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febrile infectious disease caused by pseudorabies virus (PRV), a member of the family Herpesviridae. Currently, PRV is predominantly endemoepidemic and has caused significant economic losses among domestic pigs. Other animals have been proven to be susceptible to PRV, with a mortality rate of 100%. In addition, 30 human cases of PRV infection have been reported in China since 2017, and all patients have shown severe neurological symptoms and eventually died or developed various neurological sequelae. In these cases, broad-spectrum anti-herpesvirus drugs and integrated treatments were mostly applied. However, the inhibitory effect of the commonly used anti-herpesvirus drugs (e.g., acyclovir, etc.) against PRV were evaluated and found to be limited in this study. It is therefore urgent and important to develop drugs that are clinically effective against PRV infection. Here, we constructed a high-throughput method for screening antiviral drugs based on fluorescence-tagged PRV strains and multi-modal microplate readers that detect fluorescence intensity to account for virus proliferation. A total of 2104 small molecule drugs approved by the U.S. Food and Drug Administration (FDA) were studied and validated by applying this screening model, and 104 drugs providing more than 75% inhibition of fluorescence intensity were selected. Furthermore, 10 drugs that could significantly inhibit PRV proliferation in vitro were strictly identified based on their cytopathic effects, virus titer, and viral gene expression, etc. Based on the determined 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50), the selectivity index (SI) was calculated to be 26.3\u20133937.2 for these 10 drugs, indicating excellent drugability. The antiviral effects of the 10 drugs were then assessed in a mouse model. It was found that 10 mg\/kg brincidofovir administered continuously for 5 days provided 100% protection in mice challenged with lethal doses of the human-origin PRV strain hSD-1\/2019. Brincidofovir significantly attenuated symptoms and pathological changes in infected mice. Additionally, time-of-addition experiments confirmed that brincidofovir inhibited the proliferation of PRV mainly by interfering with the viral replication stage. Therefore, this study confirms that brincidofovir can significantly inhibit PRV both in vitro and in vivo and is expected to be an effective drug candidate for the clinical treatment of PRV infections.<\/jats:p>","DOI":"10.3390\/v16030464","type":"journal-article","created":{"date-parts":[[2024,3,18]],"date-time":"2024-03-18T10:54:38Z","timestamp":1710759278000},"page":"464","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Brincidofovir Effectively Inhibits Proliferation of Pseudorabies Virus by Disrupting Viral Replication"],"prefix":"10.3390","volume":"16","author":[{"given":"Huihui","family":"Guo","sequence":"first","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingyun","family":"Liu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Yang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Kuang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yafei","family":"Li","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huanchun","family":"Chen","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Frontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"International Research Center for Animal Disease, Ministry of Science and Technology of China, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0191-1378","authenticated-orcid":false,"given":"Xiangru","family":"Wang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"Frontiers Science Center for Animal Breeding and Sustainable Production, Huazhong Agricultural University, Wuhan 430070, China"},{"name":"International Research Center for Animal Disease, Ministry of Science and Technology of China, Wuhan 430070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1128\/MMBR.69.3.462-500.2005","article-title":"Molecular biology of pseudorabies virus: Impact on neurovirology and veterinary medicine","volume":"69","author":"Pomeranz","year":"2005","journal-title":"Microbiol. 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