Influenza N1 Research Articles

Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study.

Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.

Protective effect of Asarum polysaccharide on H1N1 influenza virus infection and expression of inflammatory factors

In this paper, Asarum polysaccharides(AP) were extracted, and its composition was analyzed to study the activity against H1 N1 influenza virus in vitro and its intervention effect on mice with kidney Yang deficiency syndrome. AP was prepared by the strategy of water extraction and alcohol precipitation, the content was determined, and its monosaccharide composition was analyzed. The cell Real-time monitoring system and Reed-Muench model were adopted to evaluate the antiviral activity of AP in vitro. And the mouse model of kidney Yang deficiency syndrome was established in vivo to compare the efficacy of Mahuang Xixin Fuzi Decoction(MXF) and AP. MXF group and AP group were treated with clinical equivalent doses of 1.8 g·kg~(-1)·d~(-1) and 0.077 g·kg~(-1)·d~(-1) respectively, once a day for 6 consecutive days. Real-time PCR was used to detect the relative expression of M gene of H1 N1 influenza virus and cytokines in lung tissue. The content of AP in Asarum was 25.22%, and the protein content was 0.8%. And the monosaccharide composition was identified as L-rhamnose, D-arabinose, D-xylose, D-glucose, D-galactose and D-mannose. TI values of Tamiflu, MXF and AP were 30.00, 8.06 and 10.33, respectively. Three different doses of AP could significantly reduce the concentration of virus in supernatant. Compared with the model mice, lung indexes of MXF group and AP group decreased significantly(P<0.05), and the relative expression of M gene decreased significantly(P<0.05). The relative expressions of IL-10 and IFN-γ were up-regulated to varying degrees, while the relative gene expressions of IL-1β, IL-6 and MCP-1 were down-regulated to different degrees. In addition, AP could significantly enhance the expression of TNF-α(P<0.01). AP had a good anti-influenza virus activity in vitro, and could protect mice with kidney Yang deficiency syndrome by reducing the viral load in lung tissue, decreasing inflammation damage in lung tissue, and regulating the expression of inflammatory cytokines. Compared with the prescription of MXF, AP had a better antiviral activity.

Impact of tetramerization on the ligand recognition of N1 influenza neuraminidase via MMGBSA approach.

Influenza virus neuraminidase (NA) is a homotetrameric surface protein that, in contrast to other non-influenza NAs, requires a quaternary assembly to exhibit enzymatic activity, suggesting that the oligomeric state significantly impacts the active site of influenza NA. Nevertheless, most structure-based drug design studies have been reported by employing the monomeric state in the closed or open-loop due to the computational cost of employing the tetrameric NA. In this work, we present MD simulations coupled to the MMGBSA approach of avian N1 type NA in its monomeric and tetrameric closed and open-loop state both with and without the inhibitor oseltamivir and its natural substrate, sialic acid. Structural and energetic analyses revealed that the tetrameric state impacts flexibility as well as the map of interactions participating in stabilizing the protein-ligand complexes with respect to the monomeric state. It was observed that the tetrameric state exerts dissimilar effects in binding affinity, characteristic of positive and negative cooperativity for oseltamivir and sialic acid, respectively. Based on our results, to perform a confident structure-based drug design, as well as to evaluate the impact of key mutations through MD simulations, it is important to consider the tetrameric state closed-loop state.

Ancestral and Compensatory Mutations that Promote Antiviral Resistance in InfluenzaN1 Neuraminidase Revealed by a Phylonumerics Approach.

Implementation of a new phylonumerics approach to construct a mass tree representing over 6000 H1N1 human influenza strains has enabled ancestral and compensatory descendant mutations to be identified in N1 neuraminidase that promote antiviral resistance and restore viral fitness. Adjacent to the H275Y resistance mutation site, mutations S299A and S247N, respectively, lead the evolution of oseltamivir-resistant strains and restore viral fitness to those strains thereafter. Importantly the mass tree phylonumerics approach can identify such mutations globally, without any positional bias, so that functionally linked or compensatory mutations remote in the sequence or structure of the protein can be identified and interrogated. This is achieved using mass map datasets commonly employed for protein identification in proteomics applications, thus avoiding the need for either gene or protein sequences that are central to other phylogenetic methods.

Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets

Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses.

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.

Molecular Basis for Differential Patterns of Drug Resistance in Influenza N1 and N2 Neuraminidase.

