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

Primary aortic smooth muscle cells were isolated from the aorta of a dead spotted seal. Isolated cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (cat. no. 189-02145; Fujifilm Wako, Osaka, Japan) supplemented with 15% cosmic calf serum (HyClone, Provo, UT, USA), 1% non-essential amino acids (Gibco, Grand Island, NY, USA), 1 mM sodium pyruvate (Gibco), and ZellShield (Minerva Biolabs, Berlin, Germany). During cell passages, endothelial cells were eliminated due to slower growth than smooth muscle cells. After four passages, pLNCLT plasmid DNA encoding the large T antigen replication origin-defective simian virus 40 (23) was transfected into the cells. Transfected cells were selected by addition of G418 (cat. no. 04727878001; Roche, Mannheim, Germany) and maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (cat no. A15-701; PAA Laboratories, Pasching, Austria), 100 mg/mL of streptomycin, and 100 U/mL of penicillin. The established smooth muscle cells were designated as Phocid Smooth Muscle SV40T (PhoMT) cells.

The major envelope protein (B2L protein) of SPPV was chosen as the ELISA antigen because of its high immunogenicity (40). As SPPV has not previously been isolated and the live virus is not currently available in Japan, a DNA fragment encoding the whole nucleotide sequence of the gene encoding SPPV major envelope protein (complete genome SPPV isolate AFK76s1, GenBank accession number KY382358) (12), was synthesised using GeneArt Strings DNA Fragments (Invitrogen, Regensburg, Germany). The DNA fragment was cloned into the Xho I and EcoR I sites of the eukaryotic expression vector, pAcGFP1-N1 (cat. no. 632469; Takara, Kusatsu, Japan), which encodes the green fluorescence protein (GFP) sequence after the multiple cloning site of the vector. This step was executed using NEBuilder HiFi DNA Assembly Master Mix (cat. no. E2621; New England BioLabs, Ipswich, MA, USA). In the cloned vector, the envelope gene was attached in-frame to the N-terminus of the GFP gene, leading to the expression of a fusion protein (Env-GFP). The cloned and parental (empty) vectors were used for subsequent transfection experiments.

Transfection of PhoMT cells was with cloned (Env-GFP) or empty (GFP) vectors or was sham transfection using Lipofectamine LTX (cat. no. 15338-100, Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. The transfected cells were observed 48 hours post transfection (hpt) with an LSM700 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) to note protein expression through green fluorescence. Additionally, the intracellular locations of the GFP and Env-GFP proteins were investigated as follows: cells were grown on cover glasses in six-well plates (cat. no. 140675; Thermo Fisher Scientific, Jiangsu, China), transfected, fixed at 48 hpt using 4% paraformaldehyde, quenched by 50 mM NH⁴Cl, mounted using 50% glycerol in distilled water, and examined using a 488 mm LED laser and a 20 × or 63 × oil immersion objective confocal microscope lens.

At 48 hpt, PhoMT cells were collected and DNA was extracted from them using a DNeasy Blood & Tissue Kit (cat. no. 69506; Qiagen, Hilden, Germany). Forward and reverse primers (5'-AGTACATCAATGGGCGTGG-3' and 5'-CTGCTTCATGTGATCGGGG-3', respectively) were designed to confirm cloning with a 535 base pairs (bp) PCR product being generated for empty vectors and a 1,655 bp product for cloned vectors. The reaction was performed using GoTaq Hot Start Green Master Mix (cat. no. M5123; Promega, Madison, WI, USA) in a Veriti thermal cycler (Applied Biosystems, Foster City, CA, USA). The constituent volumes in the PCR reactions were: 2 μL of extracted DNA, 10 μL of 2 × GoTaq Hot Start Green Master Mix, 1 μL of each primer (10 μM), and 6 μL of nuclease free water for a total volume of 20 μL. The thermal cycling conditions were as follows: initial denaturation at 95°C for 2 minutes (min) followed by 30 cycles of denaturation at 95°C for 30 seconds (s), annealing at 58°C for 30 s, extension at 72°C for 1 min and 40 s, and a final extension step at 72°C for 7 min. The PCR products generated were sequenced directly using the same primer set.

The same DNA samples were examined by PCR using forward and reverse primers (5'-GCCAAAAGG GTCATCATCTC-3' and 5'-GGGGCCATCCACAGT CTTCT-3', respectively) capable of amplifying the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (3, 10), which was detected as an internal control for the extracted DNA samples.

