Immune responses and protective efficacy of American eel (Anguilla rostrata) immunized with a formalin-inactivated vaccine against Anguillid herpesvirus.

Anguillid herpesvirus 1 (AngHV), the causative agent of "mucus sloughing and hemorrhagic septicemia disease", causes serious infectious diseases in farmed eel. Among the effective prevention and control strategies, vaccination is one of the most effective approaches. However, no vaccine for AngHV is available. Our study developed a formalin-inactivated AngHV vaccine and evaluated its performance in American eels. Initially, AngHV-FJ, a strain of AngHV, was inactivated completely by 0.1 % formaldehyde, mixed with adjuvant Montanide ISA 763 A VG (763A). Then, vaccines containing different amount of antigen (3 × 106 PFU, 3 × 105 PFU, 3 × 104 PFU, 3 × 103 PFU) were immunized in each American eels. The results showed that the 3 × 105 PFU/fish was the proper dose. The inactivated AngHV vaccine was proven safe for American eels by back intramuscular injection. The results of twice immunization showed that antibody production peaked in the 8th week after the first immunization, and the antibody titer was 1:64,000. Furthermore, the immunized fishes challenged with AngHV (105 PFU/ml immersion) showed a significantly lower incidence rate (33.33 %) than the control group (95.65 %). The survival of the fish in the vaccine group (94.44 %) was significantly higher than the control group (60.87 %). The relative survival rate of the vaccinated group was 85.80 %. Also, vaccine group tissue collected at 7th d post-challenge showed reduced tissue damage and a lower virus load than the control group. The expression of cytokines of IL-1ß, IFN-α, IFN-γ, Mx1, RIG-1, and IRF-3, were significantly lower in the vaccine group than the control group at the 7th and 14th d post-challenge. Overall, the formalin-inactivated AngHV vaccine was safe and had immune protective effects against AngHV infection.

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel, Anguilla anguilla.

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (ß-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.

Proteomic Profiling Skin Mucus of European Eel Anguilla anguilla Infected with Anguillid Herpesvirus.

Anguillid herpesvirus 1 (AngHV) is an important viral pathogen affecting eel. This study was designed to investigate the potential molecular mechanisms and immune response elicited at the protein levels in the skin mucus of AngHV-infected Anguilla anguilla. Tandem mass tag (TMT)-labelling proteomics with the liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for performing quantitative identification of the proteins. In addition, the quantitative protein amount was detected by parallel reaction monitoring (PRM) analysis. A total of 3486 proteins were identified, of which 2935 were quantified. When a protein fold change was greater than 1.3 or less than 0.76, it indicated a differentially expressed protein (DEP). Overall, 187 up-regulated proteins and 126 down-regulated proteins were detected, and most of the DEPs were enriched in the CAMs pathway, intestinal immune pathway, herpes simplex virus 1 infection pathway, phagosome pathway and p53 signaling pathway. The results of the DEPs detected by PRM were highly consistent with the results of the TMT-labelled quantitative proteomic analysis. The findings of this study provide an important research basis for further understanding the pathogenesis of AngHV.

Isolation of a novel adomavirus from cultured American eels, Anguilla rostrata, with haemorrhagic gill necrosis disease.

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.

Anguillid herpesvirus 1 (AngHV) ORF95 encodes a late, structural envelope protein.

Anguillid herpesvirus 1 (AngHV) is one of the vital pathogenic agents found in the wild and cultured eel populations, which has brought significant losses to eel culture industry in China. In this study, AngHV ORF95 was characterized. Bioinformatics analysis showed that ORF95 putatively encodes a structural protein that is homologous to hemagglutinin-esterase (HE) protein of infectious salmon anemia virus (ISAV). Temporal transcription and expression analysis indicated that ORF95 is a viral late gene. Subcellular localization analysis revealed that ORF95 was predominantly localized in the cytoplasm. Further, western blot analysis indicated that ORF95 is a structural protein of virion envelope. These results provide a novel basis to make further efforts to clarify the function of ORF95 in the process of AngHV infection and the possibility to use ORF95 as antigen to develop AngHV subunit vaccine.

The effect of surface poly(ethylene glycol) length on in vivo drug delivery behaviors of polymeric nanoparticles.

Engineering nanoparticles of reasonable surface poly(ethylene glycol) (PEG) length is important for designing efficient drug delivery systems. Eliminating the disturbance by other nanoproperties, such as size, PEG density, etc., is crucial for systemically investigating the impact of surface PEG length on the biological behavior of nanoparticles. In the present study, nanoparticles with different surface PEG length but similar other nanoproperties were prepared by using poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) copolymers of different molecular weights and incorporating different contents of PCL3500 homopolymer. The molecular weight of PEG block in PEG-PCL was between 3400 and 8000 Da, the sizes of nanoparticles were around 100 nm, the terminal PEG density was controlled at 0.4 PEG/nm2 (or the frontal PEG density was controlled at 0.16 PEG/nm2). Using these nanoproperties well-designed nanoparticles, we demonstrated PEG length-dependent changes in the biological behaviors of nanoparticles and exhibited nonmonotonic improvements as the PEG molecular weight increased from 3400 to 8000 Da. Moreover, under the experimental conditions, we found nanoparticles with a surface PEG length of 13.8 nm (MW = 5000 Da) significantly decreased the absorption with serum protein and interaction with macrophages, which led to prolonged blood circulation time, enhanced tumor accumulation and improved antitumor efficacy. The present study will help to establish a relatively precise relationship between surface PEG length and the in vivo behavior of nanoparticles.

