Shun Chen

Sichuan Agricultural University, Hua-yang, Sichuan, China

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Publications (37)76.5 Total impact

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    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.
    Avian Pathology 02/2015; DOI:10.1080/03079457.2015.1019828 · 2.04 Impact Factor
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    ABSTRACT: The cDNAs encoding two distinct typeIinterferon receptors were firstly cloned from the spleen of white goose (the Chinese goose, A. cygnoides). The cDNA of goose IFNAR1 consisted of 1616bp and encoded 406 amino acids with a predicted molecular weight of 46.4kDa, while the cDNA of goose IFNAR2 consisted of 1525bp and encoded 294 amino acids with a predicted molecular weight of 32.6kDa. The IFNAR1 shared 85.4% identity in deduced amino acid sequence with duck IFNAR1, while IFNAR2 amino acid sequence showed 86% identity with that of duck IFNAR2. The age-related analysis of gene expression revealed that goose IFNα and IFNARs were all highly transcribed in pancreas, which may due to a reasonable amount of dendritic cells aggregated in pancreas. And goose IFNα and its cognate receptors had different structural features and tissue expression patterns during the period from embryonic goose to adult goose, suggesting that IFNα and IFNARs may maintain a developmental dynamic immune competence in unstimulated states. The data provided in this study may contribute to future understanding of the interaction between interferon and interferon receptors in immune mechanism. And it also helps us to understand the age-related susceptibility to pathogens in birds better. Copyright © 2015. Published by Elsevier B.V.
    Gene 01/2015; DOI:10.1016/j.gene.2015.01.040 · 2.20 Impact Factor
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    ABSTRACT: The CD8 molecule is a cell membrane glycoprotein expressed on cytotoxic T lymphocytes, which are involved in the clearance of viruses. However, the functional characterization of goose CD8α is still unclear. The immunobiological characterization of goose CD8α in goose spleen mononuclear cells (MNCs) was examined by real-time quantitative PCR (qPCR). It was shown that CD8α mRNA levels were significantly up-regulated by in vitro treatment of MNCs with phytohemagglutinin (PHA), concanavalin A (ConA), and polyinosinic-polycytidylic acid (poly I:C) in a dose-dependent way, but lipopolysaccharides (LPSs) did not have this same effect. Moreover, the time-course effect of CD8α expression in response to mitogens (PHA, ConA, and poly I:C) was evaluated in MNCs. A significant increase in the transcriptional levels of CD8α was detected in new type gosling viral enteritis virus (NGVEV)-infected goose MNCs at 48 h postinfection (PI) and in goose parvovirus (GPV)-infected MNCs at 72 h PI. Also, the number of CD8α(+) cells was significantly increased during viral infection from 72 h on. The seminal changes in mRNA profiles of antiviral cytokines (IFN-α, IFN-γ, and IL-18) were observed and were significantly increased during late phases of NGVEV and GPV infection. Accordingly, our data not only contribute to the understanding of the immune characteristics of goose CD8α, but they also provide new insight into the innate antiviral immunity of geese. © 2015 Poultry Science Association Inc.
    Poultry Science 01/2015; 94(1):17-24. DOI:10.3382/ps/peu024 · 1.54 Impact Factor
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    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.
