Journal:

Article Title: Detection and Identification of Mammalian Reoviruses in Surface Water by Combined Cell Culture and Reverse Transcription-PCR

doi: 10.1128/AEM.67.7.3016-3020.2001

Figure Lengend Snippet: Similarity dendrogram for environmental water sample combined cell culture–RT-PCR reovirus genotypes. The GenBank L3 gene sequences for reovirus types 1, 2, and 3 (GBRV1, GBRV2, and GBRV3, respectively) and sequences obtained in this study from ATCC reovirus types 1, 2 and 3 (RV1, RV2, and RV3, respectively) are included.

Article Snippet: The GenBank L3 gene sequences for reovirus types 1, 2, and 3 (GBRV1, GBRV2, and GBRV3, respectively) and sequences obtained in this study from ATCC reovirus types 1, 2 and 3 (RV1, RV2, and RV3, respectively) are included. table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Isolate % Homology RV3 GBRV3 RV1 GBRV1 RV-E RV-A RV-B RV-C RV-D RV-F RV-G RV-H RV2 GBRV2 RV3 100 GBRV3 100 100 RV1 97.5 97.5 100 GBRV1 97.5 97.8 100 100 RV-E 97.2 97.2 96.8 96.8 100 RV-A 95.8 95.2 96.2 95.5 95.2 100 RV-B 95.6 95.6 96.0 96.0 95.2 100 100 RV-C 87.9 88.3 88.7 89.1 88.3 87.9 87.6 100 RV-D 88.1 87.5 88.8 88.2 88.3 88.3 87.6 100 100 RV-F 87.6 87.6 88.3 88.3 88.3 87.6 87.6 100 100 100 RV-G 85.9 85.9 86.3 86.3 87.2 86.8 86.8 85.1 85.1 85.1 100 RV-H 80.7 80.7 80.7 80.7 81.5 81.1 81.1 81.5 81.5 81.5 82.7 100 RV2 79.2 79.2 78.8 78.8 79.1 79.6 78.3 78.4 78.4 77.1 75.5 77.1 100 GBRV2 79.1 80.3 78.7 80.0 79.1 79.1 78.7 78.5 78.6 77.1 75.9 77.5 100 100 Open in a separate window Levels of L3 RT-PCR amplicon homology for reference reovirus strains and environmental isolates There were nucleotide differences between each environmental reovirus RT-PCR product and the sequences of the three reference reovirus strains.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Discovery

Article Title: Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869

doi: 10.1038/s41421-018-0017-2

Figure Lengend Snippet: Human fetal astrocytes were infected with ZIKV strains MR766 or PRVABC59 at the indicated MOI for 24 h, washed, then cultured for another 24 h. Supernatant from uninfected Vero cells was used for mock-infected control. a – h At the experimental endpoint, ICC of flavivirus antigen (green color) was performed. DAPI (blue color) was used as a nuclear counterstain. Panels are representative of three separate donors. Scale bar: 100 µm. Images were acquired through a Zeiss LSM 710 confocal microscope. i ZIKV-positive cells in a – h were quantified and shown as percentage of total cells in the culture. j RNA was isolated and expression ZIKV RNA was determined through real-time RT-PCR. Data were normalized to GAPDH and presented as fold change compared to mock-infected control. ** p < 0.001; *** p < 0.0001 as compared to mock-infected control

Article Snippet: Quantitative Genomic RNA from ZIKV (ATCC) was used as standard for viral copy determination. f After GW4869 treatment, cell-free culture supernatants were added to Vero cells and overlaid with Agar gel.

Techniques: Infection, Cell Culture, Control, Microscopy, Isolation, Expressing, Quantitative RT-PCR

