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Establishment of serological test to detect antibody against ferret coronavirus

MATERIALS AND METHODS

Samples from domestic ferrets: From animal hospitals in Japan, 9 serum and 26 plasma samples

were collected from domestic ferrets between Aug 1st, 2012 and Feb 4th, 2014 and used for ELISA and immunoblot

analysis. We analyzed and reported the results for 79 of the feces samples in our previous study. One fecal sample from a ferret in our animal facility was used to amplify

the N gene of the FRCoV Yamaguchi-1 strain.

Amplification of N genes: RNA of the Yamaguchi-1 strain was extracted from feces using a

QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. N genes of the

Yamaguchi-1 strain was amplified by RT-PCR using TaKaRa RNA LA PCRTM Kit (AMV) Ver. 1.1 (TaKaRa,

Otsu, Japan). RT was performed using random 9-mer oligonucleotide primers, and PCR was performed using primer

pairs, NF2 (5′-TTA CAT ATG GTA TAA GAA CTA AAC-3′) and NR2

(5′-CGA TGT AGG AAC CTT CAA AAT A-3′). PCR products were

electrophoresed on a 0.8% gel and extracted using a QIAEX II Gel Extraction Kit (QIAGEN).

Construction of expression plasmids: Yamaguchi-1 strain fragments were amplified using primer

pairs, N1F (5′-TGG GAT CCA TGG CTG GAA ACG GAC CAC-3′) and N179R

(5′-GAC TCG AGT TAG TTA TTG GAT CTA TTG TTG GAC-3′) for nt 1–537

encoding a.a. 1–179, and N180F (5′-TGG GAT CCA TTA ACA GTA ACA GTG GTG ATA

T-3′) and N374R (5′-GAC TCG AGT TAG TTT AGT TCA TCA ATA ATT

TCA-3′) for nt 538–1125 encoding a.a. 180–374. These forward and reverse primers

contained BamHI and XhoI sites at the 5′-end, respectively. Fragments were

purified using a MinElute PCR purification Kit (QIAGEN) and digested with restriction enzymes,

BamHI and XhoI. Two fragments of the Yamaguchi-1 strain were electrophoresed

on a 0.8% gel and extracted using a QIAEX II Gel Extraction Kit (QIAGEN). Fragments were then cloned into

BamHI and XhoI sites of the expression plasmid pGEX-6P-1 vector (GE

Healthcare, Piscataway, NJ, U.S.A.) using a DNA Ligation Kit Ver. 2.1 (TaKaRa). Plasmids were transformed into

Escherichia (E.) coli strain DH5α (TOYOBO, Osaka, Japan).

Expression and purification of glutathione-S transferase (GST)-fusion proteins: Two N protein

fragments, N1-179 and N180-374, were expressed as fusion proteins with GST, GST-N (1-179) and GST-N (180-374),

respectively. E. coli containing recombinant or control plasmid was cultured in 2 × yeast

extract and tryptone (YT) medium (1.6% tryptone, 1% yeast extract and 0.5% NaCl, pH 7.0) containing 50

µg ampicillin ml−1. Expression of recombinant proteins was

induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (Wako, Osaka, Japan) for 4 hr. The

bacterial cells were suspended in sonication buffer (50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA and 1 mM

dithiothreitol) and lysed using a Multi-beads shocker (YASUI KIKAI, Osaka, Japan). After centrifugation,

supernatants were mixed with Triton X-100 at a final concentration of 1% for 30 min and then centrifuged at

20,630 × g at 4°C for 30 min. The supernatants were collected, mixed with glutathione sepharose 4B beads (GE

Healthcare) and incubated at 4°C for 30 min. After centrifugation, beads were washed four times with

phosphate-buffered saline (PBS) containing 0.5% Triton X-100 and once with sonication buffer. The beads were

mixed with 300 µl of 10 mM glutathione and incubated at 4°C for 1 hr. After incubation,

supernatants were harvested as purified recombinant proteins and used for ELISA and immunoblot analysis. The

purified proteins were confirmed to be single bands by coomassie-brilliant blue (CBB) staining after sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

SDS-PAGE analysis of recombinant proteins: Purified recombinant proteins were mixed in equal

volumes of 2 × sample buffer (125 mM Tris–HCl, pH 6.8, 40% glycerol, 4% SDS, 0.002% bromophenol blue and 10%

2-mercaptoethanol) and boiled for 3 min. Samples were electrophoresed by SDS-PAGE and stained with CBB.

