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Virology Journal
BioMed Central
Open Access
Case Report
Detection and phylogenetic analysis of Orf virus from sheep in
Brazil: a case report
Jônatas S Abrahão1, Rafael K Campos1, Giliane S Trindade1,
Maria IM Guedes1, Zélia IP Lobato2, Carlos Mazur3, Paulo CP Ferreira1,
Cláudio A Bonjardim1 and Erna G Kroon*1
Address: 1Laboratório 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, 2Departamento de Medicina Veterinária Preventiva, Escola
de Veterinária, Universidade Federal de Minas Gerais. Av. Antônio Carlos, 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil and 3Departamento
de Microbiologia e Imunologia Veterinária, Universidade Federal Rural do Rio de Janeiro. BR465, Km07, Boa Esperança. CEP: 23890-000,
Seropedica, Rio de Janeiro, Brazil
Email: Jônatas S Abrahão - jonatas.abrahao@gmail.com; Rafael K Campos - rafaklugleafar@msn.com;
Giliane S Trindade - gitrindade@yahoo.com.br; Maria IM Guedes - isabelguedes@icb.ufmg.br; Zélia IP Lobato - ziplobat@vet.ufmg.br;
Carlos Mazur - mazur@ufrj.br; Paulo CP Ferreira - pauloferreira120@hotmail.com; Cláudio A Bonjardim - claudio.bonjardim@pq.cnpq.br;
Erna G Kroon* - kroone@icb.ufmg.br
* Corresponding author
Published: 4 May 2009
Virology Journal 2009, 6:47
doi:10.1186/1743-422X-6-47
Received: 2 March 2009
Accepted: 4 May 2009
This article is available from: http://www.virologyj.com/content/6/1/47
© 2009 Abrahão et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Orf virus (ORFV), the prototype of the genus Parapoxvirus (PPV), is the etiological
agent of contagious ecthyma, a severe exanthematic dermatitis that afflicts domestic and wild small
ruminants. Although South American ORFV outbreaks have occurred and diagnosed there are no
South American PPV major membrane glycoprotein B2L gene nucleotide sequences available.
Case presentation: an outbreak of ovine contagious ecthyma in Midwest Brazil was investigated.
The diagnosis was based on clinical examinations and molecular biology techniques. The molecular
characterization of the virus was done using PCR amplification, cloning and DNA sequencing of the
B2L gene. The phylogenetic analysis demonstrated a high degree of identity with ORFV strains, and
the isolate was closest to the ORFV-India 82/04 isolate. Another Brazilian ORFV isolate, NE1, was
sequenced for comparative analysis and also showed a high degree of identity with an Asian ORFV
strain.
Conclusion: Distinct ORFV strains are circulating in Brazil. This is the first report on the
phylogenetic analysis of an ORFV in South America.
Background
Orf virus (ORFV), the prototype of the genus Parapoxvirus
(PPV), is the etiological agent of contagious ecthyma, a
severe exanthematic dermatitis that afflicts domestic and
wild small ruminants [1]. The disease is usually characterized by highly infectious pustules on the lips, tongue and
around the mouth. The transmission occurs by direct contact or via environmental contamination [2,3]. A decrease
in host fitness is observed, since the lesions lead to the
underfeeding of young lambs. Contagious ecthyma is a
zoonosis, and the human disease consists of acute skin
lesions, malaise and lymphadenopathy [4,5]. ImmunodePage 1 of 4
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Virology Journal 2009, 6:47
ficient people, however, can develop serious infections
[6].
In the last several years, several ORFV outbreaks have been
occurred worldwide [7-11]. Although clinical diagnosis
and electron microscopy have been used for viral identification, only PCR and genomic analyses can distinguish
ORFV from other PPV species [12]. The PPV major membrane glycoprotein (B2L) gene has been used in the
molecular characterization and phylogenetic analysis [811]. Although South American ORFV outbreaks have
occurred and diagnosed, there are no South American
ORFV B2L nucleotide sequences available [13]. Here we
described the detection and partial sequencing of the B2L
gene of a Brazilian ORFV isolate. This is the first report on
the phylogenetic analysis of ORFV in Brazil.
Case presentation
In June of 2005, an exanthematic outbreak occurred during an ovine exposition in Mato Grosso State (15°36'S
and 56°06'W), Brazil. Three sheep (Ovis aries) presented
wartlike lesions (dried-scabs) on the lips, tongue and
around the mouth. The clinical diagnosis was contagious
ecthyma. The outbreak area was isolated, and biological
specimens were collected. Dried scabs were collected
using a pair of sterilized tweezers and stored in a -70°C
freezer until the samples were processed. The tissue samples (25 mg) were mechanically homogenized in 250 μl
of phosphate buffered saline (PBS) in a tube using a pellet
pestle device, the homogenates were centrifuged at 2000
× g for 3 min, and the supernatant was collected.
Virus was detected using PCR amplification of the B2L
internal region (PPP-1 and PPP-4 primers) as previously
described, using 2 μL of the supernatants, with no DNA
extraction, as a template [14]. Water and scabs collected
from bovine vaccinia lesions were used as negative controls. Brazilian goat ORFV scabs, NE1, were used as PCR
positive controls [15,16]. All of the experimental and control samples were screened for orthopoxviruses (OPV)
using PCR and virus growth factor-specific primers [17].