Journal: Cell Discovery

Article Title: Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869

doi: 10.1038/s41421-018-0017-2

Figure Lengend Snippet: Astrocytes were infected with ZIKV stains PRVABC59 and treated with doses of GW4869 ranging from 2 μM to 10 μM. After 24 h, the cultures were washed and treated with same doses of GW4869 in fresh medium for another 24 h. a Experimental scheme. b – d EVs were isolated from culture supernatants and visualized through NanoSight ( b ). Quantifications of NanoSight data. *** p < 0.0001 in comparison to ZIKV group (ANOVA, n = 5) ( c ). The levels of flotillin-2 and Alix in EVs, as well as the levels of GFAP and β-actin in WCL were determined by Western blot ( d ). e ZIKA RNA was detected in total cellular RNA through real-time RT-PCR. Data were normalized to GAPDH and presented as fold change compared to ZIKV group. f – i Immunocytochemistry of flavivirus antigen was performed on the ZIKV-infected astrocytes. DAPI was used as a nuclear counterstain. Results are representative of three independent experiments. Scale bar: 50 μm. j Quantification of immunofluorescence data was performed with counting flavivirus antigen in infected cells. k ZIKA RNA was detected in total RNA isolated from cell-free supernatants through real-time RT-PCR. l , m After GW4869 treatment, cell-free culture supernatants were added to Vero cells and overlaid with Agar gel. Viral PFU was determined at 4-day post inoculation through crystal violet staining ( l ). Viral plaques were manually counted and calculated as PFU/ml ( m ). * p < 0.05; *** p < 0. 001, as compared to the ZIKV group

Article Snippet: Quantitative Genomic RNA from ZIKV (ATCC) was used as standard for viral copy determination. f After GW4869 treatment, cell-free culture supernatants were added to Vero cells and overlaid with Agar gel.

Techniques: Infection, Isolation, Comparison, Western Blot, Quantitative RT-PCR, Immunocytochemistry, Immunofluorescence, Staining

Journal: Cell Discovery

Article Title: Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869

doi: 10.1038/s41421-018-0017-2

Figure Lengend Snippet: Astrocytes were infected with ZIKV stains MR766 and treated with doses of GW4869 ranging from 2 μM to 10 μM. After 24 h, the cultures were washed and treated with same doses of GW4869 in fresh medium for another 24 h. a – c EVs were isolated from culture supernatants and visualized through NanoSight ( a ). Quantifications of NanoSight data. *** p < 0.0001 in comparison to ZIKV group (ANOVA, n = 5) ( b ). The levels of flotillin-2 and tTG in EVs, as well as the levels of β-actin in WCL were determined by Western blot ( c ). d ZIKA RNA was detected in total cellular RNA through real-time RT-PCR. Data were normalized to GAPDH and presented as fold change compared to ZIKV group. e ZIKA RNA was detected in total RNA isolated from cell-free supernatants through real-time RT-PCR. Quantitative Genomic RNA from ZIKV (ATCC) was used as standard for viral copy determination. f After GW4869 treatment, cell-free culture supernatants were added to Vero cells and overlaid with Agar gel. Viral PFU were determined at 4-day post inoculation through crystal violet staining

Article Snippet: Quantitative Genomic RNA from ZIKV (ATCC) was used as standard for viral copy determination. f After GW4869 treatment, cell-free culture supernatants were added to Vero cells and overlaid with Agar gel.

Techniques: Infection, Isolation, Comparison, Western Blot, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: (A) H1299 cells were infected with reovirus at an MOI of 3 before fixation at 14-20 hpi. Immunofluorescence staining was conducted with antibodies specific to OC proteins indicated in green. The OC proteins were detected with secondary antibodies conjugated to Alexa 488 (pseudo colored green). Co-immunofluorescence in the same cells was conducted using polyclonal rabbit antibodies raised against reovirus cores (α-Core) detected with secondary antibodies conjugated to Alexa 647 (red). In the merged images and their corresponding zoomed-in regions, white arrows show example regions of core-only staining, while cyan arrows indicate core-positive but μl-negative regions. Similar results were also obtained with monoclonal 10C1 and 4F2 for σ3, 8H6 for µ1, and rabbit σ1-specific polyclonal serum (data not shown). (B) Represented 3D images created from Z-stacks of reovirus infected H1299 cells probed against σ3 (10C1, red), core (α-Core, green), and nuclei (Hoechst dye, blue). (C) Represented 3D images created from Z-stacks of reovirus infected H1299 cells probed against μl (10F6, green), LDs (BODIPY dye, red), and nuclei (Hoechst dye, blue). (bottom) The BODIPY channel showing LDs is toggled off (left) and on (right). (D) Similar to (A) but rabbit polyclonal antibodies to non-structural protein μNS (α-μNS, red) were used instead of α-Core for co-immunofluorescence with antibodies directed towards the OC proteins indicated [deleted words here]. Magenta arrows indicate regions that are μNS-positive but OC-negative, and yellow arrows indicated μNS-positive but μl-negative regions. (E) Represented 3D images created from Z-stacks of reovirus infected H1299 cells probed against σNS (mouse monoclonal 2A9, red), core (α-Core, green), and LDs (BODIPY dye, magenta). (A-E) All images were acquired using spinning disk confocal microscopy and analyzed with Volocity software. Images are representative of at least four images captured for each condition from seven biologically independent experiments.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Immunofluorescence, Staining, Confocal Microscopy, Software

