Ting Yu Wang

An integrated microfluidic loop-mediated-isothermal-amplification system for rapid sample pre-treatment and detection of viruses.

This study presents a novel automatic assay for targeted ribonucleic acid (RNA) extraction and a one-step reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) process for the rapid detection of viruses from tissue samples by utilizing an integrated microfluidic system. By utilizing specific probe-conjugated magnetic beads, target RNA samples can be specifically recognized and hybridized onto the surface of the magnetic beads which are mixed with whole tissue lysates, followed by the synthesis of complementary deoxyribonucleic acid (cDNA) and isothermal amplification of target genes simultaneously with the incorporation of two specific primer sets. The nervous necrosis virus (NNV), the most common aquaculture pathogen, with a mortality rate in infected fish ranging from 80% to 100%, has been selected to verify the performance of the developed miniature system. Experimental results showed that the sensitivity of the integrated microfluidic LAMP system is about 100-fold higher when compared to a conventional one-step reverse-transcript polymerase chain reaction (RT-PCR) process. Significantly, the entire protocol from sample pre-treatment to target gene amplification can be completed within 60 min in an automatic manner without cross-reactions with other tested virus, bacteria and eukaryotic cells. Consequently, this integrated microfluidic LAMP system may provide a powerful platform for rapid purification and detection of virus samples.

Real-Time Quantitative PCR Assay for Monitoring of Nervous Necrosis Virus Infection in Grouper Aquaculture

ABSTRACT Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r 2 = 0.99) between threshold cycle (CT ) and RNA quantities, which allowed identification of infected groupers by the CT value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca2+ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.

The search for the IFN-γ receptor in fish: functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss.

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFNgammaR1 and IFNgammaR2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFNgammaRs in fish. They are widely expressed in tissues, with IFNgammaR1 typically more highly expressed than IFNgammaR2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1beta down regulating IFNgammaR1 expression but up regulating IFNgammaR2 expression. Over-expression of the two receptor chains in RTG-2 cells revealed that the level of IFNgammaR2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gammaIP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFNgammaR may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction.

Immunity to betanodavirus infections of marine fish

Betanodaviruses cause viral nervous necrosis in numerous fish species, but some species are resistant to infection by these viruses. It is essential to fully characterize the immune responses that underlie this protective response. Complete characterization of the immune responses against nodaviruses may allow the development of methods that stimulate fish immunity and of an effective betanodavirus vaccine. Such strategies could include stimulation of specific immune system responses or blockage of factors that decrease the immune response. The innate immune system clearly provides a front-line defense, and this includes the production of interferons and other cytokines. Interferons that are released inside infected cells and that suppress viral replication may be the most ancient form of innate immunity. This review focuses on the immune responses of fish to betanodavirus infection.

Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper

Infection of virus (such as nodavirus and iridovirus) and bacteria (such as Vibrio anguillarum) in farmed grouper has been widely reported and caused large economic losses to Taiwanese fish aquaculture industry since 1979. The multiplex assay was used to detect dual viral infection and showed that only nervous necrosis virus (NNV) can be detected till the end of experiments (100% mortality) once it appeared. In addition, iridovirus can be detected in a certain period of rearing. The results of real-time PCR and in situ PCR indicated that NNV, in fact, was not on the surface of the eggs but present in the embryo, which can continue to replicate during the embryo development. The virus may be vertically transmitted by packing into eggs during egg development (formation) or delivering into eggs by sperm during fertilization. The ozone treatment of eggs may fail to remove the virus, so a new strategy to prevent NNV is needed.

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.

Insights into the fish thioredoxin system: Expression profile of thioredoxin and thioredoxin reductase in rainbow trout (Oncorhynchus mykiss) during infection and in vitro stimulation

Production of reactive oxygen species (ROS) is the first biological response during a disease outbreak and after injury. ROS are highly reactive molecules that can either endanger cell homeostasis or mediate cell signaling in several physiological pathways, including the immune response. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are the essential components of the thioredoxin system, one of the main intracellular redox systems and are therefore important regulators of ROS accumulation. Through the regulation of the intracellular redox milieu, the thioredoxin system plays a key role within the immune system, linking immunology and free radical science. In this study we have firstly identified TrxRs in fish and used this new sequence information to reevaluate the evolution of the thioredoxin system within the vertebrate lineage. We next measured the expression of rainbow trout (Oncorhynchus mykiss) Trx and TrxR transcripts during infection in vivo and in vitro after stimulation of a macrophage cell line and primary macrophage cultures with pathogen associated molecular patterns (PAMPs). Our results showed that both Trx and TrxR were induced during infection at the transcriptional level, confirming their likely involvement in the innate immune response of fish. Since TrxRs are selenium-containing proteins (selenoproteins), we also measured the modulation of their expression upon organic and inorganic selenium exposure in vitro. TrxR was found to be responsive to selenium exposure in vitro, suggesting that it may represent a key mediator in the selenium modulation of innate immunity. In conclusion, our study highlights the need to investigate the involvement of the cell antioxidant pathways, especially the thioredoxin system, within the immune system of vertebrate species.

Characterization and gene expression analysis of the two main Th17 cytokines (IL-17A/F and IL-22) in turbot, Scophthalmus maximus

This report describes the cloning, characterization and gene expression pattern of two Th17 cytokines, interleukin (IL)-17A/F and -22, in turbot Scophthalmus maximus. The turbot IL-17A/F cDNA contains a 516 bp open reading frame encoding a deduced IL-17A/F protein of 171 amino acid (aa) residues, containing a predicted signal peptide of 31 aa. Turbot IL-22 had a 564 bp ORF coding for a 187 aa protein with a 33 aa signal peptide. The turbot IL-22 protein contained a typical IL-10 family signature. Both cytokines had highest expression levels in the intestine followed by head kidney and gills. Stimulation with the Gram negative bacterium Aeromonas salmonicida was able to modulate IL-17A/F and IL-22 expression in head kidney, spleen and liver but not the intestine. PMA and PHA were also able to induce the expression of both cytokines, suggesting that, as expected, T-cells are likely the main producers of these molecules in turbot as in mammals.

Red mark syndrome in rainbow trout Oncorhynchus mykiss: investigation of immune responses in lesions using histology, immunohistochemistry and analysis of immune gene expression.

Red mark syndrome (RMS) is an economically significant disease which affects farmed rainbow trout in the United Kingdom, in the US and in mainland Europe. From the pattern of incidence, it appears to be transmissable, although no causative agent has yet been identified. RMS presents as a severe lymphocytic infiltration centred on the dermis and an alternative, host-focused approach was taken to understand the disease through investigating immune responses occurring in the lesion. Lesion and non-lesion skin at different stages of lesion development were examined using histochemistry and immunohistochemistry on paraffin sections. Expression of immune-related genes was compared between lesion and non-lesion skin. Investigation of early stage lesions suggested that the initial immune response is targeted at the region of the scale pocket, with lymphocyte infiltration and anti-tumour necrosis factor (TNF)-α staining of the stratum spongiosum, and increased numbers of major histocompatibility complex (MHC) II-positive cells immediately adjacent to the scale pocket. Gene expression analysis suggested a counterbalancing T helper (Th)1 and T regulatory (Treg) - type response is occurring in the lesion, with repression of Th2 and Th17-type responses.