A Non Mouse-Adapted Dengue Virus Strain as a New Model of Severe Dengue Infection in AG129 Mice

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing

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Vascular leakage in D2Y98P-infected mice.

<p>AG129 mice were inoculated ip. with 10⁷ or 10⁴ PFU of D2Y98P. At day 6 p.i. (10⁴ PFU dose) or at moribund state (both doses), mice were intravenously administered with Evans blue. After 2 hours, they were perfused extensively with PBS and assessed for Evans Blue extravasation in tissues. (A) Evan's blue extravasation in the peritoneal cavity (top panel) and intestines (bottom panel) of uninfected or D2Y98P-infected mouse at moribund state. (B) Quantification of Evans blue dye in the intestine, liver and spleen from mice. Five animals per group per time point were individually processed. Data are expressed as the [mean ± SD] of fold increases in OD_620nm per gram of wet tissue compared to uninfected controls. (C) Serum albumin concentration. Results are expressed as the [mean ± SD] of 5 animals per time point per group. *p<0.05. Results are representative of 2 independent experiments.</p

Viremia and antibody titres in D2Y98P-infected mice.

<p>Mice were ip. infected with 10⁷ (A, C, E) or 10⁴ (B, D, F) PFU of D2Y98P. At the indicated time points, five infected animals were bled and euthanized immediately. Viremia titres (A, B), specific anti-IgM (black circle) and IgG (open circle) titers (C, D), and PRNT₅₀ (E, F) were determined for each individual serum. Results are representative of 2 independent experiments.</p

Hematology in D2Y98P-infected mice.

<p>AG129 mice were ip. infected with 10⁷ or 10⁴ PFU of D2Y98P. At the indicated time points, 5 mice per group per time point were bled and euthanized. Blood samples were processed to determine white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM), red blood cells (RBC), hematocrit (HCT), and platelets (PLT) counts. A group of uninfected mice was included as control. WBC, NEU, LYM and PLT counts are given in K/uL (10³ cells/ul), RBC count in M/uL (10⁶ cells/ul), and HCT in percentage (%). Data are expressed as the [mean ± SD] of individual measurements and are representative of 2 independent experiments. * p<0.05 compared to uninfected controls.</p

Pathology and virus titres in the liver, spleen and brain of D2Y98P-infected mice.

<p>(A) Mice were ip. infected with 10⁷ PFU of D2Y98P, and were sacrificed at moribund state and perfused extensively with PBS. Representative gross appearance of organs in the intraperitoneal cavity of uninfected (left panel) and ip. infected (right panel) mice. Insets highlight the difference in the spleen size between both animal groups. Virus titres were determined in the liver (B), spleen (C) and brain (D) from AG129 mice ip. infected with 10⁷ (black circle) or 10⁴ (open circle) PFU of D2Y98P virus. Results are expressed as log₁₀ [mean ± SD] in PFU per gram of tissue. Five mice per time point per group were assessed. Results are representative of 2 independent experiments.</p

Pro-inflammatory cytokine expression in D2Y98P-infected mice.

<p>AG129 mice were ip. infected with 10⁷ or 10⁴ PFU of D2Y98P, bled at the indicated time points and immediately euthanized. Serum levels of IFN-γ (A), IL-6 (B) and TNF-α (C) were quantified. Results are expressed in pg/ml as the [mean ± SD] of 5 mice per time point and per group.</p

Survival rates in AG129 mice infected with a dose range of D2Y98P virus.

<p>AG129 mice were infected intraperitoneally (ip.) with 10-fold serially diluted viral doses of D2Y98P ranging from 10⁷ to 10² PFU. Ten mice per group were used. Data are representative of at least 3 independent experiments.</p

Body weight changes in D2Y98P-infected mice.

<p>Mice were ip. infected with 10⁷ or 10⁶ (A), or with 10⁵ to 10² (B) PFU of D2Y98P. Body weight changes were monitored daily (A) or every other day (B) post-infection (p.i.). Results are expressed as the [mean ± SD] of body weight loss in percentage compared to initial body weight. Ten mice per group were monitored. Results are representative of 2 independent experiments.</p