Open Access

Identification of in vitro and in vivo oncolytic effect in colorectal cancer cells by Orf virus strain NA1/11

  • Authors:
    • Daxiang Chen
    • Ruixue Wang
    • Mingjian Long
    • Wei Li
    • Bin Xiao
    • Hao Deng
    • Kongyan Weng
    • Daoyuan Gong
    • Fang Liu
    • Shuhong Luo
    • Wenbo Hao
  • View Affiliations

  • Published online on: December 8, 2020
  • Pages: 535-546
  • Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Orf virus (ORFV) is a favorable oncolytic viral carrier in research, and ORFV strain NZ2 has been revealed to have antitumor effects in animal models mediated by immunoregulation profile. However, the antitumor effects triggered by the ORFV in colorectal cancer (CRC) cells is poorly characterized. The in vivo and in vitro roles of ORFV in CRC were determined using western blotting, colony formation, CCK‑8, wound scratch assay, qPCR, and animal models. Furthermore, cytokine antibody chip assay, flow cytometry, western blotting, and immunohistochemical (IHC) assays were conducted to explore the potential mechanism of ORFV. The present data revealed that ORFV strain NA1/11 infected and inhibited the in vitro growth and migration of CRC cells. By establishing a CRC model in Balb/c mice, it was revealed that ORFV strain NA1/11 significantly inhibited the in vivo growth and migration of CRC cells. A cytokine antibody array was utilized to obtain a more comprehensive profile revealing the differentially expressed cytokines in ORFV infection. Cytokines, such as IL‑7, IL‑13, IL‑15, CD27, CD30, pentraxin 3, and B lymphocyte chemoattractant (BLC), were upregulated. Axl, CXCL16, ANG‑3, MMP10, IFN‑γ R1 and VEGF‑B were downregulated. The results indicated that ORFV played roles in the regulation of key factors relevant to apoptosis, autoimmunity/inflammation, angiogenesis, and the cell cycle. Finally, data was presented to validate that ORFV infection induces oncolytic activity by enhancing apoptosis in vivo and in vitro. In conclusion, ORFV could be an oncolytic virus for CRC therapy.


Oncolytic viruses, characterized by their preferential infection, replication and destruction of tumor cells and cancer-associated endothelial cells, have become a major topic for basic research and consequent clinical applications in novel tumor therapy strategies (1). Concomitant antitumor immune responses often exist as well (2). In recent decades, oncolytic viral therapy has aroused widespread interest and great progress has been achieved (3). Various oncolytic viruses have been studied and some have already been developed for treatments agents, such as adenovirus, herpes simplex virus type I, and the vaccinia virus (4,5). Imlygic (talimogene laherparepvec; T-VEC) is the first oncolytic virus approved for melanoma therapy by the USFDA for cases where lesions are in the skin and lymph nodes and cannot be completely removed by surgery (6). The safety and effectiveness of oncolytic viruses have been widely accepted (6).

Orf virus (ORFV) is a prototype member of the genus Parapoxvirus, belonging to Poxviridae, which can cause contagious pustular stomatitis, an acute mucocutaneous infection in sheep and goats, and is usually self-limiting (7,8). With an increasing number of comprehensive studies on its molecular, biological and immunological characteristics, especially given its vigorous and particular immunomodulatory properties, ORFV has been proposed as a potential oncolytic virus vector (810).

ORFV is known to have a strong immunomodulating activity against the host. ORFV can rapidly mediate innate and adaptive immune responses in the body. Upon infection by ORFV, neutrophils, natural killer (NK) cells and dendritic cells (DC) are enriched at the infection sites, various types of cells in the innate immune system are activated, and several key cytokines are secreted (8). Notably, ORFV induces mainly the Th1 immune response in the early stages of infection where the classical cytokines IFN-γ, TNF, IL-6, IL-8, IL-12 and IL-18 are secreted from the peripheral immune cells (8,11). Then, the Th2 type immune response appears with the secretion of IL-4, IL-1 receptor antagonist (IL-1RA), and IL-10 (12).

