Development and immune evaluation of LAMP1 chimeric DNA vaccine against Singapore grouper iridovirus in orange-spotted grouper, Epinephelus coioides.

Grouper is one of the most important and valuable mariculture fish in China, with a high economic value. As the production of grouper has increased, massive outbreaks of epidemic diseases have limited the development of the industry. Singapore grouper iridovirus (SGIV) is one of the most serious infectious viral pathogens and has caused huge economic losses to grouper farming worldwide due to its rapid spread and high lethality. To find new strategies for the effective prevention and control of SGIV, we constructed two chimeric DNA vaccines using Lysosome-associated membrane protein 1 (LAMP1) fused with major capsid proteins (MCP) against SGIV. In addition, we evaluated the immune protective effects of vaccines including pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, chimeric DNA vaccine pcDNA3.1-MLAMP and pcDNA3.1-LAMCP by intramuscular injection. Our results showed that compared with groups injected with PBS, pcDNA3.1-3HA, pcDNA3.1-LAMP1 or pcDNA3.1-MCP, the antibody titer significantly increased in the chimeric vaccine groups. Moreover, the mRNA levels of immune-related factors in groupers, including IRF3, MHC-I, TNF-α, and CD8, showed the same trend. However, MHC-II and CD4 were significantly increased only in the chimeric vaccine groups. After 28 days of vaccination, groupers were challenged with SGIV, and mortality was documented for each group within 14 days. The data showed that two chimeric DNA vaccines provided 87 % and 91 % immune protection for groupers which were significantly higher than the 52 % protection rate of pcDNA3.1-MCP group, indicating that both forms of LAMP1 chimeric vaccines possessed higher immune protection against SGIV, providing the theoretical foundation for the creation of novel DNA vaccines for fish.

Oral immunizations with Bacillus subtilis spores displaying VP19 protein provide protection against Singapore grouper iridovirus (SGIV) infection in grouper.

Disease caused by Singapore grouper iridovirus (SGIV) results in major economic losses in the global grouper aquaculture industry. Vaccination is considered to be the most effective way to protect grouper from SGIV. In this study, the spores of Bacillus subtilis (B.subtilis) WB600 were utilized as the vehicle that the VP19 protein was displayed on the spores surface. To further investigate the effect of oral vaccination, the grouper were orally immunized with B.s-CotC-19 spores. After challenged, the survival rate of grouper orally vaccinated with B.s-CotC-19 spores was 34.5% and the relative percent survival (RPS) was 28.7% compared to the PBS group. Moreover, the viral load in the tissues of the B.s-CotC-19 group was significantly lower than that of the PBS group. The histopathological sections of head kidney and liver tissue from the B.s-CotC-19 group showed significantly less histopathology compared to the PBS group. In addition, the specific IgM levels in serum in the B.s-CotC-19 group was higher than those in the PBS group. In the hindgut tissue, the immune-related gene expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the B.s-CotC-19 group, suggesting that the innate and adaptive immune responses were activated. These results indicated that the oral administration of recombinant B.subtilis spores was effective for preventing SGIV infection. This study provided a feasible strategy for the controlling of fish virus diseases.

A recombinant baculovirus vector vaccine (BacMCP) against the infectious spleen and kidney necrosis virus (ISKNV).

The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-ß, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.

