Purification of Crimean–Congo hemorrhagic fever virus nucleoprotein and its utility for serological diagnosis

Lombe, Boniface Pongombo; Miyamoto, Hiroko; Saito, Takeshi; Yoshida, Reiko; Manzoor, Rashid; Kajihara, Masahiro; Shimojima, Masayuki; Fukushi, Shuetsu; Morikawa, Shigeru; Yoshikawa, Tomoki; Kurosu, Takeshi; Saijo, Masayuki; Tang, Qing; Masumu, Justin; Hawman, David W.; Feldmann, Heinz; Takada, Ayato

doi:10.1038/s41598-021-81752-0

Cited by 15 publications

(18 citation statements)

References 47 publications

(43 reference statements)

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“…The hyperimmunized calf was reactive, however, which might be due to higher N protein-specific antibody levels (NSDV N protein being the main immunogen in the vaccine and CCHFV N protein antigen in the CCHFV ELISAs). Cross-reactions, albeit at low levels, have also been observed recently by another group, when they tested their In-house N protein-based CCHFV ELISA with a NSDV hyperimmune mouse serum [ 26 ].…”

Section: Discussionmentioning

confidence: 78%

Deciphering Antibody Responses to Orthonairoviruses in Ruminants

2021

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Antibody cross-reactivities between related viruses are common diagnostic challenges, resulting in reduced diagnostic specificities and sensitivities. In this study, antibody cross-reactions between neglected members of the genus Orthonairovirus—Hazara (HAZV), Dugbe (DUGV), and Nairobi sheep disease orthonairovirus (NSDV)—were investigated. Mono-specific ovine and bovine sera following experimental infections as well immunization trials with HAZV, DUGV, and NSDV were tested in homologous and heterologous virus-specific assays, namely indirect ELISAs based on recombinant N protein, indirect immunofluorescence assays (iIFA), and two neutralization test formats (plaque reduction neutralization test (PRNT) and micro-virus neutralization test (mVNT)). The highest specificities were achieved with the ELISAs, followed by the mVNT, iIFA, and PRNT. Cross-reactivities were mainly observed within the Nairobi sheep disease serogroup–but surprisingly, HAZV antibodies in PRNT did also neutralize NSDV and DUGV. In conclusion, we recommend ELISAs and mVNTs for a discriminative diagnostic approach to differentiate between these antibodies. NSDV antisera were also used in serological assays for the detection of antibodies against the human pathogen Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). Interestingly, all CCHFV ELISAs (In-house and commercial) achieved high diagnostic specificities, whereas significant cross-reactivities were observed in a CCHFV iIFA. Previously, similar results were obtained when analyzing the HAZV and DUGV antisera.

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Section: Discussionmentioning

confidence: 78%

Deciphering Antibody Responses to Orthonairoviruses in Ruminants

2021

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“…CCHFV nucleoprotein expression in BHK-21 cells transfected with the recombinant plasmid, pSinCCHF-52S, was evaluated by immunofluorescence staining of expressing cells using anti-His tag antibodies (Figure 1A,B) and further confirmed by immunofluorescence staining using anti-CCHF IgG from CCHF survivors (Figure 1C,D). SDS-PAGE, followed by transfer to the PVDF membrane and Western blotting with anti-His 6 tag mouse monoclonal antibody, revealed a protein band of approximately 52 kDa corresponding to the estimated molecular mass of CCHFV NP [44] (Figures 1E and S5).…”

Section: Expression Of Cchfv Np By Sindbis Replicon Vectormentioning

confidence: 99%

Immunogenicity of a DNA-Based Sindbis Replicon Expressing Crimean–Congo Hemorrhagic Fever Virus Nucleoprotein

