1 Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Caixa postal 486, CEP 31270-901, Belo Horizonte, MG, Brazil
2 Laboratório de Imunologia Celular e Molecular, Centro de Pesquisas René Rachou FIOCRUZ, Avenida Augusto de Lima 1715, CEP 30190-002, Belo Horizonte, MG, Brazil
3 Laboratório de Biologia de Microrganismos, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Caixa postal 486, CEP 31270-901, Belo Horizonte, MG, Brazil
4 Department of Cancer Biology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA
5 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Ghent, Belgium
Correspondence
Erna G. Kroon
kroone{at}icb.ufmg.br
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this article are AF163843, AF163845, AF501620 and AY542799.
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MAIN TEXT |
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Most strategies for fast taxonomic analysis of poxviruses involve rapid-screening techniques for identification of unknown isolates, based on sequencing or restriction fragment-length polymorphism (RFLP) of different genes (Roop et al., 1995; Marques et al., 2001
; da Fonseca et al., 2002
). Meyer et al. (1994
, 1997)
proposed PCR amplification of the A-type inclusion body gene (ati) followed by restriction analysis as a rapid approach to differentiate orthopoxviruses. Indeed, characterization of genes such as haemagglutinin (ha) and ati has been used for identification of orthopoxvirus isolates including Monkeypox virus (MPXV), Ectromelia virus (ECTV), and VACV associated with cowpox-like outbreaks (Meyer et al., 1997
; Neubauer et al., 1997
; Damaso et al., 2000
; da Fonseca et al., 2002
; Trindade et al., 2003
).
Little is known about how orthopoxviruses are maintained in nature or about the role of wild reservoirs. It has been demonstrated that, despite its wide host range, different species of wild rodents are the natural reservoir hosts for Cowpox virus (CPXV) in Great Britain and probably in other parts of Europe, where CPXV is endemic (Chantrey et al., 1999; Hazel et al., 2000
). Similarly to CPXV, and unlike VARV, VACV has a wide host range and is able to infect humans, cattle and rodents (Fenner et al., 1989
; da Fonseca et al., 1999
). Very little is known about the occurrence and circulation of orthopoxviruses in Brazil, but in recent years many isolates of vaccinia-like viruses have been reported (Damaso et al., 2000
; da Fonseca et al., 2002
; Trindade et al., 2003
). At a time when the possible recurrence of poxvirus diseases is being considered and discussed, the increasing number of isolations of vaccinia-like viruses in Brazil (some causing illness in humans) imply a genuine public health threat.
Here we report the molecular characterization of a naturally occurring A-type inclusion body (ATI)-negative vaccinia-like virus isolated from a mousepox-like outbreak that took place in the animal facility of the Biological Institute of the University of Minas Gerais, Brazil. Mice were obtained from the University of Campinas, State of São Paulo, Brazil, and were apparently healthy on arrival. After a few days, some animals died and others presented characteristic skin lesions, developing a generalized skin rash. A virus was isolated from clinical specimens after inoculation onto chorioallantoic membranes (CAMs) of chick embryonated eggs, and named Belo Horizonte virus (VBH) (Diniz et al., 2001). After isolation, the virus was propagated and titrated in Vero cells as described by Campos & Kroon (1993)
, purified in sucrose gradients as described by Joklik (1962)
, and identified by conventional methods that included pock morphology in CAMs, electron microscopy and neutralization tests using anti-VACV polyclonal antibodies (Diniz et al., 2001
).
DNA was extracted from purified virus stocks by treatment with proteinase K, SDS and -mercaptoethanol followed by phenol extraction, as described by Massung & Moyer (1991)
. Purified virus DNA (2 µg) was digested with HindIII enzyme (Promega), separated by electrophoresis on a 0·4 % (w/v) agarose gel and stained with ethidium bromide. Surprisingly, the digestion pattern obtained did not match that of ECTV (Esposito & Knight, 1985
), a natural candidate due to the nature of the outbreak, but closely resembled digested patterns of VACV DNA (data not shown).
The digested DNA was transferred to a nylon membrane (Hybond-N; Amersham Pharmacia) using modified Southern-blot protocols (Sambrook et al., 1989) and cross-hybridized with VACV strain Western Reserve (VACV WR) total genome as probe (Meyer et al., 1997
). The probe was labelled by nick translation (Nick Translation System; Promega) with [
-32P]CTP according to the manufacturer's protocol. Samples were hybridized for 16 h at 65 °C and processed as described by Church & Gilbert (1984)
. After washing, membranes were exposed to X-Omat Kodak film. DNA from VACV WR (virus obtained from the National Institute for Medical Research, Mill Hill, London, UK) was also digested, transferred to membranes and hybridized with the same probe.
