Acute Respiratory Distress Syndrome Induced by a Swine 2009 H1N1 Variant in Mice

Citation: Zhang Y, Sun H, Fan L, Ma Y, Sun Y, et al. ( Acute Respiratory Distress Syndrome Induced by a Swine 2009 H1N1 Variant in Mice Yi Zhang 0 Honglei Sun 0 Lihong Fan 0 Yuan Ma 0 Yipeng Sun 0 Juan Pu 0 Jun Yang 0 Jian Qiao 0 Guangpeng Ma 0 Jinhua Liu 0 Rory Edward Morty, University of Giessen Lung Center, Germany 0 1 Key Laboratory of Animal Epidemiology and Zoonosis, College of Veterinary Medicine, China Agricultural University, Ministry of Agriculture , Beijing , China , 2 China Rural Technology Development Center , Beijing, China, 3 Shandong Animal Disease Control Center, Shandong , China Background: Acute respiratory distress syndrome (ARDS) induced by pandemic 2009 H1N1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 H1N1 infection. However, no animal model has been developed for ARDS caused by infection with 2009 H1N1 virus. Here, we present a mouse model of ARDS induced by 2009 H1N1 virus. Methodology Principal Findings: Mice were inoculated with A/swine/Shandong/731/2009 (SD/09), which was a 2009 H1N1 influenza variant with a G222D mutation in the hemagglutinin. Clinical symptoms were recorded every day. Lung injury was assessed by lung water content and histopathological observation. Arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. Mice infected with SD/09 virus showed typical ARDS symptoms characterized by 60% lethality on days 8-10 postinoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines. Conclusions/Significance: These results suggested that we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ARDS caused by 2009 H1N1 virus. - Funding: This work was supported by the National Natural Science Foundation of China (No. 31172324, 31172323), the National Basic Research Program (973 Program) (No. 2011CB504702), the National Key Technologies Research and Development Program (2010BAD04B01), China Postdoctoral Science Foundation funded project (2011M500456), and Chinese Universities Scientific Fund (2011BH041). Dr. Liu was funded by the Taishan Scholar Foundation. 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. . These authors contributed equally to this work. A novel influenza A (H1N1) virus of swine origin emerged among humans in Mexico during the spring of 2009 and rapidly spread worldwide [1]. The pandemic prompted the World Health Organization (WHO) to raise the alert level to the highest rating of six, the pandemic phase, within 2 months [2]. In August 2010, WHO officially declared that the disease was in the post-pandemic period [3]; however, it is still circulating among humans, together with seasonal viruses. Although most influenza cases caused by 2009 H1N1 virus infection typically display mild upper respiratory tract syndrome, some cases progress to severe pneumonia and acute respiratory distress syndrome (ARDS) [4,5]. Many studies have shown that ARDS caused by 2009 H1N1 virus results in 17.356% mortality [4,6,7,8], which was regarded as the major cause of death by 2009 H1N1 virus infection [9]. ARDS is the result of acute injury to lung tissue, commonly resulting from sepsis, trauma, and severe pulmonary infections [10]. Infectious factors, most of which are viruses, have become one of the most important causes of ARDS in humans [11,12,13]. Clinical cases and established animal models have revealed that the pathogenesis and pathological features of ARDS induced by different viral pathogens are distinct [14,15]. However, knowledge of the pathogenesis of 2009 H1N1 virus, especially ARDS induced by 2009 H1N1 virus, is still limited and hinders therapeutic strategies. Therefore, it is necessary to evaluate the pathogenesis of ARDS caused by 2009 H1N1 virus infection in an appropriate animal model to assess potential therapies. Mice are a good model for evaluating the pathogenesis and antiviral therapy of influenza pneumonia, due to the general fidelity of the illness in mice to the human disease [16]. Moreover, a mouse model of ARDS caused by highly pathogenic H5N1 avian influenza virus infection has been well established [13]. The typical 2009 H1N1 virus, such as A/California/04/2009 (CA/04), can efficiently replicate in mouse lungs without prior host adaptation. However, it only causes moderate lung lesions and no mortality, even when inoculated at a high dose of 106 pfu [17,18]. Thus, such typical 2009 H1N1 viruses may not be able to induce ARDS in a mouse model. In the present study, we used a virulent variant 2009 H1N1 virus, which was isolated from a pig and possessed a virulenceassociated HA-D222G mutation, to establish an ARDS mouse model. The model established here provides a useful tool to explore the mechanism of ARDS, as well as screening and therapeutic options. Clinical and gross pathologic observation Six-week-old female mice were infected intranasally (i.n.) with 102.5 pfu SD/09 virus. Some of the infected mice showed signs of illness, such as altered gait, inactivity, ruffled fur, and anorexia on day 2 post-infection (p.i.). From day 2 p.i., the body weight of most mice significantly decreased (Figure S1). By day 6 p.i., most mice presented with more severe clinical signs of respiratory disease, including labored respiration and respiratory distress, and most mice lost almost 20% of their initial body weight. On day 8 p.i., most mice were nearly unable to respond to exterior stimuli, and acute respiratory rates and labored respiration were observed (Video S1, and Video S2 for control). Approximately 60% of mice died between days 8 and 10 p.i. Gross observation of infected mice showed that the lungs were highly edematous, with profuse areas of hemorrhage and consolidation. No obvious gross lesions were observed in the kidneys, liver, spleen or brain of infected mice. Replication kinetics of SD/09 virus in mouse tissues Mice were infected i.n. with 102.5 pfu SD/09 virus, and three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the virus titers in viscera were determined. As shown in Figure 1A, the virus titer in the lung gradually increased between days 2 and 6 p.i., and reached a peak on day 6 p.i. The virus titers in the lung gradually decreased from day 6 p.i., and only one of three mice possessed detectable virus in the lungs on day 10 p.i. No viruses were detected in other organs, including heart, spleen, liver, kidneys, blood and brain, at the (...truncated)