Identification of Amino Acid Residues in Influenza A Virus PA-X That Contribute to Enhanced Shutoff Activity

The influenza virus protein PA-X modulates the host immune responses and viral pathogenicity through suppression of host protein expression. The endonuclease active site in the N-terminal region, the basic amino acid cluster in the C-terminal PA-X-specific region, and N-terminal acetylation of PA-X by NatB are important for the shutoff activity of PA-X. Here, we focused on the shutoff activity of PA-X derived from the A/California/04/2009 and A/WSN/33 viruses because these two PA-X proteins differ in their shutoff activity. Mutagenesis analysis revealed that proline and serine at positions 28 and 65, respectively, play a central role in this difference. Furthermore, we found that P28 and S65 also affect the shutoff activity of PA-X derived from other influenza virus subtypes. These data demonstrate that P28 and S65 contribute to enhanced shutoff activity of PA-X

Isolation and Characterization of Human Monoclonal Antibodies That Recognize the Influenza A(H1N1)pdm09 Virus Hemagglutinin Receptor-Binding Site and Rarely Yield Escape Mutant Viruses

The influenza A virus rapidly mutates to escape from antibodies. Here, we isolated and characterized three human monoclonal antibodies (mAbs) that neutralize A(H1N1)pdm09 viruses. Generation of escape mutant viruses suggested that these antibodies recognized conserved residues of the receptor-binding site (RBS) of hemagglutinin (HA) and that mutant viruses that escaped from these mAbs rarely appeared. Moreover, the escape mutant viruses grew significantly slower than wild-type virus, indicating their reduced fitness. These results indicate that these three human mAbs against the RBS of HA have the potential to be anti-influenza agents with a low propensity for the development of resistant viruses

The Cellular DExD/H-Box RNA Helicase UAP56 Co-localizes With the Influenza A Virus NS1 Protein

UAP56, a member of the DExD/H-box RNA helicase family, is essential for pre-mRNA splicing and mRNA export in eukaryotic cells. In influenza A virus-infected cells, UAP56 mediates viral mRNA nuclear export, facilitates viral ribonucleoprotein complex formation through direct interaction with the viral nucleoprotein, and may indirectly affect antiviral host responses by binding to and/or facilitating the activation of the antiviral host factors MxA and PKR. Here, we demonstrate that UAP56 also co-localizes with the influenza A viral NS1 protein, which counteracts host cell innate immune responses stimulated by virus infection. The UAP56–NS1 association relies on the RNA-binding residues R38 and K41 in NS1 and may be mediated by single-stranded RNA. UAP56 association with NS1 does not affect the NS1-mediated downregulation of cellular innate immune pathways in reporter gene assays, leaving in question the exact biological role and relevance of the UAP56–NS1 association

C57BL/6J and C57BL/6NJ Mice Are Differentially Susceptible to Inflammation-Associated Disease Caused by Influenza A Virus

Influenza viruses cause seasonal epidemics and sporadic pandemics, and are a major burden on human health. To develop better countermeasures and improve influenza disease outcomes, a clearer understanding of influenza pathogenesis is necessary. Host genetic factors have emerged as potential regulators of human influenza disease susceptibility, and in the mouse model, genetic background has been clearly linked to influenza pathogenicity. Here, we show that C57BL/6J mice are significantly more susceptible to disease caused by a 2009 pandemic H1N1 virus, an H7N9 virus, and a highly pathogenic H5N1 influenza virus compared to the closely related substrain, C57BL/6NJ. Mechanistically, influenza virus infection in C57BL/6J mice results in earlier presentation of edema, increased immune cell infiltration, higher levels of inflammatory cytokines, greater tissue damage, and delayed activation of regenerative processes in infected lung tissues compared to C57BL/6NJ mice. These differences are not dependent on virus replication levels. Six genes with known coding region differences between C57BL/6J and C57BL/6NJ strains exhibit increased transcript levels in influenza virus-infected mouse lungs, suggesting potential contributions to regulation of disease susceptibility. This work uncovers a previously unappreciated difference in disease susceptibility between the closely related C57BL/6J and C57BL/6NJ mice, which may be exploited in future studies to identify host factors and/or specific genetic elements that regulate host-dependent inflammatory mechanisms involved in influenza virus pathogenicity

