Title Avian Influenza A H 7 N 9 Virus Induces Severe Pneumonia in Mice without Prior Adaptation and Responds to a Combination of Zanamivir and COX-2 Inhibitor

Background: Human infection caused by the avian influenza A H7N9 virus has a case-fatality rate of over 30%. Systematic study of the pathogenesis of avian H7N9 isolate and effective therapeutic strategies are needed. Methods: BALB/c mice were inoculated intranasally with an H7N9 virus isolated from a chicken in a wet market epidemiologically linked to a fatal human case, (A/chicken/Zhejiang/DTID-ZJU01/2013 [CK1]), and with an H7N9 virus isolated from a human (A/Anhui/01/2013 [AH1]). The pulmonary viral loads, cytokine/chemokine profiles and histopathological changes of the infected mice were compared. The therapeutic efficacy of a non-steroidal antiinflammatory drug (NSAID), celecoxib, was assessed. Results: Without prior adaptation, intranasal inoculation of 10 plaque forming units (PFUs) of CK1 caused a mortality rate of 82% (14/17) in mice. Viral nucleoprotein and RNA expression were limited to the respiratory system and no viral RNA could be detected from brain, liver and kidney tissues. CK1 caused heavy alveolar inflammatory exudation and pulmonary hemorrhage, associated with high pulmonary levels of proinflammatory cytokines. In the mouse lung cell line LA-4, CK1 also induced high levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA. Administration of the antiviral zanamivir did not significantly improve survival in mice infected with CK1, but co-administration of the non-steroidal anti-inflammatory drug (NSAID) celecoxib in combination with zanamivir improved survival and lung pathology. Conclusions: Our findings suggested that H7N9 viruses isolated from chicken without preceding trans-species adaptation can cause lethal mammalian pulmonary infection. The severe proinflammatory responses might be a factor contributing to the mortality. Treatment with combination of antiviral and NSAID could ameliorate pulmonary inflammation and may improve survival. Citation: Li C, Li C, Zhang AJX, To KKW, Lee ACY, et al. (2014) Avian Influenza A H7N9 Virus Induces Severe Pneumonia in Mice without Prior Adaptation and Responds to a Combination of Zanamivir and COX-2 Inhibitor. PLoS ONE 9(9): e107966. doi:10.1371/journal.pone. 0107966 Editor: Kevan L. Hartshorn, Boston University School of Medicine, United States of America Received March 24, 2014; Accepted August 16, 2014; Published September 18, 2014 Copyright: 2014 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Providence Foundation Ltd, in memory of the late Lui Hac Minh, and a donation from Larry Chi-Kin Yung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: kyyuen@hkucc.hku.hk (K-YY); zhangajx@hku.hk (AJXZ) . These authors contributed equally to this work.