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.

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.

An insect nucleoside diphosphate kinase (NDK) functions as an effector protein in wheat - Hessian fly interactions.

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.

Analysis of the immune response in infections of the goldfish (Carassius auratus L.) with Mycobacterium marinum.

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-1ß1) and cytokine receptors (IFNGR1-1, TNFR2) at 7 dpi. Conversely, the gene expression levels of key anti-inflammatory cytokines TGFß 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.

Ozone treatment ameliorates oil sands process water toxicity to the mammalian immune system.

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.

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus.

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 encodes an occlusion-derived virus protein and is not essential for budded virus production.

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.

Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67.

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.