RNA Sequencing Reveals circRNA Expression Profiles in Chicken DF1 Cells Infected with H5N1 Influenza Virus

H5N1, a highly pathogenic avian influenza virus that is prevalent in Asia, seriously harms the poultry industry and global public health. However, its pathogenesis is still not well understood. Circular RNAs (circRNAs), a newly identified type of RNA, reportedly play crucial roles in various pathogenic processes. In this study, RNA sequencing was performed to analyze the expression profile of circRNAs in H5N1-infected chicken embryo fibroblast (DF1) cells. A total of 14,586 circRNAs were identified. The expression profiles of infected cells changed more significantly, relative to uninfected cells, as the infection period was extended; namely, 261, 626, and 1103 circRNAs exhibited differential expression in cells infected for 6 h, 12 h, and 20 h, respectively. GO and KEGG enrichment analysis revealed significant enrichment of the parental genes of the differentially expressed circRNAs for viral replication and immune response-related pathways, such as positive regulation of transcription from the RNA polymerase II promoter, positive regulation of I-kappaB kinase/NF-kappaB signaling, innate immune response, and ubiquitin protein ligase activity. In conclusion, we identified the expression profile of circRNAs in H5N1-infected chicken DF1 cells. Bioinformatic analyses of the dysregulated circRNAs suggest that circRNAs might play important roles in the pathogenesis of H5N1 infection, offering new insights into the mechanisms underlying H5N1–host interaction.

SUMOylation of matrix protein M1 and filamentous morphology collectively contribute to the replication and virulence of highly pathogenic H5N1 avian influenza viruses in mammals

The matrix protein (M1) of influenza A virus plays an important role in replication, assembly, and budding. A previous study found that aspartic acid (D) at position 30 and alanine (A) at position 215 of M1 contribute to the high pathogenicity of H5N1 viruses in mice, and double mutations of D to asparagine (N) at position 30 (D30N) and A to threonine (T) at position 215 (A215T) in M1 dramatically attenuate H5N1 viruses in mice. However, the underlying mechanisms by which these M1 mutations attenuate the virulence of H5N1 viruses are unknown. Here, we found that the amino acid mutation A215T eliminates the SUMOylation of M1 by reducing its interaction with the host SUMO1 protein, significantly reducing the stability of M1, slowing the export of the M1-vRNP complex from the nucleus to the cytoplasm, and reducing viral replication in MDCK cells. We further found that the D30N mutation in M1 alters the shape of progeny viruses from filamentous to spherical virions. Our findings reveal an essential role for M1 215A SUMOylation and M1 30D-related filamentous morphology in the pathogenesis of avian influenza viruses, which could be targeted in novel antiviral drug designs. Importance Identification of the pathogenic mechanism of highly pathogenic avian influenza viruses in mammals is helpful to develop novel anti-influenza virus strategies. Two amino acid mutations (D30N and A215T) in M1 were found to collectively attenuate H5N1 influenza viruses in mice, but the underlying mechanism remained unknown. This study found that the A215T mutation significantly decreases the SUMOylation of M1, which in turn attenuates the replication of H5N1 virus in mammalian cells. The D30N mutation in M1 was found to change the virion shape from filamentous to spherical. These findings are important for understanding the molecular mechanism of virulence of highly pathogenic avian influenza viruses in mammals.

Safety, Immunogenicity, and Protective Efficacy of an H5N1 Chimeric Cold-Adapted Attenuated Virus Vaccine in a Mouse Model

H5N1 influenza virus is a threat to public health worldwide. The virus can cause severe morbidity and mortality in humans. We constructed an H5N1 influenza candidate virus vaccine from the A/chicken/Guizhou/1153/2016 strain that was recommended by the World Health Organization. In this study, we designed an H5N1 chimeric influenza A/B vaccine based on a cold-adapted (ca) influenza B virus B/Vienna/1/99 backbone. We modified the ectodomain of H5N1 hemagglutinin (HA) protein, while retaining the packaging signals of influenza B virus, and then rescued a chimeric cold-adapted H5N1 candidate influenza vaccine through a reverse genetic system. The chimeric H5N1 vaccine replicated well in eggs and the Madin-Darby Canine Kidney cells. It maintained a temperature-sensitive and cold-adapted phenotype. The H5N1 vaccine was attenuated in mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H5N1 HA protein. The chimeric H5N1 candidate vaccine protected mice against lethal challenge with a wild-type highly pathogenic avian H5N1 influenza virus. The chimeric H5 candidate vaccine is thus a potentially safe, attenuated, and reassortment-incompetent vaccine with circulating A viruses.

