[Show abstract][Hide abstract] ABSTRACT: Influenza virus A(H7N9) is associated with a high case fatality rate in humans. Multiple viral factors have been postulated to account for high virulence of the virus. It has been reported that patients with influenza A(H7N9) virus infection have relatively low titers of neutralizing antibody than those with seasonal influenza virus infections. In this study, we compared serum hemagglutination inhibition (HI) and microneutralization (MN) antibody titers of mice challenged with wild-type A(H7N9) viruses (H7N9[Anhui] and H7N9[Zhejiang]), an A(H1N1)pdm09 virus (pH1N1), and a recombinant A(H7N9) virus with PR8/H1N1 internal genes (rg-PR8-H7-N9). All mice infected by H7N9(Anhui) and H7N9(Zhejiang) developed serum HI antibody at 14 days post-infection (dpi), but no detectable MN antibody even at 28 dpi. A low level of neutralizing activity was detected in H7N9(Anhui)- and H7N9(Zhejiang)-infected mice using fluorescent foci MN assay, but convalescent sera obtained from H7N9(Anhui)-infected mice did not reduce mortality of naïve mice after homologous virus challenge. Re-infection with homologous A(H7N9) virus induced higher HI and MN titers. In contrast, pH1N1(2009) virus infection induced robust HI and MN antibody responses even during the first infection. Moreover, rg-PR8-H7-N9 induced significantly higher HI and MN antibody titers. In conclusion, the internal genes of A(H7N9) virus can affect the humoral immune response against homologous viral surface proteins which may also contribute to the virulence of A(H7N9) virus.
Full-text · Article · Oct 2015 · Clinical and vaccine Immunology: CVI
[Show abstract][Hide abstract] ABSTRACT: Importance:
The non-structural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and NS1 deleted live attenuated viruses show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication competent DelNS1 virus which carries an A14U substitution in the 3' non-coding region of the M vRNA segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent NS1-deleted strains for use in functional and vaccine studies.
Full-text · Article · Jul 2015 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970. Polymerase complexes containing PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K.
Full-text · Article · Nov 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: NF90 was shown to exhibit broad antiviral activity against several viruses, but detailed mechanisms remain unclear. In this study, we examined the molecular basis for the inhibitory effect of NF90 on virus replication mediated through protein kinase (PKR)-associated translational regulation. We first verified the interaction between NF90 and PKR in mammalian cells and showed that NF90 interacts with PKR through its C-terminal and that the interaction is independent of NF90 RNA-binding properties. We further showed that knockdown of NF90 resulted in significantly lower levels of PKR phosphorylation in response to dsRNA induction and influenza virus infection. We also showed that high concentrations of NF90 exhibit negative regulatory effects on PKR phosphorylation, presumably through competition for dsRNA via the C-terminal RNA-binding domain. PKR activation is essential for the formation of stress granules in response to dsRNA induction. Our results showed that NF90 is a component of stress granules. In NF90-knockdown cells, dsRNA treatment induced significantly lower levels of stress granules than in control cells. Further evidence for an NF90-PKR antiviral pathway was obtained using an NS1 mutated influenza A virus specifically attenuated in its ability to inhibit PKR activation. This mutant virus replicated indistinguishably from wild-type virus in NF90-knockdown cells, but not in scrambled control cells or Vero cells, indicating that NF90's antiviral function occurs through interaction with PKR. Taken together, these results reveal a yet-to-be defined host antiviral mechanism in which NF90 upregulation of PKR phosphorylation restricts virus infection.
