atcc vr  (ATCC)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Schematic representation of the SARS-CoV-2 genome and crRNA design (A) The SARS-CoV-2 genome is a single-stranded, positive-sense RNA. Its first two-thirds comprise overlapping open reading frames, ORF1a and ORF1b, which are translated into 16 nonstructural proteins (nsp1–nsp16). The remaining genome encodes structural proteins—spike (S), envelope (E), membrane (M), and nucleocapsid (N)—along with 8 accessory proteins: ORF3a, 3c, 6, 7a, 7b, 8, and 9b. (B) crRNA design. A highly conserved 26-nucleotide target region was identified within the viral genome of human coronaviruses. Based on this, four overlapping 23-nucleotides crRNAs were designed indicated in red. (C) The designed crRNAs were aligned to the genomes of all seven human coronavirus species. Mismatches relative to the SARS-CoV-2 reference sequence are highlighted in red. Each number on the right represents a single mismatch, with the value indicating its position within the target sequence. The Cas13d seed region, critical for target recognition and cleavage, is marked with a green rectangle.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Membrane, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Broad antiviral CRISPR-Cas13d-crRNA activity against human coronaviruses genome (A) Schematic diagrams illustrating the luciferase reporter system and the CRISPR-Cas13d expression cassette. The luciferase reporter gene is driven by the Pol II SV40 promoter, with the target sequence cloned downstream of the luciferase gene. The CRISPR-Cas13d expression cassette includes a U6 promoter for crRNA expression and an EF1α core promoter driving the expression of Cas13d and GFP. (B) Cas13d/crRNA construct was used to assess the inhibition of luciferase expression by various crRNAs. The tested crRNAs included those targeting the nsp12 genome (crRNA1-4) and a positive control Luc crRNA are shown relative to the Ctrl. Numbers above the bars indicate mismatch positions, with mismatches within the Cas13d seed region highlighted in green. Each number corresponds to a single mismatch. The data represent mean values (±SD) from three independent experiments each performed in duplicate. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. Significant differences are indicated (∗∗∗∗ p < 0.0001).

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: CRISPR, Activity Assay, Luciferase, Expressing, Sequencing, Clone Assay, Construct, Inhibition, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Cas13d broadly inhibits the replication of human coronavirus replicons (A) Genomic diagrams of the replicons for SARS-CoV-2, SARS-CoV, and MERS-CoV. The replicons contain the 5′ and 3′ cis -acting signals required for viral replication, the large ORFs 1a and 1b encoding the replicase non-structural proteins (nsps), and the N gene, which is essential for efficient coronavirus RNA synthesis. The SARS-CoV-2 and SARS-CoV replicons, generated by in vitro ligation, also include a mNeonGreen (mNG) or GFP gene, respectively, positioned downstream of ORF1b and regulated by the S gene transcription regulatory sequence (TRS-M). (B) Inhibition of human coronavirus replicons by different crRNAs. Subgenomic mRNA (sgmRNA) levels were measured by RT-qPCR 2 days after transfection. A non-targeting crRNA (Ctrl) was included as a negative control. The horizontal axis shows the tested crRNAs, and the vertical axis shows relative viral RNA levels normalized to Ctrl. Data are presented as mean values (±SD) from three independent experiments each performed in duplicate. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test, with significance levels indicated as ∗∗∗∗ p < 0.0001.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Generated, In Vitro, Ligation, Sequencing, Inhibition, Quantitative RT-PCR, Transfection, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Cas13d inhibits the replication of various human coronavirus species when applied prior and post-infection (A) Relative subgenomic mRNA (sgmRNA) levels at 2 days post-infection are shown as the ratio of viral RNA measured in the presence of different crRNAs relative to the control (Ctrl). (B) The antiviral effects of 4 crRNAs were evaluated by quantifying genomic RNA (gRNA) in the supernatant every 24 h, up to the peak of infection, with each crRNA represented by a different color. Viral gRNA levels, including those treated with the control crRNA, were quantified by RT-qPCR. (C) Relative sgmRNA levels are shown for cells treated with different Cas13d/crRNAs. A non-targeting crRNA (Ctrl) is included as a negative control and set to 100%. The horizontal axis indicates the tested crRNAs, and the vertical axis shows relative viral RNA expression. Data represent mean ± SD from three independent experiments. Statistical significance is indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Infection, Control, Quantitative RT-PCR, Negative Control, RNA Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Cas13d-based SHERLOCK detection assay (A) Detection workflow. Infectious samples are collected, and RNA is extracted. The extracted RNA is reverse transcribed into DNA and then amplified using RPA. The amplified DNA is subsequently transcribed by T7 RNA polymerase to generate RNA targets. These RNA targets are recognized and cleaved by CRISPR-Cas13d guided by crRNA. Upon activation, Cas13d triggers collateral cleavage activity, degrading all surrounding RNAs, including fluorescent RNA reporters. Detection is performed using a lateral flow strip, where a single band indicates a negative result and two bands indicate a positive result. (B) Broad detection of human coronaviruses using Cas13d. Either crRNA2 or crRNA3 was used to broadly detect five human coronavirus species: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. C, control line; T, test line; NC, negative control. (C) Specificity testing against non-coronavirus respiratory viruses. Influenza A and Influenza B samples were tested under the same conditions used for SARS-CoV-2 detection. C, control line; T, test line; PC, positive control (SARS-CoV-2).

