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.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The ΔSH and ΔG deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the ΔG-85473 and especially ΔSH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

The role of signaling pathway through macrophage colony-stimulating factor (mCSF) and its receptor CSF1R during experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE) was studied by two different approaches. Firstly, we evaluated the effect of a stimulation of mCSF/CSF1R axis before infection. Exogenous mCSF (40 μg/kg, i.p.) was administered once daily to BALB/c mice on days 4 and 2 before intranasal infection with 2,500 plaque forming units (PFUs) of HSV-1. mCSF treatment significantly increased mouse survival compared to saline (50% versus 10%; P=0.0169). On day 6 post-infection (p.i.), brain viral titers were significantly decreased whereas IFN-β was significantly increased in mice treated with mCSF compared to saline. The number of CD68+ (a phagocytosis marker) microglial cells was significantly increased in mCSF-treated mice compared to the saline group. Secondly, we conditionally depleted CSF1R on microglial cells of CSF1R-loxP-CX3CR1-cre/ERT2 mice (in a C57BL/6 background) through induction with tamoxifen. Mice were then infected intranasally with 600,000 PFUs of HSV-1. The survival rates of mice depleted in CSF1R (KO) was significantly lower than that of wild type (WT) (0% versus 67%). Brain viral titers and cytokines/chemokines were significantly higher in KO than in WT animals on day 6 p.i. Furthermore, an increased infiltration of monocytes into the brains of WT was seen on day 6 p.i. but not in KO mice. Our results suggest that microglial cells are essential to control HSE at early stages of the disease and that the mCSF/CSF1R axis could be a therapeutic target to regulate their response to infection. Importance Microglia appear to be one of the principal regulators of neuro-inflammation in the central nervous system (CNS). An increasing number of studies demonstrated that the activation of microglia could either result in beneficial or detrimental effects in different CNS disorders. Hence, the role of microglia during herpes simplex virus encephalitis (HSE) has not been fully characterized. Using experimental mouse models, we showed that an early activation of the mCSF/CSF1R axis improved the outcome of the disease possibly by inducing a proliferation of microglia. In contrast, depletion of microglia before HSV-1 infection worsened the prognosis of HSE. Thus, an early microglial response followed by a sustained infiltration of monocytes and T cells into the brain seem to be key components for a better clinical outcome. These data suggest that microglia could be a potential target for immunomodulatory strategies combined with antiviral therapy to better control the outcome of this devastating disease.