Diagnostic potential of parechovirus capsid proteins

Abstract

To study humoral and cellular immunity against human parechovirus type 1 (HPEV1), the viral capsid proteins VP0, VP1, and VP3 were expressed and purified as glutathione S-transferase (GST)-tagged recombinant proteins. The fusion proteins were used to raise antisera in rabbits. VP0 and VP1 antisera specifically detected HPEV1-infected cells in culture by immunoperoxidase staining and immunofluorescence. Furthermore, antisera against the VP0 and VP1 proteins had neutralizing effects against HPEV1 infection. When the HPEV1 antibody titers of 20 adults and 55 children were determined by a microneutralization test, the prevalence of HPEV1 antibodies in the adult population was 96%, while 50% of children were seropositive. Selected sera were used to evaluate HPEV1 fusion proteins as antigens in an enzyme immunoassay. The VP3 capsid protein appeared to be suitable for the purpose, with specificity of 100% and sensitivity of 96% compared to the neutralization test. Furthermore, T-cell responses to the purified HPEV1 and HPEV1 capsid fusion proteins were studied in 20 adults. Sixty percent of the subjects had T-cell proliferation responses to purified HPEV1, and 90% of the subjects also had positive T-cell responses to at least one of the GST capsid proteins.

FIG. 1.

(A) Genome localization of the genes for the HPEV1 capsid proteins (VP0, VP3, and VP1) expressed as GST fusion polypeptides. (B) SDS-PAGE (left) and immunoblotting (right) analysis of the VP0-GST fusion protein. Lanes: M, molecular size markers; 1, uninduced E. coli cells; 2, induced E. coli cells expressing GST; 3, purified GST (26 kDa); 4, induced E. coli cells expressing VP0-GST; 5, purified VP0-GST (58 kDa). After separation by SDS-PAGE, the proteins were blotted onto a nitrocellulose filter and detected by primary antibodies (GST antiserum diluted 1:3,000 and HPEV1 antiserum diluted 1:1,000) and secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG). The samples analyzed in the right panel are VP0-GST and GST.

FIG. 2.

Detection of HPEV1 antigens in virus-infected A549-4A cells by immunoperoxidase (IP) staining and immunofluorescence (IF). The cell monolayers were infected with ∼200 PFU of HPEV1, and HPEV1 antiserum and capsid protein antisera were used at a dilution of 1:300.

FIG. 3.

Neutralization of HPEV1 infection with rabbit antisera against the GST fusion proteins (VP0-GST, VP3-GST, and VP1-GST) and against GST (dilution, 1:100) and purified HPEV1 (dilution, 1:1,000). The open bars indicate the effects of the preimmune serum, and the shaded bars show the effects of the specific antisera.

FIG. 4.

(A) Reactivities of the VP3 fusion protein (dotted lines) and purified HPEV1 (solid lines) as antigens in EIA. The microtiter wells were coated with 100 ng of the VP3-GST protein or HPEV1, and the reactivities of two positive sera (diamonds and triangles) and one negative serum (circles) for HPEV1 antibodies in the microneutralization assay were measured. (B) Correlation of the neutralization titer with the VP3-GST antigen in EIA in human serum samples known to be positive (n = 19) or negative (n = 5) for HPEV1 antibodies as determined by the microneutralization test. (C) Correlation of the HPEV1 neutralization antibody titer with the reactivity with purified HPEV1 antigen in EIA in human serum samples known to be positive (n = 19) or negative (n = 5) for HPEV1 antibodies.