Characterization of the in Vitro and in Vivo Efficacy of Baloxavir Marboxil against h5 Highly Pathogenic Avian Influenza Virus Infection

Human infections caused by the H5 highly pathogenic avian influenza virus (HPAIV) sporadically threaten public health. The susceptibility of HPAIVs to baloxavir acid (BXA), a new class of inhibitors for the influenza virus cap-dependent endonuclease, has been confirmed in vitro, but it has not yet been fully characterized. Here, the efficacy of BXA against HPAIVs, including recent H5N8 variants, was assessed in vitro. The antiviral efficacy of baloxavir marboxil (BXM) in H5N1 virus-infected mice was also investigated. BXA exhibited similar in vitro activities against H5N1, H5N6, and H5N8 variants tested in comparison with seasonal and other zoonotic strains. Compared with oseltamivir phosphate (OSP), BXM monotherapy in mice infected with the H5N1 HPAIV clinical isolate, the A/Hong Kong/483/1997 strain, also caused a significant reduction in viral titers in the lungs, brains, and kidneys, thereby preventing acute lung inflammation and reducing mortality. Furthermore, compared with BXM or OSP monotherapy, combination treatments with BXM and OSP using a 48-hour delayed treatment model showed a more potent effect on viral replication in the organs, accompanied by improved survival. In conclusion, BXM has a potent antiviral efficacy against H5 HPAIV infections.

Type I IFN promotes pathogenic inflammatory monocyte maturation during H5N1 infection

Ly6Chi inflammatory monocytes show high IFN responses, and contribute to both protective and pathogenic functions following influenza virus infection. In order to understand the significance of IFN responses in this subset, we examined monocytes during infection with a lethal H5N1 virus that induces high levels of IFN and a low-pathogenicity H1N1 virus that induces low levels of IFN. We show that H5N1 infection results in early recruitment of high numbers of Ly6Chi monocytes and induction of chemokines and Ifnb1. Using unbiased transcriptomic and proteomic approaches, we also find that monocytes are significantly enriched during H5N1 infection and are associated with chemokine and IFN signatures in mice, and with severity of symptoms after influenza virus infection in humans. Recruited Ly6Chi monocytes subsequently become infected in the lung, produce IFN-β, and mature into FasL+ monocyte-derived cells (FasL+MCs) expressing dendritic cell markers. Both Ccr2-/- and Faslgld mice are protected from lethal infection, indicating that monocytes contribute to pathogenesis. Global loss of type I and type III IFN signaling in Stat2-/- mice results in loss of monocyte recruitment, likely reflecting a requirement for IFN-dependent chemokine induction. Here we show that IFN is not directly required for monocyte recruitment on an IFN-sufficient background, but is required for maturation to FasL+MCs. Loss of IFN signaling skews to a Ly6Clo phenotype associated with tissue repair, suggesting that IFN signaling in monocytes is a critical determinant of influenza virus pathogenesis.

Characterization of the In Vitro and In Vivo Efficacy of Baloxavir Marboxil against H5 Highly Pathogenic Avian Influenza Virus Infection

Human infections caused by the H5 highly pathogenic avian influenza virus (HPAIV) sporadically threaten public health. The susceptibility of HPAIVs to baloxavir acid (BXA), a new class of inhibitors for the influenza virus cap-dependent endonuclease, has been confirmed in vitro, but it has not yet been fully characterized. Here, the efficacy of BXA against HPAIVs, including recent H5N8 variants, was assessed in vitro. The antiviral efficacy of baloxavir marboxil (BXM) in H5N1 virus-infected mice was also investigated. BXA exhibited similar in vitro activities against H5N1, H5N6, and H5N8 variants tested in comparison with seasonal and other zoonotic strains. Compared with oseltamivir phosphate (OSP), BXM monotherapy in mice infected with the H5N1 HPAIV clinical isolate, the A/Hong Kong/483/1997 strain, also caused a significant reduction in viral titers in the lungs, brains, and kidneys, thereby preventing acute lung inflammation and reducing mortality. Furthermore, compared with BXM or OSP monotherapy, combination treatments with BXM and OSP using a 48-h delayed treatment model showed a more potent effect on viral replication in the organs, accompanied by improved survival. In conclusion, BXM has a potent antiviral efficacy against H5 HPAIV infections.