Quantification of recombinant proteins: Concentration of purified proteins was measured using

Bio-Rad Protein Assay Dye Reagent Concentrate (BIO-RAD, Hercules, CA, U.S.A.) according to the manufacturer’s

instructions. A standard curve was constructed using bovine serum albumin (Sigma-Aldrich, St. Louis, MO,

U.S.A.). The absorbance was measured using a spectrophotometer (BIO-RAD) at 595 nm.

ELISA: The concentration of purified recombinant proteins was adjusted to 5

µg ml−1 with adsorption buffer (0.05 M carbonate-bicarbonate

buffer, pH 9.6). GST was used as a control at 5µg ml−1. One hundred

microliters of purified recombinant proteins and GST were added to 96-well microplates (Maxisorp; Nunc,

Roskilde, Denmark). After incubation at 37°C for 2 hr, plates were placed at 4°C overnight. The wells were

washed three times with PBS containing 0.05% Tween 20 (PBS-T) and then incubated with 200 µl of

1% Block Ace (Dainippon Pharmaceutical, Osaka, Japan) in PBS at 37°C for 30 min. After washing three times with

PBS-T, 100 µl of diluted sera or plasma were added to duplicate wells and incubated at 37°C for

30 min. Sera or plasma was diluted to 1:100 or 1:500 with PBS-T containing 0.4% Block Ace. Subsequently, wells

were washed three times with PBS-T before 100 µl of peroxidase-conjugated anti-ferret

immunoglobulin (ROCKLAND, Limerick, PA, U.S.A.) diluted with PBS-T containing 0.4% Block Ace was added and

incubated at 37°C for 30 min. Following three washes with PBS-T, 100 µl of Horseradish

Peroxidase Substrate (BIO-RAD) was added to each well. After incubation at room temperature for 30 min, the

enzymatic reaction was stopped by adding 100 µl of 2% oxalic acid to each well. The absorbance

was measured using a spectrophotometer (BIO-RAD) at 415 nm. All results were subtracted from the value for GST,

and the cut-off value was arbitrarily set at 0.5.

Immunoblot analysis: Recombinant proteins mixed with 2 × sample buffer were

electrophoretically separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes

(Millipore, Bedford, MA, U.S.A.). After transferring, the membranes were incubated with Tris-buffered saline

(TBS) (20 mM Tris-HCl and 150 mM NaCl, pH 7.5) containing 3% gelatin (BIO-RAD) at 37°C for 45 min. After washing

three times with TBS containing 0.05% Tween 20 (T-TBS), membranes were incubated with 2 ml of

ferret serum or plasma diluted to 1:1,000 in T-TBS containing 1% gelatin (BIO-RAD) at 37°C for 45 min. After

three washes with T-TBS, membranes were incubated with 2 ml of peroxidase-conjugated

anti-ferret immunoglobulins with T-TBS containing 1% gelatin at 37°C for 45 min. The membranes were washed three

times with T-TBS and then three times with TBS. The reaction was visualized using 3,3′-diaminobenzidine

tetrahydrochloride (Wako).

Sequence analysis: Nucleotide sequences were determined using a BigDye Terminator Ver. 3.1

Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, U.S.A.) according to the manufacturer’s instructions.

The deduced amino acid sequences of the N protein were compared with FRECV strain MSU-2 (GenBank accession no.

GU338457), FRECV strain MSU-1 (DQ340562) and FRSCV strain MSU-1 (GU338456). The nucleotide sequences of N gene

of the Yamaguchi-1 strain were deposited into DDBJ (accession no. LC029423).

Phylogenetic analysis: A phylogenetic tree was constructed using the program MrBayes Ver.

3.2.2 for MrModeltest analysis with a WAG substitution matrix. We referred to the following sequences to construct the phylogenetic

tree of N protein sequences; FRECV strain MSU-2 (GU338457), FRECV strain MSU-1 (DQ340562), FRSCV strain MSU-1

(GU338456), mink CoV strain WD1127 (HM245925), mink CoV strain WD1133 (HM245926), CCoV type II strain fc1

(AB781790), FCoV type II strain M91-267 (AB781788), FCoV type I strain C3663 (AB535528), SARS-CoV strain

BJ182-12 (EU371564) and FRCoV strain Yamaguchi-1 (LC029423). The tree was represented graphically using FigTree

Ver. 1.4.2.