A live or inactivated ORFV can induce a strong antitumor immune response in murine models of multiple tumors. Fiebig et al (9) were the first to report that inactivated ORFV mediates antitumor effects in various tumor models, including the murine syngeneic B16F10 melanoma and MDA-MB-231 human breast cancer xenograft, and revealed that NK cells play an important role in the antitumor effects of ORFV. Anti-mouse NK-1.1 antibody partially inhibited its antitumor activity by inhibiting the activity of NK and NKT cells. Moreover, when IFN-γ was neutralized, the inhibitory effects of ORFV disappeared (9). Inactivated ORFV inhibited tumor growth of MDA-MB-231 tumor-bearing NOD/LtSz-scid/j mice which lacked NK and functional T- and B-lymphocytes (9). Rintoul et al (11) confirmed that live ORFV possessed antitumor effects through activation of NK cells and by inducing secretion of cytokines IFN-γ and granzyme B. A previous study has found that surgery mediates the disfunction of NK cells, but ORFV injection during surgery can improve the function of NK cells, thereby reducing intra-operative metastasis and prolonging the survival time (13). A better understanding of the molecular mechanisms underlying the antitumor effects of ORFV will be beneficial to further develop ORFV as oncolytic vectors for human tumor treatment.

In prior studies, researchers have revealed some mechanisms through which oncolytic viruses exert oncolytic functions (1416): direct tumoricidal cytotoxicity and activation of host antitumor immune responses. Referring to functional characteristics of oncolytic viruses and complicated immunoregulation function of ORFV (1719), the present study used high-throughput screening methods to investigate the ORFV-mediated regulation of cytokine expression. In the present study, the antitumor activity of ORFV strain NA1/11 was investigated through in vitro cell experiments and in vivo animal studies.

Materials and methods


The antibodies for cleaved caspase-3 (product no. 9664), cleaved caspase-9 (product no. 7237), Smac (product no. 15108), β-tubulin (product no. 2146), PARP (product no. 9542) were obtained from Cell Signaling Technology, Inc. Annexin V/7-AAD Apoptosis Detection Kit was obtained from Nanjing KeyGen Biotech Co., Ltd. The enhanced chemiluminescence (ECL) detection kit was acquired from Pierce; Thermo Fisher Scientific, Inc. The Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo Molecular Technologies, Inc. Polyvinylidene difluoride (PVDF) membranes were obtained from EMD Millipore. Fetal bovine serum, MEM, DMEM, streptomycin and penicillin were from Gibco; Thermo Fisher Scientific, Inc. All other chemicals and solvents were of analytical grade.

Cell lines and virus

CRC cell lines (LoVo, HCT116, RKO, SW480, SW1116, Caco-2 and CT26) were purchased from the cell bank of the Chinese Academy of Sciences. All CRC cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Ovine fetal turbinate (OFTu) cells were prepared as previously described (20), and maintained in MEM supplemented with 10% FBS. Orf virus (NA1/11) was isolated in our previous study (21). NA1/11∆132-GFP is a recombinant Orf virus with the deletion of the ORFV132 gene by homologous recombination. OFTu cells were infected with NA1/11 or NA1/11∆132-GFP (MOI =0.1) and were harvested when approximately 80-90% of the cells exhibited cytopathogenic effects (CPE). After repeated freezing and thawing, cellular debris was removed by centrifugation at 800 × g for 5 min at 4°C, and supernatants were purified by sucrose gradient ultracentrifugation. Pellets were suspended in PBS (when used for animal research), or MEM (when used for virus proliferation in cells) aliquots were frozen at −80°C. Viral titers were obtained by the median tissue culture infective dose (TCID50) method (22).

Preparation of NA1/11Δ132-GFP recombinant ORFV

A virus recombinant transfer vector pSPV-EGFP with a high expression of green fluorescent protein (GFP) was successfully constructed (23). With pSPV-EGFP, the plasmids ORFV132F-pSPV-EGFP-ORFV132R were constructed by molecular cloning and used to transfect OFTu cells using Lipofectamine 3000 at room temperature (Invitrogen; Thermo Fisher Scientific, Inc.), after OFTu cells were infected with NA1/11 (MOI =1) for 2 h. For each well of a 6-well plate, plasmid DNA and lipid were mixed gently in 200 µl Opti-MEM media by adding 2.5 µg plasmid DNA and 5 µl Lipofectamine Reagent and incubated for 20 min at room temperature. It was necessary to replace the medium with fresh medium 4-6 h after transfection. Screening for fluorescent viral plaques was performed under a fluorescence microscope (Nikon Eclipse E400; Nikon Corporation) at a magnification of ×100. After 15 rounds of plaque purification, NA1/11Δ132-GFP was obtained.

Wound-healing assay

Cells were seeded in 6-well plates at a density of 1×106 cells/well and cultured for 24 h. A wound was scratched in the cell lawn with a sterile 200-µl pipette tip and the cells were washed with serum-free DMEM three times to remove debris. The cells were exposed to ORFV at designated multiplicities of infection (MOI =0, 1, 5, 10). Images of wound healing progression were captured at 24 h post infection under a light microscope (Nikon Eclipse E400) at a magnification of ×100.

