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Herein, moribund mud crab (Scylla serrata) samples were collected from various farming systems, brooders from wild and hatchery quarantine section, located in Tamil Nadu, Andhra Pradesh, and Odisha. The infected crabs showed clinical signs of lethargic movement with the sleeping position in the culture ponds, tanks, and wild-caught. In the wet mount study, massive numbers of viral giant cells were observed in the infected crab hepatopancreas and connective tissue. Mud crab reovirus (MCRV) inclusion bodies were noticed in the connective tissue of hepatopancreas, gill lamellae, muscle and gonads in histopathology analysis. Also, viral particles proliferated into the cytoplasm of the cells, but they did not affect the host nuclei. The infected crab samples were confirmed in the PCR using specific primers (ReoF and ReoR) and rDNA sequence analysis showed 99% similarity with mud crab reovirus and Scylla serrata reovirus (OL466868 and OL466869). The ultrastructural study (TEM) revealed that MCRV viral particles ranged between 70 and 75 nm in diameter, icosahedral shape, and non-enveloped with two capsid layers located in the cytoplasm of the cells. The prevalence of mud crab reovirus infection was recorded between 80 and 100% in cultured farms within 25 days and 19–33% in the wild from the collected samples. In the pathogenicity study, intramuscular injection bioassay reached 100% mortality and a co-habitation assay was 70% mortality on 14 days of MCRV post-infection. Thus, our results suggest that MCRV is highly pathogenic to mud crab culture. [ABSTRACT FROM AUTHOR]