Influenza A virus is a major human pathogen responsible for seasonal epidemics as well as pandemic outbreaks. Due to the continuing burden on human health, the need for new tools to study influenza virus pathogenesis as well as to evaluate new therapeutics is paramount. We report the development of a stable, replication-competent luciferase reporter influenza A virus that can be used for in vivo imaging of viral replication. This imaging is noninvasive and allows for the longitudinal monitoring of infection in living animals. We used this tool to characterize novel monoclonal antibodies that bind the conserved stalk domain of the viral hemagglutinin of H1 and H5 subtypes and protect mice from lethal disease. The use of luciferase reporter influenza viruses allows for new mechanistic studies to expand our knowledge of virus-induced disease and provides a new quantitative method to evaluate future antiviral therapies.

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higherorder NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.antiinfluenza | oligomerization | polymerase inhibitor | protein-protein interaction | cooperative inhibition

Without baseline human immunity to the emergent avian influenza A(H7N9) virus, neuraminidase inhibitors are vital for controlling viral replication in severe infections. An amino acid change in the viral neuraminidase associated with drug resistance, NA-R292K (N2 numbering), has been found in some H7N9 clinical isolates. Here we assess the impact of the NA-R292K substitution on antiviral sensitivity and viral replication, pathogenicity and transmissibility of H7N9 viruses. Our data indicate that an H7N9 isolate encoding the NA-R292K substitution is highly resistant to oseltamivir and peramivir and partially resistant to zanamivir. Furthermore, H7N9 reassortants with and without the resistance mutation demonstrate comparable viral replication in primary human respiratory cells, virulence in mice and transmissibility in guinea pigs. Thus, in stark contrast to oseltamivir-resistant seasonal influenza A(H3N2) viruses, H7N9 virus replication and pathogenicity in these models are not substantially altered by the acquisition of high-level oseltamivir resistance due to the NA-R292K mutation.

Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus hemagglutinin and stalk-based universal influenza virus vaccine strategies are being developed as promising countermeasures for influenza virus infections. The pan-H1-reactive monoclonal antibody 6F12 has been extensively characterized and shows broad efficacy against divergent H1N1 strains in the mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret model of influenza disease. Furthermore, we recently developed a universal influenza virus vaccine strategy based on chimeric hemagglutinin constructs that focuses the immune response on the conserved stalk domain of the hemagglutinin. Here we set out to test this vaccination strategy in the ferret model. Both strategies, pretreatment of animals with a stalk-reactive monoclonal antibody and vaccination with chimeric hemagglutinin-based constructs, were able to significantly reduce viral titers in nasal turbinates, lungs, and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal wash viral titers. In summary, both strategies showed efficacy in reducing viral loads after an influenza virus challenge in the ferret model. IMPORTANCEInfluenza virus hemagglutinin stalk-reactive antibodies tend to be less potent yet are more broadly reactive and can neutralize seasonal and pandemic influenza virus strains. The ferret model was used to assess the potential of hemagglutinin stalk-based immunity to provide protection against influenza virus infection. The novelty and significance of the findings described in this report support the development of vaccines stimulating stalk-specific antibody responses.

Zika virus is a mosquito-borne flavivirus closely related to dengue virus that can cause severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Specific treatments and vaccines for Zika virus are not currently available. Here, we isolate and characterize four monoclonal antibodies (mAbs) from an infected patient that target the non-structural protein NS1. We show that while these antibodies are non-neutralizing, NS1-specific mAbs can engage FcγR without inducing antibody dependent enhancement (ADE) of infection in vitro. Moreover, we demonstrate that mAb AA12 has protective efficacy against lethal challenges of African and Asian lineage strains of Zika virus in Stat2–/– mice. Protection is Fc-dependent, as a mutated antibody unable to activate known Fc effector functions or complement is not protective in vivo. This study highlights the importance of the ZIKV NS1 protein as a potential vaccine antigen.