Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus.

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Does the Profit Motive Matter? COVID-19 Prevention and Management in Ontario Long-Term-Care Homes.

We introduce evidence that for-profit long-term-care providers are associated with less successful outcomes in coronavirus disease 2019 outbreak management. We introduce two sets of theoretical arguments that predict variation in service quality by provider type: those that deal with the institution of contracting (innovative competition vs. erosive competition) and those that address organizational features of for-profit, non-profit, and government actors (profit seeking, cross-subsidization, and future investment). We contextualize these arguments through a discussion of how contracting operates in Ontario long-term care. That discussion leads us to exclude the institutional arguments while retaining the arguments about organizational features as our three hypotheses. Using outbreak data as of February 2021, we find that government-run long-term-care homes surpassed for-profit and non-profit homes in outbreak management, consistent with an earlier finding from Stall et al. (2020). Non-profit homes outperform for-profit homes but are outperformed by government-run homes. These results are consistent with the expectations derived from two theoretical arguments-profit seeking and cross-subsidization-and inconsistent with a third-capacity for future investment.

Dans cet article, nous présentons quelques éléments de preuve que les fournisseurs de soins de longue durée à but lucratif ont eu de moins bons résultats dans la gestion de la pandémie de la COVID-19. Nous avançons deux séries d'arguments théoriques qui prédisent la variation dans la qualité du service selon le type de fournisseur : ceux qui ont trait à l'institution contractante (concurrence novatrice versus concurrence érosive) et ceux qui s'intéressent aux caractéristiques organisationnelles des acteurs à but lucratif, sans but lucratif et gouvernementaux (recherche de profit, interfinancement et investissement futur). Nous mettons ces arguments en contexte en discutant les façons dont les contrats sont attribués dans le cas des soins de longue durée en Ontario. Cette analyse nous pousse à exclure les arguments institutionnels et à conserver, comme nos trois hypothèses, les arguments sur les caractéristiques organisationnelles. En utilisant les données relatives à l'épidémie à partir de février 2021, nous constatons que les établissements de soins de longue durée gouvernementaux ont surpassé les établissements à but lucratif et sans but lucratif dans la gestion de l'épidémie, ce qui concorde avec une conclusion antérieure par Stall et ses collègues (2020). Ces résultats concordent avec les attentes dérivées de deux arguments théoriques ­ la recherche du profit et l'interfinancement ­ mais pas avec le troisième, celui de l'investissement futur.

Determinants of attenuation in the envelope protein of the flavivirus Alfuy.

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM-E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273-277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD(50). Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.