Antigenic characterization of Hantaan and Seoul virus nucleocapsid proteins expressed by recombinant baculovirus: application of a truncated protein, lacking an antigenic region common to the two viruses, as a serotyping antigen

Abstract

Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus Hantavirus in the family Bunyaviridae and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.

FIG. 1

Detection of rNP and trNP with Western blots stained with MAb E5/G6 (a), mouse sera infected with HTN virus (b), or rat sera infected with SEO virus (c). Lane 1, HTN rNP (whole); lane 2, SEO rNP (whole); lane 3, trNP HTN 155-429; lane 4, trNP SEO 155-429; lane 5, uninfected High Five cells. In each lane, 3.0 × 10³ cells were applied.

FIG. 2

IFA profiles of patient sera with High Five cells expressing whole or truncated HTN or SEO rNP. Panels were stained with HTN (a through e) or SEO (f through j) patient serum. High Five cells expressed whole HTN rNP (a and f), whole SEO rNP (b and g), trNP HTN 155-429 (c and h), trNP SEO 155-429 (d and i), or recombinant p40 of Borna disease virus (16) (e and j).

FIG. 3

Serial dilutions of the sera of standard patients in the ELISA. Optical densities at 490 to 600 nm (OD₄₉₀–OD₆₀₀) are given for sera of patients infected with HTN (⧫) or SEO (■) and for negative human sera (+ and ×). High Five cells expressed the NPs shown above the panels.

FIG. 4

Reactivities of serial patients’ sera in ELISA. OD₄₉₀–OD₆₀₀ were plotted for acute-phase (○) and convalescent-phase (•) sera of patients from Anhui province and for acute-phase (□) and convalescent-phase (■) sera of patients from Henan province. Comparisons of ELISA ODs were made between whole HTN rNP and whole SEO rNP (A), between trNP HTN 155-429 and trNP SEO 155-429 (B), and between whole HTN rNP and trNP HTN 1-103.