Lingli Zhou

Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus (GNNV) with antiviral activities and delivery potential in grouper cells

Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.

Establishment and characterization of a novel cell line from the brain of golden pompano (Trachinotus ovatus)

Golden pompano is a commercially important marine fish that is widely cultured in China, Japan, and Southeast Asian countries but has been seriously threatened by pathogen. A novel cell line (TOGB) derived from the brain of golden pompano Trachinotus ovatus was established and characterized in this study. TOGB cell line showed high virus susceptibility, especially grouper nervous necrosis virus (GNNV) and Singapore grouper iridovirus (SGIV). As one of the most devastating viruses in marine fish aquaculture, nervous necrosis virus (NNV) causes high mortality rates exceeding 95% in severe outbreaks. Then, TOGB cell line was a useful tool for propagating viruses and provides a potentially valuable resource for the study of viral pathogenesis, the development of antiviral strategies. The TOGB cell lines showed potential application in environmental monitoring. The extracellular products from Vibrio anguillarum and Vibrio alginolyticus demonstrated cytotoxic effects in TOGB cells. TOGB cells grew most rapidly at 28°C, with an optimal concentration of 10% fetal bovine serum in L-15 medium. TOGB cells were diploid (2N = 54). The transfection efficiencies of TOGB cells were 8.6% at the 15th passage and 64.8% at the 45th passage, indicating that the cells are suitable for foreign gene expression.

Selection and characterization of novel DNA aptamers specifically recognized by Singapore grouper iridovirus-infected fish cells.

Singapore grouper iridovirus (SGIV) is a major viral pathogen of grouper aquaculture, and has caused heavy economic losses in China and South-east Asia. In this study, we generated four ssDNA aptamers against SGIV-infected grouper spleen (GS) cells using SELEX (systematic evolution of ligands by exponential enrichment) technology. Four aptamers exhibited high affinity to SGIV-infected GS cells, in particular the Q2 aptamer. Q2 had a binding affinity of 12.09 nM, the highest of the four aptamers. These aptamers also recognized SGIV-infected tissues with high levels of specificity. Protease treatment and flow cytometry analysis of SGIV-infected cells revealed that the target molecules of the Q3, Q4 and Q5 aptamers were trypsin-sensitive proteins, whilst the target molecules of Q2 might be membrane lipids or surface proteins that were not trypsin-sensitive. The generated aptamers appeared to inhibit SGIV infection in vitro. Aptamer Q2 conferred the highest levels of protection against SGIV and was able to inhibit SGIV infection in a dose-dependent manner. In addition, Q2 was efficiently internalized by SGIV-infected GS cells and localized at the viral assembly sites. Our results demonstrated that the four novel aptamers we generated were specific for SGIV-infected cells and could potentially be applied as rapid molecular diagnostic test reagents or therapeutic drugs targeting SGIV.

A tumour necrosis factor receptor-like protein encoded by Singapore grouper iridovirus modulates cell proliferation, apoptosis and viral replication.

It has been demonstrated that tumour necrosis factor receptor (TNFR) homologues encoded by viruses are usually involved in virus immune evasion by regulating the host immune response or mediating apoptotic cell death. Here, a novel TNFR-like protein encoded by Singapore grouper iridovirus (SGIV VP51) was cloned and characterized. Amino acid analysis showed that VP51 contained three cysteine-rich domains (CRDs) and a transmembrane domain at its C terminus. The expression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection via inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral titre and the expression of viral structural protein gene MCP and cell proliferation promoting gene ICP-18. In contrast, the expression of the viral apoptosis inducing gene, LITAF, was significantly decreased. Although all three CRDs were essential for the action of VP51, CRD2 and CRD3 exerted more crucial roles on virus-induced apoptosis, viral gene transcription and virus production, while CRD1 was more crucial for cell proliferation. Together, SGIV TNFR-like products not only affected cell cycle progression and enhanced cell growth by increasing the expression of the virus encoded cell proliferation gene, but also inhibited virus-induced apoptotic cell death by decreasing the expression of the viral apoptosis inducing gene. Our results provided new insights into understanding the underlying mechanism by which iridovirus regulated the apoptotic pathway to complete its life cycle.

Singapore grouper iridovirus (SGIV) TNFR homolog VP51 functions as a virulence factor via modulating host inflammation response.

Virus encoded tumor necrosis factor receptor (TNFR) homologues are usually involved in immune evasion by regulating host immune response or cell death. Singapore grouper iridovirus (SGIV) is a novel ranavirus which causes great economic losses in aquaculture industry. Previous studies demonstrated that SGIV VP51, a TNFR-like protein regulated apoptotic process in VP51 overexpression cells. Here, we developed a VP51-deleted recombinant virus Δ51-SGIV by replacing VP51 with puroR-GFP. Deletion of VP51 resulted in the decrease of SGIV virulence, evidenced by the reduced replication in vitro and the decreased cumulative mortalities in Δ51-SGIV challenged grouper compared to WT-SGIV. Moreover, VP51 deletion significantly increased virus induced apoptosis, and reduced the expression of pro-inflammatory cytokines in vitro. In addition, the expression of several pro-inflammatory genes were decreased in Δ51-SGIV infected grouper compared to WT-SGIV. Thus, we speculate that SGIV VP51 functions as a critical virulence factor via regulating host cell apoptosis and inflammation response.

