Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Phosphorylation of the PA subunit of influenza polymerase at Y393 prevents binding of the 5'-termini of RNA and polymerase function.

The influenza A virus (IAV) polymerase is a multifunctional machine that can adopt alternative configurations to perform transcription and replication of the viral RNA genome in a temporally ordered manner. Although the structure of polymerase is well understood, our knowledge of its regulation by phosphorylation is still incomplete. The heterotrimeric polymerase can be regulated by posttranslational modifications, but the endogenously occurring phosphorylations at the PA and PB2 subunits of the IAV polymerase have not been studied. Mutation of phosphosites in PB2 and PA subunits revealed that PA mutants resembling constitutive phosphorylation have a partial (S395) or complete (Y393) defect in the ability to synthesize mRNA and cRNA. As PA phosphorylation at Y393 prevents binding of the 5' promoter of the genomic RNA, recombinant viruses harboring such a mutation could not be rescued. These data show the functional relevance of PA phosphorylations to control the activity of viral polymerase during the influenza infectious cycle.

Assessing compatibility and viral fitness between poultry-adapted H9N2 and wild bird-derived neuraminidases.

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2Δ(H6), rH9N6Δ and rH9N9Δ compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

MEK inhibitors as novel host-targeted antivirals with a dual-benefit mode of action against hyperinflammatory respiratory viral diseases.

Acute hyperinflammatory virus infections, such as influenza or coronavirus disease-19, are still a major health burden worldwide. In these diseases, a massive overproduction of pro-inflammatory cytokines and chemokines (cytokine storm syndrome) determine the severity of the disease, especially in late stages. Direct-acting antivirals against these pathogens have to be administered very early after infection to be effective and may induce viral resistance. Here, we summarize data on a host-targeted strategy using inhibitors of the cellular Raf/MEK/ERK kinase cascade that not only block replication of different RNA viruses but also suppress the hyperinflammatory cytokine response upon infection. In the first phase-II clinical trial of that approach, the MEK inhibitor Zapnometinib shows evidence of clinical benefit.

Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex.

The influenza A virus (IAV)-encoded matrix protein 1 (M1) acts as a master regulator of virus replication and fulfills multiple structural and regulatory functions in different cell compartments. Therefore, the spatiotemporal regulation of M1 is achieved by different mechanisms, including its structural and pH-dependent flexibility, differential association with cellular factors, and posttranslational modifications. Here, we investigated the function of M1 phosphorylation at the evolutionarily conserved threonine 108 (T108) and found that its mutation to a nonphosphorylatable alanine prohibited virus replication. Absent T108, phosphorylation led to strongly increased self-association of M1 at the cell membrane and consequently prohibited its ability to enter the nucleus and to contribute to viral ribonucleoprotein nuclear export. M1 T108 phosphorylation also controls the binding affinity to the cellular STRIPAK (striatin-interacting phosphatases and kinases) complex, which contains different kinases and the phosphatase PP2A to shape phosphorylation-dependent signaling networks. IAV infection led to the redistribution of the STRIPAK scaffolding subunits STRN and STRN3 from the cell membrane to cytosolic and perinuclear clusters, where it colocalized with M1. Inactivation of the STRIPAK complex resulted in compromised M1 polymerization and IAV replication. IMPORTANCE Influenza viruses pose a major threat to human health and cause annual epidemics and occasional pandemics. Many virus-encoded proteins exert various functions in different subcellular compartments, as exemplified by the M1 protein, but the molecular mechanisms endowing the multiplicity of functions remain incompletely understood. Here, we report that phosphorylation of M1 at T108 is essential for virus replication and controls its propensity for self-association and nuclear localization. This phosphorylation also controls binding affinity of the M1 protein to the STRIPAK complex, which contributes to M1 polymerization and virus replication.

Differential Impact of Specific Amino Acid Residues on the Characteristics of Avian Influenza Viruses in Mammalian Systems.