    Immunobiology 01/2015; DOI:10.1016/j.imbio.2014.12.020 · 2.81 Impact Factor
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    ABSTRACT: Duck circovirus (DuCV) disease causes a long-term immunosuppressive and multiple secondary infection in ducks. In this study, relative synonymous codon usage values (RSCU), nucleotide contents and effective number of codons (ENC) values were calculated and compared among open reading frames (ORFs) of 53 DuCV genomes. The results reveal that the most of codons are ended with C and the overall bias is no remarkable in DuCV. A comparative analysis of codon contents and ENC values indicates that mutation pressure is the most significant factor responsible for the evolutional processes of codon usage bias in DuCV. However, other factors, such as composition constraints, translation selection, hydrophobicity and aromaticity should not be ignored. Finally principal component analysis (PCA) and hierarchical clustering method were performed based on RSCU. The significant difference of codon usage bias exists in DuCV-1 and DuCV-2 genotypes, but codon usage pattern of DuCV from the different epidemic areas or subtypes fails to influence the formation of codon usage bias. Analysis of the relationship of synonymous codon usage variation based on the two genotypes suggests that DuCV-2 is more conservative than DuCV-1, which may because of recombination events. Moreover, there are distinct differences in the degree of codon usage pattern evolution in different function genes, rep and cap. Therefore, the genotypes, subtypes and different functional genes also relate to the pattern of synonymous codon usage. The main objective of this study is to provide some sight into synonymous codon usage characteristics and the evolutionary relationship of DuCV. Copyright © 2014. Published by Elsevier B.V.
    Gene 12/2014; 557(2). DOI:10.1016/j.gene.2014.12.019 · 2.20 Impact Factor
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    ABSTRACT: TLR7 is a transmembrane endosomal protein that plays an essential role in innate antiviral responses via the recognition of conserved viral molecular patterns. Here, we cloned the full-length cDNA of goose TLR7 and carried out a molecular characterization of goose TLR7. The goose TLR7 gene is 3900bp and encodes a 1045 amino acid protein with high homology to poultry (93% to duck and 83% to chicken). Similar conclusions were made by phylogenetic analysis. The predicted protein secondary structure of goose TLR7 contained a conserved Toll/interleukin-1 receptor domain and characteristic leucine-rich repeat regions, which has also been reported for duck TLR7. Additionally, the tissue distribution of goose TLR7 suggests that immune-associated tissues, especially the cecal tonsil and bursa of Fabricius, have high goose TLR7 expression levels. Goose TLR7 is abundantly expressed in lung tissues, which is distinct from its expression in chickens. Similar to duck TLR7, goose spleen mononuclear cells (MNCs) exposed to the mammalian TLR7 agonists R848 and Imiquimod showed significant induction of the production of proinflammatory cytokines and IFN-α. New type gosling viral enteritis virus (NGVEV) infection resulted in high mRNA expression levels of goose TLR7 in the spleen. By contrast, no direct interaction between NGVEV and goose TLR7 was detected after infecting goose spleen MNCs with NGVEV in vitro. However, triggering of goose TLR7 resulted in the rapid up-regulation of proinflammatory cytokines and anti-viral molecules, suggesting that goose TLR7 plays an important role in anti-viral defense. Copyright © 2014. Published by Elsevier B.V.
    Immunology Letters 12/2014; 163(2). DOI:10.1016/j.imlet.2014.11.017 · 2.37 Impact Factor
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    ABSTRACT: Over the course of duck hepatitis B virus (DHBV) replication, one type of RNA (pregenome/C RNA, 3.5kb) that corresponds to the whole genome of DHBV is generated from the transcription of viral cccDNA. Previous work has proposed three functions for the pregenome/C RNA: it can serve as the pregenome and be packaged into the core protein during the process of replication, and it encodes the mRNA for both the capsid protein and the viral polymerase. However, little is known about the timing of these functions during the different stages of viral infection. In this study, a reverse transcription quantitative real-time PCR assay was developed to analyze the dynamic transcription process of the pregenome/C RNA. The dynamic expression of the core protein was investigated using an indirect immunofluorescence assay (IFA) and by western blot analysis. The generation of pregenome/C RNA began at 12h post infection and peaked at 20h post infection; however, the core protein was not detectable until 24h post infection. These results demonstrate that the core protein appeared approximately 12h later than the pregenome/C RNA. These results suggest that the DHBV pregenome/C RNA is not used for the translation of the viral core protein during the early stages of infection. Copyright © 2014 Elsevier B.V. All rights reserved.