An antibody against the SPPV major envelope protein was produced as the primary antibody in the positive control reactions for the ELISA. A synthetic peptide comprising CAMITPTATDFHMNHSGGGV (Fig. 1) as 20 amino acids selected from the sequence of the SPPV envelope protein was used to immunise a rabbit, and the rabbit serum was then collected as the anti-envelope serum. Rabbit pre-serum (collected before immunisation) was used as the negative control serum. Production of the synthetic peptide as well as rabbit sera was performed by Eurofins Genomics (Tokyo, Japan).

Fig. 1

Serum samples were collected from marine mammal species inhabiting the Port of Nagoya Public Aquarium in Japan as shown in Table 1. Samples were collected from each animal twice, in 2019 and 2020. A female spotted seal (PL1) had previously been infected with SPPV in 2010 (29), and a plasma sample that was collected from this animal at approximately 75 days after the onset of clinical signs (hereafter referred as PL1-2010 plasma) was used to validate the ELISA.

Assays were also performed to confirm expression of the proteins. Transfected PhoMT cells cultured in six-well plates were collected at 48 hpt using 25 μL/well of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 5 min. To examine marine mammals’ sera for SPPV antibodies, the ELISA antigen was prepared using pellets obtained from cells that were transfected in 100 mm dishes (cat. no. 430167; Corning, Oneonta, NY, USA), lysed with radioimmunoprecipitation (RIPA) buffer (1% sodium deoxycholate, 1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.5 mM ethylenediaminetetraacetic acid (EDTA)), and pelleted by centrifugation. The pellets were suspended in 100 μL of SDS-PAGE sample buffer, boiled for 15 min with periodic vortex mixing, and electrophoresed in 10% SDS-PAGE. Separated proteins were blotted onto an Immobilon-P polyvinylidene difluoride membrane (Merck Millipore, Cork, Ireland). Blots were blocked with 5% skimmed milk, washed using Tris-buffered saline (TBS) (100 mM Tris-HCl, pH 8.0 and 30 mM NaCl) containing 0.1% Tween-20 (TBS-T) and incubated. This operation was with either rabbit sera or marine mammal sera/plasma as primary antibodies diluted at 1:4,000 in TBS-T containing 1% skim milk or with anti-GFP-Tag rabbit polyclonal antibody (cat. no. 29779; AnaSpec, Fremont, CA, USA) diluted at 1:1,000. Next, the blots were washed and incubated with horseradish peroxidase (HRP)-conjugated protein A/G (cat. no. 32490; Thermo Fisher Scientific, Rockford, IL, USA) diluted at 1:7,500 or peroxidase-linked ECL anti-rabbit IgG (cat. no. NA9340; GE Healthcare, Buckinghamshire, UK) diluted at 1:2,000. The chimeric protein A/G conjugate was used as a probe replacing the secondary antibody in the WB and ELISA assays because of its non-species-specific binding with the Fc region of antibodies, which commends its use in detection of antibodies from a wide variety of wild animals, including marine mammal species (28). The bound antibodies were detected using a Pierce ECL Plus Western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA) and visualised using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA).

For establishment of the ELISA, several experimental conditions were compared to find the optimal components and parameters (Table 2). The conditions showing the largest difference in optical density (OD) values between Env-GFP- and GFP-coated wells incubated with rabbit anti-envelope serum or PL1-2010 plasma and the lowest OD values in both types of cell lysate-coated wells incubated with rabbit pre-serum were chosen. The assay as finally developed is as follows: PhoMT cells in 100 mm dishes were transfected, collected at 48 hpt using 500 μL of RIPA buffer containing protease inhibitor cocktail set I (cat. no. 165-26021; Wako), incubated on ice for 30 min, sonicated, and then incubated on ice again for 15 min. Cells were centrifuged at 13,000 x g for 5 min at 4°C, the supernatant was removed, 100 μL of the denaturing lysis buffer (1) was added to the cell pellet, and the buffer and pellet were boiled for 15 min with periodical vortex mixing. Protein concentrations were measured by a Lowry assay (21) using the DC Protein Assay (Bio-Rad). Each well pair of a 96-well plate (U96 MAXISORP, NUNC-Immuno plate, Thermo Fisher Scientific, Roskilde, Denmark) was coated in parallel with cell lysates diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6): one of the wells was coated with Env-GFP cell lysates and the other with GFP cell lysates. The plates were incubated for 2 hours (h) at 37°C and then overnight at 4°C. They were then washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-T), blocked with 200 μL/well of PBS containing 1% Block ACE (UK-B40, DS Pharma Biomedica, Sapporo, Japan) for 1 h at 37°C, washed, incubated with serum/plasma samples for 1 h at 37°C, washed again, incubated with HRP-conjugated protein A/G for 1 h at 37°C, and washed a final time. The reaction was colourised using 100 μL/well of KPL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) peroxidase substrate system (cat. no. 5120-0032; SeraCare Life Sciences, Milford, MA, USA) and incubated for 30 min at 37°C. Finally, the reaction was stopped by adding 100 μL/well of 5% SDS, and the OD values were measured at 405 nm using a Multiscan FC plate reader (Thermo Fisher Scientific, Shanghai, China). The adjusted OD value of each sample was calculated by subtracting the OD value of GFP-coated wells from that of the corresponding Env-GFP-coated wells. Rabbit anti-envelope serum and preserum were included as positive and negative control reactions, respectively. Additionally, the PL1-2010 plasma was included as another positive control.

