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Abstract Influenza A(H7N9) virus showed high pathogenicity in humans when it emerged in 2013. Cigarette smoke (CS) causes pulmonary diseases including bronchitis, emphysema, and lung cancer. Although habitual smoking is thought to increase the risk of severe seasonal influenza virus infection, its effect on A(H7N9) virus infection is poorly understood. Here, we employed a mouse model of long-term exposure to CS to investigate the effect of CS on the pathogenicity of A(H7N9) virus infection. Unexpectedly, body weight loss for mice exposed to CS was milder than that for mock-treated mice upon A(H7N9) virus infection. CS exposure improved the survival rate of A(H7N9) virus-infected mice even though virus titers and pathological changes in the lungs were not significantly different between CS-exposed and control mice. Microarray analysis showed that CS-exposure activates cytokine/chemokine activity, immune response, and cell cycle activities that resemble reactivities against A(H7N9) virus infection. Therefore, under conditions where cytokine and chemokine expression in the lungs is already high due to CS exposure, the enhanced expression of cytokines and chemokines caused by A(H7N9) virus infection might be less harmful to the organs compared to the rapid increase in cytokine and chemokine expression in the air-exposed mice due to the infection. CS may thus induce immunoregulatory effects that attenuate severe pulmonary disease during A(H7N9) virus infection. However, these findings do not support CS exposure due to its many other proven negative health effects.

Abstract During the Omicron wave, previous variants such as BA.2, BA.4, and BA.5 were replaced by newer variants with additional mutations in the spike protein. These variants, BA.4.6, BQ.1.1, and XBB, have spread in different countries with different degrees of success. Here, we evaluated the replicative ability and pathogenicity of BA.4.6, BQ1.1, and XBB clinical isolates in male Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with these Omicron subvariants, the replicative ability of BQ.1.1 and XBB in lung tissue was higher than that of BA.4.6 and BA.5. Of note, BQ.1.1 was lethal in both male and female transgenic human ACE2 hamsters. In competition assays, XBB replicated better than BQ.1.1 in the nasal turbinate tissues of female hamsters previously infected with Omicron BA.2. These results suggest that newer Omicron subvariants in the XBB family are still evolving and should be closely monitored.

Abstract Here, we assessed the efficacy of a lipid nanoparticle-based mRNA vaccine candidate encoding the receptor-binding domain (LNP-mRNA-RBD) in mice. Mice immunized with LNP-mRNA-RBD based on the ancestral strain (ancestral-type LNP-mRNA-RBD) showed similar cellular responses against the ancestral strain and BA.5, but their neutralizing activity against BA.5 was lower than that against the ancestral strain. The ancestral-type LNP-mRNA-RBD protected mice from the ancestral strain or BA.5 challenge; however, its ability to reduce the viral burdens after BA.5 challenge was limited. In contrast, immunization with bivalent LNP-mRNA-RBD consisting of the ancestral-type and BA.4/5-type LNP-mRNA-RBD or monovalent BA.4/5-type LNP-mRNA-RBD elicited robust cellular responses, as well as high and moderate neutralizing titers against BA.5 and XBB.1.5, respectively. Furthermore, the vaccines containing BA.4/5-type LNP-mRNA-RBD remarkably reduced the viral burdens following BA.5 or XBB.1.5 challenge. Overall, our findings suggest that LNP-mRNA-RBD is effective against SARS-CoV-2 infection.

Abstract Background People living with HIV (PLWH) with chronic inflammation may have an increasing risk for coronavirus disease 2019 (COVID-19) severity; however, the impact of their gut microbiota on COVID-19 is not fully elucidated. Here, we analyzed the temporal changes in the gut microbiota composition of hospitalized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected PLWH (PLWH-CoV) and their correlation with COVID-19 severity. Result The 16S rRNA analysis results using stool samples (along the timeline from disease onset) from 12 hospitalized PLWH-CoV, whose median CD4 + T cell count was 671 cells/µl, were compared to those of 19 healthy people and 25 PLWH. Bacterial diversity in PLWH-CoV is not significantly different from that of healthy people and SARS-CoV-2 non-infected PLWH, but a significant difference in the microbiota diversity was observed in the classification according to the disease severity. Immediately after the disease onset, remarkable changes were observed in the gut microbiota of PLWH-CoV, and the changing with a decrease in some short-chain fatty acid-producing bacteria and an increase in colitis-related pathobiont. In the second week after disease onset, relative amounts of specific bacteria distinguished between disease severity. One month after the disease onset, dysbiosis of the gut microbiota persisted, and the number of Enterobacteriaceae, mainly Escherichia-Shigella, which is potentially pathogenic, increased and were enriched in patients who developed post-acute sequelae of COVID-19 (PASC). Conclusion The changes in the gut microbiota associated with SARS-CoV-2 infection observed in PLWH in this study indicated a persistent decrease in SCFA-producing bacteria and an intestinal environment with an increase in opportunistic pathogens associated with enteritis. This report demonstrates that the intestinal environment in PLWH tends to show delayed improvement even after COVID-19 recovery, and highlights the importance of the dysbiosis associated with SARS-CoV-2 infection as a potential factor in the COVID-19 severity and the PASC in PLWH.

