[Show abstract][Hide abstract] ABSTRACT: Riemerella 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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; · 2.60 Impact Factor
[Show abstract][Hide abstract] 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.
International journal of clinical and experimental pathology. 01/2014; 7(8):4938-44.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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; · 2.28 Impact Factor
[Show abstract][Hide abstract] 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. · 3.43 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: CD4 protein is a single chain transmembrane glycoprotein belonging to the immunoglobin superfamily that recognize the major histocompatibility complex (MHC) class II molecules. It plays an important role in cell-mediated immunity. Here, the full-length cDNA of CD4 in Chinese goose (Anser cygnoides) was cloned and identified. The goose CD4 is 1,940bp in length and encodes a single open-reading frame of 480 amino acids. The putative amino acid sequence of goose CD4 is consisted of a signal peptide, four potential N-glycosylation sites, a transmembrane region and a cytoplasmic tail. The multiple sequence alignment and phylogenetic analysis suggested that goose CD4 shared a higher similarity with avian than other vertebrates. Semi-quantitative RT-PCR analysis showed that the highest level of CD4 mRNA transcripts was presented in thymus, and relative lower in spleen, small intestine, brain and trachea. Low expression was seen in bursa of fabricius, cecal tonsil, cecum, skin, lung, kidney and liver. In gosling, the CD4 transcript was expressed with high abundance in thymus, relative lower in spleen, cecal tonsil and small intestine. However, in adult goose, high expression was seen in thymus, spleen and cecum, relative lower in cecal tonsil and small intestine. During the course of NGVEV infection, the obviously increase of CD4 gene expression were observed in spleen, bursa of fabricius and harderian gland. Interestingly, a notable decrease of CD4 mRNA in the small intestine at 5 d PI and followed by increase of that at 19 d PI were showed.
[Show abstract][Hide abstract] ABSTRACT: In this study, a recombinant fusion antigen of duck enteritis virus (DEV) UL16 protein was expressed in Escherichia coli Rossetta (DE3). This target protein was used as a coating antigen to establish an indirect ELISA for detecting anti-DEV antibodies in serum samples from ducks. In the optimal method for the UL16-ELISA, the fusion protein was coated at 1.25μg/ml and duck serum samples were diluted at 1:160. The endpoint cut-off value of this assay was 0.598. The inter-assay and intra-assay coefficients of variation (CVs) were both lower than 10%. There was no cross-reaction with duck positive sera of either DHBV, DHV, RA, E. coli, S. anatum, H5N1 or DSHDV. The assay was applied successfully to examine the suspected duck serum samples and showed 95.5% (73/76) identity with the serum neutralization test (SNT). The results showed that recombinant DEV UL16 protein could be used as a coating antigen and the developed UL16-ELISA approach was rapid, specific, sensitive and repetitive for detection of duck enteritis virus- specific antibodies.
Journal of virological methods 01/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel duck circovirus (DuCV) strain, designated GH01, was isolated from ducklings in southwestern China. We report the genome sequence of GH01 and the genomic organization and genetic relationship to other DuCVs. The availability of the genome sequence will be helpful to investigations of epidemiology and the evolutionary biology of this organism and the development of preventive vaccines.
[Show abstract][Hide abstract] ABSTRACT: The icosahedral virion of duck enteritis virus (DEV) is roughly spherical and approximately 150 nm in diameter. Here, we describe the genomic features of DEV CHv together with a draft genome sequence and its annotation, highlighting the homogeneity and heterogeneity of this genome in comparison with its reference genomes.
Journal of Virology 12/2012; 86(24):13841-2. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: UL22 gene, also named gH gene(GenBank accession No. EU195089) from duck plague virus (DPV) CHv strain which was isolated in our laboratory, is a 2505bp segment. The analysis about the characteristics of this gene may improve our understanding for the evolution and classification of DPV. In this study, the structure of phylogenetic tree and the prediction of gH functional sites depended upon MegAlign procedure and some online analysis softwares. The results revealed that this gene sequence was quite conservative in DPV. The phylogenetic tree of the gH protein and its homologs of other 20 reference herpesviruses showed glycoprotein H had a close evolutionary relationship with certain avian herpesviruses, such as MeHV-1, GaHV-2 and GaHV-3 which just were classified to Mardivirus genus. Besides, 8 N-glycosylation sites and 52 potential phosphorylation sites were found in gH and they maybe play an important role in regulating the function of DPV glycoprotein H.
Proceedings of the Third international conference on Information Computing and Applications; 09/2012
[Show abstract][Hide abstract] ABSTRACT: Orally delivered DNA vaccines against duck enteritis virus (DEV) were developed using live attenuated Salmonella typhimurium (SL7207) as a carrier and Escherichia coli heat labile enterotoxin B subunit (LTB) as a mucosal adjuvant. DNA vaccine plasmids pVAX-UL24 and pVAX-LTB-UL24 were constructed and transformed into attenuated Salmonella typhimurium SL7207 resulting SL7207 (pVAX-UL24) and SL7207 (pVAX-LTB-UL24) respectively. After ducklings were orally inoculated with SL7207 (pVAX-UL24) or SL7207 (pVAX-LTB-UL24), the anti-DEV mucosal and systemic immune responses were recorded. To identify the optimum dose that confers maximum protection, we used different doses of the candidate vaccine SL7207 (pVAX-LTB-UL24) during oral immunization. The strongest mucosal and systemic immune responses developed in the SL7207 (pVAX-LTB-UL24) (1011 CFU) immunized group. Accordingly, oral immunization of ducklings with SL7207 (pVAX-LTB-UL24) showed superior efficacy of protection (60-80%) against a lethal DEV challenge (1000 LD50), compared with the limited survival rate (40%) of ducklings immunized with SL7207 (pVAX-UL24). Our study suggests that the SL7207 (pVAX-LTB-UL24) can be a candidate DEV vaccine.
Veterinary Research 07/2012; 43(1):56. · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a group of short (~22 nt) noncoding RNAs that specifically regulate gene expression at the post-transcriptional level. miRNA precursors (pre-miRNAs), which are imperfect stem loop structures of ~70 nt, are processed into mature miRNAs by cellular RNases III. To date, thousands of miRNAs have been identified in different organisms. Several viruses have been reported to encode miRNAs.
Here, we extended the analysis of miRNA-encoding potential to the Anatid herpesvirus 1 (AHV-1). Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs. We then compared the 12 mature miRNAs candidates with the all known miRNAs of the herpesvirus family. Interestingly, the "seed sequences" (nt 2 to 8) of 2 miRNAs were predicted to have the high conservation in position and/or sequence with the 2 miRNAs of Marek's disease virus type 1 (MDV-1). Additionally, we searched the targets from viral mRNAs.
Using computational approaches, we found that AHV-1 putatively encodes 12 mature miRNAs and 2 miRNAs have the high conservation with the 2 miRNAs of MDV-1. The result suggested that AHV-1 and MDV-1 should have closed evolutionary relation, which provides a valuable evidence of classification of AHV-1. Additionally, seven viral gene targets were found, which suggested that AHV-1 miRNAs could affect its own gene expression.
[Show abstract][Hide abstract] ABSTRACT: The Chinese virulent (CHv) strain of duck enteritis virus (DEV) has a genome of approximately 162,175 nucleotides with a GC content of 44.89%. Here we report the complete genomic sequence and annotation of DEV CHv, which offer an effective platform for providing authentic research experiences to novice scientists. In addition, knowledge of this virus will extend our general knowledge of DEV and will be useful for further studies of the mechanisms of virus replication and pathogenesis.
Journal of Virology 05/2012; 86(10):5965. · 5.08 Impact Factor