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A new enzyme-linked immunosorbent assay for serological diagnosis of seal parapoxvirus infection in marine mammals

Yassien Badr
Yassien Badr
, 
Md. Matiur Rahman
Md. Matiur Rahman
, 
Yoshito Ohno
Yoshito Ohno
, 
Keita Ishijima
Keita Ishijima
, 
Ken Maeda
Ken Maeda
, 
Kaoru Kohyama
Kaoru Kohyama
, 
Yuji O. Kamatari
Yuji O. Kamatari
, 
Kaori Shimizu
Kaori Shimizu
, 
Ayaka Okada
Ayaka Okada
 e 
Yasuo Inoshima
Yasuo Inoshima
   | 01 mar 2022
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Pubblicato online: 01 mar 2022
Pagine: 43 - 52
Ricevuto: 06 set 2021
Accettato: 24 gen 2022
DOI: https://doi.org/10.2478/jvetres-2022-0005
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© 2022 Y. Badr et al. published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Selection of the amino acids of the synthetic peptide used for the immunisation of a rabbit (for anti-envelope antibody production). A – Amino acid sequence of the seal parapoxvirus (SPPV) major envelope protein and the selected 20-long sequence (from the 183rd to 202nd amino acid) (bold and underlined). B – The predicted 3D structure of the SPPV major envelope protein was generated by SWISS-MODE (39) and the image was created by using PyMol (www.pymol.org). The coloured balls (four different colours) represent the atoms composing the selected amino acids
Selection of the amino acids of the synthetic peptide used for the immunisation of a rabbit (for anti-envelope antibody production). A – Amino acid sequence of the seal parapoxvirus (SPPV) major envelope protein and the selected 20-long sequence (from the 183rd to 202nd amino acid) (bold and underlined). B – The predicted 3D structure of the SPPV major envelope protein was generated by SWISS-MODE (39) and the image was created by using PyMol (www.pymol.org). The coloured balls (four different colours) represent the atoms composing the selected amino acids

Fig. 2

Confirmation of seal parapoxvirus envelope gene cloning and expression of the green fluorescence protein (GFP) and fusion protein (Env-GFP) in transfected cells. A – Results of the PCR using DNA extracted from transfected cells (mock, empty, and cloned vectors) at 48 hours post transfection (hpt); B – Upper panels: confocal microscopic photos of transfected cells (mock, empty, and cloned vectors) at 48 hpt showing the expression of GFP and Env-GFP (low magnification, scale bar 200 μm). Lower panels: the distribution of GFP and Env-GFP inside transfected cells (high magnification, scale bar 30 μm); C – 48 hpt results of Western blotting using the transfected cell lysates (mock, empty, and cloned vectors) and the rabbit anti-envelope serum, anti-GFP antibody, or PL1-2010 plasma, illustrating the expression of GFP and Env-GFP in transfected cells by the detection of specific bands of both proteins
Confirmation of seal parapoxvirus envelope gene cloning and expression of the green fluorescence protein (GFP) and fusion protein (Env-GFP) in transfected cells. A – Results of the PCR using DNA extracted from transfected cells (mock, empty, and cloned vectors) at 48 hours post transfection (hpt); B – Upper panels: confocal microscopic photos of transfected cells (mock, empty, and cloned vectors) at 48 hpt showing the expression of GFP and Env-GFP (low magnification, scale bar 200 μm). Lower panels: the distribution of GFP and Env-GFP inside transfected cells (high magnification, scale bar 30 μm); C – 48 hpt results of Western blotting using the transfected cell lysates (mock, empty, and cloned vectors) and the rabbit anti-envelope serum, anti-GFP antibody, or PL1-2010 plasma, illustrating the expression of GFP and Env-GFP in transfected cells by the detection of specific bands of both proteins

