Convergent evolution of the SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

In late 2022, although the SARS-CoV-2 Omicron subvariants have highly diversified, some lineages have convergently acquired amino acid substitutions at five critical residues in the spike protein. Here, we illuminated the evolutionary rules underlying the convergent evolution of Omicron subvariants and the properties of one of the latest lineages of concern, BQ.1.1. Our phylogenetic and epidemic dynamics analyses suggest that Omicron subvariants independently increased their viral fitness by acquiring the convergent substitutions. Particularly, BQ.1.1, which harbors all five convergent substitutions, shows the highest fitness among the viruses investigated. Neutralization assays show that BQ.1.1 is more resistant to breakthrough BA.2/5 infection sera than BA.5. The BQ.1.1 spike exhibits enhanced binding affinity to human ACE2 receptor and greater fusogenicity than the BA.5 spike. However, the pathogenicity of BQ.1.1 in hamsters is comparable to or even lower than that of BA.5. Our multiscale investigations provide insights into the evolutionary trajectory of Omicron subvariants.

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75

SARS-CoV-2 Omicron BA.2.75 emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically different from BA.5, the currently predominant BA.2 descendant. Here, we showed that the effective reproduction number of BA.2.75 is greater than that of BA.5. While the sensitivity of BA.2.75 to vaccination- and BA.1/2 breakthrough infection-induced humoral immunity was comparable to that of BA.2, the immunogenicity of BA.2.75 was different from that of BA.2 and BA.5. Three clinically-available antiviral drugs were effective against BA.2.75. BA.2.75 spike exhibited a profound higher affinity to human ACE2 than BA.2 and BA.5 spikes. The fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were comparable to those of BA.5 but were greater than those of BA.2. Our multiscale investigations suggest that BA.2.75 acquired virological properties independently of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

Unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants imposes us to continuous control measurement. Given the rapid spread, new Omicron subvariant named BA.5 is urgently required for characterization. Here we analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1 comprehensively. Although in vitro growth kinetics of BA.5 is comparable among the Omicron subvariants, BA.5 become much more fusogenic than BA.1 and BA.2. The airway-on-a-chip analysis showed that the ability of BA.5 to disrupt the respiratory epithelial and endothelial barriers is enhanced among Omicron subvariants. Furthermore, in our hamster model, in vivo replication of BA.5 is comparable with that of the other Omicrons and less than that of the ancestral B.1.1. Importantly, inflammatory response against BA.5 is strong compared with BA.1 and BA.2. Our data suggest that BA.5 is still low pathogenic compared to ancestral strain but evolved to induce enhanced inflammation when compared to prior Omicron subvariants.

Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-{beta} but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-{beta}. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

Antiviral face mask functionalized with solidified hand soap: low-cost infection prevention clothing against enveloped viruses such as SARS-CoV-2

Infection prevention clothing is becoming an essential protective tool in the current pandemic, especially because now we know that SARS-CoV-2 can easily infect humans in poorly ventilated indoor spaces. However, commercial infection prevention clothing is made of fabrics that are not capable of inactivating the virus. Therefore, viral infections of symptomatic and asymptomatic individuals wearing protective clothing such as masks can occur through aerosol transmission or by contact with the contaminated surfaces of the masks, which are suspected as an increasing source of highly infectious biological waste. Herein, we report an easy fabrication method of a novel antiviral non-woven fabric containing polymer filaments that were coated with solidified hand soap. This extra protective fabric is capable of inactivating enveloped viruses such as SARS-CoV-2 and phi 6 in one minute of contact. In this study, this antiviral fabric was used to fabricate an antiviral face mask and did not show any cytotoxic effect in human keratinocyte HaCaT cells. Furthermore, this antiviral non-woven fabric could be used for the fabrication of other infection prevention clothing such as caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons, and shoe covers. Therefore, this low-cost technology could provide a wide range of infection protective tools to combat COVID-19 and future pandemics in developed and underdeveloped countries.

