Tongyan Lu

Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry

The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

A effective DNA vaccine against diverse genotype J infectious hematopoietic necrosis virus strains prevalent in China

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In this study, a DNA vaccine, designated pIHNch-G, was constructed with the glycoprotein (G) gene of a Chinese IHNV isolate SD-12 (also called Sn1203) of genotype J. The minimal dose of vaccine required, the expression of the Mx-1 gene in the muscle (vaccine delivery site) and anterior kidney, and the titers of the neutralizing antibodies produced were used to evaluate the vaccine efficacy. To assess the potential utility of the vaccine in controlling IHNV throughout China, the cross protective efficacy of the vaccine was determined by challenging fish with a broad range of IHNV strains from different geographic locations in China. A single 100ng dose of the vaccine conferred almost full protection to rainbow trout fry (3g) against waterborne or intraperitoneal injection challenge with IHNV strain SD-12 as early as 4days post-vaccination (d.p.v.), and significant protection was still observed at 180d.p.v. Intragenogroup challenges showed that the DNA vaccine provided similar protection to the fish against all the Chinese IHNV isolates tested, suggesting that the vaccine can be widely used in China. Mx-1 gene expression was significantly upregulated in the muscle tissue (vaccine delivery site) and anterior kidney in the vaccinated rainbow trout at both 4 and 7d.p.v. Similar levels of neutralizing antibodies were determined with each of the Chinese IHNV strains at 60 and 180d.p.v. This DNA vaccine should play an important role in the control of IHN in China.

Preliminary study of an oral vaccine against infectious hematopoietic necrosis virus using improved yeast surface display technology

&NA; Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Because oral vaccines induce more efficient mucosal immunity than parenteral immunization, an oral vaccine was developed with an improved yeast cell surface display technology to induce an immune response to IHNV. The oral yeast vaccine, designated EBY100/pYD1‐bi‐G, was delivered orally to rainbow trout (Oncorhynchus mykiss) on days 1 and 32, and the nonspecific and specific immune responses were measured 50 days after the first vaccination. In the hindgut, spleen, and head kidney, the expression of IFN‐1 and Mx‐1 was significantly upregulated after oral vaccination with EBY100/pYD1‐bi‐G, and the highest expression of IFN‐1 and Mx‐1 was observed in the spleen (7.5‐fold higher than the control group) and head kidney (3.9‐fold higher than the control group), respectively. Several markers of the adaptive immune response (IgM, IgT, CD4, and CD8) were also significantly upregulated, and the highest expression of these markers was observed in the hindgut, suggesting that the mucosal immune response was successfully induced by oral vaccination with EBY100/pYD1‐bi‐G. Sera from the orally vaccinated rainbow trout showed higher anti‐IHNV neutralizing antibody titers (antibody titer 81 ± 4) than the control sera (antibody titer 7 ± 3), and the relative percentage survival after IHNV challenge was 45.8% compared with 2% in the control group. Although the protection afforded by this orally delivered vaccine was lower than that of a DNA vaccine (83%–98%), it is a promising candidate vaccine with which to protect larval fish against IHNV, which are most susceptible to the virus and difficult to inject with a DNA vaccine. HighlightsPreliminary study of an oral vaccine against infectious hematopoietic necrosis virus.The oral vaccine was designed using improved yeast surface display technology.The yeast oral vaccine induced an efficient mucosal immune response.It can be used as primer or booster vaccination before or after DNA vaccination.