Abstract Background The mucosa serves as the first defence against pathogens and facilitates the surveillance and elimination of symbiotic bacteria by mucosal immunity. Recently, the mRNA vaccine against SARS-CoV-2 has been demonstrated to induce secretory antibodies in the oral and nasal cavities in addition to a systemic immune response. However, the mechanism of induced immune stimulation effect on mucosal immunity and commensal bacteria profile remains unclear. Methods Here, we longitudinally analysed the changing nasal microbiota and both systemic and nasal immune response upon SARS-CoV-2 mRNA vaccination, and evaluated how mRNA vaccination influenced nasal microbiota in 18 healthy participants who had received the third BNT162b. Results The nasal S-RBD IgG level correlated significantly with plasma IgG levels until 1 month and the levels were sustained for 3 months post-vaccination. In contrast, nasal S-RBD IgA induction peaked at 1 month, albeit slightly, and correlated only with plasma IgA, but the induction level decreased markedly at 3 months post-vaccination. 16 S rRNA sequencing of the nasal microbiota post-vaccination revealed not an overall change, but a decrease in certain opportunistic bacteria, mainly Fusobacterium. The decrease in these bacteria was more pronounced in those who exhibited nasal S-RBD IgA induction, and those with higher S-RBD IgA induction had lower relative amounts of potentially pathogenic bacteria such as Pseudomonas pre-vaccination. In addition, plasma and mucosal S-RBD IgG levels correlated with decreased commensal pathogens such as Finegoldia. Conclusions These findings suggest that the third dose of SARS-CoV-2 mRNA vaccination induced S-RBD antibodies in the nasal mucosa and may have stimulated mucosal immunity against opportunistic bacterial pathogens. This effect, albeit probably secondary, may be considered one of the benefits of mRNA vaccination. Furthermore, our data suggest that a cooperative function of mucosal and systemic immunity in the reduction of bacteria and provides a better understanding of the symbiotic relationship between the host and bacteria in the nasal mucosa.

Abstract Ensitrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro or Nsp5), is clinically useful against SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to most monoclonal antibody therapies, SARS-CoV-2 resistance to other antivirals including main protease inhibitors such as ensitrelvir is a major public health concern. Here, repeating passages of SARS-CoV-2 in the presence of ensitrelvir revealed that the M49L and E166A substitutions in Nsp5 are responsible for reduced sensitivity to ensitrelvir. Both substitutions reduced in vitro virus growth in the absence of ensitrelvir. The combination of the M49L and E166A substitutions allowed the virus to largely evade the suppressive effect of ensitrelvir in vitro. The virus possessing Nsp5-M49L showed similar pathogenicity to wild-type virus, whereas the virus possessing Nsp5-E166A or Nsp5-M49L/E166A slightly attenuated. Ensitrelvir treatment of hamsters infected with the virus possessing Nsp5-M49L/E166A was ineffective; however, nirmatrelvir or molnupiravir treatment was effective. Therefore, it is important to closely monitor the emergence of ensitrelvir-resistant SARS-CoV-2 variants to guide antiviral treatment selection.

Abstract Nirmatrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro), is clinically useful against infection with SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to many monoclonal antibody therapies, potential SARS-CoV-2 resistance to nirmatrelvir is a major public health concern. Several amino acid substitutions have been identified as being responsible for reduced susceptibility to nirmatrelvir. Among them, we selected L50F/E166V and L50F/E166A/L167F in the 3CLpro because these combinations of substitutions are unlikely to affect virus fitness. We prepared and characterized delta variants possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F. Both mutant viruses showed decreased susceptibility to nirmatrelvir and their growth in VeroE6/TMPRSS2 cells was delayed. Both mutant viruses showed attenuated phenotypes in a male hamster infection model, maintained airborne transmissibility, and were outcompeted by wild-type virus in co-infection experiments in the absence of nirmatrelvir, but less so in the presence of the drug. These results suggest that viruses possessing Nsp5-L50F/E166V and Nsp5-L50F/E166A/L167F do not become dominant in nature. However, it is important to closely monitor the emergence of nirmatrelvir-resistant SARS-CoV-2 variants because resistant viruses with additional compensatory mutations could emerge, outcompete the wild-type virus, and become dominant.

Summary: EG.5.1 is a subvariant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB variant that is rapidly increasing in prevalence worldwide. However, the pathogenicity, transmissibility, and immune evasion properties of isolates of EG.5.1 are largely unknown. Here, we show that there are no obvious differences in growth ability and pathogenicity between EG.5.1 and XBB.1.5 in hamsters. We also demonstrate that, like XBB.1.5, EG.5.1 is transmitted more efficiently between hamsters compared to its predecessor, BA.2. In contrast, unlike XBB.1.5, we detect EG.5.1 in the lungs of four of six exposed hamsters, suggesting that the virus properties of EG.5.1 are different from those of XBB.1.5. Finally, we find that the neutralizing activity of plasma from convalescent individuals against EG.5.1 was slightly, but significantly, lower than that against XBB.1.5 or XBB.1.9.2. Our data suggest that the different virus properties after transmission and the altered antigenicity of EG.5.1 may be driving its increasing prevalence over XBB.1.5 in humans.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to circulate in humans since its emergence in 2019. While infection in humans continues, numerous spillover events to at least 32 animal species, including companion and zoo animals, have been reported. Since dogs and cats are highly susceptible to SARS-CoV-2 and have direct contact with their owners and other household members, it is important to know the prevalence of SARS-CoV-2 in dogs and cats. Here, we established an ELISA to detect serum antibodies against the receptor-binding domain and the ectodomain of the SARS-CoV-2 spike and nucleocapsid proteins. Using this ELISA, we assessed seroprevalence in 488 dog serum samples and 355 cat serum samples that were collected during the early pandemic period (between May and June of 2020) and 312 dog serum samples and 251 cat serum samples that were collected during the mid-pandemic period (between October 2021 and January 2022). We found that two dog serum samples (0.41%) collected in 2020, one cat serum sample (0.28%) collected in 2020, and four cat serum samples (1.6%) collected in 2021 were positive for antibodies against SARS-CoV-2. No dog serum samples collected in 2021 were positive for these antibodies. We conclude that the seroprevalence of SARS-CoV-2 antibodies in dogs and cats in Japan is low, suggesting that these animals are not a major SARS-CoV-2 reservoir.