Neuraminidase (NA) inhibitors are used for the prevention and treatment of influenza A virus infections. Two subtypes of NA, N1 and N2, predominate in viruses that infect humans, but differential patterns of drug resistance have emerged in each subtype despite highly homologous active sites. To understand the molecular basis for the selection of these drug resistance mutations, structural and dynamic analyses on complexes of N1 and N2 NA with substrates and inhibitors were performed. Comparison of dynamic substrate and inhibitor envelopes and interactions at the active site revealed how differential patterns of drug resistance have emerged for specific drug resistance mutations, at residues I222, S246, and H274 in N1 and E119 in N2. Our results show that the differences in intermolecular interactions, especially van der Waals contacts, of the inhibitors versus substrates at the NA active site effectively explain the selection of resistance mutations in the two subtypes. Avoiding such contacts that render inhibitors vulnerable to resistance by better mimicking the dynamics and intermolecular interactions of substrates can lead to the development of novel inhibitors that avoid drug resistance in both subtypes.

Cellular Interferon Gamma and Granzyme B Responses to Cytomegalovirus-pp65 and Influenza N1 Are Positively Associated in Elderly.

Morbidity and mortality in the elderly are associated with viral infections, including influenza and cytomegalovirus (CMV). With increasing age, cellular immunity gains importance in protection to influenza, but latent CMV is highly prevalent and associated with immune dysfunction. An insight into the association between immunity against influenza and CMV adds to the understanding of healthy aging. Here we first aimed to measure influenza-specific cellular immunity using granzyme B (GrzB) and interferon-gamma (IFN-γ)-ELISpot. Next, influenza-specific cellular immunity was associated with humoral and cellular CMV-specific immunity in healthy 65+ elderly. Vaccine trial participants gave additional blood samples 3 weeks after receiving a H1N1 containing vaccine. CMV serology was determined and peripheral blood mononuclear cells were stimulated with influenza N1 or CMV pp65-derived peptide pools for 7 days and rechallenged to assess antigen-specific GgrzB and IFN-γ responses using ELISpot assays. Results were compared using chi-square and correlation analysis. Eighty-three individuals (60% men, 65% CMV IgG+, age range 65-78y) participated. We found significant positive associations between IFN-γ and GrzB responses to both influenza and CMV, but also between a positive CMV serostatus and an influenza N1-specific activation marker response (p = 0.013). CMV pp65 responses were detected in CMV IgG+ individuals, but remarkably also in CMV IgG- individuals (27%). In this study, following influenza vaccination, elderly with cellular immunity against CMV were more likely to have cellular immunity against influenza vaccine N1 antigen. These findings stress the need to continue exploring the possible role of CMV in immunosenescence.

Numerical study of a diffusive epidemic model of influenza with variable transmission coefficient

A diffusive epidemic model for H1 N1 influenza is formulated with a view to gain basic understanding of the virus behavior. All newborns are assumed to be susceptible. Mortality rate for infective individuals in the population is assumed to be greater than natural mortality rate. Latent, infectious and immune periods are assumed to be constants throughout this study. The numerical solutions of this model are carried out under three different initial populations distribution. In order to investigate the effect of the disease transmission coefficient on the spread of disease, β is taken to be constant as well as a function of seasonally varying time t and a function of spatial variable x. The threshold quantity ( R 0 ) that governs the disease dynamics is derived. Numerical simulation shows that the system supports the existence of sustained and damped oscillations depending on initial populations distribution, the disease transmission rate and diffusion.

Molecular Dynamics Simulations Suggest that Electrostatic Funnel Directs Binding of Tamiflu to Influenza N1 Neuraminidases

Oseltamivir (Tamiflu) is currently the frontline antiviral drug employed to fight the flu virus in infected individuals by inhibiting neuraminidase, a flu protein responsible for the release of newly synthesized virions. However, oseltamivir resistance has become a critical problem due to rapid mutation of the flu virus. Unfortunately, how mutations actually confer drug resistance is not well understood. In this study, we employ molecular dynamics (MD) and steered molecular dynamics (SMD) simulations, as well as graphics processing unit (GPU)-accelerated electrostatic mapping, to uncover the mechanism behind point mutation induced oseltamivir-resistance in both H5N1 “avian” and H1N1pdm “swine” flu N1-subtype neuraminidases. The simulations reveal an electrostatic binding funnel that plays a key role in directing oseltamivir into and out of its binding site on N1 neuraminidase. The binding pathway for oseltamivir suggests how mutations disrupt drug binding and how new drugs may circumvent the resistance mechanisms.

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium-bound and calcium-free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley-Liss, Inc.

