Ningqiu Li

A shared antigen among Vibrio species: outer membrane protein-OmpK as a versatile Vibriosis vaccine candidate in Orange-spotted grouper (Epinephelus coioides).

The outer membrane protein-OmpK has been considered as a vaccine candidate for the prevention of infections due to Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus in fish. Interestingly, the polyclonal antibody raised against the recombinant OmpK from V. harveyi strain EcGs020802 recognized the OmpK homologues from other strains of Vibrio species by immunoblotting. The ompK genes from 19 Vibrio strains including V. harveyi (11), V. alginolyticus (6) and V. parahaemolyticus (2) were then cloned and sequenced. Alignment analysis based on the amino acid sequences indicated that the OmpK from V. harveyi strain EcGs020802 had 71.7-99.2% of identities with those from V. harveyi, V. alginolyticus and V. parahaemolyticus. Western blot analysis revealed that the corresponding native proteins ranged between 28 and 31 kDa, consistent with predicated molecular weight of OmpK in Vibrio strains. Furthermore, the cross-protective property of recombinant OmpK was evaluated through challenge with heterogeneous virulent Vibrio strains in Orange-spotted groupers (Epinephelus coioides). Orange-spotted groupers vaccinated with recombinant OmpK were more tolerant of the infection by virulent Vibrio strains and their relative percentage survival (RPS) was correlative with the degree of the identity of deduced amino acid sequences of their OmpK. Taken together, the OmpK is a conserved protective antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of infections due to V. harveyi, V. alginolyticus and V. parahaemolyticus.

Genotype and host range analysis of infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease causing high mortality in mandarin fish, Siniperca chuatsi. In this study, complete major capsid protein (MCP) genes of nine ISKNV isolates were sequenced and compared with other known megalocytiviruses to evaluate genetic variation and host range of the viruses. Comparison of nucleotide sequences of MCP gene revealed 92.6–100% identity among nine ISKNV isolates. A phylogenetic tree revealed that 33 megalocytiviruses were divided into three genotypes, and there was a strong host species signal in three genotypes: for genotype I, the host was mainly marine fish; for genotype II, the host was freshwater fish; and for genotype III, the host was mainly flatfish. Nine ISKNV isolates belonged to genotype I or genotype II, suggesting mandarin fish may be a mixing vessel host for megalocytivirus.

Protective immunity against infectious spleen and kidney necrosis virus induced by immunization with DNA plasmid containing mcp gene in Chinese perch Siniperca chuatsi.

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease leading to high mortality and economic losses in Chinese perch, Siniperca chuatsi. There is an urgent need to develop an effective vaccine against this fatal disease. In this study, the mcp gene encoding the major capsid protein, the predominant structural component of the iridovirus particles, was cloned into a eukaryotic expression vector pcDNA3.1+, and the recombinant plasmid, designated as pcMCP, was constructed. Expression of the mcp gene was confirmed in transfected cells and muscle tissues of vaccinated fish by RT-PCR, immunodot blot and western blot. Immune response was induced by intramuscular injection of Chinese perch with pcMCP added QCDC adjuvant. The expression levels of type I IFN system genes including IRF-7, IRAK1, Mx and Viperin were up-regulated at 6 h, and reached a peak at 48 h. In addition, there was a second peak of the expression levels of IRF-7 and Mx gene on the 21st day post-vaccination. Before the 21st day post-vaccination, the levels of IgM did not show a significant difference among all groups, but there was a remarkable increase on the 28th day post-vaccination. The relative percent survival (RPS) of Chinese perch vaccinated with pcMCP added QCDC adjuvant was 80% in a challenge trial on the 28th day post-vaccination. Moreover, real-time PCR demonstrated that the levels of viral load in the dead fish of the vaccinated group were significantly higher than those in mock-vaccinated fish. Together, these results indicate that pcMCP is a potential candidate DNA vaccine against ISKNV disease.

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.

An outbreak of granulomatous inflammation associated with Francisella noatunensis subsp. orientalis in farmed tilapia (Oreochromis niloticus × O. aureus) in China

In 2013, a novel disease was detected in tilapia (Oreochromis niloticus × O. aureus) in Guangzhou, South China. To identify the causative pathogen, we conducted histological examination, bacteria isolation, and purification, and sequenced the 16S rRNA gene of isolates. Infected fish had a large number of white granulomas in the kidney, liver, heart, and spleen. The head kidney and spleen were markedly swollen. A bacterium strain designated as gz201301 was recovered from the spleen of infected tilapia. The 16S rRNA sequence of gz201301 revealed that it was highly similar to F. noatunensis subsp. orientalis. This is the first report of a Francisella-like infection in farmed tilapia in China.

