Loss of ENTV-2 RNA and Point Mutation of Env Gene in ENA-1 Cells During Successive Passages.

PURPOSE: This study aimed to monitor the presence of enzootic nasal tumor virus 2 (ENTV-2) and the dynamic changes of the viral env gene during the successive passages of enzootic nasal adenocarcinoma (ENA) cells. MATERIALS AND METHODS: ENA cells were passaged for 24 generations, and the supernatants and cell pellets were collected to extract RNA for monitoring viral presence. The ENTV-2 env gene was amplified by PCR, cloned, and sequenced to analyze nucleotide mutations in the env gene sequence across different passages. RESULTS: The results showed that ENTV-2 RNA was consistently detected in ENA cells up to the 22nd passage, indicating sustained viral persistence within the cells, while its presence in the supernatant was unstable, suggesting possible regulation of viral release, although the underlying mechanism was not investigated. Further cloning and sequencing of the env gene from 11 passages revealed no deletions or insertions, but point mutations were observed, predominantly A ⟶ G substitutions, some of which led to amino acid changes. Notably, nonsense mutations W231 and W19 occurred in the 7th and 15th passages, respectively, resulting in premature truncation of the Env protein, while the Q168R substitution was consistently identified in the sequenced clones from all passages from Passage 5 through Passage 22. Structural predictions indicated that the Env protein maintained a conserved core structure with localized variations in non-transmembrane regions. These observations are consistent with possible structural fine-tuning, but functional validation is required. CONCLUSION: This study provides a descriptive account of sequence changes in the ENTV-2 env gene during serial passaging of ENA cells. The findings are hypothesis-generating and await experimental verification of their biological significance.