Crystal violet stain

CT26 and LoVo cells were seeded in 24-well plates at a density of 1×104 cells/well and infected with ORFV at designated multiplicities of infection (MOI =0, 1, 5, 10). Living cells were stained with crystal violet solution (0.5% crystal violet) at room temperature for 0, 24, 48 and 72 h. Cells were washed with PBS three times to remove the debris and fixed with 500 µl methanol for 5 min at room temperature. After being washed with PBS three times to remove methanol, the cells were stained with crystal violet solution for 10 min at room temperature. The stain was removed and the plates were washed thrice with PBS. Images of the crystal violet stain were captured under a light microscope (Nikon Eclipse E400) at a magnification of ×100.

CCK-8 assay

Cells were seeded at 1×103 cells/well in a 96-well plate. Cells were exposed to designated multiplicities of infection (MOI =0, 1, 5, 10) for various time-points (0, 24, 48 and 72 h). The cell supernatant was removed, the CCK-8 solution (10% CCK-8 reagent diluted in DMEM supplemented with 10% FBS) was added and the cells were incubated for 2 h at 37°C in an atmosphere of 5% CO2. The optical density (OD) was measured at 450 nm using an iMark microplate reader (iMark™; Bio-Rad Laboratories, Inc.).

Reverse transcription-quantitative (RT-q)PCR

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc.). RT-qPCR was carried out with Fast SYBR™ Master Mix (Thermo Fisher Scientific, Inc.) on ABI StepOne Real-Time PCR platform according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.). The primers used were as follows: E-cadherin forward, 5′-TACACTGCCCAGGAGCCAGA-3′ and reverse, 5′-TGGCACCAGTGTCCGGATTA-3′; N-cadherin forward, 5′-ACCTGAACGACTGGGGGCCA-3′ and reverse, 5′-TGCCAAAGCCTCCAGCAAGCA-3′; β-actin forward, 5′-TGAAGGTGACAGCAGTCGGTTG-3′ and reverse, 5′-GGCTTTTAGGATGGCAAGGGAC-3′. The reaction protocol was 30 sec at 94°C, 43 cycles of 5 sec at 94°C and 34 sec at 60°C. Data were analyzed as Relative Quantitation (RQ) with respect to a calibrator sample using the 2−ΔΔCq method (24). The results were presented as the mean ± SD from three experiments.

In vivo tumor model

In total, 12 healthy male Balb/c mice, aged from 6 to 8 weeks (body weight of 18±1.3 g), were purchased from the Laboratory Animal Center of the Southern Medical University. Animals were housed 6 per cage in the animal facility. The room temperature was maintained constant at 22±1°C and the humidity was controlled at 50-60% on a 12-h light/dark cycle. The animals had access to food and water ad libitum. In the CRC mouse model, CT26 cells (5×105 in 50 µl PBS) were subcutaneously injected into Balb/c mice. When the tumor volume reached 25-30 mm3 [tumor volume (mm3)=(1/6) × π × a2 × b (a=width, b=length)], twelve mice were randomly divided into two groups and administrated with the following agents by intratumoral injection for a total of 4 injections in 12 days: i) PBS control; and ii) ORFV (1×108 TCID50). At the end of the experiment, animals were sacrificed by cervical dislocation. Excised tumors were measured. The organs and tumor tissues were fixed in 4% paraformaldehyde for subsequent IHC assay at room temperature for 24 h. For mouse lung metastasis models, 5×105 CT26 cells were injected intravenously (i.v.) into 12 Balb/c mice. Mice were then treated i.v. with ORFV (1×107 TCID50) in 100 µl of PBS, or control-treated (100 µl PBS), on days 1, 3 and 8. At 11 days after CT26 injection, mice were anesthetized with 60-120 µg/g Euthanyl (Sigma-Aldrich; Merck KGaA) and their blood and lungs were harvested. The serum was stored at −20°C for cytokine antibody array. The lung tissues were fixed in 4% paraformaldehyde at room temperature for 24 h for hematoxylin and eosin (H&E) staining, respectively. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Foshan University (Foshan, China) (approval no. 20170311).


