PA Protein Research Articles

Synthesis and anti-influenza virus activity of substituted dibenzoxepine-based baloxavir derivatives

Seasonal influenza poses a significant threat to global public health, driving the need for effective anti-influenza agents. The PA protein, which captures the pre-mRNA cap structure, is crucial for the replication of the influenza virus and serves as an important target for developing such agents. Baloxavir, a PA inhibitor, has shown excellent activity against influenza A and B viruses. In this study, its structure was optimized using bioisosteric replacement to develop novel dibenzoxepine-based derivatives for combating influenza. As the lead compounds, ATV03 (EC50 = 0.78 ± 0.10 nM, SI > 64103) and ATV07 (EC50 = 0.78 ± 0.01 nM, SI = 31603) demonstrated excellent anti-influenza A (H3N2) activity and SI, and possessed favorable anti-influenza B activity, with 2.02 ± 0.40 nM and 2.32 ± 0.29 nM of EC50 respectively. They showed improved bioavailability and metabolic stability. Mechanism studies revealed that ATV03 and ATV07 both possessed significant activity in inhibiting PA and RdRp as well as disturbing NP. Consequently, ATV03 was selected for further investigation in the fight against seasonal and pandemic influenza due to its superior bioavailability, metabolic stability, and efficacy against multiple influenza A viruses.

Detection of influenza A(H3N2) viruses with polymerase acidic subunit substitutions after and prior to baloxavir marboxil treatment during the 2022–2023 influenza season in Japan

Baloxavir marboxil (baloxavir), approved as an anti-influenza drug in Japan in March 2018, can induce reduced therapeutic effectiveness due to PA protein substitutions. We assessed PA substitutions in clinical samples from influenza-infected children and adults pre- and post-baloxavir treatment, examining their impact on fever and symptom duration. During the 2022–2023 influenza season, the predominant circulating influenza subtype detected by cycling-probe RT-PCR was A(H3N2) (n = 234), with a minor circulation of A(H1N1)pdm09 (n = 10). Of the 234 influenza A(H3N2) viruses collected prior to baloxavir treatment, 2 (0.8%) viruses carry PA/I38T substitution. One virus was collected from a toddler and one from an adult, indicating the presence of viruses with reduced susceptibility to baloxavir, without prior exposure to the drug. Of the 54 paired influenza A(H3N2) viruses collected following baloxavir treatment, 8 (14.8%) viruses carried E23 K/G, or I38 M/T substitutions in PA. Variant calling through next-generation sequencing (NGS) showed varying proportions (6–100 %), a polymorphism and a mixture of PA/E23 K/G, and I38 M/T substitutions in the clinical samples. These eight viruses were obtained from children aged 7–14 years, with a median fever duration of 16.7 h and a median symptom duration of 93.7 h, which were similar to those of the wild type. However, the delayed viral clearance associated with the emergence of PA substitutions was observed. No substitutions conferring resistance to neuraminidase inhibitors were detected in 37 paired samples collected before and following oseltamivir treatment. These findings underscore the need for ongoing antiviral surveillance, informing public health strategies and clinical antiviral recommendations for seasonal influenza.

Exploration of influenza A virus PA protein-associated cellular proteins discloses its impact on mitochondrial function

Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LC‒MS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Molecular characterization of the whole genome of H9N2 avian influenza virus isolated from Egyptian poultry farms

H9N2 avian influenza viruses (AIVs) affect both poultry and humans on a global level, and they are especially prevalent in Egypt. In this study, we sequenced the entire genome of AIV H9N2 isolated from chickens in Egypt in 2021, using next-generation sequencing (NGS) technology. Phylogenetic analysis of the resulting sequences showed that the studied strain was generally monophyletic and grouped within the G1 sublineage of the Eurasian lineage. Four segments (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acidic [PA], and non-structural [NS]) were related to Egyptian genotype II, while the nucleoprotein (NP), neuraminidase (NA), matrix (M), and haemagglutinin (HA) segments were related to Egyptian genotype I. Molecular analysis revealed that HA protein contained amino acid residues (191H and 234L) that suggested a predilection for attaching to human-like receptors. The antigenic sites of HA had two nonsynonymous mutations: V194I at antigenic site A and M40K at antigenic site B. Furthermore, the R403W and S372A mutations, which have been observed in H3N2 and H2N2 strains that caused human pandemics, were found in the NA protein of the detected strain. The internal proteins contained virulence markers: 504V in the PB2 protein, 622G, 436Y, 207K, and 677T in the PB1 protein, 127V, 550L, and 672L in PA protein, and 64F and 69P in the M protein. These results show that the detected strain had undergone intrasubtype reassortment. Furthermore, it contains changes in the viral proteins that make it more likely to be virulent, raising a question about the tendency of AIV H9N2 to become highly pathogenic in the future for both poultry and humans.

Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

The interaction of influenza A virus RNA polymerase PA subunit with the human β-actin protein

Influenza A viruses are enveloped viruses with a genome of eight single-stranded negative-sense RNA molecules. In virions, RNA segments are found as vRNPs associated with NP proteins. The RdRp enzyme, which catalyzes the replication/transcription of the viral genome, is carried as attached to vRNPs. In this study, it was demonstrated that the PA subunit of the viral RdRp interacts with β-actin proteins by the yeast two-hybrid assay. It was shown that the amino-terminal domains of the β-actin protein bind to the carboxy-terminal moiety of the viral PA protein in the mammalian cells. The results were supported by in silico analysis. Over-expression of the β-actin protein was found to have a negative effect on the viral RdRp activity in mini-replicon, but its mechanism of action has remained unknown. The results suggest that the interaction of β-actin and PA protein, a component of vRNPs, may have a role in the intracellular trafficking of the influenza vRNPs and/or viral transcription.

Naturally occurring PAE206K point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century. In this study, we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients. Sequence comparison of the three main protein subunits (PB2, PB1, and PA) of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution (E206K) was responsible for the observed impaired replication phenotype. Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo. Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature (39.5 ​°C). Hence, this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Mutations in HA and PA affect the transmissibility of H7N9 avian influenza virus in chickens

Low pathogenic (LP) H7N9 avian influenza virus (AIV) emerged in 2013 and had spread widely over several months in China, experienced a noteworthy reduction in isolation rate in poultry and human since 2017. Here, we examined the transmission of H7N9 viruses to better understand viral spread and dissemination mechanisms. Three out of four viruses (2013–2016) could transmit in chickens through direct contact, and airborne transmission was confirmed in the JT157 (2016) virus. However, we did not detect the transmission of the two 2017 viruses, WF69 and AH395, through either direct or airborne exposure. Molecular analysis of genome sequence of two viruses identified eleven mutations located in viral proteins (except for matrix protein), such as PA (K362R and S364N) and HA (D167N, H7 numbering), etc. We explored the genetic determinants that contributed to the difference in transmissibility of the viruses in chickens by generating a series of reassortants in the JT157 background. We found that the replacement of HA gene in JT157 by that of WF69 abrogated the airborne transmission in recipient chickens, whereas the combination of HA and PA replacement led to the loss of airborne and direct contact transmission. Failure with contact transmission of the viruses has been associated with the emergence of the mutations D167N in HA and K362R and S364N in PA. Furthermore, the HA D167N mutation significantly reduced viral attachment to chicken lung and trachea tissues, while mutations K362R and S364N in PA reduced the nuclear transport efficiency and the PA protein expression levels in both cytoplasm and nucleus of CEF cells. The D167N substitution in HA reduced the H7N9 viral acid stability and avian-like receptor binding, while enhanced human-like receptor binding. Further analysis revealed these mutants grew poorly in vitro and in vivo. To conclude, H7N9 AIVs that contain mutations in the HA and PA protein reduced the viral transmissibility in chicken, and may pose a reduced threat for poultry but remain a heightened public health risk.

Genetic characteristics of the first human infection with the G4 genotype eurasian avian-like H1N1 swine influenza virus in Shaanxi Province,China

Objective: To analyze the genetic characteristics of the first human infection with the G4 genotype of Eurasian avian H1N1 swine influenza virus (EA H1N1 SIV) in Shaanxi Province. Methods: The patient's throat swab samples were collected, and MDCK cells were inoculated for virus isolation to obtain the virus strain. The whole genome deep sequencing method was used to obtain the eight gene segments of the isolated strain. The nucleotide homology analysis was conducted through the Blast program in the GenBank database, and a phylogenetic tree was constructed to analyze the genetic characteristics of the virus. Results: The throat swab specimens of the case were confirmed as EA H1N1 SIV in the laboratory, and the isolated strain was named A/Shaanxi-Weicheng/1351/2022(H1N1v). Homology analysis found that the PB2, NP, HA, NA, and M genes of this isolate had the highest nucleotide homology with A/swing/Beijing/0301/2018 (H1N1), about 98.29%, 98.73%, 97.41%, 97.52%, and 99.08%, respectively. The phylogenetic tree showed that the isolate belonged to G4 genotype EA H1N1 SIV, with PB2, PB1, PA, NP and M genes from pdm/09 H1N1, HA and NA genes from EA H1N1, and NS gene from Triple-reassortant H1N1. The cleavage site of the HA protein was IPSIQSR↓G, which was the molecular characteristic of the low pathogenic influenza virus. No amino acid mutations associated with neuraminidase inhibitors were found in the NA protein. PB2 protein 701N mutation, PA protein P224S mutation, NP protein Q357K mutation, M protein P41A mutation, and NS protein 92D all indicated its enhanced adaptability to mammals. Conclusion: The patient is the first human infection with G4 genotype EA H1N1 SIV in Shaanxi province. The virus is low pathogenic, but its adaptability to mammals is enhanced. Therefore, it is necessary to strengthen the monitoring of such SIVs.

