MicroRNA Regulation of Human Genes Essential for Influenza A (H7N9) Replication

May MicroRNA Regulation of Human Genes Essential for Influenza A (H7N9) Replication Stefan Wolf 0 1 2 Weilin Wu 1 2 Cheryl Jones 1 2 Olivia Perwitasari 1 2 Suresh Mahalingam 0 2 Ralph A. Tripp 1 2 0 Institute for Glycomics, Griffith University , Gold Coast, Southport, QLD , Australia 1 Department of Infectious Diseases, University of Georgia , Athens, GA , United States of America 2 Editor: Michael CW Chan, Centre of Influenza Research, The University of Hong Kong , HONG KONG Influenza A viruses are important pathogens of humans and animals. While seasonal influenza viruses infect humans every year, occasionally animal-origin viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. In March 2013, the public health authorities of China reported three cases of laboratory confirmed human infection with avian influenza A (H7N9) virus, and subsequently there have been many cases reported across South East Asia and recently in North America. Most patients experience severe respiratory illness, and morbidity with mortality rates near 40%. No vaccine is currently available and the use of antivirals is complicated due the frequent emergence of drug resistant strains. Thus, there is an imminent need to identify new drug targets for therapeutic intervention. In the current study, a high-throughput screening (HTS) assay was performed using microRNA (miRNA) inhibitors to identify new host miRNA targets that reduce influenza H7N9 replication in human respiratory (A549) cells. Validation studies lead to a top hit, hsa-miR-664a-3p, that had potent antiviral effects in reducing H7N9 replication (TCID50 titers) by two logs. In silico pathway analysis revealed that this microRNA targeted the LIF and NEK7 genes with effects on pro-inflammatory factors. In follow up studies using siRNAs, anti-viral properties were shown for LIF. Furthermore, inhibition of hsa-miR-664a-3p also reduced virus replication of pandemic influenza A strains H1N1 and H3N2. - OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: This work was supported by the National Institute of Allergy and Infectious 473 Diseases, National Institutes of Health (HHSN272201400004C), Georgia Research Alliance, and Griffith University (GUPRS/GUPIRS scholarship). 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. Introduction Influenza virus is still a serious global health threat affecting humans, wildlife and agricultural species. Human infection with avian influenza A H7N9 virus (H7N9) were first reported in China in March 2013 [ 1 ] Most of the infections are believed to have resulted from exposure to infected poultry or contaminated environments, as H7N9 viruses have been found in poultry in China. While some mild illnesses in humans infected with H7N9 has been reported, most patients experienced severe respiratory illness, such as pneumonia (97.3%) and acute respiratory distress syndrome (71.2%), leading to high rates of intensive care unit admissions [ 2 ]. Human mortality attributed to influenza H7N9 is over 38% with 175 deaths from 450 confirmed cases within a 20-month period [ 3 ]. No evidence of sustained human-to-human transmission of H7N9 has been recorded; however, there was some evidence for limited person-to person spread under rare circumstances [ 4 ]. H7N9 began in China, but now has rapidly spread to other countries [ 5 ]. Recently, the first documented case of H7N9 in humans was reported for North America in Canada [ 6 ] No vaccine is currently available for H7N9 [ 7 ]. There are several drugs available for the treatment of influenza infections including the M2 ion channel inhibitors amantadine and rimantadine, and the neuraminidase inhibitors, zanamivir and oseltamivir [ 8, 9 ]. Early treatment with these antiviral drugs has been shown to reduce the duration of symptoms and time to recovery, however, the use of antiviral drugs is complicated by the emergence of drug resistant viruses [ 10, 11 ]. Consequently, oseltamivir-resistant H7N9 strains have already been described in recent reports from Taiwan [ 12 ]. In addition, the use of antiviral drugs may have an effect on population vulnerability due to lack of seroconversion, as well as driving drug resistance among circulating strains [ 13 ]. To prevent the spread of infection, new drug and vaccine development is needed. However, difficulties include a lack of understanding of the host factors required for replication, and unusual mutations that occur in the virus that differ from other avian influenza viruses [ 14 ]. Linking high-throughput screening (HTS) with RNA interference (RNAi) allows for the rapid discovery of the molecular basis of disease pathogenesis, and the identification of potential pathways for (...truncated)