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

Percent mortality and pathogenicity in DBA/2J mice infected with various subtypes of avian influenza A viruses (at 10⁶ EID₅₀).

a<p>The survival score was calculated as 0.8× (survival AUC/maximum AUC).</p>b<p>The weight loss score was calculated as 0.2× (weight loss AUC/maximum AUC).</p>c<p>The total pathogenicity score is the sum of the survival and weight loss scores.</p>d<p>Pathogenicity indexes were classified as follows: 3, moderately pathogenic; 2, low pathogenic;1, least pathogenic; and 0, nonpathogenic.</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

Percent mortality and pathogenicity in DBA/2J mice infected with avian H1N1 virus isolates or pandemic H1N1 viruses (at 10⁶ EID₅₀).

a<p>The survival score was calculated as 0.8× (survival AUC/maximum AUC).</p>b<p>The weight loss score was calculated as 0.2× (weight loss AUC/maximum AUC).</p>c<p>The total pathogenicity score is the sum of the survival and weight loss scores.</p>d<p>Pathogenicity indexes were classified as follows: 4, most pathogenic; 3, moderately pathogenic; 2, low pathogenic; and 1, least pathogenic.</p

Transmissibility of A/shorebird/DE/300/2009 in ferrets.

<p>Viral titers were detected in (A) nasal washes and (B) rectal swabs of donor ferrets (red), direct-contact ferrets (DC, green) and respiratory-droplet–contact ferrets (RDC, blue) 14 dpi. Data from each animal are shown. Viral titers are given in terms of EID₅₀.</p

Comparison of the mean pathogenicity scores of each pathogenicity index (PI) category.

<p>Each avian H1N1 virus and the 2009 pandemic strains were assigned to a PI category based on their total pathogenicity score in DBA/2J mice. The PI-4 category includes the most pathogenic isolates; PI-3, moderately pathogenic isolates; PI-2, low pathogenic isolates; and PI-1, least pathogenic isolates. (*<i>P<0.05</i>).</p

Fifty percent mouse lethal dose (MLD₅₀) is inversely related to the pathogenicity index of avian H1N1 isolates.

<p>Fifty percent mouse lethal dose (MLD₅₀) is inversely related to the pathogenicity index of avian H1N1 isolates.</p

Amino acid divergence observed between sister subtypes over time.

<p>Estimates of evolutionary divergence for representative amino acid sequences between influenza A virus HA sister subtype lineages from the past (<1989) and present (>2009). Distances represent the average pairwise amino acid substitutions per site over all sequence pairs between groups. Standard error estimates were obtained by using a bootstrap procedure (1000 replicates). A total of 592 positions (non-ambiguous) over 604 sequences were used. A Poisson correction model was implemented. All analyses were conducted in MEGA5.</p

Evidence for the Circulation and Inter-Hemispheric Movement of the H14 Subtype Influenza A Virus

<div><p>Three H14 influenza A virus (IAV) isolates recovered in 2010 during routine virus surveillance along the Mississippi Migratory Bird Flyway in Wisconsin, U.S.A. raised questions about the natural history of these rare viruses. These were the first H14 IAV isolates recovered in the Western Hemisphere and the only H14 IAV isolates recovered since the original four isolates in 1982 in Asia. Full length genomic sequencing of the 2010 H14 isolates demonstrated the hemagglutinin (HA) gene from the 1982 and 2010 H14 isolates showed 89.6% nucleotide and 95.6% amino acid similarity and phylogenetic analysis of these viruses placed them with strong support within the H14 subtype lineage. The level of genomic divergence observed between the 1982 and 2010 viruses provides evidence that the H14 HA segment was circulating undetected in hosts and was not maintained in environmental stasis. Further, the evolutionary relationship observed between 1982 H14 and the closely related H4 subtype HA segments were similar to contemporary comparisons suggesting limited adaptive divergence between these sister subtypes. The nonstructural (NS) segment of one 2010 isolate was placed in a NS clade isolated infrequently over the last several decades that includes the NS segment from a previously reported 1982 H14 isolate indicating the existence of an unidentified pool of genomic diversity. An additional neuraminidase reassortment event indicated a recent inter-hemispheric gene flow from Asia into the center of North America. These results demonstrate temporal and spatial gaps in the understanding of IAV natural history. Additionally, the reassortment history of these viruses raises concern for the inter-continental spread of IAVs and the efficacy of current IAV surveillance efforts in detecting genomic diversity of viruses circulating in wild birds.</p> </div

Distribution of high-identity sequences in relation to the study site and the natural distribution of ruddy turnstones and major flyways of migratory aquatic birds.

<p>High-identity density areas are represented in shades of blue, with more intense tones representing states or provinces where a higher proportion of high-identity sequences were identified (values represent the percentage of the 400 high-identity sequences identified).</p