Polymerase Basic Protein Research Articles

Evolution and Adaptation of the Avian H7N9 Virus into the Human Host.

Influenza viruses arise from animal reservoirs, and have the potential to cause pandemics. In 2013, low pathogenic novel avian influenza A(H7N9) viruses emerged in China, resulting from the reassortment of avian-origin viruses. Following evolutionary changes, highly pathogenic strains of avian influenza A(H7N9) viruses emerged in late 2016. Changes in pathogenicity and virulence of H7N9 viruses have been linked to potential mutations in the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA), as well as the viral polymerase basic protein 2 (PB2). Recognizing that effective viral transmission of the influenza A virus (IAV) between humans requires efficient attachment to the upper respiratory tract and replication through the viral polymerase complex, experimental evidence demonstrates the potential H7N9 has for increased binding affinity and replication, following specific amino acid substitutions in HA and PB2. Additionally, the deletion of extended amino acid sequences in the NA stalk length was shown to produce a significant increase in pathogenicity in mice. Research shows that significant changes in transmissibility, pathogenicity and virulence are possible after one or a few amino acid substitutions. This review aims to summarise key findings from that research. To date, all strains of H7N9 viruses remain restricted to avian reservoirs, with no evidence of sustained human-to-human transmission, although mutations in specific viral proteins reveal the efficacy with which these viruses could evolve into a highly virulent and infectious, human-to-human transmitted virus.

Influenza Polymerase Inhibitors: Mechanisms of Action and Resistance.

The influenza virus RNA-dependent RNA polymerase is highly conserved among influenza A, B, C, and D viruses. It comprises three subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA) in influenza A and B viruses or polymerase 3 protein (P3) in influenza C and D viruses. Because this polymerase is essential for influenza virus replication, it has been considered as a target for antiviral agents. Recently, several polymerase inhibitors that target each subunit have been developed. This review discusses the mechanism of action, antiviral activity, and emergence of resistance to three inhibitors approved for the treatment of influenza or in late-phase clinical trials: the PB1 inhibitor favipiravir, the PB2 inhibitor pimodivir, and the PA inhibitor baloxavir marboxil.

Probing the effect of quercetin 3-glucoside from Dianthus superbus L against influenza virus infection- In vitro and in silico biochemical and toxicological screening

Investigation of antiviral and cytotoxic effect of quercetin 3-glucoside (Q3G) from Dianthus superbus L over influenza virus infection and replication were studied. Moreover, anti-influenza mechanism was screened by time-dependent antiviral assay, virus-induced symptoms and related gene expressions. The blockade of cap-binding domain of polymerase basic protein subunit were analysed by molecular docking study. The Q3G demonstrated potent antiviral activity showing 4.93, 6.43, 9.94, 8.3, and 7.1 μg/mL of IC50 for A/PR/8/34, A/Victoria/3/75, A/WS/33, B/Maryland/1/59, and B/Lee/40, respectively. The cellular toxicity of Q3G and oseltamivir (control) were tested and >100 μg/mL of CC50 value considered as nontoxic. Influenza A virus infection induced a higher ROS production, however potentially reduced by Q3G treatment and significantly blocked virus infection induced acidic vesicular organelles (AVO). Moreover, Q3G has no inhibitory effect for neuraminidase activity but blocked virus replication through time dependent assay and showed more competitive binding affinity (−8.0 kcal/mal) than GTP (−7.0 kcal/mol) to block polymerase basic protein-2 subunit of influenza virus. Q3G from D. superbus showed potent antiviral activity against influenza A and B viruses with suppressive effect on virus-induced cellular ROS generation and AVO formation. Thus, this study provided a new line of research for Q3G to develop possible natural anti-influenza drug.

Molecular Characterization of a Novel Avian Influenza A (H2N9) Strain Isolated from Wild Duck in Korea in 2018.