Summary: The emergence and spread of new SARS-CoV-2 variants with mutations in the spike protein, such as the XBB.1.5 and XBB.1.9.1 sublineages, raise concerns about the efficacy of current COVID-19 vaccines and therapeutic monoclonal antibodies (mAbs). In this study, none of the mAbs we tested neutralized XBB.1.9.1 or XBB.1.5, even at the highest concentration used. We also found that the bivalent mRNA vaccine could enhance humoral immunity against XBB.1.9.1, but that XBB.1.9.1 and XBB.1.5 still evaded humoral immunity induced by vaccination or infection. Moreover, the susceptibility of XBB.1.9.1 to remdesivir, molnupiravir, nirmatrelvir, and ensitrelvir was similar to that of the ancestral strain and the XBB.1.5 isolate in vitro. Finally, we found the replicative fitness of XBB.1.9.1 to be similar to that of XBB.1.5 in hamsters. Our results suggest that XBB.1.9.1 and XBB.1.5 have similar antigenicity and replicative ability, and that the currently available COVID-19 antivirals remain effective against XBB.1.9.1.

Omicron subvariants may differ in their replicative fitness and their potential to cause more severe disease. In this study, the authors characterized Omicron BA.2.75 in a hamster model and found that it replicates more efficiently in the lungs than BA.2 and BA.5.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) often causes severe viral pneumonia. Although many studies using mouse models have examined the pathogenicity of SARS-CoV-2, COVID-19 pathogenesis remains poorly understood. In vivo imaging analysis using two-photon excitation microscopy (TPEM) is useful for elucidating the pathology of COVID-19, providing pathological insights that are not available from conventional histological analysis. However, there is no reporter SARS-CoV-2 that demonstrates pathogenicity in C57BL/6 mice and emits sufficient light intensity for two-photon in vivo imaging. Here, we generated a mouse-adapted strain of SARS-CoV-2 (named MASCV2-p25) and demonstrated its efficient replication in the lungs of C57BL/6 mice, causing fatal pneumonia. Histopathologic analysis revealed the severe inflammation and infiltration of immune cells in the lungs of MASCV2-p25-infected C57BL/6 mice, not unlike that observed in COVID-19 patients with severe pneumonia. Subsequently, we generated a mouse-adapted reporter SARS-CoV-2 (named MASCV-Venus-p9) by inserting the fluorescent protein-encoding gene Venus into MASCV2-p25 and sequential lung-to-lung passages in C57BL/6 mice. C57BL/6 mice infected with MASCV2-Venus-p9 exhibited severe pneumonia. In addition, the TPEM of the lungs of the infected C57BL/6J mice showed that the infected cells emitted sufficient levels of fluorescence for easy observation. These findings suggest that MASCV2-Venus-p9 will be useful for two-photon in vivo imaging studies of the pathogenesis of severe COVID-19 pneumonia.

Summary: SARS-CoV-2 has gradually acquired amino acid substitutions in its S protein that reduce the potency of neutralizing antibodies, leading to decreased vaccine efficacy. Here, we attempted to obtain mutant viruses by passaging SARS-CoV-2 in the presence of plasma samples from convalescent patients or vaccinees to determine which amino acid substitutions affect the antigenicity of SARS-CoV-2. Several amino acid substitutions in the S2 region, as well as the N-terminal domain (NTD) and receptor-binding domain (RBD), affected the neutralization potency of plasma samples collected from vaccinees, indicating that amino acid substitutions in the S2 region as well as those in the NTD and RBD affect neutralization by vaccine-induced antibodies. Furthermore, the neutralizing potency of vaccinee plasma samples against mutant viruses we obtained or circulating viruses differed among individuals. These findings suggest that genetic backgrounds of vaccinees influence the recognition of neutralizing epitopes.

