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Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus

Martyna Krejmer-Rąbalska
Martyna Krejmer-Rąbalska
, 
Marta Peplińska
Marta Peplińska
, 
Bogusław Szewczyk
Bogusław Szewczyk
 et 
Andrzej Fitzner
Andrzej Fitzner
   | 23 mars 2024
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Publié en ligne: 23 mars 2024
Pages: 9 - 17
Reçu: 19 oct. 2023
Accepté: 14 mars 2024
DOI: https://doi.org/10.2478/jvetres-2024-0019
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© 2024 Martyna Krejmer-Rąbalska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

The alignment of six amino acid sequences of virus capsid protein 60 used in this study. These were rabbit haemorrhagic disease virus 2 (RHDV2)-wild type (WT), -127KKK129, -305RGD307 and -305KKK307 and European brown hare syndrome virus (EBHSV)-WT and -301KKK303. The position of the three-lysine (KKK) (or Arg-Gly-Asp (RGD)) substitution is marked with a black rectangle. Amino acids 127–129 in RHDV2 are in the S domain, while aa 305–307 in RHDV2 and aa 303–305 in EBHSV are in the P2 subdomain
The alignment of six amino acid sequences of virus capsid protein 60 used in this study. These were rabbit haemorrhagic disease virus 2 (RHDV2)-wild type (WT), -127KKK129, -305RGD307 and -305KKK307 and European brown hare syndrome virus (EBHSV)-WT and -301KKK303. The position of the three-lysine (KKK) (or Arg-Gly-Asp (RGD)) substitution is marked with a black rectangle. Amino acids 127–129 in RHDV2 are in the S domain, while aa 305–307 in RHDV2 and aa 303–305 in EBHSV are in the P2 subdomain

Fig. 2.

Western blot analysis of rabbit haemorrhagic disease virus 2 (RHDV2) virus-like particles (VLPs) based on virus capsid protein 60 (purified from the medium) produced in a baculovirus expression system. Lanes: 1 – Page Ruler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA); 2 – non-infected control (NC) medium (mock); 3 – medium from cells infected with baculovirus harbouring the beta-glucuronidase gene as a negative control; 4 – wild type (WT) RHDV2 VLPs; 5 – P2 subdomain Arg-Gly-Asp (RGD motif)–substituted RHDV2-305RGD307 VLPs; 6 – S-domain three-lysine (KKK)–substituted RHDV2-127KKK129 VLPs; 7 – P2 subdomain–substituted RHDV2-305KKK307 VLPs. S1/201609 – serum from a rabbit not vaccinated against RHD which survived an outbreak caused by RHDV2; S3/201609 and S6/201609 – sera from two rabbits vaccinated against RHD which survived an outbreak caused by RHDV2 infection
Western blot analysis of rabbit haemorrhagic disease virus 2 (RHDV2) virus-like particles (VLPs) based on virus capsid protein 60 (purified from the medium) produced in a baculovirus expression system. Lanes: 1 – Page Ruler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA); 2 – non-infected control (NC) medium (mock); 3 – medium from cells infected with baculovirus harbouring the beta-glucuronidase gene as a negative control; 4 – wild type (WT) RHDV2 VLPs; 5 – P2 subdomain Arg-Gly-Asp (RGD motif)–substituted RHDV2-305RGD307 VLPs; 6 – S-domain three-lysine (KKK)–substituted RHDV2-127KKK129 VLPs; 7 – P2 subdomain–substituted RHDV2-305KKK307 VLPs. S1/201609 – serum from a rabbit not vaccinated against RHD which survived an outbreak caused by RHDV2; S3/201609 and S6/201609 – sera from two rabbits vaccinated against RHD which survived an outbreak caused by RHDV2 infection

Fig. 3.

Western blot analysis of European brown hare syndrome virus (EBHSV) virus-like particles (VLPs) based on virus capsid protein 60 (purified from the medium) produced in a baculovirus expression system. Lanes: 1 – Page Ruler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA); 2 – non-infected control (NC) medium (mock); 3 – medium from cells infected with baculovirus harbouring the beta-glucuronidase gene as a negative control; 4 – wild-type (WT) EBHSV VLPs; 5 – P2 subdomain three-lysine (KKK)–substituted EBHSV-301KKK303 VLPs. S213/III – serum from an EBHSV-positive hare
Western blot analysis of European brown hare syndrome virus (EBHSV) virus-like particles (VLPs) based on virus capsid protein 60 (purified from the medium) produced in a baculovirus expression system. Lanes: 1 – Page Ruler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA); 2 – non-infected control (NC) medium (mock); 3 – medium from cells infected with baculovirus harbouring the beta-glucuronidase gene as a negative control; 4 – wild-type (WT) EBHSV VLPs; 5 – P2 subdomain three-lysine (KKK)–substituted EBHSV-301KKK303 VLPs. S213/III – serum from an EBHSV-positive hare

Fig. 4.

