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ATCC mink lung host cells
Mink Lung Host Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mv+1+lu/Mv+1+Lu/pmc13055148-57-8-12
Average 95 stars, based on 516 article reviews

mink lung host cells - by Bioz Stars, 2026-09

95/100 stars

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Article Title: Rapid inactivation kinetics of a broad range of viruses on a continuously self-cleaning polymer surface
Article Snippet: ory (BEI Resources, NR-52725), and the SARS-CoV-2 variant XBB.1.5 was obtained from BEI Resources (NR-59105), courtesy of Dr. William Florence at the National Institutes of Health. Mink cells from the American Type Culture Collection (ATCC [Manassas, VA]; Mv 1 Lu, CCL-64) were used for OC43 plaque assays, and Vero E6 cells (African Green Monkey, BEI Resources, NR-596) were used for the SARS-CoV-2 v

Article Title: The pan-variant potential of light: 425 nm light inactivates SARS-CoV-2 variants of concern and non-cytotoxic doses reduce viral titers in human airway epithelial cells
Article Snippet: Vero E6 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained as previously described ( ).Vero E6 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained as previously described ( ).. Mv 1 Lu (CCL-64), LLC-MK2 (CCL-7.1), and A549 (CCL-185) cells were purchased from the ATCC.. Mv 1 Lu and A549 cells were maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich #D6429-500mL) supplemented with 10% heat-inactivated FetalCloneII (Cytiva #SH30066.03hi) and 1% antibiotic/antimycotic (Gibco #15240-0620).Mv 1 Lu and A549 cells were maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich #D6429-500mL) supplemented with 10% heat-inactivated FetalCloneII (Cytiva #SH30066.03hi) and 1% antibiotic/antimycotic (Gibco #15240-0620).

Activity Assay:

Cell Culture:

Immunofluorescence:

Western Blot:

Produced:

Plaque Assay:

Stable Transfection:

Image Search Results

Images of cellular mats taken with an Evos FL digital inverted fluorescence microscope. ( A ) MNT1; ( B ) A549; ( C ) combo MNT1/H292; ( D ) Mv1Lu; ( E ) A549; ( F ) combo Mv1Lu/A549; ( G ) Ve6TMPRSS2; ( H ) MDCK; ( I ) ccombo Ve6TMPRSS2/MDCK; ( J ) Caco-2; ( K ) MRC5; ( L ) combo Caco-2/MRC5; ( M ) BHK-21; ( N ) L929; ( O ) combo BHK-21/L929. Pictures are taken at magnification ×20; scale bars indicate 400 µm.

Journal: Microbiology Spectrum

Article Title: Development of cell combos micromethod to isolate respiratory viruses not detected by molecular techniques

doi: 10.1128/spectrum.02571-25

Figure Lengend Snippet: Images of cellular mats taken with an Evos FL digital inverted fluorescence microscope. ( A ) MNT1; ( B ) A549; ( C ) combo MNT1/H292; ( D ) Mv1Lu; ( E ) A549; ( F ) combo Mv1Lu/A549; ( G ) Ve6TMPRSS2; ( H ) MDCK; ( I ) ccombo Ve6TMPRSS2/MDCK; ( J ) Caco-2; ( K ) MRC5; ( L ) combo Caco-2/MRC5; ( M ) BHK-21; ( N ) L929; ( O ) combo BHK-21/L929. Pictures are taken at magnification ×20; scale bars indicate 400 µm.

Article Snippet: Mv1Lu cells (ATCC-CCL-64) were associated with A549 cells (ATCC-CCL-185) to create the second combo, which we named Mv1Lu/A549.

