MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies

Diverse Heterologous Primary Infections Radically Alter Immunodominance Hierarchies and Clinical Outcomes Following H7N9 Influenza Challenge in Mice

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8(+) cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K(b)PB(1703, D(b)PA(224), and D(b)NP(366) epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines

Human H7N9 and H5N1 influenza viruses differ in induction of cytokines and tissue tropism.

Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models

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

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