Vaccination Route Determines the Kinetics and Magnitude of Nasal Innate Immune Responses in Rainbow Trout (Oncorhynchus mykiss).

Many pathogens infect animal hosts via the nasal route. Thus, understanding how vaccination stimulates early nasal immune responses is critical for animal and human health. Vaccination is the most effective method to prevent disease outbreaks in farmed fish. Nasal vaccination induces strong innate and adaptive immune responses in rainbow trout and was shown to be highly effective against infectious hematopoietic necrosis (IHN). However, direct comparisons between intranasal, injection and immersion vaccination routes have not been conducted in any fish species. Moreover, whether injection or immersion routes induce nasal innate immune responses is unknown. The goal of this study is to compare the effects of three different vaccine delivery routes, including intranasal (IN), intramuscular (i.m.) injection and immersion (imm) routes on the trout nasal innate immune response. Expression analyses of 13 immune-related genes in trout nasopharynx-associated lymphoid tissue (NALT), detected significant changes in immune expression in all genes analyzed in response to the three vaccination routes. However, nasal vaccination induced the strongest and fastest changes in innate immune gene expression compared to the other two routes. Challenge experiments 7 days post-vaccination (dpv) show the highest survival rates in the IN- and imm-vaccinated groups. However, survival rates in the imm group were significantly lower than the IN- and i.m.-vaccinated groups 28 dpv. Our results confirm that nasal vaccination of rainbow trout with live attenuated IHNV is highly effective and that the protection conferred by immersion vaccination is transient. These results also demonstrate for the first time that immersion vaccines stimulate NALT immune responses in salmonids.

Flagellar regulation mediated by the Rcs pathway is required for virulence in the fish pathogen Yersinia ruckeri.

The flagellum is a complex surface structure necessary for a number of activities including motility, chemotaxis, biofilm formation and host attachment. Flagellin, the primary structural protein making up the flagellum, is an abundant and potent activator of innate and adaptive immunity and therefore expression of flagellin during infection could be deleterious to the infection process due to flagellin-mediated host recognition. Here, we use quantitative RT-PCR to demonstrate that expression of the flagellin locus fliC is repressed during the course of infection and subsequently up-regulated upon host mortality in a motile strain of Yersinia ruckeri. The kinetics of fliC repression during the infection process is relatively slow as full repression occurs 7-days after the initiation of infection and after approximately 3-logs of bacterial growth in vivo. These results suggests that Y. ruckeri possesses a regulatory system capable of sensing host and modulating the expression of motility in response. Examination of the master flagellar operon (flhDC) promoter region for evidence of transcriptional regulation and regulatory binding sites revealed potential interaction with the Rcs pathway through an Rcs(A)B Box. Deletion of rcsB (ΔrcsB) by marker-exchange mutagenesis resulted in overproduction of flagellin and unregulated motility, showing that the Rcs pathway negatively regulates biosynthesis of the flagellar apparatus. Experimental challenge with ΔrcsB and ΔrcsBΔfliC1ΔfliC2 mutants revealed that mutation of the Rcs pathway results in virulence attenuation which is dependent on presence of the flagellin gene. These results suggest that the inappropriate expression of flagellin during infection triggers host recognition and thus immune stimulation resulting in attenuation of virulence. In addition, RNAseq analyses of the ΔrcsB mutant strain verified the role of this gene as a negative regulator of the flagellar motility system and identified several additional genes regulated by the Rcs pathway.

Draft Genome Sequence of the Fish Pathogen Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare MS-FC-4 is a highly virulent genetic group 1 (formerly genomovar I) strain isolated from rainbow trout (Oncorhynchus mykiss). The draft genome consists of three contigs totaling 3,449,277 bp with 2,811 predicted open reading frames. F. columnare MS-FC-4 is a model strain for functional genomic analyses.

CK12a, a CCL19-like Chemokine That Orchestrates both Nasal and Systemic Antiviral Immune Responses in Rainbow Trout.

