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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Establishment of an immortal chicken embryo liver-derived cell line Jeongyoon Lee,*† Douglas N. Foster,‡ Walter G. Bottje,*† Hyeon-Min Jang,* Yohanna G. Chandra,*† Lauren E. Gentles,§ and Byung-Whi Kong*†1 *Department of Poultry Science, and †Cell and Molecular Biology Graduate Program, University of Arkansas, Fayetteville 72701; ‡Department of Animal Science, University of Minnesota, St. Paul 55108; and §Department of Biology, University of Arkansas, Fayetteville 72701 ABSTRACT A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek’s disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>105 pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CELim cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21WAF), and CDK inhibitor p16 (p16INK4) were downregulated in the CELim cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16INK4 (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV. Key words: chicken embryo liver, immortal cell line, gene expression 2013 Poultry Science 92:1604–1612 http://dx.doi.org/10.3382/ps.2012-02582 INTRODUCTION Primary chicken embryo kidney, chicken embryo fibroblast (CEF), and chicken embryo liver (CEL) cells have been preferred for virus propagation, detection, and subsequent vaccine production (Kawamura and Tsubahara, 1968; Yamaguchi and Kawamura, 1977; Barta et al., 1984; Myers and Schat, 1989; Pfirschke, 1989; Kibenge and McKenna, 1992). However, the use of primary cultured cells prepared from live organ tissues for virus propagation has limitations such as the limited life-span of the cells, the time consuming and labor intensive preparation, heterogeneous cell populations, and the potential for microbial contamination. Thus, continuously growing immortal cell lines can ©2013 Poultry Science Association Inc. Received July 2, 2012. Accepted February 10, 2013. 1 Corresponding author: [email protected] serve as stable cell substrates for virus propagation. Two immortalized avian cell lines, the DF-1 line, which was derived spontaneously from CEF cells (Himly et al., 1998) and the Leghorn male hepatoma (LMH) chemically induced chicken hepatocellular carcinoma cell line (Kawaguchi et al., 1987), have been widely used for the propagation of avian infectious viruses including avian influenza, avian infectious bronchitis virus, Marek’s disease virus serotype 1 (MDV-1), avian metapneumovirus (AMPV), and infectious laryngotracheitis virus (ILTV; Schnitzlein et al., 1994). For virus propagation and vaccine production, immortalized cell lines should not harbor existing endogenous and exogenous viral genomes, should serve as a continuous supply of homogenous cells, and should overcome disadvantages found in the use of primary cells such as virus titer fluctuation. To date, only 2 immortal CEF cell lines noted as DF-1 and SC-1 have been established spontaneously without the use of tumorigenic viruses or oncogenic chemicals (Himly et al., 1998; Christman et al., 2005). In addi- 1604 CHICKEN EMBRYO LIVER-DERIVED CELL LINE tion, other immortal CEF cell lines such as breast CEF and heart CEF cell lines were established, although not spontaneously (Kim et al., 2001c). Although many chicken embryo cell lines have been established and reported, various cellular characteristics including cellular growth rate (rapid vs. slow), morphology (piling up vs. contact inhibition), and the steady state expression levels (up- vs. downregulation) for various cell cycle regulatory genes are critical to determine whether an established cell line is tumorigenic or not, and in turn, whether a cell line can serve as a suitable substrate for production of vaccines. A variety of key cell cycle regulatory factors that have been well characterized in mammalian cells, including tumor suppressor protein p53 (p53), retinoblastoma (Rb), mouse double minute 2 (Mdm2), transcription factor E2F, member 1 (E2F-1), cyclin-dependent kinase (CDK) inhibitor p21 (p21WAF), CDK inhibitor p16 (p16INK4), alternative reading