Baculovirus Expression of the Small Genome Segment of Hantaan Virus and Potential Use of the Expressed Nucleocapsid Protein as a Diagnostic Antigen

Schmaljohn, Connie S.; Sugiyama, Kazuyoshi; Schmaljohn, Alan L.; Bishop, David H. L.

doi:10.1099/0022-1317-69-4-777

Cited by 54 publications

(29 citation statements)

References 13 publications

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“…Recombinant baculoviruses (Autographa californica nuclear polyhedrosis virus) containing coding information for the NP of HTNV or SEOV and the HTNV envelope glycoprotein were supplied by C. S. Schmaljohn of USAMRIID, Frederick, Md. (4,32). The recombinant baculoviruses for expressing various truncated nucleocapsid proteins were described previously (21).…”

Section: Methodsmentioning

confidence: 99%

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

Yoshimatsu¹,

Lee²,

Araki

et al. 2003

J Virol

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Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs ofHantaviruses, which are classified in the family Bunyaviridae, genus Hantavirus, are the causative agents of two rodent-borne viral zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (22,30). Each hantavirus appears to have a single predominant rodent species that serves as its natural reservoir (31), which is thought to coevolve with its host rodent (25). Consequently, hantaviruses form three large groups according to their host rodents: Murinae-associated, Arvicolinae-associated, and Sigmodontinae-associated hantaviruses.The Murinae-associated hantaviruses include three viruses that cause HFRS: Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV). HTNV is carried by Apodemus agrarius and A. peninsulae, DOBV is carried by A. agrarius and A. flavicollis, and SEOV is carried by Rattus norvegicus and R. rattus. Although HTNV, DOBV, and SEOV have similar antigenicities (12), their reservoir rodents differ. Both HTNV and SEOV occur in Asia, due to the spread of their specific host rodents. Therefore, it is important to differentiate HTNV and SEOV infections in order to control the reservoir rodents.Hantaviruses have a segmented negative-sense RNA genome; S (small), M (medium), and L (large) segments encode the nucleocapsid protein (NP), two envelope glycoproteins (G1 and G2), and viral polymerase, respectively. Generally, focus reduction neutralization tests are needed to serotype HTNV, SEOV, and DOBV infections. To differentiate HTNV and SEOV infections, we tried to establish serotyping antigens for enzyme-linked immunosorbent assay (ELISA) by using recombinant NP. The NP molecules of HTNV, SEOV, and DOBV consist of 429 amino acid (aa) residues. The major linear epitope common to HTNV, SEOV, and DOBV is located in the 100 aa at the N-terminal end of the NP (13,39,40). We tried to develop a serotyping antigen by deleting the major linear epitope common to HTNV and SEOV. Truncated NPs lacking 154 aa at the N-terminal end were generated (155-429 antigen) from the entire cDNA for HTNV and SEOV NP and expressed in a baculovirus system. As we expected, the SEOV 155-429 antigen proved to be a useful ELISA antigen for detecting serotype-specific antibody in the sera of HFRS patients. However, HTNV 155-429 antigen showed extremely low antigenicity, especially in ELISAs (21). Next, we generated truncated NP antigens lacking 49 aa of the N-terminal end (50-429 antigen) for HTNV, SEOV, and DOBV. The 50-429 antigens were useful ELISA antigens for serotyping HTNV, SEOV, and DOBV infections (3). In these trials, it was not clear why the HTNV 155-429 antigen lost its antigenicity. We postulated that the HTNV 155-429 antigen had undergone a structural alteration.Although hantaviruses are classified in the family Bunyaviridae, they are distinct from other members of the B...

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Section: Methodsmentioning

confidence: 99%

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

Yoshimatsu¹,

Lee²,

Araki

et al. 2003

J Virol

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“…Recombinant baculoviruses (Autographa californica nuclear polyhedrosis virus) containing coding information for the NPs of HTNV strain 76-118 and SEOV strain SR-11 were kindly supplied by C. S. Schmaljohn of the U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Md. (23). The baculovirusexpressing PUUV strain Sotkamo NP was kindly supplied by A. Vaheri of Helsinki University, Helsinki, Finland (25).…”

Section: Methodsmentioning

confidence: 99%

Truncated Hantavirus Nucleocapsid Proteins for Serotyping Hantaan, Seoul, and Dobrava Hantavirus Infections

et al. 2001

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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...

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“…Recombinant N proteins, expressed in insect cells for PUU (see above), TUL (Plyusnin et al, 1994 ;Lundkvist et al, 1996), SEO and HTN (Schmaljohn et al, 1988) and in E. coli for SN (Feldmann et al, 1993), were run in SDS-PAGE for immunoblotting as described (Towbin et al, 1979).…”

Section: Methodsmentioning

confidence: 99%

Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.

Salonen

Parren

Graus

et al. 1998

Journal of General Virology

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Baculovirus Expression of the Small Genome Segment of Hantaan Virus and Potential Use of the Expressed Nucleocapsid Protein as a Diagnostic Antigen

Cited by 54 publications

References 13 publications

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

Truncated Hantavirus Nucleocapsid Proteins for Serotyping Hantaan, Seoul, and Dobrava Hantavirus Infections

Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.

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