Identification of MA-104 cells as a novel continuous cell line for the direct primary isolation of lumpy skin disease virus.

Abstract

Lumpy skin disease (LSD) poses a significant threat to cattle populations in India and worldwide, owing to its rapid spread, high morbidity rate, and continuous emergence of new variants. Reliable in vitro systems for LSD virus (LSDV) isolation are essential for diagnostic applications, virological studies, and vaccine development. Primary ruminant cells are commonly used for initial virus isolation; however, they suffer from limited availability and poor reproducibility. In this study, we systematically evaluated the susceptibility of five cell lines, Vero, MA-104, MDBK, BHK-21, and goat testis (GT) cells for the direct isolation and propagation of LSDV. Viral replication and infectivity were assessed using cytopathic effect (CPE) monitoring, conventional multi-target PCR, 50% tissue culture infectious dose (TCID₅₀) assay, and an indirect immunoperoxidase test (IPT) for antigen detection. Direct virus isolation from clinical scab material was successful only in MA-104 and GT cells. In contrast, Vero, MDBK, and BHK-21 cells did not support primary isolation but readily propagated LSDV after prior adaptation to GT cells. Subsequent one-step growth kinetics, performed at multiplicities of infection (MOI) of 0.1 and 0.01, further elucidated the distinct cell line-dependent replication patterns. Vero cells yielded the highest titers (10TCIDmL), whereas MA-104 cells supported sustained replication with peak titers of 10TCIDmL, demonstrating their strong capacity for propagation, comparable to other permissive continuous cell lines (MDBK and BHK-21 cells), which required prior viral adaptation. Unlike primary GT cells, MA-104 is a novel, permissive continuous cell line offering benefits such as reproducibility, ready availability, and reduced ethical concerns, making it a robust alternative for primary cell isolation. This study provides practical insights into the selection of appropriate cell lines and determination of optimal harvesting time points for efficient LSDV isolation and propagation, as well as downstream applications, such as pathogenesis studies and vaccine development.