Chicken Embryo Fibroblast Cultures Research Articles

The Interaction between Newcastle Disease Virus and Escherichia coli Endotoxin in Chickens

The interaction between Newcastle disease virus (NDV) and Escherichia coli endotoxin was studied in cell cultures, embryonated chicken eggs, and 8-wk-old chickens. These interactions were evaluated according to the induction of specific or nonspecific resistance in the host system and the virus titer produced in both chicken embryos and chickens. The endotoxin of E. coli induced a decrease in the size of the bursa of Fabricius in live chickens. Escherichia coli endotoxin given intravenously induced plasma antiviral activity in chickens that was interpreted to be interferon, as detected in a vesicular stomatitis virus plaque reduction assay. Endotoxin failed to produced toxic effects in the chicken embryo fibroblasts (CEFs) or to result in any antiviral effect because no change was noted in the number of NDV plaques formed in CEF cultures. When endotoxin was given 3 days before NDV exposure in chickens, the virus titers were significantly (P < 0.05) decreased from a peak of 10(2) to 10(0.18), 10(2.5) to 10(0.18), and 10(2.5) to 0 in the spleens, lungs, and kidneys, respectively, at 72 hr post-NDV inoculation. When endotoxin was given 24 hr after NDV inoculation, the NDV titer significantly (P < 0.05) increased from 10(2.0) to 10(3.5), 10(2.5) to 10(6.5), 10(2.5) to 10(4.5), 0 to 10(2.5) in the spleen, lungs, kidneys, and liver, respectively, at 72 hr after NDV inoculation. In chicken sera, hemagglutination inhibition (HI) titer to NDV was significantly (P < 0.05) enhanced from 1164 to 3127 when endotoxin was given prior to virus inoculation. However, there was a decrease in HI to NDV from 1164 to 727 without a significant difference in chicken sera when NDV was given prior to endotoxin inoculation.

Adaptation of Marek’s disease virus to the Vero continuous cell line

Marek’s disease virus (MDV) is a highly infectious, cell-associated oncogenic herpesvirus. Production of MD vaccines has been limited to primary chicken and duck embryo fibroblast (CEF and DEF) cultures. These have a limited life span and cannot be readily stored in liquid nitrogen. Moreover, the need to prepare CEF and DEF cells on a regular basis from 10 to 11 day-old embryos derived from a flock that must be tested continuously for the presence of avian pathogens adds to the cost of vaccine production. A continuous cell line that would support MDV replication could have significant advantages for the rapid large-scale preparation of MD vaccines. In this report, we describe the adaptation to growth of CEF-grown preparations of serotype 1 and serotype 3 (herpesvirus of turkeys; HVT) strains of MDV in cells of the Vero continuous cell line. Although both viruses produced typical CPE, higher levels of infectious progeny and more extensive virus-specific immunofluorescence were obtained for HVT than for the serotype 1 virus. PCR and pulsed field electrophoresis (PFE) analysis of the DNA from Vero cells infected with either virus confirmed the presence of virus-specific DNA.

Inhibition of Eimeria tenella replication after recombinant IFN-γ activation in chicken macrophages, fibroblasts and epithelial cells

We have previously shown that activation of primary cultures of chicken bone-marrow macrophages and embryo fibroblasts with supernatants of concanavaline A-stimulated or reticuloendotheliosis virus (REV)-transformed chicken spleen cells as source of IFN-γ significantly decreases Eimeria tenella growth in vitro. In the present study, we used various chicken cell lines, HD11 macrophages and DU24 fibroblasts, both virally transformed, CHCC-OU2 fibroblasts and LMH hepatic epithelial cells, both chemically transformed, to replicate E. tenella in vitro. We confirmed the previous results by showing that HD11 macrophages pre-treated for 24 h with recombinant chicken IFN-γ (either produced in E. coli or by transfected COS cells), at doses ranging from 1000 to 10 U/ml, drastically inhibited E. tenella replication as measured by [ 3H] uracil uptake after a further 70 h of culture, as when treated with REV supernatant. Likewise the fibroblast and epithelial cell lines exhibited significant inhibitory activity on E. tenella replication after pre-treatment with recombinant chicken IFN-γ, but were less sensitive (1000–100 U/ml) than when treated with REV supernatant. Recombinant chicken IFN-α pre-treatment of all cell lines had no inhibitory effect on parasite development.

Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures.

