Primary OFTu cells,Vero cells and HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 mg/ml streptomycin. The WT ORFV strain OV-SY17 was isolated and characterized in our previous work (11). The OV-SY17-Δ120, in which the virulence gene ORF120 was replaced with EGFP, was designed and modified as described in our previous work (8).

Human telomerase reverse transcriptase (hTERT) can activate telomerase activity, therefore maintains telomere length and extends cell lifespan (12, 13). The pCI-neo-hTERT was transfected into primary OFTu cells by Lipofectamine 3000 (#L3000015; ThermoFisher). 48 h after transfection, the cells were selected with G418 under the concentration of 300 μg/ mL. One week later, the concentration was reduced to 150 μg/ mL. Finally, we picked single cell clones and performed cell expansion.

Total RNA was extracted from cells according to the RNAiso Plus protocols (9108; Takara), and First-strand cDNA was synthesized using the RNA to cDNA EcoDry Premix (639549; Clontech). The sequences of specific primers targeting hTERT were prepared as follows: forward, 5'-TCCGAGGTGTCCCTGAGTAT-3'; reverse, 5'-GACCCCAAAGAGTTTGCGAC-3'. The PCR conditions were as follows: 30 cycles of 94°C for 30s, 55°C for 30s and 72°C for 1 min using the TaKaRa Ex Taq^® (RR001A; Takara). The PCR products were loaded on 1% agarose gel along with a DL2000 DNA marker (3427A; Takara).

The continuous cell cultivation was performed with immortalized OFTu cells, and the passage ratio was gradually changed from 1:3 to 1:10. Each passage of cells was grown to a confluent monolayer, and the growth conditions of the passaged cells were recorded in Table 1.

Cell viability assessed by CCK8 (HY-K0301; MCE) assay was performed according to the manufacturer's instructions, and detected the viability of immortalized OFTu cells (passage 15, 20) and primary cells (passage 15, 20).

1 × 10⁶ cells were seeded in a 10 cm dish for 48 h, then incubated with 0.1 μg/mL colchicine (HY-16569; MCE) for 4 h. After trypsin digestion cells were treated with low osmotic pressure for 20 min at 37°C, and fixed by fixative solution (methanol: acetic acid =3:1). The fixed cells were dripped to the slide and stained by Giemsa, then observed by microscope.

twelve female BALB/c nude mice were randomly divided into 4 groups and acclimatized for a week prior to the processing. The primary OFTu, immortalized OFTu and HeLa cells were hypodermically injected into the right dorsal of the mice at 1 × 10⁷ cells per mouse, and the same volume of PBS was injected as a blank control. The mice were monitored and measured once tumors were visible. The mice were sacrificed after 1 month.

All animal experiments in this study were conducted in strict accordance with the guidelines established by the Regulations on Requirements for Laboratory Animals and Laboratory Animal Environment and Housing Facilities of China and were approved by the College of Veterinary Medicine of Jilin University.

Primary OFTu cells, immortalized OFTu cells and Vero cells were cultured in 96-well plates and incubated at 37°C and 5% CO₂ to 70–80% confluency. Every column of 96-well plates was infected with 10-fold-dilution virus. Then the cells were observed daily for CPEs. The Karber's method was applied for calculating the TCID₅₀ of this virus.

Primary OFTu cells and immortalized OFTu cells were infected with virus and harvested after 75% CPE, then applied three freeze-thaw cycles. In order to remove cellular debris, the viral supernatants were concentrated at 2000rpm for 20 min at 4°C. Then the viral supernatants were concentrated by ultracentrifuge (himac CP100WX) at 20000rpm for 2 h. Moreover, we performed density gradient centrifugation to obtain a high-purity virus solution. Finally, negative-stain transmission electron microscopy (TEM) was used to observing virions.

All animals were randomly grouped, and experiments were performed 2–3 times. Data are expressed as the means ± SD of the results of 3 separate experiments and analyzed by one-way analysis of variance (ANOVA) with uncorrelated Fisher's LSD multiple comparisons test with GraphPad Prism 8. Significance is presented as ^***P < 0.001 and ^****P < 0.0001. Other details of the statistical tests are described in the individual figure legends.

In order to establish immortalized OFTu cell line, we collected ovine fetal turbinate and further isolated the cells. The morphology of primary OFTu cells is shown in Figure 1A. These primary cells were passaged for 3–5 generations before the challenge with transgenes. Then, primary OFTu cells were transfected with hTERT-expressing plasmid, which was followed by the selection of geneticin sulfate (G418) until the formation of single cell clones (Figure 1B). Furthermore, one of the selected OFTu cell clones was passaged and hTERT transcription levels from different passages (10, 20, 30, 40, 50, 60, 70, and 80 passages) were examined. Compared with control groups (empty plasmid transfected and non-transfected cells), hTERT mRNA level can be detected in hTERT-transfected OFTu cells (Figure 1C). The above data demonstrate that we have obtained hTERT stably expressed OFTu cells.

We further performed cell viability assay and found hTERT-expressing cells from passage 15 and 20 grew significantly faster than corresponding primary OFTu cells (Figure 2A). While there was no significant difference between the hTERT-expressing OFTu cells from passage 15 and 20 (Figure 2A).