Trancriptome profiles of Amur sturgeon spleen in response to Yersinia ruckeri infection

ABSTRACT Yersinia ruckeri (YR) is the causative agent of yersiniosis which has caused significant economic losses in fish culture worldwide, including in Amur sturgeon (Acipenser schrenckii) culture. To better understand the mechanism of the immune responses to YR in Amur sturgeon, the transcriptomic profiles of the spleens from YR‐infected and non‐infected groups were obtained using RNA‐seq techniques. The de novo assemblies yielded totally 145 670 unigenes from the two libraries. The total numbers of transcripts in YR‐infected and non‐infected groups were from 110 893 to 147 336, with the mean length varying from 560 to 631 (N50: from 882 to 1083). GO analysis revealed that 10 038 unigenes were categorized into 26 biological processes subcategories, 17 cellular components subcategories and 19 molecular functions subcategories. A total of 59 487 unigenes were annotated in the KEGG pathway and 20 pathways were related to the immune system. 1465 differently expressed genes (DEGs) were identified, including 377 up‐regulated genes and 1088 down‐regulated genes. 125 DEGs were found to be related to immune responses of Amur sturgeon and further divided into 16 immune‐related KEGG pathways, including antigen processing and presentation, complement and coagulation cascades, T cell receptor signaling pathway, B cell receptor signaling pathway, NOD‐like receptor signaling pathway, chemokine signaling pathway, etc. Eight of the DEGs were further validated by qRT‐PCR. Altogether, the results obtained in this study will provide insight into the immune response of Amur sturgeon against Y. ruckeri infection. HighlightsTranscriptome of Amur sturgeon infected with Yersinia ruckeri was analyzed.Totally 145 670 unigenes were yielded by de novo assembly.Totally 1465 DGEs were identified under Y. ruckeri challenge.125 DEGs were divided into 16 immune‐related KEGG pathways.Eight of DEGs were chosen for further validation using qRT‐PCR.

Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens of salmon and trout. An active bivalent DNA vaccine was constructed with the glycoprotein gene of Chinese IHNV isolate Sn1203 and VP2–VP3 gene of Chinese IPNV isolate ChRtm213. Rainbow trout (5 g) were vaccinated by intramuscular injection with 1.0 µg of the bivalent DNA vaccine and then challenged with an intraperitoneal injection of IHNV, IPNV, or both, at 30 and 60 days post-vaccination (d.p.v.). High protection rates against IHNV were observed, with 6% and 10% cumulative mortality, respectively, compared with 90–94% in the mock-vaccinated groups. IPNV loads (531-fold and 135-fold, respectively) were significantly reduced in the anterior kidneys of the vaccinated trout. Significant protection against co-infection with IHNV and IPNV was observed, with cumulative mortality rates of 6.67% and 3.33%, respectively, compared with 50.0% and 43.3%, respectively, in the mock-vaccinated groups. No detectable infective IHNV or IPNV was recovered from vaccinated trout co-infected with IHNV and IPNV. The bivalent DNA vaccine increased the expression of Mx-1 and IFN-γ at 4, 7, and 15 d.p.v, and IgM at 21 d.p.v., and induced high titres (≥160) of IHNV and IPNV neutralizing antibodies at 30 and 60 d.p.v.

The kinetics and protection of the antiviral state induced by recombinant iIFN1a in rainbow trout against infectious hematopoietic necrosis virus

The iIFN1a (intracellular IFN-a1), that is one of the IFN-a1 variants, was shown to be functional intracellularly and act as a novel defense against an infectious hematopoietic necrosis virus (IHNV). To determine its antiviral properties, a recombinant iIFN1a was generated in Escherichia coli. Its antiviral activity against IHNV was 1.69×10(7)U/mg in CHSE-214 cells. Additionally, iIFN1a was capable of inducing comparable levels of IRF-1, IRF-2, IFN-I, IFN-γ and Mx transcription in head kidney, spleen and liver tissues at an early time point (6h), that was followed by a rapid decline 24h after induction. The recombinant protein also elicited protection against IHNV in vivo. At 6 and 24h after induction there was 100% protection against the virus, however, at 48 and 72h the protection decreased to 57 and 40%, respectively. The in vivo protection kinetics correlated with the kinetics of gene expression. The results of this study provide details of the antiviral state that was induced by iIFN1a in vivo for the first time. Additionally, this information will facilitate the development of this recombinant protein as a potential anti-viral treatment and/or adjuvant.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China.

