Ginette Dambrine

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeor selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%.Neor andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Marek's disease virus microRNA designated Mdv1-pre-miR-M4 targets both cellular and viral genes.

Mdv1-miR-M4 is one of 25 microRNAs (miRNAs) expressed by Marek’s disease virus (MDV-1), an oncogenic alphaherpesvirus that induces fatal T-cell lymphoma in chickens. Mdv1-miR-M4 was shown to be the second functional viral ortholog of miR-155, a cellular miRNA that plays a crucial role in several physiological and pathological processes in lymphocyte biology. In this study, we investigated a panel of putative mdv1-miR-M4 targets involved in gene networks affecting both cellular and viral life cycles. Using luciferase reporter assays, we showed that mdv1-miR-M4-5P and miR-155 efficiently targeted a common set of 3′ untranslated regions (3′UTR) of six cellular genes (GPM6B, RREB1, c-Myb, MAP3K7IP2, PU.1 and C/EBP). In addition, we also investigated the interactions between mdv1-miR-M4-5P and mdv1-miR-M43P and viral mRNAs encoding UL28 and UL32 in both reporter and western blot assays. Mdv1-miR-M4 specifically inhibited the translation of these two viral proteins, which are involved in the cleavage/packaging of herpesvirus DNA.

Involvement of the Oncoprotein c-Myc in Viral Telomerase RNA Gene Regulation during Marek's Disease Virus-Induced Lymphomagenesis

ABSTRACT Mareks disease virus (MDV) is an alphaherpesvirus that induces a highly malignant T-lymphoma in chickens. The viral genome encodes two identical copies of a viral telomerase RNA subunit (vTR) that exhibits 88% sequence identity to its chicken ortholog chTR. The minimal telomerase ribonucleoprotein complex consists of a protein subunit with reverse transcriptase activity (TERT) and an RNA subunit (TR). The active complex compensates for the progressive telomere shortening that occurs during mitosis and is involved in the cell immortalization process. We show here that the upregulation of telomerase activity is associated with an increase in vTR gene expression in chickens infected with the highly oncogenic MDV strain RB-1B. A comparative functional analysis of the viral and chicken TR promoters, based on luciferase reporter assays, revealed that the vTR promoter was up to threefold more efficient than the chTR promoter in avian cells. We demonstrated, by directed mutagenesis of the vTR promoter region, that the stronger transcriptional activity of the vTR promoter resulted largely from an E-box located two nucleotides downstream from the transcriptional start site of the vTR gene. Furthermore, transactivation assays and chromatin immunoprecipitation assays demonstrated the involvement of the c-Myc oncoprotein in the transcriptional regulation of vTR. Finally, an Ets binding site was specifically implicated in the transcriptional regulation of vTR in the MDV-transformed lymphoblastoid cell line MSB-1.

Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity

The chicken major histocompatibility complex (MHC) genes are organized into two genetically independent clusters which both possess class I and class IIβ genes: the classical B complex and the Restriction fragment pattern-Y (Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I (YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.

Renal Cryptosporidiosis (Cryptosporidium baileyi) in Specific-Pathogen-Free Chickens Experimentally Coinfected with Marek's Disease Virus

Renal Cryptosporidiosis was experimentally induced during a study to investigate the pathogenicity of Cryptosporidium baileyi in specific-pathogen-free (SPF) chickens coinfected with Mareks disease virus (MDV). Cryptosporidium baileyi was administered orally at 4 days of age to chickens previously infected at hatching (day 0) with the HPRS 16 strain of oncogenic MDV. Three control groups received MDV at hatching, C. baileyi on day 4, or placebo consisting of distilled water. Renal cryptosporidiosis lesions were induced in the group coinfected with MDV and C. baileyi. The kidneys were markedly swollen and pale, with visible urate crystals in the ureters and surface tubules. Oocysts of C. baileyi were demonstrated in six of seven cases tested by a scoring method with modified Sheathers sugar solution on renal tissue scrapings and were confirmed in three cases by histologic examination of paraffin-embedded kidney sections. Histologic study also revealed subacute interstitial nephritis, acute ureteritis, and attachment of cryptosporidia on the epithelial cell surface of the ureters and collecting ducts, collecting tubules, and distal convoluted tubules. Various developmental stages of the parasite were present in the kidney sections. To our knowledge, this is the first report of experimentally induced renal cryptosporidiosis in SPF chickens coinfected with MDV.

ICP27 protein of Marek's disease virus interacts with SR proteins and inhibits the splicing of cellular telomerase chTERT and viral vIL8 transcripts

All herpesviruses have a post-transcriptional regulatory protein that prevents precursor mRNA splicing and leads to the shutting off of host protein synthesis. The ICP27 protein of herpes simplex virus 1 (HSV-1) is the prototype of these proteins. Mareks disease virus (MDV-1), an alphaherpesvirus that induces lymphoma in birds, also has an ICP27 protein that is produced in lytic MDV-1-infected cells. We characterized this protein. We demonstrated ICP27 production in latently infected MSB-1 cells, but only on MDV-1 reactivation. ICP27 was found predominantly in specific structures within the nucleus. The ICP27 of MDV-1 colocalized and interacted with SR proteins. We demonstrated inhibitory effects of MDV-1 ICP27 on the splicing of both the viral vIL8 and cellular chTERT (telomerase reverse transcriptase) genes. Thus, the ICP27 of MDV-1 plays a similar role to the ICP27 of HSV-1 and may be involved in MDV-1 replication and the development of Mareks disease.

