Journal: Virology

Article Title: Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells

doi: 10.1016/j.virol.2022.01.014

Figure Lengend Snippet: SARS-CoV-2-induced cytopathic effect and viral detection by immunohistochemistry in cells expressing human ACE2 and TMPRSS2 . Vero, DF1, DF1 expressing both human ACE2 and TMPRSS2 (++), MDCK, and MDCK expressing both human ACE2 and TMPRSS2 (++) were grown at 37C in 5% CO 2 on glass chamber slides. Cells were inoculated with SARS-CoV-2 at MOI of 1. At 48 h post inoculation monolayers were examined for cytopathic effect and detection of virus with rabbit monoclonal antibodies against SARS-CoV-2 spike and nucleoprotein. Cells were washed 3 times with PBS and incubated in the secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor® 555) for 1 h at room temperature. Cells were then washed counterstained with DAPI. Immunofluorescence was visualized with an EVOS 5000.

Article Snippet: Primary antibodies against SARS-CoV-2 included rabbit anti-Nucleoprotein MAb (Origene) and rabbit anti-Spike MAb (Origene), diluted as above, were added for 1 h at room temperature.

Techniques: Immunohistochemistry, Expressing, Virus, Bioprocessing, Incubation, Immunofluorescence

Journal: Virology

Article Title: Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells

doi: 10.1016/j.virol.2022.01.014

Figure Lengend Snippet: SARS-CoV-2 induced cytopathic effect and viral detection by immunohistochemistry in DF1 cells expressing animal species ACE2 and TMPRSS2 . DF1 cells expressing animal ACE2 and TMPRSS2 were grown at 37C in 5% CO 2 on glass chamber slides. Cells were inoculated with SARS-CoV-2 at MOI of 1. At 48 h post inoculation monolayers were examined for cytopathic effect and detection of virus with rabbit monoclonal antibodies against SARS-CoV-2 spike and nucleoprotein. Cells were washed 3 times with PBS and incubated in the secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor® 555) for 1 h at room temperature. Cells were then washed counterstained with DAPI. Immunofluorescence was visualized with an EVOS 5000.

Article Snippet: Primary antibodies against SARS-CoV-2 included rabbit anti-Nucleoprotein MAb (Origene) and rabbit anti-Spike MAb (Origene), diluted as above, were added for 1 h at room temperature.

Techniques: Immunohistochemistry, Expressing, Virus, Bioprocessing, Incubation, Immunofluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome

doi: 10.1101/2020.12.20.423533

Figure Lengend Snippet: A . Protein alignment of SARS-Cov-ORF3a and SARS-Cov-2-ORF3a. Dark blue shading shows identical residues, light blue shading shows similar residues. B . PCR of genomic DNA from ORF3a two independent transgenic lines. ORF3a band is indicated with red arrow. C . RT-PCR of ORF3a mRNA shows ORF3a expression is induced in muscle ( Mef2 . Gal4 ) and in the nervous system ( GMR . Gal4 ). ORF3a band is shown (red arrow). D . Representative result of a climbing assay. elav > ORF3a flies (right) had reduced motor function. E . 6d old female flies. elav > ORF3a flies (right) showed severely swollen abdomens. F . Survival curves of Mef2 . Gal4 (control) and Mef2 > ORF3a adult flies. Mef2 > ORF3a median lifespan was not significantly different than controls. n>60 flies per genotype. G . Longitudinal study of climbing ability. Locomotor activity was unaffected in Mef2 > ORF3a flies. Each data point represents percent of flies that climbed above 5 cm, averaged for 3 independent trials. H . Quantification of embryonic body wall muscle phenotypes from . Diagram shows the 30 muscles per embryonic segment; blue muscles were 100% normal in >60 Mef2 > ORF3a embryonic segments, red muscles showed a developmental phenotype in at least 1 of the 60 segments. Dot plot shows frequency of muscle phenotypes is <9.0%. (ns) not significant.

Article Snippet: Antibodies used include anti-SARS-CoV-2-ORF3a (1:200, 101AP, FabGennix International Inc), anti-Tropomyosin (1:600, MAC141, Abcam).

