Oseltamivir treatment improves clinical symptoms of infected ferrets despite no reduction in viral load.

<p>(A) Cell number and (B) protein concentration in nasal washes. (C) Body weight. (D) Body temperature; mean temperature versus day 0 temperature: *P<0.05, **P<0.01, Mann-Whitney U test. (E) Virus titre in nasal wash. (F) Serum antibody titre. ND: Non-detected. OST: Oseltamivir. Data shown are mean±SEM from the same experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118780#pone.0118780.g002" target="_blank">Fig. 2</a>. Data comparison in (A—C): *P<0.05, **P<0.01, Kruskal-Wallis test.</p

A Novel Video Tracking Method to Evaluate the Effect of Influenza Infection and Antiviral Treatment on Ferret Activity

<div><p>Ferrets are the preferred animal model to assess influenza virus infection, virulence and transmission as they display similar clinical symptoms and pathogenesis to those of humans. Measures of disease severity in the ferret include weight loss, temperature rise, sneezing, viral shedding and reduced activity. To date, the only available method for activity measurement has been the assignment of an arbitrary score by a ‘blind’ observer based on pre-defined responsiveness scale. This manual scoring method is subjective and can be prone to bias. In this study, we described a novel video-tracking methodology for determining activity changes in a ferret model of influenza infection. This method eliminates the various limitations of manual scoring, which include the need for a sole ‘blind’ observer and the requirement to recognise the ‘normal’ activity of ferrets in order to assign relative activity scores. In ferrets infected with an A(H1N1)pdm09 virus, video-tracking was more sensitive than manual scoring in detecting ferret activity changes. Using this video-tracking method, oseltamivir treatment was found to ameliorate the effect of influenza infection on activity in ferret. Oseltamivir treatment of animals was associated with an improvement in clinical symptoms, including reduced inflammatory responses in the upper respiratory tract, lower body weight loss and a smaller rise in body temperature, despite there being no significant reduction in viral shedding. In summary, this novel video-tracking is an easy-to-use, objective and sensitive methodology for measuring ferret activity.</p></div

Mean manual activity score of non-infected ferrets and infected ferrets with or without oseltamivir treatment from day 0 to day 11 post-infection.

<p>OST: Oseltamivir</p><p>Mean manual activity score of non-infected ferrets and infected ferrets with or without oseltamivir treatment from day 0 to day 11 post-infection.</p

Comparison of video-tracking with manual scoring for assessing ferret activity.

<p>(A—C) Video-tracking analysis and (D—F) manual scoring of the mean activity of non-infected ferrets (n = 4), untreated ferrets (n = 4) and oseltamivir (OST)-treated ferret (n = 4). Untreated and OST-treated ferrets were both intranasally-infected with 10³ TCID₅₀ (500 μL; 250 μL per nostril) A(H1N1)pdm09. Untreated ferrets were given sugar solution as a treatment control. Data shown are mean±SEM of an experiment independent to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118780#pone.0118780.g002" target="_blank">Fig. 2</a>. Dotted line is the baseline average of the daily activity level prior to infection on day 0. Mean versus baseline average: *P<0.05, **P<0.01, ***P<0.001, Mann-Whitney U test.</p

Reducing Uncertainty in Within-Host Parameter Estimates of Influenza Infection by Measuring Both Infectious and Total Viral Load

<div><p>For <i>in vivo</i> studies of influenza dynamics where within-host measurements are fit with a mathematical model, infectivity assays (e.g. 50% tissue culture infectious dose; TCID₅₀) are often used to estimate the infectious virion concentration over time. Less frequently, measurements of the total (infectious and non-infectious) viral particle concentration (obtained using real-time reverse transcription-polymerase chain reaction; rRT-PCR) have been used as an alternative to infectivity assays. We investigated the degree to which measuring both infectious (via TCID₅₀) and total (via rRT-PCR) viral load allows within-host model parameters to be estimated with greater consistency and reduced uncertainty, compared with fitting to TCID₅₀ data alone. We applied our models to viral load data from an experimental ferret infection study. Best-fit parameter estimates for the “dual-measurement” model are similar to those from the TCID₅₀-only model, with greater consistency in best-fit estimates across different experiments, as well as reduced uncertainty in some parameter estimates. Our results also highlight how variation in TCID₅₀ assay sensitivity and calibration may hinder model interpretation, as some parameter estimates systematically vary with known uncontrolled variations in the assay. Our techniques may aid in drawing stronger quantitative inferences from <i>in vivo</i> studies of influenza virus dynamics.</p></div

Three-dimensional neuraminidase (NA) protein structure of the influenza A/Tanzania/205/2010 (H3N2) strain in complex with oseltamivir carboxylate (depicted as the central mesh), downloaded from the RCSB Protein Data Bank (PDB ID: 4GZP).

<p>The ribbons representation of the NA protein structure was generated and edited with the UCSF Chimera software package. The positions of the amino acids, D93, Y155, I222, G248, K249, and D251, were highlighted in red.</p

Three-dimensional proton channel matrix 2 (M2) tetrameric structure of the influenza A/Udorn/307/1972 (H3N2) strain in complex with rimantadine (depicted as the 4 individual meshes), downloaded from the RCSB Protein Data Bank (PDB ID: 2RLF).

<p>The ribbons representation of the four tightly packed transmembrane helices (each consisted of 15 M2 primary polypeptide chains) were generated and edited with the UCSF Chimera software package. The positions of the amino acids L26, V27, and S31 were highlighted in green, blue, and red, respectively.</p

Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses

<div><p>CD8⁺ T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8⁺ T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8⁺ T cell responses and the establishment of immunological CD8⁺ T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8⁺ T cell responses. Importantly, functional memory CD8⁺ T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4⁺ T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8⁺ T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.</p></div

Oseltamivir treatment decreases the magnitude of the effector influenza-specific CD8⁺ T cell responses.

<p>BL/6 mice administered with oseltamivir or PBS were assessed for CD8⁺ T cell responses to the influenza A viral epitopes (A) D^bNP₃₆₆, (B) D^bPA₂₂₄, (C) K^bPB1_703-711, (D) D^bPB1-F2_62-70 and (E) K^bNS2_114-121 by intracellular IFN-γ staining after a five hour stimulation of splenocytes with cognate peptide. Data represent 9–10 mice per group, analysed over two independent experiments. Epitope-specific CD8⁺ T cells directed at (FG) D^bNP₃₆₆, (HI) D^bPA₂₂₄ were enumerated by tetramer staining in both spleen (GI) and at the site of infection (FH). Data represent 9–10 mice per group, analysed over two independent experiments. The mean and standard deviation is shown. (JK) Representative dotplots for the D^bNP₃₆₆⁺CD8⁺ and D^bPA₂₂₄⁺CD8⁺ T cell populations on d10 (acute phase) and d40 (memory) are shown; populations are gated on CD8⁺ T cells. The mean and standard deviation is shown. *P<0.05; **P<0.01.</p

Normal influenza-specific CD4⁺ T cell responses in oseltamivir-treated mice after secondary infection.

<p>Mice were treated with PBS or oseltamivir and infected with influenza virus X31, then rested for 40 days. At this time point, mice were infected i.n. with 600 pfu of influenza PR8. At 8 days post-secondary infection, (A) MedLN and (B) BAL fluid were harvested and assessed for the total number of influenza IA^b- NP₃₁₁-specific CD4⁺ T cells by intracellular cytokine staining after <i>ex vivo</i> stimulation. These data represent 2 individual experiments with 4–5 mice per group. Error bars represent standard error of the mean. n.s., not significantly different.</p