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Since 1999, an increasing number of episodes of eruptive skin disease affecting dairy cattle and humans have been reported in Brazil.1–6 Our research team has been working on these zoonotic outbreaks, and in October of 2001, we studied an outbreak that occurred in Guarani town, Minas Gerais State, in the southeast region of the country.
An epidemiologic study was conducted on the affected area involving 72 visited properties, which comprised an area covered by ~5,700 km of roads. The study revealed that a total of 1,020 lactating cows presented lesions on the teats characterized by the presence of papules that evolved to ulcers. After some time, most of these lesions naturally progressed to healing. In 83% of the farms, human cases were registered, and ~110 persons were infected. Most sick humans were milkers that were contaminated after contact with lesions on cow’s teats. These milkers presented pleiomorphic lesions on the hands (mainly papules and painful ulcers), fever, lymphadenitis with enlarged lymph nodes, and eventually secondary bacterial infection on the lesions. Also, in some farms, milkers reported transmission from person to person. The time-course of the infection was between 15 and 30 days. Initial diagnosis was done based on the observed clinical and epidemiologic features.3
To attempt laboratory diagnosis and virus isolation, two samples of dried scabs from two affected cows were collected. Each cow belonged to a different neighboring farm, and these farms were localized 10 km apart from each other. To avoid any possibility of laboratory cross-contamination, the different samples were never manipulated simultaneously. Two viral isolates were obtained and named Guarani P1 virus (GP1V) and Guarani P2 virus (GP2V). Virus isolation was performed using conventional methods including inoculation onto chorioallantoic membrane (CAM) of chicken embryonated eggs and plaque purification cloning on Vero cells to assure genetic homogeneity. These viruses were grown and purified in sucrose gradient as described elsewhere.2,3
Biologic diagnosis was based on pock morphology in CAM and a serum neutralization test using anti-VACV polyclonal rabbit serum as a positive control.2,3 In addition, atomic force microscopy was also used to look for typical poxviruses particles on the samples (data not shown).7
Molecular diagnosis of the isolated viruses was performed by polymerase chain reaction (PCR) amplification of the A type inclusion body gene (A26L). The A26L gene analysis, based on partial amplification of this gene followed by digestion using XbaI, has been widely used as a tool for rapid screening and taxonomic differentiation of Orthopoxvirus.8,9 The GP2V A26L gene was amplified, and the amplicon was similar in size to those described for Araçatuba virus (ARAV) and Passatempo virus (PSTV), other VACV strains isolated under similar circumstances in 1999 and 2003, respectively.2,6 When the A26L amplicon of GP2V was digested with XbaI, it presented the same restriction pattern as in ARAV, which was also similar but not identical to those patterns obtained for known VACV strains, such as Western Reserve (WR) and Lister (LST) (Figure 1A
). The main difference observed for the GP2V and ARAV A26L gene amplicon digests was that the larger fragment generated after XbaI digestion migrated faster on the agarose gel than the VACV-WR and VACV-LST analogous fragments (Figure 1A). Attempts to amplify the A26L gene of GP1V, using the ATI up and ATI low primers, were unsuccessful (data not shown).8 The lack of this amplicon could be caused by a deletion at the A26L gene of the GP1V, a fact previously observed for other Brazilian VACV strains, such as BeAn58058 virus (BAV) and Belo Horizonte Virus (VBH).10,11 To verify the extension of the probable GP1V A26L deletion, we performed a PCR amplification using oligonucleotides P4c1 (located within the p4c1 gene) and RNApol, designed based on Cowpox virus Brighton Red (CPXV BR) as described elsewhere.11 For CPXV, VACV-WR, and GP2V, a product of ~4.300 bp was obtained. However, for GP1V, a fragment of 300 bp (Figure 1B) was generated and sequenced (GenBank accession no. DQ363383), and it proved that, in GP1V, as well in VBH and BAV, a major portion of the A26L gene is missing (Figure 1C). Additionally, it is important to point out the usefulness of the A26L PCR-restriction fragment length polymorphism (PCR-RFLP) as a molecular diagnosis approach and for differentiation of Brazilian Orthopoxvirus species.10,11
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Amplicons were cloned into pGEM-T vector (Promega Corp., Madison, WI) and sequenced in both orientations by the dideoxynucleoside method, using M13 universal primers and ET Dynamic Terminator for MegaBACE (GE HEALTHCARE, UK).17 Nucleotide sequences were assembled using the CAP3 Sequence Assembling Program and deposited in GenBank.18 Nucleotide and inferred amino acids sequences were aligned with other Orthopoxvirus sequences using CLUSTAL W and LALIGN.19,20 J2R sequences of GP1V and GP2V (GenBank accession no. DQ206438 and DQ206439, respectively) were identical to each other, presenting high levels of similarity with VACV sequences. These sequences were also identical to VACV-WR and other Brazilian VACV strains, such as BAV, SPAn232, ARAV, and PSTV (data not shown).2,6,12,13 C11R sequences of GP1V and GP2V (GenBank accession no. DQ206440 and DQ206441, respectively) presented 99.4% of identity compared with each other and high levels of similarity among other VACV strains.
