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China has the largest aquaculture eel production in the world. High-density cultivation pattern often results in an outbreak of epidemic diseases. Since the 1990s, eel "mucus sloughing and hemorrhagic septicemia disease" was often broke out in China, and brought huge economic losses to eel breeders. Anguillid herpesvirus 1 (AngHV) was detected and isolated from the diseased eel, and proved to be the pathogen of the disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive, and specific detection of AngHV. A set of six primers targeting the ORF51 gene of AngHV was designed, which could effectively detect purified AngHV virions, AngHV-infected cells, or eel tissue samples. The suitable reaction temperature is 63℃, and the reaction time is 40 min. There was no cross-reaction with eel and other fish viruses, including Infectious pancreatic necrosis virus (IPNV), Marine birnavirus (MABV), Rana grylio virus (RGV), Cyprinid herpesvirus 3 (CyHV-3), and Eel iridovirus (EIV). The lower detection limit of the AngHV LAMP assay is 10 copies of AngHV genome DNA, which is at least 100 times more sensitive than conventional PCR in detecting AngHV. The assay could effectively detect AngHV from collected samples with typical clinical symptoms of AngHV infection. It suggested that the LAMP assay could be used in specific detection of AngHV and has great potential for early diagnosis of AngHV infection in the farm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The TRIANGLE operation involves the removal of all tissues within the triangle bounded by the portal vein-superior mesenteric vein, celiac axis-common hepatic artery, and superior mesenteric artery to improve patient prognosis. Although previously promising in patients with locally advanced pancreatic ductal adenocarcinoma (PDAC), data are limited regarding the long-term oncological outcomes of the TRIANGLE operation among resectable PDAC patients undergoing pancreaticoduodenectomy (PD)., Aim: To evaluate the safety of the TRIANGLE operation during PD and the prognosis in patients with resectable PDAC., Methods: This retrospective cohort study included patients who underwent PD for pancreatic head cancer between January 2017 and April 2023, with or without the TRIANGLE operation. Patients were divided into the PD TRIANGLE and PD non-TRIANGLE groups. Surgical and survival outcomes were compared between the two groups. Adequate adjuvant chemotherapy was defined as adjuvant chemotherapy ≥ 6 months., Results: The PD TRIANGLE and PD non-TRIANGLE groups included 52 and 55 patients, respectively. There were no significant differences in the baseline characteristics or perioperative indexes between the two groups. Furthermore, the recurrence rate was lower in the PD TRIANGLE group than in the PD non-TRIANGLE group (48.1% vs 81.8%, P < 0.001), and the local recurrence rate of PDAC decreased from 37.8% to 16.0%. Multivariate Cox regression analysis revealed that PD TRIANGLE (HR = 0.424; 95%CI: 0.256-0.702; P = 0.001), adequate adjuvant chemotherapy ≥ 6 months (HR = 0.370; 95%CI: 0.222-0.618; P < 0.001) and margin status (HR = 2.255; 95%CI: 1.252-4.064; P = 0.007) were found to be independent factors for the recurrence rate., Conclusion: The TRIANGLE operation is safe for PDAC patients undergoing PD. Moreover, it reduces the local recurrence rate of PDAC and may improve survival in patients who receive adequate adjuvant chemotherapy., Competing Interests: Conflict-of-interest statement: The authors have no conficts of interest to declare., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Eel (Anguilla sp.) is an important freshwater-cultured species with high economic value in China. Anguillid herpesvirus 1 (AngHV-1) has been proven to be the pathogen of "mucus sloughing and haemorrhagic septicaemia disease" in eels, resulting in significant mortality and substantial losses to the eel industry. Current diagnostic methods for detecting AngHV-1 are limited to laboratory-based tests, for example, conventional end-point PCR and qPCR. Therefore, there is an urgent need to develop an accurate, rapid, and simple detection method for on-site diagnosis of AngHV-1. In this study, we developed a recombinase-aided amplification combined lateral flow dipstick (RAA-LFD) assay for the detection of AngHV-1. The RAA-LFD assay can be performed within a temperature range of 18-45°C, with a reaction time of just 10 min for amplification. Importantly, the established RAA-LFD assay exhibited no reactivity with other common aquatic viral pathogens, indicating its high specificity. The limit of detection for this method is 10 2 copies of AngHV-1, which is more sensitive than the established conventional end-point PCR method similarly targeting ORF95. Clinical detection of the diseased samples demonstrated that the accuracy of RAA-LFD was significantly higher than that of the conventional end-point PCR. In conclusion, the developed RAA-LFD assay has proven to be a convenient, rapid, sensitive, and reliable tool for on-site diagnosis of AngHV-1. This advancement will be invaluable for the prevention and control of AngHV-1 in the eel farming industry., (© 2023 John Wiley & Sons Ltd.)

