A molecular survey of Marek’s Disease virus in Italy.

Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus 2, also called Marek’s disease virus serotype 1 (MDV-1). The virus induces a range of pathological conditions such as neurological disorders, tumors and immunosuppression. On the basis of the continue evolution of the virus, four pathotypes of MDV-1 are currently recognized: mild, virulent, very virulent and very virulent plus. Marek’s disease Eco RI-Q (meq) gene is the principal putative MDV-1 oncogene. It encodes for Meq protein, a transcription factor that recognises different isoforms according to the pathotype. Molecular features of the protein, such as point mutations in the amino terminus and proline-rich reiterations (PPPP) in the transactivation domain, seems to be related to the level of virulence. Meq isoforms with a higher number of PPPP repetitions in their transactivation domains seems to be more active repressors of gene transcription, conferring a lower oncogenic potential to the virus. In the current study eighteen MDV-1 strains collected from clinical cases of Marek’s disease occurred from 2014 to 2016 in Italy, were analyzed. Eight out of eighteen strains were from vaccinated industrial chicken flocks affected by acute MD, while the remaining were from unvaccinated backyard birds affected by classical nervous (n.8) or acute MD (n.2). Viruses were detected from feathers, spleen or tumor samples by a PCR protocol targeting the meq gene. Nucleotide and amino acid sequences were edited and assembled using BioEdit software, then, using Clustal W, were aligned against and compared with complete homologous sequences of selected MDV-1 strains with different degrees of pathogenicity retrieved from GenBank. The number of PPPP repetitions (in the transactivation domain), the proline content (%) and the presence of amino acid substitutions were determined. Phylogenetic analysis was performed using neighbor-joining clustering method including sequences of vaccine strains currently used in Italy. All the analysed field MDV-1 strains showed meq gene sequences different from vaccine homologous sequences. All strains detected in birds affected all by MD acute form (8 from vaccinated commercial flocks and 2 from unvaccinated backyard chickens) clustered together and with MDV-1 strains isolated in Poland in the last decade. Those viruses, all showing amino acid sequence with 4 to 5 PPPP repetitions in the transactivation domain, may be ascribed to virulent or very virulent MDVs. MDV-1 strains detected in unvaccinated backyard chickens with classical nervous form, were closely related to mild and attenuated MDVs, and possessed 8 to 9 PPPP repeats in the amino acid sequence of the transactivation domain. In conclusion high virulence MDVs seems to circulate mostly in Italian commercial compartment where vaccination is routinely applied, while the rural sector is still affected by mild viruses probably due to the absent or lower vaccine pressure. Circulation of MDV-1 strains closely related in both industrial and rural compartments may reveal the occurrence of biosecurity breaches.