Bruce, K. | Search bioRxiv

bioRxiv2020

Olfactory host entry supports herpesvirus recombination

Herpesvirus genomes record abundant recombination. Its impact on infection remains ill-defined. When co-infecting mice by the natural olfactory route, individually incapacitated Murid Herpesvirus-4 (MuHV-4) mutants routinely recombined to restore normal host colonization. Lung infection rescued much less well. Murine cytomegalovirus mutants deficient in salivary gland colonization also showed rescue via the nose but not the lungs. As nose and lung infections show similar spread, efficient recombination seemed specific to olfactory entry. Rescue of replication-deficient MuHV-4 implied co-infection of the first encountered cells, and this worked also with asynchronous inoculation, suggesting that latent virus could lie in wait for later reactivation. Inhaled MuHV-4 is commonly caught on respiratory mucus, which epithelial cilia push back towards the olfactory surface, and infection was correspondingly frequent at the anterior olfactory edge. Thus olfactory entry provides a general means for herpesviruses to meet. Author summaryInter-strain recombination allows viruses to optimise infection in diverse hosts. Many herpesviruses show past recombination. Yet they are ancient pathogens, so this past may be remote and recombination rare. Diverse herpesviruses enter new hosts via olfactory cells. We show that such entry routinely allows recombination between co-infecting virus strains, even when one strain cannot spread. Recombination was contrastingly rare after lung infection. Thus, entry via olfactory cells specifically supports frequent herpesvirus recombination.

microbiology↗

bioRxiv2020

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

The use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation - from field sampling to the analysis of PCR-based results - for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Hence, the general applicability of assays as well as associated uncertainties and limitations, often remain undetermined. The absence of clear guidelines for assay validation prevents targeted eDNA assays from being incorporated into species monitoring and policy; thus, their establishment is essential for realizing the potential of eDNA-based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks, (e.g. "in silico analysis"), and arranged on a 5-level validation scale from "incomplete" to "operational" with defined minimum validation criteria for each level. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. Of the 122 variables, 20% to 76% were reported; the majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterised by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling the appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.

molecular biology↗