    Virus Research 11/2014; 196C:13-19. DOI:10.1016/j.virusres.2014.10.026 · 2.83 Impact Factor
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    ABSTRACT: Interferon may be thought of as a key, with the interferon receptor as the signal lock: Crosstalk between them maintains their balance during viral infection. In this review, the protein structure of avian interferon and the interferon receptor are discussed, indicating remarkable similarity between different species. However, the structures of the interferon receptors are more sophisticated than those of the interferons, suggesting that the interferon receptor is a more complicated signal lock system and has considerable diversity in subtypes or structures. Preliminary evolutionary analysis showed that the subunits of the interferon receptor formed a distinct clade, and the orthologs may be derived from the same ancestor. Furthermore, the development of interferons and interferon receptors in birds may be related to an animal's age and the maintenance of a balanced state. In addition, the equilibrium between interferon and its receptor during pathological and physiological states revealed that the virus and the host influence this equilibrium. Birds could represent an important model for studies on interferon's antiviral activities and may provide the basis for new antiviral strategies.
    International Journal of Molecular Sciences 11/2014; 15(11):21045-21068. DOI:10.3390/ijms151121045 · 2.46 Impact Factor
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    ABSTRACT: BackgroundRiemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.ResultsIn this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.ConclusionThe genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.
    BMC Genomics 06/2014; 15(1):479. DOI:10.1186/1471-2164-15-479 · 4.04 Impact Factor
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    ABSTRACT: The UL49.5 gene of most herpesviruses has been reported as a conserved gene encoding glycoprotein N. However, duck enteritis virus (DEV) UL49.5 protein (pUL49.5) has not been reported yet. In this study, molecular characterization of DEV pUL49.5 was firstly analyzed. In order to verify the prediction of the intracellular localization, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and transfected in duck embryo fibroblasts. Then, the recombinant plasmid pDsRed1-N1/gM was also constructed and co-transfected with the pEGFP-C1/pUL49.5 to understand whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) located consistently. The prediction results showed that DEV pUL49.5 was an envelope glycoprotein with a signal peptide and two transmembrane domains. Also, it was forecasted to distribute in the cytoplasm and concentrated in endoplasmic reticulum with 66.7% probability. The fluorescence images shooted by fluorescence microscope at different time points revealed that DEV pUL49.5 and gM were both located in the cytoplasm. The overlap of the two kinds of fluorescence appeared at 12h after transfection and continued to exist until the end of the test, which suggested that the localization of DEV pUL49.5 was consistent with gM. It was an evidence to provide the possibility of an interaction between DEV pUL49.5 and gM.
    Journal of veterinary science (Suwŏn-si, Korea) 04/2014; 15(3). DOI:10.4142/jvs.2014.15.3.389 · 0.89 Impact Factor
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    ABSTRACT: Duck plague (DP) is a severe disease caused by DP virus (DPV). Control of the disease is recognized as one of the biggest challenges in avian medicine. Vaccination is an efficient way to control DPV and an attenuated vaccine is the main routine vaccine. Attenuated DPV vaccine CHa strain is a modified live vaccine, but the systemic and mucosal immune responses have been poorly understood. In this study, the immunogenicity and efficacy of the vaccine was evaluated after subcutaneous immunization of ducks. CD4+ and CD8+ T cells were counted by flow cytometry and humoral and mucosal Ig antibodies were analyzed by ELISA assay. The results showed that high levels of T cells and Ig antibodies were evaluated post immunization. Results indicated that there were more CD4+ T cells than CD8+ T cells. Titers of humoral IgG were higher than humoral IgA. Local IgA was found in each sample, whereas local IgG was only found in spleen, thymus, bursa of fabricius, harderian gland, liver, bile and lung. In protection assay, the attenuated DPV vaccine completely protected ducks against 1000 LD50 lethal DPV CHv strain via oral infection. These data suggest that this subcutaneous vaccine elicited sufficient systemic and mucosal immune responses against lethal DPV challenge to be protective in ducks. This study provides broad insights into understanding the immune responses to the attenuated DPV vaccine CHa strain through subcutaneous immunization in ducks.
