Development of a human herpesvirus 6 species-specific immunoblotting assay

Abstract

In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

Fig 1

(A) Construction of the HHV-6 U11 gene expression plasmids pET22b(+)-6AU11 and pET22b(+)-6BU11. These recombinant proteins expressed His₆ tag on the C-terminal end. (B) Expression of the recombinant proteins p100 (6A) and 101K (6B) and immunoblot analysis. Induction was carried out using 1 mM IPTG for 3 h at 30°C (+) or without 1 mM IPTG (−). Induction control (I.C*) was expressed by 1 mM IPTG for 3 h at 30°C. Molecular mass standards are indicated in kilodaltons on the left. Detection of recombinant p100 and 101K using an anti-His tag antibody. The anti-His tag antibody reacted with both recombinant p100 (6A) and 101K (6B) (lanes 1 and 3 of the left panel, respectively). Detection of recombinant 101K using an anti-101K antibody. The anti-101K antibody reacted with recombinant 101K (lane 3 of the right panel).

Fig 2

Immunoblot analysis with human sera collected from patients with various types of HHV-6 infections against recombinant p100 and 101K antigens. All human sera were diluted to 1:500 for the assay. The patient sera collected from a ci-HHV-6A patient (patient 2 [A]) and a CFS patient (patient 3 [B]) reacted with p100 (lane 1) and 101K (lane 2). (C) Paired sera obtained from exanthem subitum (primary HHV-6B infection) were used for the assay. Acute-phase serum did not react with p100 (lane 1) and 101K (lane 2). Meanwhile, convalescent-phase serum reacted with only 101K (lane 4). A healthy adult serum (patient 6) reacted with only 101K (D), and another healthy adult serum (patient 26) reacted with both p100 and 101k (E). Molecular mass standards are indicated in kilodaltons on the left. An open arrowhead indicates a specific band reacted to the p100 antigen. A solid arrowhead indicates a specific band reacted to the 101K antigen.