Marek's Disease Virus Serotype Research Articles

Antiviral Properties of Combination Peptides of HIV-1 Rev NLS and NES

Nuclear translocation signal has been identified as a mediator of protein shuttling between nuclear and cytoplasm. Here we report that the combination of peptides from nuclear localization signal (NLS) and nuclear export signal (NES) of HIV-1 Rev have an antiviral activity against the Herpes virus of turkey and Marek's disease virus serotype 1.

Marek's Disease Virus Encodes MicroRNAs That Map to meq and the Latency-Associated Transcript

MicroRNAs (miRNAs) are a class of small (approximately 22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposi's sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the meq oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The meq gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.

Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.

Absolute quantitation of Marek's disease virus and Herpesvirus of turkeys in chicken lymphocyte, feather tip and dust samples using real-time PCR

The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.

Characterization of a very Virulent Marek’s Disease Virus Mutant Expressing the pp38 Protein from the Serotype 1 Vaccine Strain CVI988/Rispens

Marek's disease virus (MDV), a highly cell-associated oncogenic chicken herpesvirus, causes Marek's disease in domestic chickens. A unique phosphoprotein of MDV, pp38, has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. However, recently, the biological properties of a deletion mutant virus (rMd5Deltapp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. Thus, pp38 is important for early cytolytic infection and not for transformation. The pp38 protein of the MDV serotype 1 vaccine strain CVI988/Rispens differs by one amino acid when compared to the pathogenic strains of MDV. Monoclonal antibody, H19, recognizes all serotype 1 MDV strains except CVI988/Rispens. Previous studies have also shown that the unique pp38 epitope in CVI988/Rispens induced high antibody response. In order to study the role of this epitope in the protective properties of CVI988/Rispens, we generated a mutant rMd5 virus in which the wild type pp38 gene has been substituted with that of CVI988/Rispens (rMd5/pp38CVI). The replication properties of rMd5/pp38CVI, both in vitro and in vivo, and tumor induction were examined. We found that the biological properties of rMd5/pp38CVI were similar to the wild type rMd5 virus with regards to in vivo replication, antibody response and tumor induction. This shows that the pp38 derived from CVI988/Rispens is not involved in protective properties as was previously suggested.

Quail Cell Lines Supporting Replication of Marek's Disease Virus Serotype 1 and 2 and Herpesvirus of Turkeys

Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus, can be isolated and propagated in chicken kidney cells (CKC) and chicken or duck embryo fibroblast cells (CEF or DEF, respectively). Two recently developed cell lines, CU447 and CU453, developed from methylcholanthrene-induced tumors in Japanese quail, were examined for their suitability to propagate the three serotypes of MDV. The MDV strain RB-1B (serotype 1) was passaged for more than 30 passages in CU447 without causing cytopathic effects (CPE). Polymerase chain reaction analysis of RB-1B-infected CU447 cells demonstrated the presence of MDV DNA using primers specific for ICP4, pp38, and gB. The 132-bp direct repeats within the BamH1-H and -D fragments were amplified to the same level as RB-1B that was passaged in CKC or CEF. Different passages of RB-1B in CU447 were examined for expression of gB and pp38 transcripts, and pp38, gB, gE, and VP22 protein expression. Irrespective of the passage level, these transcripts and proteins were detected in the RB-1B-infected CU447 cells. Infectious virus was rescued by cocultivation of RB-1B-infected CU447 with CKC. Herpes virus of turkeys was propagated in CU447 and CU453 causing CPE in both cell lines. SB-1 did not cause CPE in either cell line but a few SB-1-infected cells could be detected using a monoclonal antibody specific for serotype 2 MDV.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype.

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.

Characterization of Marek's disease virus serotype 1 (MDV-1) deletion mutants that lack UL46 to UL49 genes: MDV-1 UL49, encoding VP22, is indispensable for virus growth.

