Full length articleTranscriptome profiling analysis of grouper during nervous necrosis virus persistent infection
Introduction
Nervous necrosis virus (NNV) is highly lethal to many fish species and frequently causes a massive economic loss worldwide. NNV belongs to the viral genus, Betanodavirus, which was classified in the family Nodaviridae. NNV could cause viral encephalopathy and retinopathy (VER) and lead to some noticeable behavioral changes in infected fish. In a previous report, the necrosis and vacuolations of brain and retina tissue were observed in NNV-infected seven-band grouper. The abnormal swimming of diseased fish was also observed. These features are typical of NNV infection.
However, some reports have revealed that NNV persistent infection occurs in Atlantic halibut. The symptoms of persistent infection are different from typical acute infection. None of the diseased juvenile fish showed clinical signs of VER. Nevertheless, virus particles were detected in the brain and retina [1]. A case of Asian sea bass revealed that ovarian tissue was also NNV-immunopositive during the infection [2]. This study also highlighted that the vertical transmission of nodavirus causes newly hatched larvae infections. This feature of NNV seriously threatens the disease control of aquaculture, particularly subclinical fish, which under persistent infection could behave as healthy fish. Therefore, this persistent infection model needs further investigation. NNV acute and persistent infections occur on larvae and adult fish, respectively. These models were also demonstrated on zebrafish and revealed that the acute infection causing high mortality is due to the lack of inactive interferon response [3]. The transition from acute to persistent infection is the key to transform brood fish into NNV carriers, which has been a serious problem in aquaculture.
In addition to NNV, several references have addressed persistent viral infections in fish to date. The infectious pancreatic necrosis virus (IPNV) is reported in carrier state rainbow trout occurring after acute infection [4]. Suppression subtractive hybridization (SSH) was performed to represent the gene profiling of persistent infection [5]. IPNV infection induces an imbalance of cytokine expression on trout, which lowers the specific antibody response [6]. For infectious hematopoietic necrosis virus (IHNV), transcriptome profiling suggested that the immune response was not limited in Sockeye salmon with IHNV persistence. In a study of viral hemorrhagic septicemia virus (VHSV), the brain is one of the most important organs in which VHSV can persist in rainbow trout [7]. A similar result was observed in Pacific herring [8]. Overall, the mechanism of viral persistence and carrier state is not yet clear and could vary among different viruses and hosts. Thus, additional systematic information is needed to provide insights into the mechanism of how the virus is maintained in the host.
Recently, several next-generation sequencing (NGS) technologies, such as the 454 Life Sciences pyrosequencing platform, the Illumina Genome Analyzer, and the Applied Biosystems Solid platform, have provided rapid, cost-effective and high-throughput tools for sequencing the transcriptome (RNA-Seq). RNA-Seq permits deep, robust assessments of transcript abundances and transcript structure. These techniques also benefit non-model organism, which lack genomic information. The de novo process assembles the sequences from RNA-Seq and provides a non-model organism reference template for analysis. In our previous study, transcriptome analysis provided valuable insights into the molecular mechanism of NNV-induced ER stress in grouper kidney cell-line [9].
In the present study, we used the next-generation sequencing technique on Illumina NextSeq 500 to discover the genes involved in a persistent infection model. The transcriptome analysis was aimed at the regulation of the immune genes from the NNV infection. We prepared an inactivated-NNV vaccine for administration as a comparison group. Therefore, we could determine the different effects from live virus and vaccine on the responses of immune genes. These results are a great step in improving the current understanding of how NNV persistence occurs in the grouper fish.
