Establishment and evaluation of the goose embryo epithelial (GEE) cell line as a new model for propagation of avian viruses

Wang, Wenxiu; Said, Abdelrahman; Wang, Baoqin; Qu, Gui‐Rong; Xu, Qingqing; Liu, Bo; Zhi-qiang, Shen

doi:10.1371/journal.pone.0193876

Cited by 6 publications

(6 citation statements)

References 32 publications

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“…DHAV infection was recently identified in mallards, goslings and pigeons [7]. A goose embryonated epithelial cell line efficiently assists with the replication of DHAV, which shows a higher virus titer compared to other duck cell lines [8]. Based on phylogenetic and neutralization assays, DHAV has been divided into three distinct genotypes: DHAV-1, DHAV-2, and DHAV-3 [1,9].…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

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Background The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. Methods In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. Results Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and V max and K m values were determined to be 16.52 nmol/min and 50.78 μM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. Conclusions This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the V max and K m values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.

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Section: Introductionmentioning

confidence: 99%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

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“…The three panels in Fig 7C in [ 1 ] appear similar to the three panels in Fig 5A in [ 2 ] when colors are adjusted.…”

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confidence: 61%

“…The right two mice in the left panel of Fig 5C in [ 1 ] appear similar to the right two mice in the left panel of Fig 4B in [ 2 ] when stretched.…”

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confidence: 67%

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Retraction: Establishment and evaluation of the goose embryo epithelial (GEE) cell line as a new model for propagation of avian viruses

2024

PLoS ONE

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“…Yet, few avian cell lines have been established. Most of them are derived from domestic (chicken, duck, Japanese quail, turkey) and model (zebra finch, canary) species [5][6][7][8][9]. The usual sources of the avian cell lines are skin, feather pulp, or blood [10,11].…”

Section: Introductionmentioning

confidence: 99%

Establishment of the Primary Avian Gonadal Somatic Cell Lines for Cytogenetic Studies

Pristyazhnyuk

Malinovskaya

Бородин

2022

Animals

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The last decade was marked by a steep rise in avian studies at genomic and cellular levels. Cell lines are important tools for in vitro studies in cell biology and cytogenetics. We developed a simple method of primary somatic cell culture establishment from the ovaries of the great tits (Parus major) and testes of ten Passerine species, characterized the cellular composition of the ovary-derived lines using RT-PCR and immunolocalization of the tissue-specific markers and tested the efficiency of two methods of genetic transformation of the ovary-derived cell line. We found that the ovary-derived cell cultures of the great tit were composed of fibroblasts mainly, but also contained interstitial and granulosa cells. They were cultivated until the 10th passage without any noticeable decrease in their proliferative activity. The testis-derived cell cultures had lower proliferative potential. However, both ovary- and testis-derived cell cultures provided enough material for high quality mitotic metaphase chromosome preparations. The efficiency of its transduction with lentivirus containing a GFP reporter was very low, while electroporation with episomal vectors expressing GFP resulted in a high yield of GFP-positive cells. The proposed method could be used for the generation of high quality material for various cytogenetic and genomic studies.

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Establishment and evaluation of the goose embryo epithelial (GEE) cell line as a new model for propagation of avian viruses

Cited by 6 publications

References 32 publications

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Retraction: Establishment and evaluation of the goose embryo epithelial (GEE) cell line as a new model for propagation of avian viruses

Establishment of the Primary Avian Gonadal Somatic Cell Lines for Cytogenetic Studies

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