Protective immunity induced by ankyrin repeat-containing protein-based DNA vaccine against rock bream iridovirus (RBIV) in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) and remains an unsolved problem in Korea aquaculture industry. In this study, we assessed the potential of ankyrin repeat (ANK)-containing proteins to induce protective immunity in RBIV-infected rock bream. Rock bream administered with ankyrin repeat-containing protein-based DNA vaccine (200 ng/fish) exhibited significant protection against at 4 and 8 weeks post vaccination to infected with 6.7 × 105 RBIV at 23°C; relative percent survival (RPS) of 60.04% and 40.1%, respectively. Furthermore, survivors from the first infection were strongly protected from RBIV (1.1 × 107) re-infection at 70 days post infection, as 100% RPS was observed and without clinical signs of RBIV diseases. Moreover, TLR3 (9.5-fold), TLR9 (5.2-fold), MyD88 (15.9-fold), Mx (55.5-fold), ISG15 (19.0-fold), PKR (24.2-fold), MHC class I (5.1-fold), perforin (6.5-fold), Fas (6.4-fold), Fas ligand (7.1-fold), caspase8 (5.0-fold), caspase9 (12.5-fold), and caspase3 (6.3-fold) responses were significantly elevated in the muscle (vaccine injection site) of ANK-based DNA vaccinated fish at 7 days post vaccination. However, inflammatory cytokines (IL1ß, IL8, and TNFα) were not enhanced in the vaccinated rock bream. Moreover, ANK gene may be a good candidate to detect RBIV infection or in revealing specific information to elucidate the pathogenic mechanisms underlying RBIV infection. In summary, ANK-based DNA vaccination in rock bream induced TLR- and IFN-mediated or apoptosis-related immune responses and suggest efficient preventive measures against RBIV.

Efficacy of saponin-based inactivated rock bream iridovirus (RBIV) vaccine in rock bream (Oplegnathus fasciatus).

Rock bream iridovirus (RBIV) causes severe mortality in rock bream (Oplegnathus fasciatus) for last two decades. In view of this constant threat of RBIV to the rock bream industry, we conducted the present study with the aim to develop a safe and efficient remedial measure against the virus. In this study, we evaluated the safety and potentiality of squalene, aluminium hydroxide and saponin adjuvants, singly or in combinations, which can be used for developing an efficient inactivated (IV) vaccine to protect rock bream from RBIV infection. The evaluation results demonstrated that saponin (Sa) has the required potential in enacting the antiviral immune response in the host and in providing protection against virus mediated lethality, without causing any adverted side-effects. The study further, showed that a single primary dose of Sa-adjuvanted IV vaccine can confer moderate protections in short (60.04% relative percent mortality (RPS) at 4 wpv) and medium (53.38% RPS at 8 wpv) term post RBIV challenge; whereas, the same vaccine when administered in a prime-boost strategy, it resulted enhanced 93.34% RPS post virus challenge at 4 and 8 wpv. The moderate to high survivability demonstrated by the Sa-adjuvanted IV vaccine, was substantiated by the significant (p < 0.05) upregulation of IL-1ß, Mx and PKR gene transcript. All surviving fish from the Sa-adjuvanted IV vaccine groups were strongly protected from re-infection with RBIV (1.1 × 107) at 70 days post infection (dpi). In conclusion, it can be inferred that, Sa-adjuvanted IV RBIV vaccine can be an efficient control measure to protect the rock bream aquaculture industry against the lethal RBIV virus.

Epitope screening of the major capsid protein within grouper iridovirus of Taiwan and the immunoprotective effect with SWCNTs as the vaccine carrier.

Iridovirus can cause a mass of death in grouper, leading to huge economic loss in recent years. At present, practical vaccine is still the best way to control the outbreak of this virus. Many researches had indicated that the major capsid protein (MCP) of grouper iridovirus of Taiwan (TGIV) is an effective antigen to induce a specific immune response in grouper. However, these traditional vaccines that based on large proteins or whole organisms are faced with challenges because of the unnecessary antigenic load. Thus, in this study, we screened the dominant linear epitope within the MCP of TGIV and then, a new peptide vaccine (P2) was developed via prokaryotic expression system. Furthermore, SWCNTs was used as a vaccine carrier to enhance the immunoprotective effect. To evaluate the immunoprotective effect of this vaccine, a total of 245 fish were vaccinated with P2 (5, 10, 20 mg L-1) and SWCNTs-P2 (5, 10, 20 mg L-1) via immersion before being challenged with live TGIV at 28 days post immunization (d.p.i.). Results showed that the serum antibody titer, enzymatic activity, expression level of some immune-related genes (CC chemokine, IgM and TNF-α) and survival rate were significantly increased (SWCNTs-P2, 20 mg L-1, 100%) compared to the control group (0%). These results indicated that this peptide vaccine could effectively induce specific immune response in vaccinated groupers. Functionalized SWCNTs could serve as a carrier of the peptide vaccine to enhance the immunoprotective effect via immersion. To sum up, epitope screening might be a potential way to develop an effective vaccine nowadays, and SWCNTs might provide a practical method that can be used in large-scale vaccination, especially for juvenile fish, to fight against diseases in aquaculture industry.

