Specific-pathogen-free male BALB/c mice (age, 9 weeks; weight, 25.74±0.25 g) were purchased from the Experimental Animal Center of Lanzhou University (Lanzhou, China). Upon arrival, mice were housed in a biosafety level 3 room under the following conditions: Controlled temperature, 23±5°C; humidity 45–55%; 12-h light/dark cycle. The mice were given free access to commercial mouse chow and water. After a 1-week acclimation period, mice were randomly assigned into three groups (n=5 mice/group). All mice were handled in accordance with the Good Animal Practice Requirements of the Animal Ethics Procedures and Guidelines of the People's Republic of China (29). The present study was reviewed and approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science (Lanzhou, China; permit no. LVRIAEC2016-005).

The African green monkey kidney cell line Vero, the murine fibroblast cell line NIH3T3, and the BALB/c mouse-derived embryonic fibroblast cell line BALB/3T3 clone A31 were obtained from China Center for Type Culture Collection (Wuhan, China). All cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in the presence of 5% CO₂. The wild-type strain of ECTV was originally isolated from a naturally infected laboratory mouse and propagated in Vero cells (30). The virus was confirmed by PCR using specific primers (forward, 5′-ATGGACGGAACTCTTTTC-3′; reverse, 5′-AACTTCATCGTTGCGTTTAC-3′). The PCR was performed in a total volume of 25 µl containing 2 µl viral genomic DNA, 2 µl of each forward and reverse primers (10 µM), 4 µl PrimeSTAR Buffer (5X, including Mg²⁺; Takara Biotechnology Co., Ltd., Dalian, China), 1.5 µl dNTP mixture, 0.2 µl PrimeSTAR HS DNA Polymerase (Takara Biotechnology Co., Ltd.) and 8.3 µl diethylpyrocarbonate water. PCR conditions were as follows: Denaturation at 95°C for 4 min, 35 cycles at 98°C for 10 sec, 60°C for 15 sec and 72°C for 2 min, with final extension at 72°C for 10 min. The PCR products were analyzed by 1.0% agarose gel electrophoresis with 10 µg/ml ethidium bromide (Sangon Biotech Co., Ltd., Shanghai, China), and the positive products underwent Sanger sequencing by Genescript Co., Ltd. (Nanjing, China). Experimental infection of susceptible BALB/c mice exhibited less virulence than the ECTV-Moscow strain (30). Plaque-purified ECTV was serially passaged and viral titer was measured using a 50% tissue culture infective dose (TCID₅₀) assay.

To examine the host transcriptome profiles of cells following ECTV infection, 5×10⁵ BALB/3T3 cells were seeded in each well of a 6-well plate and were infected in triplicate with ECTV at a multiplicity of infection (MOI) of 1. Control cells were mock infected with virus-free DMEM. The infected and control cells were incubated in complete DMEM at 37°C in an atmosphere containing 5% CO₂ and were harvested for total RNA extraction at 6, 12, 24, 48 and 96 h post-infection (hpi). For cell harvesting, cells were washed three times with cold PBS and lysed with 1 ml TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

In view of the high transfection efficiency in NIH3T3 cells, NIH3T3 cells were chosen to carry out in vitro tests. Briefly, 7×10⁵ NIH3T3 cells were cultured in a 6-well plate and were infected in triplicate with ECTV (MOI, 1) for 2 h. Subsequently, the media were replaced with fresh media containing 2% FBS and cells were harvested for total RNA extraction at 2, 4, 8, 12, 24 and 48 hpi to detect the transcription levels of Hspa1b by qPCR.

With regards to the infection of BALB/c mice, 10 mice were individually anesthetized and infected subcutaneously in the abdomen with a single dose of 10⁴ TCID₅₀/ml ECTV (in 100 µl PBS). Furthermore, 5 uninfected mice served as a control group, which were euthanized by cervical dislocation, after which spleen tissues were isolated. On days 3 and 10 post-infection, 5 mice in 3 and 10 dpi group were euthanized by cervical dislocation and whole spleen tissues were harvested, respectively. All of the spleen tissues from each group were pooled in a cell culture plate and cut into sections (0.2 cm) using surgical scissors. A total of 2.0 g pooled spleen tissues used for RNA extraction with TRIzol were separately stored in four tubes and maintained at -70°C following freezing in liquid nitrogen.