Tumor tissues and organs isolated from the mice were fixed with 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin, and sliced (5-µm thick). The slides of the tumor tissues were immunohistochemically stained with cleaved caspase-3 (1:2,000 dilution in antibody diluent) according to the immunohistochemical (IHC) detection system kit manufacturer's instructions (product no. BD5001; Bioworld Technology, Inc.). Briefly, slides were heated at 55°C for 40 min and the dewaxing, rehydration, antigen retrieval and blocking were performed. The primary antibody was incubated at 4°C overnight. Immunostaining continued with HRP-conjugated secondary antibody (product no. BD5001; Bioworld Technology, Inc.) incubated at room temperature for 10 min according to the manufacturer's instructions. The DAB chomogen (product no. BD5001; Bioworld Technology, Inc.) was used for incubation at room temperature for 4 min followed by washing. The slides of the organs were stained with hematoxylin for 2 min and dehydrated with ethanol and xylenes, mounted and cover slipped. The images were obtained under a light microscope.

Flow cytometry

The flow cytometric assay was performed to detect apoptosis in tumor cells exposed to ORFV. CT26 tumor cells were seeded in a 6-well plate at a density of 1×106 cells/well and exposed to ORFV (MOI =10) or PBS for 24 h. The collected cells were processed using Annexin V-APC/7-AAD Apoptosis Detection Kit according to the manufacturer's instructions. Apoptosis of the stained cells was detected using a LSRFortessa flow cytometer (BD Biosciences). BD FACSDiva software 4.1 (BD Biosciences) was used for analysis.

Western blot analysis

Cells exposed to ORFV were lysed in RIPA lysis buffer (cat. no. P0013C; Beyotime Institute of Biotechnology) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protein inhibitors, such as sodium orthovanadate, sodium fluoride, EDTA and leupeptin. Levels of proteins were measured by bicinchoninic acid assay. The lysis was mixed with 1X SDS protein loading buffer and boiled for 15 min. For each individual sample, 30 µg of protein were loaded per lane on 12% SDS polyacrylamide gels. Proteins were electro-transferred to PVDF membranes at a constant 100 V for 60 min. The PVDF membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibody (with 1:1000 dilution, according to the manufacturer's protocol) overnight at 4°C. The PVDF membranes were washed with TBST five times for 5 min per wash and incubated with HRP-conjugated antibodies, including goat anti-rabbit IgG H&L (HRP) (product code ab6721; 1:10,000 dilution) and goat anti-mouse IgG H&L (HRP) (product code ab6789; 1:2,000 dilution; both from Abcam) for 1 h at room temperature. The membranes were washed with TBST again and visualized with ECL chemiluminescence reagent (Tanon Science and Technology Co., Ltd.) using photographic film.

Cytokine antibody array

Mouse sera of the ORFV-treated and PBS-treated subjects were collected and measured, according to the manufacturer's instructions, with a semi-quantitative mouse cytokine antibody array (RayBio Human Cytokine Antibody Array G series 4000; RayBiotech, Inc.) which detects 200 cytokines in one experiment. Briefly, the samples were added to the cytokine antibody chips, then incubated with the biotinylated antibody. Cy3 Equivalent Dye-Streptavidin was added, and the fluorescence signals were visualized through a laser scanner equipped with a Cy3 wavelength (InnoScan 300 Microarray Scanner; Innopsys). The mean fluorescent signals of each group containing three repeats were analyzed with specific software Q-Analyzer Tool (code: QAM-CAA-4000) provided by RayBiotech, Inc. The cytokines (FC ≥2 or FC ≤0.5; Table I) were noted by functional category, comprehensively referring to the COSMIC database (, GeneCards database (, NCBI Gene database (, and references (2531).

Table I.

Differentially expressed cytokines (fold change ≥2 or ≤0.5) were associated with regulation of apoptosis, angiogenesis, autoimmunity/inflammation, and the cell cycle after the ORFV infection.

Table I.

Differentially expressed cytokines (fold change ≥2 or ≤0.5) were associated with regulation of apoptosis, angiogenesis, autoimmunity/inflammation, and the cell cycle after the ORFV infection.

