Development of Recombinant Nucleoprotein-Based Diagnostic Systems for Lassa Fever

Abstract

Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzymelinked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses.Lassa fever (LF) is a viral hemorrhagic fever caused by Lassa virus (LASV), an Old World arenavirus. Many cases of LF occur in western Africa in countries such as Guinea, Sierra Leone, and Nigeria (7,23,27,(29)(30)(31). It is thought that LASV infects tens of thousands of humans annually and causes hundreds to thousands of deaths (34). Humans become infected through contact with infected excreta, tissue, or blood from the peridomestic rodent, Mastomys natalensis, the reservoir host of LASV (34). LASV can be transmitted to other humans via mucosal or cutaneous contact or through nosocomial contamination (27). More than 20 imported cases of LF have been reported outside the endemic region in areas such as the United States, Canada, Europe, and Japan (1,2,13,15,18,24,25). Recently, the potential for the use of hemorrhagic fever viruses, including LASV, as a biological weapon has been emphasized (5, 6). Therefore, the development of diagnostic systems for LF is important even in countries free from LF outbreaks to date.Manipulation of infectious LASV is necessary for the detection of specific antibodies. However, a high-containment laboratory (biosafety level 4 [BSL-4]) is required for handling infectious LASV and, therefore, the preparation of LASV antigens cannot be implemented in institutes without a BSL-4 facility. Within th...

Characterization of Monoclonal Antibodies to Junin Virus Nucleocapsid Protein and Application to the Diagnosis of Hemorrhagic Fever Caused by South American Arenaviruses

“…The transfected cells were selected with 4 g of blasticidin Shydrochloride/ml in culture medium. The HeLa cell clones were obtained by analyzing the expression of each rNP by IFA with rabbit serum raised against the JUNV, MACV, GTOV, SABV, or CHPV rNP, as previously described (20).…”

“…The genome consists of two ambisense singlestranded RNA molecules, one designated L, which encodes the RNA-dependent RNA polymerase and a zinc-binding matrix protein, Z, and the other designated S, which encodes the major structural components of the virion, i.e., the nucleocapsid protein (NP) and the envelope glycoprotein precursor (15). The arenavirus NP is the most abundant protein among the viral structural proteins both in infected cells and in virions (2) and is commonly used as a target for detecting viral antigens (Ags) (20). Moreover, arenavirus NPs have been known to be the most conserved among the same virus species and, to some extent, among different arenavirus species (3,8).…”

“…Moreover, arenavirus NPs have been known to be the most conserved among the same virus species and, to some extent, among different arenavirus species (3,8). Therefore, it seems likely that monoclonal antibodies (MAbs) raised against the NP of an arenavirus would also be useful for detecting other arenaviruses (20). Recently, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed by using a recombinant NP (rNP) of JUNV, obtained from a recombinant baculovirus system, and was proposed to be useful for etiologic confirmation of AHF in seroepidemiological studies (20,26).…”

“…Recently, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed by using a recombinant NP (rNP) of JUNV, obtained from a recombinant baculovirus system, and was proposed to be useful for etiologic confirmation of AHF in seroepidemiological studies (20,26). It is considered that an Ag capture ELISA using MAbs specific for viral Ags allows rapid diagnosis of the acute phase of viral hemorrhagic fever by detecting viral Ags in blood or tissue homogenates (20). In this study, we produced MAbs to the rNP of JUNV.…”

Non Susceptibility of Neonatal and Adult Rats against the Middle East Respiratory Syndrome Coronavirus

“…MERS-CoV S protein-specific IgG ELISA: IgG ELISA was performed as previously described, except for antigen preparation (11). Briefly, microtiter plates were coated overnight at 49 C with lysates of MERSCoV-infected or mock-infected Vero cell lysates that had been treated with 1z NP-40.…”