Corrigendum: ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

[This corrects the article DOI: 10.3389/fcell.2023.1290876.].

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.

Complete genome constellations of two bovine rotavirus A strains isolated in Japan reveal a unique T9 NSP3 genotype.

Full genome sequencing of two bovine rotavirus A (RVA) strains isolated in Japan in 2019 revealed two genotype constellations; one had a constellation of G8-P[1]-I2-R2-C2-M2-A3-N2-T9-E2-H3. Thereupon, genotype T9 carried by RVA/Bovine-tc/JPN/AH1041/2022/G8P[1], constitutes a rare NSP3 genotype, and only two unusual Japanese bovine RVA strains have thus far been reported to carry this genotype. The other RVA/Bovine-tc/JPN/AH1207/2022/G6P[5] strain possessed a constellation of G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Phylogenetic analyses indicate that the majority of gene segments were most closely related to Japanese bovine RVAs, suggesting that both strains might have derived through multiple reassortment events from RVA strains circulating within Japanese cattle. The emergence of RVA strains in Japan and their reassortment with locally circulating atypical RVAs could have implications for current vaccination strategies.

iPSC-derived mesenchymal cells that support alveolar organoid development.

Mesenchymal cells are necessary for organ development. In the lung, distal tip fibroblasts contribute to alveolar and airway epithelial cell differentiation and homeostasis. Here, we report a method for generating human induced pluripotent stem cell (iPSC)-derived mesenchymal cells (iMESs) that can induce human iPSC-derived alveolar and airway epithelial lineages in organoids via epithelial-mesenchymal interaction, without the use of allogenic fetal lung fibroblasts. Through a transcriptome comparison of dermal and lung fibroblasts with their corresponding reprogrammed iPSC-derived iMESs, we found that iMESs had features of lung mesenchyme with the potential to induce alveolar type 2 (AT2) cells. Particularly, RSPO2 and RSPO3 expressed in iMESs directly contributed to AT2 cell induction during organoid formation. We demonstrated that the total iPSC-derived alveolar organoids were useful for characterizing responses to the influenza A (H1N1) virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demonstrating their utility for disease modeling.

MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells.

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.

Complete genome sequencing and comparative plasmid analysis of KPC-2-producing Klebsiella pneumoniae isolated from hospital sewage water in Japan.

OBJECTIVES: The Klebsiella pneumoniae carbapenemase (blaKPC) gene is one of the most widespread carbapenemase genes in the world. However, there are few reports on KPC-producing bacteria in Japan. The aim of this study was therefore to investigate KPC-producing K. pneumoniae in Japan. METHODS: A KPC-2-producingK. pneumoniae strain (KAM260) was isolated from hospital sewage water in Japan in 2018. The complete genome was determined by whole-genome sequencing. Subsequent comparative sequence analysis of the blaKPC-2-carrying plasmid was performed. RESULTS: Klebsiella pneumoniae KAM260, belonging to sequence type 3026 (ST3026), harboured the blaKPC-2 gene in 114.6-kbp plasmid pKAM260_2 with IncFIB(pQIL) and IncFII(K) replicons. pKAM260_2 was highly identical to pKpQIL-like plasmids, which carry blaKPC genes and have spread worldwide. pKAM260_2 had functional conjugation-associated genes and was transferable to Escherichia coli. CONCLUSION: pKAM260_2, the self-transmissible plasmid carrying theblaKPC-2 gene, was detected from hospital sewage water in Japan and was characterised as a pKpQIL-like plasmid. This plasmid needs to be monitored in Japan in the future owing to its high diffusivity.

Bactericidal efficacies of food additive grade calcium hydroxide toward Legionella pneumophila.

Food additive grade calcium hydroxide (FdCa(OH)2) in the solution of 0.17% was evaluated for its bactericidal efficacies toward Legionella pneumophila with or without sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine, at room temperature (RT) (25°C ± 2°C) and 42°C, either with or without 5% fetal bovine serum (FBS). Besides, FdCa(OH)2 in different concentration solutions were prepared in field water samples (hot spring and bath tab water) and evaluated for their bactericidal efficacies at 42°C. FdCa(OH)2 (0.17%) inactivated the L. pneumophila to the undetectable level (<2.6 log CFU/ml) within 5 min and 3 min, respectively, at RT and 42°C, with 5% FBS. At RT and 42°C, NaOCl inactivated L. pneumophila to the undetectable level within 5 min, without 5% FBS, but with 5% FBS, it could only inactivate this bacterium effectively (≥3 log reductions). Conversely, at RT and 42°C, the mixture of 0.17% FdCa(OH)2 and 200 ppm NaOCl could inactivate L. pneumophila to the undetectable level, respectively, within 3 min and 1 min, even with 5% FBS, and it was elucidated that FdCa(OH)2 has a synergistic bactericidal effect together with NaOCl. FdCa(OH)2 0.05% solution prepared in hot spring water could inactivate L. pneumophila to the undetectable within 3 min at 42°C. So, FdCa(OH)2 alone could show nice bactericidal efficacy at 42°C, even with 5% FBS, as well as in field water samples.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

Virucidal efficacy of food additive grade calcium hydroxide against surrogate of human norovirus.

An alkaline agent, namely, food additive grade calcium hydroxide (FdCa(OH)2) in the solution, powder and suspension forms was evaluated as a virucidal agent, using a murine norovirus (MNV) as the surrogate for human norovirus. The main constituent of FdCa(OH)2 is Ca(OH)2, which has pH 13 in 0.17% solution. The results showed that 0.17% FdCa(OH)2 solution could inactivate MNV within 30s even in the presence of organic materials (5% fetal bovine serum (FBS)). In a contaminated surface experiment, MNV with 5% FBS was inoculated on rayon sheets, and the result showed FdCa(OH)2 solution could markedly reduce virus titer within 1min. When mouse feces were spiked with MNV and FdCa(OH)2 powder as 10% and 20% w/w was added to the feces, these concentrations could inactivate the virus within 30min and 15min, respectively. Whereas, FdCa(OH)2 suspension at 2.5% and 5% could inactivate the virus within 30min and at 1% within 45min. These and additional results obtained here indicate that FdCa(OH)2 is an effective virucidal agent against MNV, and can serve as a useful alternative disinfectant for inactivation and prevention of human norovirus in house and hospital.