A novel avian influenza virus (A/wild duck/Korea/K102/2018) (H2N9) was isolated from wild birds in South Korea in 2018, and phylogenetic and molecular analyses were conducted on complete gene sequences obtained by next-generation sequencing. Phylogenetic analysis indicated that the hemagglutinin (HA) and neuraminidase (NA) genes of the A/wild duck/Korea/K102/2018 (H2N9) virus belonged to the Eurasian countries, whereas other internal genes (polymerase basic protein 1 (PB1), PB2, nucleoprotein (NP), polymerase acidic protein (PA), matrix protein (M), and non-structural protein (NS)) belonged to the East Asian countries. A monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, E627 in the PB2 gene, and no deletion of the stalk region in the NA gene indicated that the A/wild duck/Korea/K102/2018 (H2N9) isolate was a typical low pathogenicity avian influenza (LPAI). Nucleotide sequence similarity analysis of HA revealed that the highest homology (98.34%) is to that of A/duck/Mongolia/482/2015 (H2N3), and amino acid sequence of NA was closely related to that of A/duck/Bangladesh/8987/2010 (H10N9) (96.45%). In contrast, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds of China or Japan in 2016–2018. The newly isolated A/wild duck/Korea/K102/2018 (H2N9) strain is the first reported avian influenza virus in Korea, and may have evolved from multiple genotypes in wild birds and ducks in Mongolia, China, and Japan.

Design, Synthesis, and Biological Evaluation of Novel Indoles Targeting the Influenza PB2 Cap Binding Region.

In the search for novel influenza inhibitors we evaluated 7-fluoro-substituted indoles as bioisosteric replacements for the 7-azaindole scaffold of Pimodivir, a PB2 (polymerase basic protein 2) inhibitor currently in clinical development. Specifically, a 5,7-difluoroindole derivative 11a was identified as a potent and metabolically stable influenza inhibitor. 11a demonstrated a favorable oral pharmacokinetic profile and in vivo efficacy in mice. In addition, it was found that 11a was not at risk of metabolism via aldehyde oxidase, an advantage over previously described inhibitors of this class. The crystal structure of 11a bound to influenza A PB2 cap region is disclosed here and deposited to the PDB.

Evidence for a novel mechanism of influenza A virus host adaptation modulated by PB2-627.

Influenza virus cross-species transmission is restricted by the host, but viruses overcome this restriction by accumulating mutations which allow them to adapt to a new host. Among the many factors which facilitate virus host adaptation, polymerase basic protein 2 (PB2) 627 plays an important role, although the underlying molecular mechanism has not been fully understood. In a previous study, we found that histone H1.2 (encoded by HIST1H1C) regulates human or avian influenza virus replication in different ways, indicating that it might be involved in virus host adaptation. Herein, we found that HIST1H1C expression, phosphorylation and methylation levels are decreased when infected with H1N1 influenza virus and increased when infected with H5N1 influenza virus. Overexpressing the eight gene segments of the influenza virus, we found that only PB2 significantly affects HIST1H1C expression and modifications. Since the 627 site is different between the H5N1 and H1N1 influenza viruses we constructed PB2-627E (avian variant) and PB2-627K (human variant) mutant viruses, and observed that the effects of the wild-type and the mutant viruses on HIST1H1C expression and modifications are the opposite of one another. Further analysis showed that influenza virus PB2-627 regulates HIST1H1C expression via Sp1, and specifically that PB2-627K down-regulates Sp1 and HIST1H1C while PB2-627E up-regulates Sp1 and HIST1H1C. In addition, HIST1H1C can feedback regulate DNA-dependent protein kinase and euchromatic histone-lysine N-methyltransferase 1/2, leading to altered HIST1H1C phosphorylation and methylation levels, and affecting influenza virus replication accordingly. In summary, this study illustrates the mechanism of PB2-627E/K-mediated regulation of influenza virus host adaptation.

Influenza virus polymerase inhibitors in clinical development.

