Zhibin Huang

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.

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.

Early protein ORF086 is an effective vaccine candidate for infectious spleen and kidney necrosis virus in mandarin fish Siniperca chuatsi.

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant loss in the Mandarin fish (Siniperca chuatsi) aquaculture industry. Vaccination is an important measure to prevent fatal ISKNV infection. In this study, the ORF086 gene encoding an early protein helicase of ISKNV was cloned into the prokaryotic pET32a (+) and eukaryotic pcDNA3.1 (+) expression vectors and designated as pET086 and pcDNA086, respectively. A recombinant 36 kDa protein was detected in Escherichia coli BL21 (harboring pET086) after isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. Polyclonal antibodies against the purified ORF086 protein were raised in rabbits. The antibody reaction and the pcDNA086 expression in muscle tissues of vaccinated fish were confirmed using Western blot analysis. The protective efficacy of ORF086 was also investigated. The cumulative mortality rates of Mandarin fish were significantly different between immune and control groups (P < 0.05) after ISKNV challenge. The relative percentage survival (RPS) values of the recombinant ORF086 protein emulsified with ISA763A adjuvant and pcDNA086 added with QCDC adjuvant were 73% and 63%, respectively. Transcriptional analysis of non-specific and specific immune related genes revealed that the expression levels of IRF-7, IRAK1, Mx, Viperin, and IgM were strongly up-regulated in the vaccinated groups post-immunization. In particular, the expression levels in the QCDC + pcDNA086 group was higher than those in the control groups (P < 0.05). These results indicated that the early protein ORF086 could be an effective antigen candidate for controlling ISKNV disease in Mandarin fish.

The pathogenicity and biological features of Santee-Cooper Ranaviruses isolated from Chinese perch and snakehead fish

Ranavirus has become a noticeable threat to both farmed and natural populations of fish and amphibians. Herein, we reported that 3 strains of novel viruses, designated as ScRIV-GM-20150902, CmRIV-XT-20150917 and ScRIV-ZS-20151201, were isolated from diseased Chinese perch and snakehead fish in China. Efficient propagation of these isolates were determined in Chinese perch brain (CPB) cell line by the means of cytopathic effect observation, PCR amplification and electron microscopy observation. And their viral titers in CPB cells reached 108.13 TCID50 ml-1, 107.71 TCID50 ml-1 and 107.94 TCID50 ml-1, respectively. While the challenge experiment results showed that 3 isolates resulted in 100% mortality of Chinese perch after virus infection. Electron microscopy analysis showed that two kinds of viral inclusion bodies (intracytoplasmic and intranuclear inclusion body) were observed in infected CPB cells. Sequence alignment and phylogenetic analysis of major capsid protein gene sequences of isolates revealed that these isolates belonged to the species Santee-Cooper Ranavirus.

Inhibition of Grass Carp Reovirus Infection by DNA Aptamers against S10 Protein

Grass Carp reovirus (GCRV) is one of the most pathogenic agents among aquareovirus isolates and has the ability to cause a severe epidemic outbreak of hemorrhagic disease, thus resulting in both a high mortality rate during the culture of Grass Carp Ctenopharyngodon idella and an enormous economic loss. Aptamers have been demonstrated to have strong promising applications in antiviral drug development. In the present study, a complementary DNA fragment encoding the S10 gene of GCRV was cloned. The S10 protein was expressed and purified. Aptamers for S10 protein were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and their characteristics and antiviral actions were examined. All targeting-selected aptamers formed a similar structure, forming a 5-7 base loop at the terminus. The results show that the aptamers could inhibit the GCRV infection. The most significant inhibitory effect was obsereved when the aptamers were added to the cell culture for 1 h before the cells were infected by GCRV. Our data showed that these novel molecular agents could be considered suitable candidates for anti-GCRV therapy. Received August 23, 2016; accepted February 5, 2017.

Mandarin fish p53: Genomic structure, alternatively spliced variant and its mRNA expression after virus challenge