frame of p16INK4 (ARF), and cellular proto-oncogene c-myc (c-myc) has also been characterized in chicken cells (Kim et al., 2001a,b,c; Christman et al., 2005). The p53 functions as a tumor suppressor in cell cycle arrest (Ko and Prives, 1996), and the downstream genes p21WAF (CDK inhibitor) and Mdm2 (ubiquitin ligase), which are transcriptionally upregulated by activated p53 protein, can regulate cellular proliferation and cell death (el-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993; Zauberman et al., 1995). The Rb protein regulates G1/S phase transition by binding to E2F, which activate G1/S transition when they are released from phosphorylated Rb (Murphree and Benedict, 1984; Wu et al., 1995; Dyson, 1998; Das et al., 2005). Both p16INK4 and ARF are encoded at the INK4/ARF locus (also known as CDKN2A; Sherr, 2006a). The ARF (CDK inhibitor) acts as a tumor suppressor for active p53dependent cell cycle arrest (Sherr, 2006b), and IKN4a (CDK inhibitor) is also a tumor suppressor regulating the cell cycle (Nobori et al., 1994). Myc (c-myc) is a well-known cellular proto-oncogene and transcriptional regulator controlling cell proliferation, cell growth, differentiation, and apoptosis (Marcu et al., 1992; Peukert et al., 1997). In this study, we established an immortal chicken embryo liver-derived cell line (CEL-im) for mainly targeting the propagation of poultry viruses. Furthermore, gene expression for cell cycle regulatory factors and telomerase activity were determined at various passages during the immortalization process of the newly established CEL-im cell line. MATERIALS AND METHODS Isolation and Culture of Chicken Cells Primary chicken embryo liver cells were isolated from 15-d-old specific-pathogen-free (SPF) chicken embryos (Charles River Avian Vaccine Services, North Franklin, CT). Embryonic liver tissues were treated with VT so- 1605 lution (1:1 ratio of 0.25% trypsin and PBS) for 30 min. Cells were plated into 100-mm tissue culture dishes (Sarstedt, Newton, NC) coated with 0.5% gelatin in PBS and were grown at 39°C in a 5% CO2 incubator. The CEL cells were cultured in Dulbecco’s modified eagle medium (0.45% glucose) with 10% fetal bovine serum (FBS), 100 units of penicillin/mL, 100 µg of streptomycin/mL, and 2 mM l-glutamine (Invitrogen Life Technologies, Carlsbad, CA). Cells (6 × 105) were transferred to new culture dishes, and medium was changed every 2 d. Two culture dishes of cells were passaged every 4 to 5 d, and cell numbers were counted at each passage. The DF-1 CEF cells were cultured using the same condition as CEL cells, whereas LMH cells were grown in Waymouth’s medium (Invitrogen Life Technologies) with 10% FBS, 100 units of penicillin / mL, 100 µg of streptomycin/mL, and 2 mM l-glutamine (Invitrogen Life Technologies). Viruses and Infections The stock viruses of AMPV/MN2A (provided by D. N. Foster), MDV-1 (purchased from American Type Culture Collection, Manassas, VA), and ILTV (purchased from National Veterinary Services Laboratory, Ames, IA) were used. The CEL-im cells were infected with AMPV at a multiplicity of infection of 0.5. After incubation for 72 h, virus-infected cells were subjected to 3 freeze-thaw cycles, then medium-containing virus was clarified by centrifugation at 3,000 × g for 15 min at room temperature. To determine AMPV titers, the supernatant fluid was collected and 10-fold dilutions were subjected to reinfection of Vero cells in 96-well plates. The AMPV titers were determined at 3 d postinfection (dpi) by scoring for cytopathic effect (CPE) and were measured as pfu per milliliter (Kong et al., 2007). The MDV infection and tittering were performed following the methods of Parcells et al. (1994) with minor modifications. To determine titers for MDV-1, CEL-im cells were infected with stock virus at 100 pfu. At 5 dpi, MDV infected cells were harvested with 0.25% trypsin treatment and 10-fold cell dilutions were inoculated onto fresh DF-1 CEF cells in 24-well dishes. The CPE shown in DF-1 CEF cells were counted at 5 dpi, and the mean number of plaques was