Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Characterization of porcine cell lines with regard to their CD46 pig expression. (A) Phenotypical characterization of porcine cell lines by immunofluorescence staining using a CD46 pig -specific mab (green, MCA2310GA) and DAPI (blue). Asterisks (*) indicate cell lines subjected to conventional RT-PCR for subsequent sequencing. (B) Immunofluorescence staining of APPV P100 (porcine APPV-specific antiserum, red), CD46 pig (green, MCA2310GA), and DAPI (blue) at 72 h after infection of NPTr cells. (C) Strategy used for genetic characterization and manipulation of the CD46 pig gene locus. The CD46 pig -encoding mRNA was amplified by two RT-PCRs (101/710 and 604/1192) for subsequent cloning and sequencing. Absence of a CD46 pig -encoding mRNA in porcine lymphoma cell line 38A 1 D was confirmed by RT-PCRs targeting the individual CD46 pig domains (primer pairs: 192/353, 387/543, 571/710, 766/937). Positions of signal peptide (SP), complement control proteins 1 to 4 (ccp1-4), serine, threonine, proline-rich region (STP), and transmembrane domain (TM) encoded by the mRNA are depicted. In addition, positions of guide RNAs (gRNA CD46-2 and -7) used for construction of CD46 pig knockout cells are indicated (for details see ).

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Expressing, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Sequencing, Infection, Amplification, Cloning, Control, Knock-Out

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Characterization of genetically engineered CD46 pig knockout cells. (A) Phenotypical characterization of CD46 pig knockout cells by immunofluorescence staining using a mab against CD46 pig (green, MCA2310GA) and DAPI (blue). Immunofluorescence staining of CD46 pig (green) from wild-type (WT) cell lines served as a control and is shown in . (B) CRISPR/Cas9 induced genome alterations on both alleles characterized by sequencing of plasmids containing PCR amplicons flanking target sites of the guide RNAs (primers 101fw/710rev). Consensus nucleotide sequences and deduced amino acid sequences of the regions encoding the C terminus of SP and the N terminus of ccp1 are shown. For comparison, nucleotide and deduced CD46 pig amino acid sequences of WT as determined for SPEV and PK15 cells are given in the top row. The border between SP/ccp1 and position of gRNAs including respective protospacer adjacent motifs (PAM, boxed) are indicated. For selected engineered CD46 pig knockout cell lines (ΔCD46) the corresponding sequences including deletions (Δ nt) and insertions (+ nt) are shown below the WT CD46 sequence.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Immunofluorescence, Staining, Control, CRISPR, Sequencing, Comparison

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Primers used in this study

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Sequencing, Plasmid Preparation

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Relevance of CD46 pig for the entry of porcine pestiviruses. Wild-type (WT) SPEV and PK15 as well as CD46 pig knockout cell lines (SPEVΔCD46 clones 2 and 7 and PK15ΔCD46 clone 2) were infected with APPV P17 , APPV P100 , BuPV, and CSFV strains Alfort-Tübingen (AlfT), Diepholz, Riems, Koslov, and Paderborn at an MOI of 1, respectively. Immunofluorescence staining was performed at 72 h p.i. using porcine APPV-specific antiserum, a porcine BuPV-specific antiserum, and a mab against CSFV, respectively. A strong reduction of APPV infection is evident on all SPEVΔCD46 cell lines in comparison to that on SPEV cells. PK15 cells display significantly lower permissivity to APPV P100 compared to that of SPEV cells. Non-culture-adapted APPV P17 obtained from early passage revealed the same CD46 pig dependency as the culture-adapted variant (APPV P100 ). With regard to infections with CSFV and BuPV, there are no differences in permissivity between the WT and the CD46 pig knockout cell lines.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Clone Assay, Infection, Immunofluorescence, Staining, Comparison, Variant Assay

