Shown by mean of triplicates EM (left y-axis; break indicates the cut-off in the assay), and emergence of substitutions for ED43-20m (lines) during adaptation following transfection (T; samples not obtainable from days 0 to 27) and serial passages (P1, 2, three, and so forth.). Only substitutions with SNP frequencies 20 at any time-point are shown (right y-axis). For substitutions that emerged with similar patterns, means EM are shown. The substitutions with similar SNP frequencies are grouped and shown together with the same colour. (C) Clonal ORF sequence analysis of ED43-20m virus obtained from the 6th and 10th passages; phylogeny was generated as described.31 Subsequently, ancestral reconstruction was performed depending on alignment and phylogeny to predict the look of respective substitutions. Only substitutions indicated in (B) are shown. (D,E) Infectivity titres (y-axis) of precise ED43 recombinant viruses at indicated days (x-axis) immediately after transfection of Huh7.five cells. J6/JFH1 was included as a handle. The data have been obtained from a separate experiment with equivalent J6/ JFH1 titres. Statistical significance (p0001, two-way ANOVA) is carried out for indicated samples and shown with asterisks (). ANOVA, evaluation of variance; FFU, focus-forming unit; ORF, open reading frame; SNP, single nucleotide polymorphism; SEM, common error in the imply.630 Pham LV, et al. Gut 2022;71:62742. doi:10.1136/gutjnl-2020-TableSNP frequencies ( ) H77 AA pos 195 271 405 458 732 753 769 797 827 848 1124 1291 1383 1465 1466 1572 1672 1786 1822 1865 1870 1909 1973 2226 2257 2262 2271 2329 2366 2388 2413 2545 2547 2565 2597 2598 2675 K D K N N D D N N E D E D G M T 2388 2416 2548 2550 2568 2600 2601 2678 T A 2369 T A 2332 I V 2274 D E 2265 17.eight 46.7 5.four 45.9 11.2 100 100 11.8 5.9 95.eight 8.six 100 A T 2261 5.3 L P 2226 7.7 A T 1973 25.8 G A 1909 38.0 S N 1870 one hundred T A 1865 100 T A 1822 100 A V 1786 one hundred 100 100 100 one hundred 87.eight 88.4 88.4 9.6 88.four 9.0 7.3 100 100 99.7 one hundred A S 1672 one hundred 100 F L 1572 45.eight 9.8 L M 1466 99.eight 100 S G 1465 100 one hundred one hundred one hundred one hundred one hundred one hundred 100 one hundred one hundred 99.Cathepsin S Protein manufacturer 9 99.9 99.five one hundred 100 99.eight 100 E K 1383 11.7 I V 1291 one hundred 100 one hundred T A 1124 11,three Y C 848 six.AXL Protein Source five 88.PMID:23453497 two 99.6 T A 827 100 one hundred 100 F S 797 11.5 100 100 100 100 100 one hundred one hundred one hundred 100 one hundred 100 100 100 100 100 one hundred 100 100 100 I T 769 10.two N D 753 16.eight S P 732 eight.six C R 458 26.two 88.9 99.9 one hundred S P 405 7.two 100 100 one hundred 100 100 one hundred one hundred one hundred one hundred 100 100 100 100 100 100 one hundred one hundred one hundred 100 100 Continued V G 271 28.1 89.1 99.9 one hundred R S 195 7.1 6,9 100 AA modify AA pos four.1 (4th, 13) five.0 (10th, six) four.3 (2nd, 8) five.1 (2nd, six) five.1 (2nd, six) ED43-20m ED43-31m ED43-31m_opt ED43ccNon-synonymous ORF mutations in ED43 recombinant viruses in the course of culture adaptationEDHCV geneNT posNT changeECAETGETCETCETCpAGpTCpTCNSAGNSAGNSAGPham LV, et al. Gut 2022;71:62742. doi:ten.1136/gutjnl-2020-NSAGNSGANSAGNSTANSTCNS4AGTNS4BCTNS4BAGNS4BAGNS4BGANS4BGCNS5AGANS5ATCNS5AGANS5ACANS5AAGNS5AAGNS5AAGNS5ATCNS5AAGNS5BCANS5BAGNS5BAGNS5BATNS5BACHepatologyNS5BTGSNP frequencies ( ) H77 AA pos 2793 2797 2806 100 2916 2931 2976 2978 2981 2982 2983 2991 2992 C Y 2995 99.three L R 2994 100 H Q 2986 6.3 S P 2985 26.0 M V 2984 100 Y F 2981 100 D G 2979 one hundred one hundred one hundred 100 90.1 7.0 100 99.1 Q R 2934 98.1 97.three A V 2919 one hundred 100 one hundred E T 2809 100 one hundred 100 one hundred 100 one hundred one hundred one hundred one hundred 100 one hundred one hundred K R 2800 100 100 100 V A 2796 6.1 88.four one hundred AA adjust AA pos 4.1 (4th, 13) 5.0 (10th, 6) four.three (2nd, eight) ED43-20m ED43-31m ED43-31m_opt five.1 (2nd, six) one hundred 100 100 100 one hundred – 100 one hundred one hundred one hundred 100 100 ED43cc five.1 (2nd, 6) 100 100 10.