Establishment and characterization of a mid-kidney cell line derived from golden pompano Trachinotus ovatus, a new cell model for virus pathogenesis and toxicology studies

Golden pompano Trachinotus ovatus, a popularly cultured and commercially important marine fish worldwide, has been recognized as a promising candidate for mariculture. However, outbreaks of infectious bacterial or viral diseases and environmental deterioration have led to great economic losses in T. ovatus aquaculture recently. In our research, we established a new mid-kidney cell line, designated as TOK, from golden pompano, T. ovatus. The optimized growth temperature and working concentration of fetal bovine serum (FBS) were 28°C and 10–20%, respectively. Foreign genes could express well in TOK cells. The modal number of TOK cells was 54. The TOK cells were susceptive to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), and the virus could propagate in cells. Propagation was verified by qRT-PCR, and virions were observed under electron microscopy. Cytotoxicity analysis revealed that TOK cells were sensitive to different concentrations of extracellular products (ECPs) from Vibrio alginolyticus and V. anguillarum. Moreover, heavy metals (Cd, Cu, and Hg) also showed dose-dependent cytotoxicity to the TOK cell line. We established a mid-kidney cell line from T. ovatus which could be applied to cytotoxicity assays of heavy metals. The newly established TOK cell line possesses great application potential in genetic manipulation, virus–host interaction studies, and toxicity assays of bacterial extracellular products and heavy metals.

Complete mitochondrial genome sequence of the pelagic chaetognath, sagitta ferox

Abstract Sagitta ferox is an important group of transparent marine metazoans in marine pelagic food webs, the diversity study of S. ferox in nature ecosystem is important. In this study, we report the complete mitochondrial genome sequences of S. ferox. Its complete mtDNA sequence is 12 153 bp in length, which contains 11 protein-coding genes, 11 transfer RNA genes, and 2 ribosomal RNA genes. The composition of A, T, G, and C in mtDNA is 30.23%, 28.39%, 22.13%, and 19.25%, respectively. The percentage of A + T is 58.62%. The complete mitogenome of S. ferox could be applied in the studies of biodiversity researches, molecular systematics, and the evaluation of marine ecological environment.

Development and characterization of aptamer‐based enzyme‐linked apta‐sorbent assay for the detection of Singapore grouper iridovirus infection

Singapore grouper iridovirus (SGIV) is a devastating aquaculture virus responsible for heavy economic losses to grouper, Epinephelus sp. aquaculture. The aim of this study was to develop a rapid and sensitive detection method for SGIV infections in infected groupers.

The mitochondrial genome of the pelagic chaetognath, Pterosagitta draco

Abstract Pterosagitta draco is an important predator in many food webs, and so maintaining the diversity study of Pterosagitta draco in marine ecosystem is essential. In this paper, the complete mitochondrial genome sequence of Pterosagitta draco was determined. Its complete mtDNA sequence is 10,426 bp in length, which contains 11 protein-coding genes (PCGs), 6 transfer RNA genes and 2 ribosomal RNA genes. The composition of A, T, G, C in mtDNA is 27.63%, 23.61%, 23.53% and 25.23%, respectively. The percentage of A + T is 51.24%. The complete mitochondrial genome sequence would be useful for further phylogenetic analysis in Pterosagitta draco.

MicroRNA-146a promotes red spotted grouper nervous necrosis virus (RGNNV) replication by targeting TRAF6 in orange spotted grouper, Epinephelus coioides

ABSTRACT MicroRNA‐146a (miR‐146a) has been demonstrated to function as a negative regulator of cellular immune responses against pathogens in mammals, however, little information focused on its functions in lower vertebrates. In this study, we investigated the regulatory roles of orange spotted grouper, Epinephelus coioides miR‐146a during red spotted grouper nervous necrosis virus (RGNNV) infection. During RGNNV infection in grouper spleen (GS) cells, the endogenous expression level of miR‐146a and tumor necrosis factor receptor‐associated factor 6 (TRAF6) significantly increased along with the infection time. Overexpression of miR‐146a significantly facilitated viral infection, evidenced by the increased transcription of viral CP and RdRp genes, while miR‐146a knockdown by specific inhibitors decreased RGNNV replication. Using pMIR‐REPORT Luciferase system, we found that the 3′ untranslated region (UTR) of grouper TRAF6 could be specifically targeted by miR‐146a. Further studies showed that its downstream target gene pro‐inflammatory cytokines, including TNF‐&agr;, IL‐8 and IL‐1&bgr;, were all significantly decreased in miR‐146a mimic transfected cells, but increased in miR‐146a inhibitors transfected cells during RGNNV infection. Thus, our results suggested and verified that holding the level of miR‐146a exerted crucial roles in RGNNV infection through TRAF6‐mediated inflammatory response. HighlightsRGNNV infection persistently induced the expression of miR‐146a in grouper cells.High level of miR‐146a significantly facilitated RGNNV infection.miR‐146a attenuated the production of pro‐inflammatory cytokines.The 3′ untranslated region of grouper TRAF6 could be targeted by miR‐146a.