Major rearrangements in the E element and minor variations in the U3 sequences of the avian leukosis subgroup J provirus isolated from field myelocytomatosis

Summary.We collected paraffin-embedded myelocytomatoses induced by subgroup J avian leukosis virus (ALV-J) in French poultry from 1992 to 2000. We used nested PCR to obtain the U3 LTR and the E element sequences that encompass putative binding sites for transcription factors. We observed minor mutations in the U3 sequences that rarely affected transcription factor binding sites, thus preserving the transcriptional potential of the U3 LTR. However, we observed a large variability in the E element sequences from both field and experimental tumor samples. This variability involved genomic rearrangements and various deletions that most often occurred between two direct repeat sequences. Moreover, in seven DNA samples of the 22 field tumors analyzed, we observed two different sequences for the E element region, suggesting that proviral genomes of two different sizes may be simultaneously present in a tumor. Even though most of the E element sequences were mutated or rearranged, all myelocytomatosis samples always exhibited one E element sequence containing at least one putative C/EBP binding site that was unaffected and still potentially functional.

Vaccination against Marek's disease reduces telomerase activity and viral gene transcription in peripheral blood leukocytes from challenged chickens.

We investigated whether telomerase activity and viral gene transcription were associated with protection against the RB-1B strain of Mareks disease virus (MDV) in chickens vaccinated with Rispens CVI988 or the herpes virus of turkey (HVT). Telomerase activity in peripheral blood leukocytes (PBLs) seemed to be an appropriate marker of lymphoma and levels of viral transcription were correlated with the virulence of MDV strains. Vaccinated protected birds had lower levels of telomerase activity and RB-1B viral gene transcription than unvaccinated chickens infected with RB-1B. The decrease in RB-1B viral transcription was more marked in chickens vaccinated with CVI988 than in those vaccinated with HVT. Indeed, RB-1B viral transcription was not detectable after 14 days post-challenge. In conclusion, telomerase activity and gene transcription in challenge MDV strains are potential new reliable criteria of protection in vaccinated chickens.

Interaction of Marek's Disease Virus and Cryptosporidium baileyi in Experimentally Infected Chickens

Histocompatible B13/B13 white specific-pathogen-free leghorn chickens were used to investigate the effect of coinfection with Cryptosporidium baileyi and the HPRS 16 strain of Mareks disease virus (MDV) in chickens and to assess the pathogenicity of C. baileyi when MDV is given before or after the parasite. Groups of chickens concurrently infected with C. baileyi orally inoculated at day (D)4 and MDV inoculated at hatching (C4M0 group) or at D8 (C4M8 group) were compared with relevant control groups inoculated with only C. baileyi at D4 (C4 group), only MDV at hatching (M0 group) or at D8 (M8 group), and an uninoculated control group (UC group). The chickens were kept in isolator units until the end of the experiment at D62. Our results showed a considerable synergistic effect in concurrently infected chickens and more severe consequences when chickens received MDV before C. baileyi infection. In fact, except for a slight transitory weakness, the chickens in C4 group remained free of overt clinical signs and there was no mortality. However, coinfection with both pathogens induced more lasting or permanent oocyst shedding. Severe clinical cryptosporidiosis with weakness, anorexia, depression, growth retardation, and chronic and severe respiratory disease causing death occurred in all chickens in the C4M0 group between D12 and D43 and in 67% of the chickens in the C4M8 group between D17 and D57. Eighty-two percent and 33%, respectively, died before the development of specific Mareks disease lesions. Mortality rates were 27% and 33% in the M0 and M8 groups, respectively. The presence of MDV enhanced the establishment of more lasting cryptosporidial infection in the respiratory tract, esophagus, crop, proventriculus, and kidneys (only in C4M0 group) as well as in bursa of Fabricius, ceca, and cloaca. Serologic analysis showed that chickens with chronic cryptosporidiosis in the C4M8 group had an increased level of C. baileyi-specific immunoglobulin A. Our results may explain some cases of mortality in chickens naturally infected with MDV and Cryptosporidium.

Effect of Cryptosporidium baileyi in specific pathogen free chickens vaccinated (CVI988/Rispens) and challenged with HPRS-16 strain of Marek's

This study was performed to examine the effect of Mareks disease virus (MDV) serotype 1 vaccine (CVI988/Rispens) on the pathogenicity of Cryptosporidium baileyi , and to determine whether C. baileyi infection could prevent the development of vaccinal Mareks disease (MD) immunity in specific pathogen free (SPF) chickens. Sixty-eight SPF homozygous B13 White Leghorn chickens were divided into seven groups. C. baileyi was orally administered at 5 days of age (day 4) in chickens infected with Rispens vaccine at day 0 or at day 8 and challenged with HPRS—16 strain of oncogenic MDV at day 15. Relevant control groups were constituted. The chickens were kept in isolators until the end of the experiment at day 62. The parameters evaluated were clinical signs, kinetics of oocyst shedding, mortality, macroscopic and microscopic lesions, cryptosporidia location in various organs and serum anti- C. baileyi antibodies at days 42 and 62. Our results show that C. baileyi , which is considered to be non-pathogenic when inoculated orally, may become highly pathogenic. It induced severe mortality and developed in organs other than classical target sites when chickens were vaccinated with Rispens vaccine and challenged with the HPRS—16 strain of MDV.However,parasite infection does not prevent the induction of vaccinal immunity for MD. Our results also show that vaccination of B13 chickens at hatching induces higher protection against challenge with HPRS—16 MDV at day 15 than vaccination at day 8.