[Show abstract][Hide abstract] ABSTRACT: Background:
The UL54 protein of Duck Enteritis Virus (DEV) is a homolog of herpes simplex virus-1 (HSV-1) immediate-early infectious cell protein 27 (ICP27), a multifunctional protein essential for viral infection. Nonetheless, there is little information on the UL54 protein of DEV.
The UL54 gene was cloned into the pPAL7 vector, and the recombinant protein, expressed in the E. coli Rosetta, was used to produce a specific antibody. Using this antibody, Western blotting and indirect immunofluorescence analysis (IFA) were used to analyze the expression level and intracellular localization, respectively, of UL54 in DEV-infected cells at different times. Real-time quantitative reverse transcription PCR (RT-PCR) and the pharmacological inhibition test were utilized to ascertain the kinetic class of the UL54 gene.
UL54 was expressed as a fusion protein of approximately 66.0 kDa using the prokaryotic expression system, and this protein was used to generate the specific anti-UL54 antibody. The UL54 protein was initially diffusely distributed throughout the cytoplasmic region; then, after 2 h, it gradually distributed into the nucleus, peaking at 24 h, and complete localization to the nucleus was observed thereafter. The UL54 transcript was detected as early as 0.5 h, and peak expression was observed at 24 h. The UL54 gene was insensitive to the DNA polymerase inhibitor Ganciclovir (GCV) and the protein synthesis inhibitor Cycloheximide (CHX), both of which confirmed that UL54 was an immediate early gene.
The DEV UL54 gene was expressed in a prokaryotic expression system and characterized for expression level, intracellular localization and gene kinetic class. We propose that these results will provide the foundation for further functional analyses of this gene.
[Show abstract][Hide abstract] ABSTRACT: Riemerella anatipestifer (R. anatipestifer) is among the most prevalent duck pathogens, causing acute or chronic septicemia characterized by serositis. Riemerella anatipestifer can be grown on blood-enriched media, in vitro, which provides a hemin source essential for the sustainment of R. anatipestifer and activation of hemin-uptake systems. However, the genes associated with hemin uptake cannot be identified exclusively through genome sequence analysis. Here, we show that R. anatipestifer encodes outer-membrane hemin-binding proteins. Hemin-binding proteins were identified in the cytoplasm with apparent molecular mass of ~45/37/33/23/20/13 kDa, and outer membrane with apparent molecular mass of ~90/70/60/50/15 kDa by batch affinity chromatography and hemin-blotting assays. Our results indicate that these proteins are involved in hemin acquisition. Finally, hemin-binding assay further showed that R. anatipestifer can bind hemin and this capability is increased in iron limited medium, indicating the hemin-uptake system of R. anatipestifer was regulated by iron.
No preview · Article · Nov 2015 · Current Microbiology
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) recognize components of pathogens and mediate the host innate immune response. TLR21 is a TLR that specifically recognizes exogenous double-stranded DNA and rapidly signals to downstream innate immune factors. This study reports the cDNA of goose TLR21 and identifies its immune characteristics. The goose TLR21 is 3161 base pairs and encodes a 975 amino acid protein. As predicted, the goose transmembrane protein TLR21 has a signal peptide, leucine-rich repeat regions, a transmembrane domain, and a Toll/interleukin-1 receptor domain. Multiple sequence alignments and phylogenetic analyses showed that goose TLR21 has homology to chicken TLR21. The tissue distribution of TLR21 suggested that it has high transcript levels in immune-associated tissues, especially in the bursa of Fabricius, the Hadrian gland, and the thymus. After challenge with agonist ODN2006 and new type gosling viral enteritis virus (NGVEV), significant induction of TLR21 production, pro-inflammatory cytokines IL-1β and IL-6, and interferons were observed in peripheral blood mononuclear cells. Both synthetic DNA (ODN2006) and viral DNA (NGVEV) can be recognized by goose TLR21, which leads to a rapid up-regulation of pro-inflammatory cytokines and anti-viral molecules. In vivo, avian influenza A virus H9N2 and NGVEV were used to infect goslings, which was followed by a significant up-regulation of TLR21 mRNA transcripts in multiple tissues of NGVEV-infected geese. In general, goose TLR21 plays an important role in binding invading pathogenic DNA viruses, which subsequently triggers an innate immune response; furthermore, it acts as a functional homologue of mammalian TLR9, as TLR21 recognizes a mammalian TLR9 agonist.