The role of cytokines in the pathogenesis and immunity of Marek's disease (MD), a herpesvirus-induced T-cell lymphoma in chickens, is poorly understood. Two different experiments were used to examine the potential role of particular cytokines in the pathogenesis and immune responses of MD. First, chicken embryo fibroblasts (CEF) were stimulated with lipopolysaccharide (LPS) and/or recombinant chicken interferon-gamma (rChIFN-gamma) and used to develop techniques for examining transcription of IFN-alpha, IFN-gamma, inducible nitric oxide synthase (iNOS), interleukin (IL)-1beta, IL-2, IL-6 and IL-8 by reverse transcription-polymerase chain reaction (RT-PCR). Addition of LPS and/or rChIFN-gamma resulted in the up-regulation of mRNA for iNOS, IL-1beta and IL-6, while IFN-gamma was up-regulated by LPS alone. IL-2 was down-regulated by the treatments. Second, to determine the effects of Marek's disease herpesvirus (MDV) infection on cytokine transcription in vivo, chickens were infected with MDV at 21 days of age and examined at 7 days post-infection (p.i.) (exp. 1) or were infected with MDV at 1 day of age and examined from 3 to 15 days p.i. (exp. 2). In MDV-infected chickens, IFN-gamma transcription was up-regulated as early as 3 days p.i. until the termination of the experiment at 15 days p.i., while iNOS and IL-1beta were up-regulated between 6 and 15 days p.i. Infection of 1-day-old chicks increased levels of mRNA for IFN-gamma and iNOS between 16- and 64-fold at 9 days p.i. These results suggest that IFN-gamma and iNOS may play an important role in the pathogenesis of MD.

Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus.

The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition of S-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-gamma) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition of N(G)-monomethyl L-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-gamma plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC], B(21)B(21)) and MDV-susceptible S(13) (MHC, B(13)B(13)) and P2a (MHC, B(19)B(19)) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-gamma plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-gamma plus LPS reduced the inhibition. MDV infection of chickens treated with S-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.

Down-regulation of the extracellular matrix protein SPARC in vSrc- and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo.

In vitro transformation of primary cultures of chick embryo fibroblasts by the membrane-bound vSrc or the nuclear vJun oncoproteins is correlated with a down-regulation of the secreted glycoprotein SPARC (also called BM-40 or osteonectin). This protein is a nonstructural component of the extracellular matrix that is thought to regulate cell-matrix interaction during development, wound repair, and carcinogenesis. Its precise function remains unclear. To estimate the contribution of SPARC down-regulation to the major aspects of the transformed phenotype, we have reexpressed this protein from a self-replicating retrovirus Rcas, designated R-SPARC, in the transformed cultures. These R-SPARC-infected cultures display the following main properties: (i) they accumulate the SPARC protein to a level identical to or only slightly higher than the level in normal chick embryo fibroblasts; (ii) they retain the main phenotypic properties characteristic of in vitro transformed cells, that is, altered morphology, capacity to grow in a reduced amount of serum, and capacity to develop colonies from single cells in agar; (iii) they display a clearly reduced capacity to develop local fibrosarcomas in vivo. Taken together, these data strongly suggest that down-regulation of SPARC contributes to the transformed phenotype triggered by vSrc and vJun in primary avian fibroblasts, by facilitating in vivo tumorigenesis.

Evaluation of Vaccines, Interferons and Cell Substrates for Pestivirus Contamination

Pestiviruses are potential contaminants of biological products produced in bovine or porcine cells or manufactured via processes using animal-derived raw materials such as bovine serum. In order to investigate possible contamination of products including those manufactured and/or licensed in the US, 38 lots of viral vaccines and five lots of interferon alpha (IFNα) were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of bovine viral diarrhoea virus (BVDV). All vaccines and interferons were negative for contaminating BVDV RNA when tested by RT-PCR, with the exception of an experimental live viral vaccine that had been produced in BVDV contaminated rabbit kidney cells. Cell lines commonly used to produce biological products and vaccines were experimentally infected with the NADL strain of BVDV to determine if they were permissive for virus replication. MRC-5 and Wl-38 cells were not infected. In contrast, Vero, CHO and CEF cells showed evidence of pestivirus infection. Taken together these data suggested that currently licensed viral vaccines were unlikely to be contaminated with pestiviruses. However, cell banks derived from non-human primate, hamster or rabbit kidney cell lines, or cultures of primary chick embryo fibroblasts, may be infected with BVDV if exposed to pestivirus contaminated raw materials during manufacture.