Development of a high-dose vaccine formulation for prevention of megalocytivirus infection in rock bream (Oplegnathus fasciatus).

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 1010 copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 1010 copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 109 copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 108 copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 104 copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.

Protective immunity of largemouth bass immunized with immersed DNA vaccine against largemouth bass ulcerative syndrome virus.

To reduce the largemouth bass ulcer syndrome (LBUSV) aquatic economic losses, it must take effective preventive measures and coping strategies should be urgently investigated. In this research, the effects of a functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in largemouth bass (Micropterus Salmoides) against LBUSV were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to largemouth bass against LBUSV. We found more than 10 times increase in serum antibody levels, enzyme activities and immune-related genes (IL-6, IL-8, IFN-γ, IgM and TNF-α) expression, in the SWCNTs-pcDNA-MCP immunized groups compared with PBS group and the pure SWCNTs group. The survival rates for control group (PBS), pure SWCNTs groups (40 mg L-1), four pcDNA-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) and four SWCNTs-pcDNA-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) were 0%, 0%, 0%, 2.77%, 11.11%, 19.44%, 27.78%, 38.89%, 52.78% and 61.11%, respectively. Our results demonstrate that the SWCNTs-DNA vaccine can be used as a new method against LBUSV showing protection following challenge with LBUSV.

Single-walled carbon nanotubes enhance the immune protective effect of a bath subunit vaccine for pearl gentian grouper against Iridovirus of Taiwan.

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-ß, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

Optimization of the efficacy of a SWCNTs-based subunit vaccine against infectious spleen and kidney necrosis virus in mandarin fish.

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which brings substantial economic losses to the mandarin fish culture industry in China. This study was aimed at optimizing the efficacy of a SWCNTs-based immersion subunit vaccine (SWCNTs-M-MCP) which as a promising vaccine against ISKNV. Mandarin fish were vaccinated by immersion, then we designed an orthogonal experiment to optimize different parameters affecting vaccination such as immune duration of bath immunization, immune dose, and fish density when immunized. Our results showed that the highest relative percent survival (86.7%) was found in the group 6 with 8 h of immune duration, 20 mg/L of immune dose, and 8 fish per liter of fish density. And other immune responses (serum antibody production, enzyme activities, and immune-related genes expression) also demonstrated similar results. In addition, the expression of IRF-I in group 6 (8 h, 20 mg/L, 8 fish per liter) was significant extents, and about 16-folds increases were obtained than the control group at 21 d post-vaccination. And the highest specific antibody response was significantly increased (more than 4-folds) than control group which was found in group 6. The optimum immune duration, immune dose, and fish density of SWCNTs-M-MCP were 8 h, 20 mg/L, 8 fish per liter, respectively. Importantly, our results also showed that immune duration had the greatest effect on the immune response of our vaccine, followed by immune dose. The study reported herein provides a helpful reference for the effective use of vaccine in fish farming industry.

Immune efficacy of carbon nanotubes recombinant subunit vaccine against largemouth bass ulcerative syndrome virus.

Largemouth bass ulcerative syndrome virus (LBUSV) is an important virus induce the mortality of largemouth bass (Micropterus Salmoides). In this study, we reported a single-walled carbon nanotubes (SWCNTs) containing LBUSV major capsid protein (MCP) subunit vaccine (SWCNTs-MCP) which was evaluated for its protective effect on largemouth bass by immersion immunization. We found an elevation in serum antibody levels, enzyme activities, complement C3 content and immune-related genes (IgM, TGF-ß, IL-1ß, IL-8, TNF-α and CD4) expression, in the SWCNTs-MCP immunized groups compared with the pure MCP group. The survival rates for control group, pure MCP protein groups (40 mg L-1) and four SWCNTs-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) were 0%, 27.78%, 30.56%, 50.00%, 66.67% and 80.56%, respectively. The results suggests that with the assistance of SWCNTs, the immune protection of the SWCNTs-MCP group (40 mg L-1) increased up 52.78%-80.1% compared with pure MCP group (40 mg L-1). Our results demonstrate that the single-walled carbon nanotube subunit vaccine can be used as a new immunization method against LBUSV showing protection following challenge with LBUSV. Taken together, our results demonstrate that the single-walled carbon nanotube subunit vaccine can be used as a new method against LBUSV and have a high immune protection during the largemouth bass farm.