Next generation infection prevention clothing: Non-woven Fabrics Coated with Cranberry Extracts Capable of Inactivating Enveloped Viruses such as SARS-CoV-2 and Multidrug-resistant Bacteria

The Coronavirus Disease (COVID-19) pandemic is demanding rapid action of the authorities and scientific community in order to find new antimicrobial solutions that could inactivate the pathogen SARS-CoV-2 that causes this disease. Gram-positive bacteria contribute to severe pneumonia associated with COVID-19, and their resistance to antibiotics is increasing at an alarming rate. In this regard, non-woven fabrics are currently used for the fabrication of infection prevention clothing such as face masks, caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons and shoe covers as protective tools against viral and bacterial infections. However, these non-woven fabrics are made of materials that do not possess antimicrobial activity. Thus, we have developed here non-woven fabrics with antimicrobial coatings of cranberry extracts capable of inactivating enveloped viruses such as SARS-CoV-2 and the phage phi 6, and two multidrug-resistant bacteria: the methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. The non-toxicity of these advanced technology was ensured using a Caenorhabditis elegans in vivo model. These results open up a new prevention path using natural and biodegradable compounds for the fabrication of infection prevention clothing in the current COVID-19 and future pandemics.

An Issue of Concern: Unique Truncated ORF8 Protein Variants of SARS-CoV-2

Open reading frame 8 (ORF8) protein is one of the most evolving accessory proteins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits presentation of viral antigens by the major histocompatibility complex class I (MHC-I) and interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 to evade immunity and replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein defines the B.1.1.7 lineage of SARS-CoV-2, which is engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) due to the Q27STOP mutations were identified among 49055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents which includes Africa, Asia, Europe and South America. Based on various quantitative features such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, a collection of nine possible T-ORF8 unique variants were defined. The question of whether T-ORF8 variants work similarly to ORF8 has yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.

Implications Derived from S-Protein Variants of SARS-CoV-2 from Six Continents

Spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical determinants of the infectivity and antigenicity of the virus. Several mutations in the spike protein of SARS-CoV-2 have already been detected, and their effect in immune system evasion and enhanced transmission as a cause of increased morbidity and mortality are being investigated. From pathogenic and epidemiological perspectives, spike proteins are of prime interest to researchers. This study focused on the unique variants of S proteins from six continents Asia, Africa, Europe, Oceania, South America, and North America. In comparison to the other five continents, Africa (29.065%) had the highest percentage of unique S proteins. Notably, only North America had 87% (14046) of the total (16143) specific S proteins available in the NCBI database(across all continents). Based on the amino acid frequency distributions in the S protein variants from all the continents, the phylogenetic relationship implies that unique S proteins from North America were significantly different from those of the other five continents. Overtime, the unique variants originating from North America are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. Hence it is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of COVID-19 pandemic. It is also further suggested that the efficacy of existing vaccines and future vaccine development must be reviewed with careful scrutiny, and if needed, further re-engineered based on requirements dictated by new emerging S protein variants.

Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells

Genetic differences are a primary reason for differences in the susceptibility and severity of coronavirus disease 2019 (COVID-19). Because induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Notably, undifferentiated human iPS cells themselves cannot be infected bySARS-CoV-2. Using adenovirus vectors, here we found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected with SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, the budding of viral particles, the production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We also evaluated COVID-19 therapeutic drugs in ACE2-iPS cells and confirmed the strong antiviral effects of Remdesivir, EIDD-2801, and interferon-beta. In addition, we performed SARS-CoV-2 infection experiments on ACE2-iPS/ES cells from 8 individuals. Male iPS/ES cells were more capable of producing the virus as compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro, but they are also a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/432218v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@165ec06org.highwire.dtl.DTLVardef@6a9d67org.highwire.dtl.DTLVardef@1840dd3org.highwire.dtl.DTLVardef@a7a4bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Protective face mask filter capable of inactivating SARS-CoV-2, and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating neither SARS-CoV-2 nor multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2 mediated pneumonia disease complex and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a novel protective non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating SARS-CoV-2 in one minute of contact, and the life-threatening methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many health care workers and researchers working in this urgent and challenging field.