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Detection Assay, Reverse Transcription, Amplification, CRISPR, Activation Assay, Activity Assay, Stripping Membranes, Control, Negative Control, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Limit of detection of crRNA2 and crRNA3 for various human coronaviruses (A) Detection limit of crRNA2 and (B) crRNA3 for SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. CRISPR-Cas13d, guided by a crRNA, specifically recognizes and cleaves the target RNA. Upon activation, Cas13d exhibits collateral cleavage activity, degrading nearby RNAs, including fluorescent reporters. Detection is carried out on a lateral flow strip: one band indicates a negative result, while two bands indicate a positive result. The assay was performed with serial dilutions (10-fold) ranging from 100 RNA copies down to 1 RNA copy. C, control line; T, test line; NC, negative control.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: CRISPR, Activation Assay, Activity Assay, Stripping Membranes, Control, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Mismatches of Bat-CoV relative to SARS-CoV-2 genome Positions 15–21 of the crRNA correspond to the seed region (underlined in the SARS-CoV-2 reference sequence), which differs for each crRNA. Nucleotides conserved across all four crRNA seed regions are highlighted in green in the SARS-CoV-2 sequence, whereas mismatches present in Bat-CoV are shown in bold red. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; Bat-CoV, bat coronavirus.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Mismatches of other animal coronaviruses relative to SARS-CoV-2 genome Positions 15–21 of the crRNA correspond to the seed region (underlined in the SARS-CoV-2 reference sequence), which differs for each crRNA. Nucleotides conserved across all four crRNA seed regions are highlighted in green, whereas mismatches in the animal coronavirus sequences are shown in bold red. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; PRCV, porcine respiratory coronavirus; CCoV, canine coronavirus; PEDV, porcine epidemic diarrhea virus; SADS-CoV, swine acute diarrhea syndrome coronavirus; FCoV, feline coronavirus; FRCoV, ferret coronavirus; MHV, mouse hepatitis virus; RCoV, rat coronavirus; IBV, infectious bronchitis virus; PHEV, porcine hemagglutinating encephalomyelitis virus; CRCoV, canine respiratory coronavirus; BCoV, bovine coronavirus; ECoV, equine coronavirus; TCoV, turkey coronavirus; GCoV, goose coronavirus; DCoV, duck coronavirus; BWCoV, beluga whale coronavirus; WCoV, wigeon coronavirus; MHCoV, moorhen coronavirus; MCoV, munia coronavirus; THCoV, hrush coronavirus; PDCoV, porcine deltacoronavirus.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Sequencing, Virus