Transmission electron photomicrographs of virus-like particles (VLPs) based on virus capsid protein 60 of European brown hare syndrome virus (EBHSV) and rabbit haemorrhagic disease virus 2 (RHDV2) with uranyl acetate negative staining. White triangles indicate a representative wild-type (WT) EBHSV and P2 subdomain three-lysine (KKK)–substituted EBHSV-301KKK303 VLP (A); and RHDV2-WT, S domain–substituted RHDV2-127KKK129, P2 subdomain–substituted RHDV2-305KKK307 and P2 subdomain Arg-Gly-Asp (RGD motif)–substituted 305RGD307 VLP (B). Scale bar – 50 nm
Transmission electron photomicrographs of virus-like particles (VLPs) based on virus capsid protein 60 of European brown hare syndrome virus (EBHSV) and rabbit haemorrhagic disease virus 2 (RHDV2) with uranyl acetate negative staining. White triangles indicate a representative wild-type (WT) EBHSV and P2 subdomain three-lysine (KKK)–substituted EBHSV-301KKK303 VLP (A); and RHDV2-WT, S domain–substituted RHDV2-127KKK129, P2 subdomain–substituted RHDV2-305KKK307 and P2 subdomain Arg-Gly-Asp (RGD motif)–substituted 305RGD307 VLP (B). Scale bar – 50 nm

Fig. 5.

Detection and typing of virus-like particles (VLPs) based on virus capsid protein 60 (VP60) as wild-type recombinant rabbit haemorrhagic disease virus 2 (RHDV2-WT) and P2 subdomain Arg-Gly-Asp (RGD motif)–substituted-305RGD307 VP60s (based on the Polish LIB2018 strain), and wild-type recombinant European brown hare syndrome virus (EBHSV-WT) VP60 (based on the Polish G104 strain) using a differential ELISA test and RHDV-EBHSV test OD – optical density; MAbs – monoclonal antibodies; CR – cross-reactive
Detection and typing of virus-like particles (VLPs) based on virus capsid protein 60 (VP60) as wild-type recombinant rabbit haemorrhagic disease virus 2 (RHDV2-WT) and P2 subdomain Arg-Gly-Asp (RGD motif)–substituted-305RGD307 VP60s (based on the Polish LIB2018 strain), and wild-type recombinant European brown hare syndrome virus (EBHSV-WT) VP60 (based on the Polish G104 strain) using a differential ELISA test and RHDV-EBHSV test OD – optical density; MAbs – monoclonal antibodies; CR – cross-reactive

Summary of virus-like particles (VLPs) based on wild-type or mutated variants (with Arg-Gly-Asp (RGD motif) or three-lysine (KKK) substitutions) in the VP60 protein from Polish strains of rabbit haemorrhagic virus 2 (RHDV2) or European brown hare syndrome virus (EBHSV) produced in a baculovirus expression system in Spodoptera frugiperda 9 insect cells

Sample Western blot (expression) Formation of VLP ELISA HA
RHDV2-WT VLPs +++ +++ +++ +++
RHDV2-305RGD307 VLPs +++ +++ +++ +++
RHDV2-127KKK129 VLPs +++ + +/− n/a
RHDV2-305KKK307 VLPs +++ ++ +/− n/a
EBHSV-WT VLPs +++ +++ +++ +
EBHSV-301KKK303 VLPs + + +/− n/a

Reactivity of recombinant virus-like particles (VLPs) based on virus capsid protein 60 (VP60) as wild-type European brown hare syndrome disease virus (EBHSV-WT), rabbit haemorrhagic disease virus 2 (RHDV2)-WT and P2 subdomain Arg-Gly-Asp (RGD motif)–substituted RHDV2-305RGD307 VP60s – reactivity with a single monoclonal antibody specific for EBHSV and pool of monoclonal antibodies (MAbs) specific for EBHSV-RHDV in ELISA

Sample ELISA5F5 MAb at OD 492 nm ELISACR MAbs at OD 492 nm
EBHSV-WT VLPs (G104 strain) 1.654 1.768
RHDV2-WT VLPs (LIB2018 strain) 0.065 1.628
RHDV2-305RGD307 VLPs 0.056 1.595
RHDV KGM1988 strain, native virus, positive control 0.057 0.935
RHDV2 native virus ZWO2021 strain, positive control 0.065 1.558
EBHSV native virus G104 strain, positive control 0.926 0.916
eISSN:
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Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
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