The HindIII restriction profile of VBH revealed a typical orthopoxvirus pattern (Fig. 1) and resembled that of VACV WR. Fragments shorter than 7 kb (Fig. 1i
m) derived from VBH DNA showed similarity in their migration pattern to VACV WR DNA, while small differences were detected in the migration of the larger fragments (Fig. 1a
h). Taken together, the similarity between the DNA-digested profiles from VACV and VBH and the occurrence of cross-hybridization suggested a close relationship between VBH and VACV.
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RFLP analysis using the ati gene has been used for poxvirus taxonomic characterization. Although the formation of typical A-type inclusions is restricted to cells infected with a few orthopoxviruses, such as CPXV, Raccoonpox virus and ECTV, the ati gene can be detected in the genome of other viruses (Meyer et al., 1997; Funahashi et al., 1988
). However, PCR using VBH DNA as template and the primers ATI-up and ATI-low specified by Meyer et al. (1997)
generated no products. The lack of DNA PCR amplification could be due to a deletion at the ati gene of VBH, a feature also found on the genome of certain VACV strains. To investigate this, we mapped the possible ati deletion through dot-blot hybridization employing oligonucleotides P4C1 (located within the p4c1 gene) (5'-GGAGATCTAGACCACCGTTTCCCAGACATGAATATC-3') and RNApol (located within the rpo132 gene) (5'-GGAAGCTTTCTCTCTCCTCTCTTAACAAAAATTG-3'), designed based on CPXV Brighton Red (CPXV BR). Hybridization scored positive for both primers and indicated that the flanking regions of the ati gene are present in the VBH genome (data not shown).
In order to evaluate the extent of the VBH ati gene deletion, the oligonucleotides that scored positive on the dot-blot assay were used in PCRs (Funahashi et al., 1988). Standard PCR mixtures contained 10 pmol of each primer (P4c1 and RNApol) plus 20 ng purified VBH or VACV WR DNA as templates. Annealing was performed at 58 °C. For VACV WR, as expected, a product of about 4·3 kb was obtained. However, for VBH a single DNA fragment of about 300 bp was detected (Fig. 3
a), indicating that in the VBH genome a major portion is missing between the annealing positions of primers P4c1 and RNApol of the ati gene. The PCR DNA product was cloned using the pGEM-T Easy Vector Kit (Promega) and sequenced in both orientations. The sequence obtained was deposited in GenBank under accession number AF501620 and analysed using BLASTN and BLASTX programs (Altschul et al., 1990
). It showed high similarity to equivalent regions from other orthopoxviruses, especially to VACV (Fig. 3b
). Alignment of the sequence revealed that only 112 nt of the ati gene are present, encoding only the C-terminal portion of the ATI protein (Fig. 3c
). Other VACV strains also lack the ati gene (Goebel et al., 1990
; Johnson et al., 1993
; Osterrieder et al., 1994
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
---|
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---|
Buller, R. M. L. & Palumbo, G. J. (1991). Poxvirus pathogenesis. Microbiol Rev 55, 80122.[Medline]
Campos, M. A. S. & Kroon, E. G. (1993). Critical period for reversible block of vaccinia virus replication. Rev Braz Microbiol 24, 104110.
Chantrey, J., Meyer, H., Baxby, D., Begon, M., Bown, K. J., Hazel, S. M., Jones, T., Montgomery, W. I. & Bennett, M. (1999). Cowpox: reservoir hosts and geographic range. Epidemiol Infect 122, 455460.[CrossRef][Medline]
Church, G. M. & Gilbert, W. (1984). Genomic sequencing. Proc Natl Acad Sci U S A 81, 19911995.[Abstract]
da Fonseca, F. G., Lanna, M. C. S., Campos, M. A. S., Kitajima, E. W., Peres, J. N., Golgher, R. R., Ferreira, P. C. P. & Kroon, E. G. (1998). Morphological and molecular characterization of the poxvirus BeAn 58058. Arch Virol 143, 11711186.[CrossRef][Medline]
da Fonseca, F. G., Silva, R. L. A., Marques, J. T., Ferreira, P. C. P. & Kroon, E. G. (1999). The genome of cowpox virus contains a gene related to those encoding the epidermal growth factor, transforming growth factor alpha and vaccinia growth factor. Virus Genes 18, 151160.[CrossRef][Medline]
da Fonseca, F. G., Trindade, G. S., Silva, R. L. A., Bonjardim, C. A., Ferreira, P. C. P. & Kroon, E. G. (2002). Characterization of a vaccinia-like virus isolated in a Brazilian forest. J Gen Virol 83, 223228.