Optimal conditions for the growth, purification and storage of the avian reovirus S1133

In spite of their importance as avian pathogens causing important losses in poultry farming, the biochemistry of avian reoviruses has been little investigated. In order to facilitate the handling of these agents in the laboratory, a study was carried out to establish the best conditions both for growing the avian reovirus S1133 in primary cultures of chicken embryo fibroblasts and for purification and storage of viral suspensions. The results indicate that the conditions used currently for the manipulation of mammalian reoviruses are not always the best for handling their avian counterparts. In particular, avian reoviruses are much less stable than mammalian reoviruses, and specific conditions for the purification and storage of avian reoviruses therefore should be used. Furthermore, the instability of avian reovirions may have important implications for the life cycle and pathogenesis of the virus within the animal host.

Possible involvement of the double-stranded RNA-binding core protein sigmaA in the resistance of avian reovirus to interferon.

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein sigmaA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein sigmaA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.

Combined antiinfluenza virus activity ofFlos verbasci infusion and amantadine derivatives

The infusion prepared from flowers of Verbascum thapsiforme Schrad. (Scrophulariaceae) (FVI) reduced the infectious and haemagglutination yields of a range of influenza viruses in tissue cultures. Amantadine hydrochloride is an accepted and well studied selective inhibitor of influenza virus reproduction. The combined application of the plant preparation FVI and three amantadine derivatives resulted in a marked enhancement of the inhibitory effect of FVI on the reproduction of influenza virus A/chicken/Germany/27, strain Weybridge (H7N7) in cell cultures of chicken embryo fibroblasts. The antiviral activity was determined by the difference in the infectious titres of control and treated viruses. The combined effect was defined on the basis of infectious viral yields. The most pronounced enhancement was shown for the combination of FVI and adamantanamine glucuronide.

Antiinfluenza Virus Activity of a Bacteriocin Produced by Lactobacillus delbrueckii

A novel antibacterial substance produced by Lactobacillus delbrueckii has been isolated and characterized (1). The inhibitory agent corresponded to the criteria for bacteriocins. It was active against lactic acid bacteria (LAB) species and several food-borne pathogens. The cell-free supernatant was purified by HPLC gel-filtration. Three preparations at different purification steps were tested for activity on the reproduction of influenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1) in cell cultures of chicken embryo fibroblasts (CEF). The inhibitory effect was shown to be highly selective and specific. Expression of viral glycoproteins hemagglutinin, neuraminidase, and nucleoprotein on the surface of infected cells, virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production were all reduced at nontoxic concentrations of the crude preparation (B1). B1 did not protect cells from infection, did not affect adsorption, and slightly inhibited viral penetration into infected cells. The purification did not enhance the cellular toxicity and increased about 870-fold the virus-inhibitory activity. No inactivating effect on extracellular virus was found.

SVMPA, a Mutant of Sindbis Virus Resistant to Mycophenolic Acid and Ribavirin, Shows an Increased Sensitivity to Chick Interferon

SVMPA is a mutant of Sindbis virus, selected for its ability to replicate in mycophenolic acid (MPA)-treated mosquito cells. SVMPA has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (CEF), its replication is restricted in secondary cultures prepared from aged primary CEF cultures. The mutations responsible for these phenotypes mapped to the region of the viral genome that codes for nsP1. We report here that SVMPA has yet another phenotype. Relative to our standard Sindbis virus (SVSTD) from which it was derived, SVMPA shows an increased sensitivity to chick interferon, both crude interferon prepared from virus-infected cells and recombinant interferon. Characterization of viral mutants obtained after site-directed mutagenesis indicated that the same mutations responsible for the host restriction of SVMPA in secondary cultures of CEF were also responsible for its increased sensitivity to chick interferon.

Detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.

The akt kinase: molecular determinants of oncogenicity.

The serine-threonine kinase Akt is a downstream target of phosphoinositide 3-kinase (PI 3-kinase); it is activated by the phosphoinositide 3-phosphate-dependent kinases PDK1 and PDK2. Certain mutated forms of Akt induce oncogenic transformation in chicken embryo fibroblast cultures and hemangiosarcomas in young chickens. This ability to transform cells depends on localization of Akt at the plasma membrane and on the kinase activity of Akt. A transdominant negative form of Akt interferes with oncogenic transformation induced by the p3k oncogene, which codes for an activated form of PI 3-kinase. Akt is therefore an essential mediator of p3k-induced oncogenicity.