Statistical analysis: Significant differences were statistically analyzed using Chi-square and

Fisher’s exact probability tests. P values of <0.05 were considered to be statistically

significant.

RESULTS

Antigenic comparison of GST fused recombinant proteins, GST-N (1-179) and GST-N (180-374):

Nucleotide sequence of the Yamaguchi-1 strain N gene (1,125 bp) was determined, and the deduced amino acid

sequence of N protein (374 amino acids) was phylogenetically analyzed (Fig.

1). Two recombinant N proteins, GST-N (1-179) and GST-N (180-374), based on the Yamaguchi-1 strain were

expressed as GST fusion proteins in E. coli and used as ELISA antigens with 7 sera and 15

plasma samples from ferrets. Although most samples reacted to both recombinant proteins, the plasma of ferret

No.10 and serum of ferret No.22 only reacted to GST-N (1-179) and did not recognize GST-N (180-374) (Fig. 2). These results indicated that GST-N (1-179) was suitable for detection of antibodies to FRCoVs.

Therefore, we decided to use GST-N (1-179) in the subsequent investigation. In addition, a cut-off value was

arbitrarily set at OD=0.5.

Comparison of the antigenic differences between GST-N (1-179) and GST-N (180-374) by immunoblot

analysis: The plasma of No.10 and serum of ferret No.22 showed different reactivities from the other

samples in ELISA (Fig. 2). To confirm the different antigenicity,

immunoblot analysis was carried out using serum of ferret No.22. Plasma of ferret No.48 was used to compare with

serum of ferret No.22. The purified proteins were confirmed to be single bands by CBB staining after SDS-PAGE

analysis and used (Fig. 3A). Plasma of ferret No.48 and serum of ferret No.22 reacted with recombinant protein GST-N (1-179), but

only plasma of ferret No.48 also reacted with GST-N (180-374) (Fig. 3B and

3C). The results of the immunoblot analysis were consistent with those of the ELISA.

Seroprevalence of FRCoV infection in ferrets in Japan: ELISA using GST-N (1-179) was carried

out with 1:100 dilutions of nine sera and 26 plasma samples from domestic ferrets in 12 animal hospitals in five

prefectures in Japan. The results showed that 31 of the 35 (89%) ferrets were seropositive for FRCoV infection.

There was no significant difference between seropositivity and age or sex (Table 1).

DISCUSSION

In this study, we attempted to clarify the seroprevalence of FRCoV in Japan and developed an ELISA using two

Yamaguchi-1 strain recombinant N proteins, GST-N (1-179) and GST-N (180-374). More ferret serum samples

recognized GST-N (1-179) than GST-N (180-374) (Fig. 2). In addition,

identities of N (1-179) between Yamaguchi-1 and the other FRCoVs (96.6–98.3%) were higher than those of N

(180-374) (90.7–93.8%) (data not shown). Therefore, we selected GST-N (1-179) as the ELISA antigen for our

serosurvey. Surprisingly, we found that 89% (31/35) of domestic ferrets were seropositive to this antigen by

ELISA (Table 1). There are reports of FRCoV gene detection in

56%-61% of ferrets in Japan and the Netherlands [6, 9]. These data indicate that FRCoV has already spread within the ferret population and that

many ferrets may be persistently infected with FRCoV. However, there was no significant difference between

seropositivity and symptoms, age or sex. Further studies are required to clarify the pathogenesis of FRCoV in

ferrets.

Plasma from ferret No.10 and serum from ferret No.22 showed different reactivities from those of other ferret

samples in ELISA, reacting only with GST-N (1-179), but not with GST-N (180-374) (Fig. 2). The different reactivity of ferret No. 22 serum was also confirmed by immunoblot

analysis using GST-N (180-374) (Fig. 3C). These results indicated that

GST-N (1-179) is a better choice of antigen for ELISA than GST-N (180-374). ELISA using GST-N (1-179) will be

useful for serological surveys for FRCoV. In future studies, this FRCoV infected with ferret No.22 should be

analyzed closely.

In conclusion, a new ELISA system using the recombinant N protein of FRCoV, GST-N (1-179), was established.

This ELISA will be useful for diagnosis and epidemiological studies on FRCoV infection in ferrets.