Experiments were conducted to investigate the roles of Marek's disease virus serotype 1 (MDV-1) major tegument proteins VP11/12, VP13/14, VP16, and VP22 in viral growth in cultured cells. Based on a bacterial artificial chromosome clone of MDV-1 (BAC20), mutant viruses were constructed in which the MDV-1 homologs of UL46, UL47, UL48, or UL49 were deleted alone and in various combinations. It could be demonstrated that the UL46, UL47, and UL48 genes are dispensable for MDV-1 growth in chicken embryonic skin and quail muscle QM7 cells, although the generated virus mutants exhibited reduced plaque sizes in all cell types investigated. In contrast, a UL49-negative MDV-1 (20 Delta 49) and a UL48-UL49 (20 Delta 48-49) doubly negative mutant were not able to produce MDV-1-specific plaques on either cell type. It was confirmed that this growth restriction is dependent on the absence of VP22 expression, because growth of these mutant viruses could be partially restored on cells that were cotransfected with a UL49 expression plasmid. In addition, we were able to demonstrate that cell-to-cell spread of MDV-1 conferred by VP22 is dependent on the expression of amino acids 37 to 187 of MDV-1 VP22, because expression plasmids containing MDV-1 UL49 mutant genes with deletions of amino acids 1 to 37 or 188 to 250 were still able to restore partial growth of the 20 Delta 49 and 20 Delta 48-49 viruses. These results demonstrate for the first time that an alphaherpesvirus UL49-homologous gene is essential for virus growth in cell culture.

Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines.

The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1.

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.

Suppression of transcription activity of the MEQ protein of oncogenic Marek's disease virus serotype 1 (MDV1) by L-MEQ of non-oncogenic MDV1.

Meq is one of the candidate oncogenes in the MDV1 genome. We previously reported a difference in the meq open reading frame (ORF) between oncogenic and non-oncogenic MDV1: L-meq, in which a 180-bp sequence is inserted into the meq ORF, is detected in non-oncogenic MDV1. To study the functions of a gene product of L-meq (L-MEQ), transactivation by L-MEQ was analyzed by dual luciferase assay using a reporter gene under the control of long (-1--873 bp) and short (-1 - -355 bp) meq promoter (LMP and SMP, respectively). LMP showed higher promoter function than SMP. L-MEQ transactivated the expression of the reporter gene, but less than MEQ did. In the presence of SMP or the cytomegalovirus immediate-early promoter, the same or slightly higher transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq. However, in the presence of LMP, lower transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq, suggesting that L-MEQ can be a transrepressor. Replication of a vvMDV1 was enhanced in the cells with meq. Interestingly, however, replication of vvMDV1 was suppressed in the cells with L-meq or with both L-meq and meq, compared to untransfected cells. Thus, L-MEQ could suppress replication of vvMDV1 displaying the meq gene in coinfected cells.

Glycoproteins E and I of Marek's disease virus serotype 1 are essential for virus growth in cultured cells.

The role of glycoprotein E (gE) and gI of Marek's disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20DeltagE), gI (20DeltagI), or both gE and gI (20DeltagEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088-11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20DeltagE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20DeltagI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20DeltagEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the Alphaherpesvirinae.

Cellular responses in chickens treated with IFN-alpha orally or inoculated with recombinant Marek's disease virus expressing IFN-alpha.

Mammalian type I interferons (IFN-alpha/beta) are potent mediators of innate antiviral immune responses, in particular through enhancement of natural killer (NK) cell cytotoxicity. Recently, chicken IFN-alpha (ChIFN-alpha) has been identified and shown to ameliorate Newcastle disease virus (NDV) infection when given to chickens at relatively high concentrations in the drinking water. In this report, the effect of recombinant ChIFN-alpha (rChIFN-alpha) on NK cell cytotoxicity was examined using (51)Cr-release assays. NK cell cytotoxic activity was also analyzed following inoculation with attenuated Marek's disease virus (MDV) serotype 1 strain R2/23 and a recombinant MDV (parent strain R2/23)-expressing ChIFN-alpha [rMDV(IFN-alpha)]. Treatment of chickens with high doses of rChIFN-alpha in the drinking water significantly decreased NK cell cytotoxicity compared with untreated chickens over a 7-day period. Inoculation of chickens with R2/23 significantly decreased NK cell cytotoxicity as well, whereas the rMDV(IFN-alpha) had no effect on NK cell cytotoxicity. Treatment of chicken embryo cell cultures with rChIFN-alpha inhibited replication of the very virulent MDV RB-1B strain in vitro, and oral treatment of chickens with rChIFN-alpha reduced MDV R2/23 replication in vivo.