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 × 10 6  PFU, 3 × 10 5  PFU, 3 × 10 4  PFU, 3 × 10 3  PFU) were immunized in each American eels. The results showed that the 3 × 10 5  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 (10 5  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-1β, 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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Porcine enteric diseases including swine dysentery involves a wide range of possible aetiologies and seriously damages the intestine of pigs of all ages. Metagenomic next-generation sequencing is commonly used in research for detecting and analyzing pathogens. In this study, the feces of pigs from a commercial swine farm with dysentery-like diarrhea was collected and used for microbiota analysis by next-generation sequencing. While Brachyspira spp. was not detected in diarrheal pig fecal samples, indicating that the disease was not swine dysentery. The quantity of microbial population was extremely lowered, and the bacterial composition was altered with a reduction in the relative abundance of the probiotics organisms, Firmicutes and Bacteroidetes, with an increase in pathogens like Fusobacterium and Proteobacteria, in which the specific bacteria were identified at species-level. Viral pathogens, porcine circovirus type 2, porcine lymphotropic herpesviruses 1, and porcine mastadenovirus A were also detected at pretty low levels. Carbohydrate-active enzymes (CAZy) analysis indicated that the constitute of Firmicutes and Bacteroidete were also changed. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) alignment analysis indicated that the microbiota of diarrheal pigs had a lower ability in utilizing energy sources but were enriched in multi-drug resistance pathways. Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factors of Pathogenic Bacteria (VFDB) analysis indicated that genes for elfamycin and sulfonamide resistance and the iron uptake system were enriched in diarrheal pigs. This revealed potential bacterial infection and can guide antibiotic selection for treating dysentery. Overall, our data suggested that alterations in both the population and functional attributes of microbiota in diarrheal pigs with decreased probiotic and increased pathogenic microorganisms. These results will help elucidate the mechanism of dysentery-like diarrhea and the development of approaches to control the disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Guo, Li, Song and Ge.)

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 (β-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., (© 2022 Fisheries Society of the British Isles.)

Lysozyme is a crucial component of the host's innate immune system. Due to its natural non-toxic and harmless characteristics, lysozyme is considered to be an ideal antibiotic substitute. In this study, we analyzed the expression profiles of lysozymes from zebrafish ( Danio rerio ) in uninfected or V. vulnificus -infected tissues using real-time quantitative PCR (qPCR). Furthermore, lysozymes that might be involved in the defense against V. vulnificus were selected for over-expression, and the antibacterial activity of over-expressed lysozyme proteins were evaluated using V. vulnificus . The results showed that three types of zebrafish lysozyme, i.e., c-type lysozyme (DrLysC), g1-type lysozyme (DrLysG1), and g2-type lysozyme (DrLysG2), were identified, and V. vulnificus infection significantly changed the expression levels of DrLysC and DrLysG1. Then, DrLysC and DrLysG1 were over-expressed in E. coli , and the purified recombinant DrLysC (rDrLysC) showed more potent antibacterial activity against V. vulnificus . This finding lays the foundation for further application of rDrLysC to treat V. vulnificus infection.

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.

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 TCID 50 /ml of 10 6.29 ± 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., (© 2021 John Wiley & Sons Ltd.)

