24 25 Orthopoxviruses (OPXVs) have a broad host range in mammalian cells, but 26 Chinese hamster ovary (CHO) cells are non-permissive for vaccinia virus (VACV). Here, 27 IMPORTANCE: 47 Several OPXV species, including monkeypox virus and cowpox virus, cause 48 zoonotic infection in humans. They are believed to use wild rodents as the reservoir or 49 intermediate hosts, but the host or viral factors that are important for OPXV host range in 50 rodents are unknown. Here, we showed that the abortive replication of several OPXV 51 species in a Chinese hamster cell line was caused by a species-specific difference in the 52 host antiviral factor SAMD9L, indicating that SAMD9L divergence in different rodent 53 species poses a barrier for cross-species OPXV infection. While the Chinese hamster 54 SAMD9L could not be inhibited by two previously identified OPXV inhibitors of human 55 and mouse SAMD9L, it can be inhibited by cowpox virus CP77, indicating that OPXVs 56 encode three SAMD9L inhibitors with different specificity. Our data suggest that OPXV 57 host range in broad rodent species depends on three SAMD9L inhibitors with different 58 specificities. 59 60 (VPXV) are recognized as the North American OPXVs for being endemic in North 70America (2). VARV has been eradicated from nature, but the cessation of smallpox 71 vaccination and the waning of anti-OPXV herd immunity have increased the risk of 72 zoonotic OPXV infections. MPXV is highly virulent in humans (3), and the recent 73 increase in human monkeypox cases across a wide geographic area is a concern for 74 global health security (4). CPXV and VACV are responsible for zoonoses in Europe, 75Asia and South America (5, 6). Novel OPXV species have also been discovered in recent 76 human cases (7, 8), including Akhmeta virus (AKMV) isolated from the town of 77 Akhmeta, Georgia (country). 78OPXVs vary greatly in their animal host range (2), with some, such as VARV, 79 CMLV and TATV, only known to infect a single mammalian species, while others, such 80 as MPXV, CPXV and VACV, capable of infecting a wide variety of mammalian hosts. 81