Insect-specific viruses (ISVs) are integral components of mosquito viromes, influencing virus evolution, vector competence, and arbovirus ecology. Papua New Guinea (PNG) is a region of exceptional biodiversity, yet ISVs in local mosquito populations remain poorly characterized. Here, we present the first comprehensive metagenomic survey of ISVs in Culex mosquitoes collected across five provinces of PNG between 2019 and 2021. Using unbiased shotgun metagenomic sequencing, 13,871 female Culex mosquitoes representing five species were analyzed across 86 samples. Approximately 1.6 billion paired-end reads were generated, yielding \(\sim\)20 million viral read pairs. Overall, 231 viral species representing 38 viral families were detected. Of these, 120 were ISV species spanning 25 viral families, and ISVs accounted for 93.8% of species-assigned viral reads, with RNA viruses predominating. The most abundant families included Partitiviridae, Birnaviridae, Rhabdoviridae, and Iflaviridae. Approximately 40% of viral reads corresponded to viruses that are not currently assigned to recognized ICTV families. More than 150 complete viral genomes or genome segments were recovered, including divergent sequences that share <90% amino acid identity with known references. Phylogenetic analyses revealed several divergent viral lineages across major ISV groups. Within the Partitiviridae, previously unrecognized RNA-dependent RNA polymerase (RdRP)-capsid pairings enabled reconstruction of complete genome architectures for Broome partiti-like virus 1 and Sonnbo virus for the first time, together with additional divergent partitivirus lineages. Cambodia Culex birnavirus sequences from PNG, clustered within the known lineage but formed a distinct regional clade. Rhabdovirus analyses identified host-restricted lineages, including Culex tritaeniorhynchus rhabdovirus-like sequences confined to Culex annulirostris. Within the Iflaviridae, several viruses were detected for the first time in PNG. Hubei virga-like virus 2 was the most abundant ISV detected and formed a PNG-specific clade, suggesting local diversification. ISVs were detected in both blood-fed and unfed mosquitoes, indicating that they are stable constituents of the mosquito virome. Multiple viral species from the same family were detected within pooled samples, consistent with either co-infection of individual mosquitoes or heterogeneous infections among pooled individuals. Overall, this study reveals a diverse ISV landscape in PNG Culex mosquitoes, expanding the known geographic range and genetic diversity of multiple viral groups and providing a baseline for future investigations into ISV ecology and evolution in the Indo-Pacific region and mosquito-virus interactions globally.