Bombyx mori nucleopolyhedrovirus (BmNPV) p26 is conserved among all Lepidoptera baculoviruses that have been completely sequenced thus far, and some baculoviruses even have two copies of p26 , which suggested that p26 may play an important role in the virus infection cycle. This study aimed to characterize BmNPV p26 . We found that BmNPV p26 transcripts were detectable as early as 3 h post-infection (hpi), and the transcript levels rapidly increased starting from 12 hpi. Western blot analysis using an anti- p26 polyclonal antibody demonstrated that the corresponding protein was also detectable from 6 hpi in BmNPV-infected cell lysates. Immunofluorescence analysis demonstrated that p26 was mainly dispersed in the infected cell cytoplasm, whereas the over-expressed fusion protein EGFP-p26 also accumulated in the nucleus. These results indicated that p26 is an early BmNPV gene and has functions both in the cytoplasm and the nucleus. RNAi-based knockdown of p26 could produce infectious virus and normal-appearing virions but decreased budded virus (BV) production in BmNPV-infected cells at 72 hpi. Moreover, the results of further quantitative PCR (Q-PCR) analysis indicated that the gp64 and p74 transcripts levels decreased significantly. These results indicated that BmNPV p26 may be associated with BmNPV replication during the late infection stage.

A ligand-based and docking-based virtual screening was carried out to identify novel MDM2 inhibitors. A pharmacophore model with four features was used for virtual screening, followed by molecular docking. Seventeen compounds were selected for an in vitro MDM2 inhibition assay, and compounds AO-476/43250177, AG-690/37072075, AK-968/15254441, AO-022/43452814, and AF-399/25108021 showed promising MDM2 inhibition activities with K i values of 9.5, 8.5, 23.4, 3.2, and 23.1  μ M, respectively. Four compounds also showed antiproliferative activity, and compound AO-022/43452814 was the most potent hit with IC 50 values of 19.35, 26.73, 12.63, and 24.14  μ M against MCF7 (p53 +/+), MCF7 (p53 -/-), HCT116 (p53 +/+), and HCT116 (p53 -/-) cell lines, respectively. Compound AO-022/43452814 could be used as a scaffold for the development of anticancer agents targeting MDM2., Competing Interests: The authors declare no conflict of interests., (Copyright © 2021 Bing-Hui Li et al.)

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.

This study used a reducing agent to regulate the activity of heavy metals in soil, with the aim of enhancing the washing efficiency of heavy metals in soil by ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA). The effects of EGTA dosage, reductant type, liquid-solid ratio, and leaching time on washing efficiency were studied. The heavy metal concentrations in the washing solution and the chemical specification under different washing conditions were analyzed. Scanning electron microscopy/energy-dispersive X-ray spectroscopy and Fourier-transform infrared spectroscopy were used to characterize the soil samples under different treatments. The results showed that the washing efficiencies of Cu, Zn, Pb, and Cd were 85.0%, 60.7%, 88.6%, and 66.4%, respectively, when the liquid-solid ratio was 7.5:1, the leaching time was 2 h, and the dosage of EGTA and sodium thiosulfate (Na 2 S 2 O 3 ) was 5 g·L -1 . Compared with single EGTA treatment, the leaching concentration of the four heavy metals all decreased, with the Pb decreasing most significantly. The soil particles became smaller, the aggregate structure was destroyed, the O and Al content decreased, the Si content increased, and therefore the adsorption capacity of soil mineral components was supposed to decrease. The soil organic matter content decreased significantly, and the -OH vibration was significantly weakened, indicating that the organic functional groups with stronger coordination ability were reduced, and the adsorption of organic components in the soil was reduced. The decrease in content of strongly adsorbed components in soil is an important mechanism for Na 2 S 2 O 3 to enhance heavy metal elution by EGTA.

Like pathogens, galling insects deliver effectors into plant tissues that induce gall formation. The gall midge Mayetiola destructor, also called Hessian fly, can convert a whole wheat seedling into a gall by inducing the formation of nutritive cells at the feeding site, inhibiting wheat growth, and reprogramming metabolic pathways of the attacked plants. Here we demonstrated the identification of a secreted Hessian fly protein, the nucleoside diphosphate kinase (NDK), in infested wheat plants through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blots. In association with the NDK presence, enzymatic activity of NDK increased significantly in wheat tissues at the feeding site. In addition, there was a sudden increase in ATP abundance at the feeding site of infested susceptible wheat seedlings 24 h following Hessian fly larval infestation. Even though a direct link between cause and effect remains to be established, our data points to the direction that Hessian fly larvae injected NDK into wheat tissues as an effector protein, which plays a role in manipulating host plants and converting the plants into galls., (Copyright © 2018. Published by Elsevier Ltd.)

