Anchun Cheng

Complete Genomic Sequence of Chinese Virulent Duck Enteritis Virus

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.

Electron Microscopic Studies of the Morphogenesis of Duck Enteritis Virus

Abstract The morphogenesis of duck enteritis virus (DEV) and distribution in vivo were observed by electron microscopy after ducks were infected experimentally with DEV virulent strain. The investigation showed that a few typical herpesvirus virions and nucleocapsids were first observed in the spleen, thymus, and bursa of Fabricius (BF), and many nucleocapsids, mature viruses, and viral inclusion bodies could be found in the nucleus and cytoplasm of infected liver, small intestine, spleen, thymus, and BF when the ducks died. Nucleocapsids assembled both in nucleus and cytoplasm and could be divided into four different types according to their structures. Typical herpesvirus, light particles (L-particles), and virions without tegument could be observed at the same time. With the replication, assembly, and maturation of the viruses, intracytoplasmic and intranuclear inclusion bodies, electron-density particles, microtubules, hollow tubes, and coated electron-density bodies were observed in infected cells.

Characterization of Synonymous Codon Usage Bias in the Duck Plague Virus UL35 Gene

Objective: The aim was to identify the codon usage bias between the newly identified duck plague virus (DPV) UL35 gene (GenBank accession No. EF643558) and the UL35-like genes of 27 other reference herpesviruses. Methods: A comparative analysis of the codon usage bias of the 28 herpesviruses was performed by using the CodonW 1.4 program and CUSP (create a codon usage table) program of EMBOSS (The European Molecular Biology Open Software Suite). Results: The results showed obvious differences of the synonymous codon usage bias in the 28 herpesviruses indicated by the Codon Adaptation Index, effective number of codons (ENc), and the value of G + C content at the 3rd codon position. The codon usage pattern of the DPV UL35 gene was phylogenetically conserved and similar to that of the UL35-like genes of the avian α-herpesvirus, with a strong bias towards the codons with A and T at the 3rd codon position. A cluster analysis of codon usage pattern of the DPV UL35 gene with other reference herpesviruses demonstrated that the codon usage bias of the UL35 genes of the 28 herpesviruses had a very close relation with their gene function. The ENc-plot revealed that the genetic heterogeneity in the DPV UL35 gene and the 27 reference herpesviruses were constrained by G + C content, while gene length exerted relatively weaker influences. In addition, comparisons of the codon preferences in the UL35 gene of DPV with those of Escherichia coli, yeast and humans revealed that there were 33 codons showing distinct usage differences between the DPV and yeast, and 38 between the DPV and humans, but only 31 between the DPV and E. coli. Therefore, the E. coli system may be more suitable for the expression of the DPV UL35 gene. Conclusion: Together, these results may improve our understanding of the evolution, pathogenesis and functional studies of DPV, as well as contribute significantly to the area of herpesvirus research and possibly studies with other viruses.

Identification and Characterization of Duck Enteritis Virus dUTPase Gene

Abstract Deoxyuridine triphosphatase (dUTPase) is a ubiquitous and important enzyme that hydrolyzes dUTP to dUMP. Many viruses encode virus-specific dUTPase, which plays an essential role in maintaining the integrity of the viral DNA both by reducing the dUTP levels and by providing the substrate for the thymidylate synthase. A 1344-bp gene of duck enteritis virus (DEV) homologous to herpesviral dUTPase was first reported in this paper. The gene encodes a protein of 477 amino acids, with a predicted molecular mass of 49.7 kDa. Multiple sequence alignment suggested that DEV dUTPase was quite similar to other identified herpesviral dUTPase and functioned as a homotrimer. The five conserved motifs of DEV dUTPase with 3-1-2-4-5 arrangement have been recognized, and the phylogenetic analysis showed that DEV dUTPase was genetically close to the avian herpesvirus. Furthermore, RNA dot blot, western blot, and immunofluorescence analysis indicated that the enzyme was expressed at early and late stages after infection. Immunofluorescence also confirmed that DEV dUTPase localized in the cytoplasm of DEV-infected duck embryo fibroblasts as early as 4 hr postinfection (hpi). Later, the enzyme transferred from cytoplasm to nucleus at 8 hpi, and then reached its expression peak at 12 hpi, both in the cytoplasm and nucleus. The results suggested that the DEV dUTPase gene might be an early viral gene in DEV vitro infection and contribute to ensuring the fidelity of genome replication.

