DF-1 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% inactivated fetal calf serum (FCS) in 5% CO₂ at 37℃. TMUV JS804 was isolated by our laboratory and propagated in DF-1 cells as previously described [11]. All the experiments were approved by the ethical committee of Jiangsu Academy of Agricultural Sciences (approval No. SYXK-2015-0020). The titer of virus is calculated as TCID₅₀ (50% tissue culture infective dose) according to the Reed-Muench method [26]. In brief, Serial 10-fold dilutions of the stock virus were inoculated into a monolayer of DF-1. After two-hour incubation at 37℃, inoculum was removed and cells were overlaid with a mixture of 1% agarose and cell culture media. Plaque formation was continuously observed daily for four days and TCID₅₀ was calculated according to the method of Reed-Muench. TMUV-monoclonal antibody was generated and maintained by our laboratory as previously described [8]. Alkaline phosphatase and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG were purchased from the Beyotime Institute of Biotechnology (China). Anti-HSPA9 (ab171089, mouse) was purchased from Abcam (UK).

The DI (1–50 aa, 132–197 aa, 282–298 aa), DII (51–131 aa, 198–281 aa), and HSPA9 gene were synthesized by Nanjing Genscript Biotechnology (China). A synthesized gene fragment of DI or DII was subcloned into EcoR I and Sal I sites of bacterial expression vector pGEX-4t-1 (pGEX-DI or pGEX-DII). The HSPA9 gene was ligated into pET32a vector between Bam HI and Xho I sites (32a-A9). DIII was amplified by polymerase chain reaction (PCR). Primers used for cloning were: forward primer, EDIIIF, 5′-GAATTCATGGACTACAAAGACGACGACGACAAAAAGCTGAAAGGAATGACC-3′ and reverse primer, EDIIIR, 5′-GCGGCCGCTCATTTGTCGTCGTCGTCTTTGTAGTCTTGTGCTCCTTTGAGTGTT-3′. The EcoR I and Not I restriction sites are underlined and the FLAG tag (DYKDDDDK) sequences are italicized. The PCR product was ligated into pET32a vector between the EcoR I and Not I sites (32a-DIII).

Proteins were expressed in BL21 (DE3) transformed with pGEX-DI, pGEX-DII, 32a-DIII or 32a-A9. Transformed BL21 was grown in LB with shaking at 37℃. When the optical density at 600 nm (OD₆₀₀) of the bacteria culture reached 0.4 to 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM for an additional 5 h. After induction by IPTG, cells were harvested by centrifugation. The recombinant proteins were purified by glutathione-agarose (Sigma, USA), anti-FLAG M2 Affinity Gel (Sigma) or High-Affinity Ni-NTA Resin (Novagen, USA) according to the manufacturer's instructions. After purification, the proteins were detected by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting. The protein concentration was detected by using a BCA kit according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Proteins were then quickly frozen and stored at −80℃.

The HSPA9-binding domains in E protein were determined by Western blotting. The purified recombinant HSPA9 protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane. The membrane containing the transferred proteins was blocked with 5% bovine serum albumin (BSA) in PBST (phosphate buffered saline with Tween 20; NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 4.3 mM, KH₂PO₄ 1.4 mM, pH to 7.4, with 0.05% Tween 20) at 37℃ for 2 h and then incubated with purified recombinant DI, DII, or DIII protein overnight at 4℃. After being washed three times with PBST, TMUV-positive serum was added to the membrane and incubated at 37℃ for 1 h. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG. Finally, the signal was developed using BeyoECL Plus Kit (Beyotime Institute of Biotechnology) in accordance with the manufacturer's protocol.

To confirm which domain in E protein participated in HSPA9 binding, a co-immunoprecipitation assay was carried out as previously described [16]. Briefly, the purified recombinant DI, DII, or DIII protein was incubated with purified recombinant HSPA9 protein on a rocker at 4℃ overnight followed by incubation with anti-HSPA9 antibody for 5 h. Protein A/G agarose beads were added to each reaction tube and incubated on a rotator at 4℃ for 3 h. After centrifugation, the beads were collected and washed by PBS for five times. Subsequently, beads-bound immune complexes were resuspended in 4× SDS-PAGE loading buffer, boiled for 5 min, and analyzed by SDS-PAGE.

A library of 16-mer peptides, overlapping by four amino acids, was derived from DI and DII. The sequences and positions of these peptides are shown in Table 1. All peptides were synthesized and purified by Nanjing Genscript Biotechnology. The peptide purities were > 95%.

The HSPA9-binding peptides were detected by indirect ELISA as previously described [8]. The 96-well ELISA plate was coated with purified recombinant HSPA9 (4 µg per well) at 4℃ overnight and blocked with 1% BSA in PBST at 37℃ for 2 h. The plate was incubated with different overlapping peptides in triplicate for 1 h at 37℃. After being washed three times with PBST, the plate was incubated with TMUV-positive serum followed by incubation with HRP-conjugated goat anti-mouse IgG. Then, the plate was washed three times and subsequently incubated with TMB substrate. The reaction was stopped with 2 M H₂SO₄, and the OD_{450 nm} value of each well was determined by using a BioTek (USA) microplate reader.

A series of truncated peptides derived from peptides 2 and 21 was designed and synthesized by Nanjing Genscript Biotechnology. The purities of the peptides were > 95%. The sequences of these peptides are shown in Table 2. Peptides A to H were derived from peptide 2 and peptides I to P were derived from peptide 21.

