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
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
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
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
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
Table 1.
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 | ELISA 5F5 MAb at OD 492 nm | ELISA CR 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 |
Table 2.
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 |