Influenza A Virus in Pigs in Senegal and Risk Assessment of Avian Influenza Virus (AIV) Emergence and Transmission to Human.

We conducted an active influenza surveillance in the single pig slaughterhouse in Dakar to investigate the epidemiology and genetic characteristics of influenza A viruses (IAVs) and to provide serologic evidence of avian influenza virus (AIV) infection in pigs at interfaces with human populations in Senegal. Nasal swab and blood samples were collected on a weekly basis from the same animal immediately after slaughter. Influenza A viruses were diagnosed using RT-qPCR and a subset of positive samples for H3 and H1 subtypes were selected for full genome amplification and NGS sequencing. Serum samples were tested by HI assay for the detection of antibodies recognizing four AIVs, including H9N2, H5N1, H7N7 and H5N2. Between September 2018 and December 2019, 1691 swine nasal swabs were collected and tested. Influenza A virus was detected in 30.7% (520/1691), and A/H1N1pdm09 virus was the most commonly identified subtype with 38.07% (198/520), followed by A/H1N2 (16.3%) and A/H3N2 (5.2%). Year-round influenza activity was noted in pigs, with the highest incidence between June and September. Phylogenetic analyses revealed that the IAVs were closely related to human IAV strains belonging to A/H1N1pdm09 and seasonal H3N2 lineages. Genetic analysis revealed that Senegalese strains possessed several key amino acid changes, including D204 and N241D in the receptor binding site, S31N in the M2 gene and P560S in the PA protein. Serological analyses revealed that 83.5% (95%CI = 81.6-85.3) of the 1636 sera tested were positive for the presence of antibodies against either H9N2, H5N1, H7N7 or H5N2. Influenza H7N7 (54.3%) and H9N2 (53.6%) were the dominant avian subtypes detected in Senegalese pigs. Given the co-circulation of multiple subtypes of influenza viruses among Senegalese pigs, the potential exists for the emergence of new hybrid viruses of unpredictable zoonotic and pandemic potential in the future.

The synergistic effect of residues 32T and 550L in the PA protein of H5 subtype avian influenza virus contributes to viral pathogenicity in mice.

The avian influenza virus (AIV) PA protein contributes to viral replication and pathogenicity; however, its interaction with innate immunity is not well understood. Here, we report that the H5 subtype AIV PA protein strongly suppresses host antiviral defense by interacting with and degrading a key protein in interferon (IFN) signaling, Janus kinase 1 (JAK1). Specifically, the AIV PA protein catalyzes the K48-linked polyubiquitination and degradation of JAK1 at lysine residue 249. Importantly, the AIV PA protein harboring 32T/550L degrades both avian and mammalian JAK1, while the AIV PA protein with residues 32M/550I degrades avian JAK1 only. Furthermore, the residues 32T/550L in PA protein confer optimum polymerase activity and AIV growth in mammalian cells. Notably, the replication and virulence of the AIV PA T32M/L550I mutant are attenuated in infected mice. Collectively, these data reveal an interference role for H5 subtype AIV PA protein in host innate immunity, which can be targeted for the development of specific and effective anti-influenza therapeutics.

Genetic characterization and whole-genome sequencing-based genetic analysis of influenza virus in Jining City during 2021-2022.