Purpose of reviewWe review antivirals inhibiting subunits of the influenza polymerase complex that are advancing in clinical development.Recent findingsFavipiravir, pimodivir, and baloxavir are inhibitory in preclinical models for influenza A viruses, including pandemic threat viruses and those resistant to currently approved antivirals, and two (favipiravir and baloxavir) also inhibit influenza B viruses. All are orally administered, although the dosing regimens vary. The polymerase basic protein 1 transcriptase inhibitor favipiravir has shown inconsistent clinical effects in uncomplicated influenza, and is teratogenic effects in multiple species, contraindicating its use in pregnancy. The polymerase basic protein 2 cap-binding inhibitor pimodivir displays antiviral effects alone and in combination with oseltamivir in uncomplicated influenza, although variants with reduced susceptibility emerge frequently during monotherapy. Single doses of the polymerase acidic protein cap-dependent endonuclease inhibitor baloxavir are effective in alleviating symptoms and rapidly inhibiting viral replication in otherwise healthy and higher risk patients with acute influenza, although variants with reduced susceptibility emerge frequently during monotherapy. Combinations of newer polymerase inhibitors with neuraminidase inhibitors show synergy in preclinical models and are currently undergoing clinical testing in hospitalized patients.SummaryThese new polymerase inhibitors promise to add to the clinical management options and overall control strategies for influenza virus infections.

Pimodivir. Polymerase basic protein 2 (PB2) (influenza virus) inhibitor, Treatment of influenza A infection

Pimodivir. Polymerase basic protein 2 (PB2) (influenza virus) inhibitor, Treatment of influenza A infection

Codon usage characteristics of PB2 gene in influenza A H7N9 virus from different host species

The influenza A H7N9 virus is a highly contagious virus which can only infect poultry before early 2013. But after that time, it widely caused human infections in China and brought Southeast Asia great threaten in the public health area. The coding gene for polymerase basic protein 2 (PB2) in influenza A H7N9 virus encodes the PB2 protein, which is a part of the RNA polymerase. The enzyme lacks a correction function during its own replication process, so the mutation frequency of the influenza A H7N9 virus gene is high and the PB2 gene is also included. To investigate the codon usages characteristics of PB2 gene, gene sequences of 12 kinds of poultry are downloaded form the gene bank (NCBI) and their codon usage characteristics such as the effective number of codons (ENC), the evolutionary relationship of the sequences, the codon adaptation index (CAI), the correspondence analysis (COA), the relative synonymous codon usage (RSCU) and their PR2-bias are compared and studied. The value of these reults showed that there is a low codon usage bias in the PB2 gene. Then, the differences between the codon usages of PB2 gene from 12 kinds of poultry are compared and their potential applications are discussed. These results could lay a foundation for other further study on the evolution of H7N9.

Genome-wide profiling of microRNAs reveals novel insights into the interactions between H9N2 avian influenza virus and avian dendritic cells.

The antigen-presenting ability of dendritic cells (DCs) plays an important and irreplaceable role in recognising and clearing viruses. Antiviral responses must rapidly defend against infection while minimising inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. MicroRNAs (microRNAs), small non-coding RNAs, can regulate mouse or avian DCs to inhibit the infection and replication of avian influenza virus (AIV). Here, we performed a global analysis to understand how avian DCs respond to H9N2 AIV and provide a potential mechanism to explain how avian microRNAs can defend against H9N2 AIV replication. First, we found that both active and inactive H9N2 AIV enhanced the ability of DCs to present antigens and activate T lymphocytes. Next, total microarray analyses suggested that H9N2 AIV stimulation involved protein localisation, nucleotide binding, leucocyte transendothelial migration and MAPK signalling. Moreover, we constructed 551 transcription factor (TF)-miRNA-mRNA loops based on the above analyses. Furthermore, we found that the haemagglutinin (HA) fragment, neither H5N1-HA or H9N2-HA, could not activate DCs, while truncated HA greatly increased the immune function of DCs by activating ERK and STAT3 signalling pathways. Lastly, our results not only suggested that gga-miR1644 targets muscleblind-like protein 2 (MBNL2) to enhance the ability of avian DCs to inhibit virus replication, but also suggested that gga-miR6675 targets the nuclear localisation sequence of polymerase basic protein 1 (PB1) to trigger the silencing of PB1 genes, resulting in the inhibition of H9N2 AIV replication. Altogether, our innovative study will shed new light on the role of avian microRNAs in evoking avian DCs and inhibiting virus replication.

