Investigating the association between koala retrovirus and primary bone neoplasia in koalas ( <i>Phascolarctos cinereus)</i> using real-time PCR and a novel immunohistochemistry assay

Koala retrovirus (KoRV) is a significant infectious agent impacting the health of wild and captive koala populations worldwide. While previous studies have explored its association with neoplastic diseases such as leukemia and lymphoma, its potential involvement in primary bone tumors remains unclear. This study aimed to expand our understanding of KoRV's disease spectrum by examining its potential association with primary bone tumors in koalas. Koala retrovirus proviral DNA load was analyzed in neoplastic bone using real-time quantitative polymerase chain reaction (PCR) and compared to healthy bone samples across 4 different gene targets: KoRV <i>pol</i>, KoRV-A <i>env</i>, KoRV-B <i>env</i>, and KoRV-D <i>env</i>. The relative KoRV subtype A proviral load was significantly lower in bone tumor samples (<i>n</i> = 14) when compared to healthy bone samples (<i>n</i> = 11) (<i>P</i> = .025), while other subtype-specific proviral loads did not differ significantly between tumor and healthy controls. In addition, we developed a novel immunohistochemistry assay to detect the KoRV capsid protein. Immunolabeling revealed KoRV capsid protein expression in all bone tumor samples (14/14, 100%), with an overall mean positive immunolabeling of 50.4% of tumor cells. The bone tumor group had a higher median H-score compared to the control group (<i>P</i> < .001). Among tumor subtypes, the highest mean percentage of tumor cell labeling was observed in osteosarcomas (73.0%), followed by chondrosarcomas (51.6%) and osteochondromas (38.0%). Collectively, these findings suggest that KoRV may have an important role in koala bone tumor oncogenesis, warranting further investigation into its potential as a contributing factor in tumor development.