Cytopathy And Innate Responses Of Glial Cells Induced By Influenza Virus Infection

Background and aimsInfluenza virus infection can cause human respiratory illnesses and occasional central nervous system (CNS) disorders. Influenza virus can infect the CNS. Besides, pro-inflammatory cytokines are reportedly increased in the cerebrospinal fluids and plasma of patients with influenza encephalopathy and encephalitis. Microglia and astrocytes are capable of producing cytokines in the CNS. Nevertheless, the role(s) of microglia and astrocytes in acute encephalopathy and encephalitis caused by influenza virus infection remains unexplored. Therefore, to understand the immune responses mediated by microglia and astrocytes after influenza virus infection and the mechanism underlying the associated encephalopathy and encephalitis, mouse microglia and astrocytes were isolated and infected with human H1N1 and avian H5N1 influenza viruses, then their viral susceptibility and productivity, and cytopathic, apoptotic, and pro-inflammatory cytokine responses were examined.A steady increase of uracil (U) and adenine (A) along with the decrease in cytosine (C) and guanine (G) was detected in human influenza virus genomes but not in the avian influenza virus genomes. One of those speculations is that humans have a native defense against RNA viruses similar to APOBEC proteins. Based on other reports and our previous results, we speculated that APOBEC might be involved in the inhibition or hypermutation of influenza virus. There is no report on whether APOBEC enzymes can inhibit or hypermutate human IAV until now. It will be beneficial to explore and understand the novel potential inhibitors and hyper-mutagenesis factors among cytidine deaminase or intercellular immunity mediators might be found.Methods1. To clarity the genetics background and construct the reverse genetics system, the universal reverse genetics vector was constructed. All the eight genome-fragments of human H1N1 and avian H5N1 influenza viruses were cloned using RT-PCR and sequenced. Then the fragments were inserted into the universal vectors.2. To investigate the cytopathy and immune reactions of glial cells infected influenza viruses, mouse microglia and astrocytes were isolated and infected by human H1N1 and avian H5N1 influenza viruses. Then their viral receptors distribution, infection, apoptosis, cytopathy, and immune responses were examined using affinity fluorescence, immuno- fluorescence, semi-quantitative RT-PCR, ELISA and other assays. The mRNA levels of APOBEC members in infected human glial cells were determined by semi-quantitative RT-PCR.3. To clarify the role of APOBEC3 members in cell defenses against influenza A virus, the effects of APOBEC members on influenza A virus replication and hypermutation were investigated. The APOBEC members were cloned and expressed in transfected cells and confirmed by Western blotting and FCM. The viral replication in APOBEC expressed cells were examined by TCID50 and PFU assay. The viral mutations were checked by sequencing.Results1. The genome fragments of H1N1 and H5N1 strains were cloned and sequenced successfully;2. The universal reverse genetics vector was constructed and confirmed by sequencing;3. Viral receptors, sialic acid (SA)-α2,3-Galactose(Gal) and SA-α2,6-Gal, were both observed to be homogeneously distributed on microglia and astrocytes;4. Both H1N1 and H5N1 viruses were replicative and productive in microglia and astrocytes;5. Virus-induced apoptosis and cytopathy in infected cells were observed at 24 h post-infection (p.i.);6. The mRNA expression of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α) and chemokines (CCL-2, -3, -5, and CXCL-2, -9, -10) examined at 6 h and 24 h p.i. was up-regulated, and their expression levels were considerably higher in H5N1 infection. The protein levels of secreted pro-inflammatory IL-1β, IL-6 and TNF-αat 6 h and 24 h p.i. were also induced, with greater induction by H5N1 infection;7. Mouse APOBEC3 and human APOBEC-3B, -3C, -3F, and -3G were detected in mouse and human glial cells, respectively. Their mRNA levels were up-regulated after influenza virus infection;8. The APOBEC members, including human APOBEC-1, -2, -3A, -3B, -3C, -3F, -3G, -3H, -4, AID, mouse APOBEC-3, and rat APOBEC-1, were cloned and confirmed by sequencing, and expressed in transfected cells.9. The results of viral titers assay showed that there were no significant differences in replication of influenza virus between APOBEC proteins expressed cells and the control cells. It indicated that APOBEC3F and 3G could inhibit the replication of HBV in HepG2.2.15 cells. Western blotting indicated that APOBEC3F and 3G proteins were both over-expressed during the infection of influenza virus. DNA sequencing showed that there was no hypermutation in influenza virus genome induced by APOBEC3F or 3G.Conclusions1. The genome fragments of H1N1 and H5N1 strains were cloned and sequenced successfully; The universal reverse genetics vector was constructed and confirmed by sequencing;2. This study firstly demonstrated that both human H1N1 and avian H5N1 influenza viruses could infect and induce apoptosis, cytopathy, and pro-inflammatory cytokine production in mouse microglia and astrocytes in vitro. The results suggest that direct cellular damage and the consequences of immunopathological injuries in the CNS contribute to the influenza viral pathogenesis.3. APOBEC3F and 3G could effectively inhibit HBV replication, but could not inhibit human influenza A virus replication or induce its hypermutation. And other APOBEC members, including human AID, APOBEC1, APOBEC3A, 3B, 3C, 3H and rat APOBEC1, have no inhibition effect on the replication of influenza A virus, too.