T189. PEPTIDE SHARING BETWEEN SCHIZOPHRENIA-RELATED PROTEINS AND THE INFLUENZA A VIRUS MAY OFFER A WINDOW INTO THE IMMUNE AETIOLOGY OF PSYCHOTIC DISORDERS

BackgroundSchizophrenia is a complex disorder in which infection and immune mechanisms are thought to play a role. Epidemiological and ecological studies have implicated influenza infection in particular and it is possible that cross-reactivity, or molecular mimicry, between the influenza virus and brain proteins underlies this association. Proteins might share amino acid sequences, which could thus provide the basis for an autoimmune response that targets endogenous proteins. This study is the first to characterise sequence alignment between schizophrenia-related brain proteins and the proteome of the influenza A virus, and comparing it with sequence alignment in proteins not implicated in schizophrenia.MethodsThe software Peptide Match Service (https://research.bioinformatics.udel.edu/peptidematch/index.jsp; Protein Information Resource, University of Delaware and Georgetown University Medical Center) was used to obtain sequence alignments between protein sequences. A case-control study design was used to compare schizophrenia-related proteins to proteins not involved in schizophrenia. Schizophrenia-related proteins were operationalised as proteins found significant in the Psychiatric Genomics Consortium schizophrenia genome-wide association studies (GWAS). The control group consisted of null proteins (p-value > .75) in the GWAS. Null proteins were also selected to represent genes expressed in tissues other than central nervous system tissues. Both groups were equalised for the total amino acid count. Perfect pentapeptide matches (i.e. 5 amino acids) in proteins and the influenza proteome were explored.ResultsThere was a link between schizophrenia-related (GWAS-significant) proteins and presence of perfect matches between proteins and the influenza proteins polymerase acidic protein (χ2 (1) = 5.284, p = .022, two-sided) and RNA-directed RNA polymerase catalytic subunit (χ2 (1) = 6.132, p = .013, two-sided). Pentapeptide-sharing was found to be highly significant between schizophrenia-related proteins and the hemagglutinin precursor (χ2 (1) = 17.723, p = .000026, two-sided). There was no significant difference (p > .05) between schizophrenia-related proteins and proteins not implicated in schizophrenia (GWAS-null proteins) in the frequency of proteins having perfect matches with the influenza A proteins PB2-S1, polymerase basic protein 2, matrix protein 1 and 2, and neuraminidase. However, the result for matrix protein 1 approached statistical significance (χ2 (1) = 3.319, p = .068, two-sided).DiscussionWe find evidence to suggest there is significant overlap between the linear structures of proteins involved in schizophrenia and those integral to the influenza virus. Future research should establish the biological relevance of this finding, particularly regarding the antigenicity of the peptide sequences which we have identified. Extra studies should also go beyond sequences and address structural homologies. Future research could assess whether an immune reaction against particular schizophrenia-related proteins is a plausible mechanism contributing to psychotic disorders. Also, exploring peptide sharing in different influenza strains could offer insights into links between influenza pandemics, maternal infection, and psychosis. Elucidating peptide sharing might have implications for schizophrenia risk management and safe influenza prevention.

Amino acid substitutions in low pathogenic avian influenza virus strains isolated from wild birds in Korea.

Wild birds are natural hosts and reservoirs for influenza A viruses. However, many species, such as many waterfowl, are asymptomatic when infected and so facilitate the generation of viral genetic diversity. Mutations of key genes affect the replicability, pathogenicity, transmissibility, and antiviral resistance of influenza A viruses. In this study, we isolated avian influenza (AI) viruses from wild bird fecal samples and analyzed changes in amino acids over time and geographic region to monitor the biological change of the AI virus. Between 2014 and 2016, we collected 38,921 fresh fecal samples from major wild bird habitats located throughout Korea and isolated 123 AI viruses. We subsequently selected 22 amino acid sites to analyze for changes. These sites included ten sites associated with replication, ten sites associated with pathogenicity, three sites associated with transmission, and seven sites associated with antiviral resistance. We found substitution rates of 71.7% at the C38Y amino acid site within the polymerase basic protein 1 (PB1) gene, 66.7% at the D222G site within the hemagglutinin (HA) 1 gene, and 75.6% at the A184 site within the nucleoprotein (NP) gene. Alterations of the PB1, HA1, and NP genes are closely associated with increased pathogenicity in chickens and mammals. The remaining sites of interest exhibited few modifications. In this study, we confirmed that AI viruses circulating among wild birds in Korea consistently exhibit modifications at amino acid sites linked with replication and pathogenicity.

Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

PACT is a double-stranded RNA-binding protein that has been implicated in host-influenza A virus (IAV) interaction. PACT facilitates the action of RIG-I in the activation of the type I IFN response, which is suppressed by the viral nonstructural protein NS1. PACT is also known to interact with the IAV RNA polymerase subunit PA. Exactly how PACT exerts its antiviral activity during IAV infection remains to be elucidated. In the current study, we demonstrated the interplay between PACT and IAV polymerase. Induction of IFN-β by the IAV RNP complex was most robust when both RIG-I and PACT were expressed. PACT-dependent activation of IFN-β production was suppressed by the IAV polymerase subunits, polymerase acidic protein, polymerase basic protein 1 (PB1), and PB2. PACT associated with PA, PB1, and PB2. Compromising PACT in IAV-infected A549 cells resulted in the augmentation of viral RNA (vRNA) transcription and replication and IFN-β production. Furthermore, vRNA replication was boosted by knockdown of PACT in both A549 cells and IFN-deficient Vero cells. Thus, the antiviral activity of PACT is mediated primarily via its interaction with and inhibition of IAV polymerase. Taken together, our findings reveal a new facet of the host-IAV interaction in which the interplay between PACT and IAV polymerase affects the outcome of viral infection and antiviral response.-Chan, C.-P., Yuen, C.-K., Cheung, P.-H. H., Fung, S.-Y., Lui, P.-Y., Chen, H., Kok, K.-H., Jin, D.-Y. Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin–Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks.

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014-2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.

Current Drug Design Strategies for Fighting Against Swine Influenza

Background: Swine influenza is a seasonal health threat due to global outbreak of H1N1 pandemic in 2009 leading to death of millions of people because of antigenic drift of the dangerous influenza A viral strains. Scarcity of specific chemotherapeutics emphasizes the priority of drug design and discovery of new anti-influenza leads having less toxicity and resistant to the dynamic viral strains. Objective: The present review is an update in the current trends in drug design from the stand point of ligand and structure based screening of potent swine influenza inhibitors. Method: In quest of different drug targets, ligand and structure based chemometric screening tools are the major platform for the design of potent chemotherapeutics having greater affinity towards various targets including polymerase basic protein 1 (PB1), PB1- F2, PB2, polymerase acidic protein (PA), surface glycoproteins hemagglutinin and neuraminidase, nucleocapsid protein (NP), nuclear matrix proteins including M1, Ion pore protein—M2 as well as two nonstructural proteins NS1 and NS2, respectively. Results: Ligand based screening deals with QSAR and pharmacophore modeling whereas structure based virtual screening deal with crystallography and molecular docking. Conclusion: The study in this direction can increase the search of hit rates and decrease in cost of drug design and development prior to experiment by producing potent chemotherapeutics against swine influenza. Keywords: H1N1 swine influenza A, QSAR, pharmacophore, structure based screening, molecular docking.

Inhibition of Avian Influenza A Virus Replication in Human Cells by Host Restriction Factor TUFM Is Correlated with Autophagy.

ABSTRACTAvian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2627E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2627K (human signature) and PB2627E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2627E than PB2627K in transfected human cells. Stronger binding of TUFM to avian-signature PB2590G/591Q and PB2627E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2627E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2627E viruses, but not PB2627K viruses. In addition, enhanced levels of interaction between TUFM and PB2627E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2627E virus; however, autophagy remained consistent in PB2627K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy.

