Comparative genomic analysis of Wolbachia strain wCin2USA1 reveals two pairs of cytoplasmic incompatibility factor (cif) genes and multiple intact prophage regions

Dengue fever poses an escalating global public health challenge, causing substantial morbidity and economic burden, particularly in tropical and subtropical regions. Wolbachia -mediated biocontrol has emerged as a cost-effective and environmentally sustainable strategy to suppress dengue transmission. However, the vulnerability of currently deployed Wolbachia strains in Ae. aegypti underscores the need to explore novel strains with enhanced stability and efficacy. This study aimed to identify alternative Wolbachia candidates for dengue control through comparative genomic analyses of eight Wolbachia pipientis strains: wMel, wAlbB, wlrr, wHm C, wCin2USA1, wAnm, ant7, and a wMel variant (wMel_Lab) isolated from Ae. aegypti . Comprehensive analyses were conducted, including phylogenetic reconstruction, metabolic pathway profiling, and detailed characterization of Cytoplasmic Incompatibility Factor (Cif) genes. Among the strains examined, wCin2USA1 was distinguished by its cif gene and prophage complement. This strain exhibited substantial genomic similarity to wMel (98% average nucleotide identity), the strain currently used in Ae. aegypti release programs. Notably, wCin2USA1 harbored two distinct pairs of Cif genes from different monophyletic clades, homologous to those found in wMel (Type I) and wAlbB (Type III), respectively. Both pairs are syntenic and comprise uninterrupted open reading frames. This architecture is of a type associated with cytoplasmic incompatibility in other characterized strains, although the CI phenotype of wCin2USA1 itself remains experimentally untested. Additionally, seven prophage regions were identified in wCin2USA1, indicating genomic plasticity; notably, none of the cif loci was located within a predicted prophage region. Collectively, these findings characterize the genomic architecture of an understudied Wolbachia strain and identify wCin2USA1 as warranting experimental investigation; its potential as a biocontrol agent remains a prospect contingent on functional validation. Future studies should prioritize transinfection into Ae. aegypti, crossing and embryonic hatch-rate assays to establish CI phenotype, compatibility testing against wMel- and wAlbB-infected lines, and dengue virus challenge assays.