Permissive secondary mutations enable the evolution of influenza oseltamivir resistance.

The His274-->Tyr274 (H274Y) mutation confers oseltamivir resistance on N1 influenza neuraminidase but had long been thought to compromise viral fitness. However, beginning in 2007-2008, viruses containing H274Y rapidly became predominant among human seasonal H1N1 isolates. We show that H274Y decreases the amount of neuraminidase that reaches the cell surface and that this defect can be counteracted by secondary mutations that also restore viral fitness. Two such mutations occurred in seasonal H1N1 shortly before the widespread appearance of H274Y. The evolution of oseltamivir resistance was therefore enabled by "permissive" mutations that allowed the virus to tolerate subsequent occurrences of H274Y. An understanding of this process may provide a basis for predicting the evolution of oseltamivir resistance in other influenza strains.

Eleven fatal cases with A H1N1 influenza in Tianjin

To describe the clinical characteristics of fatal cases with confirmed A H1N1 influenza so as to improve the diagnosis and treatment of this severe disease. We retrospectively analyzed the medical records of 11 fatal cases with A H1N1 influenza in Tianjin between September 1 and November 4 2009. There were 6 males and 5 females with a median age of 25 (21 - 36) years old. Two cases were pregnant women and 3 patients suffered such concurrent disorders as dilated cardiomyopathy, multiple myeloma or atrophic gastritis. Over 3 lung fields were involved on chest imaging studies and the oxygenation index was less than 300 mm Hg at admission. The therapies of oseltamivir and methylprednisolone were administered and mechanical ventilation was initiated within 24 hours. Refractory hypoxemia and a higher level of lactate dehydrogenase were present during treatment. Pneumothorax or mediastinal emphysema occurred in 4 patients, acute renal failure in 1 and pneumopyothorax in 1. The culture of airway secretion at 3 - 7 days after mechanical ventilation showed Staphylococcus aureus in 4 patients and Aspergillus in 2. The progress of disease was so quick that the duration from onset of clinical symptoms to hospitalization was a median of 4 (3 - 6) days and the duration from onset of clinical symptoms to death a median of 12 days. The fatal cases with A H1N1 influenza in Tianjin occurred mostly in young individuals and pregnant women. This severe disease had a rapid progression. And bacterial co-infections were quite common. Refractory hypoxemia resulting in respiratory failure was the main mortality reason.

Role of Secondary Sialic Acid Binding Sites in Influenza N1 Neuraminidase

Within influenza viral particles, the intricate balance between host cell binding and sialic acid receptor destruction is carefully maintained by the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, respectively. A major outstanding question in influenza biology is the function of a secondary sialic acid binding site on the NA enzyme. Through a series of Brownian dynamics (BD) simulations of the avian N1, human pandemic N2, and currently circulating pandemic (H1)N1 enzymes, we have probed the role of this secondary sialic acid binding site in the avian N1 subtype. Our results suggest that electrostatic interactions at the secondary and primary sites in avian NA may play a key role in the recognition process of the sialic acid receptors and catalytic efficiency of NA. This secondary site appears to facilitate the formation of complexes with the NA protein and the sialic acid receptors, as well as provide HA activity to a lesser extent. Moreover, this site is able to steer inhibitor binding as well, albeit with reduced capacity in N1, and may have potential implications for drug resistance or optimal inhibitor design. Although the secondary sialic acid binding site has previously been shown to be nonconserved in swine NA strains, our investigations of the currently circulating pandemic H1N1 strain of swine origin appears to have retained some of the key features of the secondary sialic acid binding site. Our results indicate possible lowered HA activity for this secondary sialic acid site, which may be an important event in the emergence of the current pandemic strain.

Neuraminidase inhibitor drug susceptibility differs between influenza N1 and N2 neuraminidase following mutagenesis of two conserved residues

Neuraminidase (NA) inhibitors are a class of antivirals designed to target the conserved residues of the influenza NA active site. While there are many conserved residues in the NA active site that are involved in NA inhibitor binding, only a few have been demonstrated to confer resistance. As such, little is known regarding the potential of the other conserved residues in the NA active site to cause NA inhibitor resistance. Two conserved residues (E227 and E276) of an N1 NA that have not previously been associated with resistance to NA inhibitors were investigated. Site-directed mutagenesis was used to generate three alternative amino acids at each residue. Reverse genetics was used to generate recombinant mutant viruses which were characterized for growth, NA activity and NA inhibitor sensitivity. Of the six recombinant viruses expressing NA with mutations at either E227 or E276, only the E227D and E276D viruses were able to grow without supplementary NA activity, and all mutant viruses had a significant reduction in NA activity. The E227D virus demonstrated significantly reduced sensitivity to zanamivir while the E276D virus did not demonstrate any significant changes in NA inhibitor sensitivity. Interestingly, the resistance profiles of E227D and E276D in N1 NA were significantly different from these sites that have been reported for N2 NA. This study confirmed the essential role of NA active site residues in viral fitness, and identified clear differences in the role of residues E227 and E276 in NA inhibitor resistance with N1 and N2 neuraminidases.