Two microliters of 5 mg/mL truncated peptides were spotted on a nitrocellulose membrane. The membrane was blocked by 5% BSA at 37℃ for 2 h. Purified recombinant HSPA9 protein was added to the membrane and incubated at 4℃ overnight. Unbound HSPA9 protein was removed by washing three times with PBST. The membrane was then incubated with anti-HSPA9 antibody followed by incubation with alkaline phosphatase conjugated goat anti-mouse IgG. The signal was detected using Alkaline Phosphatase Assay Kit (Beyotime Institute of Biotechnology) in accordance with the manufacturer's protocol.

Peptide cytotoxicity was measured by using Cell Counting Kit-8 (CCK-8; Dojindo, China) according to the manufacturer's protocol. Briefly, 100 µg/mL of peptides were added to DF-1 cells in a 96-well plate and incubated for 1 h at 4℃. After being washed three times by PBS, DMEM with 10% FCS (100 µL per well) was added. Simultaneously, 10 µL CCK-8 was added per well and incubated with the cells at 37℃ for 2 h. The OD_{450 nm} value at was measured by using a BioTek microplate reader.

DF-1 cells were incubated with 50 µg/mL of peptides or BSA (as control) at 4℃ for 1 h. Unbound peptides were removed by washing the cells twice with chilled PBS. The cells were then infected with 200 TCID₅₀ TMUV for 30 min at 4℃ followed by 1 h at 37℃. Unbound virus was removed by washing twice with PBS. Virus RNA was extracted by using a QIAamp Viral RNA Mini Kit (QIAGEN, Germany), and TMUV RNA levels were determined by quantitative reverse transcriptase (qRT)-PCR as described previously [39]. The primers used for real-time RT-PCR were: EF primer (forward, 5′-GTGAGATCTTACTGCTATGAG-3′) and the ER primer (reverse, 5′-ACTTGGCACATGTCTGTATGC-3′). Real-time RT-PCR data were analyzed using the comparative cycle threshold (CT) method (ΔΔCT). Beta-actin was chosen as a reference gene for internal control. Differences between the CT values of the target gene and the internal control (ΔCT = CT_target − CT_{internal control}) were calculated to normalize the differences in the amount of total complementary DNA added to each reaction and the efficiency of the real-time RT-PCR. The negative control was used as a reference for each comparison. Differences between the ΔCT of each sample and the reference sample (ΔΔCT = [CT_target − CT_{internal control}] sample − [CT_target − CT_{internal control}] negative control) were then calculated. Three independent experiments were carried out.

There were at least three replicates for each assay. Error bars drawn represent SD from the mean. Values of p < 0.05 were considered to be significant. The software used was IBM SPSS Statistics (ver. 19.0; IBM, USA).

To test which E protein domain participates in the HSPA9 binding process, we performed Western blotting using recombinant HSPA9. Purified recombinant DI, DII, or DIII protein was incubated with the membrane containing HSPA9. Domains bound to HSPA9 were detected by the TMUV-positive serum. As shown in panel A in Fig. 1, a distinct band with an approximate 90 kDa molecular mass was observed in the DI and DII incubated membranes, while no band was visualized in the DIII incubated membrane, suggesting that DI and DII were able to bind to HSPA9. In order to validate the binding between DI or DII and HSPA9, co-immunoprecipitation assays were carried out. As shown in panel B in Fig. 1, DI or DII was co-immunoprecipitated with HSPA9 by the anti-HSPA9 antibody. Consistent with the Western blotting results, the band of DIII was not observed in the co-immunoprecipitated mixture containing DIII and HSPA9, confirming that only DI and DII were involved in TMUV–HSPA9 binding.

The 16-mer overlapping peptides derived from DI and DII were synthesized and analyzed for their interaction with HSPA9 by using indirect ELISA. Fig. 2 showed that two (i.e., peptides 2 and 21) of the overlapping peptides interacted with HSPA9 and exhibited the highest binding indices. Other peptides did not show the capacity to bind to HSPA9. Removing the overlapping sequence, the core sequences of peptides 2 and 21, which were involved in HSPA9 binding, were GVEWIDVV and AHATKQSV, respectively.

Truncated peptides were synthesized according to core sequence of peptides 2 (peptides A to H) and 21 (peptides I to P). Dot blots were used to test whether truncated peptides could bind to HSPA9. The results demonstrated that peptides F and G derived from peptide 2 exhibited positive reactivity to HSPA9 (panel A in Fig. 3). The sequence shared by peptides F and G was EWID (E residues 19 to 22), which is located within loop B0 of E protein. However, the truncated protein derived from peptide 21 showed negative reactivity to HSPA9, suggesting that reduction of residues of AHATKQSV can lead to failure in HSPA9 binding. In order to validate further the minimal HSPA9-binding motif, peptides EWID and AHATKQSV were synthesized and analyzed. The Dot blot results showed that EWID and AHATKQSV bind to HSAP9 (panel B in Fig. 3). Therefore, it is concluded that EWID and AHATKQSV are required for TMUV–HSPA9 binding.

It is possible that peptides can induce cellular toxicity, which can block TMUV binding. To address this possibility, DF-1 cells were exposed to peptides at a 100 µg/mL concentration. Cell viability was determined by using CCK-8. There was no statistical difference in viable cell numbers between control cells and cells exposed to peptides (Fig. 4), indicating that the peptides are non-toxic to DF-1 cells.

To test whether peptides binding to HSPA9 can compete with endogenous E in mediating virus binding, we performed virus binding assays and determined the viral RNA level by using qRT-PCR. As shown in Fig. 5, the viral RNA level was significantly lower in peptide 2-, 21-, F-, G-, EWID-, and AHATKQSV-treated DF-1 cells compared with that in BSA-treated cells, indicating that virus binding to DF-1 cells was competitively inhibited by peptides 2, 21, F, G, EWID and AHATKQSV.