Next, the life span of hTERT-expressing cells by continuously passaging was recorded. We observed that non-transfected primary OFTu cells gradually stopped proliferating at around 30 passages (Figure 2B), while the hTERT-expressing cells could grow over 80 passages with normal cellular morphology (Figure 2C). The above data confirmed that immortalized OFTu cell line was established successfully. And compared with primary OFTu cells, there were no obvious cell morphological changes of hTERT-expressing cells under the observation of optical microscope (Figures 2B, C).

To determine the proliferative capacity of immortalized OFTu cells, continuous cell cultivation was performed. Interestingly, we were able to gradually change the passage ratio of immortalized OFTu cells from 1:3 to 1:10, while the cell growth time (to a confluent monolayer, h) was delayed from about 48 h to 72 h (Table 1). In contrast, the passaging ratio of primary OFTu cells was only 1:3 at most, otherwise the cells grew much slower and could not reach the expected confluence when the ratio was higher than 1:3 (data not shown).

In order to examine the chromosomes stability of immortalized OFTu cell line, karyotype analysis was performed. We found the immortalized OFTu cell line presented 27 pairs of chromosomes (Figure 3A), which was the same as those in primary OFTu cells (Figure 3B). The above data means that the chromosomes in immortalized OFTu cell line are stable.

To further examine whether immortalized OFTu cell has the ability of malignant transformation, we performed xenograft experiment by engrafting the immortalized OFTu cells into nude mice, and primary OFTu cell engraftment and PBS injection were performed as controls. Of note, Hela cells were also engrafted as the positive control. After cell engrafting, we have found that PBS group (Figure 4A), primary OFTu cell group (Figure 4B) and immortalized OFTu cell group (Figure 4C) were incapable of triggering the occurrence of tumors. In contract, mice inoculated with HeLa cells developed significant tumor masses (Figure 4D). Consequently, the immortalized OFTu cell line was not available with tumorigenic ability.

It was reported that primary OFTu cells were sensitive to ORFV infection, which indicate that primary OFTu cell has the capacity to produce ORFV (1, 2). Here, we wonder whether or not immortalized OFTu was able to produce wild-type ORFV. To prove this, both immortalized and primary OFTu cells were infected with wild-type ORFV, which was followed by daily observations. Two days after ORFV infection, similar cytopathic effects (CPEs) were observed in the ORFV-infected immortalized and primary OFTu cells (Figures 5A, B). Subsequently, when 70–80 % OFTu cells underwent CPE, cell cultures were collected for TCID₅₀ examination, with primary OFTu and Vero cells as controls. The results showed that there were no significant differences between immortalized and primary OFTu cells for ORFV production, as their TCID₅₀ levels were almost the same (Figure 5C). While ORFV production from the above two kinds of cells were significantly more than that from Vero cells (Figure 5C). Furthermore, we collected all cell cultures and concentrated viral supernatants from immortalized and primary OFTu cells by ultracentrifuge, and then density gradient centrifugation was performed to obtain high-purity viruses. After observing viral particles through TEM, we found that viral particles from the immortalized OFTu cell line had similar size and morphology as those from primary OFTu cells (Figures 5D, E). The above data indicate that our established immortalized OFTu cell line is sensitive to ORFV and is able to produce high-quality ORFV particles.

Establishing gene-modified viruses is a core process for developing live recombinant vaccines. Therefore, we wonder whether or not immortalized OFTu cell line could be an appropriate platform for ORFV recombination. We firstly determined the transfection efficiency of immortalized OFTu cell line. pEGFP-C3 plasmid was transfected into the cells, which was followed by the observation of green fluorescence signal appearance. We found stronger green signal in plasmid-transfected immortalized OFTu cells than that in plasmid-transfected primary OFTu cells (Figures 6A, B). The above data indicates that the transfection efficiency is higher in immortalized OFTu cells, which provides a competitive advantage for the application of this cell line.

We have previously reported the generation of a kind of ORFV recombinant-OV-SY17-Δ120 in primary OFTu cell line. Briefly, a cassette containing an EGFP reporter and VV7.5 promoter has been used to replace ORF120 gene in ORFV through homologous recombination, making the recombinant virus displaying green fluorescence in infected cells (8). Here, we wonder whether ORFV recombinant can also be generated in immortalized OFTu cell line. To prove this, we performed immortalized OFTu cell transfection, viral infection and virus recombinant selection as shown in the schematic illustration (Figure 6C). Interestingly, we found that it took around 48 h for the immortalized OFTu cell to generate GFP-positive recombinant viruses after ORFV infection (around 6 h after cassette transfection) (Figure 6D). In contrast, the quantity of GFP signal-positive primary cells was less than GFP signal-positive immortalized OFTu cells, which may be related to the difference in transfection efficiency (Figure 6E). After the generation of ORFV recombinant, we further infected the immortalized OFTu cell line with the viral recombinant, and found strong GFP signal in the cell line (Figure 6F). GFP signal with similar fluorescence intensity can also be found in ORFV recombinant-infected primary OFTu cells (Figure 6G). Next, we compared recombinant virus-caused CPE in the cells, and found that immortalized OFTu cell-derived GFP-positive recombinant virus was able to cause similar degree of CPE in both of these two kinds of cells (Figures 6H, I). The above data suggest the established immortalized OFTu cell line could be a suitable tool for generating and amplifying gene-modified ORFV recombinants.