determined. The ILTV infection and titering was performed following the methods of Fuchs et al. (2012). The ILTV stock virus was used to infect CEL-im cells at 100 pfu. Infected cells were incubated at 37°C for 1 h with gentle rocking every 15 min. After the incubation, 10 mL of fresh media was added, and the cells were incubated for 5 d. After incubation, infected cells and supernatant were harvested and lysed by 3 freezethaw cycles. To determine ILTV titer, 10-fold dilutions of ILTV were subjected to re-infection of LMH cells in 24-well plates. The ILTV titers were determined at 5 dpi by scoring CPE and were measured as pfu per milliliter. Three replicates for titering were conducted for all viruses examined. 1606 Lee et al. RNA Extraction The RNA samples were prepared at passages of 20, 50, 70, and 90 for CEL-im in addition to primary passage 2 CEL cells in addition to DF-1 CEF and LMH cells using TRIzol reagent (Invitrogen Life Technologies) following manufacturer’s instructions. The total RNA was treated with DNase I (New England BioLabs Inc., Ipswich, MA) and repurified using TRIzol reagent. The quantity of the repurified total RNA was measured using a Nanodrop1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and quality was assessed by agarose gel electrophoresis (data not shown). Quantitative Reverse-Transcription PCR Reverse-transcription reactions were performed with SuperScript II RTase (Invitrogen Life Technologies) using 3 μg of total RNA, and the reverse-transcription products were diluted to 1:10 with diethylpyrocarbonate (DEPC)-water. The quantitative reverse-transcription PCR (qPCR) reactions were performed by the following condition: denaturing 95°C for 30 s, annealing at 62°C for 1 min, extension at 72°C for 30 s with a final extension at 72°C for 10 min in 40 cycles. The SYBR and reference ROX dyes (Invitrogen Life Technologies) were used for quantification. A nontemplate control and chicken glyceraldehyde 3-phosphate dehydrogenase (chGAPDH) as a loading control were used for the relative quantification. The fold change values for target genes compared with the samples from primary passage 2 CEL cells were measured by the 2− ΔΔCT method (Livak and Schmittgen, 2001). Genespecific primer sets were designed by Primer3 software (http://primer3.wi.mit.edu/) and were synthesized by Integrated DNA Technologies (Coralville, IA). Primer sequences are shown in Table 1. Telomeric Repeat Amplification Protocol Assay Telomerase activity was analyzed using the TRAPeze Telomerase Detection Kit (EMD Millipore, Billerica, MA) following the manufacturer’s instructions. Briefly, 1 × 106 CEL-im cells at passage 60, 80, 100 and LMH, DF-1 cells in addition to HeLa (human cervical carcinoma cell line) positive control cells were lysed in CHAPS {3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate} buffer, and 2 µg of protein were used for the PCR-based telomeric repeat amplification protocol (TRAP) assay. RESULTS AND DISCUSSION Cellular Proliferation During Immortalizing Process Because primary CEL cells, freshly isolated from chicken embryo liver, did not proliferate rapidly, cells were passaged once every week with no increase in cell number (data not shown). At the 13th passage, several proliferating foci of cells were observed and the cell numbers began to be counted to determine the growth rates when cells were passaged. Population doublings (PD) per day for the repopulating primary CEL cells were determined at every passage by passaging cells every 4 to 5 d to the 100th passage (Figure 1). The PD/day for these cells fluctuated between 0.2 and 1.1 until passage 85, and became stable after passage 100 (Figure 1), resulting in cells that were considered to be immortal. The spontaneously immortalized CEL cells were designated as the CEL-im cell line. Currently, the CEL-im cell line grows continuously and has reached 120 passages at between 0.8 to 1.1 PD/day. The Morphology and Growth Rates of the CEL-im Cell Line The morphology and growth rates of the CEL-im cell line at passage 100 are shown in Figure 2. When