Full-text · Article · Mar 2014 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China. METHODS: Between March 7 and April 8, 2013, we included hospital inpatients if they had new-onset respiratory symptoms, unexplained radiographic infiltrate, and laboratory-confirmed H7N9 virus infection. We recorded histories and results of haematological, biochemical, radiological, and microbiological investigations. We took throat and sputum samples, used RT-PCR to detect M, H7, and N9 genes, and cultured samples in Madin-Darby canine kidney cells. We tested for co-infections and monitored serum concentrations of six cytokines and chemokines. We collected cloacal swabs from 86 birds from epidemiologically linked wet markets and inoculated embryonated chicken eggs with the samples. We identified and subtyped isolates by RT-PCR sequencing. RNA extraction, complementary DNA synthesis, and PCR sequencing were done for one human and one chicken isolate. We characterised and phylogenetically analysed the eight gene segments of the viruses in the patient's and the chicken's isolates, and constructed phylogenetic trees of H, N, PB2, and NS genes. FINDINGS: We identified four patients (mean age 56 years), all of whom had contact with poultry 3-8 days before disease onset. They presented with fever and rapidly progressive pneumonia that did not respond to antibiotics. Patients were leucopenic and lymphopenic, and had impaired liver or renal function, substantially increased serum cytokine or chemokine concentrations, and disseminated intravascular coagulation with disease progression. Two patients died. Sputum specimens were more likely to test positive for the H7N9 virus than were samples from throat swabs. The viral isolate from the patient was closely similar to that from an epidemiologically linked market chicken. All viral gene segments were of avian origin. The H7 of the isolated viruses was closest to that of the H7N3 virus from domestic ducks in Zhejiang, whereas the N9 was closest to that of the wild bird H7N9 virus in South Korea. We noted Gln226Leu and Gly186Val substitutions in human virus H7 (associated with increased affinity for α-2,6-linked sialic acid receptors) and the PB2 Asp701Asn mutation (associated with mammalian adaptation). Ser31Asn mutation, which is associated with adamantane resistance, was noted in viral M2. INTERPRETATION: Cross species poultry-to-person transmission of this new reassortant H7N9 virus is associated with severe pneumonia and multiorgan dysfunction in human beings. Monitoring of the viral evolution and further study of disease pathogenesis will improve disease management, epidemic control, and pandemic preparedness. FUNDING: Larry Chi-Kin Yung, National Key Program for Infectious Diseases of China.
[Show abstract][Hide abstract] ABSTRACT: The non-structural protein (NS1) of influenza A virus exhibits multiple functions in the virus life cycle. Proteomics screening for cellular proteins which interact with NS1 identified the cellular protein, RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1 and RAP55 and other P-body associated proteins (Ago1, Ago2 and DCP1a) were confirmed using co-immunoprecipitation and cellular co-localization assays. Over-expression of RAP55 induces RAP55-associated stress granule formation and suppresses virus replication. Knockdown of RAP55 with siRNA or expression of a dominant negative mutant RAP55 with defective interaction with P-bodies blocks NS1 co-localization to P-bodies in cells. Expression of NS1 inhibits RAP55 expression and formation of RAP55-associated P-bodies/stress granules. Viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was co-expressed. Restriction of virus replication via P-bodies occurs in the early phases of infection, as the numbers of RAP55-associated P-bodies in cells diminish over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules is RNA binding associated and mediated via a PKR-interacting viral element. Mutations introduced into either RNA binding sites, namely R38/K41, or PKR interaction sites, namely I123/M124/K126/N127, cause NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules, while NS1 counteracts this host restriction mechanism.
Full-text · Article · Sep 2012 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008-2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.
Full-text · Article · Nov 2011 · Antiviral research
[Show abstract][Hide abstract] ABSTRACT: Metabolomics is the downstream of systems biology and has drawn significant interest for studying the metabolic networks from cells to organisms. To profile the metabolites in two different cell lines (A549 and AGS) infected with influenza A virus, gas chromatography coupled with mass spectrometry (GC/MS) was employed. Some differentiating metabolites in the cell lines were tentatively identified using reference library, interpreted and visualized by applying principal components analysis (PCA) and cluster heat map. Consequently, metabolic flux profiling allowed the differentiation of fatty acid biosynthesis and cholesterol metabolism during viral replication in the cell lines. The change in fatty acid turnover was also observed. Metabolomics investigation also revealed the different responses between A549 and AGS cell lines to the virus infection. From the pattern recognition results, AGS cell line might be more susceptible to influenza A virus. Regarding the fact that AGS is a poorly differentiated gastric adenocarcinoma cell line whereas A549 is a relatively differentiated lung tumor one, it is speculated that viral replication might be associated with the cell differentiations.
[Show abstract][Hide abstract] ABSTRACT: Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC(50)) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies.
Full-text · Article · Jun 2010 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS-based proteomics approach. Moreover, O-sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus-host interaction.
