Jiagang Tu

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.

Transcriptomic profiles of striped snakehead fish cells (SSN-1) infected with red-spotted grouper nervous necrosis virus (RGNNV) with an emphasis on apoptosis pathway

ABSTRACT Nervous necrosis virus (NNV), the causative agent of viral nervous necrosis (VNN) disease, has caused mass mortality of cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. However, the molecular mechanisms underlying the pathogenicity of NNV are still poorly understood. In this study, the transcriptomic profiles of striped snakehead fish (Channa striatus) cells (SSN‐1) infected with red‐spotted grouper NNV (RGNNV) were investigated using deep RNA sequencing technique. From 254,955,234 raw reads, a total of 253,338,544 clean reads were obtained and they were assembled into 93,372 unigenes. Differentially expressed genes (DEGs) were identified from RGNNV‐infected or mock‐infected SSN‐1 cells, including 1184 up‐regulated and 1456 down‐regulated genes at 3 h (h) post of infection (poi), and 1138 up‐regulated and 2073 down‐regulated genes at 24 h poi, respectively. These DEGs were involved in many pathways related to viral pathogenesis, including retinoic acid‐inducible gene I (RIG‐I) like receptors pathway, apoptosis pathway, oxidative phosphorylation, PI3K‐Akt signaling pathway, and MAPK signaling pathway. Subsequent analysis focusing on the apoptosis pathway showed that the expression of Endonuclease G (EndoG) was up‐regulated upon RGNNV infection at both 3 and 24 h poi. Therefore, EndoG gene was cloned and its function was further characterized. The results showed that over‐expression of EndoG could also induce cellular apoptosis in SSN‐1 cells, indicating that RGNNV infection might induce apoptosis of SSN‐1 cells via EndoG‐associated mitochondrial pathway. These results will shed a new light on the pathogenesis of NNV. HIGHLIGHTSTranscriptomic analysis of stripped snakehead fish cell SSN‐1 infected with RGNNV.Immune‐related pathway: apoptosis pathway.EndoG is involved in the apoptosis induced by RGNNV infection in SSN‐1 cells.

Susceptibility of Chinese Perch Brain (CPB) Cell and Mandarin Fish to Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) Infection

Nervous necrosis virus (NNV) is the causative agent of viral encephalopathy and retinopathy (VER), a neurological disease responsible for high mortality of fish species worldwide. Taking advantage of our established Chinese perch brain (CPB) cell line derived from brain tissues of Mandarin fish (Siniperca chuatsi), the susceptibility of CPB cell to Red-Spotted Grouper nervous necrosis virus (RGNNV) was evaluated. The results showed that RGNNV replicated well in CPB cells, resulting in cellular apoptosis. Moreover, the susceptibility of Mandarin fish to RGNNV was also evaluated. Abnormal swimming was observed in RGNNV-infected Mandarin fish. In addition, the cellular vacuolation and viral particles were also observed in brain tissues of RGNNV-infected Mandarin fish by Hematoxylin-eosin staining or electronic microscopy. The established RGNNV susceptible brain cell line from freshwater fish will pave a new way for the study of the pathogenicity and replication of NNV in the future.

Characterization of microRNAs in orange-spotted grouper (Epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection

Abstract Nervous necrosis virus (NNV), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (VNN) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. However, the molecular mechanisms underlying the pathogenicity of NNV remain elusive. In this study, the expression profiles of microRNA (miRNA) were investigated in grouper fin (GF‐1) cells infected with red‐spotted grouper nervous necrosis virus (RGNNV) via deep sequencing technique. The results showed that a total of 220 miRNAs were identified by aligning the small RNA sequences with the miRNA database of zebrafish, and 18 novel miRNAs were predicted using miRDeep2 software. Compared with the non‐infected groups, 51 and 16 differentially expressed miRNAs (DE‐miRNAs) were identified in the samples infected with RGNNV at 3 and 24 h, respectively. Six DE‐miRNAs were randomly selected to validate their expressions using quantitative reverse transcription polymerase chain reaction (qRT‐PCR), the results showed that their expression profiles were consistent with those obtained by deep sequencing. The target genes of the DE‐miRNAs covered a wide range of functions, such as regulation of transcription, oxidation‐reduction process, proteolysis, regulation of apoptotic process, and immune response. In addition, the effects of four DE‐miRNAs including miR‐1, miR‐30b, miR‐150, and miR‐184 on RGNNV replication were evaluated, and the results showed that over‐expression of each of the four miRNAs promoted the replication of RGNNV. These data provide insight into the molecular mechanism of RGNNV infection, and will benefit for the development of effective strategies to control RGNNV infection. HighlightsThe molecular mechanism on the pathogenicity of nervous necrosis virus (NNV) is poorly understood.This is the first study on the expression profiles of microRNA (miRNA) on NNV infection.We identified 116 new microRNAs in orange spotted grouper.

Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics.

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association

Significance Orthomyxoviruses, including the influenza viruses A–D, all encode a matrix protein M1 that forms a continuous matrix layer underneath the viral envelope. Here we report the crystal structure of a full-length M1 from an orthomyxovirus that infects fish. Consisting of two domains connected by a flexible linker, M1 forms an infinite two-dimensional lattice in crystal through parallel packing. A cryoelectron tomography reconstruction of intact virions confirmed that the M1 lattice closely mimics the viral matrix layer, with the N domain abutting the viral envelope and the C domain pointing toward the virus interior. Our findings provide valuable insights into the mechanisms of M1 in mediating self-oligomerization, membrane association, and viral genome packaging. Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1–M1, M1–membrane, and M1–RNP interactions in the virion.

Glutamine and glutaminolysis are required for efficient replication of infectious spleen and kidney necrosis virus in Chinese perch brain cells

Viruses rely on host cellular metabolism for energy and macromolecule synthesis during their replication. Infectious spleen and kidney necrosis virus (ISKNV) causes significant economic losses in the Chinese perch (Siniperca chuatsi) industry worldwide. However, little is known about the relationship between ISKNV replication and cellular metabolism. Using transcriptomic analysis, we observed that glutamine metabolism in Chinese perch brain (CPB) cells is altered during ISKNV infection. Moreover, ISKNV replication was decreased in CPB cells cultured in the glutamine-depleted medium. ISKNV replication was also inhibited in CPB cells cultured in the presence of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (an inhibitor of glutaminase), (–)-epigallocatechinmo nogallate (an inhibitor of glutamate dehydrogenase) or L-buthionine sulfoximine (an inhibitor of glutathione synthesis). However, virus replication was rescued by the addition of multiple tricarboxylic acid cycle intermediates, ATP, or glutathione reduced ethyl ester. ATP and reduced glutathione/oxidized glutathione levels were increased in CPB cells infected with ISKNV, but were decreased in CPB cells cultured in glutamine-depleted medium. These results indicate ISKNV infection induces glutaminolysis to accommodate the biosynthetic and energy needs for its efficient virus replication.

MicroRNA miR-214 inhibits snakehead vesiculovirus replication by targeting the coding regions of viral N and P

Snakeheadvesiculovirus (SHVV), a new member of the family Rhabdoviridae, has caused enormous economic losses in snakehead fish culture during the past years in China; however, little is known about the molecular mechanisms of its pathogenicity. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in virus infection. In this study, we identified that SHVV infection downregulated miR-214 in striped snakehead (SSN-1) cells in a time- and dose-dependent manner. Notably, transfecting SSN-1 cells with miR-214 mimic significantly inhibitedSHVV replication, whereas miR-214 inhibitor promoted it, suggesting that miR-214 acted as a negative regulator of SHVV replication. Our study further demonstrated that N and P of SHVV were the target genes of miR-214. Over-expression of P, but not N, inhibited IFN-α production in SHVV-infected cells, which could be restored by over-expression of miR-214. Taken together, these results suggest that miR-214 is downregulated during SHVV infection, and the downregulated miR-214 in turn increased N and P expression and decreased IFN-α production, thus facilitating SHVV replication. This study provides a better understanding of the molecular mechanisms on the pathogenesis of SHVV and a potential antiviral strategy against SHVV infection.

Transcriptomic analysis of the head kidney of Topmouth culter (Culter alburnus) infected with Flavobacterium columnare with an emphasis on phagosome pathway

Flavobacterium columnare (FC) has caused worldwide fish columnaris disease with high mortality and great economic losses in cultured fish, including Topmouth culter (Culter alburnus). However, the knowledge about the host factors involved in FC infection is little known. In this study, the transcriptomic profiles of the head kidney from Topmouth culter with or without FC infection were obtained using HiSeq™ 2500 (Illumina). Totally 79,641 unigenes with high quality were obtained. Among them, 4037 differently expressed genes, including 1217 up-regulated and 2820 down-regulated genes, were identified and enriched using databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The differently expressed genes were mainly associated with pathways such as immune response, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Since phagocytosis is a central mechanism of innate immune response by host cells to defense against infectious agents, genes related to the phagosome pathway were scrutinized and 9 differently expressed phagosome-related genes were identified including 3 up-regulated and 6 down-regulated genes. Five of them were further validated by quantitative real-time polymerase chain reaction (qRT-PCR). This transcriptomic analysis of host genes in response to FC infection provides data towards understanding the infection mechanisms and will shed a new light on the prevention of columnaris.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus)

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.