higher resolution microscopy (200×) was used, the morphology of CEL-im cells showed a smaller size (~10 µm) compared with a typical primary hepatocyte (~25 µm). After growing for 3 d, the culture dishes became confluent from 1 × 105 seeded cells in 6-cm-diameter culture dish (Figure 2A). The growth rate of CEL-im cells was compared with the primary CEL (passage 2) and the LMH hepatoma cell line (Figure 2B). Whereas primary Table 1. The primer sets for quantitative reverse-transcription PCR Gene1 Forward Reverse p53 Rb Mdm2 E2F-1 p21WAF ARF p16INK4 c-myc chGAPDH CCATCCACGGAGGATTATGG TGTGCTGAGATTGGCTCACA GCCAAATTTCGGCTTGAAAA AGCGGAAGCTGAACTTGGAG CCCGTAGACCACGAGCAGAT GGAAGACCTGGGAATGGATG GCGGGATGAACTAGCCAACG TGTCACGTCAACATCCACCA GGCACTGTCAAGGCTGAGAA TTCAGCACCGGGGAGTAAGT CTGAGAGGCGCTCTTCTTCC TGTTGTTGGCTGGGAAGTTG CAGGAGACTTTGCCCCTCTG CGTCTCGGTCTCGAAGTTGA TGATGGGTGCACCACTGAAT GTCCGACCGAAGGAGTTGAC ACCCTGCCACTGTCCAACTT TGCATCTGCCCATTTGATGT 1p53 = tumor suppressor protein p53; Rb = retinoblastoma; Mdm2 = mouse double minute 2; E2F-1 = transcription factor E2F, member 1; p21WAF = cyclin dependent kinase (CDK) inhibitor p21; ARF = alternative reading frame of p16INK4; p16INK4 = CDK inhibitor p16; c-myc = cellular proto-oncogene c-myc; chGAPDH = chicken glyceraldehyde 3-phosphate dehydrogenase. CHICKEN EMBRYO LIVER-DERIVED CELL LINE 1607 Figure 1. Growth curve during immortalization of chicken embryo liver line. Population doublings (PD) per day were determined by passaging every 4 to 5 d. CEL cells showed no increase in cell numbers and LMH cells showed growth only reaching 6 × 105 cells, respectively, CEL-im showed a much greater growth rate for 4 d. The fact that newly established CEL-im cell line can proliferate stably and rapidly suggests the CEL-im cell line can serve as an alternative cellular substrate for virus propagation. Permissiveness of CEL-im Cells to Infectious Avian Viruses To determine the viral susceptibility, the spontaneously immortalized CEL-im cells were tested for permissiveness of avian infectious viruses including AMPV, MDV-1, and ILTV (Table 2). The CEL-im cells were highly permissive for AMPV infection producing titers of ~105 pfu/mL, which is comparable with other cell substrates, such as Vero, TT-1, and DF-1 (Kong et al., 2006, 2007), whereas CEL-im cells were weakly permissive, producing low titers (~10 pfu/mL) for both MDV-1 and ILTV (Table 2). These results suggest that CEL-im cells can be used as an alternative cellular substrate for AMPV and can be tested for permissiveness for other infectious avian viruses. Altered Expression of Cell Cycle Regulatory Genes To characterize genetic alterations that led to the induction of rapid cell divisions in the CEL-im cell line, transcriptional changes of cell cycle regulatory genes such as p53, Rb, Mdm2, E2F-1, p21WAF, ARF, p16INK4, and c-myc were determined by qPCR during the immortalization process (Figure 3). The connection between cellular phenotypes and mRNA expression data may not be as strong as expected. This could be due to the potential lack of correlation between mRNA expres- sion and protein abundance [which has been reported previously in both prokaryotes and eukaryotes (Greenbaum et al., 2003; Nie et al., 2006; Guo et al., 2008; Pascal et al., 2008; Taniguchi et al., 2010)]. The mRNA expression for the cell cycle regulatory genes tested here was confirmed to be correlated with the protein abundance during either the aging process or immortalization of chicken cells (in addition to human and mouse cells) and have been reported previously (Kim et al., 2002a,b; Christman et al., 2006). Therefore, the changes in mRNA expression levels of cell cycle regulatory genes could provide insight into understanding the basic genetic alterations of the newly established CELim cell line, although further study is needed to reveal the more detailed functional roles of these alterations in the CEL-im immortalization process. The expression level of p53 mRNA in the CEL-im cell line was dramatically downregulated compared with primary passage 2 CEL