Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

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.     

Fish embryo cell cultures for derivation of stem cells and transgenic chimeras.

It has been demonstrated in mammalian systems that techniques using embryonal stem cells provide advantages over conventional injection of DNA into embryos for generation of transgenic animals. We employed cell culture approaches in an attempt to develop this technology for fish transgenesis. Using a trout embryo-derived mitogenic preparation in a specialized culture medium, we initiated replication of zebrafish blastula-derived cell cultures and expressed marker genes introduced into the cells by plasmid transfection. Reintroduction of cells from the cultures into blastula-stage embryos indicated that the cultured cells survived and may contribute to the developing organism.     

Marek's disease virus: from miasma to model

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.     

Efficacy of production of reassortant of epidemic strains and cold-adapted influenza viruses in chicken embryo and MDCK cells

Reassortant strains for modern live influenza vaccines are prepared using growing chicken embryos. It is very important to switch manufacture of influenza vaccines from chicken embryos to cell cultures, especially due to the threat of future pandemic, when there will be need of big quantities of vaccine for immunization of all age groups. Efficacy of production of reassortant strains with 6:2 vaccine formulation of genome (6 internal genes from the donor of attenuation and 2 genes coding external antigens--hemagglutinin and neuraminidase--from epidemic strain) in MDCK cell culture, using standard techniques employed for production of the vaccine in chicken embryos, was studied. It was shown that yield frequency of aforementioned reassortants of influenza A viruses did not exceed 5.7% whereas in chicken embryos vaccine 6:2 reassortants were isolated with frequency of 4%. For influenza B viruses, yield of 6:2 reassortants in growing chicken embryos exceeded 67% whereas in MDCK cell culture we were unable to produce clones with required genome composition. Thus, existing method while effective for production of vaccine reassortants in chicken embryos is low effective for isolation of 6:2 reassortants in MDCK cell culture. Fundamentally new techniques are needed for production of reassortant strains for live influenza vaccine in cell culture.     

Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

The RNA subunit of telomerase is encoded by Marek's disease virus

Marek's disease virus (MDV) is a herpesvirus of chickens that induces T lymphomas and tumors within 4 to 5 weeks of infection. Although the ability of MDV to induce tumors was demonstrated many years ago and although a number of viral oncogenic proteins have been identified, the mechanism by which the MDV is implicated in tumorigenesis is still unknown. We report the identification of a virus-encoded RNA telomerase subunit (vTR) within the genome of MDV. This gene is found in the genomic DNA of the oncogenic MDV strains, whereas it is not carried by the nononcogenic MDV strains. The vTR sequence exhibits 88% sequence identity with the chicken gene (cTR). Our functional analysis suggests that this telomerase RNA can reconstitute telomerase activity in a heterologous system (the knockout murine TR(-/-) cell line) by interacting with the telomerase protein component encoded by the host cell. We have also demonstrated that the vTR promoter region is efficient whatever the species of cell line considered and that vTR is expressed in vivo in peripheral blood leukocytes from chickens infected with the oncogenic MDV-RB1B and the vaccine MDV-Rispens strains. The functionality of the vTR gene and the potential implication of vTR in the oncogenesis induced by MDV is discussed.     

Fine structure of Plasmodium gallinaceum in embryonic and neonate chicks.

The erythrocytic stages of Plasmdoium gallinaceum in chicken embryos injected with parasited blood either from a syringe-passaged infection in chickens or from a chicken infected with sporozoites were characterized by abnormal structure. Particularly evident were large, unstained vacuoles within the cytoplasm; these occurred with greatest frequency in schizonts. The presence of myelin bodies within these vacuoles was revealed by transmission electron microscopy; abnormal cytokinesis and aberrant merozoites provided additional evidence of the parasite's inability to develop naturally within the milieu of the embryonic erythrocytes. Fifty-five passages were necessary to restore normal structure of the parasites in embryos, while only 5 passages were required for such restoration in neonate chicks. The probable adaptation of the parasite to the proportions of hemoglobin of the adult chicken may be responsible for the abnormal growth in the immature host.     