Avian influenza virus (AIV) H9N2 was declared to be endemic in birds of the Middle East, in particular in Egypt, with multiple cases of human infections. Despite concerns about the pandemic threat posed by H9N2 AIV, due to the fact that its receptor specificity is similar to that of human influenza viruses, its morbidity and mortality rates in humans are so far negligible. However, the acquisition of specific adaptive amino acid (aa) mutations in the viral polymerase can enhance cross-species transmission of the virus itself or of reassortants, which gained these changes. The polymerase basic protein 2 (PB2) is one of the key determinants for AIV adaptation towards mammals. Although mammalian pathogenicity-related mutations (MPMs) in PB2 genes were identified in different AIVs, the specific effect of single or multiple mutations on viral fitness has not been compared so far. Here, we studied the effect of the aa K at position 591, which was frequently reported in the PB2 of Egyptian H9N2 isolates, on the proliferation efficiency and polymerase activity of an H5N1 (clade 2.2.1.2) AIV already carrying the mammalian adaptive mutation 627K. Using reverse genetics, we generated a set of recombinant parental strains and H5N1 variants carrying the avian-like 591Q/627E or mammalian-like adaptive mutations 591K/627K (H5N1EGY, H9N2EGY, H5N1PB2-H9N2EGY, H5N1H9N2_PB2_K591Q, H5N1PB2_K627E, H5N1PB2_K627E/591K, H5N1PB2_627K/591K). Regardless of the avian-like 627E or the mammalian-adaptive 627K, both variants carrying the 591K (H5N1PB2_K627E/591K, H5N1PB2_627K/591K) and the reassortant H5N1PB2-H9N2EGY replicated to significantly higher levels in mammalian continuous MDCK and Calu-3 cell lines and primary normal human bronchial epithelial cells than the parental H5N1EGY virus (carrying solely the 627K adaptive mutation). Expectedly, the H5N1 variants carrying avian-like PB2 mutations (H5N1H9N2_PB2_K591Q, H5N1PB2_K627E) replicated to significantly lower levels than the parental H5N1EGY virus in the predefined primary and continuous mammalian cell line systems. Consistently, the activity of H5N1 subtype AIV polymerase complexes comprising PB2 segments with singular 591K or combined with 627K was significantly enhanced when compared to parental H5N1EGY and H9N2EGY. This study emphasizes the significant impact of 591K containing PB2 segments in the background of H5N1 polymerase on viral fitness in addition to the well-known MPM 627K in vitro.

Correction: Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

[This corrects the article DOI: 10.1371/journal.ppat.1010219.].

Broad Antiviral Effects of Echinacea purpurea against SARS-CoV-2 Variants of Concern and Potential Mechanism of Action.

SARS-CoV-2 variants of concern (VOCs) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Broad-spectrum antivirals could play an important role to control infections. The activity of Echinacea purpurea (Echinaforce® extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish), B1.525 (eta), and B.1.1.529.BA1 (omicron); (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild type SARS-CoV-2 (Hu-1) was analyzed. Molecular dynamics (MD) were applied to study the interaction of Echinacea's phytochemical markers with known pharmacological viral and host cell targets. EF extract broadly inhibited the propagation of all investigated SARS-CoV-2 VOCs as well as the entry of SARS-CoV-2 pseudoparticles at EC50's ranging from 3.62 to 12.03 µg/mL. The preventive addition of 25 µg/mL EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) and OC43. MD analyses showed constant binding affinities to VOC-typical S protein variants for alkylamides, caftaric acid, and feruloyl-tartaric acid in EF extract and interactions with serine protease TMPRSS-2. EF extract demonstrated stable virucidal activity across seven tested VOCs, likely due to the constant affinity of the contained phytochemical substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain the cell protective benefits of the extract by the inhibition of membrane fusion and cell entry. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.

Comparative kinase activity profiling of pathogenic influenza A viruses reveals new anti- and pro-viral protein kinases.

Constant evolution of influenza A viruses (IAVs) leads to the occurrence of new virus strains, which can cause epidemics and occasional pandemics. Here we compared two medically relevant IAVs, namely A/Hamburg/4/09 (H1N1pdm09) of the 2009 pandemic and the highly pathogenic avian IAV human isolate A/Thailand/1(KAN-1)/2004 (H5N1), for their ability to trigger intracellular phosphorylation patterns using a highly sensitive peptide-based kinase activity profiling approach. Virus-dependent tyrosine phosphorylations of substrate peptides largely overlap between the two viruses and are also strongly overrepresented in comparison to serine/threonine peptide phosphorylations. Both viruses trigger phosphorylations with distinct kinetics by overlapping and different kinases from which many form highly interconnected networks. As approximately half of the kinases forming a signalling hub have no known function for the IAV life cycle, we interrogated selected members of this group for their ability to interfere with IAV replication. These experiments revealed negative regulation of H1N1pdm09 and H5N1 replication by NUAK [novel (nua) kinase] kinases and by redundant ephrin A (EphA) receptor tyrosine kinases.