Effects of Synechococcus sp. PCC 7942 harboring vp19, vp28, and vp (19 + 28) on the survival and immune response of Litopenaeus vannamei infected WSSV.

This study aimed to assess the effect of oral administration of Synechococcus sp. PCC 7942 harboring vp19, vp28, and vp(19 + 28)against infection by white spot syndrome virus (WSSV) on juveniles of Litopenaeus vannamei. L. vannamei was orally administrated by feeding with different mutants of Synechococcus for 10 days, and then challenged with WSSV. The cumulative mortality of vp19, vp28, vp (19 + 28) groups was lower than that of the positive control group (57.8%, 62.2%, 71.1%, respectively); vp (19 + 28) group had a better protection rate than vp19 and vp28 groups. The analysis of shrimp immunological parameters showed that, after WSSV injection, the activity of superoxide dismutase, phenol oxidase, catalase, and lysozyme in the hepatopancreas of vp19, vp28, and vp (19 + 28) groups was higher than in the positive group; at the same time, growth performances of L. vannamei of experimental groups were better than control groups. Results showed that the Synechococcus mutants harboring vp19, vp28, and vp (19 + 28) could be used both as drug and feed to also enhance the defensive ability of juvenile shrimp against WSSV infection by increasing the activity of immune related enzymes.

Transcriptomic profiles reveal that inactivated iridovirus and rhabdovirus bivalent vaccine elicits robust adaptive immune responses against lethal challenge in marbled sleepy goby.

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effect of an immersion DNA vaccine against infectious spleen and kidney necrosis virus in mandarin fish.

As a high mortality disease, Infectious spleen and kidney necrosis virus (ISKNV) can cause massive economic damage on mandarin fish farming industry in China, which seriously hindered the development of mandarin fish farming industry. In this research, SWCNTs (single-walled carbon nanotubes) as a candidate for DNA vaccine carrier was vaccinated by immersion (1, 2, 5, 10, 20 mg/L) in juvenile mandarin fish. In muscle, spleen and kidney tissues, the results showed that transcription and expression of MCP gene can be detected in pcDNA-MCP and SWCNTs-pcDNA-MCP groups after bath immunization. The immune response (immune-related genes expression, serum antibody production, enzyme activities and C3 content) was significantly enhanced in fish which vaccinated with SWCNTs-pcDNA-MCP in comparison with those vaccinated with pcDNA-MCP alone. After 14 d challenge, the RPS (relative percentage survival) can be enhanced which using SWCNTs as a carrier in SWCNTs-pcDNA-MCP (82.4%) group at 20 mg/L (the highest vaccine dose) than the naked pcDNA-MCP (54.2%) group. This study reveals that functionalized SWCNTs could be a promising immersion DNA vaccine carrier in aquaculture.

Protective immunity induced by DNA vaccine encoding viral membrane protein against SGIV infection in grouper.

Singapore grouper iridovirus (SGIV) is the main grouper-infecting virus in southern China that causes serious economic losses. However, there is no effective way to control this viral disease. In this study, SGIV ORF19R (SGIV-19R) encoding a viral membrane protein was constructed into pcDNA3.1-HA and then was used to evaluate the immune protective effects in grouper Epinephelus coioides. Subcellular localization showed that SGIV-19R distributed in the cytoplasm and co-localization analysis indicated the protein partially co-localized with the endoplasmic reticulum (ER). RT-PCR and Western blot analyses confirmed the expression of the vaccine plasmids in grouper muscle tissues. Moreover, the transcription levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), myxovirus resistance 1 (Mx1) and immunoglobulin M (IgM) genes were significantly up-regulated in the spleen, liver and kidney of vaccinated groupers. SGIV challenge experiments showed the relative percent survival (RPS) was significantly enhanced in fish with 49.9% at the DNA dose of 45 µg pcDNA3.1-19R, while 75.0% RPS when using 90 µg pcDNA3.1-19R. Meanwhile, vaccination with pcDNA3.1-19R significantly reduced the virus replication, evidenced by a low viral load in the spleen of survivals groupers after SGIV challenge. These results imply that pcDNA3.1-19R could induce protective immunity in grouper, and might be a potential vaccine candidate for controlling SGIV disease.