Spleen tissue samples and BALB/3T3 cells were sent to Beijing CapitalBio Technology Co., Ltd. (Beijing, China) for microarray analysis. Briefly, total RNA was extracted from the samples, and the quality and quantity of RNA were assessed by formaldehyde agarose gel electrophoresis and spectrophotometry. Gene expression analysis was performed using the Affymetrix GeneChip Mouse Genome Arrays (GeneChip^® Mouse Genome 430 2.0; Affymetrix; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Microarray data were analyzed using Bio Molecule Annotation System 3.0 software (Beijing CapitalBio Technology Co., Ltd.). Using the cutoff criterion of a fold-change ≥2 or ≤0.5, differentially expressed genes were screened and clustered. All data were deposited into the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/gds/) under the accession nos. GSE100644 (30) and GSE102850. The differential expression of 10 isoforms of the HSP70 family contained in the genechip were filtered out for hierarchical clustering using Cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm), and displayed by using Java TreeView (http://jtreeview.sourceforge.net/).

The murine Hspa1b gene (National Center for Biotechnology Information accession no. NM_010478) was synthesized from C57BL/6 mouse cDNA stored in our laboratory at −80°C (29), and was cloned into the pCMV-Tag2b vector (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) with a FLAG tag at the N-terminus. Subsequently, the plasmids were transfected into Escherichia coli (Takara Biotechnology Co., Ltd.). The nucleotide sequences of the plasmids were confirmed using DNA sequencing, and the plasmid was extracted from the confirmed clone of E. coli using Endo-Free Plasmid Maxi kit (Omega Bio-Tek, Inc., Norcross, GA, USA). For transfection of cells with the plasmid, FuGENE HD transfection reagent (Promega Corporation, Madison, WI, USA) was used according to the manufacturer's protocol. A total of 3.0 µg pCMV-HSPa1b or pCMV-Tag2b plasmid mixed with 8.5 µl FuGENE HD reagent was transfected into 7×10⁵ NIH3T3 cells cultured in a 6-well plate; the control cells were incubated with 8.5 µl FuGENE HD reagent only. After 24 h transfection at 37°C, the cells were collected for western blot analysis or were infected with ECTV (MOI, 1 or 5) for 24 h. Furthermore, NIH3T3 cells were transfected with various amounts (1.0, 2.0 and 3.0 µg) of pCMV-Hspa1b plasmid or empty vector followed by ECTV infection (MOI, 1) for 24 h to explore the effects of Hspa1b on ECTV replication.

Gene silencing of Hspa1b by small interfering (si)RNA was conducted in NIH3T3 cells. siRNA sequences (Shanghai GenePharma Co., Ltd., Shanghai, China) targeting Hspa1b were as follows: S1, 5′-GAUUACUGUCAAGGUUAUUTT-3′; S2, 5′-AUCUGCUUGUCCAUGUUAATT-3′; and negative control (NC) siRNA, 5′-UUCUUCGAACGUGUCACGUTT-3′. A total of 7×10⁴ cells were seeded into 12-well plates and were transfected with a final concentration of 100 nM Hspa1b siRNA or NC siRNA using the FuGENE HD transfection reagent. Subsequently, the cells were incubated at 37°C for 36 h in an atmosphere containing 5% CO₂ prior to determination of knockdown efficiency by qPCR or infection with ECTV (MOI, 1 or 5) for 24 h.