Chemokines and chemokine receptors have rapidly diversified in teleost fish but their immune functions remain unclear. We report in this study that CCL19, a chemokine known to control lymphocyte migration and compartmentalization of lymphoid tissues in mammals, diversified in salmonids leading to the presence of six CCL19-like genes named CK10a, CK10b, CK12a, CK12b, CK13a, and CK13b. Salmonid CCL19-like genes all contain the DCCL-conserved motif but share low amino acid sequence identity. CK12 (but not CK10 or CK13) is constitutively expressed at high levels in all four trout MALT. Nasal vaccination with a live attenuated virus results in sustained upregulation of CK12 (but not CK10 or CK13) expression in trout nasopharynx-associated lymphoid tissue. Recombinant His-tagged trout CK12a (rCK12a) is not chemotactic in vitro but it increases the width of the nasal lamina propria when delivered intranasally. rCK12a delivered intranasally or i.p. stimulates the expression of CD8α, granulysin, and IFN-γ in mucosal and systemic compartments and increases nasal CD8α+ cell numbers. rCK12a is able to stimulate proliferation of head kidney leukocytes from Ag-experienced trout but not naive controls, yet it does not confer protection against viral challenge. These results show that local nasal production of CK12a contributes to antiviral immune protection both locally and systemically via stimulation of CD8 cellular immune responses and highlight a conserved role for CK12 in the orchestration of mucosal and systemic immune responses against viral pathogens in vertebrates.

Draft Genome Sequence of the Fish Pathogen Flavobacterium columnare Strain CSF-298-10.

We announce here the draft genome assembly of Flavobacterium columnare CSF-298-10, a strain isolated from an outbreak of columnaris disease at a commercial trout farm in Hagerman Valley, Idaho, USA. The complete genome consists of 13 contigs totaling 3,284,579 bp, with an average G+C content of 31.5% and 2,933 predicted coding genes.

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

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 50days 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.

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.

DNA Vaccination Partially Protects Muskellunge against Viral Hemorrhagic Septicemia Virus (VHSV-IVb).

A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 µg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 105 plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 102 pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 105 pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G-vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species. Received May 1, 2016; accepted September 1, 2016.

An efficient and simple method to increase the level of displayed protein on the yeast cell surface.

BACKGROUND: The development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response. METHODS: To address this issue, we established an efficient and simple method of increasing the level of displayed protein on the yeast cell surface. We used the single chain variable fragment (scFv) of an antibody against the infectious hematopoietic necrosis virus isolate Sn1203 as a target display protein. The yeast-derived scFv was first displayed on the yeast surface by galactose induction, and then Escherichia coli-derived scFv was also displayed on the same yeast via an artificial anchoring condition to increase the total scFv level on the yeast surface. RESULTS: The levels of yeast- and E. coli-derived scFv displayed on the yeast cell surface were analyzed by flow cytometry, western blotting, and fluorescent microscopy. The flow cytometry results indicated that when the cells were suspended in phosphate-buffered saline with 1mmol/L glutathione, 0.2mmol/L oxidized glutathione, and 5% dimethyl sulfoxide at 4°C for 6h, the E. coli-derived scFv protein was stably anchored to the yeast cell surface. The mean fluorescence intensity in these experiments, which is an indirect quantitative representation of the surface scFv expression, was three times higher in the treated cells than that in control cells. The western blotting results show two specific protein bands, the smaller of which was identified as the E. coli-derived scFv that was displayed on the yeast cell surface. Cell immunofluorescence is a more direct way to detect differentially produced proteins that are displayed on the yeast cell surface. The fluorescence microscopy results show that both fluorescence corresponding to the yeast-derived scFv and fluorescence corresponding to the E. coli-derived scFv can exist on the cell surface of same yeast cell. This confirms that the E. coli-derived scFv protein was successfully displayed on the yeast cell surface. CONCLUSIONS: This method provides a rapid, simple, and high-efficiency strategy to increase the level of displayed protein on the yeast cell surface. Application of this technique may allow the yeast surface display system to be used to generate potential oral vaccines.

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout (Oncorhynchus mykiss).

A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout (Oncorhynchus mykiss) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5' and 3'-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89-99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16-42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae. However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Transmission potential of infectious hematopoietic necrosis virus in APEX-IHN®-vaccinated Atlantic salmon.

Infectious hematopoietic necrosis virus (IHNV) outbreaks have had a significant negative impact on Atlantic salmon Salmo salar production in British Columbia, Canada, since the first outbreak was reported in 1992. In 2005, the APEX-IHN® vaccine was approved for use in Canada for prevention of IHN. The vaccine was proven to be safe and efficacious prior to approval; however, it is unknown as to whether APEX-IHN®-vaccinated Atlantic salmon infected with IHNV can support replication and virus shedding in sufficient quantities to provide an infectious dose to a nearby susceptible host. To determine whether vaccinated, infected fish are able to transmit an infectious dose of IHNV, vaccinated Atlantic salmon were injected with IHNV (104 plaque-forming units per fish) and cohabitated with either naïve Atlantic salmon or naïve sockeye salmon Oncorhynchus nerka. APEX-IHN®-vaccinated fish were significantly protected against IHNV with mortality occurring in only 2.6% of the population as opposed to 97% in unvaccinated controls. Vaccination in IHNV-infected Atlantic salmon completely abolished disease transmission to cohabitating naïve sockeye salmon and reduced virus spread among cohabitating naïve Atlantic salmon. At 7 mo post-vaccination, IHNV-neutralizing antibodies were detected in nearly all vaccinated fish (94%) with similar titer occurring between vaccinated, infected fish and vaccinated, uninfected fish, indicating APEX-IHN® vaccination induces a robust seroconversion response. Taken together, these results demonstrate that vaccination greatly reduces the infectious load and potential for IHNV transmission. As such, APEX-IHN® should be included in fish health management strategies when culturing Atlantic salmon in IHNV endemic areas.