    Clinical and vaccine Immunology: CVI 01/2014; DOI:10.1128/CVI.00605-13 · 2.37 Impact Factor
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    ABSTRACT: Duck circovirus was a newly discovered pathogen that causing ducks immunosuppression in recent years, but it can not been cultured in vitro that limited its depth study. In the present study, the Cap gene that defect the nuclear localization signal (NLS) of DuCV was amplified and connected to the express vector pET-32a (+), to express the recombinant Cap protein in the bacterium Escherichia coli Rosetta. The recombinant Cap protein was purified and an indirect ELISA method was developed based on the recombinant Cap protein. The results showed that the truncated Cap gene was 567 bp and cloned into pET-32a (+) vector successfully. The recombinant Cap protein was expressed as inclusion bodies. The results of optimization for indirect ELISA revealed that the optimal antigen and serum dilutions were selected to be 4 μg/well and 1:40, respectively; the coating condition was 37°C for 1 h and 4°C overnight; the blocking time in 1% BSA was 1 h at 37°C and the second antibody dilution was 1:800, the cut-off value was 0.352. Known anti-sera samples of other duck common pathogens were tested by the developed ELISA and the results showed it was specific for DuCV anti-sera detection. The sensitivity of indirect ELISA reached 1:2560, and the coefficient of variation between intra-assay and inter-assay were less than 10%. Compared the PCR and indirect ELISA methods, the positive compliance rate was 95.6% for detected 59 duck samples.
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    ABSTRACT: To explore and isolate genes related to duck embryonic fibroblast cells (DEFs) post-infected with duck enteritis virus (DEV), a cDNA library was established using SMART (Switching Mechanism At 5' end of the RNA Transcript) technique coupling with a homologous recombination method. The cells were harvested and total RNA was extracted at 48 h post infection. Then the mRNAs were purified and reverse transcribed to first-strand cDNAs using oligo (dT) primers (CDS III). Subsequently, long distance-PCR was performed, the double-stranded cDNAs were purified, and a transformation assay was carried out in that order. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 2.33 × 10(6) transformants/4.34 μg pGADT7-Rec (>1.0 × 10(6)). The cell density of the library was 1.75 × 10(9) cells/mL (>2×10(7) cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 × 10(5) and 2.33 × 10(7) CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 × 10(7) and 1.14 × 10(9), respectively, and the recombinant rate was 97.14 %. The sequence results of 27 randomly picked independent clones revealed the insert ranged from 0.323 to 2.017 kb with an average insert size of 0.807 kb. Full-length transcripts of DEV-CHv LORF3, UL26 and UL35 genes were acquired through sequence similarity analysis from the non-redundant nucleic acid or protein database. Five polyA sites were identified in the DEV-CHv genome. Also, a new transcript of 668 bp was found between the IRS gene and US1 gene of the DEV-CHv genome. Thus, we concluded that the constructed cDNA library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs, and discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases.
    Molecular Biology Reports 11/2013; 41(1). DOI:10.1007/s11033-013-2881-z · 1.96 Impact Factor
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    ABSTRACT: Infecting ducks with duck hepatitis B virus (DHBV) is widely accepted as a relevant model for studying aspects of human HBV infection. However, efficient and sensitive diagnostic methods for the various infection models are limited. In order to provide a more simple and convenient method for serologic diagnosis, we improved the production of recombinant DHBV viral capsid protein (core protein) and then used it to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-DHBc antibodies (DHBcAg ELISA) in DHBV-infected ducks. Given the positive/negative cut-off value, the maximum dilution of duck sera in which anti-DHBc antibodies could be detected was 1:12,800. In addition, the DHBcAg ELISA displayed no cross reactivity with duck antisera against duck circovirus (DuCV), duck plague virus (DPV), duck hepatitis virus (DHV), duck swollen head septicemia virus (DSHSV), avian influenza virus (AIV), Riemerella anatipestifer, Salmonella anatum, or Escherichia coli. Furthermore, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below than 10 %. When compared to PCR for accuracy on clinical samples from cases of suspected DHBV infection, the DHBcAg showed 95.45 % coincidence with PCR. In conclusion, recombinant DHBc was readily produced and used to establish a simple DHBcAg ELISA that provided a highly specific and sensitive method for analysis of clinical samples.
