RESUMEN
BACKGROUND: Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. METHODS: Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. RESULTS: I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. CONCLUSION: The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oxazinas/farmacología , Piridinas/farmacología , ARN Polimerasa Dependiente del ARN/genética , Tiepinas/farmacología , Triazinas/farmacología , Proteínas Virales/genética , Sustitución de Aminoácidos , Animales , Antivirales/uso terapéutico , Dibenzotiepinas , Perros , Femenino , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Concentración 50 Inhibidora , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Morfolinas , Mutación , Infecciones por Orthomyxoviridae/virología , Oxazinas/uso terapéutico , Fenotipo , Piridinas/uso terapéutico , Piridonas , ARN Polimerasa Dependiente del ARN/metabolismo , Tiepinas/uso terapéutico , Triazinas/uso terapéutico , Carga Viral/efectos de los fármacos , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.