Protein encoded by ORF093 is an effective vaccine candidate for infectious spleen and kidney necrosis virus in Chinese perch Siniperca chuatsi

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a disease causing high mortality and economic losses in Chinese perch, Siniperca chuatsi in China. Little information about the antigenicity of ISKNV proteins is available. In this study the ORF093 gene of ISKNV was cloned into the prokaryotic expression vector pET32a(+) and eukaryotic expression vector pcDNA3.1(+), and designated as pET-093 and pcDNA-093, respectively. A recombinant 51-kDa protein was detected in Escherichia coli BL21 (harboring pET-093) after IPTG inducement. Polyclonal antibodies were raised in rabbits against the purified ORF093 protein and the reaction of the antibodies was confirmed by western blotting using the purified recombinant protein. Expression of the pcDNA-093 in muscle tissue of vaccinated fish was confirmed by western blotting. The protection efficacy of ORF093 was investigated and results showed that cumulative mortality of Chinese perch was significant differences between immune groups and control (P<0.05) after ISKNV challenge, and the RPS value of 093 recombinant protein, pcDNA093 and pcDNA093+MCP was 47%, 50% and 57%. The results suggested that ORF093 is an effective vaccine candidate for ISKNV and it can be used in the control of ISKNV disease in Chinese perch.

The biological features and genetic diversity of novel fish rhabdovirus isolates in China

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses which infects mammals, birds, reptiles, fish, insects and plants. Herein, we reported the isolation and characterization of 6 novel viruses from diseased fish collected from China including SCRV-QY, SCRV-SS, SCRV-GM, CmRV-FS, MsRV-SS, OmbRV-JM. The typical clinical symptom of diseased fish was hemorrhaging. Efficient propagation of these isolates in a Chinese perch brain cell line was determined by means of observation of cytopathic effect, RT-PCR and electron microscopy. Sequence alignment and phylogenetic analysis of the complete G protein sequences revealed that these isolates were clustered into one monophyletic lineage belonging to the species Siniperca chuatsi rhabdovirus.

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.

A TFPI-1 peptide that induces degradation of bacterial nucleic acids, and inhibits bacterial and viral infection in half-smooth tongue sole, Cynoglossus semilaevis.

ABSTRACT Tissue factor pathway inhibitor 1 (TFPI‐1) is a serine protease inhibitor that inhibits tissue factor (TF)‐mediated coagulation. The C‐terminal region of TFPI‐1 could be cleaved off and proved to be antimicrobial against a broad‐spectrum of microorganism. In a previous study, a C‐terminal peptide, TC24 (with 24 amino acids), derived from tongue sole (Cynoglossus semilaevis) TFPI‐1, was synthesized and found antibacterial against Micrococcus luteus. In the present study, the antibacterial spectrum and the action mode of TC24 was further examined, and its in vivo function was analyzed. Our results showed that TC24 also possesses bactericidal activity against Staphylococcus aureus and Vibrio vulnificus. During its interaction with the target bacterial cells, TC24 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA and total RNA. In vivo study showed that administration of tongue sole with TC24 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. These results indicated that TC24 is a novel antimicrobial peptide against bacterial and viral pathogens, and that the observed effect of TC24 on bacterial RNA adds new insights to the action mechanism of fish antimicrobial peptides. Moreover, TC24 may play an important role in fighting pathogenic infection in aquaculture. HIGHLIGHTSA tongue sole TFPI‐1 peptide TC24 displayed bactericidal activity against Staphylococcus aureus and Vibrio vulnificus.TC24 could destroy cell membrane integrity, penetrated into the cytoplasm, and induced degradation of nucleic acids.TC24 could inhibit bacterial or viral invasion in tongue sole.

Autophagy promoted infectious kidney and spleen necrosis virus replication and decreased infectious virus yields in CPB cell line

ABSTRACT Autophagy plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of infectious kidney and spleen necrosis virus (ISKNV), an agent that has caused devastating losses in Chinese perch (Siniperca chuatsi) industry. We found that ISKNV infection triggered the complete autophagic process, as demonstrated by microtubule‐associated protein 1 light chain 3B II (LC3B‐II) conversion, an increased accumulation of punctate GFP‐LC3‐expressing cells, a higher number of autophagosome‐double‐membrane vesicles in the cytoplasm, and increased levels of autophagic flux in CPB cells. Then, we investigated the role of autophagy in the process of ISKNV replication. Results showed that inducing autophagy by rapamycin promoted ISKNV replication and proteins synthesis but decreased extracellular virus yields. While, blocking autophagosome‐lysosome fusion by chloroquine (CQ) promoted infectious virus yields in culture supernatant. These results offer insight into the complex interactions between ISKNV and host cell, providing new insights into viral pathogenesis and antiviral treatment strategies. HIGHLIGHTSAutophagy was triggered by ISKNV infection in CPB cells.ISKNV infection increased the levels of autophagic flux.Induction of autophagy promoted ISKNV replication.Inhibition of autophagy increased extracellular ISKNV yields.