Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice

Jin et al. Virology Journal (2017) 14:243 DOI 10.1186/s12985-017-0911-5 RESEARCH Open Access Pulmonary edema following central nervous system lesions induced by a non- mouse-adapted EV71 strain in neonatal BALB/c mice Yuefei Jin1, Chao Zhang1, Rongguang Zhang1,2, Jingchao Ren2,3, Shuaiyin Chen1, Meili Sui1, Guangyuan Zhou3, Dejian Dang1, Jiehui Zhu1, Huifen Feng4, Yuanlin Xi1, Haiyan Yang1,2 and Guangcai Duan1* Abstract Background: Enterovirus (EV) infection has been a serious health issue in Asia-Pacific region. It has been indicated that the occurrence of fatal hand foot and mouth disease (HFMD) cases following EV71 infection is mainly attributed to pulmonary edema. However, the development of pulmonary disorders after EV71 infection remains largely unknown. To establish an EV71-infected animal model and further explore the underlying association of central nervous system (CNS) invasion with pulmonary edema, we isolated a clinical source EV71 strain (ZZ1350) from a severe case in Henan Province. Methods: We evaluated the cytotoxicity of ZZ1350 strain and the susceptibility in 3-day-old BALB/c mice with intraperitoneal, intracerebral and intramuscular inoculation. Various histopathological and immunohistochemical techniques were applied to determine the target organs or tissue damage after infection. Correlation analysis was used to identify the relationship between CNS injury and pulmonary disorders. Results: Our experimental results suggested that ZZ1350 (C4 subtype) had high cytotoxicity against African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells and neonatal BALB/c mice were highly susceptible to the infection with ZZ1350 through three different inoculation routes (2 × 106 pfu/mouse) exhibiting severe neurological and respiratory symptoms that were similar to clinical observation. Viral replication was found in brain, spinal cord, skeletal muscle, lung, spleen, liver, heart of infected mice and these sections also showed histopathological changes. We found that brain histology score was positive correlated with lung histology score in total experimental mice and mice under the three inoculation routes (P < 0.05). At the same time, there were positive correlations between spinal cord score and lung score in total experimental mice and mice with intracerebral inoculation (P < 0.05). Conclusions: ZZ1350 strain is effective to establish animal model of EV71 infection with severe neurological and respiratory symptoms. The development of pulmonary disorders after EV71 infection is associated with severity of CNS damage. Keywords: Enterovirus 71, Hand foot and mouth disease, Pulmonary edema, Central nervous system * Correspondence: 1 Department of Epidemiology, College of Public Health, Zhengzhou University, No.100 Kexue Avenue, Zhengzhou, Henan 450001, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jin et al. Virology Journal (2017) 14:243 Page 2 of 12 Background Enterovirus (EV) genus of the Picornaviridae family containing EV71 and coxsackievirus A16 (CA16) are the causative agents of hand foot and mouth disease (HFMD). Recently, outbreak of HFMD caused by EV71 infection have been a serious threat to infants and children in East and Southeast Asia. EV71 was first isolated in California, United States in 1967 and then had been recognized as a cause of epidemics of HFMD in Japan since 1973 [1, 2]. It has been acknowledged that EV71 is responsible for HFMD contributing to severe neurological complications and even fatalities in infants and young children worldwide [3, 4]. Generally, EV71 infection is a typically mild, self-limiting childhood disorder. However, a handful of cases can develop into devastating clinical outcomes presenting as either acute flaccid paralysis, aseptic meningitis, brainstem encephalitis, encephalomyelitis or pulmonary edema, once EV71 invades into central nervous system (CNS) [4, 5]. Although, current studies involving animal models of EV71 infection are well established to explore the pathological process [6, 7], the development of EV71related pulmonary edema still remains largely unknown. Therefore, it is extremely urgent to reveal the pathogenesis of fatal EV71-induced pulmonary diseases and make great efforts to identify novel therapies for severe cases. In this study, we separated an EV71 strain from a nonfatal case with CNS involvement in Henan Province. Unlike other animal models, 3-day-old mice were directly infected with clinical source virus purified and cultured in African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells, not a mouse adapted strain. Additionally, mice in our experiments were inoculated via three infection routes and diverse outcomes were assessed. A variety of histopathological and immunohistochemical techniques were applied to demonstrate that whether EV71-induced CNS lesions are associated with occurrence of pulmonary edema. [8]. RD cells and African green monkey kidney (Vero) cells (ATCC CCL-81) were cultured in Dulbecco’s modified Eagle’s medium (Gibco Company, New York, USA) containing 10% fetal bovine serum (FBS) (Gibco Company, New York, USA). Enrichment of the virus was performed using Dulbecco’s modified Eagle’s medium (Gibco Company, New York, USA) containing 2% fetal bovine serum (FBS) (Gibco Company, New York, USA). The virus stocks were stored at −80°C. TCID50 were determined by plaque assay using RD cells [9], and viral titers were expressed as PFU per ml. Working stocks in the present study contain 108 PFU per ml. Methods Cell viability assay Virus and cells The Vero cells (2 × 104/well) were seeded in 96-well plates in a total volume of 200 ul for 24 h, and then were infected with EV71 at an MOI of 0, 0.4, 1 and 5 for 24 h. We investigated the effect of EV71 infection on the viability of Vero cells using a MTT assay kit (Vazyme Biotech Co. Ltd., Nanjing, China) at 24 h post infection (hpi). EV71 strain ZZ1350 was isolated from a nonfatal case with CNS involvement in Children’s Hospital of Zhengzhou (Zhengzhou, Henan, China). Viral growth was performed in human rhabdomyosarcoma (RD) cells, and virus purification was conducted as previously described Viral RNA isolation and quantitative real-time PCR (qRT-PCR) Sixty hour after infection, RD cells were collected and viral RNA was extracted using Hipure viral RNA (...truncated)