Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use

Abstract

Abstract. A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23-25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log 2 ) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.have the gene for the Aleutian coat color. Aleutian Aleutian disease of mink (AD) is caused by the autonomous parvovirus, Aleutian mink disease parvovirus (ADV).6 First described in 1956, 15 AD is responsible for significant economic loss in commercial mink ranching operations.1,14 The infection in adult mink is persistent and is characterized by plasmacytosis, hypergammaglobulinemia, and ultimately immune complex mediated glomerulonephritis and arteritis.12,18-21 Newborn mink kits without maternal antibodies usually develop an acute fulminant, fatal respiratory distress syndrome characterized by interstitial pneumonia and the formation of hyaline membranes. 3,4,16 The disease is most severe in mink that been unsuccessful. Consequently, the disease is controlled in mink populations by serologically identifying and subsequently removing infected mink. 1,17 Experimental studies have demonstrated that detectable levels of IgM antibody appear within 6 days after infection and can persist for at least 85 days.5,21 Detectable levels of IgG antibody usually appear within 10-15 days of infection and will persist for the life of the mink.6 ,17These immunoglobulins show reactivity for the capsid proteins VP1 and VP2 and the nonstructural proteins NS-1 and NS-2. 1The most commonly used serologic tests for detection of anti-ADV antibody are counter current immunoelectrophoresis (CIE) 10,7 and indirect counter genotype mink usually die within 12-16 weeks after mink generally occurs by 26-30 weeks after infection, infection. In contrast, 50% mortality for non-Aleutian depending upon virus strain. 12,13 current electrophoresis.2 These assays require ...

Identification of Aleutian Mink Disease Parvovirus Capsid Sequences Mediating Antibody-Dependent Enhancement of Infection, Virus Neutralization, and Immune Complex Formation

“…Crandell feline kidney cells (CrFK), K562 cells and the Spodoptera frugiperda insect cell line (Sf9) were maintained according to previously reported methods (14,29,41,63). The procedure for propagating the molecularly cloned, cell culture-adapted ADV-G has also been described (14).…”

“…The procedure for propagating the molecularly cloned, cell culture-adapted ADV-G has also been described (14). For production of recombinant ADV capsids containing only VP2 (ADV-G VP2 ), Sf9 cells were infected with recombinant baculovirus in suspension cultures at a multiplicity of 1 PFU/cell for 72 h (41,63).…”

“…90% is the 647-amino-acid major capsid protein, VP2 (8,23). The minor capsid protein, VP1, contains the entire VP2 sequence but has 43 additional unique residues at the N terminus (8,23,24,63). When VP2 from the ADV-G isolate is expressed in either recombinant vaccinia viruses (24) or baculoviruses (23,63), the proteins assemble into empty capsids.…”

“…The minor capsid protein, VP1, contains the entire VP2 sequence but has 43 additional unique residues at the N terminus (8,23,24,63). When VP2 from the ADV-G isolate is expressed in either recombinant vaccinia viruses (24) or baculoviruses (23,63), the proteins assemble into empty capsids. Recent work with prokaryotic expression vectors has localized immunodominant targets for the antibody response to specific regions of the VP2 capsid protein (16,28).…”

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Recombinant VP2 Capsids for the Detection of Antibodies to Aleutian Mink Disease Virus

“…Recombinant AMDV VP2 proteins have been expressed (13,14,34,35) and shown to be antigenic and able to form virus-like particles (VLPs) (13,14,34). However, only a few diagnostic applications have been described (3,14,35), and published comparative data are scarce.…”