Background: There has been an increased focus in recent years on antimicrobial resistance of bacteria isolated from clinical samples. However, resistance of bacteria from hospital environments has been less frequently investigated., Methods: According to hygienic standard for disinfection in hospitals, samples were collected from hospital inanimate surfaces and the hands of health care workers after daily cleaning. An automatic microorganism analyzer was used to identify bacteria and test for antimicrobial susceptibility. Polymerase chain reaction was used to detect antimicrobial resistance genes., Results: The detection rate of bacteria in general wards was significantly higher than that in intensive care units. The isolates were predominantly gram-negative (GN) bacteria, with Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella pneumoniae being the most common. P aeruginosa isolates from other surfaces were much higher than those from medical instruments. E cloacae was isolated more frequently from the hands of other staff than medical staff. Most P aeruginosa and K pneumoniae were resistant to sulfonamides and β-lactam antimicrobials. Only 1 strain of P aeruginosa and 1 strain of K pneumoniae showed multiple antimicrobials resistance., Conclusions: The GN bacteria isolated from hospital environments demonstrate variable resistance to antimicrobials., (Copyright © 2017. Published by Elsevier Inc.)

Ethnopharmacological Relevance: The ripe seeds of Herpetospermum caudigerum have been used in Tibetan folk medicine for treatment of bile or liver diseases including jaundice, hepatitis, intumescences or inflammation. Previously reports suggested that the seed oil and some lignans from H. caudigerum exhibited protective effects against carbon tetrachloride (CCl 4 )-induced hepatic damage in rats, which may be related to their free radical scavenging effect. However, the protective effect of H. caudigerum against cholestasis is still not revealed. The aim of the present study was to investigate the pharmacological effect and the chemical constituents of the petroleum ether extract (PEE) derived from H. caudigerum against α-naphthylisothiocyanate (ANIT)-induced acute cholestasis in rats., Materials and Methods: Male cholestatic Sprague-Dawley (SD) rats induced by ANIT (60mg/kg) were orally administered with PEE (350, 700 and 1400mg/kg). Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP), total bilirubin (TBIL), direct bilirubin (DBIL) and total bile acid (TBA), as well as bile flow, and histopathological assay were evaluated. Hepatic malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione S-transferase (GST), and nitric monoxide (NO) in liver were measured to explore the possible protective mechanisms. Phytochemical analysis of PEE was performed by gas chromatography-mass spectrometer (GC-MS)., Results: PEE have exhibited significant and dose-dependent protective effect on ANIT-induced liver injury by reduce the increases in serum levels of ALT, AST, ALP, γ-GTP, TBIL, DBIL and TBA, restore the bile flow in cholestatic rats, and reduce the severity of the pathological tissue damage induced by ANIT. Hepatic MDA, MPO and NO contents in liver tissue were reduced, while SOD and GST activities were elevated in liver tissue. 49 compounds were detected and 39 of them were identified by GC-MS analysis, in which long-chain fatty acids were the main constituents., Conclusions: PEE exhibited a dose-dependently protective effect on ANIT-induced liver injury in cholestatic rats with the potential mechanism of attenuated oxidative stress in the liver tissue, and the possible active compounds were long-chain fatty acids., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Phytochemical investigation on the stems of Kadsura renchangiana led to the isolation of two new sesquiterpenoids, renchangianins F and G (1 and 2). Their structures were elucidated by spectroscopic methods, including 2D NMR techniques. The in vitro cytotoxic activities of the isolates were studied against HepG2, A549, and LN229 cancer cell lines.

One new cyanoside, rhobupcyanoside B (1), together with 7 known ones, was isolated from the 70% ethanol extract of the roots and rhizomes of Rhodiola bupleuroides. Their structures were determined by spectroscopic methods, including 2D NMR techniques. Compound 1 was evaluated for its inhibitory activity against α-glucosidase with IC50 value of 278.28 ± 0.55 μM by comparing with the positive control (acarbose) at 210.40 ± 0.32 μM.

