The Marek’s disease virus (MDV) protein encoded by the UL17 ortholog is essential for virus growth

Marek’s disease virus type 1 (MDV-1) shows a strict dependency on the direct cell-to-cell spread for its propagation in cell culture. As MDV-1 shows an impaired nuclear egress in cell culture, we wished to address the characterization of capsid/tegument genes which may intervene in the maturation of intranuclear capsids. Orthologs of UL17 are present in all herpesviruses and, in all reported case, were shown to be essential for viral growth, playing a role in capsid maturation and DNA packaging. As only HSV-1 and PrV UL17 proteins have been characterized so far, we wished to examine the role of MDV-1 pUL17 in virus replication. To analyze MDV-1 UL17 gene function, we created deletion mutants or point mutated the open reading frame (ORF) to interrupt its coding phase. We established that a functional ORF UL17 is indispensable for MDV-1 growth. We chose to characterize the virally encoded protein by tagging the 729 amino-acid long protein with a repeat of the HA peptide that was fused to its C-terminus. Protein pUL17 was identified in infected cell extracts as an 82 kDa protein which localized to the nucleus, colocalizing with VP5, the major capsid protein, and VP13/14, a major tegument protein. By using green fluorescent protein fusion and HA tagged proteins expressed under the cytomegalovirus IE gene enhancer/promoter (PCMV IE), we showed that MDV-1 pUL17 nuclear distribution in infected cells is not an intrinsic property. Although our results strongly suggest that another viral protein retains (or relocate) pUL17 to the nucleus, we report that none of the tegument protein tested so far were able to mediate pUL17 relocation to the nucleus

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges

Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia

Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells

Réplication et morphogenèse du virus MDV-1 (caractérisation d'une protéine de tégument produit du gène UL17 essentiel à la réplication virale)

This work aimed at studying the Marek's Disease virus (MDV) UL17 protein which is homologous to the capsid and tegument protein of HSV-1 virus. For this purpose, we used a bacterial artificial chromosome (BAC) of the highly pathogenic MDV strain RB-1b to generate mutant viruses in which the UL17 gene was either deleted or tagged with the HA peptide. The results showed that MDV pUL17 is a phosphoprotein (82 kDa) essential for viral replication. During the infection, pUL17 localizes in the nuclear compartment. This nuclear localization is not an intrinsic property of pUL17 and implies a viral factor. The co-localization of pUL17 with the major capsid protein VP5 and its influence on the cellular distribution of the tegument protein pUL14 favour the hypothesis that pUL17 participates in early tegumentation.L'étude a porté sur la protéine pUL17 du virus MDV (Marek's Disease Virus), homologue à la protéine de capside et de tégument d'HSV-1. La stratégie adoptée repose sur la délétion et l'étiquetage du gène UL17 par recombinaison homologue en utilisant un chromosome bactérien artificiel (BAC) contenant le génome de la souche pathogène RB-1b du virus MDV (BAC-RB-1b). La délétion du gène UL17 ou la mutagenèse de l'ATG sont létales. La restauration du gène permet au virus de retrouver sa capacité de réplication. Lors de l'infection virale, la phosphoprotéine pUL17 (82 kDa) est localisée au noyau, pour partie associée à la membrane nucléaire. Cette localisation nucléaire n'est pas une propriété intrinsèque de la protéine et implique un facteur viral. La co-localisation partielle de pUL17 avec la protéine majeure de capside VP5 et l'influence qu'elle exerce sur la relocalisation de la protéine de tégument pUL14 semblent confirmer l'intervention de pUL17 dans la constitution du tégument viral.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Differential expression of Marek's disease virus (MDV) late proteins during in vitro and in situ replication: Role for pUL47 in regulation of the MDV UL46-UL49 gene locus

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that replicates in a highly cell-associated manner in vitro. Production of infectious cell-free virus only occurs in feather follicle epithelial (FFE) cells of infected chicken skins. Previously, we described differential expression for a core alphaherpesvirus protein, pUL47 that was found to be abundantly expressed in FFE cells of infected chickens, while barely detectable during in vitro propagation. Here, we further examined the dynamics of expression of four tegument proteins within the UL46-49 locus during in vitro and in situ replication. All four proteins examined were expressed abundantly in situ, whereas both pUL47 and pUL48 expression were barely detectable in vitro. Replacement of the putative UL47 and UL48 promoters with the minimal cytomegalovirus promoter enhanced mRNA and protein expression in vitro. Interestingly, enhanced expression of pUL47 resulted in increased UL46, UL48, and UL49 transcripts that resulted in increased pUL46 and pUL48 expression

11th International Symposium on Marek's Diseases and Avian Herpesviruses

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Statistical methods for the comparison of antibody levels in serums assayed by enzyme linked immunosorbent assay

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High-Level Expression of Marek's Disease Virus Glycoprotein C Is Detrimental to Virus Growth In Vitro

Expression levels of Marek's disease virus (MDV) glycoprotein C (gC) are significantly reduced after serial virus passage in cell culture. Reduced gC expression coincides with enhanced MDV growth in vitro and attenuation. To analyze this phenomenon in detail, a full-length infectious MDV clone was modified by Red-based and shuttle mutagenesis in Escherichia coli. Besides a gC-negative deletion mutant harboring a kanamycin resistance gene, a markerless mutant with the U(L)44 gene deleted was constructed. On the basis of this deletion mutant, the original or a modified U(L)44 gene with a mutated start codon (AUG→ACG) was reinserted into the authentic locus. Similarly, mutants expressing authentic gC or the start codon mutation under the control of a strong constitutive promoter were generated. In vitro studies demonstrated that gC deletion mutants induced twofold-larger plaques than the parental virus did, whereas constitutive overexpression of the glycoprotein resulted in a more than twofold reduction in plaque size. In addition, plaque sizes of the gC deletion mutant were reduced when virus was grown using supernatants from cells infected with parental virus, but supernatants obtained from cells infected with the gC deletion mutant had no measurable effect on plaque size. The results indicated that (i) expression of MDV gC, albeit at low levels in a highly passaged virus, had a significant negative impact on the cell-to-cell spread capabilities of the virus, which was alleviated in its absence and exacerbated by its overexpression, and that (ii) this activity was mediated by the secreted form of MDV gC

ESCDL-1 cell-line derived from chicken ES cells is fully permissive to Mardiviruses

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