Total RNA was extracted from cellular or tissue samples using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and first-strand cDNA was synthesized using PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The mRNA expression levels of Hspa1b were detected using specific primers (sense, 5′-TGG TGC AGT CCG ACA TGA AG-3′ and antisense, 5′-AGGTCGGTGTGAACGGATTTG-3′). GAPDH (sense, 5′-TCTTCCAGCCCTCCTTCCT-3′ and antisense, 5′-TGTAGACCATGTAGTTGAGGTCA-3′) was used as the internal amplification control. qPCR was performed in a final volume of 20 µl containing 10 µl 2X SYBR-Green qPCR Master Mix (Takara Biotechnology Co., Ltd.), 0.8 µl each primer (10 µM), 7.2 µl nuclease-free water and 2 µl cDNA. PCR amplification was performed as described previously (30). Relative gene expression levels were calculated using the 2^−ΔΔCq method (31).

To determine viral titers at various time-points post-infection, cells were lysed by three cycles of freeze-thawing and the supernatant was clarified by centrifugation at 1,000 × g for 10 min. Viral titers were determined in Vero cells according to the TCID₅₀ assay (32). Briefly, 2×10⁴ Vero cells in 100 µl DMEM supplemented with 10% FBS were seeded in each well of a 96-well plate and incubated at 37°C in an atmosphere containing 5% CO₂. After 24 h of incubation, 25 µl serial 10-fold dilutions of viral samples were added to each well, with eight replicates per dilution. The plates were incubated for 7 days at 37°C and checked daily for characteristic cytopathic effects. TCID₅₀ end point titers were calculated according to the Reed and Muench method (32).

Genomic DNA was extracted from the NIH3T3 cell culture supernatants and whole-cell lysates using a viral RNA/DNA extraction kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. ECTV genomic DNA copy numbers were determined by qPCR using ECTV P4b gene specific primers: Forward, 5′-GTAGAACGACGCCAGAATAAGATA-3′ and reverse, 5′-AGAAGATATCAGACGATCCACAATC-3′. PCR amplification was performed as described previously (33). A standard curve was established from a cloned DNA fragment of the ECTV P4b gene (33). Quantification cycle values obtained by qPCR were plotted on the standard curve to calculate the viral DNA copy number.

Protein expression levels of Hspa1b were determined through the anti-FLAG tag by western blot analysis of total cell proteins. Cells were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Beijing, China) and the protein concentration of cell lysates was determined using the bicinchoninic acid (BCA) assay (QuantiPRO BCA Assay kit; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol. Equal amounts of total protein (20 µg) were resolved by electrophoresis on 10% bis-Tris polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 h in 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) and were then probed with the following primary antibodies: Anti-FLAG (F3165; 1:10,000; Sigma-Aldrich; Merck KGaA) and anti-β-actin (60008-1; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA). Membranes were incubated with the primary antibodies diluted in 5% (wt/vol) bovine serum albumin (Sangon Biotech Co., Ltd.) and 1X TSBT at 4°C overnight. Antibody signals were detected by enhanced chemiluminescence detection kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following incubation with secondary antibodies conjugated to horseradish peroxidase (A9044; 1:8,000; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min.

Quercetin (Sigma-Aldrich; Merck KGaA) and VER155008 (VER; Sigma-Aldrich; Merck KGaA) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) to produce a 10 mM stock solution, which was further diluted in DMEM supplemented with 2% FBS to the appropriate working concentrations. NIH3T3 or Vero cells were seeded in 12-well plates and incubated at 37°C until the cells reached 60–80% confluence. Subsequently, cells were infected with ECTV at an MOI of 1 for 2 h, after which the media were replaced with fresh media containing the appropriate concentrations (10, 50 and 100 µM in NIH3T3 cells; 10, 20 and 50 µM in Vero cells) of quercetin and VER. After 24 h incubation, cell culture supernatants and whole-cell lysates were harvested for virus titration. To investigate the effects of the two Hspa1b inhibitors on viral growth kinetics, NIH3T3 cells were infected with ECTV at an MOI of 1 for 2 h, after which the media were replaced with fresh media containing 50 µM quercetin, VER, DMSO or DMEM media (Mock). Subsequently, cell culture supernatants and whole-cell lysates were harvested at the indicated time points.

