Pathogen surveillance by agnostic high-throughput sequencing in sand flies collected in the country of Georgia

Introduction Sand flies are known vectors of human pathogens that cause acute febrile illness (AFI). Understanding the pathogen landscape within these insects is crucial for biosurveillance and prevention of infectious diseases in U.S forces deployed to regions where sand flies are present. Methods We used agnostic high-throughput sequencing to perform genomic surveillance for the presence of known human pathogens associated with AFI and novel relatives thereof in sand flies ( Psychodidae: Phlebotominae ) collected in the country of Georgia between August 2017 and August 2023. A total of 102 sand fly pools, containing 1-37 specimens each and in some cases sorted by sex, were homogenized. Of those, 86 produced Illumina RNA libraries suitable for sequencing. The resulting data were queried in an entirely pathogen agnostic manner: sequencing reads were processed through two bioinformatic pipelines, VirusSeeker 2.0 and MetaDetector, to perform quality control steps, de novo assembly, and a DIAMOND analysis on reads and assembled contigs. Manual analysis was performed to identify expected pathogens and any other pathogens of notable abundance. Results with similarity to pathogens of human or veterinary importance, as well as low confidence results represented by a low number of reads were also subjected to in-depth analysis. Results This pathogen-agnostic assessment revealed both expected and unexpected results. We detected novel viruses consistent with insect vector viromes, including novel orthomyxoviruses that could merit further exploration and some vesiculovirus sequences that may represent endogenous viral elements. Although Leishmania was an expected finding, it was not detected with high confidence. A significant unexpected result was the detection of Rickettsia felis , a pathogen typically vectored by fleas and occasionally mosquitoes, in two separate pools, with 51% of the genome covered in one pool. Furthermore, we identified two distinct and complete novel phlebovirus genomes. Their genome divergence from previously known phleboviruses is so significant that they are not predicted to be amplified using any of the five published phlebovirus assays. Discussion These results are achieved through a combination of agnostic sequencing and detailed bioinformatic analyses, enabling the detection of novel viruses and an expansion of our understanding of vector-borne disease ecology.