Histone Methylation Analysis and Pathway Predictions in Chickens after MDV Infection

Marek's disease (MD) is a lymphoproliferative disease in chicken induced by Marek's disease virus (MDV). Although studies have focused on the genetic differences between the resistant and susceptible chicken, less is known about the role of epigenetic factors in MD. In this study, genome-wide histone modifications in the non-MHC-associated resistant and susceptible chicken lines were examined. We found that tri-methylation at histone H3 Lys4 (H3K4me3) enrichment is positively correlated with the expression of protein coding genes as well as microRNA (miRNA) genes, whereas tri-methylation at histone H3 Lys27 (H3K27me3) exhibits a negative correlation. By identifying line-specific histone modifications in MDV infection, we found unique H3K4me3 islands in the resistant chicken activated genes, which are related to immune response and cell adhesion. Interestingly, we also found some miRNAs from unique H3K27me3 patterns in the susceptible chickens that targeted genes involved in 5-hydroxytryptamine (5-HT)-receptor and adrenergic receptor pathways. In conclusion, dynamic line-specific histone modifications in response to MDV infection suggested that intrinsic epigenetic mechanisms may play a role in MD-resistance and -susceptibility.     

Fine Structure of Plasmodium gallinaceum in Embryonic and Neonate Chicks*

SYNOPSIS. The erythrocytic stages of Plasmodium gallinaceum in chicken embryos injected with parasitized blood either from a syringe-passaged infection in chickens or from a chicken infected with sporozoites, were characterized by abnormal structure. Particularly evident were large, unstained vacuoles within the cytoplasm; these occurred with greatest frequency in schizonts. The presence of myelin bodies within these vacuoles was revealed by transmission electron microscopy; abnormal cytokinesis and aberrant merozoites provided additional evidence of the parasite's inability to develop naturally within the milieu of the embryonic erythrocytes. Fifty-five passages were necessary to restore normal structure of the parasites in embryos, while only 5 passages were required for such restoration in neonate chicks. The probable adaptation of the parasite to the proportions of hemoglobin of the adult chicken may be responsible for the abnormal growth in the immature host.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.     

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.     

Production of Sindbis,influenza, and vesicular stomatitis viruses in chicken embryo and rat embryo cell suspensions

Studies were carried out on the production of Sindbis, influenza and vesicular stomatitis viruses in suspensions of chicken embryo and rat embryo cells. The yield of Sindbis virus in chicken embryo cell suspensions was independent of the multiplicity of infection over the range 0.0001 to 0.01 although reduction in multiplicity caused a delay in virus production. With influenza virus the use of higher multiplicities gave increased virus yields possibly due to the very slow production at low multiplicities. In both monolayer and suspension cultures of chicken embryo cells addition of serum or use of media richer than minimum essential medium (Eagle) had little effect on Sindbis virus production, but if the glucose were omitted the virus yield was markedly reduced. In cell suspensions, a marked reduction in virus yield occurred if infection were delayed more than 24 hr after cell preparation whereas in monolayers the delay of infection allowed cell propagation and hence a higher yield of virus. It was also shown that vesicular stomatitis virus can be produced in chicken embryo cell suspensions, and that rat embryo primary cell suspensions can be used to prepare both Sindbis and vesicular stomatitis viruses. Sindbis virus obtained from chicken embryo cell suspensions was purified by polyethylene glycol precipitation and sucrose density gradient centrifugation and shown to contain only those proteins previously identified as viral, without any contamination from chicken cell proteins. The relative ease and economics of virus production by cell suspension and monolayer methods is compared.     

Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek's Disease Virus

Marek's Disease Virus (MDV) is an avian alpha-herpesvirus that only spreads from cell-to-cell in cell culture. While its cell-to-cell spread has been shown to be dependent on actin filament dynamics, the mechanisms regulating this spread remain largely unknown. Using a recombinant BAC20 virus expressing an EGFPVP22 tegument protein, we found that the actin cytoskeleton arrangements and cell-cell contacts differ in the center and periphery of MDV infection plaques, with cells in the latter areas showing stress fibers and rare cellular projections. Using specific inhibitors and activators, we determined that Rho-ROCK pathway, known to regulate stress fiber formation, and Rac-PAK, known to promote lamellipodia formation and destabilize stress fibers, had strong contrasting effects on MDV cell-to-cell spread in primary chicken embryo skin cells (CESCs). Inhibition of Rho and its ROCKs effectors led to reduced plaque sizes whereas inhibition of Rac or its group I-PAKs effectors had the adverse effect. Importantly, we observed that the shape of MDV plaques is related to the semi-ordered arrangement of the elongated cells, at the monolayer level in the vicinity of the plaques. Inhibition of Rho-ROCK signaling also resulted in a perturbation of the cell arrangement and a rounding of plaques. These opposing effects of Rho and Rac pathways in MDV cell-to-cell spread were validated for two parental MDV recombinant viruses with different ex vivo spread efficiencies. Finally, we demonstrated that Rho/Rac pathways have opposing effects on the accumulation of N-cadherin at cell-cell contact regions between CESCs, and defined these contacts as adherens junctions. Considering the importance of adherens junctions in HSV-1 cell-to-cell spread in some cell types, this result makes of adherens junctions maintenance one potential and attractive hypothesis to explain the Rho/Rac effects on MDV cell-to-cell spread. Our study provides the first evidence that MDV cell-to-cell spread is regulated by Rho/Rac signaling.     

