Exploring associations between viral titer measurements and disease outcomes in ferrets inoculated with 125 contemporary influenza A viruses

Abstract

ABSTRACT As use of the ferret model to study influenza A virus (IAV) pathogenicity increases, periodic assessment of data generated in this model is warranted, to identify features associated with virus replication throughout the respiratory tract and to refine future analyses. However, protocol-specific differences present between independent laboratories limit easy aggregation of virological data. We compiled viral titer and clinical data from >1,000 ferrets inoculated with 125 contemporary IAV under a consistent experimental protocol (including high- and low-pathogenicity avian, swine-origin, and human viruses, spanning H1, H2, H3, H5, H7, and H9 subtypes) and examined which meaningful and statistically supported associations were present among numerous quantitative measurements. Viral titers correlated positively between ferret nasal turbinate tissue, lung tissue, and nasal wash specimens, though the strength of the associations varied, notably regarding the particular nasal wash summary measure employed and properties of the virus itself. Use of correlation coefficients and mediation analyses further supported the interconnectedness of viral titer measurements taken at different sites throughout the respiratory tract. IAV possessing mammalian host adaptation markers in the HA and PB2 exhibited more rapid growth in the ferret upper respiratory tract early after infection, supported by quantities derived from infectious titer data to capture infection progression, compared with viruses bearing hallmarks of avian IAV. Collectively, this work identifies summary metrics most closely linked with virological and phenotypic outcomes in ferrets, supporting continued refinement of data analyzed from in vivo experimentation, notably from studies conducted to evaluate the public health risk posed by novel and emerging IAV. IMPORTANCE Ferrets are frequently employed to study the pandemic potential of novel and emerging influenza A viruses. However, systematic retrospective analyses of data generated from these experiments are rarely performed, limiting our ability to identify trends in this data and explore how analyses can be refined. Using logarithmic viral titer and clinical data aggregated from one research group over 20 years, we assessed which meaningful and statistically supported associations were present among numerous quantitative measurements obtained from influenza A virus (IAV)-infected ferrets, including those capturing viral titers, infection progression, and disease severity. We identified numerous linear correlations between parameters assessing virus replication at discrete sites in vivo , including parameters capturing infection progression not frequently employed in the field, and sought to investigate the interconnected nature of these associations. This work supports continued refinement of data analyzed from in vivo experimentation, notably from studies which evaluate the public health risk posed by IAV.