Summary: Background: Like its predecessors in the XBB family, XBB.1.5 is highly immune evasive from therapeutic monoclonal antibodies and neutralizing antibodies generated by vaccination and/or infection. However, there is a lack of in vivo data on XBB.1.5 in animal models such as Syrian hamsters. Methods: Syrian hamsters (females) were used to examine airborne transmission along with virus replication of XBB.1.5 in naïve animals and human ACE2 hamsters with pre-existing immunity from a previous infection with Omicron BA.1. Assays were performed to determine neutralizing antibody responses, and virus titers were determined by standard plaque assays. Findings: Unlike earlier Omicron subvariants, such as BA.1 and BA.2, XBB.1.5 transmitted more efficiently in the hamster model. In addition, XBB.1.5 partially escaped BA.1-immunity from a previous infection with XBB.1.5 replicating in the nasal turbinate tissues and to a lesser extend in the lung tissues of previously infected hamsters. Interpretation: Our in vivo data showing better airborne transmissibility of the Omicron subvariant XBB.1.5 than its predecessor, BA.2, in Syrian hamsters will allow researchers to further investigate amino acid substitutions that give XBB.1.5 a fitness advantage over BA.2 in transmission, data that may be important in studies of SARS-CoV-2 transmission in humans. Funding: This research is supported by grants from the Center for Research on Influenza Pathogenesis and Transmission (CRIPT; 75N93021C00014), funded by the National Institute of Allergy and Infectious Diseases and by a Research Program on Emerging and Reemerging Infectious Diseases (JP21fk0108552 and JP21fk0108615), a Project Promoting Support for Drug Discovery (JP21nf0101632), the Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125002), and The University of Tokyo Pandemic Preparedness, Infection and Advanced Research Center (UTOPIA) grant (JP223fa627001) from the Japan Agency for Medical Research and Development.

Broadly protective antibodies targeting influenza viruses are of interest as potential therapeutics or to inform vaccine development. Here the authors characterize a human mAb (DA03E17) with heterosubtypic binding to neuraminidases from IAVs and IBVs that provides broad protection in vitro and in vivo.

Japan has reported a relatively small number of COVID-19 cases. Because not all infected persons receive diagnostic tests for COVID-19, the reported number must be lower than the actual number of infections. We assessed SARS-CoV-2 seroprevalence by analyzing >60,000 samples collected in Japan (Tokyo Metropolitan Area and Hokkaido Prefecture) during February 2020–March 2022. The results showed that ≈3.8% of the population had become seropositive by January 2021. The seroprevalence increased with the administration of vaccinations; however, among the elderly, seroprevalence was not as high as the vaccination rate. Among children, who were not eligible for vaccination, infection was spread during the epidemic waves caused by the SARS-CoV-2 Delta and Omicron variants. Nevertheless, seroprevalence for unvaccinated children

Summary: Several antibody therapeutics have been developed against SARS-CoV-2; however, they have attenuated neutralizing ability against variants. In this study, we generated multiple broadly neutralizing antibodies from B cells of convalescents, by using two types of receptor-binding domains, Wuhan strain and the Gamma variant as bait. From 172 antibodies generated, six antibodies neutralized all strains prior to the Omicron variant, and the five antibodies were able to neutralize some of the Omicron sub-strains. Structural analysis showed that these antibodies have a variety of characteristic binding modes, such as ACE2 mimicry. We subjected a representative antibody to the hamster infection model after introduction of the N297A modification, and observed a dose-dependent reduction of the lung viral titer, even at a dose of 2 mg/kg. These results demonstrated that our antibodies have certain antiviral activity as therapeutics, and highlighted the importance of initial cell-screening strategy for the efficient development of therapeutic antibodies.

Summary: Background: The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized. Methods: We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics in vitro and in vivo. Findings: S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3 cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected. Interpretation: Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity. Funding: A full list of funding bodies that contributed to this study can be found under Acknowledgments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing coronavirus pandemic. Besides vaccines and antiviral drugs, probiotics have attracted attention for prevention of SARS-CoV-2 infection. Here, we examined the efficacy of heat-killed Lactiplantibacillus pentosus ONRICb0240 (b240) against SARS-CoV-2 infection in mice. We observed that oral intake of heat-killed b240 did not affect virus titers in the respiratory organs of SARS-CoV-2-infected mice, but did provide partial protection against SARS-CoV-2 infection. In addition, heat-killed b240 treatment suppressed the expression of IL-6, a key proinflammatory cytokine, on Day 2 post-infection. Our results highlight the promising protective role of heat-killed b240 and suggest a possible mechanism by which heat-killed b240 partially protects against SARS-CoV-2 infection by modulating host responses.

Summary: The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from patients with COVID-19-convalescent, and identified antibodies that exhibited the comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced. Our antibodies showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) is an acute respiratory infection transmitted by droplets, aerosols, and contact. Controlling the spread of COVID-19 and developing effective decontamination options are urgent issues for the international community. Here, we report the quantitative inactivation of SARS-CoV-2 in liquid and aerosolized samples by a state-of-the-art, high-power, AlGaN-based, single-chip compact deep-UV (DUV) light-emitting diode (LED) that produces a record continuous-wave output power of 500 mW at its peak emission wavelength of 265 nm. Using this DUV-LED, we observed a greater-than-5-log reduction in infectious SARS-CoV-2 in liquid samples within very short irradiation times (