Techniques: Fluorescence, Microscopy

Examples of cytopathic effects with various viruses on each cell combo developed. ( A ) MNT1/H292 negative control, ( B ) HCoV-OC43 on MNT1/H292, ( C ) Mv1Lu/A549 negative control, ( D ) HRSV-B on Mv1Lu/A549, ( E ) Ve6TMPRSS2/MDCK negative control, ( F ) HPIV-1 on Ve6TMPRSS2/MDCK, ( G ) Caco-2/MRC5 negative control, ( H ) HCoV-229E on Caco-2/MRC5, ( I ) BHK-21/L929 negative control, ( J ) adenovirus on BHK-21/L929. Pictures are taken at magnification ×10; scale bars indicate 400 µm.

Journal: Microbiology Spectrum

Article Title: Development of cell combos micromethod to isolate respiratory viruses not detected by molecular techniques

doi: 10.1128/spectrum.02571-25

Figure Lengend Snippet: Examples of cytopathic effects with various viruses on each cell combo developed. ( A ) MNT1/H292 negative control, ( B ) HCoV-OC43 on MNT1/H292, ( C ) Mv1Lu/A549 negative control, ( D ) HRSV-B on Mv1Lu/A549, ( E ) Ve6TMPRSS2/MDCK negative control, ( F ) HPIV-1 on Ve6TMPRSS2/MDCK, ( G ) Caco-2/MRC5 negative control, ( H ) HCoV-229E on Caco-2/MRC5, ( I ) BHK-21/L929 negative control, ( J ) adenovirus on BHK-21/L929. Pictures are taken at magnification ×10; scale bars indicate 400 µm.

Article Snippet: Mv1Lu cells (ATCC-CCL-64) were associated with A549 cells (ATCC-CCL-185) to create the second combo, which we named Mv1Lu/A549.

Techniques: Negative Control

Respiratory virus growths on each cell combo developed. ( A ) Combo MNT1/H292; ( B ) combo Mv1Lu/A549; ( C ) combo VE6TMPRSS2/MDCK; ( D ) combo Caco-2/MRC5; ( E ) combo BHK21/L929. Dashed line indicates significant growth (3.3 Ct that is a proxy of a 1.0 log 10 variation). Ct, real-time PCR cycle threshold value; HCoV, human coronavirus; HPIV, human parainfluenza virus; HRSV, human respiratory syncytial virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Journal: Microbiology Spectrum

Article Title: Development of cell combos micromethod to isolate respiratory viruses not detected by molecular techniques

doi: 10.1128/spectrum.02571-25

Figure Lengend Snippet: Respiratory virus growths on each cell combo developed. ( A ) Combo MNT1/H292; ( B ) combo Mv1Lu/A549; ( C ) combo VE6TMPRSS2/MDCK; ( D ) combo Caco-2/MRC5; ( E ) combo BHK21/L929. Dashed line indicates significant growth (3.3 Ct that is a proxy of a 1.0 log 10 variation). Ct, real-time PCR cycle threshold value; HCoV, human coronavirus; HPIV, human parainfluenza virus; HRSV, human respiratory syncytial virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: Mv1Lu cells (ATCC-CCL-64) were associated with A549 cells (ATCC-CCL-185) to create the second combo, which we named Mv1Lu/A549.

