The Entry Route Of Egg Drop Syndrome Virus Enters Into Host Cell

Egg drop syndrome is caused by egg drop syndrome virus(EDSV), which is characterized by sudden severe drop in egg production, egg deformity as well as egg inferior quality in apparently healthy laying birds. Currently, limited researchs on EDSV mainly focus on serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV remains poorly understood. In the present study, we aimed to explore the entry pathway of EDSV into host cells, as well as the viral infection characteristics and proliferation on primary duck embryonic fibroblast(DEF) and primary chick embryo liver(CEL) cells. Our purpose is to provide theoretical basis and technical support for study the infection mechanism of DEF cells infected with EDSV.1. One pairs of primers specific for penton gene of EDSV were designed according to the gene sequences(No.Y09598.1) published on the National Center for Biotechnology Information(NCBI) GenBank database. A fragment of penton gene was amplified and inserted into pMD-19 T plasmid. The standard plasmid DNA was diluted in a series of 10-fold dilution and measured. A strand curve of quantitative real-time PCR to detect EDSV was established. The result showed that the developed quantitative real-time PCR was sensitive and reproducible, which can be used for the quantitative detection of EDSV particles. DEF and CEL cells were prepared to study EDSV infection characteristics. Furthermore, the proliferation of EDSV on DEF and CEL cells was measured by quantitative real-time PCR. The results showed that EDSV can cause cytopathic effect(CPE) of DEF and CEL cells. The virus copy numbers peaked at 72 h post inoculation(p.i.), and the virus HA titer were 212 and 211, respectively. In addition, the viral copy numbers shed from DEF cells were higher than those from CEL cells at all time points.2. DEF cells were infected with EDSV for 0 min, 10 min, 20 min, 30 min, 2 h and 72 h. Ultra-thin sections(70 nm) of cells were prepared and stained with 4% uranyl acetate for observation under a Hitachi HT 7700 transmission electron microscope. The images revealed that the viruses triggered DEF cell membrane invagination as early as 10 min p.i., and formed integrated endocytic vesicles at 20 min p.i., followed by transported the vesicle to the cell interior. In addition, at 72 h p.i., a large number of virus particles accumulated in the cell followed by the cell ruptures and the viruses are released.3. DEF cells were pretreated with various concentrations of CPZ(an inhibitor blocking the clathrin-mediated endocytosis), sucrose, MβCD(a drug disrupting both the caveolin-dependent endocytosis and the caveolin-independent lipid raft-dependent endocytosis), and NH4Cl(a drug interfering with endosome acidification) and then infected with EDSV for 24 h. The changes of virus copy number were measured by quantitative real-time PCR. The results showed that CPZ and sucrose markedly inhibited the progeny viral copy number in a dose-dependent manner as compared to mock-treated controls, while MβCD treatment did not cause a significant difference from controls. Furthermore, NH4 Cl also resulted in a strong inhibitory effect on the progeny viral copy number of EDSV.4. To observe the inhibitory effect of chemicals on viral entry, DEF cells were pretreated with CPZ(50 μmol/L) and sucrose(100 mmol/L) for 1 h at 37°C and then infected with EDSV. Untreated DEF cells infected without EDSV were as a negative control, while untreated DEF cells infected with EDSV were as a positive control. The indirect immunofluorescence images showed that the viruses clustered on DEF cell surface after cells pretreated with CPZ and sucrose. The data obtained in this study showed that EDSV can infect DEF cells through a clathrin-mediated endocytosis pathway in a pH-dependent manner.