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In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.

Structural polypeptides from a number of poxviruses were analysed by SDS-polyacrylamide gel electrophoresis. The patterns obtained with orthopoxviruses were generally quite similar to one another, but variola, monkeypox, cowpox and vaccinia viruses could be distinquished by their profiles in the molecular weight (mol. wt.) region around 30,000 to 40,000; some additional variation was found amongst cowpox and vaccinia strains. Whitepox virus was shown to have structural polypeptides indistinguishable from those of variola virus. The structural polypeptides of poxviruses belonging to other genera were different from those of the orthopoxvirus, except those of mol. wt. about 122,000 and 97,000, which were common to all viruses irrespective of genus. A polypeptide of mol. wt. about 25,000 was also observed in all cases, though its position varied slightly with the individual virus.

The poxvirus Cotia was studied by electron microscopy and by serological and biochemical analyses. Thin-sectioned preparations of infected Vero cells indicated that Cotia virus morphogenesis was similar to other mammalian poxviruses; unique filamentous structures and inclusion matrices were apparent in the cytoplasm. Complement fixation tests that included purified Cotia virions showed a reciprocal cross-reaction with rabbit myxoma virus and no cross-reaction with vaccinia virus. Serological results coupled with gradient polyacrylamide gel electropherograms of the structural proteins of purified Cotia, vaccinia, myxoma and fibroma viruses suggested that Cotia virus was similar to the latter two viruses. Agarose gel electropherograms of cleavage fragments of each of these virus DNAs digested with three separate restriction endonucleases showed that each of these viruses had a unique DNA gel profile.

Cleavage sites for the restriction endonucleases EcoRI, KpnI and XhoI were mapped on rabbit poxvirus and vaccinia virus DNA. These physical maps were used to analyse the structural variations between the two DNAs. Two specific heterologous regions, symmetrically arranged at each end of the genomes, have been identified. Region 1, representing the exterior part of the terminal repetition, appears to contain unrelated sequences in each DNA and accounts for the difference in length of the two genomes. Region 2, separated from region 1 by a conserved part of the terminal repetition, is located at the transition from repeated to unique DNA sequences. Its overall length of about 4 megadaltons is well conserved and it contains individual DNA-specific as well as conserved restriction sites. The major central part of the genomes (over 100 megadaltons) contains very few, widely dispersed restriction site variations.

Orthopoxvirus DNA from representative strains of rabbitpox, vaccinia, monkeypox, variola, cowpox and ectromelia viruses was analysed by cleavage with restriction endonucleases HindIII, XhoI or SmaI. Genome mol. wt. vary from about 120 x 10(6) for rabbitpox to about 145 x 10(6) for cowpox. Physical maps of cleavage sites are similar and characteristic for strains of the same Orthopoxvirus type. The distribution of HindIII sites suggests that an internal region of mol. wt. about 30 x 10(6) is highly conserved between Orthopoxvirus genomes although some type-specific differences occur within this region, especially with strains of ectromelia virus. Conservation of internal sequences is less marked following analysis with XhoI although cleavages within this central region of particular genomes appear to represent a subset of preferred sites. Endonuclease SmaI cleaves exceptionally infrequently and distinguishes variola, monkeypox, vaccinia, cowpox or ectromelia viruses. Type specific differences result largely from extensive, near terminal variations in length and sequence. Representative Orthopoxvirus genomes have rapidly renaturing terminal restriction fragments confirming the presence of near terminal, covalent cross-links. Terminal restriction fragments from the same or different genomes generally cross hybridize indicating the presence of near terminal repetitions of mol. wt. up to 6 x 10(6) and which share at least a subset of common sequences. Variola strains however, appear to lack such sequences from one specific terminus which maps shorter than that of related viruses.

Radioactively labeled vaccinia, cowpox and Shope fibroma virions free from any detectable contamination with host cell protein, were dissociated into their constituent polypeptides, and these were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The profiles of constituent polypeptide bands of four strains of vaccinia virus (IHD-W, IHD-J, Lister and DIs) were almost the same, except that a polypeptide of about 41,000 daltons was not detectable in the autoradiogram of strain IHD-W which has no hemagglutinin. The profile of polypeptide bands of cowpox virions was also almost the same as that of vaccinia virions, except for several polypeptides of about 40,000 to 50,000 daltons, but the profile of Shope fibroma virions differed considerably from that of vaccinia or cowpox virions.

The presence of cellular material in purified preparations of Shope fibroma virus (SFV) and two orthopoxviruses (vaccinia and Indiana), was investigated. Mitochondria were observed in purified preparations of SFV by electron microscopy and mitochondrial (mt) DNA was identified in restricted viral DNA by Southern blot hybridization with cloned mouse mt DNA. Mitochondrial DNA was also detected in vaccinia and SFV DNAs extracted from purified virions treated with DNase I followed by core isolation. The viral and mt DNAs could be separated on the basis of their size by agarose gel electrophoresis, but not by their buoyant density by centrifugation in cesium chloride gradients. These findings led us to re-examine previously reported results showing some homology between SFV, a leporipoxvirus and Indiana, an Orthopoxvirus (Berkowitz and Pogo, Virology 142, 437-440, 1985) using cloned fragments of SFV DNA instead of the entire viral DNA. The results indicated that cross-hybridization between SFV and Indiana DNAs was due in part to mt DNA but they also revealed an unrecognized region of homology between the two poxvirus genera.

Homology between DNAs of some orthopoxviruses: vaccinia, neurovaccinia, cowpox, ectromelia, variola and white-pox was studied. By the method of molecular hybridization of labelled virion DNAs of the viruses studied with an excess of unlabelled vaccinia DNA and further treatment of the hybridized products by the single-stranded-specific nuclease S1, it was shown that the concentration of unlabelled vaccinia virus DNA, converting 50% of homologous labelled DNA into a hybrid, converted under similar experimental conditions into a hybrid 50% of labelled neurovaccinia virus DNA, 48% of white-pox virus DNA, 43% of variola virus DNA, about 30% of cowpox virus DNA and 27% of ectromelia virus DNA. Based on the quantitative data obtained the relationship between viruses of this subgroup and the possible origin of vaccinia virus is discussed.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.

Deoxyribonucleic acid (DNA) extracted from purified virions of Shope fibroma virus (SFV) (by using DNA from Microccocus lysodeikticus as marker) had a buoyant density of 1.6996 +/- 0.0003 g/ml), hence a guanine plus cytosine (G + C) content of 40.4 +/- 0.3%, which is close to the G + C content of the DNA of susceptible rabbit cells (40.9 +/- 0.4%) and different from that of vaccinia virus DNA (35.5 +/- 0.4%). For the determination of the molecular weight of DNA, SFV and vaccinia purified virions, treated with Pronase and detergent, were cosedimented in sucrose density gradients. Results showed that SFV-DNA has a molecular weight of about 153 x 10(6) daltons. By electron microscopy, only one molecule corresponding to this value was observed (its length was 80.3 mum). The others had a median size of 49.8 mum +/- 0.9.