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): A549 clone 2E10 was treated with IFN⍺/IFNγ for 16h then infected as indicated. Infection was assessed 72 hpi by flow cytometry. (B): RNA-seq analysis heatmap depicting normalized expression of selected ISGs across four A549 SCCs (1B7, 1C10, 2E10, 3G7) after 16h mock/IFNα/IFNγ treatment. TPM; transcripts per million. (C): Venn diagram of overlap between differentially expressed genes (>4-fold change and padj < 0.05) following IFNα/IFNγ treatment. (D): Schematic of screen setup. (E): Screen results, where the x axis corresponds to each unique gene in the library, and the y axis denotes the relative fold change. Differentially represented genes in infected versus uninfected populations (p<0.01) are labeled in magenta, non-targeting controls are labeled in blue. (F): Flow cytometry of knockout cells treated with IFNα or IFNγ for 16h and infected with HCoV-OC43 for 72h (normalized to EV control).

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Infection, Flow Cytometry, RNA Sequencing, Expressing, Labeling, Knock-Out, Control

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): RNF213 KO (n=20) or EV (n=7) A549 SCCs were treated as indicated for 16h, infected with HCoV-OC43 at MOI 0.1 for 72h and percent infected cells determined by flow cytometry. Each dot represents an independent SCC (representative experiment, n=3; statistics used each clone as an data point, +/- SD, p values shown: student’s t-test). (B) : Growth curve of HCoV-OC43 in A549 EV or RNF213 KO cells pretreated, or not, for 16h with IFNγ and infected at MOI 0.1 (n=3). (C): Cells were pretreated with IFNγ for 16h and infected with HCoV-OC43 at MOI 5 for 24h. Labeled extracellular virions were run on an SDS-PAGE gel (representative image, n=3). (D): A549 cells were transfected with siRNAs against RNF213 or a non-targeting control (NT), pretreated with IFNγ for 16h, and plaque assay performed for rHCoV-OC43/eGFP (representative image, n=3, scale 1 mm). (E): Quantification of plaque size (representative experiment shown +/- SD, p values shown: 2-way ANOVA). (F): Quantification of plaque number (n=3, +/- SD, p values shown: 2-way ANOVA). (G): siRNA transfected A549 cells were pretreated or not with IFNγ and infected with HCoV-OC43. Percentage infected cells at 72 hpi was determined by flow cytometry (n=3 +/-SD), (H): Same as (G) , in Vero cells (I): Schematic of human RNF213 and locations of CRISPR-mediated editing. (J): Flow cytometry of A549 SCCs containing the indicated RNF213 mutations, pretreated with IFNγ (16h) and infected with HCoV-OC43 at MOI 0.1 for 72h. Each dot represents an independent SCC (representative experiment, n=3; statistics based on each clone as an independent experiment, +/- SD, p values shown: 2-way ANOVA). EV: empty vector. KO: knockout. MOI: multiplicity of infection. PFU: plaque forming units. Tri: Trimer. Mono: Monomer. SCC: single-cell clone.

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Infection, Flow Cytometry, Labeling, SDS Page, Transfection, Control, Plaque Assay, CRISPR, Plasmid Preparation, Knock-Out, Single Cell

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): Quantification of fluorescent signal for each of the indicated bands from (n=3, +/- SD, p-values displayed: student’s t-test). (B): Calculation of the ratio of each other protein to M (n=3, +/- SD, p-values displayed: student’s t-test). (C): Flow cytometry analysis of A549 RNF213 knock-out cells containing knock-in mutations in the guide RNA target sequence. Cells were pretreated for 16h with IFNγ and infected with HCoV-OC43 at MOI 0.1 for 72 hours (n=3, +/- SD, p-values shown: 2-way ANOVA). (D): Western blot of EV or RNF213 KO A549 cells pretreated with IFN for 24 hours (representative images shown, n=3). (E): Western blot of the indicated cells transfected with siRNAs against RNF213 or a non-targeting control, then pretreated with IFNγ for an additional 24 hours (representative images shown, n=3). (F-G): Flow cytometry of the indicated cells transfected with siRNAs, pretreated with IFNγ for 16 hours, and infected with HCoV-OC43 (F) or the indicated virus (G) at the indicated MOI for 72h, except VSV which was infected for 24h (n=3. +/- SD). (H): Western blot of A549 cells with the indicated point mutations pretreated with IFNγ for 24 hours (representative images shown, n=3).