[Show abstract][Hide abstract] ABSTRACT: The goose (Anser cygnoides), having high nutritional value, high-quality feathers and high economic benefit, is an economically important poultry species. However, the molecular mechanisms underlying the higher susceptibility to pathogens in goslings than in adult geese remains poorly understood. In this study, the histological sections of spleen tissue from a two-week-old gosling and an adult goose, respectively, were subjected to comparative analysis. The spleen of gosling was mainly composed of mesenchyma, accompanied by scattered lymphocytes, whereas the spleen parenchyma was well developed in the adult goose. To investigate goose immune-related genes, we performed deep transcriptome and gene expression analyses of the spleen samples using paired-end sequencing technology (Illumina). In total, 50,390 unigenes were assembled using Trinity software and TGICL software. Moreover, these assembled unigenes were annotated with gene descriptions and gene ontology (GO) analysis was performed. Through Kyoto encyclopedia of genes and genomes (KEGG) analysis, we investigated 558 important immune-relevant unigenes and 23 predicted cytokines. In addition, 22 immune-related genes with differential expression between gosling and adult goose were identified, among which the three genes showing largest differences in expression were immunoglobulin alpha heavy chain (IgH), mannan-binding lectin serine protease 1 isoform X1 (MASP1) and C-X-C chemokine receptor type 4 (CXCR4). Finally, of these 22 differentially expressed immune-related genes, seven genes, including tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), C-C motif chemokine 4-like (CCL4), CXCR4, interleukin 2 receptor alpha (IL2RA), MHC class I heavy chain (MHCIα), transporter of antigen processing 2 (TAP2) IgH, were confirmed by quantitative real-time PCR (qRT-PCR). The expression levels of all the candidate unigenes were up-regulated in adult geese other than that of TNFRSF13B. The comparative analysis of the spleen transcriptomes of gosling and adult goose may promote better understanding of immune molecular development in goose.
Full-text · Article · Sep 2015 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: Domestic ducks remain a major source of zoonotic Salmonella enterica infections for man worldwide and approaches to protection should include vaccine-mediated immunity. With this in mind we developed several genetically defined mutants in a virulent duck Salmonella typhimurium isolate TT-1. From initial tests for virulence in day-old ducks, ΔrpoS, ΔhilA, and ΔslyA mutants retained some virulence so were not studied further. Amongst the mutants showing greater attenuation, ΔssrB, ΔphoPQ, ΔompR, and ΔclpP also showed high levels of protection when 1-day-old ducks, which were vaccinated orally, were challenged 1 week later demonstrating the capacity to protect ducks in the first few weeks of life when they are most susceptible and when the risk of infection is greatest. Immunized ducks triggered Omp-specific IgG, IgM, and IgA responses and raised IL-2 and IFN-γ levels in the serum coupled with IL-4 suppression.
