Citation: Cruz, N.V.G.; Luques, M.N.; Castiñeiras, T.M.P.P.; Costa Ferreira Jr, O.; Peralta, R.H.S.; Costa, L.J.d.; Damaso, C.R. Genomic Characterization of the Historical Smallpox Vaccine Strain Wyeth Isolated from a 1971 Seed Vial. Viruses 2023, 15, 83. https:// doi.org/10.3390/v15010083 Academic Editors: Sergei N. Shchelkunov and Larisa N. Shishkina Received: 24 November 2022 Revised: 24 December 2022 Accepted: 27 December 2022 Published: 28 December 2022 Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article Genomic Characterization of the Historical Smallpox Vaccine Strain Wyeth Isolated from a 1971 Seed Vial Nádia Vaez G. Cruz 1,2 , Matheus Nobrega Luques 2 , Terezinha Marta Pereira P. Castiñeiras 3 , Orlando Costa Ferreira Jr 4 , Regina Helena S. Peralta 5 , Luciana J. da Costa 6 and Clarissa R. Damaso 2, * 1 Instituto de Biologia do Exército, Rio de Janeiro 20911-270, Brazil 2 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil 3 Núcleo de Enfrentamento e Estudo de Doenças Infecciosas Emergentes e Reemergentes (NEEDIER), Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil 4 Departamento de Genética, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil 5 Faculdade de Medicina, Universidade Federal Fluminense, Niterói 24220-000, Brazil 6 Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil * Correspondence: damasoc@biof.ufrj.br; Tel.: +55-(21)-3938-6510 Abstract: The Wyeth strain of vaccinia virus (VACV) produced by Wyeth Pharmaceuticals was supposedly used to manufacture the old freeze-dried American smallpox vaccine, Dryvax, until its discontinuation in 2008. Although the genomic sequences of numerous Dryvax clones have been reported, data on VACV-Wyeth genomes are still lacking. Genomic analysis of old VACV strains is relevant to understand the evolutionary relationships of smallpox vaccines, particularly with the recent resumption of smallpox vaccination in certain population groups as an attempt to control the worldwide monkeypox outbreak. Here we analyzed the complete genome sequences of three VACV-Wyeth clonal isolates obtained from a single seed vial donated to the Brazilian eradication program in the 1970s. Wyeth clones show >99.3% similarity to each other and >95.3% similarity with Dryvax clones, mapping together in clade I of the vaccinia group. Although the patterns of SNPs and INDELs comparing Dryvax and Wyeth clones are overall uniform, important differences were detected particularly at the ends of the genome. In addition, we detected recombinant events of clone Wyeth A111 and the Dryvax clone Acam2000, suggesting that other regions of the genomes may have similar patchy patterns of recombination. A small-scale serological survey using VACV- Wyeth as antigen in ELISA assays revealed that 63 of the 65 individuals born before the end of smallpox vaccination in Brazil still have anti-VACV IgG antibodies, demonstrating the usefulness of the VACV-Wyeth strain in future extended serological studies of the Brazilian population. Keywords: smallpox vaccine; vaccinia virus; monkeypox virus; poxvirus 1. Introduction In 1980, WHO declared the eradication of smallpox after implementing an intensified eradication program, which achieved high levels of vaccination coverage worldwide and widespread surveillance. The WHO program also contributed to the expansion of national vaccination programs and to the robust production of the smallpox vaccine by several countries [1]. Different strains of vaccinia virus (VACV; genus Orthopoxvirus, family Poxviridae) were used as the smallpox vaccine during the eradication era. The strains differed in reactogenicity and immunogenicity and were used in the production mostly of calf-based and egg-based vaccines [1–3]. Countries were free to choose the VACV strains used in their national vaccination program. However, two main strains, Lister and the New York City Viruses 2023, 15, 83. https://doi.org/10.3390/v15010083 https://www.mdpi.com/journal/viruses