Complete genomic sequence of an infectious pancreatic necrosis virus isolated from rainbow trout (Oncorhynchus mykiss) in China

Infectious pancreatic necrosis (IPN) is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality in China. However, no gene sequence of any Chinese infectious pancreatic necrosis virus (IPNV) isolates was available. In the study, moribund rainbow trout fry samples were collected during an outbreak of IPN in Yunnan province of southwest China in 2013. An IPNV was isolated and tentatively named ChRtm213. We determined the full genome sequence of the IPNV ChRtm213 and compared it with previously identified IPNV sequences worldwide. The sequences of different structural and non-structural protein genes were compared to those of other aquatic birnaviruses sequenced to date. The results indicated that the complete genome sequence of ChRtm213 strain contains a segment A (3099 nucleotides) coding a polyprotein VP2–VP4–VP3, and a segment B (2789 nucleotides) coding a RNA-dependent RNA polymerase VP1. The phylogenetic analyses showed that ChRtm213 strain fell within genogroup 1, serotype A9 (Jasper), having similarities of 96.3% (segment A) and 97.3% (segment B) with the IPNV strain AM98 from Japan. The results suggest that the Chinese IPNV isolate has relative closer relationship with Japanese IPNV strains. The sequence of ChRtm213 was the first gene sequence of IPNV isolates in China. This study provided a robust reference for diagnosis and/or control of IPNV prevalent in China.

Effects of different cytokines on immune responses of rainbow trout in a virus DNA vaccination model

Seven rainbow trout cytokine genes (interleukin (IL)-2, IL-8, IL-15, IL-17, IL-1β, intracellular interferon (iIFN) 1a, and IFN-γ2) were evaluated for their adjuvant effects on a DNA vaccine, called pG, containing the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV). Distinct DNA constructs in expression plasmid pcDNA3.1 encoding a cytokine gene were generated. Immunofluorescence assays in rainbow trout gonadal cells demonstrated successful protein expression from all these constructs. Subsequently, fish were immunized with pG alone or together with a cytokine expression plasmid. Results showed that each cytokine plasmids at an appropriate dose showed notable effects on immune gene expression. IL-17 and IFN-γ2 can enhance early specific IgM response. All cytokines, except IL-8, can benefit initial neutralizing antibody (NAb) titers. At 35 days post immunization (dpi), NAb titers of fish immunized with pG and IL-2, iIFN1a, or IFN-γ2 plasmids remained at high levels (1:160). NAb titers of fish immunized with pG alone decreased to 1:40. IL-8 or IL-1β can enhance antigen-specific proliferative T-cell responses at 14 dpi. At 28 dpi, coinjection of pG with IL-2, IL-8, IL-15, or IL-17 plasmids induced considerably stronger lymphocyte proliferation than that with injection of pG alone. All cytokine plasmids delivered with pG plasmid enhanced protection of trout against IHNV-mediated mortality. These results indicate that the type and dose of trout cytokine genes injected into fish affect quality of immune response to DNA vaccination.

Autophagy induced by infectious hematopoietic necrosis virus inhibits intracellular viral replication and extracellular viral yields in epithelioma papulosum cyprini cell line

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Recent work demonstrated that autophagy plays an important role in pathogen invasion by activating innate and adaptive immunity. This study investigated the relationship between IHNV and autophagy in epithelioma papulosum cyprini cells. The electron microscopy results show that IHNV infection can induce typical autophagosomes which are representative structures of autophagy activation. The punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II were respectively confirmed by confocal fluorescence microscopy and western blotting. Furthermore, the effects of autophagy on IHNV replication were also clarified by altering the autophagy pathway. The results showed that rapamycin induced autophagy can inhibit both intracellular viral replication and extracellular viral yields, while autophagy inhibitor produced the opposite results. These findings demonstrated that autophagy plays an antiviral role during IHNV infection.