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: T47D cells were infected with reovirus at an MOI of 3 before fixation at 22 hpi. Immunofluorescence staining was conducted with antibodies specific to OC proteins indicated in green (monoclonal 10G10 for σ3, monoclonal G5 for σl, or monoclonal 10F6 for μl as indicated) or σNS (monoclonal 2A9, bottom). The OC proteins were detected with secondary antibodies conjugated to Alexa 488 (pseudo colored green) or Alexa 647 (pseudo-colored red). Co-immunofluorescence in the same cells was conducted using polyclonal rabbit antibodies raised against reovirus cores (α-Core) detected with secondary antibodies conjugated to Alexa 647 (red). In the merged images, white arrows show example regions of core-only staining, while cyan arrows indicate regions of core-positive but μl-negative. Similar results were also obtained with monoclonal 10OC1 and F42 for σ3, 8H6 for μl, and rabbit σ1-specific polyclonal serum (data not shown).

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: (A) H1299 cells were transfected with S1pcDNA3 (σ1), S4pcDNA3 (σ3), and M3pcDNA3 (μNS) with (left) or without M2pcDNA3 (μ1) (right). Immunofluorescence staining was conducted with antibodies specific to OC proteins μ1 (monoclonal 10F6) and σ1 (monoclonal G5 directly labelled with AlexaFluor 647) (top) or μ1 (10F6) and σ3 (monoclonal 10C1 directly labelled with AlexaFluor 647) (bottom), BODIPY for LDs, and DAPI staining for nuclei. μ1 was detected with secondary antibodies conjugated to AlexaFluor 488. (B) H1299 cells were transfected with S2pcDNA3 (σ2), S3pcDNA3 (σNS), S4pcDNA3 (σ3), and M3pcDNA3 (μNS) with (left) or without M2pcDNA3 (μ1) (right). Immunofluorescence staining was conducted with antibodies specific to OC protein μ1 (monoclonal 10F6) and polyclonal rabbit antibodies raised against reovirus cores (α-Core), BODIPY for LDs, and DAPI staining for nuclei. μ1 was detected with secondary antibodies conjugated to AlexaFluor 647, and core protein σ2 with secondary antibodies conjugates to AlexaFluor 488. Represented images were created from Z-stacks acquired using immunofluorescent spinning disk confocal microscopy. Images are representative of at least five images captured for each condition from three biologically independent experiments.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Transfection, Immunofluorescence, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: (A) Representative images of H1299 cells that were infected with reovirus at an MOI of 3 and fixed at the indicated timepoints. Immunofluorescence spinning disk confocal microscopy was used to capture images of cells stained with monoclonal mouse α-σ3 (10G10, Alexa Fluor 647, red) and polyclonal rabbit α-core (Alexa Fluor 488, green). Cell nuclei were stained with DAPI (blue). White arrows indicate example regions of core-only staining. Images are representative of at least five images for each time point from 3 biological experiments. (B-E) σ3/core and σ1/core data were pooled together. At 8hpi there were no visible core- and OC (shared) factories hence labelled “none detected”. (B) The number of core-alone or core- and OC (shared) viral objects were quantified and graphed as a percentage of total viral objects within the cell at each timepoint. Statistical analysis is reported as a two-way ANOVA with multiple comparisons between the mean of each column. (C) The volume of core-alone or shared factory objects were independently added together for each time point and graphed as a percentage of total viral volume within the cell. Statistical analysis is reported as a two-way ANOVA with multiple comparisons between the mean of each column. (D) The factory volume for core-only objects or shared factory objects was found and plotted as individual points at each timepoint. (E) The edge-to-edge distance for pixels in each factory type was calculated and plotted at each time point by object identity. (F) Representative images of H1299 cells infected with reovirus at an MOI of 3 and fixed at the indicated timepoints. Immunofluorescence confocal microscopy was used to capture cells stained with monoclonal mouse α-μ1 (10F6, Alexa Fluor 647, green). Cell LDs were stained with BODIPY 493/503 dye (red) and nuclei were stained with DAPI (blue). Images representative of at least 5 images for each time point and three biological experiments. (G) The percent of LDs within the cell that co-stain with μ1 over the time course. Statistical analysis is reported as aone-way ANOVA with multiple comparisons, comparing the mean of the 8 hpi group to all the others. (H) The mean distance between cellular LDs at each time point. (I) The edge-to-edge distance of LDs from the nucleus. Statistical analysis was done by one-way ANOVA with multiple comparisons, comparing the mean of the mock group to all the others. Each point represents an individual value from n=5-6 images for each timepoint. (J) H1299 cells were infected with T3D PL at an MOI of 3 for 6 hours in the presence of 35 S-methionine/cysteine. Cell lysates were immunoprecipitated using 5μL of the indicated monoclonal antibodies (σ3: 10C1, 5C3, 10G10, 4F2.) or no-antibody control (No-Ab) prior to resolving by SDS-PAGE. (K) Summary diagram depicting factory/protein and LD localization during reovirus infection at ∼8, 12, and 16 hpi. Figure created using Biorender.com. All graphs are plotted as mean +/- 95% CI. Statistical analysis is reported as one-way ANOVA with multiple comparisons between the mean of each column, unless otherwise indicated. **** p<0.0001, *** p<0.001, **p<0.05, ns > 0.05.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Immunofluorescence, Confocal Microscopy, Staining, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: Representative images of H1299 cells that were infected with reovirus at an MOI of 3 and fixed at the indicated timepoints. (A) Immunofluorescence spinning disk confocal microscopy with monoclonal mouse α-σ3 (10G10, Alcxa Fluor 647, red) and polyclonal rabbit α-core (Alexa Fluor 488, green). Cell nuclei were stained with DAPI (blue). (B) Immunofluorescence spinning disk confocal microscopy with monoclonal mouse α-σ1 (G5, Alcxa Fluor 647, red) and polyclonal rabbit α-core (Alexa Fluor 488, green). Cell nuclei were stained with DAPI (blue). (C) Immunofluorescence spinning disk confocal microscopy with monoclonal mouse α-μl (10F6, Alcxa Fluor 488, green) and BODIPY (lipid droplets, red). Cell nuclei were stained with DAPI (blue).

