Low dose influenza virus challenge in the ferret leads to increased virus shedding and greater sensitivity to oseltamivir

Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 106 plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 102 pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 104 pfu gave an infection that was intermediate between those of the 106 pfu and 102 pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (106 pfu) and low (102 pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines

Chikungunya Virus and Central Nervous System Infections in Children, India

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus best known for causing fever, rash, arthralgia, and occasional neurologic disease. By using real-time reverse transcription–PCR, we detected CHIKV in plasma samples of 8 (14%) of 58 children with suspected central nervous system infection in Bellary, India. CHIKV was also detected in the cerebrospinal fluid of 3 children

Relatório de estágio em farmácia comunitária

Relatório de estágio realizado no âmbito do Mestrado Integrado em Ciências Farmacêuticas, apresentado à Faculdade de Farmácia da Universidade de Coimbr

Nasal wash cell counts in the presence or absence of oseltamivir treatment.

<p>A, prophylactic oseltamivir regimen from 2; B, therapeutic oseltamivir regimen from 6 hr post-infection. • (red) high dose (10⁶ pfu); ▴ (blue) low dose (10² pfu); ★ mock infected animals; open symbols represent oseltamivir-treated animals. Means from 5 ferrets (A) or 3–9 ferrets per group (B).</p

Effect of virus dose on parameters of infection with influenza A/California/04/09.

1<p>Mean clinical score is calculated as described in Materials and Methods, and expressed as mean score per ferret per day ± standard error of the mean.</p>2<p>Day of onset refers to median onset of respiratory signs and inactivity from four studies.</p><p>ND, not done.</p

Viral RNA loads in ferret respiratory tract tissues.

<p>Ferrets were infected intra-nasally with 10⁶ or 10² pfu Cal/04 and, where indicated, treated with oseltamivir from 6 hr post-infection. Circles show RNA loads for individual animals. Horizontal lines show group means. Filled circles, no treatment; open circles, oseltamivir treated. A, nasal turbinate; B, trachea; C, lung. High, 10⁶ pfu inoculum; Low, 10² pfu inoculum. Samples were taken from 2 ferrets on days 1 to 4, and 3 ferrets on day 5. The sensitivity of the assay was approximately 10³ copies/mg.</p

Summary of severity of pathological changes in untreated or oseltamivir treated ferret tissues.

<p>A. Nasal cavity, B. lung. In each case, changes were scored as minimal (min), mild, moderate (mod), or marked, and were summed for each group of ferrets on each day post-infection. Group size was 2 ferrets, except day 5 which was groups of 3 ferrets. The day 5 summed frequencies have been normalised to facilitate comparison to the other days.</p

Effect of decreasing infectious dose on virus shedding.

<p>Ferrets were infected intra-nasally, and nasal washes were collected at the intervals shown for virus plaque assay. Markers show geometric mean nasal wash titre from groups of 5 or 8 ferrets; error bars show standard deviation. For days 10 and 14 post-infection, 2 ferrets per group were used. • (red) high dose (10⁶ pfu), ▪ (green) medium dose (104 pfu), ▴ (blue) low dose (102 pfu) inoculum. The lower limit of detection was 10 pfu/ml.</p

Microscopic changes in ferret nasal cavity and lung, with and without oseltamivir treatment.

<p>A. Nasal cavity, low dose, untreated group, 4 dpi. Propria mucosa is infiltrated by marked numbers of mixed inflammatory cells. Overlying respiratory epithelium comprises attenuated, pre-ciliated, regenerating cells (arrow). B. Nasal cavity, low dose with oseltamivir-treated group, 4 dpi. A mild, mononuclear cell infiltrate within the nasal propria mucosa underlying a normal, pseudostratified, columnar, ciliated epithelium (arrow). C. Lung, low dose, untreated group, 5 dpi. Multifocally extensive, mononuclear cell infiltration of parenchyma. Inset: focal, bronchial gland necrosis (asterisk). D. Lung, high dose, untreated group, 5 dpi. Marked infiltration of parenchyma with inflammatory cells by peribronchial and perivascular oedema (Oe) and bronchial gland necrosis (arrow). E. Lung, low dose, oseltamivir-treated group, 5 dpi. Minimal, parenchymal, mononuclear cell infiltrate (arrow). F. Lung, high dose, oseltamivir-treated group, 5 dpi. Mild, peribronchiolar, mononuclear cell infiltration (arrow). Haematoxylin and eosin.</p