Keming Xie ¹ Benfu Lin ² Xinyu Sun ¹ Peng Zhu ³ Chang Liu ³ Guangfeng Liu ³ Xudong Cao ⁴ Jingqi Pan ¹ Suiping Qiu ¹ Xiaoqi Yuan ¹ Mengshi Liang ¹ Jingzhe Jiang ^3* Lihong Yuan ^1*

• ¹ Guangdong Pharmaceutical University, Guangzhou, Guangdong Province, China
• ² Huadu District Animal Health Supervision Institution, Guangzhou, China
• ³ Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, South China Sea Fisheries Research Institute (CAFS), Guangzhou, China
• ⁴ University of Ottawa, Ottawa, Ontario, Canada

Microviridae is a family of phages with circular ssDNA genomes and they are widely found in various environments and organisms. In this study, virome techniques were employed to explore potential members of Microviridae in a poultry slaughterhouse, leading to the identification of 98 novel and complete microvirus genomes. Using a similarity clustering network classification approach, these viruses were found to belong to at least 6 new subfamilies within Microviridae and 3 higher-level taxonomic units. Genome size, GC content and genome structure of these new taxa showed evident regularities, validating the rationality of our classification method. Our method can divide microviruses into about 45 additional detailed clusters, which may serve as a new standard for classifying Microviridae members. Furthermore, by addressing the scarcity of host information for microviruses, the current study significantly broadened their host range and discovered over 20 possible new hosts, including important pathogenic bacteria such as Helicobacter pylori and Vibrio cholerae, as well as different taxa demonstrated different host specificities. The findings of this study effectively expand the diversity of the Microviridae family, providing new insights for their classification and identification. Additionally, it offers a novel perspective for monitoring and controlling pathogenic microorganisms in poultry slaughterhouse environments.