    Archives of Virology 10/2013; 159(5). DOI:10.1007/s00705-013-1897-y · 2.28 Impact Factor
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    ABSTRACT: : Similar to mammals, several viral-sensing pattern recognition receptors (PRR) have been identified in birds including Toll-like receptors (TLR) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR). Avian TLR are slightly different from their mammalian counterparts, including the pseudogene TLR8, the absence of TLR9, and the presence of TLR1La, TLR1Lb, TLR15, and TLR21. Avian TLR3 and TLR7 are involved in RNA virus recognition, especially highly pathogenic avian influenza virus (HPAIV), while TLR15 and TLR21 are potential sensors that recognize both RNA viruses and bacteria. However, the agonist of TLR15 is still unknown. Interestingly, chickens, unlike ducks, geese and finches, lack RIG-I, however they do express melanoma differentiation-associated gene 5 (MDA5) which functionally compensates for the absence of RIG-I. Duck RIG-I is the cytosolic recognition element for HPAIV recognition, while chicken cells sense HPAIV through MDA5. However, the contributions of MDA5 and RIG-I to IFN-beta induction upon HPAIV infection is different, and this may contribute to the chicken's susceptibility to highly pathogenic influenza. It is noteworthy that the interactions between avian DNA viruses and PRR have not yet been reported. Furthermore, the role for avian Nod-like receptors (NLR) in viral immunity is largely unknown. In this review, recent advances in the field of viral recognition by different types of PRR in birds are summarized. In particular, the tissue and cellular distribution of avian PRR, the recognition and activation of PRR by viruses, and the subsequent expression of innate antiviral genes such as type I IFN and proinflammatory cytokines are discussed.
    Veterinary Research 09/2013; 44(1):82. DOI:10.1186/1297-9716-44-82 · 3.38 Impact Factor
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    ABSTRACT: CD8 molecule is a cell membrane glycoprotein, which plays an important role in cell-mediated immunity. Here, we identified Chinese goose CD8α (goCD8α) gene for the first time. The full-length cDNA of goCD8α is 1459bp in length and contains a 711bp open reading frame. Phylogenetic analysis shows that the waterfowl CD8α formed a monophyletic group. Semi-quantitative RT-PCR analysis showed transcripts of goCD8α mRNA were high in the immune-related organs and mucosal immune system in gosling, and high in thymus and spleen comparing to other immune-related tissues in goose. The obviously increase of CD8α expression was observed in spleen of acute new type gosling viral enteritis virus (NGVEV) infected bird, while the increase of CD8α were observed in the thymus, bursa of fabricius, and cecum of chronic infected bird. The CD8α mRNA transcription level in spleen mononuclear cells was significantly up-regulated when stimulated by phytohemagglutinin, but not by lipopolysaccharide in vitro.
    Gene 08/2013; DOI:10.1016/j.gene.2013.07.104 · 2.20 Impact Factor
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    ABSTRACT: Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8-99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparisons analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.
    Gene 08/2013; DOI:10.1016/j.gene.2013.07.028 · 2.20 Impact Factor
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    ABSTRACT: Duck enteritis virus (DEV) UL49.5 encoding glycoprotein N was a conserved gene. The transcription dynamic process of UL49.5 homologous genes in herpesviruses was reported. However, the transcription dynamic process of DEV UL49.5 gene has not yet been established. In this study, a real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay was established to test the transcription dynamic process of DEV UL49.5 gene, and the recombinant plasmid pUCm-T/UL49.5 was constructed as the standard DNA. The samples prepared from DEV-infected (at different time points) and uninfected cell were detected and calculated. The results demonstrated that the real-time qRT-PCR assay was successfully established. The transcription product of DEV UL49.5 gene was first detected at 0.5 h post infection (p.i.), increased at 8 h p.i. and reached a peak at 60 h p.i. Our results illustrated that DEV UL49.5 gene could be regarded as a late gene. The transcription dynamic process of DEV UL49.5 gene may provide a significant clue for further studies of DEV UL49.5 gene.
