Journal: bioRxiv

Article Title: Powassan Viruses Spread Cell to Cell During Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

doi: 10.1101/2021.09.30.462684

Figure Lengend Snippet: (A) I. scapularis ticks were collected in Long Island, N.Y. Tick homogenates from groups of 10 ticks were added to wells of 24-well plates containing VeroE6 cells. After 7 days, cells were fixed and immunoperoxidase stained using a specific anti-POWV HMAF (1:10,000). Wells #9 and # 41 were positive for POWV antigen and formed distinct infected cell foci. First passage (P1) virus was inoculated into a well of 24-well plate, and cells were fixed and immunoperoxidase stained using a specific anti-POWV HMAF (1:10,000) after 7 days (left panels). Right panels show the magnification of the cell foci from initial viral isolation and P1 infection of VeroE6 cells. Bars represent 50 μm. (B) VeroE6 were infected with POWV-LI9 (P3) at an MOI of 5 or mock-infected, cells were fixed at 1, 3, 5, 8, and 10 dpi, and immunoperoxidase stained using anti-POWV HMAF (1:10,000). Bars represent 50 μm. (C) Viral titration from POWV-infected VeroE6 supernatants was determined by FFU assay at 1 dpi in Vero E6 cells.

Article Snippet: One week after inoculation, infected cell supernatants were harvested and cell monolayers were immunoperoxidase stained using anti-POWV HMAF antibody (ATCC).

Techniques: Staining, Infection, Virus, Isolation, Titration

Journal: bioRxiv

Article Title: Powassan Viruses Spread Cell to Cell During Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

doi: 10.1101/2021.09.30.462684

Figure Lengend Snippet: (A) Maximum-likelihood tree analysis between POWV-LI9 and POWV-related strains using complete genome sequences . Branch numbers are bootstrap confidence estimates based on 100 replicates. Tick-borne encephalitis virus (TBEV) is shown as an outgroup. (B) Multiple-sequence analysis comparison using Clustal Omega between POWV-LI9 and other POWV sequences deposited in the GenBank. POWV genomes were divided into three groups based on the genetic identity compared with POWV-LI9: sequences from the viruses collected in the Northeastern U.S. (group A), in Wisconsin (group B), and sequences from lineage I POWV (group C). Black bars within the sequences represent nucleotide differences compared to POWV-LI9. (C) Among 5 residue differences with Northeastern POWVs there are 2 not conserved residues in the Envelope (Env), including Domain II residues S119 and K151 in POWV-LI9 and additional unique residues in the NS2B (F125), NS3 (D402) and NS5 (T282) proteins of POWV-LI9.

Article Snippet: One week after inoculation, infected cell supernatants were harvested and cell monolayers were immunoperoxidase stained using anti-POWV HMAF antibody (ATCC).

Techniques: Virus, Sequencing, Comparison, Residue

Journal: bioRxiv

Article Title: Powassan Viruses Spread Cell to Cell During Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

doi: 10.1101/2021.09.30.462684

Figure Lengend Snippet: (A) hBMECs were infected with POWV-LI9 (P3) at an MOI of 5 or mock-infected, cells were fixed at 1, 2, 3, 6, and 8 dpi, and immunoperoxidase stained using anti-POWV HMAF (1:10,000). Bars represent 50 μm. (B) Viral titration from POWV-infected hBMECs supernatants was determined by FFU assay at 1 dpi in Vero E6 cells. (C) POWV-LI9 was pretreated with WGA (1 μg/mL) for 5 min. Control mock WGA treated media (1 μg/mL), or WGA treated POWV was adsorbed to hBMECs for 1 hour, and subsequently cells were PBS washed and resupplemented with EBM2-2% FBS media. Cells were fixed and immunoperoxidase stained using anti-POWV HMAF (1:10,000) at 1 dpi. (D) Human brain pericytes (hPCs) were infected with POWV-LI9 (P3) at an MOI of 5 or mock-infected, cells were fixed at 1, 2, 3, 6, and 8 dpi, and immunoperoxidase stained using a specific anti-POWV HMAF (1:10,000). Bars represent 50 μm. (E) Viral titers from POWV-infected pericyte supernatants were determined by FFU assay at 1 dpi in Vero E6 cells.

Article Snippet: One week after inoculation, infected cell supernatants were harvested and cell monolayers were immunoperoxidase stained using anti-POWV HMAF antibody (ATCC).

