Simultaneous Tracking of Capsid, Envelope Protein Localization in Living Cells Infected with Double Fluorescent Duck Enteritis Virus

2020 ◽  
Author(s):  
Liu Chen ◽  
Zheng Ni ◽  
Jionggang Hua ◽  
Weicheng Ye ◽  
Keshu Liu ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Subcellular Location ◽  
Duck Enteritis Virus ◽  
Transmission Electron ◽  
Infected Cells ◽  
Enteritis Virus ◽  
Calcium Phosphate Precipitation ◽  
Chicken Embryo Fibroblasts

Abstract Background Duck enteritis virus can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of duck enteritis virus (DEV) vaccine strain pDEV-EF1 has been constructed in our previous study. To visually studying DEV, a double-labeled recombinant virus have been constructed. Methods based on pDEV-EF1, a recombinant mutated clone pDEV-UL35(c)CFP-gC(c)mRFP which carries a red fluorescent protein(mRFP) gene attached to the viral envelope protein gC, combined with a cyan fluorescent protein (CFP) gene fused to the viral capsid UL35 gene was constructed by two-step Red/ET recombination and the recombinant virus rDEV-UL35(c)CFP-gC(c)mRFP was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Then protein expression were detected by Western blot analysis, and subcellular location of gC and UL35 were observed by confocal microscopy. Viral morphogenesis were observed by transmission electron microscopy (TEM). Results The recombinant virus rDEV-UL35(c)CFP-gC(c)mRFP was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed UL35-CFP and gC-mRFP are both expressed in fusion forms in rDEV-UL35(c)CFP-gC(c)mRFP-infected CEFs, and subcellular location study showed gC-mRFP was mainly localized in whole cell at 36 h.p.i. and 48 h.p.i.; and then mostly migrated to cytoplasm after 60 h.p.i.; UL35-CFP was localized in nucleus in all stages of virus infection. Transmission electron microscopy indicated that viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells. However,mature C capsids was less in rDEV-UL35(c)CFP-gC(c)mRFP-infected cells than rDEV-dEF1GFP and rDEV-gC(c)mRFP-infected samples. Conclusions This study has laid a foundation for visually studying localization, transportation of DEV capsid protein and envelope glycoprotein, as well as virus assembly, virion movement and virus-cell interaction.

scholarly journals Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

2003 ◽  
Vol 77 (10) ◽  
pp. 6070-6075 ◽  
Author(s):  
Dirk Spitzer ◽  
Kurt E. J. Dittmar ◽  
Manfred Rohde ◽  
Hansjörg Hauser ◽  
Dagmar Wirth
Keyword(s):  
Green Fluorescent Protein ◽  
Envelope Protein ◽  
Fluorescent Protein ◽  
Inverse Correlation ◽  
Cell Interactions ◽  
Virus Particles ◽  
Viral Particles ◽  
Infected Cells ◽  
Env Protein ◽  
Green Fluorescent

ABSTRACT Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions.

scholarly journals Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-β signalling by interacting with STAT1

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Tianqiong He ◽  
Mingshu Wang ◽  
Anchun Cheng ◽  
Qiao Yang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Duck Enteritis Virus ◽  
Poly I:C ◽  
Nuclear Localization Sequence ◽  
Heterologous Proteins ◽  
Structural Protein ◽  
Oligoadenylate Synthetase ◽  
Enteritis Virus

Abstract Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae subfamily. The characteristics of some DEV genes have been reported. However, information regarding the DEV UL47 gene is limited. In this study, we identified the DEV UL47 gene encoding a late structural protein located in the nucleus of infected cells. We further found that two domains of DEV pUL47, amino acids (aa) 40 to 50 and 768 to 777, could function as nuclear localization sequence (NLS) to guide the nuclear localization of pUL47 and nuclear translocation of heterologous proteins, including enhanced green fluorescent protein (EGFP) and beta-galactosidase (β-Gal). Moreover, pUL47 significantly inhibited polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced interferon beta (IFN-β) production and downregulated interferon-stimulated gene (ISG) expression, such as Mx and oligoadenylate synthetase-like (OASL), by interacting with signal transducer and activator of transcription-1 (STAT1).

scholarly journals Two nuclear localization signals regulate intracellular localization of the Duck enteritis virus UL13 protein

2020 ◽  
Author(s):  
Linjiang Yang ◽  
Xixia Hu ◽  
Anchun Cheng ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Duck Enteritis Virus ◽  
Significant Information ◽  
Nuclear Localization Signals ◽  
Dependent Process ◽  
Importin Α ◽  
Localization Signal ◽  
Enteritis Virus

Abstract Background UL13 multifunctional tegument protein of duck enteritis virus (DEV) is predicted as protein kinase (CHPK); however, little is known concerning its subcellular localization signal. Results In this study, by transfection with two predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein (EGFP), two bipartite nuclear localization signal (NLS) was identified. We identified the nuclear localization signals (NLSs) that control its nuclear importing using fluorescence assay and proved that nuclear localization of DEV UL13 is a classical importin α/β-dependent process. And we constructed the mutant DEV, with the NLSs of UL13 deleted, to explore whether it will affect the replication of virus particles. Conclusions DEV UL13 protein is directed by amino acids 4 to 7 and 90 to 96, and proved that this nuclear import occurs via the classical importin α/β-dependent process. And Entry nucleus of UL13 protein has no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results would provide significant information for the stud y of the biological function of UL13 during DEV infection.

Proteomic analysis of host cellular proteins co-immunoprecipitated with duck enteritis virus gC

2021 ◽  
Vol 245 ◽  
pp. 104281
Author(s):  
Liu Chen ◽  
Zheng Ni ◽  
Jionggang Hua ◽  
Weicheng Ye ◽  
Keshu Liu ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Duck Enteritis Virus ◽  
Cellular Proteins ◽  
Enteritis Virus

scholarly journals A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 157
Author(s):  
Kinga Böszörményi ◽  
Janet Hirsch ◽  
Gwendoline Kiemenyi Kayere ◽  
Zahra Fagrouch ◽  
Nicole Heijmans ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Neutralizing Antibodies ◽  
Size Exclusion ◽  
Vitro Assay ◽  
Usutu Virus ◽  
Transmission Electron ◽  
Exclusion Chromatography ◽  
Efficacious Vaccine ◽  
Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

scholarly journals Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 365
Author(s):  
Fengli Liu ◽  
Yanxin Cao ◽  
Maokai Yan ◽  
Mengxu Sun ◽  
Qingshui Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colloidal Gold ◽  
Preventive Intervention ◽  
Virus Detection ◽  
Cross Reactivity ◽  
Duck Enteritis Virus ◽  
Detection Sensitivity ◽  
Immunochromatographic Assay ◽  
Efficient Detection ◽  
Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

scholarly journals Bacmid Expression of Granulovirus Enhancin En3 Accumulates in Cell Soluble Fraction to Potentiate Nucleopolyhedrovirus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1233
Author(s):  
Adriana Ricarte-Bermejo ◽  
Oihane Simón ◽  
Ana Beatriz Fernández ◽  
Trevor Williams ◽  
Primitivo Caballero
Keyword(s):  
Trichoplusia Ni ◽  
Recombinant Virus ◽  
Spodoptera Exigua ◽  
Insect Cells ◽  
Soluble Fraction ◽  
Autographa Californica ◽  
Insect Midgut ◽  
Baculovirus Infection ◽  
Occlusion Bodies ◽  
Infected Cells

Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy.

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