Triple helix formation with the promoter of human alpha1(I) procollagen gene by an antiparallel triplex-forming oligodeoxyribonucleotide.

The promoters of alpha1(I) procollagen genes of vertebrates contain two contiguous stretches of polypyrimidine/polypurine sequences, referred to as C1 (-140 to -170) and C2 (-171 to -200). Antiparallel triplex-forming upstream oligonucleotides form efficient triplexes with C1. The C1 tract of human differs from rodent alpha1(I) promoters by 7 nt which are mainly A-->G transitions. Human triplex-forming oligodeoxyribonucleotide (TFO) formed stable triplexes efficiently with a K d of approximately 10-20 nM compared with a K d of approximately 100 nM for rodent TFO. Mutational analysis indicated that 3 or 4 nt (-153 to -155) are sufficient for this higher affinity. TFOs specific for human C1 inhibited transcription from human promoter both in vitro in HeLa cell nuclear extracts and in vivo in cultured chick embryo fibroblasts.

The development and evaluation of two tissue culture‐grown Marek's disease challenge viruses

Standardized challenge viruses are essential for the evaluation of Marek's disease (MD) vaccines with many MD challenge preparations consisting of lymphocytes or whole blood from infected birds. Virus present in these preparations is difficult to quantify by tissue culture assays and, therefore, the infectious bird dose and long‐term storage viability cannot be assured. We report on the properties of two low‐passage virulent Australian MD viruses, the Woodlands No. 1 strain and strain MPF 57. Both strains were isolated in chicken embryo kidney cultures and adapted to grow in chicken embryo fibroblast cultures for a maximum of 14 passages. Both strains could be readily assayed in tissue culture and produced titres of 103 to 104 50% tissue culture infectious doses per ml (TCID50). Birds inoculated at three different doses were observed over 10 weeks, and tissues examined for gross and histological lesions, bursarbody weight ratios and the presence of viraemia. Tissue culture‐grown preparations of both strains were only slightly less virulent than the original lymphocyte challenge material and produced similar pathological responses and around 80% death or gross lesions. From bursarbody weight ratios strain MPF 57 appeared to be more virulent than the Woodlands No. 1 strain.

Effects of Site-Directed Mutations of Transmembrane Cysteines in Sindbis Virus E1 and E2 Glycoproteins on Palmitylation and Virus Replication

The two glycoproteins that form the external spikes of the alphaviruses are type 1 membrane proteins whose transmembrane domains of hydrophobic amino acids are close to the carboxyl termini of the polypeptides and anchor the proteins in the lipid bilayer. Most of the members of the alphavirus genus contain within this transmembrane sequence one or more highly conserved cysteines, which are positioned close to the cytoplasmic face of the lipid bilayer. Cysteines in the cytoplasmic domains of the alphavirus glycoproteins and other enveloped viruses have been shown to be modified by palmitylation. To determine whether the transmembranal cysteines in Sindbis virus also were palmitylated, we used site-directed mutation to change the single transmembranal cysteine in the E1 glycoprotein and two of the transmembranal cysteines in the E2 glycoprotein to alanines. Transfection of RNA transcribed from the differently mutated Sindbis virus cDNAs led to production of infectious virus. Cells infected with the mutant virions and labeled with [3H]-palmitic acid showed that the E1 mutant no longer contained fatty acid in the E1 glycoprotein and that the extent of palmitylation was reduced about twofold in the E2 glycoprotein of virions containing the E2 mutations. At early times postinfection, the mutants grew slightly slower than the wild type in cultures of chicken embryo fibroblasts and secreted about half the amount of virus particles as wild type, but little difference was found at later time points. A triple mutant containing both the E1 and E2 mutations formed virions deficient in palmitylation of both glycoproteins, and this mutant had growth properties that were similar to those of the independent E1 and E2 mutants. Virions with the mutated glycoproteins that were deficient in fatty acid were more susceptible than the wild-type virions to inactivation by the detergent Triton X-100.

The reverse transcriptase activity in cell-free medium of chicken embryo fibroblast cultures is not associated with a replication-competent retrovirus.