Techniques: Infection, Staining, Titration, Control

Journal: bioRxiv

Article Title: Powassan Viruses Spread Cell to Cell During Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

doi: 10.1101/2021.09.30.462684

Figure Lengend Snippet: (A) Polarized hBMECs and VeroE6 cells grown for 5 days in Transwell inserts, were apically infected with POWV-LI9 (MOI 5). Cells were visualized by phase contrast microscopy for CPE and costained with Calcein-AM (green-live cells) and propidium iodide (red-dead cells) to visualize cell viability 3 dpi. (B) Confocal immunofluorescence. VeroE6 grown on microslides were infected with POWV-LI9 (MOI, 5). Cells were fixed 2 dpi, immunostained with anti-POWV HMAF and anti-ZO-1 antibodies, DAPI counterstained, and visualized by confocal microscopy. Experiments were done in triplicate, repeated at least three times, and representative data are presented. Bars represent 10 μm. (C) Polarized hBMECs and VeroE6 cells grown for 5 days in Transwell inserts, were apically infected with POWV-LI9 (MOI, 5) in triplicate, and TEER was measured 1 to 3 dpi. (D) hBMECs and (E) VeroE6, grown for 5 days in Transwell inserts, were apically infected with POWV-LI9 (MOI, 5) in triplicate. Cells were washed and viral titers from both apical or basolateral compartments were titered 3 dpi. (F) Schematic representation of hBMECs and human pericyte co-culture model (left); hBMECs were seeded on the apical side of Transwell inserts, grown for 7 days confluent by TEER resistance. hBMECs were mock or POWV infected from the apical side for 1 h, cells were washed, and 1 dpi Transwell inserts were transferred to 24-well plates containing monolayers of primary human pericytes. Pericytes were immunoperoxidase stained for POWV antigen 3 dpi using anti-POWV HMAF (1:10,000). Bars represent 50 μm.

Article Snippet: One week after inoculation, infected cell supernatants were harvested and cell monolayers were immunoperoxidase stained using anti-POWV HMAF antibody (ATCC).

Techniques: Infection, Microscopy, Immunofluorescence, Confocal Microscopy, Co-Culture Assay, Staining

Journal: bioRxiv

Article Title: Powassan Viruses Spread Cell to Cell During Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

doi: 10.1101/2021.09.30.462684

Figure Lengend Snippet: (A) Vero E6, hBMECs and human brain pericytes (hPCs) were pretreated with IFN-α (1,000 U/ml) for 2 or 18 h and before POWV infection (MOI, 5). One day post-infection cells were methanol fixed, immunoperoxidase stained using anti-POWV HMAF (1:10,000) and the number of infected cells quantitated and compared untreated controls. (B) hBMECs and (C) hPCs were mock or POWV-LI9 infected (MOI 5) and 1-3 dpi supernatants of cells were assayed for secreted IFNβ by ELISA (R&D systems).

Article Snippet: One week after inoculation, infected cell supernatants were harvested and cell monolayers were immunoperoxidase stained using anti-POWV HMAF antibody (ATCC).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Powassan Viruses Spread Cell to Cell During Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes

doi: 10.1101/2021.09.30.462684

Figure Lengend Snippet: (A) POWV-LI9 was pretreated with WGA (1 μg/mL) for 5 min. Control mock WGA treated media (1 μg/mL), or WGA treated POWV was adsorbed to hBMECs for 1 hour, and subsequently cells were PBS washed resupplemented with EBM2-5% FBS media and grown for 1-3 days. Total cellular RNA from mock-WGA-treated and WGA-treated POWV infected hBMECs was harvested in RLT buffer, purified, DNAse treated and subjected to Affymetrix array analysis. Log 2 -induced genes from POWV-infected samples were compared to respective mock-infected time points, generating a heatmap with 0-to 100-fold induction scale, where induced genes above 100-fold were in black. Genes were separated into functional categories as chemokines, interferons (IFN), ISGs, IRFs, cytokines (Cytok.), transcriptional factors (TFs), apoptosis-related genes (Apop.), and ISGylation (ISGyl.) genes. (B) Selected genes from the Affymetrix array analysis were confirmed by qRT-PCR. (C) Total RNA from mock- and POWV-infected hPCs was isolated at 1-3 dpi and submitted to Affymetrix array analysis as in A. Log 2 -induced genes from POWV-infected samples were compared against its respective mock-infected timepoint, generating a heatmap with 0-to 500-fold (left graph) or 100-fold (right graph) induction scale, where induced genes above 500- or 100-fold were represented as black color. Genes were separated in functional categories as chemokines, interferons (IFN), ISGs, IRFs, cytokines (Cytok.), transcriptional factors (TFs), apoptosis-related (Apop.), ISGylation-related (ISGyl.), and complement related genes. (D) Selected genes induced by POWV infection of pericytes by Affymetrix array were confirmed by qRT-PCR. (E) Gene Ontology (GO) enrichment analysis was conducted by querying the upregulated genes from infected hBMECs or hPCs 1, 2, and 3 dpi using STRING v11 . GO terms from selected biological processes were presented.

Article Snippet: One week after inoculation, infected cell supernatants were harvested and cell monolayers were immunoperoxidase stained using anti-POWV HMAF antibody (ATCC).

Techniques: Control, Infection, Purification, Functional Assay, Quantitative RT-PCR, Isolation