Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants

AbstractIn the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential

Pathogenicity and Transmissibility of North American Triple Reassortant Swine Influenza A Viruses in Ferrets

<div><p>North American triple reassortant swine (TRS) influenza A viruses have caused sporadic human infections since 2005, but human-to-human transmission has not been documented. These viruses have six gene segments (PB2, PB1, PA, HA, NP, and NS) closely related to those of the 2009 H1N1 pandemic viruses. Therefore, understanding of these viruses' pathogenicity and transmissibility may help to identify determinants of virulence of the 2009 H1N1 pandemic viruses and to elucidate potential human health threats posed by the TRS viruses. Here we evaluated in a ferret model the pathogenicity and transmissibility of three groups of North American TRS viruses containing swine-like and/or human-like HA and NA gene segments. The study was designed only to detect informative and significant patterns in the transmissibility and pathogenicity of these three groups of viruses. We observed that irrespective of their HA and NA lineages, the TRS viruses were moderately pathogenic in ferrets and grew efficiently in both the upper and lower respiratory tracts. All North American TRS viruses studied were transmitted between ferrets via direct contact. However, their transmissibility by respiratory droplets was related to their HA and NA lineages: TRS viruses with human-like HA and NA were transmitted most efficiently, those with swine-like HA and NA were transmitted minimally or not transmitted, and those with swine-like HA and human-like NA (N2) showed intermediate transmissibility. We conclude that the lineages of HA and NA may play a crucial role in the respiratory droplet transmissibility of these viruses. These findings have important implications for pandemic planning and warrant confirmation.</p> </div

Growth characteristics of North American TRS viruses in MDCK cells.

<p>Cells were inoculated with the respective viruses at an MOI of 0.001 and incubated at the indicated temperatures. At the indicated h p.i., supernatants were harvested and virus was titrated by pfu assay. At the higher temperatures (37°C and 39.5°C), cytopathic effects caused detachment of most infected cells from the dish after 38 h p.i.; therefore, virus release was not examined beyond that point. Values are the mean of two independent experiments performed in duplicate (n = 4).</p

Transmission of North American TRS viruses in ferrets via direct contact (co-housing) and respiratory droplets.

<p>Sw, swine; hu, human.</p>a<p>In nasal wash specimens.</p>b<p>Delayed transmission: virus detected on day 7 post-exposure (day 8 p.i.).</p

Histopathology of ferret lung tissue.

<p>Lung tissue of control (un-inoculated) and virus-inoculated ferrets was collected on day 5 p.i. Formalin-fixed, paraffin-embedded 5-µm sections were stained with hematoxylin and eosin and microscopically examined in a blinded fashion. Representative images show bronchi (A–D), bronchioles (E–H), and alveoli (I–L) from un-inoculated (A,E,I) and virus-inoculated ferrets. The two North American TRS viruses (C,G,K and D,H,L) caused bronchitis, bronchiolitis, alveolitis, and alveolar wall interstitial changes. The bronchitis featured intraluminal granulocytes and/or mucus, bronchial epithelial hyperplasia with submucosal mucus gland loss, and mixed inflammatory-cell infiltrates. The bronchiolitis featured intraluminal cellular debris, sloughed epithelial cells, and inflammatory cells (macrophages and/or granulocytes). The peribronchiolar alveoli contained mixed inflammatory cell infiltrates and foci of pneumocyte hyperplasia. The Eurasian avian-like swine virus (B,F,J) caused morphologic changes similar to those caused by the TRS viruses but far less severe.</p