Development of porcine rotavirus vp6 protein based ELISA for differentiation of this virus and other viruses

Background The context and purpose of the study included 1) bacterial expression of viral protein 6 (VP6) of porcine rotavirus (PRV) and generation of rabbit polyclonal antiserum to the VP6 protein; 3) establishment of a discrimination ELISA to distinguish PRV from a panel of other porcine viruses. Results The VP6 gene of PRV isolate DN30209 amplified by reverse transcription-PCR was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. Sequence comparison and phylogenetic analysis indicated that PRV DN30209 may belong to group A of rotavirus. Bacterially expressed VP6 was expressed in E.coli and anti-VP6 antibody was capable of distinguishing PRV from Porcine transmissible gastroenteritis virus, Porcine epidemic diarrhea virus, Porcine circovirus type II, Porcine reproductive and respiratory syndrome virus, Porcine pseudorabies virus and Porcine parvovirus. Conclusions PRV VP6 expressed in E. coli can be used to generate antibodies in rabbit; anti-VP6 serum antibody can be used as good diagnostic reagents for detection of PRV.


Background
Rotavirus, a member of family Reoviridae, is one of the causative agents of viral diarrhea in young children and animals worldwide [1][2][3]. Rotavirus is approximately 75 nm in diameter and has multilayered icosahedral protein capsid composed of an outer layer, an inner layer, and a core [4]. The virus genome is comprised of 11 segments of double-stranded RNA, encoding six structural proteins (VP1-VP4, VP6, VP7) and six nonstructural proteins (NSP) [5,6]. The outer capsid layer proteins of rotavirus, VP4 and VP7, may elicit neutralizing antibodies independently; in addition, the two proteins can be used to classify rotaviruses into P and G genotypes, respectively [7][8][9]. VP1, VP2 and VP3 proteins consist of the rotavirus core particles. VP1 was partially accessible to iodination in single-shelled particles [10]. The accession degree of VP1 to the immune system may affect the reaction of anti-PRV to VP1 protein [4]. VP2 is the most abundant structural protein in core particles [11]. Anti-VP2 serum is a good indicator of prior infection due to its high immunogenicity [12]. VP3 is a minor structural protein that may comigrate with the outer capsid protein VP4 in many gel systems and it may be involved in RNA replication; VP4 is cleaved to form VP5 and VP8 [4,11].
VP6 is the major structural protein in virus particles located on the outer surface of single-shelled particles, in addition, VP6 is known as a subgroup antigen [4]. VP6 is required not only for polymerase activity and but also for maintaining the proper structure of the viral core or the core protein-based transcriptional complex. Myristylation of VP6 is required for virus particle formation as well as targeting single-shelled particles to the ER membrane for budding [13]. As VP6 is both highly immunogenic and antigenic, it is the most frequently used in diagnostic assays to detect PRV [14]. Herein, we expressed the VP6 gene from a PRV Chinese isolate DN30209 by cloning this gene into a bacterial expression system. We then generated rabbit polyclonal anti-serum and ascertained its immunoreactivity to VP6 protein.
Using the anti-VP6 antibody, we established a differentiating ELISA for PRV. These experiments provided basis for determination of PRV.

Results and discussion
Cloning and phylogeny of VP6 gene of PRV PRV may cause piglet diarrhea andV P6 is an important structural component of viral particles. In this study, we cloned the VP6 gene from a Chinese PRV isolate DN30209. The sequencing results showed that the cloned VP6 gene was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. The sequence has been submitted to GenBank and was allocated a GenBank accession number (JN977137). Compared with PRV JL94, there were three mutations at N 94 H, G 126 R and I 182 V; compared with PRV GD, three mutations N 94 H, I 182 V and T 244 A were identified. As DN30209, JL94 and GD all were isolated from northern China, these viruses may be geographical prevalent in northern areas in China. Based on the randomly selected PRV reference strains, the VP6 amino acid sequences were compared. As shown in Figure 1, PRVs DN30209 and several PRV group A strains OSU, JL94 GD, CRW-8, 4F and 4S etc. had more than 90% homologous identity at amino acid level. RNA-PAGE analysis showed that the rotavirus represented long electropherotype migration pattern of porcine group A rotaviruses ( Figure 2). Further phylogenetic tree analysis also indicated that PRV DN30203 located in the same clade with above-mentioned PRV group A strains ( Figure 3). Therefore, we speculate that PRV DN30209 also belongs to group A of rotavirus. Nevertheless, it is necessary to do more serological and epidemiological investigations to get full understanding on the evolution of PRV in the future.