Variability of Accessory Proteins Rules the SARS-CoV-2 Pathogenicity

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) which is pandemic with an estimated fatality rate less than 1% is ongoing. SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 with putative functions to manipulate host immune mechanisms such as interferons, immune signaling receptor NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) inflammasome, inflammatory cytokines such as interleukin 1{beta} (IL-1{beta}) are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins of all complete proteomes (available as of October 26, 2020, in the National Center for Biotechnology Information depository) of SARS-CoV-2, were observed across six continents. Across all continents, the decreasing order of percentage of unique variations in the accessory proteins was found to be ORF3a>ORF8>ORF7a>ORF6>ORF10>ORF7b. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. This finding suggests that the wide variations of accessory proteins seem to govern the pathogenicity of SARS-CoV-2, and consequently, certain propositions and recommendations can be made in the public interest.

Possible transmission flow of SARS-CoV-2 based on ACE2 features

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Generation of human bronchial organoids for SARS-CoV-2 research

Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that can reproduce the viral life cycle and evaluate the drug efficacy of anti-viral drugs is essential. In this study, we established a method to generate human bronchial organoids (hBO) from commercially available cryopreserved human bronchial epithelial cells and examined whether they could be used as a model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research. Our hBO contain basal, club, ciliated, and goblet cells. Angiotensin-converting enzyme 2 (ACE2), which is a receptor for SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an essential serine protease for priming spike (S) protein of SARS-CoV-2, were highly expressed. After SARS-CoV-2 infection, not only the intracellular viral genome, but also progeny virus, cytotoxicity, pyknotic cells, and moderate increases of the type I interferon signal could be observed. Treatment with camostat, an inhibitor of TMPRSS2, reduced the viral copy number to 2% of the control group. Furthermore, the gene expression profile in SARS-CoV-2-infected hBO was obtained by performing RNA-seq analysis. In conclusion, we succeeded in generating hBO that can be used for SARS-CoV-2 research and COVID-19 drug discovery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/115600v2_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@13a6908org.highwire.dtl.DTLVardef@1c59300org.highwire.dtl.DTLVardef@362167org.highwire.dtl.DTLVardef@1cb31ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cough related to swallowing in asthma patients

BACKGROUND: Occurrence of cough during swallowing is common among asthma patients, but has not been investigated in detail. OBJECTIVE: We conducted an observational study to determine the prevalence of swallowing-related cough (SRC) and its characteristics in asthma patients. METHODS: Asthma patients attending our outpatient department between May 2005 and April 2007 were interviewed to investigate if they had ever experienced SRC, as well as postnasal drip or heartburn and cough related to these conditions. RESULTS: Among 417 patients who completed the questionnaire, 121 patients (29.0%) had experienced SRC. Spicy and sour foods were the most frequent tussigenic foods, causing cough in 76.0% and 53.7% of the 121 patients, respectively. In patients without SRC, the prevalence rates of postnasal drip and postnasal drip-induced cough were 35.8% (106 of 296) and 7.8% (23 of 296), respectively. The corresponding prevalence rates in patients with SRC were 50.4% (61 of 121) and 37.2% (45 of 121), which were both significantly higher than in patients without cough (p = 0.006 and p < 0.001 respectively). In patients without SRC, the prevalence rates of heartburn and heartburn-induced cough were 22.2% (66 of 296) and 2.4% (7 of 296), respectively. The corresponding prevalence rates in patients with SRC were 45.5% (55 of 121) and 16.5% (20 of 121), with both being significantly higher than in patients without cough (p = 0.002 and p < 0.001, respectively). CONCLUSION: SRC was frequent in asthma patients, and was closely related to postnasal drip and heartburn. Irritable larynx is one of the possible underlying mechanisms of SRC. This study was registered with the University Hospital Medical Information Network clinical trials registry (registration number: UMIN000017426).