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Flow Cytometry, Knock-Out, Knock-In, Sequencing, Infection, Western Blot, Transfection, Control, Virus

Journal: bioRxiv

Article Title: Interferon-gamma induced RNF213 targets nascent double membrane vesicles to inhibit coronavirus primary translation

doi: 10.64898/2026.06.02.729663

Figure Lengend Snippet: (A): Flow cytometry analysis of A549 cells transfected with siRNAs, treated with IFNγ for 16 hours, and infected with the indicated virus at the indicated MOI for 72h (n=3, +/- SD). (B): Same in Vero cells, except rHCoV-OC43/NLuc-NSP3/eGFP was infected for 48h. (C): Flow cytometry analysis of 293T cells infected with rHCoV-OC43/eGFP at MOI 0.1 for 72h, with normal human serum added at 4 hpi (representative experiment, n=3). (D): Same with A549 cells. (E): Flow cytometry analysis of Vero cells infected with rHCoV-OC43/eGFP at MOI 1 for 24h with soluble heparin added concurrently with infection. (F): Vero cells expressing LgBiT were infected with rHCoV-OC43/M-HiBiT at MOI 1 for 24h in the presence of the luciferase detection reagent endurazine. Soluble heparin was added at 0 hpi and/or at 24 hpi, and luminescence detected at 25 hpi (n=3. +/-SD). (G): qPCR of A549 cells infected with HCoV-OC43 at MOI 0.1 for 24h in the presence of Molnupiravir (representative images, n=3). (H): Western blot of EV or RNF213 KO A549 cells infected for the indicated time with HcoV-OC43 and 200 μM Molnupiravir (representative images, n=3.

Article Snippet: HCoV-OC43 (ATCC VR-1588) and HCoV-229E (ATCC VR-740) were obtained from Zeptometrix Corporation.

Techniques: Flow Cytometry, Transfection, Infection, Virus, Expressing, Luciferase, Western Blot

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: Overexpression of SERINC1, 3, and 5 impairs infectivity of hCoV‐OC43 but not hCoV‐229E. (A and B) Huh7 cells transiently transfected with empty or SERINC encoding pBJ6 vectors were infected with hCoV‐229E (A) or hCoV‐OC43 (B) at the indicated MOIs. At 30 hpi (A) or 48 hpi (B), supernatants and cell lysates were collected for qRT‐PCR and western blot analysis, respectively. (C) Supernatants of Huh7 cells treated as described in (A) were harvested for TCID 50 determination at 30 h postinfection. (D) Huh7 cells were treated as described in (B) and supernatants harvested for TCID 50 assay at 72 h postinfection. Shown are mean values ± SEM from three or four independent experiments. Statistical significance compared with the mock control was assessed using unpaired t ‐test with Welch's correction. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Over Expression, Infection, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: Endogenous SERINC1, 3, 4, and 5 restrict infectivity of hCoV‐OC43 but not hCoV‐229E. (A) Huh7 cells transfected with nontargeting or SERINC‐specific siRNA were infected with hCoV‐229E at an MOI of 0.001 or hCoV‐OC43 at MOI 0.01. Supernatants were harvested at 30 or 72 hpi for qRT‐PCR and TCID 50 . Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. (B) NHLF cells transfected with nontargeting or SERINC‐specific siRNAs were infected with hCoV‐229E (MOI 0.02)) or hCoV‐OC43 (MOI 0.002). Supernatants were collected at 36 hpi (hCoV‐229E) or 72 hpi (hCoV‐OC43) for qRT‐PCR and TCID 50 . Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. (C) Supernatants from (B) were used to infect A549 cells. Viral infection was assessed by immunofluorescent staining of the hCoV‐OC43 nucleocapsid (purple) and DAPI‐stained nuclei (blue). Scale bar: 40 µm. Infection rates were quantified using image J. A total of 28 images (seven images from each independent experiment) per condition were included for analysis. Shown are mean values ± SEM from three or four independent experiments. Statistical comparisons with mock control were performed using unpaired t ‐test with Welch's correction. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Infection, Transfection, Quantitative RT-PCR, Plasmid Preparation, Control, Staining