Analysis of VP6 expression and immunoreactivities of its antibody
It is clear that E. coli system has advantages such as low costs, high production, and manipulation convenience, etc. [15]. In our laboratory, we have expressed several heterologous proteins in this system [16][17][18][19]. In this study, we analyzed the bacterial expression of VP6 protein in E.coli. SDS-PAGE showed that fused VP6 protein was approx. 48 kDa as expected and accumulated in the bacteria as inclusion bodies ( Figure 4). In this study, a rabbit anti-VP6 antibody was generated by conventional animal immunization. The optimization of the ELISA was determined. As shown in Figure 5, after the fixation of the concentration of PRV VP6 protein, the optimal antibody dilution was1:2 9 . In addition to reaction with VP6 protein, at such dilution, the anti-VP6 serum also had the best reactivity to PRV. The optimization of the ELISA may be useful reference to detect PRV from clinical samples in the future. The reactivity of the protein to anti-VP6 antibody was then analyzed by Western blot. Our result showed that the bacterially expressed VP6 can be recognized by the antiserum, confirming that the VP6 is both highly immunogenic and antigenic ( Figure 6). The immunoreactivity of the anti-VP6 antibody to Figure 1 Homologous identity comparison of PRV VP6 gene. VP6 sequences of PRV isolates used in this study were subjected to multiple sequence alignments using the DNAStar software. Their grouping, name and origin are shown in Table 1.
eukaryotically expressed VP6 was further analyzed by IFA. As shown in Figure 7, the antibody reacted with VP6-expressing cells; in contrast, no green signal was detected in mock-transfected cells, confirming the specific recognition of the anti-VP6 antibody. In this study, the anti-VP6 antibody was generated by inoculating rabbit with VP6 expressed from E.coli, however, this antibody reacted with VP6 protein transiently expressed on cells. These data further confirm that bacterially expressed VP6 can be used as a good immunogen and the antibody can be used as good diagnostic reagent for detection of PRV VP6 protein.

Discrimination ELISA for detection of PRV
Like PRV, TGEV and PEDV are capable of causing diarrhea symptoms in pigs. Other porcine viruses may cause co-infection with PRV, thus, discrimination between PRV and other viruses is important for clinical diagnosis purpose. Recently, we have confirmed that the antiserum to VP7 protein of PRV may also serve as a diagnostic agent for detection of PRV (unpublished data). Nevertheless, it has been pointed out that PRV VP6 protein is the most frequently target protein in diagnostic assays to detect virus particles. As ELISA is simple, convenient and sensitive immunological assays suitable for detection of pathogens [19][20][21][22], a discrimination ELISA for detection of PRV was established using the anti-VP6 antibody. Several control viruses were included in the ELISA to determine the specificity of the ELISA. The result showed that the anti-VP6 antibody had significant reactivity with PRV; in contrast, there was no positive P/N value among other controls (p<0.01) (Figure 8). Our results have demonstrated clearly that the VP6-antibody based ELISA works well in our laboratory, however, validation of the test using clinical samples are important so that the test can be applied in the field level for detection of PRVs. Thus, we will evaluate the utility of this VP6-antibody based ELISA in nature by including PRV clinical samples in the future.

Methods
Amplification of VP6 gene of PRV by PCR PRV Chinese isolate named DN30209 was recently isolated in a pig farm in Heilongjiang Province, northeastern China. The virus was propagated in MA104 cells in presence of trypsin at a final concentration of 3 μg/mL (w/v). The viral total RNA was extracted with a commercial kit according to the manufacturer's instructions (HaiGene, China). Sense primer (VP6-1): 5'-GGCTTTTAAACGAAGTCTTC and antisense primer (VP6-2): 5'-GGTCACATCCTCTCACTA were published primers [23] and used to amplify the VP6 gene for sequencing. The reverse transcription mixture included 5 μL of RNA (1 μg), 1 μL of primer VP6-2 (10 pmol), 4 μL of 5×RT-PCR buffer (TaKaRa, China),0.5 μL of RNase inhibitor, 1 μL of dNTP (10 mM/each), 1 μL sucrose and 0.04% bromophenol blue) followed by phenol-chloroform extraction at 12, 000 rpm and 4°C for 10 min. The resulting supernatant was subjected to RNA-PAGE to detect and analyze the genome of rotavirus. The RNA-PAGE was performed in 10% polyacrylamide slab gel using discontinuous buffer system without SDS as described by Laemmli [24] for 13 h at 10 mA. Viral RNA segments were visualized in silver staining.

Sequence comparison and phylogenetic analysis
The obtained VP6 sequence was deposited in GenBank database and compared with reference PRV VP6 sequences  using DNASTAR software. The name, GenBank accession number and virus isolation location of these genes are shown in Table 1. At the same time, a phylogenetic tree was constructed by neighbor-joining method base on these VP6 amino acid sequences using the DNASTAR program [25][26][27]. The bootstrap probabilities of each node were calculated using 1,000 replicates.