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Immunofluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: (A, B) Representative images of H1299 cells were infected with reovirus at an MOI of 3, and at 10 hours post infection (hpi) cells were either fixed (A) or treated with 100μg/mL cycloheximide or DMSO (B) . (A) Cells were immunofluorescently labelled with monoclonal mouse α-σNS (3E10 directly conjugated to AlexaFluor 568, red) in combination with DAPI for nuclei staining (blue). (B) At 16hpi, cells were fixed and immunofluorescently labelled with monoclonal mouse α-σ3 (10G10, AlexaFluor 647, red), monoclonal mouse α-σNS (3E10 directly conjugated to AlexaFluor 568, cyan) and DAPI for nuclei staining (blue). All images were acquired via immunofluorescence spinning disk confocal microscopy.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Staining, Immunofluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: (A, B) Representative images of Hl 299 cells were infected with reovirus at an MOI of 3, and at IO hpi cells were either fixed (A, B left) or treated with K)0μg/mL cyclohcximidc or DMSO (B right). (A) Cells were immunofluorcsccntly labelled with monoclonal mouse α-3E10 (σNS directly conjugated to AlcxaFluor 568, red) in combination with DAPI for nuclei staining (blue). (B) Cells were fixed and immunofluorescently labelled with monoclonal mouse α-σ3 (10G10, AlcxaFluor 647, red), monoclonal mouse α-3E10 (σNS directly conjugated to AlcxaFluor 568, cyan), polyclonal rabbit α-core (Alexa Fluor 488, green) and DAPI for nuclei staining (blue). All images were acquired via spinning disk confocal microscopy.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: (A-F) Prior to infecting H1299 cells with reovirus at an MOI of 3, cells were transfected with DsiRNAs: an irrelevant control DsiRNA (IRR), a positive control DsiRNA towards an essential core protein (S2 gene; σ2 reovirus core protein) or test condition (M2 gene; μ1 reovirus outercapsid protein). (A) Cell lysates were subjected to SDS-PAGE followed by Western blot analysis to evaluate the extent of gene silencing. (B) Lysates from cells infected and pre-treated with DsiRNAs were collected at 18 hpi and viral titers for each condition were assessed by plaque assay. Statistical analysis is reported as a one-way ANOVA with multiple comparisons between the mean of each column. **** p<0.0001, *** p<0.001, **p<0.05, ns > 0.05. (C) Cells were fixed at 17 hpi and imaged by TEM. Example images from various regions around a representative M2/μ1 (bottom) DsiRNA-transfected cell. ( D) Close up of dominant particles found in IRR-versus M2/μ1 DsiRNA-treated cells. Violin plot shows size (area) of reovirus particles imaged by TEM of IRR DsiRNA versus M2/μ1 DsiRNA transfected cells. Statistical analysis is reported as a two-tailed student t-test and graph is plotted as mean +/- 95% CI. **** p<0.0001, *** p<0.001, **p<0.05, ns > 0.05. (E) Representative images of DsiRNA-treated infected cells at 12hpi and 16hpi. Cells were immunofluorescently labelled with monoclonal mouse α-μ1 (10F6, Alexa Fluor 647, red) or monoclonal mouse α-σ3 (10G10, Alexa Fluor 647, red) in combination with polyclonal rabbit α-core (Alexa Fluor 488, green), Tye563 for DsiRNA staining (cyan), and DAPI for nuclei staining (blue). (C-E) Data from μ1/core and σ3/core co-staining were pooled. (F) The volume and distance from the nucleus was measured for each factory in IRR (blue squares) versus M2/μ1 (red circles) siRNA-treated cells, using the α-core channel to capture both core-only and core+OC shared factories. Quadrants were established based arbitrarily at 10µm 3 volume and 10µm distance from the nucleus to compare ratio of factories between IRR- and M2/μ1 siRNA-treated cells. Data represents eight images per condition and is representative of two independent experiments.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Transfection, Positive Control, SDS Page, Western Blot, Infection, Plaque Assay, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly

doi: 10.1101/2022.06.06.494974

Figure Lengend Snippet: Representative images of DsiRNA-treated infected cells at 12hpi and 16hpi. Cells were immunofluorescently labelled with monoclonal mouse α-μ1 (Top, 10F6, Alcxa Fluor 647, red in merged images) or monoclonal mouse α-σ3 (Bottom, 10G10, Alcxa Fluor 647, red in merged images) in combination with polyclonal rabbit α-core (Alexa Fluor 488, green in merged images), Tye563 for siRNA staining (cyan), and DAPI for nuclei staining (blue). All images were acquired via immunofluorescence spinning disk confocal microscopy.

Article Snippet: Then, blocking solution was removed and cells were incubated with primary antibody solutions (block buffer containing: rabbit pAB against whole virus at 1:1000 (Biologics International Corp), rabbit pAB against core at 1:1000 (Biologics International Corp), mouse α-σ3 (4F2) at 1:250 (DSHB), mouse α-σ3 (10G10) at 1:250 (DSHB), mouse α-μ1 (10F6) at 1:1000 (DSHB), rabbit α-μNS at 1:1000 (Biologics International Corp), mouse α-σNS (2F5) at 1:500 (DSHB), mouse α-σ1 (G5) at 1:250 (DSHB), mouse α-σ3 (10C1 or 10G10) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit (A10475, Invitrogen) at 1:100, mouse α-σ1 (G5) directly labelled with AlexaFluor 647 using the Apex AlexFluor 647 Ab-Labelling Kit, (Invitrogen) at 1:100, or mouse α-σNS (2A9) directly labelled with AlexaFluor 568 using the Apex AlexFluor 568 Ab-Labelling Kit, (A10494, Invitrogen) at 1:100 overnight at 4°C.

Techniques: Infection, Staining, Immunofluorescence, Confocal Microscopy