The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The rapid doubling time and genetic relatedness of the fish pathogen Mycobacterium marinum to Mycobacterium tuberculosis has rendered the former an attractive model for investigating mycobacterial host-pathogen interactions. We employed the M. marinum-goldfish infection model to investigate the in vivo immune responses to this pathogen in the context of a natural host. Histological analysis revealed mycobacterial infiltrates in goldfish kidney and spleen tissues, peaking 28 days post infections (dpi). Quantitative gene expression analysis showed significant increases of mRNA levels of pro-inflammatory cytokines (IFNγ, IL-12p40, IL-1β1) and cytokine receptors (IFNGR1-1, TNFR2) at 7 dpi. Conversely, the gene expression levels of key anti-inflammatory cytokines TGFβ and IL-10 were elevated at 14 dpi. Furthermore, M. marinum infections markedly increased the cytokine-primed oxidative burst responses of isolated kidney phagocytes at 7 but not 56 dpi. We believe that the M. marinum-goldfish infection model will be invaluable in furthering the understanding of the mycobacterium host-pathogen interface., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We evaluated whether ozonation ameliorated the effects of the organic fraction of oil sands process water (OSPW) on immune functions of mice. Ozonation of OSPW eliminated the capacity of its organic fraction to affect various mouse bone marrow-derived macrophage (BMDM) functions in vitro. These included the production of nitric oxide and the expression of inducible nitric oxide synthase, the production of reactive oxygen intermediates and the expression of NADPH oxidase subunits, phagocytosis, and the expression of pro-inflammatory cytokine genes. Ozone treatment also eliminated the ability of OSPW organic fraction to down-regulate the expression of various pro-inflammatory cytokine and chemokine genes in the liver of mice, one week after oral exposure. We conclude that ozone treatment may be a valuable process for the remediation of large volumes of OSPW., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Open reading frame 75 (Bm-p33) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homologue of Autographa californica multiple nucleopolyhedrovirus ORF92. The gene is conserved among all baculoviruses that have been completely sequenced to date and is considered to be a baculovirus core set gene. No amino acid mutation was found in Bm-p33 sequences among six BmNPV strains differing in geography, phenotype, or host. The Bm-p33 transcript can be detected as early as 12 h post infection (h p.i.) and remains detectable until 96 h p.i. The Bm-p33 protein was detected in cell lysates from 18 h p.i. through 96 h p.i., and no positive band could be detected in budded viruses (BVs) and occlusion-derived viruses (ODVs) by western blot using anti-Bm-p33 serum. Immunofluorescence microscopy indicated that Bm-p33 accumulated in the nuclear membrane and the intranuclear region, especially near the nuclear membrane of the virus-infected cells. Bm75 RNAi significantly decreased the mRNA level. However, no obvious effects on ODV formation and BV production in BmNPV-infected cells could be detected. Bm-p33 is a BmNPV late gene encoding a nonstructural protein which may function mainly in the nucleus of the infected cells.

Objective: To investigate the prevalence of Anaplasma phagocytophilum in small mammals from the forest area of Hengduan Mountains in southwestern China., Methods: Small mammals captured from Gaoligong and Xianggelila mountainous area of Yunnan province were detected by PCR amplification. The sequences of 16S rRNA and Msp4 gene fragments from positive samples were compared with corresponding sequences deposited in GenBank., Results: A total number of 436 small animals, which belongs to 5 orders 18 genera 35 species were tested, 32 (7.34%) were positive in 6 genera 11 species. There were 8.64% (26/301) positive in 25 species at Goligong mountainous areas, and 4.44% (6/135) were positive in 19 species at the Xianggelila mountainous areas. Positive small mammals were most rodents. The nucleotide sequences of A.phagocytophilum 16S rRNA gene amplified from small mammals varied from 99% - 100% and were 99% - 100% similar with the corresponding segments of A. phagocytophilum from Jilin deposited in GeneBank. The sequences of A. phagocytophilum Msp4 gene showed that there was 95% - 97% similarity with the corresponding sequences registered in GenBank., Conclusion: A. phagocytophilum was firstly identified in 6 genera 11 species small mammals from a forest area of Hengduan Mountainous areas in southwestern China. Rodents might serve as the primary hosts indicating the potential risk to the domestic animals and human beings in this area.