[Show abstract][Hide abstract] ABSTRACT: The replication activity of 2009 pandemic H1N1 influenza virus in human lung cells was evaluated in this study. Twentytwo
surgically removed human lung tissue samples were infected ex vivo with pandemic H1N1 influenza virus (A/California/ 04/2009),
seasonal human H1N1 influenza virus (A/ Shantou/92/09), or a highly pathogenic H5N1 influenza virus (A/Vietnam/1194/04). Examination
of nucleoprotein expression and viral RNA replication in the infected human lung tissue samples showed that whereas the replication
of pandemic H1N1 influenza virus varied between tissue samples, overall this virus replicated more efficiently than seasonal
H1N1 influenza virus but less efficiently than H5N1 influenza virus. Double-immunostaining for viral antigens and cellular
markers indicated that pandemic H1N1 influenza virus replicates in type 2 alveolar epithelial cells.
Preview · Article · Apr 2010 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: The D225G (aspartic acid to glycine) substitution in the hemagglutinin of H1N1 influenza virus may alter its receptor-binding specificity. Direct analysis of polymorphisms in 126 amino acids spanning the receptor-binding site in the hemagglutinin of pandemic H1N1 2009 virus from 117 clinical specimens in Hong Kong found the D225G substitution for 7 (12.5%) of 57 patients with severe disease and for 0 (0%) of 60 patients with mild disease. D225G quasispecies were identified mainly in endotracheal aspirate samples and were identified less frequently in nasopharyngeal aspirate samples from patients with severe disease. Continuous monitoring of the prevalence and tissue tropism of this variant during its circulation among humans is important.
Preview · Article · Apr 2010 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: A lysine at the 627 position (627K) of PB2 protein of influenza virus has been recognized as a determinant for host adaptation and a virulent element for some influenza viruses. While seasonal influenza viruses exclusively contained 627K, the pandemic (H1N1) 2009 possessed a glutamic acid (627E), even after circulation in humans for more than 6months. To explore the potential role of E627K substitution in PB2 in the pandemic (H1N1) 2009 virus, we compared pathogenicity and growth properties between a recombinant virus containing 627K PB2 gene and the parental A/California/4/2009 strain containing 627E. Our results showed that substitution of 627K in PB2 gene does not confer higher virulence and growth rate for the pandemic (H1N1) 2009 virus in mice and cell culture respectively, suggesting 627K is not required for human adaptation of the pandemic (H1N1) 2009 virus.
[Show abstract][Hide abstract] ABSTRACT: Nucleoprotein (NP), the structural component of ribonucleoprotein complex of avian influenza virus, performs multiple essential functions in the regulation of viral RNA synthesis and in the control of nuclear traffic of viral proteins. Mutations have often been found in NP, some of which are relevant to viral survival strategies. In this study, we used nanospray-MS/MS to analyze tryptic digestion of nucleoprotein of avian influenza virus (H5N1) and to identify three mutated peptides. The MS/MS analyses allowed the confident determination of the three mutated amino acid residues F313Y, I194V and V408I/L in the mutated peptides of LLQNSQVYSLIRPNENPAHK, GVGTMVMELVR and ASAGQI/LSVQPTFSVQR, respectively.
[Show abstract][Hide abstract] ABSTRACT: Interactions between host factors and the viral replication complex play important roles in host adaptation and regulation of influenza virus replication. A cellular protein, nuclear factor 90 (NF90), was copurified with H5N1 viral nucleoprotein (NP) from human cells in which NP was transiently expressed and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. In vitro coimmunoprecipitation of NF90 and NP coexpressed in HEK 293T cells or individually expressed in bacterial and HEK 293T cells, respectively, confirmed a direct interaction between NF90 and NP, independent of other subunits of the ribonucleoprotein complex. This interaction was prevented by a mutation, F412A, in the C-terminal region of the NP, indicating that the C-terminal of NP is required for NF90 binding. RNase V treatment did not prevent coprecipitation of NP and NF90, which demonstrates that the interaction is RNA binding independent. After small interfering RNA knockdown of NF90 expression in A549 and HeLa cells, viral polymerase complex activity and virus replication were significantly increased, suggesting that NF90 negatively affects viral replication. Both NP and NF90 colocalized in the nucleus of virus-infected cells during the early phase of infection, suggesting that the interaction between NF90 and NP is an early event in virus replication. Quantitative reverse transcription-PCR showed that NF90 downregulates both viral genome replication and mRNA transcription in infected cells. These results suggest that NF90 inhibits influenza virus replication during the early phase of infection through direct interaction with viral NP.