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity.

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5'-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5'-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.

Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

Improved in vitro Efficacy of Baloxavir Marboxil Against Influenza A Virus Infection by Combination Treatment With the MEK Inhibitor ATR-002.

Currently, all available antiviral drugs against influenza virus (IV) that target the virus proteins directly, like Baloxavir acid (BXA), lead to viral resistance. Therefore, cellular mechanisms and factors essential for IV replication are promising antiviral targets. As IV strongly depends on the virus-induced Raf/MEK/ERK signal pathway for efficient generation of infectious progeny virions, this pathway represents an important target. We aimed to determine whether the MEK inhibitor ATR-002 (PD0184264) is able to impair replication of BXA-resistant influenza A virus (IAV) and whether a treatment combining BXA and ATR-002 improves the therapeutic efficiency in vitro. A549 cells infected with different IAV strains including BXA-resistant variants were treated with ATR-002 or BXA and the effect on virus titer reduction was determined. The synergistic effect of ATR-002 and BXA was also analyzed using different evaluation methods. The data demonstrated that ATR-002 has a significant and dose-dependent inhibitory effect on IAV replication across different strains and subtypes. IAV with the PA-I38T mutation shows resistance against BXA, but is still susceptible toward ATR-002. The combination of ATR-002 and BXA exhibited a synergistic potency reflected by low combination index values. In conclusion, we show that ATR-002 permits to counteract the limitations of BXA against BXA-resistant IAV. Moreover, the results support the use of ATR-002 (i) in a mono-therapy, as well as (ii) in a combined approach together with BXA. These findings might also apply to the treatment of infections with IAV, resistant against other direct-acting antiviral compounds.

The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a severe respiratory disease with varying clinical presentations and outcomes, and responsible for a major pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against multiple RNA viruses including coronaviruses. Specifically, rocaglates inhibit eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. Here, we assessed the antiviral activity of the synthetic rocaglate CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In primary human airway epithelial cells, CR-31-B (-) reduced viral titers to undetectable levels at a concentration of 100 nM. Reduced virus reproduction was accompanied by substantially reduced viral protein accumulation and replication/transcription complex formation. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

Genetic incompatibilities and reduced transmission in chickens may limit the evolution of reassortants between H9N2 and panzootic H5N8 clade 2.3.4.4 avian influenza virus showing high virulence for mammals.

The unprecedented spread of H5N8- and H9N2-subtype avian influenza virus (AIV) in birds across Asia, Europe, Africa, and North America poses a serious public health threat with a permanent risk of reassortment and the possible emergence of novel virus variants with high virulence in mammals. To gain information on this risk, we studied the potential for reassortment between two contemporary H9N2 and H5N8 viruses. While the replacement of the PB2, PA, and NS genes of highly pathogenic H5N8 by homologous segments from H9N2 produced infectious H5N8 progeny, PB1 and NP of H9N2 were not able to replace the respective segments from H5N8 due to residues outside the packaging region. Furthermore, exchange of the PB2, PA, and NS segments of H5N8 by those of H9N2 increased replication, polymerase activity and interferon antagonism of the H5N8 reassortants in human cells. Notably, H5N8 reassortants carrying the H9N2-subtype PB2 segment and to lesser extent the PA or NS segments showed remarkably increased virulence in mice as indicated by rapid onset of mortality, reduced mean time to death and increased body weight loss. Simultaneously, we observed that in chickens the H5N8 reassortants, particularly with the H9N2 NS segment, demonstrated significantly reduced transmission to co-housed chickens. Together, while the limited capacity for reassortment between co-circulating H9N2 and H5N8 viruses and the reduced bird-to-bird transmission of possible H5N8 reassortants in chickens may limit the evolution of such reassortant viruses, they show a higher replication potential in human cells and increased virulence in mammals.

Anti-Influenza Activity of the Ribonuclease Binase: Cellular Targets Detected by Quantitative Proteomics.