Techniques: Virus, Real-time Polymerase Chain Reaction

Replication of bovine- and mink-derived H5N1 viruses in mammalian cell lines with and without raw milk supplementation. Viral replication levels expressed as log 10 TCID50 equivalents per mL from RT-qPCR targeting the influenza A matrix gene in MDCK, A549, MDBK, Vero, and MV1 cells at 24 h post-infection (MOI = 0.01) with bovine H5N1 (A/dairy cattle/Kansas/5/2024) or mink H5N1 (A/Mink/Spain/3691-8_22VIR10586-10/2022). Cells were incubated in standard medium (without milk) or raw milk–milk-supplemented medium (with milk). Data represent individual technical replicates ( n = 3). Viral loads were calculated using a validated standard curve correlating Ct values to known viral titers (R 2 = 0.9779, linear range 10 2 –10 8 TCID50/mL). Results represent RNA levels as log 10 TCID50 equivalents; correlation with infectious virus production requires validation.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Replication of bovine- and mink-derived H5N1 viruses in mammalian cell lines with and without raw milk supplementation. Viral replication levels expressed as log 10 TCID50 equivalents per mL from RT-qPCR targeting the influenza A matrix gene in MDCK, A549, MDBK, Vero, and MV1 cells at 24 h post-infection (MOI = 0.01) with bovine H5N1 (A/dairy cattle/Kansas/5/2024) or mink H5N1 (A/Mink/Spain/3691-8_22VIR10586-10/2022). Cells were incubated in standard medium (without milk) or raw milk–milk-supplemented medium (with milk). Data represent individual technical replicates ( n = 3). Viral loads were calculated using a validated standard curve correlating Ct values to known viral titers (R 2 = 0.9779, linear range 10 2 –10 8 TCID50/mL). Results represent RNA levels as log 10 TCID50 equivalents; correlation with infectious virus production requires validation.

Article Snippet: Madin-Darby Canine Kidney (MDCK, ATCC, cat# CCL-34), Madin-Darby Bovine Kidney (MDBK, ATCC, cat# CCL-22), A549 (human lung carcinoma, ATCC, cat# CCL-185), MV1 (mink lung epithelial, ATCC, cat# CCL-64), and Vero (African green monkey kidney, ATCC, cat# CCL-81) cells were obtained from ATCC.

Techniques: Derivative Assay, Quantitative RT-PCR, Infection, Incubation, Virus, Biomarker Discovery

Host-and condition-specific amino acid substitutions in bovine- and mink-derived H5N1 viruses. Heatmaps of substitutions (≥5% frequency) in viral populations recovered 24 h post-infection from MDCK, A549, MDBK, Vero, and MV1 cells under standard (−milk) or raw milk-supplemented (+milk) conditions. ( A ) Bovine-H5N1 (A/dairy cattle/Kansas/5/2024). ( B ) Mink-H5N1 (A/Mink/Spain/3691-8_22VIR10586-10/2022). Purple boxes indicate the presence of the substitution.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Host-and condition-specific amino acid substitutions in bovine- and mink-derived H5N1 viruses. Heatmaps of substitutions (≥5% frequency) in viral populations recovered 24 h post-infection from MDCK, A549, MDBK, Vero, and MV1 cells under standard (−milk) or raw milk-supplemented (+milk) conditions. ( A ) Bovine-H5N1 (A/dairy cattle/Kansas/5/2024). ( B ) Mink-H5N1 (A/Mink/Spain/3691-8_22VIR10586-10/2022). Purple boxes indicate the presence of the substitution.

Techniques: Derivative Assay, Infection

Transcriptional profiling of cell type- and environment-dependent responses to H5N1 infection. ( A ) Principal component analysis (PCA) of variance Stabilizing Transformation (VST)-normalized RNA-seq counts from A549, MDCK, MDBK, MV1, and Vero cells at 24 h post-infection with bovine-H5N1, mink-H5N1, or mock, with or without raw milk supplementation. Shapes denote infection type (bovine-H5N1 = circle; mink-H5N1 = triangle; mock = square) and colors indicate the cell lines with or without milk. ( B ) Gene Ontology (GO) enrichment for genes upregulated in bovine-H5N1 (gray) and mink-H5N1 (dark blue) infections. Points are sized by enrichment score and ordered by −log 10 ( p -value). Enrichment was computed in Metascape and visualized in R (ggplot2).

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Transcriptional profiling of cell type- and environment-dependent responses to H5N1 infection. ( A ) Principal component analysis (PCA) of variance Stabilizing Transformation (VST)-normalized RNA-seq counts from A549, MDCK, MDBK, MV1, and Vero cells at 24 h post-infection with bovine-H5N1, mink-H5N1, or mock, with or without raw milk supplementation. Shapes denote infection type (bovine-H5N1 = circle; mink-H5N1 = triangle; mock = square) and colors indicate the cell lines with or without milk. ( B ) Gene Ontology (GO) enrichment for genes upregulated in bovine-H5N1 (gray) and mink-H5N1 (dark blue) infections. Points are sized by enrichment score and ordered by −log 10 ( p -value). Enrichment was computed in Metascape and visualized in R (ggplot2).

