Sustained circulation of Aedes albopictus-derived novel almendraviruses in the urban parks

The Rhabdoviridae family comprises a diverse range of negative-sense single-stranded ribonucleic acid (RNA) viruses, including significant human and mammalian viruses transmitted by various arthropod species. Herein, using Aedes albopictus (Ae. albopictus) samples collected in two urban parks during 2023 and 2024, through metagenomics sequencing, 16 sequences were identified as putative novel viruses, showing closest homology to insect-specific viruses, mycoviruses, or plant-associated viruses. Notably, two novel viruses, Aedes albopictus almendravirus GCCDC15 (Aealb-AlmV GCCDC15) and Aedes albopictus almendravirus GCCDC16 (Aealb-AlmV GCCDC16) were identified and successfully isolated. Both of these viruses belong to the genus Almendravirus within the Rhabdoviridae family. Phylogenetic analysis revealed that Aealb-AlmV GCCDC15 and GCCDC16 are distantly related to Coot Bay virus (the United States of America, 2013) and Menghai rhabdovirus (Yunnan Province, China, 2017). The genetic distances between these two viruses and their most similar viruses are marked by 59.85 % and 87.20 % of amino acid identity in the L protein, respectively, supporting their classification as two new species in the Rhabdoviridae family. Cytopathic effects and rod-like virions were observed in mosquito cells (C6/36) after inoculating with supernatants from the Ae. albopictus samples. To investigate the natural distribution and persistence of the novel almendraviruses, we conducted a specific reverse transcription-polymerase chain reaction (RT-PCR) screening of Ae. albopictus mosquitoes collected from two urban parks across different time points. The assays confirmed the presence of both Aealb-AlmV GCCDC15 and GCCDC16 in mosquito populations. Critically, these viruses were detected repeatedly over successive sampling periods and in mosquitoes from geographically distinct sites within the urban environment. In summary, our study delineates the virome characteristics of Aedes mosquitoes in the urban ecosystem and successfully isolated two novel rhabdoviruses. The recurrent detection provides clear evidence for the sustained circulation of Ae. albopictus-derived almendraviruses in urban parks, highlighting their ongoing transmission and establishment in these habitats.