Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells

Figure 1. Genome copy numbers measured by RT-qPCR from cell culture supernatants collected from persistent infection models over time. In all the models the copy number eventually stabilized to approximately 108 to 109 copies/mL. Addition of fresh cells into three of the models is indicated by an arrow.

Figure 2. (a) Immunostaining of persistently CVB1 infected PANC-1 and 1.1B4 cells from two-timepoints (3.4–4.7 months post-infection in top row and 12.7–14.2 months post-infection in bottom row, corresponding to TP1 and TP2 respectively) and of uninfected control cells (CNTRL). VP1 protein (red) and dsRNA (yellow), and the average proportion of cells positive for viral VP1 protein was 22.9 ± 13.7% at the later time point (12.7–14.2 months post-infection) ranging from 7.1 ± 0.5% in the CVB1 10796 persistent infection model in 1.1B4 cells to 41.7 ± 9.8% in CVB1 10797 model in PANC-1 cells (altogether three microscopic fields were counted in each model including a total of 16,338 cells). (b) The virus amino acid sequence of the epitope region of the 3A6 antibody used in VP1 staining is shown for each PIDV. The red frames mark the mutations observed in CVB strains at last time point.

Figure 3. Plaque morphology of each PIDV over time. Plaque size reduces over time during the development of persistence in PANC-1 and 1.1B4 cell lines with the exception of ATCC-PIDV[1.1B4] (6th column).

Figure 4. Lolliplots of each PIDV at the last timepoint. Single nucleotide polymorphism (SNP) are indicated by lollipop. All non-synonymous SNPs, and SNPs at both UTR are indicated. Grey bar below the genome bar characterizes the read depth (>10). (a) ATCC E1-PIDV[PANC-1]. (b) ATCC E2-PIDV[PANC-1]. (c) 10796-PIDV[PANC-1]. (d) 10797-PIDV[PANC-1]. (e) 10802-PIDV[PANC-1]. (f) ATCC-PIDV[1.1B4]. (g) 10796-PIDV[1.1B4]. (h) 10802-PIDV[1.1B4].

Figure 5. Mutation in 5′-UTR. SNPs are color coded indicating different PIDVs. Six distinct domains of the 5′-UTR are indicated by horizontal bars. Domain I is the cloverleaf structure and the IRES covers the domains II-V. Domain I: base pairs 2–87; Domain II: base pairs 105–181; Domain III: base pairs 189–209; Domain IV: base pairs 213–456; Domain V: base pairs 466–636 and Domain VI: base pairs 651–669 [54].

Figure 6. Non-synonymous mutations in PIDVs, marked with red frames, accumulated in the BC loop, DE loop and EF loop regions and at the end of the VP1 protein at TP2. Mutation K257R is indicated with an arrow.

Figure 7. Mutations of PIDVs in capsid proteins VP2-4 in TP2. (a) Mutations on VP2 puff region. (b) Mutations on VP3 knob region. (c) Mutations on VP4 infection-enhancing epitope AA 11-30.

Figure 8. ORF2p and its conserved WIGHPV domain in TP2. One mutation was observed in conserved WIGHPV domain in 10802-PIDV[1.1B4].

Figure 9. (a) 3D model of CVB1 virus pentameric structure displayed in ChimeraX from TP2. One pentamer consists of five protomers, each constructed by viral proteins VP1-4. Blue indicates mutation in one strain; yellow, mutations in 2–3 strains; orange, mutations in 4–5 strains; purple, mutations in 6 strains; and red, mutations (K257R) in the AA in all PIDVs. (b) 3D model of CVB1 with CAR binding site marked with blue color and DAF binding site with green color, shared AAs are marked with yellow color. (c) Mutations in CAR and DAF binding site are marked with red. Number 1. is mutation on AA202 in VP1 shared by three PIDVs, number 2. is mutation on AA 162 in VP2 shared by five PIDVs, number 3. is mutation on AA 165 in VP2 shared by three PIDVs and number 4. is AA 237 in VP3 shared by three PIDVs. AA257 in VP1, which has K257R mutation found in all studied PIDV strains, is also indicated.