Techniques: Infection, Transformation Assay, RNA Sequencing

Impact of H5N1 strain and milk supplementation on host genes involved in influenza A entry, adsorption, and uncoating. VST normalized RNA-seq counts for six entry/adsorption/uncoating-associated genes ( GNE, SLC35A1, ATP1B1, ATP1B4, ATP1A1, ATP1B3 ) were measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Impact of H5N1 strain and milk supplementation on host genes involved in influenza A entry, adsorption, and uncoating. VST normalized RNA-seq counts for six entry/adsorption/uncoating-associated genes ( GNE, SLC35A1, ATP1B1, ATP1B4, ATP1A1, ATP1B3 ) were measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Techniques: Adsorption, RNA Sequencing, Infection, Incubation, Expressing

Impact of H5N1 strain and milk supplementation on host genes involved in influenza A genome replication, trafficking, and protein synthesis. VST normalized RNA-seq counts for genome replication, trafficking, and protein synthesis-associated genes ( ANP32B, EIF4A2, NUP98, NXF1, NUP205, KPNB1 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Impact of H5N1 strain and milk supplementation on host genes involved in influenza A genome replication, trafficking, and protein synthesis. VST normalized RNA-seq counts for genome replication, trafficking, and protein synthesis-associated genes ( ANP32B, EIF4A2, NUP98, NXF1, NUP205, KPNB1 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Techniques: RNA Sequencing, Infection, Incubation, Expressing

Impact of H5N1strain and milk supplementation on host genes involved in host antiviral responses against influenza A virus. VST normalized RNA-seq counts for host antiviral responses-associated genes ( ISG15, RSAD2, MOV10, ZC3HAV1, SPOCK2, ISG20 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Impact of H5N1strain and milk supplementation on host genes involved in host antiviral responses against influenza A virus. VST normalized RNA-seq counts for host antiviral responses-associated genes ( ISG15, RSAD2, MOV10, ZC3HAV1, SPOCK2, ISG20 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Techniques: Virus, RNA Sequencing, Infection, Incubation, Expressing

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Impact of H5N1 strain and milk supplementation on host genes involved in host antiviral responses against influenza A virus. VST normalized RNA-seq counts for host antiviral responses-associated genes ( TRIM14, TRIM21, TRIM25, TRIM35, OAS2, OASL ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Techniques: Virus, RNA Sequencing, Infection, Incubation, Expressing

Impact of H5N1 strain and milk supplementation on host genes involved in host signaling. VST normalized RNA-seq counts for genes associated with host signaling ( CLK1, PLK3, FGFR2, MAP2K3, TNK2, DCLK2 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Impact of H5N1 strain and milk supplementation on host genes involved in host signaling. VST normalized RNA-seq counts for genes associated with host signaling ( CLK1, PLK3, FGFR2, MAP2K3, TNK2, DCLK2 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Techniques: RNA Sequencing, Infection, Incubation, Expressing

Impact of H5N1 strain and milk supplementation on host genes involved in virus assembly and trafficking. VST normalized RNA-seq counts for host genes associated with virus assembly and trafficking ( ARCN1, COPA, COPB1, COPB2, COPE, GBF1 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Journal: Life