Marek's Disease Virus Down-Regulates Surface Expression of MHC (B Complex) Class I (BF) Glycoproteins during Active but not Latent Infection of Chicken Cells

Infection of chicken cells with three Marek's disease virus (MDV) serotypes interferes with expression of the major histocompatibility complex (MHC or B complex) class I (BF) glycoproteins. BF surface expression is blocked after infection of OU2 cells with MDV serotypes 1, 2, and 3. MDV-induced T-cell tumors suffer a nearly complete loss of cell surface BF upon virus reactivation with 5-bromo-2′-deoxyuridine (BUdR). The recombinant virus (RB1BUS2gfpΔ) transforming the MDCC-UA04 cell line expresses green fluorescent protein (GFP) during the immediate early phase of viral gene expression. Of the UA04 cells induced to express the immediate early GFP, approximately 60% have reduced levels of BF expression. All of the reactivated UA04 and MSB1 tumor cells expressing the major early viral protein pp38 display reduced levels of BF. Thus, BF down-regulation begins in the immediate early phase and is complete by the early phase of viral gene expression. The intracellular pool of BF is not appreciably affected, indicating that the likely mechanism is a block in BF transport and not the result of transcriptional or translational regulation.

The genome of turkey herpesvirus.

Here we present the first complete genomic sequence of Marek's disease virus serotype 3 (MDV3), also known as turkey herpesvirus (HVT). The 159,160-bp genome encodes an estimated 99 putative proteins and resembles alphaherpesviruses in genomic organization and gene content. HVT is very similar to MDV1 and MDV2 within the unique long (UL) and unique short (US) genomic regions, where homologous genes share a high degree of colinearity and their proteins share a high level of amino acid identity. Within the UL region, HVT contains 57 genes with homologues found in herpes simplex virus type 1 (HSV-1), six genes with homologues found only in MDV, and two genes (HVT068 and HVT070 genes) which are unique to HVT. The HVT US region is 2.2 kb shorter than that of MDV1 (Md5 strain) due to the absence of an MDV093 (SORF4) homologue and to differences at the UL/short repeat (RS) boundary. HVT lacks a homologue of MDV087, a protein encoded at the UL/RS boundary of MDV1 (Md5), and it contains two homologues of MDV096 (glycoprotein E) in the RS. HVT RS are 1,039 bp longer than those in MDV1, and with the exception of an ICP4 gene homologue, the gene content is different from that of MDV1. Six unique genes, including a homologue of the antiapoptotic gene Bcl-2, are found in the RS. This is the first reported Bcl-2 homologue in an alphaherpesvirus. HVT long repeats (RL) are 7,407 bp shorter than those in MDV1 and do not contain homologues of MDV1 genes with functions involving virulence, oncogenicity, and immune evasion. HVT lacks homologues of MDV1 oncoprotein MEQ, CxC chemokine, oncogenicity-associated phosphoprotein pp24, and conserved domains of phosphoprotein pp38. These significant genomic differences in and adjacent to RS and RL regions likely account for the differences in host range, virulence, and oncogenicity between nonpathogenic HVT and highly pathogenic MDV1.

Protein-coding content of the sequence of Marek's disease virus serotype 1.

MDV is classified as a member of the family Herpesviridae (Roizman et al. 1992). Because of its lymphotropism, MDV was originally classified as a γ-herpesvirus (Roizman et al. 1981). However, later on, based on its genomic organization and significant sequence similarity to other α-herpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), it was classified in the subfamily Alphaherpesvirinae (Buckmaster et al. 1988; Roizman et al. 1992). Like other α-herpesviruses, MDV establishes a life-long latent infection, but differs from other members of the subfamily in that it goes latent not in sensory nerve ganglia but in activated T cells.KeywordsHerpes Simplex Virus TypeThymidine KinaseTegument ProteinUnique LongUnique ShortThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant.

The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.

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 Marek's 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 Marek's 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.

The genome of a very virulent Marek's disease virus.

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.