ยฉ 2016, American Society for Microbiology. Bats harbor severe acute respiratory syndrome (SARS)-like coronaviruses (SL-Co. Vs) from which the causative agent of the 2002-2003 SARS pandemic is thought to have originated. However, despite the fact that a large number of genetically diverse SL-CoV sequences have been detected in bats, only two strains (named WIV1 and WIV16) have been successfully cultured in vitro. These two strains differ from SARS-CoV only in containing an extra open reading frame (ORF) (named ORFX), between ORF6 and ORF7, which has no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-ฮ”X), and a green fluorescent protein (GFP)-expressing mutant (rWIV1-GFP-ฮ”X). Northern blotting and fluorescence microscopy indicate that ORFX was expressed during WIV1 infection. A virus infection assay showed that rWIV1-ฮ”X replicated as efficiently as rWIV1 in Vero E6, Calu-3, and He. La-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activate NF-ฮบB. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of the host immune response but is not essential for in vitro viral replication.