Article Title: Differential Host Responses and Viral Replication of Highly Pathogenic Avian Influenza H5N1 Strains in Diverse Cell Lines with a Raw Milk Supplement

doi: 10.3390/life15101625

Figure Lengend Snippet: Impact of H5N1 strain and milk supplementation on host genes involved in virus assembly and trafficking. VST normalized RNA-seq counts for host genes associated with virus assembly and trafficking ( ARCN1, COPA, COPB1, COPB2, COPE, GBF1 ) measured at 24 h post-infection in five mammalian cell lines (MDCK, MDBK, A549, MV1, and Vero). Cells were infected with either bovine-H5N1 or mink-H5N1 (MOI = 0.01) and incubated in infection medium with or without raw milk. Boxplots display the distribution of expression values ( n = 3 technical replicates): box = interquartile range (25th–75th percentiles), horizontal line = median, whiskers = 1.5 × IQR, and individual points = values beyond whiskers.

Techniques: Virus, RNA Sequencing, Infection, Incubation, Expressing

Optimizing hCoV-OC43 NG-ns12.9 ds-circDNA transfection and co-cultivation for infectious NG virus production. ( A ) The N protein promotes virus production from CPER-derived ds-circDNAs. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an hCoV-OC43 N expression vector (pCOC42) or a control vector (pFLAG-CMV-5.1) and then co-cultivated with HCT-8 for 8 days. The numbers of NG + HCT-8 cells were counted and averaged from 10 random microscopic fields on days 6, 7, and 8 (D6–D8) post-transfection. ( B ) A representative microscopic field showing the NG + HCT-8 cells co-transfected by an FL hCoV-OC43 NG-ns12.9 ds-circDNA with an hCoV-OC43 N expression vector (pCOC42) or a control vector pFLAG-CMV-5.1 on D6 and D8. ( C, D ) Fetal bovine serum (FBS), but not new calf serum (NCS), is required for efficient virus production in HCT-8 cells. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an N expression vector pCOC42 and maintained in DMEM supplemented with 2% FBS overnight. The transfected cells were then co-cultivated by the addition of HCT-8 cells with cell passage every 3 days for a total of 10 days in DMEM supplemented with 10% FBS or 10% NCS. The number of NG + HCT-8 cells was counted and averaged from 10 random microscopic fields on D8–D10 ( C ). Significantly more NG + HCT-8 cells were shown from the cells with an FL NG-ns12.9 ds-circDNA on D8–D10 when growing in the DMEM containing 10% FBS when compared with 10% NCS ( D ). ( E ) hCoV-OC43 induced visible and well-defined cytopathic effect (CPE) in both LLC-MK2 and Mv1Lu cells. The monolayer of LLC-MK2 or Mv1Lu cells in ~70% confluence was infected with WT hCoV-OC43 (100 µL supernatant of infected HCT-8 cells). One representative microscopic field is shown for each cell type. ( F ) Mv1Lu cells are more sensitive than LLC-MK2 cells for hCoV-OC43 infection and plaque formation. Plaque assays of LLC-MK2 and Mv1Lu cells were infected with 100 µL of each diluent after serial 10-fold dilutions of WT hCoV-OC43 virus and overlayed with semisolid media (1× DMEM, 0.5% methylcellulose and 10% FBS) for 8 days. The plaques were fixed for 30 min by 3.7% formaldehyde solution and stained with 1% crystal violet.

