Studies by electron microscopy on the assembly of duck hepatitis B virus in the liver

Liver specimens from 1-day-old ducklings infected in ovo with maternally transmitted duck hepatitis B virus (DHBV) were examined by electron microscopy. Complete and incomplete DHBV particles were located within hypertrophied cisternae of the endoplasmic reticulum of the hepatocytes. The complete viral particles found intracellularly have inner cores with a diameter ranging from 35 to 37.5 nm and an outer coat or envelope. The whole particle measures approximately 45-65 nm in diameter. Naked core particles were located in the nuclei, free in the cytoplasm, and also near or on the cisternal membrane of the endoplasmic reticulum on the cytoplasmic face. Duck hepatitis B virions appear to share morphological characteristics with the viral coat and core of human hepatitis B virus (HBV). Electron microscopy suggested that the core particles of DHBV migrate from the nucleus into the cytoplasm through the nuclear pores. The complete viral particles are probably formed by protrusion of the core particles through the endoplasmic reticulum and by simultaneous encapsulation with a coat derived from the endoplasmic reticulum.     

Experimental in ovo transmission of duck hepatitis B virus

Inoculation of fertile Pekin duck eggs with diluted serum containing DHBV into eggs incubated for 24 h and into the extra-embryonic cavities of 14-day-old embryos resulted in a high proportion of viraemic ducklings irrespective of the route of inoculation. Long-term observation of som of the ducks established that the viraemia induced experimentally is long-lasting and has persisted for periods up to 16 mth post-hatch. Separation of DHBV from the plasma of carrier ducks by rate zonal centrifugation was examined by DNA polymerase (DNAP) activity. Particles in the fraction with peak DNAP activity had a buoyant density of 1.16 g X cm-3 in sucrose and an estimated sedimentation coefficient, S20.w of 77. DHBV particles, the morphology of which could be resolved under the electron microscope, consisted of a coat (about 10 nm in thickness) surrounding a core with a diameter measuring 40 nm but not 27 nm as previously reported. Spike-like projections were found on the surface of the core as described previously by W.S. Mason, G. Seal and J. Summers, 1980, J. Virol. 36, 829-836.     

广东省绿头鸭场鸭乙肝病毒自然携带状况调查

目的　调查两个鸭场携带乙肝病毒(DHBV)状况。方法　在对广东省家养绿头鸭分布状况进行系统调查的基础上,对南海市和顺镇、里水镇两个绿头鸭场作了随机抽样调查,采集了雏鸭、青年鸭和种鸭的血标本共519份,应用斑点分子杂交(DotBlot)测定法观察绿头鸭乙型肝炎病毒(DHBV)自然携带率。结果　两鸭场绿头鸭的DHBV自然携带率为36.0%(187/519),其95%可信限为31.0%～44.0%。不同鸭龄组绿头鸭DHBV自然携带比较,两鸭场同日龄绿头鸭DHBV自然携带率比较,绿头鸭DHBV自然携带率的性别比较,差异均无显著意义。不同鸭龄的绿头鸭DHBV-DNA含量定量分析(A值),差异有非常显著性(P＜0.01),其含量由高到低顺序为雏鸭＞种鸭＞青年鸭,和顺鸭场的绿头鸭DHBV-DNA含量高于里水鸭场。结论　两鸭场绿头鸭携带的DHBV可能源于不同的亚株。     

白背叶根抗鸭乙型肝炎病毒的实验研究

目的:观察白背叶根体内抗鸭乙型肝炎病毒(duck hepatitis B virus,D-HBV)的作用。方法:采用PCR技术筛选D-HBV阳性3日龄广州麻鸭40只。将麻鸭随机分为空白对照组,白背叶根高、中、低剂量组和拉米夫定组,分别给予相应药物进行干预。用药前、用药第7、14、21天及停药第7天,取各组麻鸭静脉血,采用实时定量荧光PCR技术检测血清D-HBV DNA含量;治疗前后取肝脏组织进行病理组织学观察。结果:拉米夫定组于用药后,血清病毒水平迅速降低,但停药后存在反跳现象。白背叶根高、中剂量组分别于治疗第14、21天其血清病毒含量明显下降,低剂量组治疗期间未见明显的抑制病毒作用。停药后,白背叶根高、中剂量组与空白对照组比较仍表现出一定的病毒抑制作用。白背叶根高剂量组对肝脏炎症的改善作用较拉米夫定组明显。结论:白背叶根有抑制体内D-HBV复制的作用,其作用大小与剂量及用药时间相关;其治疗作用较拉米夫定弱,但作用维持时间长,且用药较为安全。     

Molecular Characterization of Duck Hepatitis B Virus Isolated from Hubei Brown Ducks

The objective of this study was to characterize the genome structure of duck hepatitis B virus (DHBV) isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction (PCR) and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 %. This strain (GenBank accession number DQ276978) had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the "Chinese" DHBV strains than those of the "Western" country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.     

Novel transparent collagen film patch derived from duck’s feet for tympanic membrane perforation

Abstract

To increase healing rate of tympanic membrane (TM) perforations, patching procedure has been commonly conducted. Biocompatible, biodegradable patching materials which is not limited across cultures is needed. The authors evaluated the effectiveness of novel transparent duck’s feet collagen film (DCF) patch in acute traumatic TM perforation. This procedure was compared with spontaneous healing and paper patching. Cell proliferation features were observed in paper and DCF patches. Forty-eight TMs of 24 rats were used for animal experiment, perforations were made on each TMs, and divided into three groups according to treatment modality. Sixteen were spontaneously healed, 16 were paper patched and 16 were DCF patched. The gross and histological healing results were analyzed. Both paper and DCF patch showed no cytotoxicity, but cell proliferations were more active in DCF than paper in early stage. In animal study, the healing of TM perforations were completed within 14 days in all three groups, but found to be faster in DCF patch group than paper patch or spontaneous healing group. The DCF patches were transparent and size of DCF patches were gradually decreased, so there were no need to remove the DCF patches to check the wound status or after the completion of healing. According to this result, authors concluded that DCF patch is transparent, biocompatible and biodegradable material, and can induce fast healing in acute traumatic TM perforations.     

Efficient Strategy to Generate a Vectored Duck Enteritis Virus Delivering Envelope of Duck Tembusu Virus

Duck Tembusu virus (DTMUV) is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV) was examined as a candidate vaccine vector to deliver the envelope (E) of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The envelope (E) gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC) strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs). Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.