Full-text · Article · Jul 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Matrix protein 1 (M1), the major structural protein of the avian influenza virus, plays a critical role in regulation of viral RNA transcription via interaction with RNA and transportation of RNP cores. Mutations in M1 have been frequently observed in the highly virulent avian influenza H5N1 virus, which might be crucial to the pathogenic function. Here we report the characterization of mutated peptides in M1 purified from highly pathogenic avian influenza virus H5N1 by nanoelectrospray MS and MS/MS analyses on a quadrupole-time-of-flight mass spectrometer (Q-TOFMS). The specificity of tandem mass spectrometry allowed the identification of six amino acid (AA) substitutions in M1, including R95K, A166V, I168T, N207S, N224S, and R230K. Two commonly observed modifications such as oxidation and deamidation were accurately assigned in the protein. Bioinformatics analysis suggested some relationship between the amino acid substitution and structural property of M1 protein. Discussions on de novo sequencing of MS/MS spectra, especially in dealing with the AA substitutions, were provided.
Full-text · Article · Nov 2008 · Journal of the American Society for Mass Spectrometry
[Show abstract][Hide abstract] ABSTRACT: We present the first proteomic analysis on the cellular responses to avian influenza virus (H9N2) infection in a human cell line in different time courses in order to search for target proteins for viral pathogenesis/adaptation studies. By using 2-DE coupled with MALDI-TOF MS and nano-ESI-MS/MS, we identified a set of differentially expressed cellular proteins, including cytoplasmic actin, cytokeratin, prohibitin, enoyl-CoA hydratase, peptide-prolyl cis-trans isomerase A (PPIase A), chloride intracellular channel protein 1, pyruvate dehydrogenase E1 component subunit beta, adenine phosphoribosyltransferase, guanine nucleotide-binding protein subunit beta, nucleoside diphosphate kinase A, elongation factor 1-beta and splicing factor, arginine/serine rich 1. The most significant changes in different time courses were found in cytoplasmic actin and cytokeratin, both of which constituted the major components of cytoskeleton network in the cells. The obtained data suggested a possible role of the cytoskeleton during avian influenza virus infection of mammalian cells, which might help for better understanding of the dynamics of avian influenza virus and host interaction in mammalian cell setting.
[Show abstract][Hide abstract] ABSTRACT: Antigenic profiles of post-2002 H5N1 viruses representing major genetic clades and various geographic sources were investigated
using a panel of 17 monoclonal antibodies raised from five H5N1 strains. Four antigenic groups from seven clades of H5N1 virus
were distinguished and characterized based on their cross-reactivity to the monoclonal antibodies in hemagglutination inhibition
and cell-based neutralization assays. Genetic polymorphisms associated with the variation of antigenicity of H5N1 strains
were identified and further verified in antigenic analysis with recombinant H5N1 viruses carrying specific mutations in the
hemagglutinin protein. Modification of some of these genetic variations produced marked improvement to the immunogenicity
and cross-reactivity of H5N1 strains in assays utilizing monoclonal antibodies and ferret antisera raised against clade 1
and 2 H5N1 viruses, suggesting that these sites represent antigenically significant amino acids. These results provide a comprehensive
antigenic profile for H5N1 virus strains circulating in recent years and will facilitate the recognition of emerging antigenic
variants of H5N1 virus and aid in the selection of vaccine strains.
Full-text · Article · Mar 2008 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: We examined the distribution of genetic mutations associated with resistance to the M2 ion channel–blocking adamantane derivatives,
amantadine and rimantadine, among H5N1 viruses isolated in Vietnam, Thailand, Cambodia, Indonesia, Hong Kong, and China. More
than 95% of the viruses isolated in Vietnam and Thailand contained resistance mutations, but resistant mutants were less commonly
isolated in Indonesia (6.3% of isolates) and China (8.9% of isolates), where human infection was recently reported. The dual
mutation motif Leu26Ile–Ser31Asn (leucine→isoleucine at aa 26 and serine→asparagine at aa 31) was found almost exclusively
in all resistant isolates from Vietnam, Thailand, and Cambodia, suggesting the biological selection of these mutations
Preview · Article · Jul 2006 · The Journal of Infectious Diseases