Cell viability was determined using the MTT Cell Proliferation and Cytotoxicity Assay kit (Beyotime Institute of Biotechnology). Briefly, NIH3T3 cells were incubated in a 96-well plate and the medium was replaced with fresh medium containing various concentrations of quercetin, VER or DMSO. After 24 h, cell culture supernatants were replaced with 50 µl MTT (2 µg/ml) and incubated for 4 h at 37°C. Subsequently, 100 µl formazan-dissolving solution (Beyotime Institute of Biotechnology) was added to each well and incubated at 37°C until the crystals dissolved. Finally, the absorbance of the cell solution was measured using a universal microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 570 nm wavelength.

Data are presented as the means ± standard deviation from at least three independent experiments and were analyzed using Student's t-test, or one-way analysis of variance followed by Duncan's multiple range test. Statistical analyses were conducted using SPSS software (SPSS 18.0 for Windows; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

ECTV has a restricted host range and infection of BALB/c mice with ECTV is associated with a high mortality rate. The natural route of infection is believed to be through abrasions in the skin, after which the virus uncontrollably replicates in the spleen and the liver (10). In the present study, infected mice began to exhibit disease symptoms starting at 7 days post-infection (dpi) and one mouse succumbed to the disease at 10 dpi (data not shown). To elucidate alterations in the host transcriptome profile caused by ECTV infection, microarray analysis was used to examine the spleen cells from ECTV-infected BALB/c mice and BALB/3T3 cells. Following normalization, the results demonstrated that Hspa1b was one of the most upregulated genes in spleen tissues of infected mice and BALB/3T3 cells (30). To examine whether other isoforms of the Hsp70 family were upregulated during ECTV infection, the differential expression of 10 isoforms of the HSP70 family contained in the genechip were analyzed. Compared with in the mock control groups, the heat maps demonstrated that the expression levels of numerous Hsp70 isoforms were induced in vivo (Fig. 1A) and in vitro (Fig. 1B), including Hspa1b, Hspa5, Hspa8 and Hspa9. To examine the level of induction, the ratio of Hsp70 isoform mRNA expression levels in the infected samples compared with the mock control samples was determined; the results indicated that Hspa1b was the most highly induced isoform in vivo and in vitro (Fig. 1C). To validate the microarray data, the transcription levels of Hspa1b were determined in the spleen tissues by RT-qPCR analysis. As shown in Fig. 1D, the results of RT-qPCR were consistent with the microarray data. Furthermore, the transcription levels of Hspa1b were detected in NIH3T3 cells at various time-points. As demonstrated in Fig. 1E, the expression levels of Hspa1b were upregulated post-infection; the highest expression was detected at 48 hpi.

To investigate the effects of Hspa1b on ECTV replication, Hspa1b was overexpressed in NIH3T3 cells using the pCMV-Hspa1b plasmid; the empty pCMV-Tag2b vector was used as a negative control. A total of 24 h post-transfection, cells were infected with ECTV (MOI, 1 or 5), and the protein expression levels of FLAG (Fig. 2A) and viral titers were determined. As shown in Fig. 2B, the viral yields from Hspa1b-transfected cells were significantly higher compared with the control cells. Cells overexpressing Hspa1b that were infected with ECTV at an MOI of 5 also exhibited a significantly higher viral yield (Fig. 2C). To further confirm the positive effects of Hspa1b on ECTV replication, NIH3T3 cells were transfected with various amounts of pCMV-Hspa1b plasmid, followed by ECTV infection (MOI, 1). As shown in Fig. 2D (lower panel), the protein expression levels of FLAG were increased with the amount of transfected pCMV-Hspa1b plasmid. The results demonstrated that Hspa1b enhanced ECTV replication in a dose-dependent manner (Fig. 2D, upper panel).

To further investigate the effects of Hspa1b on ECTV replication, Hspa1b expression in NIH3T3 cells was suppressed following transfection with siRNAs for 36 h; the effects of Hspa1b knockdown on viral yield were subsequently determined. NIH3T3 cells were transfected with siRNAs, which either targeted the Hspa1b sequence (S1 and S2) or a nonspecific sequence (NC), and knockdown efficiency was confirmed by RT-qPCR analysis. siRNAs that targeted Hspa1b significantly reduced Hspa1b mRNA expression compared with in cells transfected with NC siRNA; knockdown with S1 was more pronounced compared with in cells transfected with S2 (57% for S1 and 36% for S2; Fig. 3A). Silencing Hspa1b with two siRNAs significantly impaired ECTV replication at 24 hpi. A marked decrease in viral yield was detected in Hspa1b siRNA-transfected cells following infection with two doses of ECTV compared with in the control group (Fig. 3B and C).