2021

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Crimean–Congo hemorrhagic fever virus (CCHFV) infrequently causes hemorrhagic fever in humans with a case fatality rate of 30%. Currently, there is neither an internationally approved antiviral drug nor a vaccine against the virus. A replicon based on the Sindbis virus vector encoding the complete open reading frame of a CCHFV nucleoprotein from a South African isolate was prepared and investigated as a possible candidate vaccine. The transcription of CCHFV RNA and recombinant protein production by the replicon were characterized in transfected baby hamster kidney cells. A replicon encoding CCHFV nucleoprotein inserted in plasmid DNA, pSinCCHF-52S, directed transcription of CCHFV RNA in the transfected cells. NIH-III heterozygous mice immunized with pSinCCHF-52S generated CCHFV IgG specific antibodies with notably higher levels of IgG2a compared to IgG1. Splenocytes from mice immunized with pSinCCHF-52S secreted IFN-γ and IL-2, low levels of IL-6 or IL-10, and no IL-4. No specific cytokine production was registered in splenocytes of mock-immunized mice (p < 0.05). Thus, our study demonstrated the expression of CCHFV nucleoprotein by a Sindbis virus vector and its immunogenicity in mice. The spectrum of cytokine production and antibody profile indicated predominantly Th1-type of an anti-CCHFV immune response. Further studies in CCHFV-susceptible animals are necessary to determine whether the induced immune response is protective.

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“…Among CCHFV proteins, the nucleoprotein (NP), which is antigenically conserved among the CCHFV clades, is the most abundant primary antigen detectable during CCHFV infection and is also known to be highly immunogenic [ 9 ]. Thus, CCHFV NP serves as the principal antigen for the development of serological assays [ 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Similar to other hemorrhagic fevers, CCHF tends to be reported in remote areas lacking diagnostic capacity, which causes a delay in its clinical recognition, while retrospective serosurveys provide information on past exposure to the virus but not with active infection [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Previously, we and others showed the usefulness of recombinant CCHFV NP antigens in an enzyme-linked immunosorbent assay (ELISA) and an immunofluorescent assay, and its ability for detecting CCHFV NP-specific IgG directed against strains originating from different geographic regions [ 10 , 16 , 18 , 19 , 20 ]. However, it is known that some anti-NP antibodies are cross-reactive to multiple orthonairoviruses in ELISA and Western blotting [ 16 , 18 , 19 ]. Considering the overlapping distribution of CCHFV-related orthonairoviruses such as Nairobi sheep disease virus (NSDV), also known as Ganjam virus in Asia, and Dugbe virus (DUGV), both of which are known as zoonotic pathogens, the cross-reactivity of anti-NP antibodies is an important issue for serological diagnosis and surveillance of CCHF [ 18 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

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Mapping of Antibody Epitopes on the Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein

Lombe

Saito

Miyamoto

et al. 2022

Viruses

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Crimean-Congo hemorrhagic fever virus (CCHFV), a nairovirus, is a tick-borne zoonotic virus that causes hemorrhagic fever in humans. The CCHFV nucleoprotein (NP) is the antigen most used for serological screening of CCHFV infection in animals and humans. To gain insights into antibody epitopes on the NP molecule, we produced recombinant chimeric NPs between CCHFV and Nairobi sheep disease virus (NSDV), which is another nairovirus, and tested rabbit and mouse antisera/immune ascites, anti-NP monoclonal antibodies, and CCHFV-infected animal/human sera for their reactivities to the NP antigens. We found that the amino acids at positions 161–320 might include dominant epitopes recognized by anti-CCHFV IgG antibodies, whereas cross-reactivity between anti-CCHFV and anti-NSDV antibodies was limited. Their binding capacities were further tested using a series of synthetic peptides whose sequences were derived from CCHFV NP. IgG antibodies in CCHFV-infected monkeys and patients were reactive to some of the synthetic peptide antigens (e.g., amino acid residues at positions 131–150 and 211–230). Only a few peptides were recognized by IgG antibodies in the anti-NSDV serum. These results provide useful information to improve NP-based antibody detection assays as well as antigen detection tests relying on anti-NP monoclonal antibodies.

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Purification of Crimean–Congo hemorrhagic fever virus nucleoprotein and its utility for serological diagnosis

Cited by 15 publications

References 47 publications

Deciphering Antibody Responses to Orthonairoviruses in Ruminants

Deciphering Antibody Responses to Orthonairoviruses in Ruminants

Immunogenicity of a DNA-Based Sindbis Replicon Expressing Crimean–Congo Hemorrhagic Fever Virus Nucleoprotein

Mapping of Antibody Epitopes on the Crimean-Congo Hemorrhagic Fever Virus Nucleoprotein

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