CytokineF-valueP-valueFold changeMean (ORFV-infected, n=3)Mean (control, n=3)Functional classification
IL-150.0000.1842,189.610827.60.4Apoptosis, angiogenesis, and autoimmunity/inflammation
IL-130.0000.3421,869.543402.60.2 Autoimmunity/inflammation
RAGE0.0000.190562.57649.20.1MAPK signaling, ERK1/2 and p53/TP53 signaling and NF-κB
EDAR0.0000.282372.6563,807.410.2Apoptosis and NF-κB
MIG0.0000.114353.760359.91.0Transfection factor and TOLLR
TRAIL0.0000.250289.072321.71.1Apoptosis, angiogenesis, and cell cycle
BLC (CXCL13)0.0000.133219.855111.90.5Apoptosis and autoimmunity/inflammation
IL-70.0000.306200.395185.20.9Apoptosis and autoimmunity/inflammation
CD270.0000.080185.302471.62.5Apoptosis, Autoimmunity/inflammation, and cell cycle
Tryptase ε0.0000.197180.2941,374.27.6Immune response
DLL40.0020.01864.5176,938.8107.6Angiogenesis, Notch
Fractalkine0.0010.37048.51915,760.2324.8Immune response
TARC0.0010.19836.51256.81.6 Autoimmunity/inflammation
DAN0.0000.18434.1642,324.768.0 Autoimmunity/inflammation and cell cycle
VEGF-D0.0010.31732.31846.41.4Notch, angiogenesis, and cell cycle
OX40 ligand0.0020.20525.443245.59.6Apoptosis, autoimmunity/inflammation
Prostasin0.0130.25820.0232,087.1104.2Trypsin-like cleavage specificity
I-TAC0.0630.07111.77796.68.2Transfection factor, TOLLR, apoptosis, angiogenesis, and autoimmunity/inflammation
Leptin0.0060.42911.208960.185.7Apoptosis, autoimmunity/inflammation, and angiogenesis
TIM-10.0710.1929.311111.912.0Immune response
IL-210.2990.0029.14291.710.0 Autoimmunity/inflammation
H600.0890.0447.08495.213.4MHC I
TPO0.3700.0176.500192.729.6Growth factor
KC0.5510.0025.892234.539.8Angiogenesis, autoimmunity/inflammation
ALK-10.6380.0053.44467.219.5Protein kinase
BAFF R0.0580.0043.272100.830.8Immune response
Osteoactivin0.2910.0353.2111,234.1384.4Growth factor
Pentraxin 30.0120.1643.1155,956.91,912.4Apoptosis and autoimmunity/inflammation
Granzyme B0.1310.0962.767406.0146.7Apoptosis and autoimmunity/inflammation
Prolactin0.8050.0692.4192,615.61,081.4Insulin1 and apoptosis
VEGF R10.8410.0072.236942.3421.4Angiogenesis and protein kinase
Neprilysin0.6980.0812.1841,059.8485.2Apoptosis and angiogenesis
CD300.0490.1902.152110.551.4Apoptosis and angiogenesis
Axl0.8740.0930.469682.51,455.8Protein kinase, apoptosis, and cell cycle
ANG-30.9670.0310.369120.1325.4Tyrosine kinase signaling pathway
MMP100.6300.0350.36939.6107.4Tumor metastasis
IFN-γ R10.2250.0340.3165.316.8The JAK/STAT signaling pathway
VEGF-B0.0630.0270.0809.1113.0Angiogenesis and cell cycle

[i] ORFV, Orf virus; VEGF, vascular endothelial growth factor; DLL4, delta-like canonical Notch ligand 4; BLC, B lymphocyte chemoattractant; IL, interleukin; RAGE, receptor of advanced glycosylation end product; MMP10, matrix metallopeptidase 10.

Statistical analysis

SPSS software version 17.0 (SPSS, Inc.) was used for all analysis. Results are expressed as the means ± SEM. One-way ANOVA was performed to test the null hypothesis of group differences with post hoc tests (Dunnett's test) and unpaired Student's t-test was also performed. P<0.05 was considered to indicate a statistically significant difference. The F-value, which represents the significance of the whole fitting equation and was used to assess the differences between groups, was analyzed using One-way ANOVA.


ORFV strain NA1/11 infects CRC cells and inhibits proliferation

We used the ORFV strain NA1/11, isolated from an outbreak in northeast China, to infect human CRC cells (LoVo, Caco-2, HCT116, RKO, SW480 and SW1116) with multiplicities of infection (MOI) of 0 (mock), 1, 5, 10. After 24 h, cytopathic lesions were observed in some CRC cells, including rounding and swelling (Fig. S1). A recombinant orf virus, NA1/11∆132-GFP, was used to infect human CRC cells. Then, 24 h post infection (h.p.i.), GFP was visible in the CRC cells (Fig. 1A). In addition, it was determined, via western blotting, that both viral protein ORFV086, which is a proteolytic protein, and GFP reporter protein were expressed at 18 h.p.i in the CRC cells (Fig. 1B).

The crystal violet assay revealed that the total number of cells was decreased when LoVo or CT26 cells were infected by ORFV (Fig. 2A and B). To investigate whether inhibition of proliferation was involved in response to ORFV, CCK-8 assays were performed (Fig. 2C and D). ORFV inhibited the proliferation of LoVo and CT26 in a dose-dependent manner. Notably, the inhibitory effect was not obvious at 24 h.p.i., but increasingly significant at 48 and 72 h.p.i. ORFV also inhibited the proliferation of Caco-2, HCT116, RKO, SW480 and SW1116 cells (Fig. S2).