The PB2 mutation with lysine at 627 enhances the pathogenicity of avian influenza (H7N9) virus which belongs to a non-zoonotic lineage

A novel avian-origin influenza A (H7N9) virus emerged in China in 2013 and has caused zoonotic disease in over 1123 persons with an overall mortality around 30%. Amino acid changes at the residues 591, 627 and 701 of polymerase basic protein 2 (PB2) have been found frequently in the human H7N9 isolates but not in viruses isolated from avian species. We have recently identified a cluster of H7N9 viruses in ducks which circulated in China prior to the first recognition of zoonotic disease in 2013. These duck viruses have genetic background distinct from the zoonotic H7N9 lineage. We found that the introduction of PB2 mutation with K at 627 but not K at 591 or N at 701 to the duck H7N9 virus led to increased pathogenicity in mice. We also found that the induction of pro-inflammatory cytokines including TNF-α, IP-10, MCP-1 and MIP-1α were associated with increased severity of infection. We conclude that introduction of the mammalian adaptation mutations into the PB2 gene of duck H7N9 viruses, which are genetically unrelated to the zoonotic H7N9 lineage, can also enhance pathogenicity in mice.

In vivo inhibition of influenza A virus replication by RNA interference targeting the PB2 subunit via intratracheal delivery.

BackgroundInfluenza virus infection is a major threat to human health. Small interfering RNA (siRNA) is a promising approach for the prevention and treatment of viral infections. In this study, we constructed a series of DNA vector-based short hairpin RNAs (shRNAs) that target various genes of the influenza A virus using the polymerase III U6-RNA promoter to prevent influenza virus infection in vitro and in a mouse model.ResultsThree sets of DNA vector-based shRNA, two targeting genes encoding the polymerase acidic protein (PA) and one targeting polymerase basic protein 2 (PB2), efficiently inhibited the replication of influenza virus A/WSN/33(H1N1) in vitro. We also successfully prevented influenza virus A/WSN/33(H1N1) infection in a C57BL/6 mouse model by intratracheal delivery of anti-PB2 shRNA.ConclusionsOur findings suggest that the PB2-targeting shRNA plasmid showed potential for use as an RNAi-based therapeutic for influenza virus infection.

Ethanolic Extract of Melia Fructus Has Anti-influenza A Virus Activity by Affecting Viral Entry and Viral RNA Polymerase.

Meliae Fructus (MF) is the dried ripe fruit of Melia toosendan Siebold et Zuccarini, Meliaceae family. MF is widely used in traditional medicine to treat inflammation and helminthic infection and has anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and analgesic activities. However, potential anti-influenza properties of MF have yet to be investigated. We determined whether an ethanolic extract of MF (EMF) has anti-viral activity via an EMF pre-, co-, and post-treatment assay, using the Influenza A/PR/8/34 and H3N2 virus on Madin-Darby canine kidney (MDCK) cells. The EMF had anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cell. EMF inhibited the activity of hemagglutinin (HA) and neuraminidase (NA) of influenza virus. EMF inhibited viral HA, nucleoprotein (NP), matrix protein 2 (M2), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) mRNA synthesis at 5 h post infection (hpi), however, the levels of PA, PB1, and PB2 mRNA were increased in pre- and co-EMF treated cells compared with control virus-infected and EMF post-treated cells at 18 hpi. The level of M2 protein expression was also decreased upon pre- and co-treatment with EMF. The PA protein was accumulated and localized in not only the nucleus but also the cytoplasm of virus-infected MDCK cells at 18 hpi. Pre-EMF treatment inhibited the expression of pAKT, which is induced by influenza virus infection, at the stage of virus entry. We also found that treatment of EMF up-regulated the antiviral protein Mx1, which may play a partial role in inhibiting influenza virus infection in pre- and co-EMF treated MDCK cells. In summary, these results strongly suggested that an ethanolic extract of Meliae Fructus inhibited influenza A virus infection by affecting viral entry, PA proteins of the RNA polymerase complex, and Mx1 induction and may be a potential and novel anti-influenza agent.