Renyong Jia

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.

Toxicological assessment of combined lead and cadmium: acute and sub-chronic toxicity study in rats.

The exposure to chemical mixtures is a common and important determinant of toxicity and receives concern for their introduction by inhalation and ingestion. However, few in vivo mixture studies have been conducted to understand the health effects of chemical mixtures compared with single chemicals. In this study, the acute and 90day sub-chronic toxicity tests of combined Pb and Cd were conducted. In the acute toxicity test, the LD50 value of Pb(NO3)2 and CdCl2 mixture by the oral route was 2696.54mg/kg by Bliss method. The sub-chronic treatment revealed that the low-dose combination of Pb and Cd exposures can significantly change the physiological and biochemical parameters of the blood of Sprague-Dawley (SD) rats with dose-response relationship and causes microcytic hypochromic anemia and the damages of liver and kidney of the SD rats to various degrees. Histopathological exams showed that the target organs of Pb and Cd were testicle, liver, and kidneys. These observations suggest that Pb and Cd are practically additive-toxic for the SD rats in oral acute toxicity studies. The lowest observed adverse-effect level in rats may be lower than a dose of 29.96mg/(kgbwday) when administered orally for 90 consecutive days.

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.

Quantification of metallothionein on the liver and kidney of rats by subchronic lead and cadmium in combination

The combined subchronic effects of exposure to lead acetate and cadmium chloride on oxidative stress and metallothionein (MT) gene expression were detected in the liver and kidney of rats to investigate the hazards of environmentally relevant, low-dose exposure to these compounds. Pb and Cd co-induced oxidative stress in liver and kidney tissues. This result was indicated by a significant (P<0.01) increase in the maleic dialdehyde level and decreased levels of reduced glutathione, superoxide dismutase, catalase, and glutathione peroxidase. MT mRNA and protein significantly increased (P<0.01) in the liver and kidney of rats. Furthermore, the expression levels of MT-1 mRNA and MT-2 mRNA differed between the liver and kidney. The findings indicate that Pb combined with Cd induced oxidative damage in the liver and kidney of rats, and MT may be a biochemical environmental indicator.

Antiviral activity of sulfated Chuanminshen violaceum polysaccharide against duck enteritis virus in vitro.

Duck enteritis virus (DEV) of the family Herpesviridae is one of the main diseases in waterfowl. Despite the wide use of vaccines to control the disease, infection with the virus cannot be completely prevented. Therefore, antiviral agents against DEV should be developed. This study presents a novel sulfated polysaccharide from Chuanminshen violaceum (sCVPS), which exhibits significant antiviral activity against DEV with 50% inhibitory concentrations (IC₅₀) ranging from 77.12 μg/mL to 104.81 μg/mL. sCVPS is more effective than heparan sulfate (HS, as a positive control) with IC₅₀=132.61 μg/mL. sCVPS and HS inhibit viral activity by preventing virus adsorption with IC₅₀ values ranging from 82.83 μg/mL to 109.28 μg/mL for sCVPS and 150.22 μg/mL for HS. Direct immunofluorescence assay and transmission electron microscopy demonstrated that the mechanism of action was the interference with virus adsorption. The amount of inhibited virus during adsorption was quantified using fluorescent quantitative polymerase chain reaction, which revealed that both sCVPS and HS can significantly reduce all viruses attached to cells. sCVPS also prevented the cell-to-cell spread of DEV. These results indicated that sCVPSs perform more effectively than does HS as antiviral agents against DEV and can be further examined for potential effects as an alternative control measure for DEV infection.