ABSTRACT The role of the intestinal microbiota in coronavirus disease 2019 (COVID-19) is being elucidated. Here, we analyzed the temporal changes in microbiota composition and the correlation between inflammation biomarkers/cytokines and microbiota in hospitalized COVID-19 patients. We obtained stool specimens, blood samples, and patient records from 22 hospitalized COVID-19 patients and performed 16S rRNA metagenomic analysis of stool samples over the course of disease onset compared to 40 healthy individual stool samples. We analyzed the correlation between the changes in the gut microbiota and plasma proinflammatory cytokine levels. Immediately after admission, differences in the gut microbiota were observed between COVID-19 patients and healthy subjects, mainly including enrichment of the classes Bacilli and Coriobacteriia and decrease in abundance of the class Clostridia. The bacterial profile continued to change throughout the hospitalization, with a decrease in short-chain fatty acid-producing bacteria including Faecalibacterium and an increase in the facultatively anaerobic bacteria Escherichia-Shigella. A consistent increase in Eggerthella belonging to the class Coriobacteriia was observed. The abundance of the class Clostridia was inversely correlated with interferon-γ level and that of the phylum Actinobacteria, which was enriched in COVID-19, and was positively correlated with gp130/sIL-6Rb levels. Dysbiosis was continued even after 21 days from onset. The intestines tended to be an aerobic environment in hospitalized COVID-19 patients. Because the composition of the gut microbiota correlates with the levels of proinflammatory cytokines, this finding emphasizes the need to understand how pathology is related to the temporal changes in the specific gut microbiota observed in COVID-19 patients. IMPORTANCE There is growing evidence that the commensal microbiota of the gastrointestinal and respiratory tracts regulates local and systemic inflammation (gut-lung axis). COVID-19 is primarily a respiratory disease, but the involvement of microbiota changes in the pathogenesis of this disease remains unclear. The composition of the gut microbiota of patients with COVID-19 changed over time during hospitalization, and the intestines tended to be an aerobic environment in hospitalized COVID-19 patients. These changes in gut microbiota may induce increased intestinal permeability, called leaky gut, allowing bacteria and toxins to enter the circulatory system and further aggravate the systemic inflammatory response. Since gut microbiota composition correlates with levels of proinflammatory cytokines, this finding highlights the need to understand how pathology relates to the gut environment, including the temporal changes in specific gut microbiota observed in COVID-19 patients.

The influenza A(H1N1)pdm09 virus that emerged in 2009 causes seasonal epidemic worldwide. The virus acquired several amino acid substitutions that were responsible for antigenic drift until the 2018–2019 influenza season. Viruses possessing mutations in the NA and PA proteins that cause reduced susceptibility to NA inhibitors and baloxavir marboxil, respectively, have been detected after antiviral treatment, albeit infrequently. Here, we analyzed HA, NA, and PA sequences derived from A(H1N1)pdm09 viruses that were isolated during the 2018–2019 and 2019–2020 influenza seasons in Japan. We found that A(H1N1)pdm09 viruses possessing the D187A and Q189E substitutions in HA emerged and dominated during the 2019–2020 season; these substitutions in the antigenic site Sb, a high potency neutralizing antibody-eliciting site for humans, changed the antigenicity of A(H1N1)pdm09 viruses. Furthermore, we found that isolates possessing the N156K substitution, which was predicted to affect the antigenicity of A(H1N1)pdm09 virus at the laboratory level, were detected at a frequency of 1.0% in the 2018–2019 season but 10.1% in the 2019–2020 season. These findings indicate that two kinds of antigenically drifted viruses—N156K and D187A/Q189E viruses—co-circulated during the 2019–2020 influenza season in Japan.

Summary: CRISPR-based diagnostics (CRISPR-dx), including the Cas12-based DETECTR and Cas13-based SHERLOCK Class 2 CRISPRs, have been used to detect the presence of DNA or RNA from pathogens, such as the 2009 pandemic influenza virus A (IAV) and the 2019 novel coronavirus SARS-CoV-2. Here, we describe the collateral single-stranded DNA cleavage with Class 1 type I CRISPR-Cas3 and highlight its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection method for SARS-CoV-2. This assay, which we call Cas3-Operated Nucleic Acid detectioN (CONAN), not only detects SARS-CoV-2 in clinical samples, but also offers specific detection of single-base-pair mutations in IAV variants. This tool allows rapid and accurate point-of-care testing for patients with suspected SARS-CoV-2 or drug-resistant IAV infections in hospitals.

Summary: We developed an intranasal vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the replication-incompetent human parainfluenza virus type 2 (hPIV2) vector BC-PIV, which can deliver ectopic gene as stable RNA and ectopic protein on the envelope. BC-PIV expressing the full-length prefusion-stabilized spike gene (K986P/V987P) of SARS-CoV-2, S-2PM, possessed a corona-like viral envelope. Intranasal vaccination of mice with BC-PIV/S-2PM induced high levels of neutralizing immunoglobulin G (IgG) and mucosal IgA antibodies against the spike protein. Although BC-PIV showed hemagglutinating activity, BC-PIV/S-2PM lacked such activity, in accordance with the presence of the massive spike protein on the viral surface. Furthermore, single-dose intranasal vaccination of hamsters with BC-PIV/S-2PM completely protected the lungs from SARS-CoV-2 at 11-week post-immunization, and boost vaccination two weeks before the challenge conferred virtually complete protection of the nasal turbinates against SARS-CoV-2. Thus, this chimeric hPIV2/spike intranasal vaccine is a promising vaccine candidate for SARS-CoV-2 to curtail virus transmission.

Viruses infect cells mainly via specific receptors at the cell surface. Antibody-dependent enhancement (ADE) of infection is an alternative mechanism of infection for viruses to infect immune cells that is mediated by antibodies and IgG receptors (FcγRs).