Journal: Journal of Virology

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

doi: 10.1128/jvi.01438-25

Figure Lengend Snippet: Optimizing hCoV-OC43 NG-ns12.9 ds-circDNA transfection and co-cultivation for infectious NG virus production. ( A ) The N protein promotes virus production from CPER-derived ds-circDNAs. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an hCoV-OC43 N expression vector (pCOC42) or a control vector (pFLAG-CMV-5.1) and then co-cultivated with HCT-8 for 8 days. The numbers of NG + HCT-8 cells were counted and averaged from 10 random microscopic fields on days 6, 7, and 8 (D6–D8) post-transfection. ( B ) A representative microscopic field showing the NG + HCT-8 cells co-transfected by an FL hCoV-OC43 NG-ns12.9 ds-circDNA with an hCoV-OC43 N expression vector (pCOC42) or a control vector pFLAG-CMV-5.1 on D6 and D8. ( C, D ) Fetal bovine serum (FBS), but not new calf serum (NCS), is required for efficient virus production in HCT-8 cells. HEK293T cells were co-transfected with an FL hCoV-OC43 NG-ns12.9 ds-circDNA along with an N expression vector pCOC42 and maintained in DMEM supplemented with 2% FBS overnight. The transfected cells were then co-cultivated by the addition of HCT-8 cells with cell passage every 3 days for a total of 10 days in DMEM supplemented with 10% FBS or 10% NCS. The number of NG + HCT-8 cells was counted and averaged from 10 random microscopic fields on D8–D10 ( C ). Significantly more NG + HCT-8 cells were shown from the cells with an FL NG-ns12.9 ds-circDNA on D8–D10 when growing in the DMEM containing 10% FBS when compared with 10% NCS ( D ). ( E ) hCoV-OC43 induced visible and well-defined cytopathic effect (CPE) in both LLC-MK2 and Mv1Lu cells. The monolayer of LLC-MK2 or Mv1Lu cells in ~70% confluence was infected with WT hCoV-OC43 (100 µL supernatant of infected HCT-8 cells). One representative microscopic field is shown for each cell type. ( F ) Mv1Lu cells are more sensitive than LLC-MK2 cells for hCoV-OC43 infection and plaque formation. Plaque assays of LLC-MK2 and Mv1Lu cells were infected with 100 µL of each diluent after serial 10-fold dilutions of WT hCoV-OC43 virus and overlayed with semisolid media (1× DMEM, 0.5% methylcellulose and 10% FBS) for 8 days. The plaques were fixed for 30 min by 3.7% formaldehyde solution and stained with 1% crystal violet.

Article Snippet: The human embryonic kidney HEK293T (CRL-3216), human colorectal adenocarcinoma HCT-8 (CCL-244), monkey kidney epithelial LLC-MK2 (CCL-7) and American mink lung epithelial Mv1Lu (CCL-64) cells were obtained from ATCC.

Techniques: Transfection, Virus, Derivative Assay, Expressing, Plasmid Preparation, Control, Infection, Staining

Positional NG insertion into the hCoV-OC43 genome to determine hCoV-OC43 replication and synthesis direction of viral sgRNAs. ( A ) Agarose gel electrophoresis of CPER-derived hCoV-OC43 ds-circDNAs (black arrow) of NG-Δns2 (lane 1) and NG-ns12.9 (lane 2). See for details. ( B and C ) Visualization and quantification of NG + HCT-8 cells after co-cultivation with HEK293T cells transfected with CPER-derived hCoV-OC43 NG-Δns2 or NG-ns12.9 ds-circDNAs. HEK293T cells were transfected with individual FL ds-circDNA products along with an hCoV-OC43 N protein expression vector pCOC42 and then co-cultivated by addition of HCT-8 cells for the indicated days. ( B ) Representative microscopic images on D6 showing NG + HCT-8 cells from the NG-Δns2 or NG-ns12.9 ds-circDNA. ( C ) Numbers of NG + cells quantified from 10 random microscopic fields on D5 and D6, showing more NG + cells (**, P < 0.01, Student’s t -test) for the NG-ns12.9 ds-circDNA than that of the NG-Δns2 ds-circDNA. ( D ) Northern blot analysis of hCoV-OC43 RNA from HCT-8 cells in co-cultivation with HEK293T cells transfected by CPER-derived NG-Δns2 ds-circDNA (lane 1) or NG-ns12.9 ds-circDNA (lane 2) or infected by wild-type (lane 3) viruses. Total RNA extracted from the D6 cells was analyzed by Northern blot using a 32 P-labeled probe antisense to the hCoV-OC43 N ORF. The sgRNAs containing inserted NG are labeled in green. ( E, F ) Virus titration of the HCT-8 cell culture supernatant collected on D8 co-cultivation by using methylcellulose-overlay fluorescent foci ( E ) and plaque ( F ) assays in Mv1Lu cells. ( E ) Representative images of fluorescent foci on D8 of infected Mv1Lu cells (left panel). The Mv1Lu cells were infected by 100 µL of each diluent after serial 10-fold dilutions of the collected HCT-8 culture supernatant collected on D6 described above ( C ). Virus titers are calculated as fluorescent-forming units (FFU/mL) (right bar graph), showing a higher titer of hCoV-OC43 NG-ns12.9 virus (1.2 × 10 6 FFU/mL) than hCoV-OC43 NG-Δns2 virus (4.0 × 10 4 FFU/mL). ( F ) Titration of hCoV-OC43 NG-Δns2 and NG-ns12.9 virus titers by plaque assays using Mv1Lu cells infected by 100 µL of each diluent after serial 10-fold dilutions of the HCT-8 culture supernatant collected on D6 of the cell co-cultivation. The visible virus plaques of hCoV-OC43 NG-Δns2 and hCoV-OC43 NG-ns12.9 on D8 in Mv1Lu cells were visualized by crystal violet staining.

