Characterization of two novel Negevirus strains and analysis of global Negevirus distribution and host diversity

Mosquitoes and mosquito-borne viruses pose a significant global public health concern. Furthermore, mosquitoes carry a wide range of insect-specific viruses (ISVs), and studying these ISVs is important due to their potential influence on mosquito behavior and the transmission of mosquito-borne viruses. In this study, we report the first isolation of two Negevirus strains from Aedes and Culex mosquitoes in Xinjiang, China: Dezidougou virus (DEZV, isolate XJ-ALT23-420-01) and Negev-like virus (NEGLV, isolate XJ-JH20-91-01). Phylogenetically, the nucleotide sequence of DEZV exhibits high similarity (98.96%) with the DEZV isolate 8345 from Germany but shows lower similarity (< 91.91%) with other DEZV strains. In contrast, the NEGLV isolate XJ-JH20-91-01 displays significant divergence from other Negevirus, sharing only 79.39% nucleotide similarity with the most closely related strain, NEGV BeAr805514. Both DEZV/XJ-ALT23-420-01 and NEGLV/XJ-JH20-91-01 replicated rapidly in mosquito cell lines (C6/36 and Aag2), reaching viral loads of up to 10^9-10 copies/mL, causing significant cytopathic effect (CPE) in these cell lines but failing to replicate in vertebrate cells. In addition, analysis of the location and potential hosts of all published Negevirus members indicated their wide distribution and diverse host range. These findings extend the current spectrum of Negevirus group and provide deeper insights into its geographical distribution and host diversity.