Revisiting Jenner's mysteries, the role of the Beaugency lymph in the evolutionary path of ancient smallpox vaccines

The biological properties of poxvirus isolates from skin lesions on dairy cows and milkers during recent exanthem episodes in Cantagalo County, Rio de Janeiro State, Brazil, were more like vaccinia virus (VV) than cowpox virus. PCR amplification of the hemagglutinin (HA) gene substantiated the isolate classification as an Old World orthopoxvirus, and alignment of the HA sequences with those of other orthopoxviruses indicated that all the isolates represented a single strain of VV, which we have designated Cantagalo virus (CTGV). HA sequences of the Brazilian smallpox vaccine strain (VV-IOC), used over 20 years ago, and CTGV showed 98.2% identity; phylogeny inference of CTGV, VV-IOC, and 12 VV strains placed VV-IOC and CTGV together in a distinct clade. Viral DNA restriction patterns and protein profiles showed a few differences between VV-IOC and CTGV. Together, the data suggested that CTGV may have derived from VV-IOC by persisting in an indigenous animal(s), accumulating polymorphisms, and now emerging in cattle and milkers as CTGV. CTGV may represent the first case of long-term persistence of vaccinia in the New World. Copyright 2000 Academic Press.

Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses.

Cowpox virus (CPXV) is described as the source of the first vaccine used to prevent the onset and spread of an infectious disease. It is one of the earliest described members of the genus Orthopoxvirus, which includes the viruses that cause smallpox and monkeypox in humans. Both the historic and current literature describe “cowpox” as a disease with a single etiologic agent. Genotypic data presented herein indicate that CPXV is not a single species, but a composite of several (up to 5) species that can infect cows, humans, and other animals. The practice of naming agents after the host in which the resultant disease manifests obfuscates the true taxonomic relationships of “cowpox” isolates. These data support the elevation of as many as four new species within the traditional “cowpox” group and suggest that both wild and modern vaccine strains of Vaccinia virus are most closely related to CPXV of continental Europe rather than the United Kingdom, the homeland of the vaccine.