Chicken astrovirus capsid proteins produced by recombinant baculoviruses: potential use for diagnosis and vaccination.
Chicken astroviruses (CAstVs) have been characterized recently. Due to their relatively poor growth in cell culture, virus-specific antigens are not readily available for the development of diagnostic reagents and vaccines. For this purpose two capsid protein antigens, specified by the 11672 isolate of CAstV, were produced in insect cells following infection with recombinant baculoviruses. The GST-11672 capsid protein, a fusion protein comprising the capsid protein and glutathione-S-transferase (GST) as an N-terminal affinity tag, and the 11672 capsid protein alone were detected by western blotting as proteins of ~100 and 70 kDa, respectively. Immunization with the affinity-purified GST-11672 capsid protein produced a polyclonal rabbit antiserum, which reacted by indirect immunofluorescence with Group B CAstVs but which showed no reactivity with the Group A CAstV isolate, 612. When used as part of an immunoperoxidase-based immunohistochemical procedure, this rabbit antiserum facilitated the detection of CAstV antigen in formalin-fixed, paraffin-embedded kidney tissue at the sites of histopathology characteristic of nephritis. Although further evaluation with sera from commercial chickens is required, a prototype indirect antibody-detecting enzyme-linked immunosorbent assay (ELISA) based on affinity-purified GST-11672 capsid protein as coating antigen demonstrated considerable potential with low ELISA absorbance values being generated with sera from specific pathogen free (SPF) chickens, and high absorbance values being generated with serum samples from experimentally infected chickens. Immunization experiments of SPF chickens showed that, when administered as mixtures with oil adjuvant, crude cell lysates containing the GST-11672 capsid protein or the 11672 capsid protein elicited virus-specific antibody responses that were detectable by indirect immunofluorescence and by virus neutralization assays.
a School of Biological Sciences, Medical Biology Centre , Queen's University of Belfast , Belfast , UK.
A
A
Lee
M
M
Wylie
V J
VJ
Smyth
A
A
Skibinska
I A
IA
Patterson
F
F
Forster
M D
MD
Welsh
D
D
Todd
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000097:blood; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019350:Astroviridae Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D052676:Avastrovirus / Q000235:genetics / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D007106:Immune Sera / Q000276:immunology; D011201:Poultry Diseases / Q000175:diagnosis / Q000276:immunology / Q000517:prevention & control*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D061987:Sf9 Cells; D013047:Specific Pathogen-Free Organisms; D018411:Spodoptera; D014611:Vaccination / Q000662:veterinary*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D007106:Immune Sera; D011994:Recombinant Proteins