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1.
PLoS Pathog ; 18(6): e1010553, 2022 06.
Article in English | MEDLINE | ID: covidwho-1951567

ABSTRACT

Nelson Bay orthoreovirus (NBV), a member of the family Reoviridae, genus Orthoreovirus, is a bat-borne virus that causes respiratory diseases in humans. NBV encodes two unique nonstructural proteins, fusion-associated small transmembrane (FAST) protein and p17 protein, in the S1 gene segment. FAST induces cell-cell fusion between infected cells and neighboring cells and the fusogenic activity is required for efficient viral replication. However, the function of p17 in the virus cycle is not fully understood. Here, various p17 mutant viruses including p17-deficient viruses were generated by a reverse genetics system for NBV. The results demonstrated that p17 is not essential for viral replication and does not play an important role in viral pathogenesis. On the other hand, NBV p17 regulated viral replication in a bat cell line but not in other human and animal cell lines. Nuclear localization of p17 is associated with the regulation of NBV replication in bat cells. We also found that p17 dramatically enhances the cell-cell fusion activity of NBV FAST protein for efficient replication in bat cells. Furthermore, we found that a protein homologue of NBV p17 from another bat-borne orthoreovirus, but not those of avian orthoreovirus or baboon orthoreovirus, also supported efficient viral replication in bat cells using a p17-deficient virus-based complementation approach. These results provide critical insights into the functioning of the unique replication machinery of bat-borne viruses in their natural hosts.


Subject(s)
Chiroptera , Orthoreovirus , Reoviridae , Animals , Antibodies, Viral , DNA Viruses , Orthoreovirus/genetics , Virus Replication
2.
Commun Biol ; 5(1): 516, 2022 05 30.
Article in English | MEDLINE | ID: covidwho-1947507

ABSTRACT

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.


Subject(s)
COVID-19 , SARS-CoV-2 , Bronchi , Humans , Organoids
3.
J Infect Chemother ; 28(9): 1347-1351, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1867378

ABSTRACT

INTRODUCTION: Norovirus (NoV) is the most common agent causing outbreaks and sporadic cases of acute gastroenteritis among all ages, especially children under 5 years old. During the coronavirus disease 2019 (COVID-19) pandemic, NoV infection has decreased drastically in Japan due to school closures and no outbreak related to NoV infection had been reported. METHOD: In mid-September 2021, NoV outbreak occurred in kindergarten and nursery schools in Maizuru, Kyoto prefecture, Japan. Twenty-six stool samples collected from patients who were diagnosed of NoV gastroenteritis from the outbreak by an immunochromatographic (IC) kit at a pediatric outpatient clinic in Maizuru city during 3 weeks from September 13 to October 8, 2021 were examined for the presence of NoV GII by reverse transcriptase-polymerase chain reaction (RT-PCR), genome sequencing, and phylogenetic analysis. RESULT: All 26 samples were confirmed positive to NoV GII and their genotypes were identified as GII.4 Sydney[P31]. The amino acid substitutions in open reading frame1 (ORF1) and ORF2 genes were found when compared with previously detected sporadic NoV GII.4 Sydney[P31] strains isolated in Japan. The clinical characterization of infected children was described. Most of the children were mild cases and vomiting was the most frequent clinical symptom. CONCLUSION: This study reported a recent emergence of NoV GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan during the COVID-19 pandemic and suggests a need for further monitoring of NoV GII.4 variants.


Subject(s)
COVID-19 , Caliciviridae Infections , Gastroenteritis , Norovirus , COVID-19/epidemiology , Caliciviridae Infections/epidemiology , Child , Child, Preschool , Feces , Gastroenteritis/epidemiology , Genotype , Humans , Japan/epidemiology , Norovirus/genetics , Pandemics , Phylogeny
4.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-333006

ABSTRACT

Understanding the T-cell responses involved in inhibiting COVID-19 severity is crucial for developing new therapeutic and vaccine strategies. Here, we characterized SARS-CoV-2 spike-specific CD8+ T cells interacting with overlapping peptides on peripheral blood mononuclear cells from acute-phase COVID-19 patients. Relative to severe COVID-19, patients with mild COVID-19 had more frequent antigen-specific CD8+ T cells, and significantly increased SARS-CoV-2 spike-specific CD8+ T cells simultaneously expressing granzyme A, granzyme B, and perforin, suggesting that inducing highly cytotoxic CD8+ T cells during early infection suppresses COVID-19 severity. The BNT162b2 mRNA vaccine induced these antigen-specific CD8+ T cells in healthy donors, although lesser than in infected patients, and the induced subpopulation was not maintained long-term after second vaccination. Importantly, these CD8+ T cells showed cross-reactivity with the Delta and Omicron strains of SARS-CoV-2. Incorporating factors that efficiently induce CD8+ T cells with polyfunctional cytotoxic activity may improve future vaccine efficacy against such variants.