[Show abstract][Hide abstract] ABSTRACT: Interferon γ receptor 1 (IFNGR1) and IFNGR2 are two cell membrane molecules belonging to class II cytokines, which play important roles in the IFN-mediated antiviral signaling pathway. Here, goose IFNGR1 and IFNGR2 were cloned and identified for the first time. Tissue distribution analysis revealed that relatively high levels of goose IFNγ mRNA transcripts were detected in immune tissues, including the harderian gland, cecal tonsil, cecum, and thymus. Relatively high expression levels of both IFNGR1 and IFNGR2 were detected in the cecal tonsil, which implicated an important role of IFNγ in the secondary immune system of geese. No specific correlation between IFNγ, IFNGR1, and IFNGR2 expression levels was observed in the same tissues of healthy geese. IFNγ and its cognate receptors showed different expression profiles, although they appeared to maintain a relatively balanced state. Furthermore, the agonist R848 led to the upregulation of goose IFNγ but did not affect the expression of goose IFNGR1 or IFNGR2. In summary, trends in expression of goose IFNγ and its cognate receptors showed tissue specificity, as well as an age-related dependency. These findings may help us to better understand the age-related susceptibility to pathogens in birds.
[Show abstract][Hide abstract] ABSTRACT: Enteroviruses are a large group of small nonenveloped viruses that cause common and debilitating illnesses affecting humans and animals worldwide. The capsid composed by viral structural proteins packs the RNA genome. It is becoming apparent that structural proteins of enteroviruses play versatile roles in the virus-host interaction in the viral life cycle, more than just a shell. Furthermore, structural proteins to some extent may be associated with viral virulence and pathogenesis. Better understanding the roles of structural proteins in enterovirus infection may lead to the development of potential antiviral strategies. Here, we discuss recent advances from studies on the role of structural proteins in enterovirus infection and antiviral therapeutics targeted structural proteins.
[Show abstract][Hide abstract] ABSTRACT: Tembusu virus (TMUV) has caused significant economic losses in the Chinese duck industry and may have been overlooked regarding its zoonotic transmission potential. A novel TMUV isolate (named CQW1) was separated from the liver tissue of a young duck in Southwest China. The CQW1 isolate proliferated in embryonated duck eggs and led to death within 3-4 days post-inoculation. Furthermore, CQW1 replicated in duck embryo fibroblast (DEF) cells and caused a cytopathic effect (CPE). The disease emerged on a duck farm in Southwest China and was reproduced by animal experiment. We found that CQW1 was detectable by RT-PCR in brain and liver tissues of dead ducklings within 5 days after inoculation. Most importantly, concentrated nuclei, neuronophagia and microglial nodules were observed in the brain tissue of the inoculated ducklings, and additionally, the liver tissue was affected, mainly by disordered lobular architecture, degeneration, necrosis and regenerated hepatocytes. Analysis of the complete genome sequence showed that CQW1 was 10,992 nt in length with two nucleotide insertions and shared 96.8 % to 99.1 % and 98.4 % to 99.6 % identity at nucleotide and amino acid level, respectively, with Chinese isolates. Phylogenetic analysis of the nucleotide sequences demonstrated that the CQW1 isolate was closely related to other members of the genus Flavivirus and formed a new clade together with the GX2013H isolate. Also, the CQW1 isolate demonstrated the highest average pairwise distance value among the Chinese isolates. In the present study, we obtained evidence that TMUV is present in Southwest China. Extensive pathological and epidemiological studies are urgently needed.
No preview · Article · Aug 2015 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: The parvoviruses are widely spread in many species and are among the smallest DNA animal viruses. The parvovirus is composed of a single strand molecule of DNA wrapped into an icosahedral capsid. In a viral infection, the massy capsid participates in the entire viral infection process, which is summarized in this review. The capsid protein VP1 is primarily responsible for the infectivity of the virus, and the nuclear localization signal (NLS) of the VP1 serves as a guide to assist the viral genome in locating the nucleus. The dominant protein VP2 provides an "anti-receptor", which interacts with the cellular receptor and leads to the further internalization of virus, and, the N-terminal of VP2 also cooperates with the VP1 to prompt the process of nucleus translocation. Additionally, a cleavage protein VP3 is a part of the capsid, which exists only in several members of the parvovirus family; however, the function of this cleavage protein remains to be fully determined. Parvoviruses can suffer from the extreme environmental conditions such as low pH, or even escape from the recognition of pattern recognition receptors (PRRs), due to the protection of the stable capsid, which is thought to be an immune escape mechanism. The applications of the capsid proteins to the screening and the treatment of diseases are also discussed. The processes of viral infection should be noted, because understanding the virus-host interactions will contribute to the development of therapeutic vaccines.