Theoretical insights into the molecular mechanism of I117V mutation in neuraminidase mediated reduction of oseltamivir drug susceptibility in A/H5N1 influenza virus

The substitution of Ile to Val at residue 117 (I117V) of neuraminidase (NA) reduces the susceptibility of the A/H5N1 influenza virus to oseltamivir (OTV). However, the molecular mechanism by which the I117V mutation affects the intermolecular interactions between NA and OTV has not been fully elucidated. In this study, we performed molecular dynamics (MD) simulations to analyze the characteristic conformational changes that contribute to the reduced binding affinity of NA to OTV after the I117V mutation. The results of MD simulations revealed that after the I117V mutation in NA, the changes in the secondary structure around the mutation site had a noticeable effect on the residue interactions in the OTV-binding site. In the case of the WT NA-OTV complex, the positively charged side chain of R118, located in the β-sheet region, frequently interacted with the negatively charged side chain of E119, which is an amino acid residue in the OTV-binding site. This can reduce the electrostatic repulsion of E119 toward D151, which is also a negatively charged residue in the OTV-binding site, so that both E119 and D151 simultaneously form hydrogen bonds with OTV more frequently, which greatly contributes to the binding affinity of NA to OTV. After the I117V mutation in NA, the side chain of R118 interacted with the side chain of E119 less frequently, likely because of the decreased tendency of R118 to form a β-sheet structure. As a result, the electrostatic repulsion of E119 toward D151 is greater than that of the WT case, making it difficult for both E119 and D151 to simultaneously form hydrogen bonds with OTV, which in turn reduces the binding affinity of NA to OTV. Hence, after the I117V mutation in NA, influenza viruses are less susceptible to OTV because of conformational changes in residues of R118, E119, and D151 around the mutation site and in the binding site.

Influenza Antigens NP and M2 Confer Cross Protection to BALB/c Mice against Lethal Challenge with H1N1, Pandemic H1N1 or H5N1 Influenza A Viruses

Influenza hemagglutinin (HA) is considered a major protective antigen of seasonal influenza vaccine but antigenic drift of HA necessitates annual immunizations using new circulating HA versions. Low variation found within conserved non-HA influenza virus (INFV) antigens may maintain protection with less frequent immunizations. Conserved antigens of influenza A virus (INFV A) that can generate cross protection against multiple INFV strains were evaluated in BALB/c mice using modified Vaccinia virus Ankara (MVA)-vectored vaccines that expressed INFV A antigens hemagglutinin (HA), matrix protein 1 (M1), nucleoprotein (NP), matrix protein 2 (M2), repeats of the external portion of M2 (M2e) or as tandem repeats (METR), and M2e with transmembrane region and cytoplasmic loop (M2eTML). Protection by combinations of non-HA antigens was equivalent to that of subtype-matched HA. Combinations of NP and forms of M2e generated serum antibody responses and protected mice against lethal INFV A challenge using PR8, pandemic H1N1 A/Mexico/4108/2009 (pH1N1) or H5N1 A/Vietnam/1203/2004 (H5N1) viruses, as demonstrated by reduced lung viral burden and protection against weight loss. The highest levels of protection were obtained with NP and M2e antigens delivered as MVA inserts, resulting in broadly protective immunity in mice and enhancement of previous natural immunity to INFV A.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Universal influenza virus neuraminidase vaccine elicits protective immune responses against human seasonal and pre-pandemic strains

The hemagglutinin (HA) surface protein is the primary immune target for most influenza vaccines. The neuraminidase (NA) surface protein is often a secondary target for vaccine designs. In this study, computationally optimized broadly reactive antigen methodology was used to generate the N1-I NA vaccine antigen that was designed to cross-react with avian, swine, and human influenza viruses of N1 NA subtype. The elicited antibodies bound to NA proteins derived from A/California/07/2009 (H1N1)pdm09, A/Brisbane/59/2007 (H1N1), A/Swine/North Carolina/154074/2015 (H1N1) and A/Viet Nam/1203/2004 (H5N1) influenza viruses, with NA-neutralizing activity against a broad panel of HXN1 influenza strains. Mice vaccinated with the N1-I COBRA NA vaccine were protected from mortality and viral lung titers were lower when challenged with four different viral challenges: A/California/07/2009, A/Brisbane/59/2007, A/Swine/North Carolina/154074/2015 and A/Viet Nam/1203/2004. Vaccinated mice had little to no weight loss against both homologous, but also cross-NA genetic clade challenges. Lung viral titers were lower compared to the mock vaccinated mice, and at times, equivalent to the homologous control. Thus, the N1-I COBRA NA antigen has the potential to be a complimentary component in a multi-antigen universal influenza virus vaccine formulation that also contains HA antigens. Importance The development and distribution of a universal influenza vaccines would alleviate global economic and public health stress from annual influenza virus outbreaks. The influenza virus NA vaccine antigen allows for protection from multiple HA subtypes and virus host origins, but it has not been the focus of vaccine development. The N1-I NA antigen described here protected mice from direct challenge of four distinct influenza viruses and inhibited the enzymatic activity of a N1 influenza virus panel. The use of the NA antigen in combination with the HA widens the breadth of protection against various virus strains. Therefore, this research opens the door to the development of a longer lasting vaccine with increased protective breadth.