Minion, J. | bioRxiv Science

bioRxiv2024

Tiled PCR amplification-based Whole Genome Sequencing and Phylogenetic Classification Accelerate the Implementation of Respiratory Syncytial Virus Genomic surveillance in Canada as a Pilot Study

Whole genome sequencing (WGS) has emerged as a powerful tool to facilitate the study of existing and emerging infectious diseases. WGS-based genomic surveillance provides information on the genetic diversity and tracks the evolution of important viral pathogens including Respiratory Syncytial Virus (RSV). Development and implementation of robust tiled multiplex PCR amplification-based WGS assays will facilitate high-throughput RSV surveillance initiatives. In this study, we developed multiplex PCR assays for targeted enrichment of viral genomes using PrimalScheme (http://primal.zibraproject.org) to amplify over 97% of the genome in the majority of contemporaneous specimens tested. A pilot dataset comprising 52 RSVA and 37 RSVB genomes derived from Canadian clinical specimens during the 2022-2023 respiratory virus season were used to perform phylogenetic analyses using both near complete genome and Glycoprotein (G) sequences. Overall, the RSV phylogenetic tree built with whole genomes showed identical lineage clusters as that compared to the G gene, but showed more confidence and discriminatory features within individual lineage. Moreover, availability of whole genomes enabled the identification of a broader range of mutations, for instance the identified S377N, K272M, S276N, S211N, S206I and S209Q in Canadian fusion proteins that could be potentially associated with effectiveness of vaccines or antiviral-based therapeutics. In conclusion, the tiled-PCR amplification assays described offer a more streamlined approach to facilitate high-throughput, high sensitivity of RSV WGS, which is capable of supporting enhanced genomic surveillance initiatives, as well as the more comprehensive genomic analyses required to inform public health strategies for the development and usage of vaccines and antiviral drugs. IMPORTANCEWe present assays to efficiently sequence genomes of the RSVA and RSVB. This enables researchers and public health agencies to acquire high-quality genomic data using rapid and cost-effective approaches. Genomic data based comparative analysis can be used to conduct surveillance and monitor circulating isolates for efficacy of vaccines and antiviral therapeutics.

genomics↗

bioRxiv2024

Phenotypic diversity and shared genomic determinants among isolates causing a large incidence of disseminated gonococcal infections in Canada

The incidence of disseminated gonococcal infection (DGI) has remained low since the advent of antibiotics, however recent surge in DGI have inexplicably emerged within several regions during the past decade. In an effort to understand whether Neisseria gonorrhoeae that cause disseminated disease can be differentiated from non-invasive strains, we have performed a phenotypic and genotypic analysis on a selection of isolates obtained from invasive and uncomplicated infections in Canada. Phenotypic analysis of a matched subset of 19 isolates obtained since 2013 found that these varied in their capacity to aggregate in suspension and in their association with serum complement proteins, however these interactions did not discriminate between the invasive and mucosal isolates. Sequence typing of 360 Canadian isolates revealed that two porB alleles are significantly associated with the DGI strains, one of these being present throughout the past decade whereas the other became associated more recently. A PopNet-based population dynamics analysis, which instead establishes relationships based upon variance among discrete chromosomal segments, found that DGI isolates were restricted in their phylogenetic distribution. While this implies a genetically-linked potential to cause invasive disease, it cannot distinguish between an inherent difference in the phenotype of these populations or the horizontal exchange of some virulence factor among closely related strains. Regardless, a large number of genetic determinants are enriched in the DGI strains, making these enticing candidates for future work to understand how they might either promote the gonococcal capacity to cause systemic infection or reduce the presentation of clinical symptoms from localized infection so that it remains untreated. AUTHOR SUMMARYNeisseria gonorrhoeae is a sexually transmitted bacteria that causes over 82 million cases of gonorrhea each year. With its re-emergence, rising incidence rates and the prevalence of multidrug resistant strains increasing, the bacteria is considered to be a high burden threat to global public health. While disseminated gonococcal disease arising from untreated infections are uncommon, there have recently been regions with high incidence invasive disease. Here, we take advantage of the active ongoing collection of gonococcal isolates in Canada to perform a combined phenotypic and genotypic analysis that aims to understand whether certain strains are more often linked to invasive infections. We found that all disseminated isolates bound to the complement regulatory factors factor H and/or C4 binding protein to facilitate their resistance to the bactericidal activity of serum, but this was not sufficient to explain the heightened instance of invasive disease. While classical genome-based phylogenetic analysis displayed little association between invasive strains, PopNet-based analysis revealed that invasive isolates fell within defined sub-populations and indicated variant alleles enriched among the disseminating bacteria, and a broader pan-genome approach revealed genes more likely to be present in invasive strains. Our study thereby provides support for a genetic contribution to the invasive potential of gonococcal isolates and provides candidate drivers of this virulent outcome.

microbiology↗