5.
Research Square ; 2022.
Article in English | EuropePMC | ID: covidwho-1786459

ABSTRACT

Understanding the T-cell responses involved in inhibiting COVID-19 severity is crucial for developing new therapeutic and vaccine strategies. Here, we characterized SARS-CoV-2 spike-specific CD8+ T cells interacting with overlapping peptides on peripheral blood mononuclear cells from acute-phase COVID-19 patients. Relative to severe COVID-19, patients with mild COVID-19 had more frequent antigen-specific CD8+ T cells, and significantly increased SARS-CoV-2 spike-specific CD8+ T cells simultaneously expressing granzyme A, granzyme B, and perforin, suggesting that inducing highly cytotoxic CD8+ T cells during early infection suppresses COVID-19 severity. The BNT162b2 mRNA vaccine induced these antigen-specific CD8+ T cells in healthy donors, although lesser than in infected patients, and the induced subpopulation was not maintained long-term after second vaccination. Importantly, these CD8+ T cells showed cross-reactivity with the Delta and Omicron strains of SARS-CoV-2. Incorporating factors that efficiently induce CD8+ T cells with polyfunctional cytotoxic activity may improve future vaccine efficacy against such variants.

6.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-322534

ABSTRACT

High-throughput, high-accuracy detection of emerging viruses allows for pandemic prevention and control. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is used to diagnose the presence of SARS-CoV-2. The principle of the test is to detect RNA in the virus using a pair of primers that specifically binds to the base sequence of the viral RNA. However, RT-PCR is a sophisticated technique requiring a time-consuming pretreatment procedure for extracting viral RNA from clinical specimens and to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity using artificial intelligent nanopores utilizing a simple procedure that does not require RNA extraction. Artificial intelligent nanopore platform consists of machine learning software on the servers, portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. Here we show that the artificial intelligent nanopores are successful in accurate identification of four types of coronaviruses, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2, which are usually extremely difficult to detect. The positive/negative diagnostics of the new coronavirus is achieved with a sensitivity of 95 % and specificity of 92 % with a 5-minute diagnosis. The platform enables high throughput diagnostics with low false negatives for the novel coronavirus.

7.
Sci Rep ; 12(1): 1060, 2022 01 20.
Article in English | MEDLINE | ID: covidwho-1639278

ABSTRACT

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.


Subject(s)
Coronavirus Nucleocapsid Proteins , Gold/chemistry , Metal Nanoparticles/chemistry , SARS-CoV-2/chemistry , Surface Plasmon Resonance , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/chemistry , Phosphoproteins/analysis , Phosphoproteins/chemistry
8.
Nat Commun ; 12(1): 3726, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1275922

ABSTRACT

High-throughput, high-accuracy detection of emerging viruses allows for the control of disease outbreaks. Currently, reverse transcription-polymerase chain reaction (RT-PCR) is currently the most-widely used technology to diagnose the presence of SARS-CoV-2. However, RT-PCR requires the extraction of viral RNA from clinical specimens to obtain high sensitivity. Here, we report a method for detecting novel coronaviruses with high sensitivity by using nanopores together with artificial intelligence, a relatively simple procedure that does not require RNA extraction. Our final platform, which we call the artificially intelligent nanopore, consists of machine learning software on a server, a portable high-speed and high-precision current measuring instrument, and scalable, cost-effective semiconducting nanopore modules. We show that artificially intelligent nanopores are successful in accurately identifying four types of coronaviruses similar in size, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2. Detection of SARS-CoV-2 in saliva specimen is achieved with a sensitivity of 90% and specificity of 96% with a 5-minute measurement.


Subject(s)
Artificial Intelligence , COVID-19 Nucleic Acid Testing/methods , Machine Learning , Nanopores , COVID-19 Nucleic Acid Testing/instrumentation , Coronavirus 229E, Human/genetics , Equipment Design/economics , Humans , Limit of Detection , Middle East Respiratory Syndrome Coronavirus/genetics , Nanoparticles/chemistry , Polymerase Chain Reaction , SARS-CoV-2/genetics , Saliva/virology , Sensitivity and Specificity , Software
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