counterpart (Figure 3A). Similar downregulation of p53 was found in the LMH cell line. In turn, the downregulation of p53 in CEL cells was confirmed by the decreased expression of p21WAF and Mdm2 mRNA, which are the transcriptional downstream targets of p53 protein (Figure 3B and 3C). Although p53 expression in DF-1 CEF cells was not decreased, its functional activity as a transcription factor appeared to be greatly downregulated (in lieu of the dramatic downregulation of p21WAF), possibly due to genetic mutations (Figure 3A and 3B). Similar results were reported previously showing that p53 mRNA expression was greatly decreased in immortal CEF cell lines (Kim et al., 2001b,c; Christman et al., 2005, 2006). Furthermore, the downregulation of p53 has been known as a key regulatory alteration in the immortalization of human cells (Williams et al., 1994; Hahn et al., 1999). Thus, the downregulation of p53 in CEL-im cells appeared to be a typical genetic alteration process during cellular immortalization. 1608 Lee et al. Table 2. Permissiveness of an immortal chicken embryo liverderived cell line infected with avian metapneumovirus (AMPV), Marek’s disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus (ILTV) Virus AMPV MDV-1 ILTV 1++ Permissiveness1 Titer (pfu/mL) ++ + + 7 × 105 1.03 × 101 1.50 × 101 = highly permissive; + = weakly permissive. However, Rb, another tumor suppressor, was greatly upregulated in CEL-im cell line (Figure 3D), but not in LMH cells, as found previously with other immortal chicken cell lines (Kim et al., 2001c). The increased Rb expression in immortal chicken cell lines is in contrast to the decreased expression found in mammalian cancer cells (Hickman et al., 2002). In addition, the mRNA level of E2F-1, which is regulated (suppressed) by binding with Rb, was upregulated in CEL-im cells in addition to DF-1 CEF and LMH cells (Figure 3E), as reported previously in the immortal breast CEF (BCEFi) and heart CEF (HCEFi) cell lines (Kim et al., 2001c). Generally E2F, including E2F-1, function in the cellular proliferation by the cell cycle progression (Wu et al., 2001). Moreover, E2F-1 mRNA expression is downregulated in senescent cells of human diploid fibroblast (HDF), mouse embryonic fibroblast (MEF), and CEF cells (Kim et al., 2002b). Figure 2. Morphology and the growth of chicken embryo liver (CEL) cell line. (A) Cells were visualized for 4 d using a phase contrast microscope at 100× magnification. (B) Primary CEL (passage 2; diamond), Leghorn male hepatoma (LMH; square), and immortal chicken embryo liver-derived (CEL-im; circle) cells were plated at a density of 1 × 105 cells/6 cm dish, and the number of cells was calculated every day for 4 d. Each value represents the mean ± SE of 3 observations. CHICKEN EMBRYO LIVER-DERIVED CELL LINE 1609 Figure 3. The mRNA expression for cell cycle regulatory genes in chicken embryo liver immortal (CEL-im) cells. The quantitative reversetranscription PCR results were displayed for each gene examined. The x-axis indicates the passage number of CEL-im cells, and the y-axis presents fold change values of relative expression (REL). (A) tumor suppressor protein p53 (p53); (B) cyclin-dependent kinase (CDK) inhibitor p21 (p21WAF); (C) mouse double minute 2 (Mdm2); (D) retinoblastoma (Rb); (E) transcription factor E2F, member 1 (E2F1); (F) CDK inhibitor p16 (p16INK4); (G) alternative reading frame of p16INK4 (ARF); (H) cellular proto-oncogene c-myc (c-myc). Each value represents the mean ± SE of 3 replicates. CEL1° P2 = primary chicken embryo fibroblast (CEF) passage 2 cells; DF-1 = spontaneously immortalized CEF cell line; LMH = Leghorn male hepatoma. As previously reported (Kim et al., 2001c), the expression level of cyclin A, B2, B3, C, D1, and E were upregulated in immortal CEF cell lines, whereas the expression level of p16INK4, one of the cyclin-dependent kinase inhibitors, was downregulated in immortal CEF cell lines. We confirmed the downregulation of p16INK4 mRNA expression in the CEL-im cell line in addition to DF-1 CEF and LMH cells (Figure 