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: hCoV‐229E and hCoV‐OC43 reduce exogenous SERINC expression levels. (A) Huh7 cells transiently transfected with HA‐tagged SERINCs were infected with hCoV‐229E (MOI 0.001) or hCoV‐OC43 (MOI 0.01). Whole cell lysates and supernatants were harvested at 3 days postinfection (dpi) for western blot. Shown are the western blot analyses of cell lysates and purified virions representative for four independent experiments. (B) Huh7 cells transfected with GFP‐tagged SERINC1, SERINC2, SERINC3, and SERINC5 were infected with hCoV‐229E (MOI 0.01), hCoV‐OC43 (MOI 0.1), or left untreated. Immunofluorescence staining was performed at 2 dpi. Nucleocapsid proteins (purple) of hCoV‐229E or hCoV‐OC43 were stained to indicate infection; nuclei were stained with DAPI (blue). Scale bar: 20 µm. Results are representative of at least three independent experiments.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Expressing, Transfection, Infection, Western Blot, Purification, Immunofluorescence, Staining

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: SERINC5 degradation by hCoV‐229E infection is lysosome dependent. (A) Huh7 cells transiently transfected with GFP‐SERINC5 were infected with hCoV‐229E (MOI 0.01), hCoV‐OC43 (MOI 0.1), or left untreated. Immunofluorescence staining was performed at 2 dpi. Viral infection was detected via nucleocapsid staining (purple), and lysosomes were marked with LAMP1 (yellow). Colocalization of GFP‐SERINC5 with LAMP1 was quantified using Huygens software ( n = 22 cells). Scale bar: 20 µm. (B) Huh7 cells transiently transfected with vectors expressing GFP alone or GFP‐SERINC5 were infected with hCoV‐229E or left untreated. Mean GFP fluorescence intensity in live cells was measured by flow cytometry at 2 dpi. (C and D) Huh7 cells transfected with GFP‐SERINC5 were infected with hCoV‐229E (C), hCoV‐OC43 (D), or left uninfected, followed by treatment with proteasome inhibitor MG132 (20 µM, 6 h) or lysosome inhibitor NH 4 Cl (10 mM, 18 h). Cells were harvested at 48 h postinfection for flow cytometry analysis. (E) Huh7 cells transiently transfected GFP‐SERINC5 were infected with hCoV‐229E, followed by MG132 treatment for 6 h or NH 4 Cl treatment for 18 h. At 48 hpi, cells were fixed for immunofluorescence staining or harvested for western blot. Scale bar: 20 µm. A total of 45 cells were analyzed for colocalization of GFP‐SERINC5 and LAMP1 using Huygens. Shown are mean values ± SEM from three or four independent experiments. Statistical significance compared with the mock control was assessed using unpaired t ‐test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01. *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Infection, Transfection, Immunofluorescence, Staining, Software, Expressing, Fluorescence, Flow Cytometry, Western Blot, Control

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: hCoV‐229E ORF4a mediates SERINC5 relocalization and degradation. (A) Huh7 cells were cotransfected with constructs expressing GFP‐SERINC5 and the indicated hCoV‐229E proteins. At 2 dpi, cells were analyzed by flow cytometry staining or western blot. Shown are mean GFP fluorescence intensities in live cells, normalized to vector control. Data represent two biological replicates from four independent experiments. (B and C) Colocalization analysis of GFP‐SERINC5 with LAMP1 or with Strep II‐tagged viral proteins in Huh7 cells. A total of 10 to 22 cells in each group were included for analysis. Statistical significance compared with the mock control was determined using unpaired t ‐test with Welch's correction. ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Construct, Expressing, Flow Cytometry, Staining, Western Blot, Fluorescence, Plasmid Preparation, Control