B18R, E3L, and A56R sequences of GP1V and GP2V were also similar to other VACV strains sequences, although there was a higher level of genetic variance among them. GP1V and GP2V B18R sequences presented seven nucleotide substitutions that resulted in the two amino acid modifications in the inferred amino acid sequences. For GP1V, GP2V, ARAV, and BAV, in the position 346 of the B18R protein, a serine was found, whereas in VACV-WR, VACV-IOC, and CTGV, there was a threonine in the same position (data not shown). The GP1V and GP2V E3L genes presented 98.6% of identity to each other, with substitutions in eight nucleotide positions (Table 1). These mutations led to six amino acid differences observed between the GP1V and GP2V inferred amino acid sequences (data not shown).
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). As observed for ARAV, PSTV, CTGV, and VACV-IOC, GP2V also presented the same 18-nucleotide deletion signature showed previously at the A56R gene of those VACV strains. This 18-nucleotide deletion has been found in all Brazilian VACV strains isolated to date, and it has been proposed as a molecular signature of new world VACV strains. However, GP1V and VBH did not present this deletion. Additionally, the lack of this deletion in the GP1V and VBH sequences points out the necessity to review the 18-nucleotide deletion as a molecular signature of new world VACV strains.
To establish a phylogenetic relationship among GP1V, GP2V, and other VACV strains, alignments of the A56R, B18R, and E3L genes were used to construct a concatenated phylogenetic tree by the neighbor-joining method using the Tamura Nei model of nucleotide substitutions implemented in MEGA3.21,22 The tree was midpoint-rooted, and 1,000 bootstrap replicates were performed (Figure 2A).
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Our findings show that there are genetically different populations of VACV circulating in the country and even in the same outbreak. It has been proposed that the Brazilian VACV isolates obtained from cowpox-like outbreaks form a genetically homogenous population that could have been originated from the spread of a smallpox vaccine strains, particularly the VACV IOC.1 According to this theory, these strains could have escaped to wild and established circulation in some unknown host, becoming feral during this process.1,2,6 However, the characterization of two different VACV strains isolated from a single outbreak, co-circulating in Brazil, points in a different direction. One hypothesis to explain this virus diversity is that different samples, from different sources, and under different circumstances, could have established circulation in nature. Thus, these viruses would have not one but multiple origins. This does not entirely exclude the possibility that the viruses could have evolved from a common origin, derived from a escapee vaccine strain, and differentiated by means of mutation and/or recombination events during circulation in nature. However, our results suggest that multi-factor events may be the best explanation for the VACV genetic diversity observed in Brazil, including the pre-existence and circulation of a possible autochthonous poxvirus.
Received February 15, 2006. Accepted for publication May 10, 2006.
Acknowledgments: The authors thank Dr. Marieta Cristina Madureira from Instituto Mineiro de Agropecuária for support during the epidemiologic investigation. We also thank Dr. José Mário Vilela and Dr. Margareth Spangler Andrade from Nanoscopy Laboratory, Fundação Centro Tecnológico de Minas Gerais, for the AFM analysis. The aid of Dr. Fabrício dos Santos, Rodrigo Redondo, and Laboratório de Biodiversidade e Evolução Molecular where all sequences were made is gratefully acknowledged.
Financial support: The CNPq, CAPES, and FAPEMIG provided financial support. E. G. Kroon, C. A. Bonjardim, P. C. P. Ferreira, F. G. Da Fonseca, and J. R. Santos received fellowship from CNPq. M. I. M. C. Guedes received fellowship from FAPEMIG.
* Address correspondence to Erna Geessien Kroon, Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, caixa postal 486, CEP 31270-901 Belo Horizonte, MG, Brazil. E-mail: kroone{at}icb.ufmg.br 
Authors’ addresses: Giliane de Souza Trindade, Betânia Paiva Drumond, Juliana Almeida Leite, Ricardo Campos Trigueiro, Maria Isabel Maldonado Coelho Guedes, João Rodrigues dos Santos, Cláudio Antônio Bonjardim, Paulo César Peregrino Ferreira, and Erna Geessien Kroon, Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, caixa postal 486, CEP 31270-901, Belo Horizonte, MG, Brasil, Fax: 55 31 3443-6482, E-mails: gitrindade{at}yahoo.com.br, betaniadrumond{at}uol.com.br, ju{at}icb.ufmg.br, rictrigueiro{at}yahoo.com.br, isabelguedes{at}icb.ufmg.br, santosjr{at}icb.ufmg.br, claubonj{at}icb.ufmg.br, paulocpf{at}icb.ufmg.br, and kroone{at}icb.ufmg.br. Zélia Inês Portela Lobato, Departamento de Medicina Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brasil, Fax: 55 31 3443-6482, E-mail: ziplobat{at}vet.ufmg.br. Flávio Guimarães da Fonseca, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, caixa postal 486, CEP 31270-901, Belo Horizonte, MG, Brasil, Fax: 55 31 3443-6482, E-mail: fdafonseca{at}cpqrr.fiocruz.br.
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, Brazilian strains that presented partial A26L gene deletion; •, Brazilian strains that presented 18 nt deletion in the A56R gene. B, Brazilian states where and when Brazilian VACV strains were isolated. ES, Espírito Santo; GO, Goiás; MG, Minas Gerais; PA, Pará; RJ, Rio de Janeiro; SP, São Paulo. 1, BeAn 58058 virus in 1963; 2, SPAn232 virus in 1965; 3, Belo Horizonte virus in 1993; 4, ARAV in 1999; 5, CTGV in 1999; 6, GP1V and GP2V in 2001; 7, GO isolates after 2001; 8, SP isolates after 2001; 9, MG isolates after 2001; 10, PSTV in 2003; ES isolates in 2004 (unpublished data).