Lipid-mediated biosynthetic labeling strategy for in vivo dynamic tracing of avian influenza virus infection

Monitoring the infection behavior of avian influenza viruses is crucial for understanding viral pathogenesis and preventing its epidemics among people. A number of viral labeling methods have been utilized for tracking viral infection process, but most of them are laborious or decreasing viral activity. Herein we explored a lipid biosynthetic labeling strategy for dynamical tracking the infection of H5N1 pseudotype virus (H5N1p) in host. Biotinylated lipids (biotinyl Cap-PE) were successfully incorporated into viral envelope when it underwent budding process by taking advantage of host cell-derived lipid metabolism. Biotin-H5N1p virus was effectively in situ–labeled with streptavidin-modified near-infrared quantum dots (NIR SA-QDs) using streptavidin-biotin conjugation with well-preserved virus activities. Dual-labeled imaging obviously shows that H5N1p viruses are primarily taken up in host cells via clathrin-mediated endocytosis. In animal models, Virus-conjugated NIR QDs displayed extraordinary photoluminescence, superior stability, and tissue penetration in lung, allowing us to long-term monitor respiratory viral infection in a noninvasive manner. Importantly, the co-localization of viral hemagglutinin protein and QDs in infected lung further conformed the dynamic infection process of virus in vivo. Hence, this in situ QD-labeling strategy based on cell natural biosynthesis provides a brand-new and reliable tool for noninvasion visualizing viral infection in body in a real-time manner.

Influenza Virus Down-Modulates G6PD Expression and Activity to Induce Oxidative Stress and Promote Its Replication

Influenza virus infection induces oxidative stress in host cells by decreasing the intracellular content of glutathione (GSH) and increasing reactive oxygen species (ROS) level. Glucose-6-phosphate dehydrogenase (G6PD) is responsible for the production of reducing equivalents of nicotinamide adenine dinucleotide phosphate (NADPH) that is used to regenerate the reduced form of GSH, thus restoring redox homeostasis. Cells deficient in G6PD display elevated levels of ROS and an increased susceptibility to viral infection, although the consequences of G6PD modulation during viral infection remain to be elucidated. In this study, we demonstrated that influenza virus infection decreases G6PD expression and activity, resulting in an increase in oxidative stress and virus replication. Moreover, the down regulation of G6PD correlated with a decrease in the expression of nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor that regulates the expression of the antioxidant response gene network. Also down-regulated in influenza virus infected cells was sirtuin 2 (SIRT2), a NADPH-dependent deacetylase involved in the regulation of G6PD activity. Acetylation of G6PD increased during influenza virus infection in a manner that was strictly dependent on SIRT2 expression. Furthermore, the use of a pharmacological activator of SIRT2 rescued GSH production and NRF2 expression, leading to decreased influenza virus replication. Overall, these data identify a novel strategy used by influenza virus to induce oxidative stress and to favor its replication in host cells. These observations furthermore suggest that manipulation of metabolic and oxidative stress pathways could define new therapeutic strategies to interfere with influenza virus infection.

Vaccine-associated enhanced respiratory disease following influenza virus infection in ferrets recapitulates the model in pigs

