Effects of glycine 64 substitutions in RNA-dependent RNA polymerase on ribavirin sensitivity and pathogenicity of coxsackievirus A6

Fig. 1. Construction and analysis of wild-type and mutant CVA6 clones. (A) Full-length cDNAs of infectious CVA6 strain 54203/HeB/2012 was cloned in pcDNA3.1(+) and subjected to site-directed mutagenesis to obtain CVA6-G64S and CVA6-G64T mutants. (B) Live clonogenic strains were rescued by in vitro transcription and sequenced after six consecutive passages in RD cells. (C) Immunofluorescence detection of the viruses in RD cells infected at MOI = 0.01 for 24 h. (D, E) Viral growth kinetics in RD cells infected at MOI = 0.1 based on viral titers (TCID50) and RNA content measured by qRT-PCR at 2,4,8,12,24,48 h post infection, The virus titers were finally calculated using the Kärber formula. The RNA copy numbers were calculated based on the standards in our laboratory. The results are expressed as the means ± standard deviation (n = 3).

Fig. 2. Survival rate, body weight, clinical score and clinical signs results of ICR suckling mice infected with CVA6, CVA6-G64S, and CVA6-G64T. One-day-old ICR suckling mice were intramuscularly injected with 107 TCID50 of CVA6, CVA6-G64S, and CVA6-G64T and observed for survival (A), body weight (B), clinical score (C), and clinical signs of "rescue" CVA6 infection in suckling mice at 3 dpi. (D).

Fig. 3. Titers of CVA6, CVA6-G64S, and CVA6-G64T in the tissues of infected ICR mice. One-day-old ICR suckling mice were intramuscularly injected with 107 TCID50 of CVA6, CVA6-G64S, and CVA6-G64T, sacrificed 1, 3 and 4 days post infection and analyzed for virus titers in the brain (A), lung (B), heart (C), and hind limb muscle (D). The results are presented as the means ± standard deviation (n = 5). **P < 0.01, ***P < 0.001, and ****P < 0.0001 according to two-tailed Student's t-test; ns, not significant.

Fig. 4. Histopathology analysis of mouse tissues after infection with CVA6, CVA6-G64S, and CVA6-G64T. One-day-old ICR suckling pups mice were intramuscularly injected with 107 TCID50 of CVA6, CVA6-G64S, and CVA6-G64T or culture medium (control) and analyzed for pathological changes in the indicated tissues after HE staining. After 3 days of post-infection, brain, lung, heart, and muscle tissue was collected from Suckling pups mice in each group. (A1–4) Brain, (B1–4) lung, (C1–4) heart, (D1–4) hind limb muscle. Magnification, ×200. The images show representative results for 7–9 mice with the same clinical signs.

Fig. 5. Immunohistochemistry analysis of mouse tissues after infection with CVA6, CVA6-G64S and CVA6-G64T. One-day-old ICR suckling pups mice were intramuscularly injected with CVA6, CVA6-G64S, CVA6-G64T, or culture medium (control) and analyzed at day 3 post infection for virus presence in the indicated tissues after staining with an anti-CVA6 antibody. (A1–4) Brain, (C1–4) lung, (E1–4) heart, (G1–4) hind limb muscle. Magnification, ×200. (B1–4) Brain, (D1–4) lung, (F1–4) heart, (H1–4) hind limb muscle. Magnification, ×400.

Fig. 6. Ribavirin sensitivity testing of CVA6, CVA6-G64S, and CVA6-G64T strains. (A) Cytotoxicity of ribavirin, RD cells were treated with the indicated concentrations of ribavirin and analyzed for viability using the CCK-8 assay. (B) Ribavirin sensitivity of CVA6, CVA6-G64S, and CVA6-G64T. RD cells were infected with the viruses at MOI = 0.1 for 2 h, treated with ribavirin (0.8 mM), and analyzed for TCID50. (C) The ribavirin susceptibility ratio was calculated as the viral titer in the absence of ribavirin divided by the titer in the presence of ribavirin, based on the final titers shown in panel B. The results are presented as the means ± standard deviation (n = 3). **P < 0.01, ***P < 0.001, and ns, not significant.

Fig. 7. Guanidine sensitivity testing of CVA6, CVA6-G64S, and CVA6-G64T strains.(A) Cytotoxicity of guanidine, RD cells were treated with the indicated concentrations of guanidine and analyzed for viability using the CCK-8 assay. (B–D) Guanidine sensitivity of CVA6 (1–7), CVA6-G64S (1–7), and CVA6-G64T (1–7). RD cells were infected with the viruses at MOI = 0.1 for 2 h, treated with Guanidine (0.8 mM), and analyzed for TCID50. (E, F) The guanidine resistance ratio of CVA6–1 to CVA6–7, CVA6-G64S-1 to CVA6-G64S-7, and CVA6-G64T-1 to CVA6-G64T-7 were calculated as the viral titer in the presence of guanidine divided by the titer in the absence of guanidine, based on the final titers shown in panel B, C D. The results are presented as the means ± standard deviation (n = 3). **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant.

Fig. 8. Nucleotide and amino acid mutations in the 2C and 3D coding regions of CVA6, 3D-G64S, and 3D-G64T after passage in the presence of ribavirin (A) CVA6, CVA6-G64S, and CVA6-G64T were serially passaged 15 times in RD cells in the presence of 0.8 mM ribavirin. Next generation sequencing (5000reads) was used to detect the nucleotide mutations in 2C and 3D regions, and more than 5 % of the mutations were analyzed for amino acid mutations. (B) Next generation sequencing was used to detect the mutation of CVA6, CVA6-G64S and CVA6-G64T under ribavirin.