Induction of host gene expression following infection of chicken embryo fibroblasts with oncogenic Marek's disease virus

Microarrays containing 1,126 nonredundant cDNAs selected from a chicken activated T-cell expressed sequence tag database (http://chickest.udel.edu) were used to examine changes in host cell gene expression that accompany infection of chicken embryo fibroblasts (CEF) with Marek's disease virus (MDV). Host genes that were reproducibly induced by infection of CEF with the oncogenic RB1B strain of MDV included macrophage inflammatory protein, interferon response factor 1, interferon-inducible protein, quiescence-specific protein, thymic shared antigen 1, major histocompatibility complex (MHC) class I, MHC class II, beta(2)-microglobulin, clusterin, interleukin-13 receptor alpha chain, ovotransferrin, a serine/threonine kinase, and avian leukosis virus subgroup J glycoprotein.     

Effect of Conditioned Media from Chicken and Turkey Intestinal Cell Cultures on Invasion by Sporozoites of Three Species of Avian Coccidia

The effect of conditioned media from cultures of turkey and chicken intestinal cells on cellular invasion by sporozoites of avian Eimeria species was examined in vitro. Media conditioned by the growth of cells from the ceca, mid-intestine (area of the yolk stalk diverticulum), and duodenal loop were examined for their ability to enhance invasion. Conditioned medium from cultures of turkey cecal cells significantly enhanced invasion by the turkey coccidia Eimeria adenoeides, by 2.4-fold, and E. meleagrimitis , by 2.2-fold, as compared with invasion in the presence of control medium. Conditioned medium from mid-intestinal cell cultures enhanced invasion by the two coccidial species by 2.0- and 2.1-fold, respectively. The enhancement occurred with conditioned media from early (1) as well as later (11) passages of cells. This suggests that the enhancing factor was produced by fibroblast-like cells, the predominant cell type at both early and late passages, and not by epithelial-like cells that had disappeared by the first or second passage. Additionally, conditioned media from cultures of chicken cecal and duodenal loop cells significantly enhanced invasion by the turkey cecal coccidium, E. adenoeides , (1.7- and 1.6-fold, respectively). This was less enhancement than was caused by the turkey cell conditioned media. Heat treatment (56° C for 45 min) of conditioned media failed to alter the effect on invasion. Neither the turkey or chicken cecal cell media nor conditioned media from any other chicken intestinal cell cultures enhanced invasion by E. tenella , the chicken cecal coccidium. Although morphologically dissimilar when they were first plated, the gross appearance and growth of the turkey and chicken cells when conditioned media was collected was comparable.     

Cultivation of Cryptosporidium baileyi: studies with cell cultures, avian embryos, and pathogenicity of chicken embryo-passaged oocysts

Sporozoites of Cryptosporidium baileyi did not undergo development in primary cell cultures from either avian or mammalian hosts, or in mammalian cell lines. Oocysts of C. baileyi produced infections resulting in complete development to sporulated oocysts in chicken embryos and embryos of 8 other avian species examined. Inoculation of 4 X 10(5) oocysts was not pathogenic for avian embryos as evidenced by the lack of gross lesions or death. Oocysts obtained after C. baileyi had been passaged 10 times (first experiment) or 20 times (second experiment) in chicken embryos still caused clinical respiratory disease and gross airsacculitis when inoculated intratracheally into 2-day-old broiler chickens. Oocysts that had been passaged 10 times in chicken embryos were similarly pathogenic for 4-day-old turkeys after intratracheal inoculation.