Construction of recombinant plasmids bearing VP6 gene and its bacterial expression
To construct expression plasmids bearing the VP6 gene, forward primer: 5'-CCCCGGATCCATGCAAAATTACG GA(VP6F) and reverse primer (VP6R) 5'-GGGGAAGC TTGTTAGACTCGGTAATA were self designed and used to re-amplify the VP6 gene using the obtained PCR product above as template. Both primers contained BamH I and EcoR I sites, respectively. Subsequent PCR was performed as above using the full-length VP6 gene as template. The PCR profile included 95°C for 5 min; 30 cycles of 95°C for 30 s, 52.8°C for 30 s, and 72°C for 2 min as well as a final extension at 72°C for 10 min. The resulting PCR product was inserted into the prokaryotic expression vector pET30a and eukaryotic expression vector pVAX-1 to generate recombinant plasmids pET30a-VP6 and pVAX-VP6, respectively. The pET30a-VP6 was transformed into Escherichia coli (E. coli) Rosetta. The transformed E.coli was  cultured in Luria-Bertani (LB) medium supplemented with Kanamycin (50 mg/mL) with shaking at 37°C. When OD 600 reached 0.6, isopropyl beta-D-thiogalactoside (IPTG) was added to the medium to a final concentration of 1 mM to induce protein expression. Control culture containing the same bacteria transformed with empty vector was used as control. The expressing protein designated as Pro-VP6 was subjected to gel-purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and renaturation by dialysis according to our previously reported protocols [28].

Generation of polyclonal antibody to VP6
Generation of polyclonal antiserum to Pro-VP6 was processed according to references with modifications [17][18][19]28]. A New Zealand rabbit was immunized with 2 mL of gel-purified Pro-VP6 (1 mg/mL) emulsified with equal amounts of Freund's complete adjuvant via subcutaneous injection. After ten days, 2 mL of the same antigen mixed with Freund's incomplete adjuvant were injected into the rabbit weekly at two intervals. Antiserum was collected from the peripheral blood of the rabbit.
The titration of the antiserum and its reaction with VP6 protein was determined using ELISA: Purified VP6 was coated onto ELISA plates at a final concentration of 5 μg/well at 4°C overnight. The next day, the wells were incubated with blocking buffer at 37°C for 2 h, after three washes with PBS-0.1% Tween20 (PBST). The wells were incubated with serially diluted polyclonal antibodies at 37°C for 1 h. Then the plates were incubated with goat anti-rabbit IgG conjugated with peroxidase (1:5000 dilution in PBS) at 37°C for 1 h, after three washes with PBST. OPD (o-Phenylenediamine dihydrochloride) substrate (100 μL/well) was added and incubated for 15 min followed by stopping reaction with 2 M H 2 SO 4 . The OD 490 values were determined an ELISA reader. In addition, the reactivity of the anti-VP6 antibody to PRV was also tested. Briefly, the purified PRV (10 4 PFU/mL) were coated onto ELISA plates (100 μL/well) and the dilution of antiserum to Pro-VP6 or the control serum was 1:2 4 , 1:2 5 , 1:2 6 , 1:2 7 , 1:2 8 , 1:2 9 , 1:2 10 , 1:2 11 or 1:2 12 . The other experimental steps were performed as above.

Western blot
Pro-VP6-containing bacterial protein and empty vectortransforming bacterial protein (negative control) were subjected to SDS-PAGE and transferred to a nitrocellulose (NC) membrane. Rabbit antiserum to Pro-VP6 was diluted 1:2,000 in PBS and was used to incubate the membrane at room temperature for 1.5 h followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000 dilution, Boster, China) for 1.5 h. The protein bands were visualized using OPD.

Indirect immunofluorescence assay (IFA)
Baby hamster kidney (BHK) cells seeded onto 24-well plates were transfected with pVAX-VP6 using polyethylenimine (PEI) transfection agent (Sigma, China) according to the manufacturer's instructions. Empty vector-transfected cells were used as control. The cells were fixed with 4% PFA (polyformaldehyde) in PBS at The grouping, name and origin of the PRV isolates used in this study are provided. room temperature for 20 min and quenched with 0.1 M glycine in PBS. Then, the cells were washed three times with PBST and treated with or without 1% Triton X-100 for 10 min followed by incubation with anti-VP6 serum (1:200 dilution in PBS containing 0.1% BSA) for 1 h. After three washes with PBST, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (1:200 in PBS containing 1% BSA) for 1 h at 37°C. After washing with PBST, the fluorescence signals were analyzed using fluorescence microscopy.
Immunoreactivity of the anti-VP6 antibody to other control viruses  4], and 1 mmol/liter EDTA) followed by dilution in carbonate-bicarbonate buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6) and the pathogens were coated into ELISA plates (8 μg/well) at 4°C overnight. Wells coated with DMEM or lysis buffer were used as control. The anti-PRV antibody was used as primary antibody in the above-mentioned ELISA to detect the pathogens. The OD 490 value of tested samples (P)/the OD 490 value of negative control, coating buffer (N)>2 was judged as positive. The experiment was performed in triplicate. Statistical analysis of the data was processed with SPSS 11.5 software; p<0.05 and p<0.01 were defined as statistically significant and statistically very significant, respectively.