Techniques: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Climbing Assay, Activity Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome

doi: 10.1101/2020.12.20.423533

Figure Lengend Snippet: A . ORF3a localization. elav . Gal4 and elav > ORF3a adult brains labeled for ORF3a (green) and DAPI (blue). ORF3a localized to cytoplasmic foci. B . Survival curves of elav . Gal4 (control) and elav > ORF3a adult flies. elav > ORF3a median lifespan was significantly less than controls. n>60 flies per genotype. C . Longitudinal study of climbing ability. Locomotor activity was reduced in elav > ORF3a flies. Each data point represents percent of flies that climbed above 5 cm, averaged for 3 independent trials. See . D . F 1 adult progeny from elav . gal4,Sb/Tb X UAS-ORF3a/Sb F 0 parents. 3 phenotypic classes with an equivalent number of progeny (33.3%) were expected. elav > ORF3a flies were underrepresented. E . Micrographs of 3d adult eyes. GMR > ORF3a eyes were rough and disorganized. F . Stage 16 embryonic body wall muscles labeled with Tropomyosin. Mef2 > ORF3a embryos showed largely normal body wall musculature (see for quantification). G . Apoptosis assay. elav . Gal4 and elav > ORF3a adult brains labeled for cleaved Caspase-3 (green) and DAPI (blue). ORF3a induced Caspase-3 cleavage. H . Immunoblot of whole brain lysates from 3d elav . Gal4 and elav > ORF3a adults validated results shown in ( G ). I . qRT-PCR of RNA from 3d old adult heads. Transcripts encoding IMD pathway reporters ( dipt and attA ) and a Toll pathway reporter ( Drs ) were enriched in elav > ORF3a flies. n>20 unless otherwise noted. Error bars represent standard error of the mean (SEM) from at least three independent replicates. Significance was determined by log-rank test (B), two-way ANOVA (C), and student’s t-test (I). *p < 0.05, **p < 0.01, ****p < 0.0001, (ns) non-significant.

Article Snippet: Antibodies used include anti-SARS-CoV-2-ORF3a (1:200, 101AP, FabGennix International Inc), anti-Tropomyosin (1:600, MAC141, Abcam).

Techniques: Labeling, Activity Assay, Apoptosis Assay, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome

doi: 10.1101/2020.12.20.423533

Figure Lengend Snippet: A . Survival curves of elav . Gal4 (control), elav > ORF3a , and CQ treated elav > ORF3a adult flies. CQ treatment significantly extended elav > ORF3a median lifespan. n>60 flies per genotype B . Longitudinal study of climbing ability. Locomotor activity was significantly improved in elav > ORF3a flies treated with CQ. Each data point represents percent of flies that climbed above 5 cm, averaged for 3 independent trials. C . F 1 adult progeny treated with CQ from elav . gal4,Sb/Tb X UAS-ORF3a/Sb F 0 parents. 3 phenotypic classes with an equivalent number of progeny (33.3%) were expected. CQ treatment improved elav > ORF3a survivability to adulthood (compare to ). D . Immunoblot of whole brain lysates from 3d elav . Gal4, elav > ORF3a , and CQ treated elav > ORF3a adults. CQ reduced cleaved Caspase-3 levels in elav > ORF3a flies. E , F . qRT-PCR of RNA from 3d old adult heads. CQ treatment blunted the expression of the Toll pathway reporter Drs (E), but not IMD pathway reporters ( dipt and attA ; F) in elav > ORF3a flies. G . Live imaging of HELA cells transfected with CMV-GFP-ORF3a (green) for 24hr, treated with or without CQ, and labeled with Lysotracker (red). ORF3a transfected cells showed reduced Lysotracker staining (deacidified lysosomes) than untransfected controls (outlined in heat map). CQ treatment restored Lysotracker staining in ORF3a expressing cells (H) . n>20 unless otherwise noted. Error bars represent standard error of the mean (SEM) from at least three independent replicates. Significance was determined by log-rank test (A), two-way ANOVA (B), and student’s t-test (H). *p < 0.05, **p < 0.01, ****p < 0.0001, (ns) non-significant.

Article Snippet: Antibodies used include anti-SARS-CoV-2-ORF3a (1:200, 101AP, FabGennix International Inc), anti-Tropomyosin (1:600, MAC141, Abcam).

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Expressing, Imaging, Transfection, Labeling, Staining