Metagenomic analysis of the gut microbiota of hooded cranes (Grus monacha) on the Izumi plain in Japan.

Recent advances in DNA sequencing technology have dramatically improved our understanding of the gut microbiota of various animal species. However, research on the gut microbiota of birds lags behind that of many other vertebrates, and information about the gut microbiota of wild birds such as migratory waterfowl is particularly lacking. Because the ecology of migratory waterfowl (e.g., lifestyle, diet, physiological characteristics) differs from that of other birds, the gut microbiota of migratory waterfowl likely also differs, but much is still unknown. The hooded crane (Grus monacha) is an important representative migratory waterbird species and is listed as endangered on the International Union for Conservation of Nature and Natural Resources Red List of Threatened Species. In this study, we analyzed the bacterial and viral microbiota in the gut of hooded cranes by using deep sequencing data from fecal samples of hooded cranes that winter on the Izumi plain in Japan, and found that Cetobacterium, Clupeiformes, and Pbunavirus were clearly present in the fecal samples of hooded cranes. These findings advance our understanding of the ecology of hooded cranes.

Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution.

Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.

Characterization of a neutralizing antibody that recognizes a loop region adjacent to the receptor-binding interface of the SARS-CoV-2 spike receptor-binding domain.

Although the global crisis caused by the coronavirus disease 2019 (COVID-19) pandemic is over, the global epidemic of the disease continues. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19, initiates infection via the binding of the receptor-binding domain (RBD) of its spike protein to the human angiotensin-converting enzyme II (ACE2) receptor, and this interaction has been the primary target for the development of COVID-19 therapeutics. Here, we identified neutralizing antibodies against SARS-CoV-2 by screening mouse monoclonal antibodies and characterized an antibody, CSW1-1805, that targets a narrow region at the RBD ridge of the spike protein. CSW1-1805 neutralized several variants in vitro and completely protected mice from SARS-CoV-2 infection. Cryo-EM and biochemical analyses revealed that this antibody recognizes the loop region adjacent to the ACE2-binding interface with the RBD in both a receptor-inaccessible "down" state and a receptor-accessible "up" state and could stabilize the RBD conformation in the up-state. CSW1-1805 also showed different binding orientations and complementarity determining region properties compared to other RBD ridge-targeting antibodies with similar binding epitopes. It is important to continuously characterize neutralizing antibodies to address new variants that continue to emerge. Our characterization of this antibody that recognizes the RBD ridge of the spike protein will aid in the development of future neutralizing antibodies.IMPORTANCESARS-CoV-2 cell entry is initiated by the interaction of the viral spike protein with the host cell receptor. Therefore, mechanistic findings regarding receptor recognition by the spike protein help uncover the molecular mechanism of SARS-CoV-2 infection and guide neutralizing antibody development. Here, we characterized a SARS-CoV-2 neutralizing antibody that recognizes an epitope, a loop region adjacent to the receptor-binding interface, that may be involved in the conformational transition of the receptor-binding domain (RBD) of the spike protein from a receptor-inaccessible "down" state into a receptor-accessible "up" state, and also stabilizes the RBD in the up-state. Our mechanistic findings provide new insights into SARS-CoV-2 receptor recognition and guidance for neutralizing antibody development.

Sensitivity improvement of a single-NV diamond magnetometer using a chiral waveguide.

A single nitrogen-vacancy (NV) center in diamond is utilized to perform nanoscale magnetic measurements. However, the low contrast and poor collection efficiency of spin-dependent emitted photons limited the instrument sensitivity to approximately several nT/Hz. Here, we design a diamond magnetometer based on a chiral waveguide. We numerically demonstrate that the proposed device achieves a sensitivity of 170 pT/Hz owing to near-unity contrast and efficient photon collection. We also confirm that the device sensitivity is robust against position misalignment and dipole misorientation of an NV center. The proposed approach will enable the construction of a highly-sensitive magnetometer with high spatial resolution.

Genomic diversity of SARS-CoV-2 can be accelerated by mutations in the nsp14 gene.

Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a proofreading exonuclease, nonstructural protein 14 (nsp14), that helps ensure replication competence at a low evolutionary rate compared with other RNA viruses. In the current pandemic, SARS-CoV-2 has accumulated diverse genomic mutations including in nsp14. Here, to clarify whether amino acid substitutions in nsp14 affect the genomic diversity and evolution of SARS-CoV-2, we searched for amino acid substitutions in nature that may interfere with nsp14 function. We found that viruses carrying a proline-to-leucine change at position 203 (P203L) have a high evolutionary rate and that a recombinant SARS-CoV-2 virus with the P203L mutation acquired more diverse genomic mutations than wild-type virus during its replication in hamsters. Our findings suggest that substitutions, such as P203L, in nsp14 may accelerate the genomic diversity of SARS-CoV-2, contributing to virus evolution during the pandemic.

Receptor-Binding Specificity of Influenza Viruses.

Influenza A virus infection begins with the attachment of virus particles to sialic acid-containing receptors on the surface of host cells. This attachment is mediated by the viral surface glycoprotein hemagglutinin (HA). Influenza A viruses have a wide host range, meaning they are able to infect many mammal and bird species. Influenza pandemics have been caused by viruses that contain genes from avian influenza viruses. Therefore, the infection of humans with avian influenza viruses, including avian H5Nx and H7Nx viruses, poses a huge threat to public health. These avian influenza viruses can transmit directly to humans from infected poultry, but do not spread easily among people, in part, due to differences in the receptor-binding specificities of human and avian influenza viruses. Therefore, conversion from avian- to human-type receptor-binding specificity is widely believed to be necessary for the efficient transmission of avian influenza viruses among humans. Accordingly, constant monitoring of the receptor-binding specificity of avian influenza viruses is important. In this chapter, we describe the protocol for assessing the receptor-binding specificity of influenza A viruses.

H3N2 Influenza Viruses with 12- or 16-Amino Acid Deletions in the Receptor-Binding Region of Their Hemagglutinin Protein.

Human influenza viruses evade host immune responses by accumulating mutations around the receptor-binding region of the hemagglutinin (HA) protein, which is composed of three key elements, the 130-loop, the 190-helix, and the 220-loop. Here, we characterized two human H3N2 influenza viruses with 12- and 16-amino acid deletions around the HA receptor-binding site that were isolated after antigenic selection of mutated H3N2 viruses. Structural modeling suggested that the 12-amino acid deletion eliminated the 190-helix. The 16-amino acid deletion comprises two stretches of 11- and 5-amino acid deletions. As the result of a frameshift, "novel" amino acids (not found in wild-type HA at these positions) are encoded between the deleted regions. Interestingly, structural modeling predicted that the novel sequence forms a structure resembling the 190-helix. However, compared to wild-type HA, the 16-amino acid deletion mutant lacks two antiparallel beta-sheets that connect the 190-helix and the 220-loop in wild-type HA. Nonetheless, both HA deletion mutants replicated in mammalian cells, and the 16-amino acid deletion mutant (with a remodeled 190-helix) also replicated in Syrian hamsters, albeit at low titers. Wild-type virus bound preferentially to α2,6-linked sialic acids, whereas both mutants gained affinity for α2,3-linked sialic acids. Moreover, the 12- and 16-amino acid deletions may affect the antigenic properties of the viruses. Thus, viruses with sizeable deletions around the HA receptor-binding site are viable but may display altered sialic acid preferences, altered antigenic properties, and attenuated replicative ability in cultured cells and virulence in Syrian hamsters. IMPORTANCE The hemagglutinin (HA) protein of influenza viruses serves as the receptor-binding protein and is the principal target of the host immune system. The antigenic epitopes in the receptor-binding region are known to tolerate mutations, but here, we show that even deletions of 12 or 16 amino acids in this region can be accommodated. In cultured cells, 12- and 16-amino acid deletion mutants were attenuated, and the 16-amino acid deletion mutant replicated in Syrian hamsters. Compared with wild-type virus, both mutants showed changes in their reactivity to some of the sera tested and changes in their binding affinity to sialic acids, which serve as influenza virus receptors. Collectively, our findings highlight the plasticity of HA.

The hokW-sokW Locus Encodes a Type I Toxin-Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157.

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

Plasticity of the Influenza Virus H5 HA Protein.

Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants.IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development.

At the end of 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was detected in Wuhan, China, that spread rapidly around the world, with severe consequences for human health and the global economy. Here, we assessed the replicative ability and pathogenesis of SARS-CoV-2 isolates in Syrian hamsters. SARS-CoV-2 isolates replicated efficiently in the lungs of hamsters, causing severe pathological lung lesions following intranasal infection. In addition, microcomputed tomographic imaging revealed severe lung injury that shared characteristics with SARS-CoV-2-infected human lung, including severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation. SARS-CoV-2-infected hamsters mounted neutralizing antibody responses and were protected against subsequent rechallenge with SARS-CoV-2. Moreover, passive transfer of convalescent serum to naïve hamsters efficiently suppressed the replication of the virus in the lungs even when the serum was administrated 2 d postinfection of the serum-treated hamsters. Collectively, these findings demonstrate that this Syrian hamster model will be useful for understanding SARS-CoV-2 pathogenesis and testing vaccines and antiviral drugs.

Influenza A variants with reduced susceptibility to baloxavir isolated from Japanese patients are fit and transmit through respiratory droplets.

Here we report the isolation of the influenza A/H1N1 2009 pandemic (A/H1N1pdm) and A/H3N2 viruses carrying an I38T mutation in the polymerase acidic protein-a mutation that confers reduced susceptibility to baloxavir marboxil-from patients before and after treatment with baloxavir marboxil in Japan. These variants showed replicative abilities and pathogenicity that is similar to those of wild-type isolates in hamsters; they also transmitted efficiently between ferrets by respiratory droplets.

Long-term culture of human lung adenocarcinoma A549 cells enhances the replication of human influenza A viruses.

Long-term culture of the human lung adenocarcinoma cell line A549 promotes the differentiation of these cells toward an alveolar type II cell phenotype. Here, we evaluated the susceptibility of long-term cultured A549 cells to human influenza viruses. A549 cells were cultured continuously for 25 days (D25-A549) or 1 day (D1-A549) in Ham's F12K medium. Six human influenza A viruses grew much faster in D25-A549 cells than in D1-A549 cells; however, two influenza B viruses replicated poorly in both cell types. Two avian influenza viruses replicated efficiently in both cell types, with similar titres. Expression levels of human virus receptors were higher in D25-A549 cells than in D1-A549 cells. D25-A549 cells thus more efficiently support the replication of human influenza A viruses compared with D1-A549 cells. Our data suggest that long-term cultured A549 cells will be useful for influenza A virus research.

A humanized MDCK cell line for the efficient isolation and propagation of human influenza viruses.

Here, we developed hCK, a Madin-Darby canine kidney (MDCK) cell line that expresses high levels of human influenza virus receptors and low levels of avian virus receptors. hCK cells supported human A/H3N2 influenza virus isolation and growth much more effectively than conventional MDCK or human virus receptor-overexpressing (AX4) cells. A/H3N2 viruses propagated in hCK cells also maintained higher genetic stability than those propagated in MDCK and AX4 cells.

A Highly Pathogenic Avian H7N9 Influenza Virus Isolated from A Human Is Lethal in Some Ferrets Infected via Respiratory Droplets.

Low pathogenic H7N9 influenza viruses have recently evolved to become highly pathogenic, raising concerns of a pandemic, particularly if these viruses acquire efficient human-to-human transmissibility. We compared a low pathogenic H7N9 virus with a highly pathogenic isolate, and two of its variants that represent neuraminidase inhibitor-sensitive and -resistant subpopulations detected within the isolate. The highly pathogenic H7N9 viruses replicated efficiently in mice, ferrets, and/or nonhuman primates, and were more pathogenic in mice and ferrets than the low pathogenic H7N9 virus, with the exception of the neuraminidase inhibitor-resistant virus, which showed mild-to-moderate attenuation. All viruses transmitted among ferrets via respiratory droplets, and the neuraminidase-sensitive variant killed several of the infected and exposed animals. Neuraminidase inhibitors showed limited effectiveness against these viruses in vivo, but the viruses were susceptible to a polymerase inhibitor. These results suggest that the highly pathogenic H7N9 virus has pandemic potential and should be closely monitored.