ORFV strain NA1/11 inhibits the migration of CRC cells

The influence of ORFV on the migration of LoVo and CT26 cells was assessed via a wound healing assay. The cells were cultured at high density in medium containing 10% FBS. After 24 h of serum-starvation, a scratch was made in the confluent cells and the cells incubated for an additional 24 h in medium containing 10% FBS, with or without ORFV treatment. ORFV decreased the migration of the cells into the denuded area, compared to the control (P<0.05; Figs. 3A, B and S3A, B). However, the morphological changes and wound healing assay differences among multiple MOIs of virus infection were small, probably due to insufficient infection time.

Cadherin is associated with tumor progression and metastasis (32). E-cadherin homophilic binding can lead to contact-mediated inhibition of growth, thus the loss of E-cadherin expression is often associated with tumor metastasis (33). On the contrary, N-cadherin promotes the metastatic behavior of tumor cells by directly mediating cell-cell adhesion (34). Thus, it was examined whether E-cadherin and N-cadherin are involved in increased cytotoxicity of ORFV. As anticipated, ORFV treatment caused a significant increase in the expression of E-cadherin. A reduction of N-cadherin was observed in CRC cells incubated with ORFV (Figs. 3C and S3C), indicating that ORFV regulated the expression of cadherins to inhibit the migration of CRC cells.

ORFV strain NA1/11 reduces in vivo tumor growth and metastasis

To investigate the tumoricidal activity of ORFV strain NA1/11 in CRC cells, CT26 cells were injected subcutaneously into BALB/c mice for the development of transplanted tumors. When the tumor size was 25-30 mm3, the mice were treated with ORFV or PBS (Fig. 4A). Consistent with the in vitro results, ORFV strain NA1/11 significantly inhibited in vivo tumor growth (Fig. 4B) and reduced the tumor size (Fig. 4C). Compared to the PBS-treated group, the average tumor volume decreased significantly in the ORFV-treated group. In addition, CT26 cells were injected into the tail vein of Balb/c mice for the development of a CRC lung metastasis model (Fig. 4D). After three ORFV or PBS treatments, the lungs were harvested (Fig. 4E) and subjected to H&E staining. The average tumor area ratio in lung biopsies of the ORFV-treated group significantly decreased in comparison with those from PBS-treated group (Fig. 4F and G).

ORFV strain NA1/11 alters cytokine secretion

To explore the mechanism of ORFV inhibition of cancer cell growth and metastasis, the ORFV-induced cytokine expression was detected via cytokine antibody chip analysis. After three ORFV or PBS treatments, mice sera were collected from lung metastasis models on the 11th day (Fig. 4D) and subjected to semi-quantitative cytokine antibody chip assays with 200 cytokines.

The detection images (Fig. 5A) were scanned and converted to a concentration unit. Fold-changes between the ORFV-treated and PBS-treated groups were calculated. A total of 42 cytokines (FC ≥2 or FC ≤0.5; Table I) were sorted into different clusters according to functional annotation, and the forehead clusters of cytokines with the largest numbers were autoimmunity/inflammation, apoptosis, angiogenesis, and the cell cycle. The fold-change sorting of these cytokines and brief functional annotations are presented in Fig. 5B. Notably, for the individual differences in the mice, the results of cytokine antibody chips array revealed that a variety of cytokines with large fold changes were not statistically significant (Table I). The results of cytokine antibody chip assay indicated that NA1/11 may play roles in those cytokines and direct the preliminary direction for our future research. These cytokines must be further confirmed in additional studies.

ORFV strain NA1/11 induces CRC cell apoptosis

Among differentially expressed cytokines screened by cytokine antibody chip analysis, apoptosis-related cytokines were in the majority. There were 15 upregulated cytokines and 1 downregulated cytokine related to apoptosis in the ORFV-treated group. Thus, apoptosis was examined by three distinct approaches. Using flow cytometry, apoptotic cells were revealed to be significantly increased in the ORFV-stimulated group compared to the PBS-treated group (Fig. 6A). Western blot analysis revealed that apoptosis-regulated proteins Smac, cleaved caspase-3, cleaved caspase-9 and cleaved PARP were significantly enhanced in LoVo cells incubated with ORFV virus from 0 to 48 h.p.i. (Fig. 6B).

