Schematic of the evolutionary history among HA, NP, NA and M segments of H1N1 IAVs.

<p>The schematic view is based on the phylogenetic trees of avian, swine, and human origin H1N1 IAVs and the most recent common ancestor (MRCA) for (A) HA and NP gene segments and (B) NA and M gene segments. North American avian (light blue) and 2009 pandemic H1N1 viruses (pink) are highlighted.</p

The Genomic Contributions of Avian H1N1 Influenza A Viruses to the Evolution of Mammalian Strains

<div><p>Among the influenza A viruses (IAVs) in wild aquatic birds, only H1, H2, and H3 subtypes have caused epidemics in humans. H1N1 viruses of avian origin have also caused 3 of 5 pandemics. To understand the reappearance of H1N1 in the context of pandemic emergence, we investigated whether avian H1N1 IAVs have contributed to the evolution of human, swine, and 2009 pandemic H1N1 IAVs. On the basis of phylogenetic analysis, we concluded that the polymerase gene segments (especially PB2 and PA) circulating in North American avian H1N1 IAVs have been reintroduced to swine multiple times, resulting in different lineages that led to the emergence of the 2009 pandemic H1N1 IAVs. Moreover, the similar topologies of hemagglutinin and nucleoprotein and neuraminidase and matrix gene segments suggest that each surface glycoprotein coevolved with an internal gene segment within the H1N1 subtype. The genotype of avian H1N1 IAVs of Charadriiformes origin isolated in 2009 differs from that of avian H1N1 IAVs of Anseriformes origin. When the antigenic sites in the hemagglutinin of all 31 North American avian H1N1 IAVs were considered, 60%-80% of the amino acids at the antigenic sites were identical to those in 1918 and/or 2009 pandemic H1N1 viruses. Thus, although the pathogenicity of avian H1N1 IAVs could not be inferred from the phylogeny due to the small dataset, the evolutionary process within the H1N1 IAV subtype suggests that the circulation of H1N1 IAVs in wild birds poses a continuous threat for future influenza pandemics in humans.</p></div

Evolutionary relatedness of PB2 gene segments of H1N1 IAVs of avian, swine, and human origins isolated from North America and Eurasia.

<p>The taxa are colored based on their host and geography: red-North American avian isolates from the St. Jude repository, light blue-North American avian isolates, purple-Eurasian avian isolates, green- swine isolates from North America, grey- swine isolates from Eurasia (both Eurasian avian-like swine and Eurasian classical swine), dark blue-human isolates from North America; orange- human isolates from Eurasia, and pink- 2009 pandemic isolates. The nodes with spillover avian H1N1 IAVs are denoted with light blue arrows.</p

Calculation of the spectroscopic correction factor.

<p><b>Abbreviations:</b> MUNANA, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid; 4-MU, 4-methylumbelliferone; RFU, relative fluorescence units.</p>a<p>Fluorescence of MUNANA alone in enzyme buffer at 1.95–2000 µM concentrations (shown in column 1).</p>b<p>Fluorescence of 4-MU alone in enzyme buffer at 19 µM concentration. Mean (± SD) 4-MU fluorescence in enzyme buffer was 10222±90 RFU.</p>c<p>Fluorescence of 4-MU in enzyme buffer at 19 µM concentration in the presence of 1.95–2000 µM MUNANA concentrations (shown in column 1).</p>d<p>Values (in RFU) of MUNANA fluorescence (column 2) subtracted from the values (in RFU) obtained in the mixture of 4-MU and MUNANA (column 4).</p>e<p>Correction factor calculated as the ratio of 4-MU fluorescence (in RFU) in the mixture with MUNANA substrate (column 5) and 4-MU fluorescence (in RFU) alone (mean value from column 3).</p

Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate

<div><p>The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining <i>K_m</i> and <i>V_max</i> kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.</p></div

Flowchart for the determination of NA enzyme kinetic parameters using whole influenza virus preparations.

<p>Flowchart for the determination of NA enzyme kinetic parameters using whole influenza virus preparations.</p

Inner filter effect (IFE) from light absorption at the excitation and emission wavelengths of the 4-MU product.

<p>(A) Absorbance spectra of MUNANA and 4-MU at 0.01 mg/mL and 0.1 mg/mL concentrations in enzyme buffer. Optical density was measured using Synergy 2 multi-mode microplate reader in a UV-transparent 96-well plate. (B) Absorbance spectra of MUNANA and 4-MU at 0.1 mg/mL concentration. (C) 4-MU fluorescence measured in the presence of different concentrations of MUNANA (15–2000 µM) shows similar impact of MUNANA-associated spectroscopic interference across 4-MU concentrations of 10–80 µM.</p

Standard curve of 4-methylumbelliferone (4-MU) fluorescence.

<p>Fluorescence intensity was measured using a Synergy 2 multimode microplate reader at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Relative fluorescence units (RFU) obtained at low 4-MU concentrations (0–2.0 µM) are shown in the insert. Each data point represents the mean ± standard deviation (SD) of 10 independent measurements.</p

Viral titers in the lungs of DBA/2J mice infected with avian H1N1 virus isolates from different pathogenicity levels.^a

a<p>Lungs were collected from mice after natural death or euthanasia upon 25% weight loss, according to our protocol.</p><p><b>Abbreviations:</b> ND, not determined; UD, undetected.</p

Hematoxylin & eosin staining of influenza-infected lungs from DBA/2J mice.

<p>(A) In the uninfected lung, the bronchiole is lined with epithelium. (B) The infected lung shows severe bronchiole epithelial necrosis. (C) In the uninfected lung, the alveoli lumens are free of cells and have thin septal walls. (D) The infected lung shows focal alveolitis with neutrophils and mononuclear cells in the alveoli lumens, and the alveolar septal walls are thickened. Magnification: 40×(A–B) or 80×(C–D).</p