Reverse transcriptase (RT) activity has previously been reported in concentrated medium of primary chicken embryo cell cultures using the traditional RT assay. Recently, using the newly-developed and highly-sensitive product-enhanced reverse transcriptase (PERT) assay, RT activity has been detected in live, attenuated vaccines grown in chicken cell substrates. Furthermore, this activity has been associated with particles that contain RNA related to an ancient, endogenous avian retrovirus family designated as EAV-0. To investigate whether the RT activity present in vaccines produced in specific pathogen-free chicken cell substrates is associated with an infectious retrovirus that can replicate in human cells. The kinetics of RT activity produced by 10-day-old chicken embryo fibroblast (CEF) cultures was determined by analyzing cell-free medium in a PCR-based RT (PBRT) assay. Material containing the peak PBRT activity was used as the inoculum to infect various human cell lines and peripheral blood mononuclear cells. Filtered supernatants from control and test cultures were analyzed for the presence of replication-competent retroviruses by the PBRT assay. The cells were monitored for other adventitious agents by routine observation for cytopathic effect (CPE) and by transmission electron microscopy (TEM) at culture termination. The PBRT activity did not increase above the background level in the human target cells through at least five cell passages, thus indicating the absence of a replicating retrovirus. No other adventitious agents were detected based upon TEM analysis and the absence of CPE. The RT activity produced by chicken primary cell cultures is not associated with a retrovirus that can replicate in human cells.

Thrombin Aivation of the 9E3/CEF4 Chemokine Involves Tyrosine Kinases Including c-src and the Epidermal Growth Factor Receptor

The 9E3/CEF4 gene codes for a chemokine that is highly homologous to human interleukin-8 and melanoma growth-stimulating activity/groalpha. These chemokines belong to a family of molecular mediators that are importantly involved in inflammation, wound healing, tumor development, and viral entry into cells. On the chorioallantoic membrane the 9E3 protein is chemotactic for monocyte/macrophages and lymphocytes and is angiogenic. In cultured chicken embryo fibroblasts, which have many of the properties of wound fibroblasts, the gene is stimulated by a variety of agents including oncogenes, growth factors, phorbol esters, and thrombin. The strong stimulation of 9E3 by thrombin in culture correlates well with the observation that in young chicks this gene is stimulated to very high levels in fibroblasts upon wounding and remains high throughout wound repair. Activation of 9E3 by thrombin: (i) occurs very rapidly, one minute exposure to thrombin is sufficient to initiate the signals necessary for gene activation; (ii) is independent of mitogenesis; (iii) operates through the proteolytically activated receptor for thrombin; (iv) is mediated by tyrosine kinases, including c-src and the epidermal growth factor (EGF) receptor, rather than Ser/Thr kinases such as protein kinase C and protein kinase A. Inhibition of either c-src or the EGF receptor tyrosine kinase inhibits the stimulation of 9E3 by thrombin. We show here for the first time that activation of the EGF receptor through a cell-surface receptor that does not have tyrosine kinase activity can lead to expression of an immediate early response gene which encodes for a secreted protein, a chemokine. This rapidly activated tyrosine kinase pathway may be a general stress response by which in vivo a localized cell population reacts to emergency situations such as viral infection, wounding, or tumor growth.

Isolation of serotype 2 Marek's disease virus from a cell line of avian lymphoid leukosis.

To determine a serotype of Marek's disease virus (MDV) persistently infected in a lymphoid leukosis (LL)-cell line, LSCC-BK3, clone A (BK3A), and to examine the pathogenicity of the virus, an attempt was made to isolate MDV from culture fluid of the LL-cell line, using chick embryo fibroblast cultures resistant to infection with subgroup A avian leukosis virus (ALV) to eliminate subgroup A ALV. The MDV isolate, serologically identified as serotype 2 MDV and designated as the 2H strain, was free from ALV and reticuloendotheliosis virus. A serotype 2-specific antigen of MDV was detected in 44% of BK3A, but antigens of serotypes 1 and 3 were not detected in this cell line, by the indirect immunofluorescent antibody test using serotype-specific monoclonal antibodies. MDV antigens were undetectable in LSCC-BK3, clone 2C; both cell lines were established from the same chicken. Neither clinical signs nor macroscopic lesions were observed in any of 19 chicks inoculated with the 2H strain. Three out of 19 chicks histopathologically examined had no lesions. These results suggest that serotype 2 MDV can persist in B cells transformed by ALV without cytopathic effect at a high rate, and the isolate may become a candidate for MD vaccine strains.