To examine whether the antitumor activity of ORFV was due to the potentiation of apoptosis in vivo, the tumor tissues from the mouse model were analyzed by IHC assay. Consistent with the in vitro results, the expression of cleaved caspase-3 in tumor tissues from the ORFV-treated group was significantly increased compared to that of tumor tissues from the PBS-treated groups (Fig. 6C and D).


There are some unique biological characteristics which make ORFV an attractive oncolytic virus vector. First, the ORFV genes are replicated and transcribed in the cytoplasm rather than integrated into the host genome, and thus ORFV is considered relatively safe to develop as an oncolytic virus. Second, the host range of ORFV infection is constricted, its damage is limited mainly to the skin that can recover quickly, and the virus has low toxicity and shows little evidence of systemic transmission. Third, ORFV infection can rapidly mediate humoral and adaptive immune responses, but little neutralizing serum antibody is produced after stimulation by ORFV. Furthermore, attenuated virus strains can be constructed by knockout of the virulence genes. As a viral vaccine vector, the gene compatibility is large, and it can replicate and express exogenous genes. Finally, ORFV infection can cause the release of immunoregulatory molecules, including IFN, IL-2, and TNF. Therefore, ORFV has the potential to be an ideal oncolytic virus vector (9,10,17,19,35,36).

Although ORFV strain NZ2 has been reported to be able to induce anticancer effects in murine cancer models (11), it is necessary to verify the antitumor effect of the ORFV strain NA1/11 for its high variability in the terminal regions of the genome among different strains (37). In the present study, it was first confirmed that ORFV strain NA1/11 could infect and cause typical cytopathic lesions in CRC cell lines. Then, CCK-8 assay indicated that ORFV strain NA1/11 could suppress the proliferation of CRC cells. Furthermore, it was revealed that ORFV strain NA1/11 significantly inhibited the migration of CRC cells. ORFV strain NA1/11 inhibited tumor proliferation and metastasis in vivo. A cytokine antibody chip assay was performed to explore the potential mechanism of ORFV, which revealed that apoptosis may play an important role in ORFV-induced cytotoxicity.

Cytokine antibody chip assay revealed the general profile of cytokine regulation. According to functional classification, four groups containing the most numerous cytokines were apoptosis, autoimmunity/inflammation, angiogenesis, and the cell cycle. Some of the cytokines, including IL-15, IL-21, CD27, VEGF-B, delta-like canonical Notch ligand 4 (DLL4), are pleiotropic cytokines that likely play important roles in apoptosis and autoimmunity/inflammation. It is possible that cytokines, such as CCL6, TREM-1, CXCL16, could affect chemotactic activity, inhibition of tumor metastasis or regulation of the NF-κB pathway. More detailed studies are required to depict an integrated regulation network of the ORFV-mediated oncolysis.

Interleukins (ILs) were first discovered to be expressed by leukocytes, which extensively influence the functions of the immune system, such as promoting the development and differentiation of T and B lymphocytes and hematopoietic cells (26). In the present study, IL-15 was the cytokine most markedly altered in the microarray assay, with a fold-change of more than 2000. This cytokine and IL-2 share numerous common functional characteristics, including regulating the activation and proliferation of T cells and NK cells (27,31). IL-15 can also induce the activation of JAK kinases. IL-21 is another interleukin whose expression levels were upregulated in this assay, with a fold-change of 9.142, and plays a role in both the innate and adaptive immune responses. It can induce the differentiation, proliferation, and activity of various immune cells, including NK cells, macrophages, cytotoxic T cells and B cells, and functions in synergy with IL-15 in some of these regulatory roles (31). IL-21 can stimulate IFN production in T cells and NK cells, and inhibits activation and maturation of DC within T-cell mediated immune responses (28). Analogously, IL-7 may facilitate the functions of the immune system through a similar mechanism (29). These results indicated that interleukins may play a crucial role in the oncolytic effect of ORFV, possibly through regulating the proliferation and activation of T cells, NK cells and other immune cells. CD27, a member of the TNF-receptor superfamily, plays a crucial role in regulating B-cell activation and immunoglobulin synthesis via binding to ligand CD70, and can exert apoptosis-promoting effects through association with the proapoptotic CD27-binding protein (SIVA) (25, 30).

Angiogenesis plays an important role in tumor tissue development and thus has become a key target in various tumor molecular therapy strategies (38,39). The most downregulated cytokine in the microarray assay was VEGF-B, a member of the vascular endothelial growth factor (VEGF) family, which can regulate the formation of blood vessels (angiogenesis) and is involved in the growth of endothelial cells (40). DLL4 is related to the Notch signaling pathway and can negatively regulate the proliferation, migration, and angiogenic sprouting of endothelial cells (41).