Injectable Excipients as Novel Influenza Vaccine Adjuvants

Influenza outbreaks can be either seasonal or pandemic. Vaccination is an effective strategy to control influenza; however, the efficacy of the currently available inactivated influenza virus vaccines is suboptimal, especially in the elderly. Vaccine efficacy can be improved by the addition of adjuvants, but few adjuvants have been approved for human vaccines. To explore novel, safe, and effective adjuvants for influenza vaccines, here we used a mouse model to screen 46 injectable drug additives approved in Japan. Of these 46 candidates, we identified 20 compounds that enhanced the efficacy of the split influenza HA vaccine against lethal virus challenge. These 20 compounds included 15 novel adjuvant candidates and 5 compounds with previously reported adjuvant effects for other antigens but not for influenza vaccine. Given that these additives are already approved for human use, the hurdle for their clinical use as novel and effective adjuvants for influenza or other vaccines is lower than for other adjuvant candidates whose safety profiles are unknown

Influenza A Virus with Defective M2 Ion Channel Activity as a Live Vaccine

AbstractWe propose a rational approach to the design of live virus vaccines against influenza infection by alteration of the influenza A virus M2 protein, which is responsible for ion channel activity. Previously we demonstrated that a mutant A/WSN/33 (H1N1) influenza virus with defective M2 ion channel activity did not show appreciable growth defects in cell culture, although its growth was attenuated in mice (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, 2001, J. Virol. 75, 5656–5662). Here, we show that this M2 ion channel defective mutant virus, the M2del29-31, protected mice against challenge with lethal doses of influenza virus, indicating the potential of incorporating this M2 alteration in a live influenza vaccine as one of the attenuating mutations

Interaction of Polymerase Subunit PB2 and NP with Importin α1 Is a Determinant of Host Range of Influenza A Virus

We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin α1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin α1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin α1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus

Comparative Study of Influenza Virus Replication in MDCK Cells and in Primary Cells Derived from Adenoids and Airway Epithelium

Although clinical trials with human subjects are essential for determination of safety, infectivity, and immunogenicity, it is de- sirable to know in advance the infectiousness of potential candidate live attenuated influenza vaccine strains for human use. We compared the replication kinetics of wild-type and live attenuated influenza viruses, including H1N1, H3N2, H9N2, and B strains, in Madin-Darby canine kidney (MDCK) cells, primary epithelial cells derived from human adenoids, and human bron- chial epithelium (NHBE cells). Our data showed that despite the fact that all tissue culture models lack a functional adaptive im- mune system, differentiated cultures of human epithelium exhibited the greatest restriction for all H1N1, H3N2, and B vaccine viruses studied among three cell types tested and the best correlation with their levels of attenuation seen in clinical trials with humans. In contrast, the data obtained with MDCK cells were the least predictive of restricted viral replication of live attenuated vaccine viruses in humans. We were able to detect a statistically significant difference between the replication abilities of the U.S. (A/Ann Arbor/6/60) and Russian (A/Leningrad/134/17/57) cold-adapted vaccine donor strains in NHBE cultures. Since live at- tenuated pandemic influenza vaccines may potentially express a hemagglutinin and neuraminidase from a non-human influenza virus, we assessed which of the three cell cultures could be used to optimally evaluate the infectivity and cellular tropism of vi- ruses derived from different hosts. Among the three cell types tested, NHBE cultures most adequately reflected the infectivity and cellular tropism of influenza virus strains with different receptor specificities. NHBE cultures could be considered for use as a screening step for evaluating the restricted replication of influenza vaccine candidates

Replicative capacity of SARS-CoV-2 omicron variants BA.5 and BQ.1.1 at elevated temperatures

新型コロナウイルス・オミクロン株のBA.5系統およびBQ.1.1系統が、高温で増殖しづらいことを解明. 京都大学プレスリリース. 2023-04-27

Pneumo- and neurotropism of avian origin Italian highly pathogenic avian influenza H7N1 isolates in experimentally infected mice

AbstractAn experimental infection of mice was performed in order to investigate the potential for interspecies transmission in mammals of Italian HPAI viruses of the H7N1 subtype. Three avian origin isolates were selected, two strains obtained from ostrich (one of which contained a PB2-627 Lysine residue) and one from a chicken. Following intranasal infection of mice, clinical signs and mortality were recorded in the experimental groups challenged with the two ostrich isolates, while only weight loss was observed in those receiving the chicken strain. Viruses were recovered to a varying extent from respiratory and nervous tissues of infected animals. These results suggest that HPAI viruses, other than H5N1 and H7N7, may have zoonotic implications, and support the consensus that AI infections in poultry are to be eradicated rather than contained