Influenza A virus (IAV) causes respiratory disease in swine and humans. Vaccines are used to prevent influenza illness in both populations but must be frequently updated due to rapidly evolving strains. Mismatch between the circulating strains and strains contained in vaccines may cause loss in efficacy. Whole inactivated virus (WIV) vaccines with adjuvant utilized by the swine industry are effective against antigenically similar viruses; however, vaccine-associated enhanced respiratory disease (VAERD) may happen when the WIV is antigenically mismatched with the infecting virus. VAERD is a repeatable model in pigs, but had yet to be experimentally demonstrated in other mammalian species. We recapitulated VAERD in ferrets, a standard benchmark animal model for studying human influenza infection, in a direct comparison to VAERD in pigs. Both species were vaccinated with WIV with oil in water adjuvant containing a δ-1 H1N2 (1B.2.2) derived from the pre-2009 human seasonal lineage, then challenged with a 2009 pandemic H1N1 (H1N1pdm09, 1A.3.3.2) five weeks after vaccination. Nonvaccinated and challenged groups showed typical signs of influenza disease, but the mismatched vaccinated and challenged pigs and ferrets showed elevated clinical signs, despite similar viral loads. VAERD affected pigs exhibited a 2-fold increase in lung lesions, while VAERD affected ferrets showed a 4-fold increase. Similar to pigs, antibodies from VAERD affected ferrets preferentially bound to the HA2 domain of the H1N1pdm09 challenge strain. These results indicate VAERD is not limited to pigs, as demonstrated here in ferrets, and the need to consider VAERD when evaluating new vaccine platforms and strategies. Importance We demonstrated the susceptibility of ferrets, a laboratory model species for human influenza A virus research, to vaccine associated enhanced respiratory disease (VAERD) using an experimental model previously demonstrated in pigs. Ferrets developed clinical characteristics of VAERD very similar to that in pigs. The hemagglutinin (HA) stalk is a potential vaccine target to develop more efficacious, broadly reactive influenza vaccine platforms and strategies. However, non-neutralizing antibodies directed towards a conserved epitope on the HA stalk induced by an oil-in-water adjuvanted whole influenza virus vaccine were previously shown in VAERD-affected pigs and were also identified here in VAERD-affected ferrets. The induction of VAERD in ferrets highlights the potential risk of mismatched influenza vaccines to humans and the need to consider VAERD when designing and evaluating vaccine strategies.

Relapsing Neurological Complications in a Child With ATP1A3 Gene Mutation and Influenza Infection: A Case Report

Mutations in the ATP1A3 gene encoding the α3 subunit of Na+/K+-ATPase are associated with different neurological manifestations that may be elicited by febrile episodes. A recently described phenotype, linked to the p.Arg756Cys mutation, is clinically characterized by Relapsing Encephalopathy with Cerebellar Ataxia (RECA). In our case, a diagnosis of RECA has been established, and despite an alternative, reasonable cause had been already identified.We describe the case of a child with two recurrent episodes, 2 years apart, of hypotonia and ataxia. In both episodes, a laboratory-confirmed influenza virus infection suggested the diagnosis of influenza-associated encephalopathy. After the second episode, a search for genetic mutations was performed, and ATP1A3 mutation associated to RECA was found. After both episodes, the child was discharged after partial improvement of neurological conditions.The diagnosis of encephalopathy in children is often challenging. A genetic predisposition to neurological decompensation should be suspected in case of recurrent episodes, even if an alternative diagnosis has been established. Indeed, febrile infections may only represent the trigger of neurological involvement. In these patients, the knowledge of a genetic predisposing factors may help in the prevention of neurological episodes by the prompt use of anti-pyrectics and preventive measures as appropriate vaccination.

Could Interleukin-33 (IL-33) Govern the Outcome of an Equine Influenza Virus Infection? Learning from Other Species

Influenza A viruses (IAVs) are important respiratory pathogens of horses and humans. Infected individuals develop typical respiratory disorders associated with the death of airway epithelial cells (AECs) in infected areas. Virulence and risk of secondary bacterial infections vary among IAV strains. The IAV non-structural proteins, NS1, PB1-F2, and PA-X are important virulence factors controlling AEC death and host immune responses to viral and bacterial infection. Polymorphism in these proteins impacts their function. Evidence from human and mouse studies indicates that upon IAV infection, the manner of AEC death impacts disease severity. Indeed, while apoptosis is considered anti-inflammatory, necrosis is thought to cause pulmonary damage with the release of damage-associated molecular patterns (DAMPs), such as interleukin-33 (IL-33). IL-33 is a potent inflammatory mediator released by necrotic cells, playing a crucial role in anti-viral and anti-bacterial immunity. Here, we discuss studies in human and murine models which investigate how viral determinants and host immune responses control AEC death and subsequent lung IL-33 release, impacting IAV disease severity. Confirming such data in horses and improving our understanding of early immunologic responses initiated by AEC death during IAV infection will better inform the development of novel therapeutic or vaccine strategies designed to protect life-long lung health in horses and humans, following a One Health approach.