Chemokines are a family of small cytokines able to induce directed chemotaxis in adjacent responsive cells, and are classified into four main subfamilies: CXC, CC, CX3C and XC (42). In the present study, the microarray assay results indicated three chemokines, CCL6, TREM-1 and CXCL16, that may take part in ORFV-mediated oncolysis in cancer cells (4345). For instance, TREM-1 can strengthen neutrophil and monocyte-mediated inflammatory responses, which are triggered by promoting the release of pro-inflammatory chemokines and other cytokines (44). The receptor of advanced glycosylation product (RAGE), a multi-ligand cell surface receptor, was markedly upregulated when cancer cells were treated with ORFV. It can also interact with various molecules involved in homeostasis, development, and inflammation. The MAPK signaling, ERK1/2 signaling, p53/TP53 signaling and NF-κB signaling pathways are important pathways in which RAGE functions (46,47). The cytokine matrix metallopeptidase 10 (MMP10), involved in tissue remodeling and in certain disease processes, is the only cytokine identified to be related to tumor metastasis (48).

Apoptosis is an important cellular process induced or inhibited by viral infection. Apoptosis plays an important role in the antitumor effect (49), and previous studies suggested that apoptosis contributes to the oncolytic effect induced by oncolytic viruses (1,50,51). In the present study, there were 15 upregulated cytokines and 1 downregulated cytokine related to apoptosis in the ORFV strain NA1/11-treated group, consistent with literature studies (30,5255).

In conclusion, the present results indicated that ORFV strain NA1/11 could inhibit CRC growth and metastasis by inducing apoptosis of CRC cells. In addition, ORFV NA1/11s could upregulate serum levels of IL-7, IL-13, IL-15, CD27, CD30, pentraxin 3, and B lymphocyte chemoattractant (BLC or CXCL13) and downregulate Axl, CXCL16, ANG-3, MMP10, IFN-γ R1 and VEGF-B, leading to enhanced oncolytic activity.

Supplementary Material

Supporting Data


Not applicable.


The present study was supported by grants from the National Natural Science Foundation of China (NSFC) (grant nos. 81773271 and 31672536), the Key Projects of Basic and Applied Research (Natural Science Class), the Department of Education, Guangdong Province (grant nos. 2017KZDXM088 and 2018KQNCX284), the Joint Fund of Basic and Applied Basic Research Fund of Guangdong Province (grant no. 2019A1515110689), the Foshan University High-level University Fund (grant no. 20170131020) and the Foshan University Senior Talent Start Fund (grant no. 20161110004). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

WH, DC and SL conceived and designed the study. DC, RW, ML, WL, BX, HD, KW, DG and FL performed the experiments. DC, RW, ML, and SL analyzed the data. DC, RW, WH and SL wrote the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Foshan University (Foshan, China) (approval no. 20170311).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.





colorectal cancer


Orf virus


natural killer


dendritic cells


IL-1 receptor antagonist


multiplicities of infection


optical density


receptor of advanced glycosylation end product


matrix metallopeptidase 10


enhanced chemiluminescence


polyvinylidene difluoride


fetal bovine serum


ovine fetal turbinate


vascular endothelial growth factor


delta-like canonical Notch ligand 4


B lymphocyte chemoattractant



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Chen D, Wang R, Long M, Li W, Xiao B, Deng H, Weng K, Gong D, Liu F, Luo S, Luo S, et al: Identification of in vitro and in vivo oncolytic effect in colorectal cancer cells by Orf virus strain NA1/11. Oncol Rep 45: 535-546, 2021
Chen, D., Wang, R., Long, M., Li, W., Xiao, B., Deng, H. ... Hao, W. (2021). Identification of in vitro and in vivo oncolytic effect in colorectal cancer cells by Orf virus strain NA1/11. Oncology Reports, 45, 535-546.
Chen, D., Wang, R., Long, M., Li, W., Xiao, B., Deng, H., Weng, K., Gong, D., Liu, F., Luo, S., Hao, W."Identification of in vitro and in vivo oncolytic effect in colorectal cancer cells by Orf virus strain NA1/11". Oncology Reports 45.2 (2021): 535-546.
Chen, D., Wang, R., Long, M., Li, W., Xiao, B., Deng, H., Weng, K., Gong, D., Liu, F., Luo, S., Hao, W."Identification of in vitro and in vivo oncolytic effect in colorectal cancer cells by Orf virus strain NA1/11". Oncology Reports 45, no. 2 (2021): 535-546.