Cloning and expressional analysis of secretory and membrane-bound IgM in rock bream (Oplegnathus fasciatus) under megalocytivirus infection and vaccination.

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.

Characterization of Bacillus subtilis from gastrointestinal tract of hybrid Hulong grouper (Epinephelus fuscoguttatus × E. lanceolatus) and its effects as probiotic additives.

Probiotics are widely used for the improvement of animals' growth and health. However, few marine aquatic probiotics are applied and licensed in China. In this study, a Bacillus spp. strain was isolated from the Hulong grouper gastrointestinal tract, which was identified as a new strain of Bacillus subtilis and was named as 7k. B. subtilis 7k showed desirable capability of sporulation and resistance to heat, simulated gastric juice and simulated duodenum juice, indicating its potential as probiotics. Seven antimicrobial chemicals were found in the secretion of the B. subtilis 7k. B. subtilis 7k addition in diet promoted the growth rate of Hulong groupers. Moreover, B. subtilis 7k can inhibit infection by iridovirus, making B. subtilis 7k a suitable kind of probiotic for maintaining fishes' health. Our results also revealed that B. subtilis 7k induced non-specific immune response in Hulong grouper under virus infection. Hulong grouper fed by diets containing B. subtilis 7k at 108 and 1010 cfu g-1 for 4-8 weeks were significantly strengthened in serum lysozyme activity, serum alternative complement activity (ACH50), serum bactericidal activity, respiratory burst, superoxide dismutase activity (SOD), and phagocytic activity of head kidney leucocytes when compared with those fed by control diets. In conclusion, B. subtilis 7k was isolated and characterized to be a kind of process enduring, growth stimulating, immunity enhancing and health promoting probiotic using in grouper culture.

Viral surveillance using PCR during treatment of AML and ALL.

BACKGROUND: While viral surveillance of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus using PCR is routine in patients undergoing hematopoetic stem cell transplant and solid organ transplant, the utility in the nontransplant pediatric leukemia population is unknown. Our institution screens patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) for viral DNAemia by PCR as part of clinical care. PROCEDURE: This retrospective chart review included patients treated for newly diagnosed or relapsed AML or ALL between April 2010 and September 2014. We retrieved data for viral PCR screening, detection and quantification, duration of positivity, and prophylaxis or treatment. RESULTS: One hundred eleven patients were included in analyses. Forty (36.0%) had at least one blood PCR positive for EBV, CMV, or adenovirus. Patients with ALL had significantly higher rates of persistent viral detection and treatment than those with AML (P < 0.02, P < 0.01, respectively). International patients had significantly higher rates of viral detection (P < 0.01), persistence (P < 0.01), any treatment (P < 0.03), and antiviral treatment (P < 0.01); 16.9% of patients who received intravenous immunoglobulin (IVIG) prophylactically had viral detection compared to 63% of patients who did not receive prophylactic IVIG (P = 0.0008). CONCLUSIONS: Patients with ALL were more susceptible than those with AML to viral reactivation that was persistent or resulted in treatment. Patients with relapsed ALL, refractory ALL, or infantile ALL are most likely to benefit from asymptomatic screening for CMV and adenovirus. International patients are at higher risk for reactivation and may merit screening. EBV reactivation was not significant and does not warrant screening.

Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus.

The Chinese giant salamander iridovirus (CGSIV), belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP) was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographacalifornica nucleopolyhedrosis virus (AcNPV), expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL) and confirmed by Western blot and indirect immunofluorescence (IIF) assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9) cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

LAMP-1-chimeric DNA vaccines enhance the antibody response in Japanese flounder, Paralichthys olivaceus.

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination.