Conceptualization, N.V.G.C. and C.R.D.; methodology, N.V.G.C., R.H.S.P., T.M.P.P.C., O.C.F.J. and L.J.d.C.; software, M.N.L.; validation, N.V.G.C. and C.R.D.; formal analyses, N.V.G.C., M.N.L. and C.R.D.; investigation, N.V.G.C., M.N.L., R.H.S.P., T.M.P.P.C., O.C.F.J., L.J.d.C. and C.R.D.; resources, N.V.G.C., T.M.P.P.C., O.C.F.J. and C.R.D.; data curation, N.V.G.C., M.N.L. and C.R.D.; writing—original draft preparation, N.V.G.C. and C.R.D.; writing—review and editing, N.V.G.C., T.M.P.P.C., L.J.d.C., O.C.F.J. and C.R.D.; visualization, N.V.G.C. and C.R.D.; supervision, L.J.d.C. and C.R.D.; project administration, C.R.D.; funding acquisition, N.V.G.C. and C.R.D. All authors have read and agreed to the published version of the manuscript.

Figure 1. Clone isolation of VACV-Wyeth and plaque phenotype. (A,B) Original vial of lyophilized VACV-Wyeth propagated in LLC-MK2 in 1971. (C) Representative images of crystal-violet stained BSC-40 cells infected with 10−6 dilution of the original vial before plaque selection and stocks of Wyeth clones A111, A211, and A311. (D) Random viral plaques (n = 20) were photographed at 4× magnification, and the individual areas were measured. Circles: plaques of the original vaccine vial; diamonds: plaques of clone A111; squares: plaques of clone A211; triangles: plaques of clone A311. Asterisks: * p ≤ 0.05 and ** p ≤ 0.01; ns means non-significant.

Figure 1. Clone isolation of VACV-Wyeth and plaque phenotype. (A,B) Original vial of lyophilized VACV-Wyeth propagated in LLC-MK2 in 1971. (C) Representative images of crystal-violet stained BSC-40 cells infected with 10−6 dilution of the original vial before plaque selection and stocks of Wyeth clones A111, A211, and A311. (D) Random viral plaques (n = 20) were photographed at 4× magnification, and the individual areas were measured. Circles: plaques of the original vaccine vial; diamonds: plaques of clone A111; squares: plaques of clone A211; triangles: plaques of clone A311. Asterisks: * p ≤ 0.05 and ** p ≤ 0.01; ns means non-significant.

Figure 2. Genome-wide analysis of differences comparing the genomes of VACV-Wyeth clones A111, A211, and A311. The full-genome alignment of VACV-Wyeth clones A111, A211, and A311 was analyzed using Base-by-base [29]. (A) A graphical presentation of sequence variations per site (SNPs and INDELs) was revealed by pairwise comparison of the three Wyeth genomes. (B) Differences per site detected in the genomes of clones A211 (red line) and A311 (blue line) in relation to clone A111 (green bottom line) were scored along the genome. Figure 2. Genome-wide analysis of differences comparing the genomes of VACV-Wyeth clones A111, A211, and A311. The full-genome alignment of VACV-Wyeth clones A111, A211, and A311 was analyzed using Base-by-base [29]. (A) A graphical presentation of sequence variations per site (SNPs and INDELs) was revealed by pairwise comparison of the three Wyeth genomes. (B) Differences per site detected in the genomes of clones A211 (red line) and A311 (blue line) in relation to clone A111 (green bottom line) were scored along the genome.

Figure 3. Phylogenetic reconstruction of VACV-Wyeth clones A111, A211, and A311. A multialignment of the conserved region of 38 orthopoxvirus genomes was used to infer a maximum likelihood tree using MEGA X, opting for the General Time Reversible (GTR) model and five-category discrete gamma distributed with invariant site. Numbers next to branch nodes indicate the percentage of 1000 replicates of bootstrap support (values >70 are shown). The scale bar indicates the number of substitutions per site. Colored boxes indicate the three main clades in the vaccinia group. The Wyeth clones are indicated with red circles.

Figure 3. Phylogenetic reconstruction of VACV-Wyeth clones A111, A211, and A311. A multialignment of the conserved region of 38 orthopoxvirus genomes was used to infer a maximum likelihood tree using MEGA X, opting for the General Time Reversible (GTR) model and five-category discrete gamma distributed with invariant site. Numbers next to branch nodes indicate the percentage of 1000 replicates of bootstrap support (values >70 are shown). The scale bar indicates the number of substitutions per site. Colored boxes indicate the three main clades in the vaccinia group. The Wyeth clones are indicated with red circles.

Figure 4. Genome-wide distribution of differences and Median-joining network analysis comparing the genomes of clones of VACV-Wyeth and Dryvax. (A) A multialignment of the full genomes of VACV-Wyeth and Dryvax clones (clade I) was analyzed by using Base-by-base to generate a graphical map of genetic differences (insertions, SNPs, and deletions) in relation to the genome of VACV-Wyeth A111 used as reference. VACV-IOC clones B141 and B388 (clade II) were also included for sake of comparison. Asterisk indicates a highly conserved region of the genomes. Black arrows indicate regions in the genomes of Dryvax clones enriched in deletions in relation to Wyeth A111. Open arrows indicate deletions unique to the genome ends of the Dryvax clones. (B) Median-joining network analysis using a Jalview-edited multialignment of the full genomes of VACV-Wyeth and Dryvax clones. Branch lengths are proportional to the number of accumulated SNPs. The small red nodes (median vectors) indicate unsampled or extinct common ancestors.

Figure 4. Genome-wide distribution of differences and Median-joining network analysis comparing the genomes of clones of VACV-Wyeth and Dryvax. (A) A multialignment of the full genomes of VACV-Wyeth and Dryvax clones (clade I) was analyzed by using Base-by-base to generate a graphical map of genetic differences (insertions, SNPs, and deletions) in relation to the genome of VACV-Wyeth A111 used as reference. VACV-IOC clones B141 and B388 (clade II) were also included for sake of comparison. Asterisk indicates a highly conserved region of the genomes. Black arrows indicate regions in the genomes of Dryvax clones enriched in deletions in relation to Wyeth A111. Open arrows indicate deletions unique to the genome ends of the Dryvax clones. (B) Median-joining network analysis using a Jalview-edited multialignment of the full genomes of VACV-Wyeth and Dryvax clones. Branch lengths are proportional to the number of accumulated SNPs. The small red nodes (median vectors) indicate unsampled or extinct common ancestors.

Figure 5. Similarity profile and Bootscan analysis to detect recombination in the genomes of VACV-Wyeth clones. (A) Similarity plot of the whole-genome multi-alignment using Simplot, opting for VACV-Wyeth A111 sequence as query, window size 5000, step size 500. The asterisk and the dotted box indicate the region of lower similarity of Wyeth A111 with the Wyeth clones A211 and A311, and the Dryvax clones, except for Acam2000. (B) A 15-kb region spanning from 70 kb to 85 kb of the multi-alignment shown in A was analyzed by Bootscan, opting for Wyeth A111 as query, window size 800, and step size 70. (C) Two putative recombinant events with Acam2000 and Wyeth 311 were identified in (B), and the corresponding 2.5-kb region was extracted and reanalyzed by Bootscan, opting for Wyeth A111 as query, window size 100, and step size 20, and using Acam2000 and Wyeth 311 as donor sequences, and DPP25 as non-donor strain.

Figure 5. Similarity profile and Bootscan analysis to detect recombination in the genomes of VACV-Wyeth clones. (A) Similarity plot of the whole-genome multi-alignment using Simplot, opting for VACV-Wyeth A111 sequence as query, window size 5000, step size 500. The asterisk and the dotted box indicate the region of lower similarity of Wyeth A111 with the Wyeth clones A211 and A311, and the Dryvax clones, except for Acam2000. (B) A 15-kb region spanning from 70 kb to 85 kb of the multi-alignment shown in A was analyzed by Bootscan, opting for Wyeth A111 as query, window size 800, and step size 70. (C) Two putative recombinant events with Acam2000 and Wyeth 311 were identified in (B), and the corresponding 2.5-kb region was extracted and reanalyzed by Bootscan, opting for Wyeth A111 as query, window size 100, and step size 20, and using Acam2000 and Wyeth 311 as donor sequences, and DPP25 as non-donor strain.

Figure 6. Detection of vaccinia virus IgG antibodies in serum samples of 130 individuals born before and after the completion of smallpox vaccination in Brazil. Detection was performed by ELISA using purified inactivated VACV-Wyeth clone A111 as antigen. Results for each serum are represented by individual dots and expressed as arbitrary units. (A) Distribution of anti-VACV IgG values according to the year of birth. Black arrow points to 1979, when smallpox vaccination was ended in Brazil. (B) Serological response to VACV-Wyeth was plotted according to the presumed vaccination status. Individuals born before 1979 were considered vaccinated and individuals born after 1979 were considered unvaccinated. The blue circle refers to a laboratory worker recently and repeatedly vaccinated with the smallpox vaccine. The green circle corresponds to a pool of two convalescent sera from laboratory workers accidently infected with VACV. Dotted lines indicate the upper and lower limits of the test cut-off. **** p < 0.0001 (Mann–Whitney).

Figure 6. Detection of vaccinia virus IgG antibodies in serum samples of 130 individuals born before and after the completion of smallpox vaccination in Brazil. Detection was performed by ELISA using purified inactivated VACV-Wyeth clone A111 as antigen. Results for each serum are represented by individual dots and expressed as arbitrary units. (A) Distribution of anti-VACV IgG values according to the year of birth. Black arrow points to 1979, when smallpox vaccination was ended in Brazil. (B) Serological response to VACV-Wyeth was plotted according to the presumed vaccination status. Individuals born before 1979 were considered vaccinated and individuals born after 1979 were considered unvaccinated. The blue circle refers to a laboratory worker recently and repeatedly vaccinated with the smallpox vaccine. The green circle corresponds to a pool of two convalescent sera from laboratory workers accidently infected with VACV. Dotted lines indicate the upper and lower limits of the test cut-off. **** p < 0.0001 (Mann–Whitney).

Table 1. Genomic features of the genomes of VACV-Wyeth clones A111, A211, and A311.

Table 1. Genomic features of the genomes of VACV-Wyeth clones A111, A211, and A311.

VACV-Wyeth Clones A111A211A311Genome size (bp)200,123199,816199,892ITR size (pb)16,94516,11716,128Number of reads3,716,9243,295,1523,816,236% mapped reads 94.34%92.45%93.26%Genome coverage (x)2184.381852.412168.59Number of annotated ORFs249249246% Identity in relation to A11110099.4599.38A21199.4510099.55A31199.3899.55100