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Production of infectious particles and RNA replication of porcine pestiviruses in dependence on CD46 pig . Wild-type (WT) SPEV and PK15, as well as CD46 pig knockout cell lines (SPEVΔCD46 clones 2 and 7 and PK15ΔCD46 clone 2), were infected with APPV P100 , CSFV Alfort-Tübingen (AlfT), and BuPV at an MOI of 1, respectively. (A) Supernatants were harvested 72 h p.i. to determine virus titers by using endpoint dilution assays in quadruplicates and in three repetitions. (B) Cells were collected at 72 h p.i. for RNA preparation and subsequent RT-PCR analysis. TaqMan based qRT-PCR assays were used for detection of CSFV and APPV genomes, whereas a SYBR green-based real-time RT-PCR was performed for detection of BuPV genomes. 30 ng total RNA was used per reaction. Samples collected from three individual experiments were tested in duplicates. Mean values with standard deviations are shown. APPV genome copy numbers obtained from WT cells are significantly higher compared to those from CD46 pig knockout cells (***, P < 0.0001, highly significant; *, P < 0.01, significant). CSFV and BuPV genome levels obtained from WT cells did not show significant differences compared to genome loads detected in infected knockout cells.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Knock-Out, Clone Assay, Infection, Virus, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Impact of CD46 pig at early time points of porcine pestivirus infections. (A) Immunofluorescence analysis of CSFV- and BuPV-infected cells. Wild-type (WT) PK15 and PK15ΔCD46 clone 2 cells were infected with CSFV strains Alfort-Tübingen (AlfT), Diepholz, Riems, Koslov, Paderborn, and BuPV at an MOI of 1. Infections with different CSFV strains and BuPV showed no dependency on CD46 pig even very early after infection (16 h p.i.). (B) Fluorescence in situ hybridization (FISH) analysis of APPV-infected cells. WT SPEV and PK15 as well as CD46 pig knockout cell lines (SPEVΔCD46 clone 2 and PK15ΔCD46 clone 2) were infected with cell culture-adapted APPV P100 at an MOI of 0.5. Scale bars indicate 100 µm for lower magnification and 50 µm for higher magnification. A strong reduction of APPV P100 infection is evident on both CD46 pig knockout cell lines in comparison to that on WT cells at early time point of infection (16 h p.i.). APPV P100 genomes were observed only on single CD46 pig knockout cells within the infected wells. APPV P100 infection of CD46 pig -expressing WT SPEV cells at a later time point (72 h p.i.) and noninfected SPEV cells (NIC) served as controls.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Immunofluorescence, Infection, Fluorescence, In Situ Hybridization, Knock-Out, Cell Culture, Comparison, Expressing

Journal: Journal of Virology

Article Title: Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus

doi: 10.1128/JVI.02186-20

Figure Lengend Snippet: Comparison of E2 envelope protein sequences of pestiviruses. (A) Phylogenetic tree (maximum likelihood) based on E2 amino acid sequences of known pestivirus species (APPV: AUL76967 ; bat: AFK85014 , AYV99177 ; rodent: ATP66856 , ATP66857 , YP009109567; pangolin: QIE06437 ; LindaV: YP009407716; whale: MK910228 ; BuPV: YP008992092; BDV: AAC16444 ; Aydin: YP006860588; ovine Italy: MG770617 ; giraffe: NP620053; pronghorn: YP009026415; BVDV-1: Q01499 ; BVDV-2: YP009513240; BVDV-3: AB871953 ; CSFV: YP009508222). APPV and CSFV sequence (bold) are the same as shown in the alignment. (B) Alignment (ClustalW) of APPV (isolate L277) and CSFV (Alfort 187) E2 amino acid sequences. Highlighted is the CSFV sequence analogous to the motif in the E2 of BVDV folding into a hairpin that might serve as ligand to the CD46 bov receptor . The positions of two nonsynonymous mutations (N751K and D752N) which occurred during cell culture adaptation of APPV are highlighted by a box.

Article Snippet: Staining was performed using commercially available mabs against CD46 pig (MCA2310GA and MCA2262GA, Bio-Rad, 1:500 dilution) and a secondary mab Alexa fluor 488 goat anti-mouse IgG (A11029, Invitrogen, 1:1,000 dilution).

Techniques: Comparison, Sequencing, Cell Culture