Journal: Journal of Virology

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

doi: 10.1128/jvi.01438-25

Figure Lengend Snippet: Positional NG insertion into the hCoV-OC43 genome to determine hCoV-OC43 replication and synthesis direction of viral sgRNAs. ( A ) Agarose gel electrophoresis of CPER-derived hCoV-OC43 ds-circDNAs (black arrow) of NG-Δns2 (lane 1) and NG-ns12.9 (lane 2). See for details. ( B and C ) Visualization and quantification of NG + HCT-8 cells after co-cultivation with HEK293T cells transfected with CPER-derived hCoV-OC43 NG-Δns2 or NG-ns12.9 ds-circDNAs. HEK293T cells were transfected with individual FL ds-circDNA products along with an hCoV-OC43 N protein expression vector pCOC42 and then co-cultivated by addition of HCT-8 cells for the indicated days. ( B ) Representative microscopic images on D6 showing NG + HCT-8 cells from the NG-Δns2 or NG-ns12.9 ds-circDNA. ( C ) Numbers of NG + cells quantified from 10 random microscopic fields on D5 and D6, showing more NG + cells (**, P < 0.01, Student’s t -test) for the NG-ns12.9 ds-circDNA than that of the NG-Δns2 ds-circDNA. ( D ) Northern blot analysis of hCoV-OC43 RNA from HCT-8 cells in co-cultivation with HEK293T cells transfected by CPER-derived NG-Δns2 ds-circDNA (lane 1) or NG-ns12.9 ds-circDNA (lane 2) or infected by wild-type (lane 3) viruses. Total RNA extracted from the D6 cells was analyzed by Northern blot using a 32 P-labeled probe antisense to the hCoV-OC43 N ORF. The sgRNAs containing inserted NG are labeled in green. ( E, F ) Virus titration of the HCT-8 cell culture supernatant collected on D8 co-cultivation by using methylcellulose-overlay fluorescent foci ( E ) and plaque ( F ) assays in Mv1Lu cells. ( E ) Representative images of fluorescent foci on D8 of infected Mv1Lu cells (left panel). The Mv1Lu cells were infected by 100 µL of each diluent after serial 10-fold dilutions of the collected HCT-8 culture supernatant collected on D6 described above ( C ). Virus titers are calculated as fluorescent-forming units (FFU/mL) (right bar graph), showing a higher titer of hCoV-OC43 NG-ns12.9 virus (1.2 × 10 6 FFU/mL) than hCoV-OC43 NG-Δns2 virus (4.0 × 10 4 FFU/mL). ( F ) Titration of hCoV-OC43 NG-Δns2 and NG-ns12.9 virus titers by plaque assays using Mv1Lu cells infected by 100 µL of each diluent after serial 10-fold dilutions of the HCT-8 culture supernatant collected on D6 of the cell co-cultivation. The visible virus plaques of hCoV-OC43 NG-Δns2 and hCoV-OC43 NG-ns12.9 on D8 in Mv1Lu cells were visualized by crystal violet staining.