ABSTRACT A number of size variants of the p53 protein have been described in mammal, but little is known about alternative splicing of p53 expression and function in the fish. In our previous study, the immune defense and antiviral responses of p53 had been determined in mandarin fish (Siniperca chuatsi). However, the role of its splicing variants remains unknown. In the present study, the organization of mandarin fish p53 (Sc‐p53) genome sequence was determined and a novel splice variant was characterized. The Sc‐p53 genomic sequence was composed of 5543 bp, containing 11 exons and 10 introns, which was similar to other species. Then, a 1106 bp full‐length cDNA of a novel splice variant p53 from mandarin fish (designed as Sc‐p53I6) was cloned and characterized. Quantitative real‐time PCR assays revealed that Sc‐p53I6 was expressed in all tissues examined, and it was most abundant in the gill, hemocyte and hind kidney. Western blotting analysis revealed that Sc‐p53I6 protein was abundant in liver, trunk kidney, hind kidney, stomach and heart. In addition, the regulation of Sc‐p53I6 gene expression after virus infection was determined and characterized. The results showed twice rise expression pattern of Sc‐p53I6 in CPB cells and spleen of mandarin fish in response to infectious kidney and spleen necrosis virus (ISKNV). However, a different expression pattern, once rise, of Sc‐p53I6 in response to Siniperca chuatsi rhabdovirus (SCRV) infection was found. The mRNA expression of Sc‐p53I6 was significantly up‐regulated in CPB at 4 h and spleen of mandarin fish at 12 h post‐infection. These results will shed a new light on antiviral response mechanisms of p53 in mandarin fish. HighlightsThe genome of mandarin fish p53 was cloned and analyzed.A novel isoform splice variant of mandarin fish 53 (designed as Sc‐p53I6) was cloned and characterized.The tissue distribution of Sc‐p53I6 in mandarin fish was determined by qRT‐PCR.The mRNA expression patterns of Sc‐p53I6 were determined in CPB cell line and mandarin fish following virus challenge.

Molecular cloning and expression of a heat-shock cognate 70 (hsc70) gene from swordtail fish (Xiphophorus helleri)

Heat shock proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In the present study, a full-length cDNA, encoding the constitutively expressed 70-kDa heat shock cognate protein (Hsc70), was isolated from swordtail fish (Xiphophorus helleri) and designated as XheHsc70. The Xhehsc70 cDNA was 2 104 bp long with an open reading frame of 1 941 bp, and it encoded a protein of 646 amino acids with a theoretical molecular weight of 70.77 kDa and an isoelectric point of 5.04. The deduced amino acid sequence shared 94.1%-98.6% identities with the Hsc70s from a number of other fish species. Tissue distribution results show that the Xhehsc70 mRNA was expressed in brain, heart, head kidney, kidney, spleen, liver, muscle, gill, and peripheral blood. After immunization with formalin-killed Vibrio alginolyticus cells there was a significant increase in the Xhehsc70 mRNA transcriptional level in the head kidney of the vaccinated fish compared with in the control at 6, 12, 24, and 48 h as shown by quantitative real time RT-PCR. Based on an analysis of the amino acid sequence of XheHsc70, its phylogeny, and Xhehsc70 mRNA expression, XheHsc70 was identified as a member of the cytoplasmic Hsc70 (constitutive) subfamily of the Hsp70 family of heat shock proteins, suggesting that it may play a role in the immune response. The Xhehsc70 cDNA sequence reported in this study was submitted to GenBank under the accession number JF739182.

Construction of Edwardsiella tarda ghosts from eel and their optimization of preparation conditions

Edwardsiellosis caused by Edwardsiella tarda is one of the most serious diseases in the eel culture industry.Bacterial ghosts,which have native surface antigenic structures and can induce humoral and cellular immune response,have become candidate of vaccine development.To explore the feasibility of E.tarda ghost vaccine in eel,E.tarda ghosts were generated by the thermal controlled expression of the PhiX174 lysis gene E in present study.At the same time the morphology,lysis kinetics,lysis efficiency and preparation conditions were also studied.Holes were observed in Escherichia coli and E.tarda ghosts by scanning electron microscopy and the bacteria become withered due to the loss of bacterial content.The lysis kinetics in E.tarda were also compared with those in E.coli.Generation of ghosts in the transformants of E.coli and E.tarda carrying plasmid pBVlysisE was performed successfully by increasing the incubation temperature up to 42℃.Compared to E.coli,in which lysis was observed within 30 min and was completed 3 h after induction of E gene expression,onset of E.tarda lysis occurred 1 h after temperature elevation and the lysis process was completed 5 h after induction.At the end of the lysis process,the efficiency of ghost induction in non-lyophilized E.tarda was 99.99%,as the results of 5 replicate experiments showed.However,no bacterial growth was detected in lyophilized E.tarda and there is no obvious difference in bacterial morphology before and after lyophilization.The lysis kinetics and lysis efficiency had no difference between beginning of induction at OD600=0.4 and 0.6.Among LB,BHI and NB medium,LB medium is the best for preparation of E.tarda because in this medium the ghosts are more integrated and lysed completely.In the study we successfully constructed E.tarda ghosts and optimized their preparation conditions,which will provide the foundation for development of vaccine against edwardsiellosis in eel.