Section snippets
Fish
Healthy Malabar groupers, Epinephelus malabaricus, weighing approximately 100 g, were obtained from the Aquatic Animal Center of National Taiwan Ocean University (Keelung, Taiwan). The fish were maintained in a holding tank at a water temperature of 24–26 °C with constant aeration and daily fresh seawater changes. The fish use protocol in the present study has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of College of Life Sciences, National Taiwan Ocean
Validation of NGS sample preparation
To confirm the NNV infection efficacy, an NNV detection PCR was performed on sampled brain tissue from a later stage of infection. A low-level of NNV coat protein (CP) was detected only in NNV treatment group samples. Neither negative control nor inactivated NNV were detected (Fig. 1). These samples were also verified by qPCR. The results were consistent with the PCR results and showed that the average quantity of NNV is 3.9 ± 0.7 log copies/mL. These results suggested that the treatment is
Discussion
In the present study, we report the first RNA sequencing study of the transcriptome of NNV persistent infection in vivo on Malabar grouper, Epinephelus malabaricus. We successfully obtained grouper fish in the persistent stage, which survived after acute infection. The NNV capsid protein was detected at a low level in the brain tissue. The inactivated NNV vaccine group was set up as a comparison group to focus the profiling on the live virus infection in the host. We anticipated observing the
Acknowledgment
This work was financially supported through grants from the Ministry of Science and Technology (grant number: MOST106-3114-B-019-001, MOST106-3114-8-019-001 and MOST106-3114-B-020-001) in Taiwan.
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2023, Virologica SinicaCitation Excerpt :Transcriptomic analysis of the viral-infected samples showed that the severity of viral infection is usually positively correlated with the high expression of immune-relevant genes in vivo (Gupta and Rao, 2011; Rosenberg et al., 2018). Transcriptomic analysis of nervous necrosis virus (NNV)-infected Epinephelus malabaricus showed that significant differentially expressed transcripts included immune-relevant pathways (Tso and Lu, 2018), which is consistent with our data. In our research, the GCRV-SVCV co-infection resulted in a marked alleviation of zebrafish viral-associated damage and a marked inhibition of SVCV proliferation in zebrafish tissues.
Response signatures of intestinal microbiota and gene transcription of the tiger grouper Epinephelus fuscoguttatus to nervous necrosis virus infection
2022, AquacultureCitation Excerpt :For example, endoplasmic reticulum stress (ER) was induced in the NNV-infected the grouper kidney (GK) cells, and the viral capsid protein might contribute to the ER stress response (Lu et al., 2012). Highly immune cell active signaling and surface receptor expression were triggered in the brain of Malabar grouper E. malabaricus during persistent NNV infection (Tso and Lu, 2018). The collagen synthesis and adhesion molecules were triggered in orange-spotted grouper (Ge et al., 2020).
Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform
2021, GeneCitation Excerpt :In fact, in the latest study, isg15 and sacs were the most expressed genes, similarly to the results recorded in the present study. Sacsins are regulators of the heat shock protein 70 (HSP70) chaperon machinery; therefore, the up-regulation of sacs could be a response to the betanodavirus-induced stress (Tso and Lu, 2018). In the present study, expression of HSPs has not been detected; however, other authors have reported the implication of HSPs in betanodavirus infection in several fish species (Chaves-Pozo et al., 2019; Dios et al., 2007; Kim et al., 2017; Liu et al., 2016; Lu et al., 2012).
Transcriptomic analysis of red-spotted grouper nervous necrosis virus infected Asian seabass Lates calcarifer (Bloch, 1790)
2020, Aquaculture ReportsCitation Excerpt :For instance, a study in NNV- infected Senegalese sole (Solea senegalensis) demonstrated the activation of immune genes including IFN I, mediators of IFN signaling pathway (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISGs; ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1) (Labella et al., 2018). High expression levels of ISGs (GIVNP1, Mx) together with cluster of differentiation (CD; CD3A, CD4, CD8) and negative regulators of T-cell signaling (PD-L1 and LAG3) was also observed in persistently infected Malabar grouper (Epinephelus malabaricus) (Tso and Lu, 2018). In infected Epinephelus moara, MHC class I, but not immunoglobulin (IgD, IgM) and perforin (Prf1), was reported to be upregulated (Wang et al., 2019).