To further examine the importance of Hspa1b in ECTV replication, the effects of two Hspa1b inhibitors, quercetin and VER, were determined on ECTV replication. Quercetin is one of the most ubiquitous flavonoids, and as a Hsp inhibitor, its function is not limited to Hsp70 (34,35). VER is a small-molecule inhibitor of Hsp70, which can specifically bind to the ATPase domain of Hsp70 consequently inhibiting its function (36). In order to assess the effects of quercetin and VER on ECTV replication, the present study examined their cytotoxic effects on NIH3T3 and Vero cells by MTT assay. NIH3T3 or Vero cells were incubated with various concentrations of quercetin and VER. As shown in Fig. 4A, the viability of NIH3T3 cells was not significantly affected by treatment with ≤100 µM quercetin and VER. In addition, no cytotoxicity was detected following treatment of Vero cells with ≤50 µM quercetin and VER, whereas higher concentrations of quercetin and VER exhibited cytotoxic effects in Vero cells (Fig. 4B). Therefore, the present study determined the ability of VER and quercetin at 10, 20 and 50 µM to interfere with viral replication in Vero, and VER and quercetin at 10, 50 and 100 µM in NIH3T3 cells. Treatment of ECTV-infected NIH3T3 and Vero cells with various doses of quercetin or VER for 22 h resulted in a reduction of viral replication in a dose-dependent manner (Fig. 4C and D). In addition, treatment with 100 µM quercetin in NIH3T3 cells led to a significant reduction (~70-fold) in viral replication compared with in the DMSO-treated control group. Under the same conditions, VER (100 µM) only inhibited viral replication by ~15-fold. A similar inhibition was observed in Vero cells (Fig. 4D).

To further assess the effects of the two Hspa1b inhibitors on viral growth kinetics, NIH3T3 cells were infected with ECTV at an MOI of 1 for 2 h, after which the media were replaced with fresh media containing 50 µM quercetin or VER. Infected cells and supernatants were harvested for virus titration at 6, 12, 24, 36, 48 and 72 hpi. Compared with in the DMSO-treated cells, the viral titer of quercetin- or VER-treated cells began to reduce at 12 hpi; quercetin and VER treatment reduced viral titers by ~2.3- and 1.7-fold, respectively. At 72 hpi, the effects were enlarged to ~200-fold in quercetin-treated cells and ~52-fold in VER-treated cells (Fig. 4E). These results further confirmed the importance of Hspa1b in ECTV replication and indicated that quercetin is a much more potent inhibitor than VER in NIH3T3 cells.

It has previously been reported that Hsp70 is required in the genome replication of numerous viruses (18,37–39). To investigate whether Hspa1b affects the genome replication of ECTV, the present study examined the viral genome copy number of ECTV following overexpression or knockdown of Hspa1b. The viral genome copy number was significantly increased when Hspa1b was overexpressed in NIH3T3 cells (Fig. 5A). Cells transfected with two Hspa1b siRNAs exhibited a marked decrease in viral genome copy number compared with in the NC control group (Fig. 5B). Consistent with the viral yields presented in Fig. 3B, the viral genome was more impaired in S1-transfected cells compared with in cells transfected with S2. Furthermore, the present study determined the effects of quercetin and VER on viral genome replication. A significant decrease in viral genome copy number was observed in a dose-dependent manner, with ~30 and 25% reductions in viral genome following treatment with 100 µM quercetin and VER, respectively (Fig. 5C). Taken together, these results suggested that cellular Hspa1b may promote ECTV DNA replication. However, due to underdeveloped technological skills and unavailable ECTV antibodies, the exact mechanisms underlying how Hspa1b affects ECTV replication require further study.