    Virus Genes 07/2013; 47(2). DOI:10.1007/s11262-013-0949-4 · 1.84 Impact Factor
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    ABSTRACT: Riemerella anatipestifer (RA) is one of the most important pathogens of 1- to 8-wk-old ducklings that severely affects the development of the duck industry in China. Every year, antibiotic medicines including tetracycline and doxycycline are used in the duck industry. Few reports compare the expression of multidrug-resistant genes in RA before and after addition of chemical drugs. With this in mind, the direct effects of gradient concentration of tetracyclines on the expression of tetracycline resistance genes (TETr) in RA at the cDNA level were studied by using a quantitative real-time PCR method. The expression of TETr, tetA, tetC, and tetM was investigated in ATCC11845 and in 30 RA isolated from different samples. Using a range of doxycycline concentrations up to 50% of the minimum inhibitory concentration (MIC), the optimal induction concentration of 0.0625 μg/mL was selected. Under the optimal inducible expression, concentrations of TETr, tetC, and tetM cDNA were detected in all isolates, and the highest mRNA expression level of TETr genes was shown. Additionally, the expression levels of 3 TETr genes in RA14 (tetA and tetC) and RA17 (tetM and tetC) were compared. Both tetC and tetA found in isolate RA14 was found to express both tetC and tetA, and tetC cDNA was detected in isolate RA17 at all doxycycline concentrations tested, whereas tetM cDNA was not detected at any concentration. We can conclude that resistance pump is the main mechanism of tetracycline antibiotic resistance, and under the action of drug resistance pump tetC, the expression of tetM was not activated in RA17. These data suggest that the mRNA expression level of TETr genes was correlated with the MIC values, indicating that the degree of drug resistance is determined by the expression levels of TETr genes. Also, the induction of TETr is the major tetracycline resistance mechanism in RA, especially the resistance pump. However, lower concentrations of doxycycline induced higher TETr expression, and higher concentrations inhibited TETr expression. Maybe that is the reason for selection mutation to make tolerated bacteria survive.
    Poultry Science 06/2013; 92(6):1552-1559. DOI:10.3382/ps.2012-02672 · 1.54 Impact Factor
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    ABSTRACT: BACKGROUND: A eukaryotic expression plasmid encoding glycoprotein C (gC) of Anatid herpesvirus 1 (AnHV-1) (pcDNA3.1-gC) was constructed and validated. The tissue distribution of chitosan/DNA complexes, liposome/DNA complexes and pcDNA3.1-gC alone were evaluated using a quantitative real-time PCR based TaqManTM probe following intramuscular administration in ducklings. RESULTS: Compared with pcDNA3.1-gC alone, liposomes universally increased the plasmid DNA copy number at the injection sites (liver, spleen, heart, brain, bursa of Fabricius, and especially in the enteron [esophagus, duodenum, rectum, and cecum]). Chitosan also universally increased the plasmid DNA copy number at the injection sites (liver, spleen, heart, brain and esophagus). Compared with lipoplex-gC, higher chitosan-gC plasmid DNA copy numbers were detected at the injection sites (liver, spleen, heart, brain and esophagus). In contrast, compared with lipoplex-gC, lower copy numbers of chitosan-gC plasmid DNA were detected in the duodenum, rectum and cecum. CONCLUSIONS: The results of this study demonstrated that chitosan and liposomes mediated rapid and extensive plasmid distribution in duck tissues, with low levels maintained from 1 d after DNA vaccination.
    Virology Journal 03/2013; 10(1):89. DOI:10.1186/1743-422X-10-89 · 2.09 Impact Factor