Background: To develop an effective vaccine against a novel viral pathogen, it is important to understand the longitudinal antibody responses against its first infection. Here we performed a longitudinal study of antibody responses against SARS-CoV-2 in symptomatic patients. Methods: Sequential blood samples were collected from 39 individuals at various timepoints between 0 and 154 days after onset. IgG or IgM titers to the receptor binding domain (RBD) of the S protein, the ectodomain of the S protein, and the N protein were determined by using an ELISA. Neutralizing antibody titers were measured by using a plaque reduction assay. Findings: The IgG titers to the RBD of the S protein, the ectodomain of the S protein, and the N protein peaked at about 20 days after onset, gradually decreased thereafter, and were maintained for several months after onset. Extrapolation modeling analysis suggested that the IgG antibodies were maintained for this amount of time because the rate of reduction slowed after 30 days post-onset. IgM titers to the RBD decreased rapidly and disappeared in some individuals after 90 days post-onset. All patients, except one, possessed neutralizing antibodies against authentic SARS-CoV-2, which they retained at 90 days after onset. The highest antibody titers in patients with severe infections were higher than those in patients with mild or moderate infections, but the decrease in antibody titer in the severe infection cohort was more remarkable than that in the mild or moderate infection cohort. Interpretation: Although the number of patients is limited, our results show that the antibody response against the first SARS-CoV-2 infection in symptomatic patients is typical of that observed in an acute viral infection. Funding: The Japan Agency for Medical Research and Development and the National Institutes of Allergy and Infectious Diseases.

Assays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method using Bayesian hierarchical modelling to reduce the workload of these assays and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. Inflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from 3 spread-out dilutions correlated with those inferred from 8 consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. In serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

ABSTRACT Influenza virus relies heavily on cellular machinery to replicate in host cells. Therefore, to better understand the influenza virus life cycle, it is important to identify which host proteins are involved and how they function in virus replication. Previously, we identified G protein pathway suppressor 1 (GPS1) to be a matrix protein 2 (M2)-interacting host protein. GPS1 is a component of the COP9 signalosome, which regulates the NF-κB signaling pathway. Here, we found that the downregulation of GPS1 expression reduced influenza virus replication by more than 2 log units. Although GPS1 was not involved in the early and late stages of virus replication, such as viral entry, uncoating, assembly, or budding, we found that viral polymerase activity was impaired in GPS1-downregulated cells. Moreover, our results suggest that M2 activates the NF-κB signaling pathway in a GPS1-dependent manner and that activation of NF-κB signaling leads to the upregulation of influenza virus polymerase activity. Our findings indicate that GPS1 is involved in the transcription and replication of influenza virus genomic RNA through the activation of the NF-κB signaling pathway. IMPORTANCE In the present study, we identified G protein pathway suppressor 1 (GPS1) to be a host cellular protein that is important for influenza virus replication. We also found that GPS1 plays a role in viral genome transcription through the NF-κB signaling pathway. Moreover, downregulation of GPS1 also affected the growth of vesicular stomatitis virus. Therefore, GPS1 may be a host target for antiviral drugs against influenza virus and possibly other viruses.

Epidemics of seasonal influenza caused by H1N1pdm09, H3N2, and type B viruses occur throughout the world. Sporadic human H5 and H7N9 virus infections are also reported in particular regions. To treat influenza patients effectively with antivirals, sensitive and broad-reactive influenza rapid diagnostic tests (IRDTs) are required. Here, we tested the sensitivity of 23 IRDTs during the 2018–2019 influenza season for their ability to detect H1N1pdm09, H3N2, H5N1, H5N6, H7N9, and Victoria- and Yamagata-lineage type B viruses. All IRDTs detected all influenza A and B viruses tested but with different sensitivities. Several IRDTs detected the H5 and H7 viruses and the seasonal viruses with similar sensitivity. Such IRDTs might be useful for diagnosing patients infected with H5 and H7 viruses.

To clarify the threat posed by emergence of highly pathogenic influenza A(H7N9) virus infection among humans, we characterized the viral polymerase complex. Polymerase basic 2–482R, polymerase basic 2–588V, and polymerase acidic–497R individually or additively enhanced virus polymerase activity, indicating that multiple replication-enhancing mutations in 1 isolate may contribute to virulence.

Rapid antigen tests (RATs) for COVID-19 based on lateral flow immunoassays are useful for rapid diagnosis in a variety of settings. Although many kinds of RATs are available, their respective sensitivity has not been compared. Here, we examined the sensitivity of 27 RATs available in Japan for the detection of the SARS-CoV-2 delta variant. All of the RATs tested detected the delta variant albeit with different sensitivities. Nine RATs (ESPLINE SARS-CoV-2, ALSONIC COVID-19 Ag, COVID-19 and Influenza A+B Antigen Combo Rapid Test, ImmunoArrow SARS-CoV-2, Fuji Dri-chem immuno AG cartridge COVID-19 Ag, 2019-nCoV Ag rapid detection kit, Saliva SARS-CoV-2(2019-nCoV) Antigen Test Kit, and Rabliss SARS-CoV-2 antigen detection kit COVID19 AG) showed superior sensitivity to the isolated delta variant. Although actual clinical specimens were not examined, the detection level of most of the RATs was 7500 pfu, indicating that individuals whose test samples contained less virus than that would be considered negative. Therefore, it is important to bear in mind that RATs may miss individuals shedding low levels of infectious virus.

