Genomic Analysis, Phenotype, and Virulence of the Historical Brazilian Smallpox Vaccine Strain IOC: Implications for the Origins and Evolutionary Relationships of Vaccinia Virus.

UNLABELLED: Smallpox was declared eradicated in 1980 after an intensive vaccination program using different strains of vaccinia virus (VACV; Poxviridae). VACV strain IOC (VACV-IOC) was the seed strain of the smallpox vaccine manufactured by the major vaccine producer in Brazil during the smallpox eradication program. However, little is known about the biological and immunological features as well as the phylogenetic relationships of this first-generation vaccine. In this work, we present a comprehensive characterization of two clones of VACV-IOC. Both clones had low virulence in infected mice and induced a protective immune response against a lethal infection comparable to the response of the licensed vaccine ACAM2000 and the parental strain VACV-IOC. Full-genome sequencing revealed the presence of several fragmented virulence genes that probably are nonfunctional, e.g., F1L, B13R, C10L, K3L, and C3L. Most notably, phylogenetic inference supported by the structural analysis of the genome ends provides evidence of a novel, independent cluster in VACV phylogeny formed by VACV-IOC, the Brazilian field strains Cantagalo (CTGV) and Serro 2 viruses, and horsepox virus, a VACV-like virus supposedly related to an ancestor of the VACV lineage. Our data strongly support the hypothesis that CTGV-like viruses represent feral VACV that evolved in parallel with VACV-IOC after splitting from a most recent common ancestor, probably an ancient smallpox vaccine strain related to horsepox virus. Our data, together with an interesting historical investigation, revisit the origins of VACV and propose new evolutionary relationships between ancient and extant VACV strains, mainly horsepox virus, VACV-IOC/CTGV-like viruses, and Dryvax strain. IMPORTANCE: First-generation vaccines used to eradicate smallpox had rates of adverse effects that are not acceptable by current health care standards. Moreover, these vaccines are genetically heterogeneous and consist of a pool of quasispecies of VACV. Therefore, the search for new-generation smallpox vaccines that combine low pathogenicity, immune protection, and genetic homogeneity is extremely important. In addition, the phylogenetic relationships and origins of VACV strains are quite nebulous. We show the characterization of two clones of VACV-IOC, a unique smallpox vaccine strain that contributed to smallpox eradication in Brazil. The immunogenicity and reduced virulence make the IOC clones good options for alternative second-generation smallpox vaccines. More importantly, this study reveals the phylogenetic relationship between VACV-IOC, feral VACV established in nature, and the ancestor-like horsepox virus. Our data expand the discussion on the origins and evolutionary connections of VACV lineages.

One-step duplex polymerase chain reaction for the detection of swinepox and vaccinia viruses in skin lesions of swine with poxvirus-related disease.

Infection of pigs with swinepox virus (SWPV) was reported in Brazil in 2011. SWPV causes a systemic pustular disease in pigs and the symptoms are clinically indistinguishable from those caused by vaccinia virus (VACV) infection. Pigs infected with VACV have been reported in various countries; however, VACV is endemic in Brazil, India and other countries, where it affects mainly dairy cows, dairy buffaloes and dairy workers causing localized pustules. The transmission of VACV to other susceptible hosts has also been detected in Brazil. Therefore, VACV should be investigated as a possible etiologic agent of pustular skin disorders in pigs. This work describes the development of a one-step duplex assay to detect swinepox and vaccinia viruses simultaneously in skin lesions of pigs with generalized pustular disease. The investigation of VACV infection in pigs is important in countries where this zoonosis is endemic and should be differentiated from SWPV infection.

Presence of neutralizing antibodies to Orthopoxvirus in capybaras (Hydrochoerus hydrochaeris) in Brazil.

Cantagalo virus is a strain of vaccinia virus (genus Orthopoxvirus) and the etiological agent of an important vesicopustular disease that affects dairy cows and milkers in Brazil. The reservoirs involved in the maintenance of this virus in nature are unknown. In the present work, the detection of neutralizing antibodies to Orthopoxvirus in capybaras collected in São Paulo state is reported. Capybaras are the largest rodent species native to South America and have already been reported as putative reservoirs of other pathogenic microorganisms. Thirteen out of thirty-three serum samples were found positive in plaque-reduction neutralization tests, some of them showing high titers compared to positive controls. These results suggest that capybaras may play a role in the infection cycle of vaccinia virus in Brazil.

Increased susceptibility of Cantagalo virus to the antiviral effect of ST-246®.

Cantagalo virus (CTGV) is the etiologic agent of a pustular disease in dairy cows and dairy workers in Brazil with important economical and occupational impacts. Nevertheless, no antiviral therapy is currently available. ST-246 is a potent inhibitor of orthopoxvirus egress from cells and has proved its efficacy in cell culture and in animal models. In this work, we evaluated the effect of ST-246 on CTGV replication. Plaque reduction assays indicated that CTGV is 6-38 times more susceptible to the drug than VACV-WR and cowpox virus, respectively, with an EC50 of 0.0086µM and a selective index of >11,600. The analysis of ß-gal activity expressed by recombinant viruses in the presence of ST-246 confirmed these results. In addition, ST-246 had a greater effect on the reduction of CTGV spread in comet tail assays and on the production of extracellular virus relative to VACV-WR. Infection of mice with CTGV by tail scarification generated primary lesions at the site of scarification that appeared less severe than those induced by VACV-WR. Animals infected with CTGV and treated with ST-246 at 100mg/kg for 5days did not develop primary lesions and virus yields were inhibited by nearly 98%. In contrast, primary lesions induced by VACV-WR were not affected by ST-246. The analysis of F13 (p37) protein from CTGV revealed a unique substitution in residue 217 (D217N) not found in other orthopoxviruses. Construction of recombinant VACV-WR containing the D217N polymorphism did not lead to an increase in the susceptibility to ST-246. Therefore, it is still unknown why CTGV is more susceptible to the antiviral effects of ST-246 compared to VACV-WR. Nonetheless, our data demonstrates that ST-246 is a potent inhibitor of CTGV replication that should be further evaluated as a promising anti-CTGV therapy.

Animal movement and establishment of vaccinia virus Cantagalo strain in Amazon biome, Brazil.

To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008-2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondonia State, leading to the establishment and spread of this pathogen.