The open reading frame 122 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm122) has been observed to be a conserved gene in the lepidopteran baculoviruses that have been completely sequenced so far. Its transcript was detected at 3 h post infection (h p.i.) and remained detectable at up to 96 h p.i. Temporal transcription analysis indicated that Bm122 is transcribed by host RNA polymerase. The size of the translational product of the Bm122 gene in Tn5B-1-4 cells was approximately 23 kDa, which is in agreement with the predicted value of 22.9 kDa, suggesting that no major posttranslational modification occurred in the primary protein product. The subcellular localization of Bm122 was studied using EGFP-Bm122, which revealed that Bm122 protein was accumulated within the nuclear region of virus-infected BmN cells. All these results suggest that Bm122 is an early gene encoding a protein that functions in the nucleus.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF 51 (Bm51) is a gene present in many lepidopteran NPVs, but its function is unknown. In this study, Bm51 was characterized. Transcripts of Bm51 were detected from 4.5 through 72 hour post infection (h p.i.) by RT-PCR. The corresponding protein was detected from 6 to 72 h p.i. in BmNPV-infected BmN cells by western blot analysis using a polyclonal antibody against Bm51. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm51 encodes a 23-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. The protein was temporarily called BV-E23. In addition immunofluorescence microscopy demonstrated that the protein was present within the cytoplasm and nuclei in virus-infected cells. In conclusion, the available data suggest that Bm51 is a functional ORF of BmNPV and encodes a protein expressed in the early stage of the infection cycle that is associated with the BV envelope.

The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14-22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD(50) was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase-Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16-18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

Open reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of 'enveloped' nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.

B cell lymphomas in immunocompromised patients frequently contain the Epstein-Barr virus (EBV) genome (MacMahon et al, 1991), suggesting that gene therapy strategies that target EBV-positive cells for destruction might be useful for the therapy of such tumors. We have previously shown that stable expression of the cytosine deaminase (CD) gene in EBV-positive lymphoblastoid cell lines induces cell killing in the presence of the prodrug 5-fluorocytosine, with a substantial bystander killing effect (Rogers et al., 1996). To promote specific killing of EBV-positive tumor cells, we have constructed two different EBV-based vectors containing the cytosine deaminase gene. The first vector (OriP-CD), which contains the intact EBV oriP enhancer/replication element, replicates as an episome specifically in EBV-positive cells and likewise enhances transcription in an EBV-specific manner. The OriP-CD vector cannot be packaged or spread from cell to cell. The second vector (OriLyt-CD) contains the EBV lytic origin of replication (oriLyt), the EBV packaging sequences (located in the viral termini), the oriP enhancer element (but not the complete replication origin), and the EBV BZLF1 gene (which induces expression of the EBV proteins required for replication of oriLyt). The OriLyt-CD vector is replicated through the oriLyt origin specifically in EBV-positive cells and packaged as an EBV pseudovirion. The packaged oriLyt-CD virion can subsequently infect cells containing the EBV receptor, CD21, and initiate another round of replication in EBV-positive cells. Here we demonstrate that each of these two different EBV-based gene therapy strategies induces specific killing of EBV-positive B cells in vitro (in the presence of 5-FC). The advantages and disadvantages of each strategy are discussed.

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.

A simple new method is reported for the identification of arbovirus isolates and the titration of arboviruses. Horseradish peroxidase conjugated Staphylococcus aureus protein A was used for the indirect staining of virus antigens grown in an Aedes albopictus cell line, C6/36 cells. Thirty-five isolates from Culex tritaeniorhynchus mosquitoes were examined by this method and 20 of these were identified as Japanese encephalitis virus (JEV). These results were confirmed by immunofluorescent assay and plaque neutralization test. Comparative titration of JEV, Murray Valley encephalitis (MVE) and Kunjin viruses showed that this method was as sensitive as the chick embryo plaque assay. Specific enzymatic reactions on these virus-infected cells began to appear before day 3 and reached the end-point on day 5 post-infection of C6/36 cells, whereas the cytopathic effect (CPE) appeared about 2 days later than the positive enzymatic reactions. Fixation with 10% formalin for 0.5-8 h did not damage the positive reaction in infected cells and did not increase the background colour of uninfected cells.