Many broadly reactive human monoclonal antibodies against the hemagglutinin (HA) stem of influenza A virus have been developed for therapeutic applications. These antibodies typically inhibit viral entry steps, especially the HA conformational change that is required for membrane fusion. To better understand the mechanisms by which such antibodies inhibit viral replication, we established broadly reactive human anti-HA stem antibodies and determined the properties of these antibodies by examining their reactivity with 18 subtypes of HA, evaluating their in vivo protective efficacy, identifying their epitopes, and characterizing their inhibitory mechanisms. Among the eight human monoclonal antibodies we generated, which recognized at least 3 subtypes of the soluble HA antigens tested, clone S9-1-10/5-1 reacted with 18 subtypes of HA and protected mice from lethal infection with H1N1pdm09, H3N2, H5N1, and H7N9 viruses. This antibody recognized the HA2 helix A in the HA stem, and inhibited virus particle release from infected cells but did not block viral entry completely. These results show that broadly reactive human anti-HA stem antibodies can exhibit protective efficacy by inhibiting virus particle release. These findings expand our knowledge of the mechanisms by which broadly reactive stem-targeting antibodies inhibit viral replication and provide valuable information for universal vaccine development.

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.

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.

Reverse transcription-quantitative PCR (RT-qPCR)-based tests are widely used to diagnose coronavirus disease 2019 (COVID-19). As a result that these tests cannot be done in local clinics where RT-qPCR testing capability is lacking, rapid antigen tests (RATs) for COVID-19 based on lateral flow immunoassays are used for rapid diagnosis. However, their sensitivity compared with each other and with RT-qPCR and infectious virus isolation has not been examined. Here, we compared the sensitivity among four RATs by using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates and several types of COVID-19 patient specimens and compared their sensitivity with that of RT-qPCR and infectious virus isolation. Although the RATs read the samples containing large amounts of virus as positive, even the most sensitive RAT read the samples containing small amounts of virus as negative. Moreover, all RATs tested failed to detect viral antigens in several specimens from which the virus was isolated. The current RATs will likely miss some COVID-19 patients who are shedding infectious SARS-CoV-2.

Ebola virus disease is a severe disease, often fatal, with a mortality rate of up to 90%. Presently, effective treatment and safe prevention options for Ebola virus disease are not available. Therefore, there is an urgent need to develop control measures to prevent or limit future Ebola virus outbreaks. Ebola virus protein-based virus-like particle (VLP) and inactivated whole virion vaccines have demonstrated efficacy in animal models, and the addition of appropriate adjuvants may provide additional benefits to these vaccines, including enhanced immune responses. In this study, we screened 24 compounds from injectable excipients approved for human use in Japan and identified six compounds that significantly enhanced the humoral response to Ebola VLP vaccine in a murine model. Our novel adjuvant candidates for Ebola VLP vaccine have already been demonstrated to be safe when administered intramuscularly or subcutaneously, and therefore, they are closer to clinical trials than adjuvants whose safety profiles are unknown.

Viral neuraminidase inhibitors show limited efficacy in mice infected with H7N9 influenza A viruses isolated from humans. Although baloxavir marboxil protected mice from lethal challenge infection with a low pathogenic avian influenza H7N9 virus isolated from a human, its efficacy in mice infected with a recent highly pathogenic version of H7N9 human isolates is unknown. Here, we examined the efficacy of baloxavir marboxil in mice infected with a highly pathogenic human H7N9 virus, A/Guangdong/17SF003/2016. Treatment of infected mice with a single 1.5 mg/kg dose of baloxavir marboxil protected mice from the highly pathogenic human H7N9 virus infection as effectively as oseltamivir treatment at 50 mg/kg twice a day for five days. Daily treatment for five days at 15 or 50 mg/kg of baloxavir marboxil showed superior therapeutic efficacy, largely preventing virus replication in respiratory organs. These results indicate that baloxavir marboxil is a valuable candidate treatment for human patients suffering from highly pathogenic H7N9 virus infection.

Human influenza A(H2N2) viruses emerged in 1957 and were replaced by A(H3N2) viruses in 1968. The antigenicity of human H2N2 viruses has been tested by using ferret antisera or mouse and human monoclonal antibodies. Here, we examined the antigenicity of human H2N2 viruses by using human plasma samples obtained from 50 aged individuals who were born between 1928 and 1933 and from 33 younger adult individuals who were born after 1962. The aged individuals possessed higher neutralization titers against H2N2 viruses isolated in 1957 and 1963 than those against H2N2 viruses isolated in 1968, whereas the younger adults who were born between 1962 and 1968 possessed higher neutralization titers against H2N2 viruses isolated in 1963 than those against other H2N2 viruses. Antigenic cartography revealed the antigenic changes that occurred in human H2N2 viruses during circulation in humans for 11 years, as detected by ferret antisera. These results show that even though aged individuals were likely exposed to more recent H2N2 viruses that are antigenically distinct from the earlier H2N2 viruses, they did not possess high neutralizing antibody titers to the more recent viruses, suggesting immunological imprinting of these individuals with the first H2N2 viruses they encountered and that this immunological imprinting lasts for over 50 years.

Influenza A virus exploits multiple host proteins during infection. To define the virus–host interactome, our group conducted a proteomics-based screen and identified 299 genes that contributed to virus replication and 24 genes that were antiviral. Of these genes, we focused on the role during virus replication of eukaryotic elongation factor 1 gamma (eEF1G), which is a subunit of the eukaryotic elongation factor-1 complex and known to be a pro-viral host protein. Using the CRISPR/Cas9 system, we obtained two clones that were defective in eEF1G expression. In both of these clones, A/WSN/33 (H1N1) virus growth and protein expression were significantly suppressed, but viral mRNA, vRNA, and cRNA expression were not reduced. However, the replication and protein expression of A/California/04/2009 (H1N1pdm) virus in both clones were similar to those in parental cells. We found that the PB2 and PA proteins of WSN virus were responsible for the eEF1G-dependent replication. Our data show that eEF1G plays a role in the translation of virus proteins in a strain-specific manner. Additional analyses may be needed to further understand the role of strain-specific host proteins during virus replication.