Damaso, C. R. A., Esposito, J. J., Condit, R. & Moussatché, N. (2000). An emergent poxvirus from humans and cattle in Rio de Janeiro state: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology 277, 439449.[CrossRef][Medline]
Diniz, S., Trindade, G. S., da Fonseca, F. G. & Kroon, E. G. (2001). An outbreak of mousepox in Swiss mice in a laboratory animal facility case report. Arq Braz Med Vet Zootec 53, 152156.
Esposito, J. J. & Knight, J. C. (1985). Orthopoxvirus DNA: a comparison of restriction profiles and maps. Virology 143, 230251.[Medline]
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783791.
Fenner, F., Wittek, R. & Dumbell, K. R. (1989). The global spread, control and eradication of smallpox. In The Orthopoxviruses, pp. 317352. San Diego, CA: Academic Press.
Funahashi, S., Sato, T. & Shida, H. (1988). Cloning and characterization of the gene encoding the major protein of the A-type inclusion body of cowpox virus. J Gen Virol 69, 3547.[Abstract]
Goebel, S. J., Johson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P. & Paoletti, E. (1990). The complete DNA sequence of vaccinia virus. Virology 179, 247266.[Medline]
Hall, T. A. (1999). BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 9598.
Hasegawa, M., Kishino, H. & Yano, T. (1985). Dating of the humanape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 22, 160174.[Medline]
Hazel, S. M., Bennett, M., Chantrey, J., Bown, K., Cavanagh, R., Jones, T. R., Baxby, D. & Begon, M. A. (2000). A longitudinal study of an endemic disease in its wildlife reservoir: cowpox and wild rodents. Epidemiol Infect 124, 551562.[CrossRef][Medline]
Johnson, G. P., Goebel, S. J. & Paoletti, E. (1993). An update on the vaccinia virus genome. Virology 196, 381401.[CrossRef][Medline]
Joklik, W. K. (1962). The purification of four strains of poxvirus. Virology 18, 918.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21132. Edited by H. N. Munro. New York: Academic Press.
Marques, J. T., Trindade, G. S., da Fonseca, F. G., dos Santos, J. R., Bonjardim, C. A., Ferreira, P. C. P. & Kroon, E. G. (2001). Characterization of ATI, TK and IFN-alpha/betaR genes in the genome of the BeAn 58058 virus, a naturally attenuated wild orthopoxvirus. Virus Genes 23, 291301.[CrossRef][Medline]
Massung, R. F. & Moyer, R. W. (1991). The molecular biology of swinepox virus. I. A characterization of the viral DNA. Virology 180, 347354.[CrossRef][Medline]
Meyer, H., Pfeffer, M. & Rziha, H. J. (1994). Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of othopoxvirus species by polymerase chain reaction. J Gen Virol 75, 19751981.[Abstract]
Meyer, H., Roop, S. L. & Esposito, J. J. (1997). Gene for A-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxvirus. J Virol Methods 64, 217221.[CrossRef][Medline]
Neubauer, H., Pfeffer, M. & Meyer, H. (1997). Specific detection of mousepox virus by polymerase chain reaction. Lab Anim 31, 201205.[Medline]
Osterrieder, N., Meyer, H. & Pfeffer, M. (1994). Characterization of the gene encoding the A-type inclusion body protein of mousepox virus. Virus Genes 8, 125135.[Medline]
Raes, J. & Van der Peer, Y. (1999). FORCON, a tool to automatically convert sequence alignment formats. EMBnet.news 6 (1), http://www.psb.rug.ac.be/jerae/ForCon/index.html
Ronquist, F. & Huelsenbeck, J. P. (2003). MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 15721574.
Roop, S. L., Jin, Q. I., Knight, J. C., Massung, R. F. & Esposito, J. J. (1995). PCR strategy for identification and differentiation of smallpox and other orthopoxviruses. J Clin Microbiol 33, 20692076.[Abstract]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Analysis and cloning of eukaryotic genomic DNA. In Molecular Cloning: A Laboratory Manual, 2nd edn, vol. 2, pp. 9.19.62. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 54635467.[Abstract]
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol Biol Evol 13, 964969.
Swofford, D. L. (1998). PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sunderland, MA: Sinauer Associates.
Tajima, F. & Nei, M. (1984). Estimation of evolutionary distance between nucleotide sequences. Mol Biol Evol 1, 269285.[Abstract]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Trindade, G. S., Fonseca, F. G., Marques, J. T. & 8 other authors (2003). Araçatuba virus: a vaccinia-like virus associated with cattle and human infection. Emerg Infect Dis 9, 155160.[Medline]
Van de Peer, Y. & de Wachter, R. (1997). Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites. Comput Appl Biosci 13, 227230.[Abstract]
Received 24 November 2003;
accepted 2 March 2004.
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