Remarkable Loop Flexibility in Avian Influenza N1 and Its Implications for Antiviral Drug Design

The emergence and continuing global spread of the highly virulent avian influenza H5N1 has raised concerns of a possible human pandemic. Several approved anti-influenza drugs effectively target the neuraminidase (NA), a surface glycoprotein that cleaves terminal sialic acid residues and facilitates the release of viral progeny from infected cells. The first crystal structures of group-1 NAs revealed that although the binding pose of oseltamivir was similar to that seen in previous crystallographic complexes, the 150-loop adopted a distinct conformation, opening a new cavity adjacent to the active site. Here we show that the 150-loop is able to open into significantly wider conformations than seen in the crystal structures, through explicitly solvated MD simulations of the apo and oseltamivir-bound forms of tetrameric N1. We find that motion in the 150-loop is coupled to motion in the neighboring 430-loop, which expands the active site cavity even further. Furthermore, in simulations of the oseltamivir-bou...

Characterization of drug-resistant recombinant influenza A/H1N1 viruses selected in vitro with peramivir and zanamivir

There is a limited information with regard to the neuraminidase (NA) mutations conferring resistance to peramivir and zanamivir in the influenza N1 background. In this study, an influenza A/WSN/33 (H1N1) recombinant virus was passaged under peramivir or zanamivir pressure. The peramivir-selected variant had a H274Y mutation in the neuraminidase (NA) gene conferring resistance to peramivir and oseltamivir but susceptibility to zanamivir. The zanamivir-selected variant had a massive deletion in the region encoding the NA active center and an A200T hemagglutinin mutation. This variant exhibited reduced susceptibility to zanamivir with a drug-dependent phenotype.

A preliminary panorama of the diversity of N1 subtype influenza viruses

N1 subtype influenza viruses have caused many epidemics and even a few pandemics in humans, pigs and fowls including 1918 human H1N1 pandemic, which killed 20-50 million people and the current avian H5N1 pandemic in the Eastern Hemisphere, which has caused great economic losses and posed a severe threat to human public health. To elucidate the whole diversity of N1 influenza viruses from a dynamic view, 202 neuraminidase (NA) sequences of N1 subtype influenza isolates were selected and analyzed in this study. Our results showed that N1 influenza isolates could be divided into three distinct lineages (Human, Classic Swine and Avian), which largely circulated in the humans, pigs and fowls respectively, though viruses in the Avian lineage could infect mammals and even there was a sublineage in the Avian lineage wholly isolated from pigs. The Avian lineage and the Human lineage, which have existed at least for decades, possibly began divergence around in 1890 through regression analysis. Both of the Human and Avian lineages could be further divided into some sublineages, and the correlation between these lineages (or sublineages) and their isolation places, isolation time, hemagglutinin (HA) subtypes, host species, virulence, or epidemics were discussed. The panorama of the diversity of N1 influenza viruses presented in this study provided a framework for the studies on the evolution and epidemiology of N1 influenza viruses.

Rapid Detection of Avian Influenza Virus A and Subtype H5N1 by Single Step Multiplex Reverse Transcription-polymerase Chain Reaction

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

A case of avian influenza (H5N1) pulmonary infection with early expansion of lung bulla formations

Summary A 22 year old previously healthy male subject was admitted to hospital in Ho Chi Minh City, Vietnam, with fever and severe pneumonia. On interviewing the patient a recent close contact with poultry was revealed and a RT-PCR test for H5 and N1 influenza antigen was found positive following nose and throat swabs. Partial recovery was highlighted by the development of extensive bulla formations in the left lung. We herein report clinical and radiological features in a 22 year old male Vietnamese patient who developed severe acute pneumonia and in whose RT-PCR test for H5 and N1 influenza antigen was found positive following nose and throat swabs. Partial recovery was highlighted by the development of extensive bulla formations in the left lung.