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), andDobrava 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.Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome are rodent-borne viral zoonoses caused by viruses in the genus Hantavirus, family Bunyaviridae (3). Four antigenically and genetically distinct hantaviruses are known to cause HFRS. They are defined as different serotypes: Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), and Puumala virus (PUUV). Sin Nombre virus and related viruses cause hantavirus pulmonary syndrome. There is a close association between the viruses and their rodent hosts (10,14,21). At present, the only serological assay available to define the serotype of a causative hantavirus is the neutralization test (NT) (4,12,15). However, the NT needs specialized techniques and equipment, takes 1 to 2 weeks to perform, and requires a containment laboratory for virus manipulation.Hantavirus nucleocapsid protein (NP) possesses immunodominant, linear, cross-reactive epitopes within the first 100 amino acids (aa) of the N terminus (6,7,26). In addition, serotype-specific conformational epitopes have been detected in about half of the C termini of the NPs by serotype-specific monoclonal antibodies (MAbs) (20,28). Recombinant NPs (rNPs) of HTNV and SEOV that were truncated 154 aa from the N termini of the NPs (rNP155s) were previously evaluated as diagnostic antigens with expression by a baculovirus system (13). An indirect immunofluorescent-antibody (IFA) test, using the truncated rNPs HTNV rNP155 and SEOV rNP155 as antigens, was able to differentiate HTNV and SEOV infections serologically. However, at least two problems remained. (i) The IFA titers with the rNP155s were more than 10 times lower than those with authentic viruses or whole rNPs. Therefore, patient sera with low titers could not be differentiated; (ii) The antigenicity of HTNV rNP155 was too low to be applied in an enzyme-linked immunosorbent assay (ELISA). These problems were probably caused by an alteration of the antigenic structure after removing 154 aa. It was reported that the crossreactive, immunodominant epitopes of Sin Nombre virus NP were mapped to the segment between aa 17 an...

Epidemiological Study of Hantavirus Infection in the Samara Region of European Russia

“…Enzyme-linked immunosorbent assay (ELISA): Anti-hantavirus IgG in patients was detected by ELISA [2,20,27]. Briefly, 96-well plates were coated with the monoclonal antibody (MAb) E5/G6 [26,47] as a capture antibody and were treated with Block Ace (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) at 37C for 1 hr for blocking.…”

“…Briefly, 96-well plates were coated with the monoclonal antibody (MAb) E5/G6 [26,47] as a capture antibody and were treated with Block Ace (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) at 37C for 1 hr for blocking. The Baculovirus-expressed recombinant hantavirus NP of PUUV, DOBV, or HTNV [2,20] was then added to the plates and incubated at room temperature for 1 hr. Sera from clinically diagnosed HFRS patients were diluted to 1:200, added to the plates, and incubated at room temperature for 1 hr.…”

“…Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan, 2) Hantaviruses belong to the genus Hantavirus, within the family Bunyaviridae, and cause two forms of human illness. These zoonoses include hemorrhagic fever with renal syndrome (HFRS), which occurs in Asia and Europe and is caused by the Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Dobrava-Belgrade virus (DOBV), and Amur virus (AMRV).…”

“…, Vyacheslav G. MOROZOV 3) , Takahiro SETO 1) , Yoichi TANIKAWA 1) , Sergey I. KOLOMINOV 3) , Sergey N. BELOV 3) , Ichiro NAKAMURA 4) , Nobuo HASHIMOTO 1) , Alexander E. BALAKIEV 2) , Tamara K. DZAGURNOVA 2) , Nur Hardy bin Abu DAUD 1) , Daisuke MIYASHITA 1) , Olga A. MEDVEDKINA 2) , Mina NAKAUCHI 1) , Mariko ISHIZUKA 1) , Kentaro YOSHII 1) , Kumiko YOSHIMATSU 5) , Jiro ARIKAWA 5) and Ikuo TAKASHIMA 1)…”

A soluble and heat-resistant form of hantavirus nucleocapsid protein for the serodiagnosis of HFRS

“…One is located within the N-terminal 1-120 amino acids and contains linear and cross-reactive epitopes to other hantaviruses (Elgh et al, 1996b;Gott et al, 1997;Jenison et al, 1994;Wang et al, 1993;Yamada et al, 1995). The other is located within the C-terminus and contains conformation-dependent and serotypespecific epitopes (Araki et al, 2001;Morii et al, 1998;Ruo et al, 1991;Yoshimatsu et al, 1996). Previous studies have shown that the N-terminal immunoreactive region is conserved and that those of HTNV, SEOV, and DOBV are cross-reactive, and those of SN and PUUV are cross-reactive .…”