Regulating the surface poly(ethylene glycol) density of polymeric nanoparticles and evaluating its role in drug delivery in vivo.

Poly(ethylene glycol) (PEG) is usually used to protect nanoparticles from rapid clearance in blood. The effects are highly dependent on the surface PEG density of nanoparticles. However, there lacks a detailed and informative study in PEG density and in vivo drug delivery due to the critical techniques to precisely control the surface PEG density when maintaining other nano-properties. Here, we regulated the polymeric nanoparticles' size and surface PEG density by incorporating poly(ε-caprolactone) (PCL) homopolymer into poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) and adjusting the mass ratio of PCL to PEG-PCL during the nanoparticles preparation. We further developed a library of polymeric nanoparticles with different but controllable sizes and surface PEG densities by changing the molecular weight of the PCL block in PEG-PCL and tuning the molar ratio of repeating units of PCL (CL) to that of PEG (EG). We thus obtained a group of nanoparticles with variable surface PEG densities but with other nano-properties identical, and investigated the effects of surface PEG densities on the biological behaviors of nanoparticles in mice. We found that, high surface PEG density made the nanoparticles resistant to absorption of serum protein and uptake by macrophages, leading to a greater accumulation of nanoparticles in tumor tissue, which recuperated the defects of decreased internalization by tumor cells, resulting in superior antitumor efficacy when carrying docetaxel.

Efficacy and safety of intravenous stronger neo-minophagen C and S-adenosyl-L-methionine in treatment of pregnant woman with chronic hepatitis B: a pilot study.

BACKGROUND: There have been no studies evaluating the efficacy and potential risks of stronger neo-minophagen C (SNMC) in pregnant women with chronic hepatitis B CHB. MATERIAL/METHODS: A total of 36 pregnant women with CHB, but without severe complications, were randomized to intravenously receive SNMC or S-adenosyl-L-methionine (SAM) daily for 4 weeks or until birth. Normalization of serum alanine transaminase (ALT) and aspartate transaminase (AST) levels and changes in ALT and AST levels from baseline were determined. All neonates were regularly examined for up to 1 year. RESULTS: Treatment with SNMC and SAM resulted in normalization of ALT levels at 4 weeks in 64.3% and 21.4% of patients, respectively (OR=6.60, 95% CI: 1.23-35.44, P=0.0540). SNMC and SAM significantly decreased ALT (from 558.28+/-390.24 to 47.07+/-24.94 IU/L, P<0.0001 and from 525.61+/-483.87 to 117.43+/-85.44 IU/L, P=0.0041, respectively) and AST (from 419.72+/-409.49 to 38.14+/-18.87 IU/L, P=0.0016, and from 510.78+/-621.58 to 79.93+/-63.25 IU/L, P=0.0152, respectively) at 4 weeks relative to baseline values. Hypokalemia was observed in 4 SNMC-treated patients and in 2 SAM-treated patients and hypernatremia in 3 SNMC-treated and in 3 SAM-treated patients. Hypertension was observed in 1 SNMC-treated patient. There was no significant difference in the volume of amniotic fluid or meconium between SNMC-treated and SAM-treated groups. All the neonates were physically normal at birth and at the 1-year follow-up examination. CONCLUSIONS: Both SNMC and SAM improve liver function, with SNMC appearing more effective, in pregnant women with chronic hepatitis B without impact on fetal development.

Retrospective analysis of nosocomial infections in the intensive care unit of a tertiary hospital in China during 2003 and 2007.

BACKGROUND: Nosocomial infections are a major threat to patients in the intensive care unit (ICU). Limited data exist on the epidemiology of ICU-acquired infections in China. This retrospective study was carried out to determine the current status of nosocomial infection in China. METHODS: A retrospective review of nosocomial infections in the ICU of a tertiary hospital in East China between 2003 and 2007 was performed. Nosocomial infections were defined according to the definitions of Centers for Disease Control and Prevention. The overall patient nosocomial infection rate, the incidence density rate of nosocomial infections, the excess length of stay, and distribution of nosocomial infection sites were determined. Then, pathogen and antimicrobial susceptibility profiles were further investigated. RESULTS: Among 1980 patients admitted over the period of time, the overall patient nosocomial infection rate was 26.8% or 51.0 per 1000 patient days., Lower respiratory tract infections (LRTI) accounted for most of the infections (68.4%), followed by urinary tract infections (UTI, 15.9%), bloodstream (BSI, 5.9%), and gastrointestinal tract (GI, 2.5%) infections. There was no significant change in LRTI, UTI and BSI infection rates during the 5 years. However, GI rate was significantly decreased from 5.5% in 2003 to 0.4% in 2007. In addition, A. baumannii, C. albicans and S. epidermidis were the most frequent pathogens isolated in patients with LRTIs, UTIs and BSIs, respectively. The rates of isolates resistant to commonly used antibiotics ranged from 24.0% to 93.1%. CONCLUSION: There was a high and relatively stable rate of nosocomial infections in the ICU of a tertiary hospital in China through year 2003-2007, with some differences in the distribution of the infection sites, and pathogen and antibiotic susceptibility profiles from those reported from the Western countries. Guidelines for surveillance and prevention of nosocomial infections must be implemented in order to reduce the rate.