[Show abstract][Hide abstract] ABSTRACT: Interferons, as the first line of defense against the viral infection, play an important role in innate immune responses. Type III interferon (IFN-λ) was a newly identified member of IFN family, which plays IFN-like antiviral activity. Towards a better understanding of the type III interferon system in birds, type III interferon lambda receptor (IFNLR1) was first identified in the Chinese goose. In this paper, we had cloned 1952 bp for goose IFNLR1 (goIFNLR1), including an ORF of 1539 bp, encoding a 512-amino acid protein with a 20 aa predict signal peptide at its N terminal and a 23 aa transmembrane region. The predicted amino acid sequence of goIFNLR1 has 90%, 73%, and 34% identity with duck IFNLR1 (predicted sequence), chicken IFNLR1, and human IFNLR1, respectively. And the age-related tissue distribution of goIFNLR1 was identified by Real Time quantitative PCR (RT-qPCR), we found that the goIFNLR1 has a mainly expression in epithelium-rich tissues similar to other species’, such as small intestinal, lung, liver, and stomach. Moreover, a relatively high expression of goIFNLR1 was also observed in the secondary immune tissues (harderian gland and cecal tonsil). The identification and tissue distribution of goIFNLR1 will facilitate further study of the role of IFN-λ in goose antiviral defense.
[Show abstract][Hide abstract] ABSTRACT: Duck circovirus may predispose the host to immunosuppression and may serve as an immunological trigger for further complicated disease progression. Due to the lack of a cell culture system for propagating DuCV, little is known regarding the molecular biology and pathogenesis of DuCV. The aim of this study was to describe the construction and initial in vivo characterization of full-length DNA clones of DuCV (pIC-Mu2DuCV) and its infectivity under in vivo conditions.
The constructed pIC-Mu2DuCV contained two copies of the whole DuCV genome and an introduced Xho I restriction enzyme site. Eighty-one 10-day-old conventional ducklings that were free of DuCV were randomly divided equally into three groups (1, 2 and 3). The ducklings in groups 1, 2 and 3 were inoculated intramuscularly with pIC-Mu2DuCV, wild-type virus GH01 and PBS, respectively. Subsequently, all of the ducklings were examined clinically, which were each given a physical condition score, and their rectal temperatures were taken daily during the experimental period. DuCV genomes in serum samples and in various tissues from all of the ducklings at 0, 1, 3, 5, 7, 10, 15, 21 and 28 DPC were detected by PCR and real-time quantitative PCR, respectively.
The average daily weight gain (ADWG) of group 3 was significantly higher than those of groups 1 and 2, and the temperature of all ducklings was stable between 41.7 °C and 42.2 °C. The clinical values (physical condition scores) of groups 1, 2 and 3 were 12.5, 15.6 and 0, respectively. In addition, viremia occurred at 15 and 10 days post-challenge (DPC) in groups 1 and 2, and antibodies could be detected in these ducklings at 21 and 15 DPC. Proliferation ability analysis showed that the viral titers of group 1 were lower than those of their parental viruses in group 2.
This study shows that the rescued viruses are not significantly different but exhibit lower pathogenicity and proliferation ability compared with the parental virus. The results will facilitate future studies on DuCV pathogenesis and biology.
[Show abstract][Hide abstract] ABSTRACT: Riemerella anatipestifer (R. anatipestifer) is one of the most important pathogens in ducks. The bacteria causes acute or chronic septicemia characterized by fibrinous pericarditis and meningitis. The R. anatipestifer genome encodes multiple iron/hemin-uptake systems that facilitate adaptation to iron-limited host environments. These systems include several TonB-dependent transporters and three TonB proteins responsible for energy transduction. These three tonB genes are present in all the R. anatipestifer genomes sequenced so far. Two of these genes are contained within the exbB-exbD-tonB1 and exbB-exbD-exbD-tonB2 operons. The third, tonB3, forms a monocistronic transcription unit. The inability to recover derivatives deleted for this gene suggests its product is essential for R. anatipestifer growth. Here, we show that deletion of tonB1 had no effect on hemin uptake of R. anatipestifer, though disruption of tonB2 strongly decreases hemin uptake, and disruption of both tonB1 and tonB2 abolishes the transport of exogenously added hemin. The ability of R. anatipestifer to grow on iron-depleted medium is decreased by tonB2 but not tonB1 disruption. When expressed in an E. coli model strain, the TonB1 complex, TonB2 complex, and TonB3 protein from R. anatipestifer cannot energize heterologous hemin transporters. Further, only the TonB1 complex can energize a R. anatipestifer hemin transporter when co-expressed in an E. coli model strain.
[Show abstract][Hide abstract] ABSTRACT: Riemerella anatipestifer is a major bacterial pathogen of waterfowl, globally responsible for avian septicemia disease. As chemotherapy is the predominant method for the prevention and treatment of R. anatipestifer infection in poultry, the widespread use of antibiotics has favored the emergence of antibiotic resistant strains. However, little is known about R. anatipestifer susceptibility to macrolide antibiotics and its resistance mechanism. We report for the first time the identification of a macrolide resistance mechanism in R. anatipestifer that is mediated by the ribosomal RNA methyltransferase ermF. We identified the presence of the ermF gene in 64/206 (31%) R. anatipestifer isolates from different regions in China. An ermF deletion strain was constructed to investigate the function of the ermF gene on the resistance to high-levels of macrolides. The ermF mutant strain showed significantly decreased resistance to macrolide and lincosamide, exhibiting 1024-, 1024-, 4-, and >2048-fold reduction in the minimum inhibitory concentrations for erythromycin, azithromycin, tylosin, and lincomycin, respectively. Furthermore, functional analysis of ermF expression in E.coli XL1-blue showed that R. anatipestifer ermF gene was functional in E.coli XL1-blue and conferred resistance to high-levels of erythromycin (100 µg/ml), supporting the hypothesis that the ermF gene is associated with high-level macrolide resistance. Our work suggests that ribosomal RNA modification mediated by the ermF methyltransferase is the predominant mechanism of resistance to erythromycin in R. anatipestifer isolates.
[Show abstract][Hide abstract] ABSTRACT: Aquatic birds play n critical role in the transmission and dissemination of many important pathogens such as avian influenza virus. The cell-mediated immunity is very important in eliminating the intracellular antigens. Expression of CD4 and CD8 on T cell surface is essential for cell-mediated immune defence and T-cell development. However, the ontogeny of T lymphocytes in waterfowl is scarce and fragmentary. To address these questions, here we report the development and tissues distribution of CD4 and CD8α in goose embryo, gosling and goose by immunocytochemistry assay using monoclonal antibodies. Moreover, the age-related mRNA level of goose CD4 and CD8α in different immune tissues were study by real time quantitative PCR. Our results suggested that the high expression of CD4 and CD8α were readily found in thymus, which peaked at the first week post-hatch. And the highest expression level of CD4 and CD8α were detected in bursa of Fabricius, caecal tonsils, spleen and intestine at the second week, after that the expression level were gradually decreased. Interestingly, the remarkably high expression of CD4 and CD8α in Harderian gland were detected at the first week, which is about hundreds times more than that in other tissues. Our findings demonstrated that the development and the distribution of CD4 and CD8α are partly changed in an age-related way. Moreover, the histological morphogenesis of immune tissues were also discussed. Our results may shed lights on the better understand of T-cell mediate immunity in goose.