that expressed higher relatively in morbid larvae: apolipoprotein A-I, trypsinogen, pyruvate kinase, and astacin-like metalloprotease. Moreover, although most immune factors had higher expression in morbid larvae, immunoglobulin M (IgM) heavy chain gene transcription was significantly higher in survived larvae that carried high virus levels than morbid larvae, indicating that humoral immunity might protect organisms from viral infection. These results suggest that some non-immune-related genes may have played important roles in the survival event during the immune-immature larval metamorphosis stage after NNV infection. In the other hand, disease caused by grouper iridovirus (GIV) also has resulted in economic losses due to high mortality in all stages of grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using a GIV-infected grouper kidney cDNA microarray chip. Further quantitative validation was executed in the head-kidney and spleen for 24 candidate genes and 7 immune factors following GIV inoculation. Significant induction with various patterns could be seen in 30 tested genes in the spleen. However, only 23 genes had induction in the head-kidney and meanwhile 5 genes showed reduction. Transcriptional expression profiles of selected genes in response to lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (PIC) were also established to compare with the GIV-stimulated expression. The results indicated that the responses of most genes facing GIV invasion have more similarities to PIC treatment than LPS. Seven genes are thought to be interferon-related factors: RNA helicase DHX58, ISG15, viperin, HECT E3 ligase (HECT), CD9, urokinase plasminogen activator surface receptor (PLAUR) and Mx-1. Following immunization with inactivated GIV, significant induction could be seen in DHX58, viperin, IL-1beta, IL-8, COX-2, HECT, PLAUR, IgM, Mx-1, very large inducible GTPase-1 (VLIG1) and TNF-alpha in the head-kidney or spleen, and the latter 6 genes also had a gradual increasing pattern by a boosting immunization. These factors might play important roles in adaptive antiviral protection. Thus, we have characterized the temporal response patterns of virus responsive genes and have also identified several potential immune markers to further investigate host antiviral defense mechanisms.