Complete Genome Sequence of Riemerella anatipestifer Reference Strain

Riemerella anatipestifer is an infectious pathogen causing serositis in ducks. We had the genome of the R. anatipestifer reference strain ATCC 11845 sequenced. The completed draft genome consists of one circular chromosome with 2,164,087 bp. There are 2,101 genes in the draft, and its GC content is 35.01%.

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop-mediated isothermal amplification.

Aim:  The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions.

Development and evaluation of an antigen-capture ELISA for detection of the UL24 antigen of the duck enteritis virus, based on a polyclonal antibody against the UL24 expression protein

Abstract An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) method was developed for the efficient detection of the UL24 antigen of the duck enteritis virus (DEV) using polyclonal antibodies. Ducks and rabbits were immunized, respectively, with expressed UL24 recombinant protein. The IgG antibodies against UL24 from ducks and rabbits were purified and used as the capture antibodies. The specificity of the optimized AC-ELISA was evaluated by use of DEV, duck hepatitis virus (DHV), duck hepatitis B virus (DHBV), gosling plague virus (GPV), Riemerella anatipestifer (R.A.), Escherichia coli (E. coli), Pasteurella multocida (P.M.) and Salmonella Enteritidis (S.E.). Only DEV specimens yielded a specific and strong signal. The limit of the sensitivity of this method for the detection of DEV was 46ng/100μl. Compared with PCR and virus isolation, the rate of agreement for the detection of experimentally infected sera was 100%. A comparative test used on clinical specimens between the neutralization test and the AC-ELISA showed that the proportions of true positives and true negatives by the AC-ELISA were 0.90 and 0.67 respectively. These results indicated that the AC-ELISA approach is rapid, sensitive, and reliable for specific detection of DEV antigen.

Expression and characterization of the UL31 protein from duck enteritis virus.

BackgroundPrevious studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product.ResultsThe entire ORF of the UL31 was cloned into pET 32a (+) prokaryotic expression vector. Escherichia coli BL21(DE3) competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa) was produced on the SDS-PAGE. Over expressed 6×His-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs.ConclusionIn this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the Alpha herpesvirus subfamily. Furthermore, in vivo experiments with ducks infected with UL31-defective isolates of DEV will also be of importance in order to assess the possible role of the UL31 protein in viral pathogenesis. These properties of the UL31 protein provide a prerequisite for further functional analysis of this gene.

Development of TaqMan ® MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus 1

BackgroundAnatid herpesvirus 1 (AHV-1) is an alphaherpesvirus associated with latent infection and mortality in ducks and geese and is currently affecting the world-wide waterfowl production severely. Here we describe a fluorescent quantitative real-time PCR (FQ-PCR) method developed for fast measurement of AHV-1 DNA based on TaqMan MGB technology.ResultsThe detection limit of the assay was 1 × 101 standard DNA copies, with a sensitivity of 2 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation.ConclusionThe high sensitivity, specificity, simplicity and reproducibility of the AHV-1 fluorogenic PCR assay, combined with its wide dynamic range and high throughput, make this method suitable for a broad spectrum of AHV-1 etiologically related application.

Complete nucleotide sequence of the duck plague virus gE gene

Duck plague (DP), which is caused by duck plague virus (DPV), is a disease that severely affects various types of waterfowl (ducks, geese, and swans). DPV infection causes significant economic losses to the commercial duck industry because of the consequent high mortality and low egg production rates [7]. DPV has been classified as belonging to the subfamily Alphaherpesvirinae of the family Herpesviridae based on the report of the Eighth International Committee on Taxonomy of Viruses (ICTV), but it has not been grouped into any genus [4]. Currently, there is little information on the molecular characteristics of DPV, and very few DPV genomic sequences have been published. Here, we report the complete sequence of the newly isolated gE gene of DPV.