Journal: MedComm

Article Title: Human Coronavirus 229E Uses ORF4/4a to Antagonize the Host Restriction Factor SERINC5

doi: 10.1002/mco2.70785

Figure Lengend Snippet: hCoV‐229E ORF4 is critical for SERINC5 resistance of hCoV‐229E. (A) Huh7 cells were transfected with a construct expressing SERINC5‐iFLAG followed by infection with wild‐type or ORF4‐deleted GFP‐hCoV‐229E. At 2 dpi, cells were fixed for immunofluorescence staining. SERINC5‐iFLAG was detected via anti‐FLAG staining (purple); LAMP1 (yellow) marks lysosomes; DAPI (blue) stains nuclei. Scale bar: 20 µm. Colocalization of GFP‐SERINC5 with LAMP1 was quantified using Huygens ( n = 21 cells per condition). (B) Huh7 cells transiently transfected with empty or SERINC5 encoding vector were infected with wild‐type or GFP hCoV‐229E at indicated MOIs. At 36 hpi, supernatants and cell lysates were harvested for qRT‐PCR, TCID 50 , and western blot. Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. (C) Huh7 cells transfected with nontargeting or siRNA or SERINC5‐targeting siRNAs were infected with wild‐type or GFP hCoV‐229E at the indicated MOIs. Supernatants were harvested for qRT‐PCR and TCID 50 at 36 hpi. (D) NHLF transfected with nontargeting or SERINC5‐targeting siRNAs were infected with wild‐type or GFP hCoV‐229E (MOI 0.02). Supernatants were collected at 48 hpi for qRT‐PCR and TCID 50 . Normalized infectivity was calculated as TCID 50 /vRNA copy number, with the vector control set to 100%. Shown are mean values ± SEM from three to eight independent experiments. Statistical significance compared with the mock control was determined using unpaired t ‐test with Welch's correction. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: Expression constructs for the empty vector, hCoV‐229E‐ORF4, hCoV‐NL63‐ORF3, hCoV‐OC43‐Ns12.9, and SARS‐CoV‐2‐ORF3a in pTWIST backbone were obtained from TWIST Bioscience. hCoV‐229E and hCoV‐OC43 were purchased from ATCC (Cat No. VR‐740TM and Cat No. CR‐1558TM) and propagated as described [ ].

Techniques: Transfection, Construct, Expressing, Infection, Immunofluorescence, Staining, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control

Journal: Computational and Structural Biotechnology Journal

Article Title: Next-Generation Sequencing Dataset Downloader and In Silico Sequence Mining: Graphical-User-Interface-Based Tools for Accessible, Multiprobe Target Mining in Next-Generation Sequencing Data

doi: 10.34133/csbj.0095

Figure Lengend Snippet: ISSM analysis of an NGS dataset obtained from CRFK cells infected with feline coronavirus (FCoV), using probes targeting various coronavirus groups, other viruses, CRFK cell sequences, and beta-actin. (A) Heatmap output generated by the ISSM. Probe sequences are color coded according to their target: SARS-CoV-2 (red), SARS-CoV (brown), Bovine CoV (yellow), Alphacoronavirus (blue), Universal coronavirus (green), Bat-CoV (purple), Feline coronavirus (gray), other viruses (black), CRFK cell (light orange), and beta-actin (pink). Color intensity indicates the relative number of matched reads, with deeper red representing higher read counts. (B) Relationship between extraction fraction and detected matched read proportion. Matched read counts obtained at 1%, 10%, 25%, and 50% extraction were expressed as percentages relative to the corresponding matched read counts obtained under 100% extraction, and a linear regression trendline was fitted to model this relationship. (C) Back-calculated 100% equivalent matched read counts derived from the regression equation shown in panel (B), compared with the actual matched read counts obtained under 100% extraction. The actual 100% extraction values used as reference are provided in Table . (D) Reliability assessment of downsampling-based back-calculation. The relationship between the log-transformed actual matched read counts obtained under 100% extraction and the relative error of the back-calculated 100% equivalent values is shown for each extraction setting. Trend equations were used to estimate approximate matched read ranges corresponding to selected relative error thresholds, which are summarized in Table .

Article Snippet: The feline coronavirus (FCoV) type 2 strain WSU 79-1683 (American Type Culture Collection VR-989) was obtained from the American Type Culture Collection and used in this study.

Techniques: Infection, Generated, Extraction, Derivative Assay, Transformation Assay