Since the spring of 2013, human infections with H7N9 viruses have been detected in China. Some of these viruses have become highly pathogenic. Highly and low pathogenic avian influenza H7N9 viruses are currently co-circulating with the seasonal influenza A viruses H3N2 and H1N1pdm09. Prompt identification and isolation of H7N9 patients is one measure to prevent the spread of H7N9 virus and help prevent a pandemic. The majority of commercially available point-of-care rapid influenza diagnostic kits can differentiate between influenza A and B viruses, but cannot distinguish between H7N9 viruses and seasonal influenza A viruses. Accordingly, we have developed a rapid diagnostic kit specific for the H7 subtype that is accessible, easy to use. Although the detection limit of this H7 kit is one-tenth lower than that of a commercially available rapid influenza A and B diagnostic kit of similar design, except for the specificity of the monoclonal antibodies used, this kit is highly specific, detecting only H7-subtype influenza viruses, including the recent highly pathogenic H7N9 viruses from humans, and does not show any non-specific reactions with other HA subtypes. This H7 kit will be of value for the early detection of H7N9-infected patients.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease (HFMD). Occasionally, EV71 infection is associated with severe neurological diseases, such as acute encephalitis, acute flaccid paralysis and cardiopulmonary failure. Several molecules act as cell surface receptors that stimulate EV71 infection, including scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), sialylated glycan, heparan sulfate and annexin II (Anx2). SCARB2 plays critical roles in attachment, viral entry and uncoating, and it can facilitate efficient EV71 infection. The three-dimensional structures of the mature EV71 virion, procapsid and empty capsid, as well as the exofacial domain of SCARB2, have been elucidated. This structural information has greatly increased our understanding of the early steps of EV71 infection. Furthermore, SCARB2 plays essential roles in the development of EV71 neurological disease in vivo. Adult mice are not susceptible to infection by EV71, but transgenic mice that express human SCARB2 become susceptible to EV71 infection and develop similar neurological diseases to those found in humans. This mouse model facilitates the in vivo investigation of many issues related to EV71. PSGL-1, sialylated glycan, heparan sulfate and Anx2 are attachment receptors, which enhance viral infection by retaining the virus on the cell surface. These molecules also contribute to viral infection in vitro either by interacting with SCARB2 or independently of SCARB2. However, the cooperative effects of these receptors, and their contribution to EV71 pathogenicity in vivo, remain to be elucidated.

Highly pathogenic avian H5 influenza viruses persist among poultry and wild birds throughout the world. They sometimes cause interspecies transmission between avian and mammalian hosts. H5 viruses possessing the HA of subclade 2.3.4.4, 2.3.2.1, 2.2.1, or 7.2 were detected between 2015 and 2018. To understand the neutralizing epitopes of H5-HA, we characterized 15 human monoclonal antibodies (mAbs) against the HA of H5 viruses, which were obtained from volunteers who received the H5N1 vaccine that contains a subclade 2.2.1 or 2.1.3.2 virus as an antigen. Twelve mAbs were specific for the HA of subclade 2.2.1, two mAbs were specific for the HA of subclade 2.1.3.2, and one mAb was specific for the HA of both. Of the 15 mAbs analyzed, nine, which were specific for the HA of subclade 2.2.1, and shared the VH and VL genes, possessed hemagglutination inhibition and neutralizing activities, whereas the others did not. A single amino acid substitution or insertion at positions 144–147 in antigenic site A conferred resistance against these nine mAbs to the subclade 2.2.1 viruses. The amino acids at positions 144–147 are highly conserved among subclade 2.2.1, but differ from those of other subclades. These results show that the neutralizing epitope including amino acids at positions 144–147 is targeted by human antibodies, and plays a role in the antigenic difference between subclade 2.2.1 and other subclades.

Many cases of human infection with the H7N9 virus have been detected in China since 2013. H7N9 viruses are maintained in chickens and are transmitted to humans at live bird markets. During circulation in birds, H7N9 viruses have accumulated amino acid substitutions in their hemagglutinin (HA), which resulted in an antigenically change in the recent H7N9 viruses. Here, we characterized 46 mouse monoclonal antibodies against the HA of the prototype strain. 16 H7-HA-specific monoclonal antibodies (mAbs) possessed hemagglutination inhibition (HI) and neutralization activities by recognizing the major antigenic site A; four other H7-HA-specific clones also showed HI and neutralizing activities via recognition of the major antigenic sites A and D; seven mAbs that reacted with several HA subtypes and possibly recognized the HA stem partially protected mice from lethal infection with prototype H7N9 virus; and the remaining 19 mAbs had neither HI nor neutralization activity. All human H7N9 viruses tested showed a similar neutralization sensitivity to the first group of 16 mAbs, whereas human H7N9 viruses isolated in 2016⁻2017 were not neutralized by a second group of 4 mAbs. These results suggest that amino acid substitutions at the epitope of the second mAb group appear to be involved in the antigenic drift of the H7N9 viruses. Further analysis is required to fully understand the antigenic change in H7N9 viruses.