Unpredictable influenza pandemics, annual epidemics, and sporadic poultry-to-human avian influenza virus infections with high morbidity and mortality rates dictate a need to develop new antiviral approaches. Targeting cellular pathways and processes is a promising antiviral strategy shown to be effective regardless of viral subtypes or viral evolution of drug-resistant variants. Proteomics-based searches provide a tool to reveal the druggable stages of the virus life cycle and to understand the putative antiviral mode of action of the drug(s). Ribonucleases (RNases) of different origins not only demonstrate antiviral effects that are mediated by the direct RNase action on viral and cellular RNAs but can also exert their impact by signal transduction modulation. To our knowledge, studies of the RNase-affected cell proteome have not yet been performed. To reveal cellular targets and explain the mechanisms underlying the antiviral effect employed by the small extra-cellular ribonuclease of Bacillus pumilus (binase) both in vitro and in vivo, qualitative shotgun and quantitative targeted proteomic analyses of the influenza A virus (IAV) H1N1pdm09-infected A549 cells upon binase treatment were performed. We compared proteomes of mock-treated, binase-treated, virus-infected, and virus-infected binase-treated cells to determine the proteins affected by IAV and/or binase. In general, IAV demonstrated a downregulating strategy towards cellular proteins, while binase had an upregulating effect. With the help of bioinformatics approaches, coregulated cellular protein sets were defined and assigned to their biological function; a possible interconnection with the progression of viral infection was conferred. Most of the proteins downregulated by IAV (e.g., AKR1B1, AKR1C1, CCL5, PFN1, RAN, S100A4, etc.) belong to the processes of cellular metabolism, response to stimulus, biological regulation, and cellular localization. Upregulated proteins upon the binase treatment (e.g., AKR1B10, CAP1, HNRNPA2B1, PFN1, PPIA, YWHAB, etc.) are united by the processes of biological regulation, cellular localization, and immune and metabolic processes. The antiviral activity of binase against IAV was expressed by the inversion of virus-induced proteomic changes, resulting in the inhibition of virus-associated processes, including nuclear ribonucleoprotein export (NCL, NPM1, Nup205, and Bax proteins involved) and cytoskeleton remodeling (RDX, PFN1, and TUBB) induced by IAV at the middle stage of single-cycle infection in A549 cells. Modulation of the immune response could be involved as well. Overall, it seems possible that binase exerts its antiviral effects in multiple ways.

PA from a Recent H9N2 (G1-Like) Avian Influenza a Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems.

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1EGY and H9N2EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2EGY, expressed individually and in combination in the genetic background of H5N1EGY, were genetically compatible with the other H5N1EGY segments. At 37 °C the replication efficiencies of H5N1EGY reassortants expressing the H9N2EGY polymerase subunits PB2 and PA (H5N1PB2-H9N2EGY, H5N1PA-H9N2EGY) were higher than the wild-type H5N1EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1PB2-H9N2EGY, but reduced for H5N1PA-H9N2EGY. At 33 °C and 39 °C, H5N1PB2-H9N2EGY and H5N1PA-H9N2EGY replicated to higher levels than the wild-type H5N1EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1EGY. Genetic analysis of the polymerase-encoding segments revealed that the PAH9N2EGY and PB2H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1EGY enabled the mutant virus H5N1PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.

Combinatory Treatment with Oseltamivir and Itraconazole Targeting Both Virus and Host Factors in Influenza A Virus Infection.

Influenza virus infections and their associated morbidity and mortality are a major threat to global health. Vaccination is an effective influenza prevention measure; however, the effectiveness is challenged by the rapid changes in the influenza virus genome leading to viral adaptation. Emerging viral resistance to the neuraminidase inhibitor oseltamivir limits the treatment of acute influenza infections. Targeting influenza virus­host interactions is a new and emerging field, and therapies based on the combination of virus­ and host­directed drugs might significantly improve treatment success. We therefore assessed the combined treatment with oseltamivir and the repurposed antifungal drug itraconazole on infection of polarized broncho­epithelial Calu-3 cells with pdm09 or Panama influenza A virus strains. We detected significantly stronger antiviral activities in the combined treatment compared to monotherapy with oseltamivir, permitting lower concentrations of the drug than required for the single treatments. Bliss independence drug interaction analysis indicated that both drugs acted independently of each other. The additional antiviral effect of itraconazole might safeguard patients infected with influenza virus strains with heightened oseltamivir resistance.