Changes in ORF4 of HCoV-229E under different culture conditions.

The genome of human coronavirus 229E (HCoV-229E), a causative agent of human respiratory infections, encodes a unique accessory gene, ORF4. Analysis of laboratory strains and clinical specimens has suggested that HCoV-229E acquires truncating mutations in ORF4 under standard laboratory culture conditions. This study confirmed that HCoV-229E from patients with acute respiratory infections harboured a full-length ORF4 (219 amino acids). In contrast, virus stocks derived from the same patients and passaged in conventional cultured cells [LLC-MK2, human embryonic fibroblast (HEF); HEF, HeLa] exhibited truncated ORF4 of various lengths, such as 168, 143 and 16 amino acids. However, when these virus stocks were propagated in human bronchial/tracheal epithelial cells (HBTECs) cultured at the air-liquid interface (ALI), the full-length ORF4 was selected and stably maintained throughout a prolonged observation period. These findings highlight the importance of ORF4 in patients and under physiologically relevant conditions, and the HBTEC-ALI culture system is valuable for analyzing the native properties of HCoV-229E with an intact full-length ORF4.