Truncated hantavirus nucleocapsid proteins for serotyping Hantaan, Seoul, and Dobrava hantavirus infections

Abstract

Truncated recombinant nucleocapsid proteins (rNPs) of Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV) were expressed by a baculovirus system. The truncated rNPs, which lacked 49 (rNP50) or 154 (rNP155) N-terminal amino acids of the NPs of HTNV, SEOV, and DOBV, were able to differentiate HTNV-, SEOV-, and DOBV-specific immune sera. Recombinant NP50s retained higher reactivities than rNP155s and were proven useful for enzyme-linked immunosorbent assay (ELISA). The ELISAs based on the rNP50s of HTNV, SEOV, and DOBV successfully differentiated three groups of patient sera, previously defined by neutralization tests: 17 with HTNV infection, 12 with SEOV infection, and 20 with DOBV infection. The entire rNP of Puumala virus (PUUV) distinguished PUUV infection from the other types of hantavirus infection. Serotyping with these rNP50s can be recommended as a rapid and efficient system for hantavirus diagnosis.

FIG. 1

Reaction patterns of the rNPs to the MAb E5/G6 and rabbit immune sera in Western blotting. The rNPs were separated by electrophoresis and blotted onto a membrane and were detected with various antibodies. (a) MAb E5/G6; (b) serum from an HTNV-infected rabbit. Lanes 1, HTNV whole rNP; lanes 2, SEOV whole rNP; lanes 3, DOBV whole rNP; lanes 4, PUUV whole rNP; lanes 5, HTNV rNP50; lanes 6, SEOV rNP50; lanes 7, DOBV rNP50; lanes 8, HTNV rNP155; lanes 9, SEOV rNP155; lanes 10, DOBV rNP155; lanes 11, Borna disease virus p24; lanes M, molecular mass markers.

FIG. 2

Reaction patterns of rNPs with rabbit immune sera in ELISA. (A) Whole rNPs; (B) rNP50s; (C) rNP155s. HTNV (⧫), SEOV (■), DOBV (▴), and PUUV (○) rNPs were used. Each antigen was diluted from 1:10 to 1:5,120 and subjected to capture ELISA (see Materials and Methods). Capture antigens were detected with various rabbit immune sera: (a) anti-HTNV; (b) anti-SEOV; (c) anti-DOBV; (d) anti-PUUV. All sera were diluted to 1:200.

FIG. 3

Reaction patterns of representative HFRS patient sera in ELISA. Anti-HTNV, patient serum obtained from China; anti-SEOV, patient serum obtained from Korea; anti-DOBV, patient serum obtained from Bosnia; anti-PUUV, patient serum obtained from Sweden; NHS, uninfected human serum from Japan. The serotypes of the four representative sera were differentiated by the NT; the results are summarized in the lower table. (a) whole rNP; (b) rNP50. Solid bars, HTNV rNP; open bars, SEOV rNP; hatched bars, DOBV rNP; shaded bars, PUUV rNP. All sera were diluted to 1:200. All antigens were diluted to 1:10. OD₄₅₀, OD at 450 nm.

FIG. 4

Reactivities of groups of sera from hantavirus-infected patients in ELISA. The horizontal and vertical axes show the ODs at 450 to 600 nm (OD₄₅₀ to OD₆₀₀) for the sera from HTNV-infected patients (□), SEOV-infected patients (○), and DOBV-infected patients (⧫) for each antigen. ELISA ODs were compared as follows: (a) HTNV whole rNP versus SEOV whole rNP; (b) SEOV whole rNP versus DOBV whole rNP; (c) DOBV whole rNP versus HTNV whole rNP; (d) HTNV rNP50 versus SEOV rNP50; (e) SEOV rNP50 versus DOBV rNP50; and (f) DOBV rNP50 versus HTNV rNP50. The lines are the linear regression for each group of sera: solid line, sera from HTNV-infected patients; dashed line, sera from SEOV-infected patients; broken line, sera from DOBV-infected patients.