Cloning was confirmed by the PCR assay using DNA from transfected cells (Fig. 2A) and sequencing (data not shown). The two proteins in expression were confirmed by the detection of green fluorescence in transfected cells (Fig. 2B, upper panels). Expression of Env-GFP was in the cellular cytoplasm and was concentrated in localised sites, especially near the nucleus, whereas expression of GFP was in the whole cell (Fig. 2B, lower panels). The Env-GFP fusion protein (632 amino acids, with an expected size of 70.2 kDa) was detected in the Env-GFP-transfected cell lysates through WB using the rabbit anti-envelope serum, the anti-GFP antibody, or the PL1-2010 plasma (Fig. 2C). Additionally, GFP (239 amino acids, with an expected size of 26.9 kDa) was detected in GFP-transfected cell lysates using anti-GFP antibody (Fig. 2C). These results confirmed the expression of GFP and Env-GFP in the respective transfected cells.

Fig. 2

In the ELISA plates, the Env-GFP-coated wells incubated with the rabbit anti-envelope serum showed a colour reaction (Fig. 3A), whereas the GFP- and mock-coated wells did not, with higher OD values being observed in the Env-GFP-coated wells than in the GFP-coated wells (Fig. 3B). By comparing different concentrations of the cell lysates, it was found that 25 μg/well was the optimal concentration (Fig. 3A and B). These results indicated the specificity of the prepared rabbit sera. Different combinations of cell lysate concentrations and dilutions of PL1-2010 plasma were compared (Fig. 3C). As observed using rabbit anti-envelope serum, 25 μg/well cell lysate concentration using 1:50 PL1-2010 plasma dilution showed the greatest difference between the Env-GFP-and GFP-coated wells (Fig. 3C). However, the difference using PL1-2010 plasma (Fig. 3C) was much lower than that observed using rabbit anti-envelope serum (Fig. 3B). To examine the hypothesis that plasma might increase non-specific reactions compared to serum, the OD values obtained using serum and plasma samples from the same seals were compared. The OD values in both types of samples showed non-remarkable differences (data not shown), indicating that using plasma samples for ELISA is straightforward.

Fig. 3

In repeated experiments (four independent iterations) using 25 μg/well of cell lysates and 1:50 dilution of the PL1-2010 plasma, the adjusted OD values were in the range of 0.1 ≤ OD < 0.15. The lower limit, i.e. 0.1, was used as the cut-off value. Therefore, the cut-off value of the assay was set to an adjusted OD ≥ 0.1 (an adjusted OD ≥ 0.1 indicated that the animal is considered positive). The lower limit (low conservative cut-off value) was used due to the fact that this would eventually increase the sensitivity of the assay.

This established ELISA was used to examine serum samples collected from marine mammals inhabiting an aquarium. The OD values for Env-GFP-coated wells were the same as or very similar to those of the GFP-coated wells for the examined samples. None of the examined serum samples passed the pre-determined cut-off value, and these samples were therefore judged to be negative sera. Each serum sample was assayed at least twice.

To confirm the ELISA results, all seal serum samples were examined again using the WB assay, as were selected serum samples from cetacean species. The positive controls, i.e. rabbit anti-envelope serum and PL1-2010 plasma, showed bands as expected using Env-GFP cell lysates but not when using GFP cell lysates (Fig. 4A and B). These bands were not detected with the preserum (Fig. 4A and B). The examined seals’ sera (collected either in 2019 or 2020) did not show evidence of positive reactions (Fig. 4A), and the examined cetaceans’ sera were also negative (Fig. 4B). These results confirmed those obtained using the ELISA established with this research.

Fig. 4