The influenza virus poses a significant threat to global public health due to its high mutation rate. Continuous surveillance, development of new vaccines, and public health measures are crucial in managing and mitigating the impact of influenza outbreaks. Nasal swabs were collected from individuals with influenza-like symptoms in Jining City during 2021-2022. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect influenza A viruses, followed by isolation using MDCK cells. Additionally, nucleic acid detection was performed to identify influenza A H1N1, seasonal H3N2, B/Victoria, and B/Yamagata strains. Whole-genome sequencing was conducted on 24 influenza virus strains, and subsequent analyses included characterization, phylogenetic construction, mutation analysis, and assessment of nucleotide diversity. A total of 1,543 throat swab samples were collected. The study revealed the dominance of the B/Victoria influenza virus in Jining during 2021-2022. Whole-genome sequencing showed co-prevalence of B/Victoria influenza viruses in the branches of Victoria clade 1A.3a.1 and Victoria clade 1A.3a.2, with a higher incidence observed in winter and spring. Comparative analysis demonstrated lower similarity in the HA, MP, and PB2 gene segments of the 24 sequenced influenza virus strains compared to the Northern Hemisphere vaccine strain B/Washington/02/2019. Mutations were identified in all antigenic epitopes of the HA protein at R133G, N150K, and N197D, and the 17-sequence antigenic epitopes exhibited more than 4 amino acid variation sites, resulting in antigenic drift. Moreover, one sequence had a D197N mutation in the NA protein, while seven sequences had a K338R mutation in the PA protein. This study highlights the predominant presence of B/Victoria influenza strain in Jining from 2021 to 2022. The analysis also identified amino acid site variations in the antigenic epitopes, contributing to antigenic drift.

An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast-two-hybrid screening data.

The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA-splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo-cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three-dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I-TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy-terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxy-terminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610-630.

Antigenic identity of immunodominant proteins of <i>Bacillus anthracis</i> genovariants

Introduction. The main biological raw materials for the production of immunobiological preparations for identification of Bacillus anthracis are its specific antigens, the protective antigen and the EA1 protein.
 Purpose. To determine the antigenic identity of immunodominant proteins of different genovariants of B. anthracis isolated by gel chromatography and electrophoresis.
 Materials and methods. Culture filtrates of isogenic variants of B. anthracis strain 575/122 (pXO1+, pXO2+): R01 (pXO1+, pXO2); R00 (pXO1, pXO2) were used in the study. Gel chromatographic fractionation and electrophoretic separation were carried out according to standard methods. The antigenic properties of proteins isolated by gel chromatography and electrophoresis were studied by immunodiffusion with polyclonal monospecific sera against the protective antigen and the EA1 protein of the S-layer.
 Results. Gel chromatographic separation of B. anthracis 575/122 culture filtrates R01 (pXO1+, pXO2) and R00 (pXO1, pXO2)yielded fractions 1 and 5. Sera against EA1 protein and antigens of fraction 1 of strains B. anthracis 575/122 R00 and B. anthracis 575/122 R01 culture filtrates identified the identical antigens. Serum against antigens of fraction 5 of B. anthracis 575/122 R01 contained antibodies to numerous proteins, including the protective antigen isolated by electrophoresis.
 Discussion. The antigenic identity of immunodominant proteins isolated by gel chromatography and electrophoresis was identified.
 Conclusion. EA1 and PA proteins isolated by electrophoresis and gel chromatography can be used for production of monoclonal and polyclonal monospecific antibodies suitable for the design of diagnostic preparations.

Upregulation of galectin-3 in influenza A virus infection promotes viral RNA synthesis through its association with viral PA protein

BackgroundInfluenza is one of the most important viral infections globally. Viral RNA-dependent RNA polymerase (RdRp) consists of the PA, PB1, and PB2 subunits, and the amino acid residues of each subunit are highly conserved among influenza A virus (IAV) strains. Due to the high mutation rate and emergence of drug resistance, new antiviral strategies are needed. Host cell factors are involved in the transcription and replication of influenza virus. Here, we investigated the role of galectin-3, a member of the β-galactoside-binding animal lectin family, in the life cycle of IAV infection in vitro and in mice.MethodsWe used galectin-3 knockout and wild-type mice and cells to study the intracellular role of galectin-3 in influenza pathogenesis. Body weight and survival time of IAV-infected mice were analyzed, and viral production in mouse macrophages and lung fibroblasts was examined. Overexpression and knockdown of galectin-3 in A549 human lung epithelial cells were exploited to assess viral entry, viral ribonucleoprotein (vRNP) import/export, transcription, replication, virion production, as well as interactions between galectin-3 and viral proteins by immunoblotting, immunofluorescence, co-immunoprecipitation, RT-qPCR, minireplicon, and plaque assays. We also employed recombinant galectin-3 proteins to identify specific step(s) of the viral life cycle that was affected by exogenously added galectin-3 in A549 cells.ResultsGalectin-3 levels were increased in the bronchoalveolar lavage fluid and lungs of IAV-infected mice. There was a positive correlation between galectin-3 levels and viral loads. Notably, galectin-3 knockout mice were resistant to IAV infection. Knockdown of galectin-3 significantly reduced the production of viral proteins and virions in A549 cells. While intracellular galectin-3 did not affect viral entry, it increased vRNP nuclear import, RdRp activity, and viral transcription and replication, which were associated with the interaction of galectin-3 with viral PA subunit. Galectin-3 enhanced the interaction between viral PA and PB1 proteins. Moreover, exogenously added recombinant galectin-3 proteins also enhanced viral adsorption and promoted IAV infection in A549 cells.ConclusionWe demonstrate that galectin-3 enhances viral infection through increases in vRNP nuclear import and RdRp activity, thereby facilitating viral transcription and replication. Our findings also identify galectin-3 as a potential therapeutic target for influenza.

