Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, caixa postal 2496, cep: 31270-901, Belo Horizonte, MG, Brazil1
Author for correspondence: Erna Kroon. Fax +55 31 34436482. e-mail kroone{at}mono.icb.ufmg.br
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Abstract |
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Very little is known about the occurrence and ecology of veterinary poxviruses in Brazil. Many occurrences are related to mousepox virus outbreaks in animal facilities, but most cases remain unpublished. Outbreaks of parapoxviruses in goat and sheep herds have been documented also (Mazur & Machado, 1989 ; Mazur et al., 2000
). We reported the characterization of an orthopoxvirus related to VV, the BeAn 58058 virus (BAV), obtained from a wild rodent captured on the border of the Amazon rain forest (da Fonseca et al., 1998
). Similarly, the isolation and characterization of a VV-like virus, named Cantagalo virus, was reported recently. This virus was isolated from cattle and, eventually, from humans in the state of Rio de Janeiro, Southeast Brazil (Damaso et al., 2000
). Another poxvirus, the Cotia virus (CV), was isolated initially from sentinel mice in 1961, in Cotia county, São Paulo, an area of forest in the southeastern region of the country (Lopes et al., 1965
). The virus has been re-isolated consistently and it was proposed to be a recombinant between leporipoxviruses and orthopoxviruses (Ueda et al., 1978
, 1995
; Esposito et al., 1980
).
The sample of SPAn232 virus (SPAnv) was obtained during a Brazilian government effort to survey rural regions with reported circulation of unknown viruses. The sample was isolated from sentinel mice that had been exposed in the Cotia forest. It was considered initially to be another CV isolate, as the virus presented serological cross reaction with the viruses isolated previously (unpublished results). We received SPAnv in our laboratory as a CV sample and intended to use the virus as a comparison tool to characterize BAV (da Fonseca et al., 1998 ). The serological relationship between these two viruses had been described more than two decades ago (Woodall, 1967
; Ueda et al., 1978
). However, after initial experiments, we realized that the sample we were working with presented remarkable differences from the CV isolate described originally. Moreover, the virus presented extensive similarity with members of the VV subgroup.
In order to characterize the SPAnv isolate, we performed a number of analyses at the genomic level. To avoid misinterpretation due to laboratory cross contamination, two plaque-purified and independently maintained SPAnv samples were analysed throughout the study. VV samples used for comparison were the WR and Lister strains of VV. BAV was included also. All viruses were plaque purified by standard methods to assure genetic homogeneity. Viruses were grown in Vero cells supplemented with 1 or 5% foetal calf serum. When necessary, viruses were purified on sucrose gradients, as described previously (Joklik, 1962 ).
For the generation of restriction patterns and DNA cross hybridization of total genomes, DNA from purified suspensions of VV WR, VV Lister, BAV and SPAnv was extracted, as described by Massung & Moyer (1991) , and digested with HindIII (Promega). The resolved fragments were transferred onto nylon membranes and hybridized with total (non-digested) VV Lister DNA, labelled with [32P]CTP by nick translation (Nick Translation system, Promega) under stringent conditions (Sambrook et al., 1989
). The same blots were also used to detect the presence of genes homologous to the thymidine kinase (TK), vaccinia growth factor (VGF) and A-type inclusion (ATI) genes of VV using the PCR-amplified coding regions of these genes as probes (Meyer et al., 1997
; da Fonseca et al., 1998
).
The coding regions of the SPAnv TK and VGF homologous genes, cloned into pUC19 plasmids, were sequenced in both orientations (Sanger et al., 1977 ) using M13 universal primers. Several independent clones were sequenced to prevent mistakes due to Taq polymerase. Sequences were analysed by search and alignment with similar sequences from GenBank using BLAST programs (Altschul et al., 1990
). Amino acid sequences of the SPAnv TK and VGF proteins were inferred from nucleotide sequences. Phylogenetic tree analyses were performed using the TREECON program (Van de Peer & De Wachter, 1994
). Bootstrap analyses of 1000 replicates were done.
The SPAnv genome presented a profile closely similar to that obtained for VV HindIII-digested DNA (compared with both WR and Lister strains) (Fig. 1A). The main differences are located in the large upper fragments of the digested VV and SPAnv DNA. The migration patterns of the fragments labelled A and B (Fig. 1A
) from the SPAnv DNA HindIII restriction profile resemble the analogous fragments from the VV Lister DNA profile, but not those of VV WR which are differently placed. The migration pattern of fragment C from SPAnv DNA is similar to fragment C from VV WR DNA, but there is no correlated fragment in the VV Lister restriction profile. Furthermore, the SPAnv D fragment has no analogous fragment in either the Lister or the WR VV DNA profile. Smaller fragments retain the same migration pattern for each of the viruses, with little variation. The restriction pattern of SPAnv is similar also to the BAV profile and, again, differed on the larger DNA fragments. Fig. 1(A)
also illustrates the result of the cross-hybridization between the SPAnv, VV Lister, VV WR and BAV genomes. Labelled VV Lister DNA hybridizes extensively with VV WR, BAV and SPAnv DNA.
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After sequencing, both the TK and VGF homologous genes from SPAnv presented nucleotide similarity of more than 99% with the correlated VV WR genes. In the SPAnv TK gene, only two silent nucleotide substitutions were detected when genes from VV WR and SPAnv were compared (data not shown). When we compared the VGF homologous gene from SPAnv with the VGF gene from VV WR, we detected three nucleotide deletions, leading to alterations in two amino acids, SerGly and Glu
Lys, and the loss of one Asn residue. These deletions are indicated in Fig. 2(A)
. All six codons for cysteine amino acid residues, which are known to be essential for protein conformation and biological activity of this family of proteins (James & Bradshaw, 1984
), were found to be intact on the SPAnv VGF homologous gene. The nucleotide sequences were also used to draw phylogenetic trees. In both cases, when sequences from the TK or VGF genes were used, SPAnv was placed in the same branch as VV WR and BAV (Fig. 2B
). The trees drawn from the sequences of the TK and VGF genes were very similar in both shape and evolutionary distance among the viruses. Only the VGF-derived tree is shown (Fig. 2B
).
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Concerning the possible origin of SPAnv, it is tempting to associate the virus with vaccine samples that may have escaped to the wild during smallpox vaccination. This seems to be the case for Cantagalo virus, which is genetically related to the IOC strain of VV (Damaso et al., 2000 ). However, it is impossible to track back to the original vaccine strain from which SPAnv originated because many different samples were used at the same time in the region. They include the Lister, WR (Brazilian Health Ministry, personal communication) and IOC (Damaso et al., 2000
) strains of VV and even pools made of different strains. However, it is important to note that SPAnv was isolated in 1979 and the smallpox vaccination in Brazil was terminated in 1973. This goes beyond short-term infection of VV, described as the occurrence of contact between vaccinated humans and domestic animals (Lum et al., 1967
), and points to another case of VV establishment in the wild. This finding is strengthened by the detection of new VV-like outbreaks in the São Paulo State (unpublished results). Therefore, the establishment of VV in nature is not as rare as stated before, but it is possibly more complex and frequent than once thought.
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References |
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Received 9 May 2001;
accepted 12 September 2001.