High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody

Li, Chuan‐Feng; Chen, Zongyan; Chen, Meng; Lü, Li; Liu, Guangqing

doi:10.1016/j.jviromet.2013.04.004

Cited by 17 publications

(4 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Detection of VP3 expression by immunofluorescence assay. To prepare the anti-VP3 serum, each mouse was injected intramuscularly three times with 50 μg of Escherichia coli expressed DHAV VP3 protein as described previously (Li et al, 2013). Primary duck embryo fibroblast cells were seeded on 24-well plates at a density of 2×10 5 cells/well and infected with rAAAV-VP3.…”

Section: Figmentioning

confidence: 99%

Expression of duck hepatitis A virus type 1 VP3 protein mediated by avian adeno-associated virus and its immunogenicity in ducklings

Wang¹,

Liu²,

Gu³

et al. 2019

View full text Add to dashboard Cite

The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus that has been proved to be useful as a viral vector in gene delivery. In this study, the feasibility of AAAV for transgenic expression of duck hepatitis A virus (DHAV) VP3 structural protein and its ability to induce protective immunity in ducklings was assessed. The recombinant AAAV (rAAAV-VP3) expressing the VP3 protein was prepared by co-infection of Sf9 cells with recombinant baculovirus (rBac-VP3) containing VP3 gene flanked by inverted terminal repeats (ITRs) of AAAV and the other two recombinant baculovirus expressing AAAV functional and structural genes, respectively. The generation of rAAAV-VP3 was demonstrated by electron microscopy, immunofluorescence assay, and western blot analysis. One day old ducklings were inoculated with rAAAV-VP3 or commercial attenuated vaccine and then challenged with DHAV-1 strain SH two weeks post vaccination. Anti-DHAV-1 antibodies were detected in all vaccinated groups by ELISA, and the titers between the rAAAV-VP3 group and the attenuated vaccine group were not statistically significant. Real time RT-PCR analysis showed that the virus copy numbers in the livers of the PBS control group were significantly higher than that of the rAAAV-VP3 and attenuated vaccine groups. In conclusion, we demonstrated that the VP3 expression mediated by rAAAV in ducklings could induce protective immunity against DHAV challenge, and this could be a candidate vaccine for the control of duck viral hepatitis.

show abstract

Section: Figmentioning

confidence: 99%

Expression of duck hepatitis A virus type 1 VP3 protein mediated by avian adeno-associated virus and its immunogenicity in ducklings

Wang¹,

Liu²,

Gu³

et al. 2019

View full text Add to dashboard Cite

show abstract

“…paraformaldehyde and blocked for 60 min with 5% BSA in PBS. The standard indirect immunofluorescence was performed using mouse anti-ARep (1:100) or anti-AVP (1:200) serum, which was prepared by immunizing mice with E. coli-expressed AAAV Rep or Cap protein as described previously (Li et al, 2013). After incubation with FITC-labeled goat anti-mouse IgG (1:500, Jackson ImmunoResearch, USA), the cell cultures were observed under microscope for fluorescently positive cells.…”

Section: Detection Of Aaav Rep and Vp Expression In Insect Cells By Imentioning

confidence: 99%

Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system

Wang

et al. 2017

Journal of Virological Methods

View full text Add to dashboard Cite

Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene. After demonstration of the correct expression of AAAV genes, rAAAV-GFP was produced by triple infection of insect cells or triple transfection of HEK293 cells for comparison purpose. Electron microscopy revealed the formation of typical AAAV particles in the insect cells. Western blotting showed the correct assembly of rAAAV particles with a VP protein ratio similar to that of AAAV. Quantitative PCR showed that the insect cell-produced rAAAV yield was almost 25-fold higher than that produced by HEK293 cells. Fluorescent microscopy showed that the insect cell-produced rAAAV could transfer GFP reporter gene into two avian cell types with similar transfer efficiency to that of HEK293 cell-produced rAAAV. These data suggest that insect cell/baculovirus expression system could be used for scalable production of rAAAV, and the viral vector produced could be used as the gene transfer vehicle for avian cells.

show abstract

“…Duck viral hepatitis (DVH) is an acute, contagious, rapidly spreading viral infection of young ducklings that is characterized primarily by ecchymotic hemorrhage and liver necrosis Xue et al, 2019). DVH is mainly caused by duck hepatitis A virus (DHAV), which is the only member of a novel genus Avihepatovirus in the family Picornaviridae (Li et al, 2013, Wang et al, 2018Xue et al, 2019). It is caused by three types of RNA viruses, namely, duck hepatitis virus (DHV) types 1, DHV-2 and DHV-3 (Woolcock and Fabricant, 1991;McNulty, 2001;Ding and Zhang, 2007).…”

Section: Introductionmentioning

confidence: 99%

Comparative Pathogenicity of Duck Hepatitis A Virus Type 1 and 3 Infections in South Korea

Soliman¹

2019

PVJ

View full text Add to dashboard Cite

Despite the impact of duck hepatitis virus type 1 (DHAV) as economically important and devastating pathogen in duck industry, there is a scarcity of data on the pathogenesis of DHAV. In this study, 1-day old ducklings were orally inoculated with DHAV-3 isolate (DHAV-3/duck-wt/KOR/Y02-1/2012) and DRL-62 strain as control virus in addition to mock-inoculated group. These strains induced the same clinical signs and gross lesions in virus-inoculated ducklings. The clinical signs appeared at HPI 24 and the mortalities were recorded by HPI 36. Histopatholoically, both strains induced the same characteristic liver lesions: hepatocyte necrosis, lymphoid cell infiltration in the portal triad and bile duct hyperplasia. Different microscopic lesions were also observed in extra-hepatic organs and tissues. By RT-PCR and nested PCR, viral RNA was detected in liver, spleen, brain, kidney, lung, heart, pancreas, bursa of Fabricious and thymus. An immunofluorescence assay confirmed virus antigen localization in these organs and tissues. Serum biochemical analysis revealed a significant hypoalbuminemia which reached the lowest level by HPI 24. These results indicated that DHAV-1 or 3 have a strong liver tropism and a pantropic effect with wide damage to various organs and tissues. The lesions of spleen, bursa of Fabricious and thymus indicated that the immune function might be affected by the DHAV infection. ©2019 PVJ. All rights reserved Key words: Albumin Duck hepatitis virus Histopathology IFA PCR To Cite This Article: Soliman M, Park JG and Park SI, 2019. Comparative pathogenicity of duck hepatitis a virus type 1 and 3 infections in south korea. Pak Vet J, 39(2): 271-277. http://dx.

show abstract

High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody

Cited by 17 publications

References 33 publications

Expression of duck hepatitis A virus type 1 VP3 protein mediated by avian adeno-associated virus and its immunogenicity in ducklings

Expression of duck hepatitis A virus type 1 VP3 protein mediated by avian adeno-associated virus and its immunogenicity in ducklings

Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system

Comparative Pathogenicity of Duck Hepatitis A Virus Type 1 and 3 Infections in South Korea

Contact Info

Product

Resources

About