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Characterization of Influenza A(H1N1)pdm09 Viruses Isolated in the 2018-2019 and 2019-2020 Influenza Seasons in Japan.

The influenza A(H1N1)pdm09 virus that emerged in 2009 causes seasonal epidemic worldwide. The virus acquired several amino acid substitutions that were responsible for antigenic drift until the 2018-2019 influenza season. Viruses possessing mutations in the NA and PA proteins that cause reduced susceptibility to NA inhibitors and baloxavir marboxil, respectively, have been detected after antiviral treatment, albeit infrequently. Here, we analyzed HA, NA, and PA sequences derived from A(H1N1)pdm09 viruses that were isolated during the 2018-2019 and 2019-2020 influenza seasons in Japan. We found that A(H1N1)pdm09 viruses possessing the D187A and Q189E substitutions in HA emerged and dominated during the 2019-2020 season; these substitutions in the antigenic site Sb, a high potency neutralizing antibody-eliciting site for humans, changed the antigenicity of A(H1N1)pdm09 viruses. Furthermore, we found that isolates possessing the N156K substitution, which was predicted to affect the antigenicity of A(H1N1)pdm09 virus at the laboratory level, were detected at a frequency of 1.0% in the 2018-2019 season but 10.1% in the 2019-2020 season. These findings indicate that two kinds of antigenically drifted viruses-N156K and D187A/Q189E viruses-co-circulated during the 2019-2020 influenza season in Japan.

Beneficial effects of cellular coinfection resolve inefficiency in influenza A virus transcription.

For diverse viruses, cellular infection with single vs. multiple virions can yield distinct biological outcomes. We previously found that influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) virus (GFHK99) displays a particularly high reliance on multiple infection in mammalian cells. Here, we sought to uncover the viral processes underlying this phenotype. We found that the need for multiple infection maps to amino acid 26K of the viral PA protein. PA 26K suppresses endonuclease activity and viral transcription, specifically within cells infected at low multiplicity. In the context of the higher functioning PA 26E, inhibition of PA using baloxavir acid augments reliance on multiple infection. Together, these data suggest a model in which sub-optimal activity of the GFHK99 endonuclease results in inefficient priming of viral transcription, an insufficiency which can be overcome with the introduction of additional viral ribonucleoprotein templates to the cell. More broadly, the finding that deficiency in a core viral function is ameliorated through multiple infection suggests that the fitness effects of many viral mutations are likely to be modulated by multiplicity of infection, such that the shape of fitness landscapes varies with viral densities.

Synthesis and biological evaluation of baloxavir marboxil analogs for the treatment of influenza A (H1N1) virus

AbstractThe RNA‐dependent RNA polymerase (RdRp) of influenza virus, consisting of subunits of PA, PB1, and PB2 proteins, is responsible for viral mRNA transcription and vRNA replication. Cap‐dependent endonuclease (CEN) exists in PA subunit of RdRp and mediates the critical “cap‐snatching” step of viral RNA transcription, which is considered as a promising anti‐influenza target. In this manuscript, a series of novel compounds were designed, synthesized, and biologically investigated as CEN inhibitors. The cellular activities of the target compounds against influenza A/H1N1 virus were investigated and the structure–activity relationship (SAR) was summarized. The results showed that some compounds were active against influenza A/H1N1 virus with IC50 values of less than 100 μM. The microsomal metabolic stabilities were carried out in human, monkey, and rat species, and the results showed that the active ingredients of compounds 32 and 40 were stable enough to be in vivo investigated. The in vivo pharmacokinetic results showed that compound 32 had acceptable biological parameters for both ig and iv administrations. Then the in vivo toxicity study of compound 32 with the doses of 100 and 50 mg/kg BID was performed in BALB/C mice. No obvious toxicity of compound 32 was observed during the experiment. Finally, the in vivo efficacy study of compound 32 in BALB/C mice showed that it was effective in alleviating influenza symptoms and showed a significant pharmacodynamic dose dependence. These results indicated that compound 32 was a promising candidate for further development.