Fig. 3

Optimisation of the ELISA cell lysates and primary antibody concentrations. A – Colour reactions in the ELISA plate wells coated with mock, green fluorescence protein (GFP), or fusion protein (Env-GFP) cell lysates (25 μg/well) and incubated with either rabbit anti-envelope serum or pre-serum (1:50 dilutions); B – Optical density (OD) values detected using either rabbit anti-envelope serum or pre-serum at 1:50 dilution in either Env-GFP lysate– or GFP cell lysate–coated wells at different concentrations. Data are shown as means ± SD for two separate experiments; C – OD values detected using PL1-2010 plasma at either 1:50 or 1:100 dilution with different concentrations of Env-GFP lysate– or GFP cell lysate–coated wells, in addition to the OD difference in each case (subtracting the GFP OD value from the Env-GFP OD value)
Optimisation of the ELISA cell lysates and primary antibody concentrations. A – Colour reactions in the ELISA plate wells coated with mock, green fluorescence protein (GFP), or fusion protein (Env-GFP) cell lysates (25 μg/well) and incubated with either rabbit anti-envelope serum or pre-serum (1:50 dilutions); B – Optical density (OD) values detected using either rabbit anti-envelope serum or pre-serum at 1:50 dilution in either Env-GFP lysate– or GFP cell lysate–coated wells at different concentrations. Data are shown as means ± SD for two separate experiments; C – OD values detected using PL1-2010 plasma at either 1:50 or 1:100 dilution with different concentrations of Env-GFP lysate– or GFP cell lysate–coated wells, in addition to the OD difference in each case (subtracting the GFP OD value from the Env-GFP OD value)

Fig. 4

Western blotting assay to examine the reactivity of selected serum samples. Transfected cell lysates, green fluorescence protein (GFP) (G lane) and fusion protein (Env-GFP) (E lane), were loaded as the antigens, horseradish peroxidase-conjugated protein A/G was used in place of the secondary antibody, and sera from rabbit (anti-envelope and/or pre-serum) were used as primary antibodies with A – Primary antibodies from all spotted seals or; B – Primary antibodies from selected cetaceans. Arrows – Specific bands in the E lanes at approximately 70 kDa indicating positive reactions only with the rabbit anti-envelope serum and PL1-2010 plasma. A protein ladder showing different sizes is provided on the left
Western blotting assay to examine the reactivity of selected serum samples. Transfected cell lysates, green fluorescence protein (GFP) (G lane) and fusion protein (Env-GFP) (E lane), were loaded as the antigens, horseradish peroxidase-conjugated protein A/G was used in place of the secondary antibody, and sera from rabbit (anti-envelope and/or pre-serum) were used as primary antibodies with A – Primary antibodies from all spotted seals or; B – Primary antibodies from selected cetaceans. Arrows – Specific bands in the E lanes at approximately 70 kDa indicating positive reactions only with the rabbit anti-envelope serum and PL1-2010 plasma. A protein ladder showing different sizes is provided on the left

Optimisation of the enzyme-linked immunosorbent assay (ELISA) conditions

Factor Optimal condition Other evaluated but not chosen conditions
Transfection reagent Lipofectamine LTX FuGENE HD (E2311, Promega, Madison, WI, USA), and Polyethylenimine HCL MAX (24765, Polysciences, Taiwan)
Cell lysis buffer Using both RIPA and denaturing lysis buffers 1lysis % NPbuffer 40, RIPA alone buffer alone, and denaturing
Antigen preparation Pellets of lysed cells Supernatants of lysed cells, and whole cells lysed in the dish without pelleting
Time of collection of transfected cells (hours post transfection) 48 60
Antigen concentration 25 μg/well (100 μL/well at 0.25 μg/μL) 6.25, 12.5, 50, 100, and 200 μg/well
Blocking reagent PBS containing 1% Block ACE c-block-e, h-block-e, k-block-e, and b-block-e (BCL-BKSE-01, Beacle, Kyoto, Japan)
Primary sera/plasma dilution 1:50 (100 μL/well) diluted in PBS-T containing 0.4% Block ACE 1:100
HRP-conjugated protein A/G dilution 1:10,000 (100 μL/well) diluted in PBS-T containing 0.4% Block ACE 1:5,000

Marine mammal species used in this study

Species Animal identification Sex Age in years (as of October, 2020)
Spotted seal (Phoca largha) PL1PL2 FM 1110
DL1 M 42
DL5 F 21
Beluga whale (Delphinapterus leucas) DLDL69 FF 2513
DL11 M 8
DL12 M 12
LO2 M 20
Pacific white-sided dolphin (Lagenorhynchus obliquidens) LO4 F 21
LO5 F 11
TTC F 20
TTH M 4
TTL F 23
TTP M 23
Bottlenose dolphin (Tursiops truncatus) TTQTTR MF 223
TTS M 2
TTT M 25
TTW F 20
TTX F 21
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