Techniques: Agarose Gel Electrophoresis, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Northern Blot, Infection, Labeling, Virus, Titration, Cell Culture, Staining

Determination of virus infectivity and replication capacity of hCoV-OC43 NG-Δns2 and NG-ns12.9 virions by de novo virus infection of HCT-8 and Mv1Lu cells. ( A and B ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions exhibit equal infectivity to HCT-8 cells. HCT-8 cells were infected in parallel with 0.01 MOI of hCoV-OC43 NG-Δns2 or NG-ns12.9 virions titrated, as described in . NG + HCT-8 cells were quantified from five random microscopic fields at days 1, 2, and 3 post infection (D1–D3) of the indicated virus. ( A ) No difference in NG + HCT-8 cells from infection with the NG-Δns2 to NG-ns12.9 virions. ( B ) Selectively shown are one representative fluorescent image each from the HCT-8 cells infected with the NG-Δns2 or NG-ns12.9 virions at D3 post-infection. ( C and D ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions from the infected HCT-8 cell culture supernatant exhibit equal replication capacity with similar virus titers in Mv1Lu cells. The supernatant from the HCT-8 cells infected by 0.01 MOI of NG-Δns2 or NG-ns12.9 virions was collected on D3 post infection and, after serial 10-fold dilutions, 100 µL of each diluent was used to infect Mv1Lu cells, and titration of viral infectivity was carried out by the methylcellulose-overlay fluorescent foci assay ( C ). Virus titers for NG-Δns2 and NG-ns12.9 virions were calculated as fluorescent-forming units (FFU/mL) shown in a bar graph with NG-Δns2, 1.5 × 10 4 FFU/mL and NG-ns12.9, 2.0 × 10 4 FFU/mL ( D ).

Journal: Journal of Virology

Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription

doi: 10.1128/jvi.01438-25

Figure Lengend Snippet: Determination of virus infectivity and replication capacity of hCoV-OC43 NG-Δns2 and NG-ns12.9 virions by de novo virus infection of HCT-8 and Mv1Lu cells. ( A and B ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions exhibit equal infectivity to HCT-8 cells. HCT-8 cells were infected in parallel with 0.01 MOI of hCoV-OC43 NG-Δns2 or NG-ns12.9 virions titrated, as described in . NG + HCT-8 cells were quantified from five random microscopic fields at days 1, 2, and 3 post infection (D1–D3) of the indicated virus. ( A ) No difference in NG + HCT-8 cells from infection with the NG-Δns2 to NG-ns12.9 virions. ( B ) Selectively shown are one representative fluorescent image each from the HCT-8 cells infected with the NG-Δns2 or NG-ns12.9 virions at D3 post-infection. ( C and D ) hCoV-OC43 NG-Δns2 and NG-ns12.9 virions from the infected HCT-8 cell culture supernatant exhibit equal replication capacity with similar virus titers in Mv1Lu cells. The supernatant from the HCT-8 cells infected by 0.01 MOI of NG-Δns2 or NG-ns12.9 virions was collected on D3 post infection and, after serial 10-fold dilutions, 100 µL of each diluent was used to infect Mv1Lu cells, and titration of viral infectivity was carried out by the methylcellulose-overlay fluorescent foci assay ( C ). Virus titers for NG-Δns2 and NG-ns12.9 virions were calculated as fluorescent-forming units (FFU/mL) shown in a bar graph with NG-Δns2, 1.5 × 10 4 FFU/mL and NG-ns12.9, 2.0 × 10 4 FFU/mL ( D ).

Techniques: Virus, Infection, Cell Culture, Titration

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