3F). Interestingly, the opposite expression pattern between p16INK4 and E2F-1 in the CEL-im cells was similar to what was previously reported in the immortal CEF cells (Kim et al., 2002b). Taken together, in the Rb/p16INK4/E2F1 pathway, the E2F-1 protein is bound to hypophosphorylated Rb, whereas phosphorylated Rb releases 1610 Lee et al. Figure 4. Telomerase assay. Passages 60, 80, and 100 of the chicken embryo liver immortal (CEL-im) and Leghorn male hepatoma (LMH), DF-1 chicken embryo fibroblast (CEF) cells were analyzed using the PCR-based telomeric repeat amplification protocol (TRAP) assay for telomerase activity. HeLa cell extract was used as a control (positive). The sequential 6-bp increments of amplified telomeric repeats are indicated in the positive control lane and LMH sample. DF-1 = spontaneously immortalized CEF cell line; HeLa = human cervical carcinoma cell line. free E2F-1, and cells are then able to pass from G1 into the S phase of the cell cycle. The Rb phosphorylation process can be suppressed by p16INK4 (Wu et al., 2001). Although expression of Rb is upregulated, the downregulation of p16INK4 may induce hyperphosphorylation of Rb and the higher level of free E2F1, not bound to Rb, may stimulate enhanced cell cycle progression in CEL-im cells. The expression level of ARF mRNA was upregulated in the CEL-im cell line (Figure 4G). Although the major function of ARF is tumor suppression and induction of apoptosis through the binding of Mdm2 to stabilize p53 (Sherr, 2001), thereby preventing cellular proliferation (Bertwistle et al., 2004), recent findings of the p53-independent function of ARF have shown ribosome biogenesis and cell growth stimulation by binding to nucleophosmin/B23 (NPM; Bertwistle et al., 2004; Apicelli et al., 2008). Thus, the upregulated ARF mRNA may also play a role in enhanced cell growth capability of CEL-im cells. The expression level of c-myc mRNA fluctuated during the all passages in the CEL-im cell line compared with primary passage 2 CEL counterpart (Figure 3G). The c-myc oncogene has long been known to be among the most frequently deregulated genes in cancer cells. The deregulation of c-myc leads to additional mutations, such as genomic instability including single nucleotide substitutions, double-stranded breaks, and numerical chromosomal defects (Prochownik and Li, 2007; Prochownik, 2008). Although c-myc is a strong proto-oncogene and shows critical functions in cellular proliferation and genomic instability in mammals (Marcu et al., 1992; Peukert et al., 1997), functional roles of c-myc showing variable expression patterns during the immortalization process in the CEL-im remain for further study. Telomerase Activity To further verify the basic cellular characteristics of the CEL-im cell line, cells were subjected to the TRAP assay to determine telomerase activity. The CEL-im cells were not positive for telomerase activity at passages 60, 80, or 100 (Figure 4), compared with the positive control (an extract from the HeLa human cervical carcinoma cell line). Interestingly, from the comparison with other immortal avian cell lines, LMH cells showed positive activity (the sequential 6-bp increments of amplified telomeric repeats), whereas DF-1 CEF cells showed negative telomerase activity (Figure 4). In summary, a newly established immortal CELim cell line retains similar genetic alterations found in other immortal avian cell lines. The CEL-im cell line was permissive for avian infectious viruses including ILTV, MDV-1, and AMPV. The CEL-im cell line may be valuable for studying virus propagation due to the rapid proliferation potential and its morphological stability. Further research is needed to investigate the potential of the CEL-im cell line as cellular substrates for the propagation and vaccine production of infectious viruses. ACKNOWLEDGMENTS This work was supported by US Poultry and Egg Association (project number 640) and, in part, by the Arkansas Bioscience Institute and Arkansas Agricultural Experimental Station. 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