Marek's disease (MD) continues to cause significant economic losses in the global poultry industry. During routine MD surveillance in China, MD virus type 2 (MDV-2), which has the potential for widespread dissemination, was detected. In this study, 2842 suspected MD samples were collected from 237 poultry flocks vaccinated against MD across 21 provinces and four municipalities in China. Among these, 534 samples (18.79%) from 95/150 flocks (63.29%) tested positive for MDV-2, with detections reported in all provinces and municipalities. A total of 54 MDV-2 strains were isolated and further characterized. The genomes of four isolates, SW20, JLWK1501, LB3, and ZH4, were sequenced and analyzed. Comparative analysis with reference MDV-2 strains (SB-1, 301B/1, and HPRS24) showed that 33 genes in the Chinese isolates differed in length due to insertions or deletions, whereas 27 genes exhibited single-nucleotide polymorphisms (SNPs) without length variation. Homology analysis demonstrated that the four Chinese isolates were more closely related to the US strain 301B/1 than to the US strain SB-1 or the UK strain HPRS24. Point mutations resulted in SNP differences in 18 genes between strain 301B/1 and the Chinese isolates. Additionally, variations in the lengths of four genes (ORF3, ORF413, R-LORF1, and R-LORF9) were observed among the four Chinese isolates, indicating marked variability in these genomic regions. These findings align with alterations reported in circulating MDV-2 strains in China. Pathogenicity assessment of strain SW20 in specific-pathogen-free chickens showed that the strain induced inflammatory liver nodules, thymic atrophy, and splenic enlargement. This study highlights the expanding distribution of MDV-2 in China and confirms its ability to induce inflammatory responses, representing the first systematic epidemiological investigation of MDV-2 in the country. The findings provide an important reference for MD control efforts and emphasize the ongoing need for active surveillance.

Keywords: Marek  s disease virus; epidemiological survey; genetic evolutionary analysis; pathogenicity; serotype 2.