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We previously described cardiomyocyte abnormality caused by Km_5666 strain, a variant of fowl glioma-inducing virus (FGV) prototype, which is an avian leukosis virus (ALV). However, the cardiac involvement appeared to be eradicated from the flock after a few years. An epidemiological survey from 2017 to 2020 was performed to elucidate the current prevalence of the cardiopathogenic strains in this flock. Four of the 71 bantams pathologically examined showed both glioma and cardiomyocyte abnormality, from which three ALV strains were detected. DNA sequencing revealed that several different ALV strains coexisted in each bantam and that the conserved Km_5666 virus fluid also contained at least two different ALV strains. We generated three infectious molecular clones from these samples, named KmN_77_clone_A, KmN_77_clone_B, and Km_5666_clone. The  env SU of KmN_77_clone_A shared high sequence identity with that of Km_5666 (94.1%). In contrast, the  env SU of KmN_77_clone_B showed &gt;99.2% nucleotide similarity with that of an FGV variant without cardiopathogenicity. Furthermore, Km_5666_clone experimentally reproduced both gliomas and cardiomyocyte abnormality in chickens. From these results, it is suggested that the pathogenic determinant of cardiomyocyte abnormality is located in  env SU similar to that of Km_5666. The cloning technique described here is beneficial for evaluating the viral pathogenicity in cases where affected birds are coinfected with several different ALV strains.

Avian retroviral cardiomyopathy induced by infectious molecular clones of avian leukosis viruses (fowl glioma-inducing virus variants)