Zebrafish STAT6 negatively regulates IFNφ1 production by attenuating the kinase activity of TANK-binding kinase 1.

The aquatic spring viremia of carp virus (SVCV) causes significant mortality in common carp (Cyprinus carpio), and TBK1 plays a crucial role in the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system by phosphorylating its substrates to induce type I interferons (IFNs) and cellular antiviral responses. In this study, we report that zebrafish STAT6 is induced during SVCV infection and reduces IFNφ1 expression by suppressing TBK1 phosphorylation. A typical IFN stimulatory response element (ISRE) motif was found in the promoter region of zebrafish STAT6, and zebrafish STAT6 transcription was significantly upregulated in the early stages of virus infection. Overexpression of STAT6 interfered with IFNφ1 promoter activity in response to SVCV infection. Additionally, TBK1-, but not MITA-mediated activation of the IFNφ1 promoter was impaired by STAT6. Co-immunoprecipitation and Western blot experiments indicated that MITA and IRF3 were significantly phosphorylated by TBK1, and that the N-terminal kinase domain of TBK1 was critical in this process. In the final step, STAT6 interacted with the N-terminal kinase domain of TBK1 causing dephosphorylation, which resulted in reductions in the phosphorylation of IRF3 and the production of IFNφ1. These results indicate that fish STAT6 can attenuate the kinase activity of TBK1, leading to suppression of IFNφ1 expression which may in turn facilitate virus replication.

Tissue Microenvironments in the Nasal Epithelium of Rainbow Trout (Oncorhynchus mykiss) Define Two Distinct CD8α+ Cell Populations and Establish Regional Immunity.

Mucosal surfaces require balancing different physiological roles and immune functions. To effectively achieve multifunctionality, mucosal epithelia have evolved unique microenvironments that create unique regional immune responses without impairing other normal physiological functions. Whereas examples of regional immunity are known in other mucosal epithelia, to date, no immune microenvironments have been described in the nasal mucosa, a site where the complex functions of olfaction and immunity need to be orchestrated. In this study we identified the presence of CD8α+ cells in the rainbow trout (Oncorhynchus mykiss) nasal epithelium. Nasal CD8α+ cells display a distinct phenotype suggestive of CD8+ T cells with high integrin ß2 expression. Importantly, nasal CD8α+ cells are located in clusters at the mucosal tip of each olfactory lamella but scattered in the neuroepithelial region. The grouping of CD8α+ cells may be explained by the greater expression of CCL19, ICAM-1, and VCAM-1 in the mucosal tip compared with the neuroepithelium. Whereas viral Ag uptake occurred via both tip and lateral routes, tip-resident MHC class II+ cells are located significantly closer to the lumen of the nasal cavity than are their neuroepithelial counterparts, therefore having quicker access to invading pathogens. Our studies reveal compartmentalized mucosal immune responses within the nasal mucosa of a vertebrate species, a strategy that likely optimizes local immune responses while protecting olfactory sensory functions.

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.

Spring Viremia of Carp Virus N Protein Suppresses Fish IFNφ1 Production by Targeting the Mitochondrial Antiviral Signaling Protein.

For a virus to replicate efficiently, it must try and inhibit host IFN expression because IFN is an important host defense at early stages after viral infection. For aquatic viruses, the mechanisms used to escape the hosts IFN system are still unclear. In this study, we show that the N protein of spring viremia of carp virus (SVCV) inhibits zebrafish IFNφ1 production by degrading the mitochondrial antiviral signaling protein (MAVS). First, the upregulation of IFNφ1 promoter activity stimulated by polyinosinic:polycytidylic acid, retinoic acid-inducible gene I (RIG-I) or MAVS was suppressed by the SVCV infection. However, the upregulation by the downstream factor of the RIG-I-like receptor signaling pathway, TANK-binding kinase 1, was not affected. Notably, at the protein level, MAVS decreased remarkably when cells were infected with SVCV. Second, consistent with the result of the SVCV infection, overexpression of the N protein of SVCV blocked the IFNφ1 transcription activated by MAVS and downregulated MAVS expression at the protein level but not at the mRNA level. Further analysis demonstrated that the N protein targeted MAVS for K48-linked ubiquitination, which promoted the degradation of MAVS. These data indicated that fish MAVS could be degraded by the N protein of SVCV through the ubiquitin-proteasome pathway. To our knowledge, this is the first article of a fish RIG-I-like receptor pathway interfered by an aquatic virus in an ubiquitin-proteasome manner, suggesting that immune evasion of a virus also exists in lower vertebrates.

Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods.

Gas-exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin responses against pathogens in specialized respiratory organs have only been described in tetrapods. Since fish gills are considered a mucosal surface, we hypothesized that a dedicated mucosal immunoglobulin response would be generated within its mucosa on microbial exposure. Supporting this hypothesis, here we demonstrate that following pathogen exposure, IgT(+) B cells proliferate and generate pathogen-specific IgT within the gills of fish, thus providing the first example of locally induced immunoglobulin in the mucosa of a cold-blooded species. Moreover, we demonstrate that gill microbiota is predominantly coated with IgT, thus providing previously unappreciated evidence that the microbiota present at a respiratory surface of a vertebrate is recognized by a mucosal immunoglobulin. Our findings indicate that respiratory surfaces and mucosal immunoglobulins are part of an ancient association that predates the emergence of tetrapods.

An attenuated virus vaccine appears safe to the central nervous system of rainbow trout (Oncorhynchus mykiss) after intranasal delivery.

Nasal vaccines are very effective but the olfactory organ provides direct access of antigens to the brain. Infectious hematopoietic necrosis virus (IHNV) is known to cause high mortalities in salmonids. The purpose of this study is to evaluate the safety of a live attenuated IHNV nasal (I.N) vaccine in rainbow trout (Oncorhynchus mykiss). In the olfactory organ, the vaccine was detected 1 and 4 days after primary I.N vaccination but not in the intramuscular (i.m) or control groups. In the brain, IHNV was detected by RT-qPCR 4 and 21 days after i.m primary vaccination. One i.m and one I.N vaccinated trout were positive at days 4 and 28 days post-boost, respectively. Presence of IHNV in the brain of i.m vaccinated fish correlated with moderate increases in IL-1ß and TNF-α expression in this tissue. These results demonstrate that IHNV vaccine lasts for 4 days in the local nasal environment and that nasal vaccination appears to be safe to the CNS of rainbow trout.

Transcriptomic analysis of the host response to an iridovirus infection in Chinese giant salamander, Andrias davidianus.

The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus-host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is "complement and coagulation cascades", and significantly enriched diseases include "inherited thrombophilia", "immune system diseases", "primary immunodeficiency", "complement regulatory protein defects", and "disorders of nucleotide excision repair". Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander.

Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus.

The major capsid protein (MCP) is the main immunogenic protein of iridoviruses, that has been widely used as an immunogen in vaccination trials. In this study, the codon-optimized giant salamander iridovirus (GSIV) MCP gene (O-MCP) was synthesized and cloned into a pPICZα B vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The expression of the O-MCP protein was detected by the Bradford protein assay, SDS-PAGE, Western blotting and electron microscopy. The Bradford protein assay indicated that the concentration of the O-MCP expressed was about 40 µg/ml in culture supernatants. SDS-PAGE analysis revealed that the O-MCP had a molecular weight of about 66 kDa and reacted with a His-specific MAb that was confirmed by Western blotting. Electron microscopy observations revealed that the purified O-MCP could self-assemble into virus-like particles. Healthy giant salamanders were vaccinated by intramuscular injection with the O-MCP antigen at a dose of 20 µg/individual. The numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly at day 3 and reached a peak at day 5 post-immunization. Meanwhile, the differential leukocyte counts of monocytes and neutrophils increased significantly at day 5 post-immunization compared to that of the control group. The percentage of lymphocytes was 71.33 ± 3.57% at day 21 post-immunization. The neutralization assay showed that the serum neutralizing antibody titer reached 321 at day 21 post-immunization. The GSIV challenge test revealed that the relative percent survival of Chinese giant salamanders vaccinated with O-MCP was 78%. These results indicated that the O-MCP antigen expressed by the Pichia pastoris system elicited significant immune response in the Chinese giant salamander against GSIV and might represent a potential yeast-derived vaccine candidate that could be used for the control of disease caused by the giant salamander iridovirus.

Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID50/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future.