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We isolated 23 Chlorella viruses from 9 Korean cities. The viruses were initially amplified in the Chlorella strain NC64A. Pure isolates were obtained by repeated plaque isolations. A SDS-PAGE analysis revealed simi-lar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype Chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction en-zymes revealed different restriction fragment patterns among the isolates. One isolate, SS-1, was resistant to digestion with HindIII, PvuII, AluI, and HaeIII, indi-cating methylation at the AGCT or GC sequences. Some isolates reacted with antiserum against PBCV-1. The others that did not react to this PBCV-1 antibody reacted to the antibody that was raised against puri-fied HS-2 virion. The tRNA-coding regions of 8 Chlor-ella viruses were cloned and sequenced. These viruses contained 14-16 tRNA genes within a 1.2- to 2-kb re-gion, except for the SS-1 isolate, which had a 1039-bp spacer in a cluster of 11 tRNA genes. The SS-1 spacer contained an open-reading frame (ORF) of 294 amino acids. This ORF had a 51% amino acid sequence simi-larity to the PBCV-1 ORF A478L. A Southern blot analysis suggested that it was a novel gene that lacked a homologue in PBCV-1.