Nanopore adaptive sampling for targeted mitochondrial genome sequencing and bloodmeal identification in hematophagous insects.

BACKGROUND: Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. METHODS: We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. RESULTS: Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively. CONCLUSIONS: Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel 'phylogenetic capture' methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens.

Potential Novel N-Glycosylation Patterns Associated with the Emergence of New Genetic Variants of PRRSV-2 in the U.S.

Glycosylation of proteins is a post-translational process where oligosaccharides are attached to proteins, potentially altering their folding, epitope availability, and immune recognition. In Porcine reproductive and respiratory syndrome virus-type 2 (PRRSV-2), positive selection pressure acts on amino acid sites potentially associated with immune escape through glycan shielding. Here, we describe the patterns of potential N-glycosylation sites over time and across different phylogenetic lineages of PRRSV-2 to better understand how these may contribute to patterns of coexistence and emergence of different lineages. We screened 19,179 PRRSV GP5 sequences (2004−2021) in silico for potential N-glycosylated sites. The emergence of novel combinations of N-glycosylated sites coincided with past PRRSV epidemics in the U.S. For lineage L1A, glycosylation at residues 32, 33, 44, 51, and 57 first appeared in 2012, but represented >62% of all L1A sequences by 2015, coinciding with the emergence of the L1A 1-7-4 strain that increased in prevalence from 8 to 86% of all L1A sequences from 2012 to 2015. The L1C 1-4-4 strain that emerged in 2020 also had a distinct N-glycosylation pattern (residues 32, 33, 44, and 51). From 2020 to 2021, this pattern was responsible for 44−47% of the L1C sequences, contrasting to <5% in years prior. Our findings support the hypothesis that antigenic evolution contributes to the sequential dominance of different PRRSV strains and that N-glycosylation patterns may partially account for antigenic differences amongst strains. Further studies on glycosylation and its effect on PRRSV GP5 folding are needed to further understand how glycosylation patterns shape PRRSV occurrence.