The PCR B2L product was purified using the QIAquick Gel
Extraction Kit, (QIAGEN) and cloned into the pGEMTeasy vector (Promega, Madison, Wisconsin, USA). Three
clones were sequenced in both orientations using M13
universal primers (Mega-BACE sequencer, GE Healthcare,
Buckinghamshire, UK). The Brazilian ORFV NE1 was also
sequenced for comparative analysis. The sequences were
aligned with previously published PPV sequences from
GenBank by using the ClustalW method, manually
aligned using the MEGA software version 3.1(Arizona
State University, Phoenix, Arizona, USA) and adjusted to
equal length of 549 bp (ranging from nucleotides 409–
957 in the full-length ORFV B2L nucleotide sequence).
http://www.virologyj.com/content/6/1/47
Multiple alignments of deduced amino acids sequences
were generated. Phylogenetic trees were constructed by
neighbor-joining method with 1,000 bootstrap replicates
using the Tamura 3 parameters model implemented by
MEGA 3.1. The Brazilian ORFV partial B2L sequences
were deposited in GenBank, named ORFV-MT05
(FJ665818) and ORFV-NE1 (FJ665819).
The expected PPV B2L gene fragment (590 bp) was amplified from the Mato Grosso ovine samples and from the
NE1 positive control. With the exception of the bovine
vaccinia sample, the OPV PCR did not generate any specific amplicon. The comparison of the PPV B2L sequences
demonstrated a high degree of identity among our isolates
and other ORFV strains (Figure 1-A), and the paired identity at the nucleotide level ranged from 98.2% to 99.8%
and from 98% and 99.8% for the MT05 and NE1 isolates,
respectively. Nucleotide and amino acid sequences demonstrated that the MT05 isolate was closer to the ORFV
India82/04 isolate (DQ263303). The sequences of MT05
and India82/04 showed 99.8% and 100% similarities at
the nucleotide and amino acid level, respectively. Multiple alignment of nucleotide sequences revealed a MT05
singleton site (90), corresponding to a silent mutation
(Figure 2).
The other Brazilian isolate analyzed, NE1, was closer to
the ORFV Taiping isolate (EU327506), based on nucleotide and amino acid sequences. NE1 and ORFV Taiping
showed 99.8% and 100% similarities at the nucleotide
and amino acid level, respectively. Many nucleotide substitutions, including 10 singletons, were observed dispersed along B2L sequences of the different analyzed PPV
species, including the Brazilian ORFV isolates. Although
grouped in ORFV cluster, the Brazilian samples showed
variations between their sequences, and presented similarity of 98.7% at the nucleotide level.
Conclusion
An increasing number of ORFV outbreaks have occurred
worldwide in the last several years [7-11]. New molecular
diagnostic tests have been developed and used in association with traditional clinical approaches [14,18,19]. The
B2L gene is an important PPV molecular target, and several PPV B2L nucleotide and amino acids sequences are
available in GenBank. Moreover, detection of the B2L
gene is the most sensitive method for virus detection
because it harbors epidemiologically relevant sequence
information [9-11].
ORFV is endemic in Brazil [16]. Although vaccination has
been implemented in some regions, morbidity and economic losses are significant. An increase in the Brazilian
ovine and caprine market has been observed over the last
several years, leading to the emancipation of herds and
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Virology Journal 2009, 6:47
http://www.virologyj.com/content/6/1/47
Figure 1
PPV phylogenetic analysis (A) and ORFV phylogenetic analysis (B) based on the B2L gene sequence
PPV phylogenetic analysis (A) and ORFV phylogenetic analysis (B) based on the B2L gene sequence. The midpoint-rooted condensed tree (cutoff value of 70% from 1,000 bootstrap replicates) was constructed based on the B2L gene
sequences by the neighbor-joining method using the Tamura-3 parameters model of nucleotide substitution implemented in
MEGA3. Black spots indicate Brazilian ORFV isolates.
Figure 2 sequence others PPV available in GenBank
same sequences from fragment of the ORFV-MT05 and ORFV-NE1 major envelope protein gene (B2L) and comparison with
Nucleotide
Nucleotide sequence fragment of the ORFV-MT05 and ORFV-NE1 major envelope protein gene (B2L) and
comparison with same sequences from others PPV available in GenBank. Box indicates a MT05 singletone site, correspondent to a degenerated mutation. Points indicate regions conserved in all viruses.
Page 3 of 4
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Virology Journal 2009, 6:47
the intensification of the circulation of animals. In contrast with this increase, the management of small ruminants is rudimentary, uses unsophisticated technical
assistance and depends on the native vegetation. This scenario is favorable for an increased number of ORFV outbreaks. We analyzed a partial B2L gene sequence from
MT05, a new Brazilian ORFV strain, and the previously
described ORFV-NE1 isolate. Both Brazilian samples were
grouped with Asiatic isolates, ORFV-India82/04 and
ORFV-Taiping, respectively. Although the phylogenetic
analysis can indicate a hypothetical origin of viral strains,
it is difficult to determine the precise route in which the
identified ORFV strains were introduced into Brazil. However, these data suggest that the introduction of new ORFV
strains into Brazil may occur through the import of animals in order to improve herd genetics. Therefore, epidemiological surveillance can reduce ORFV outbreaks. This
study provides phylogenetic information about ORFV
strains, which is a matter for prospective public health
studies.
Competing interests
http://www.virologyj.com/content/6/1/47
10.
11.
12.
13.
14.
15.
16.
17.
The authors declare that they have no competing interests.
18.
Authors' contributions
19.
JA, GT, MG, ZL, PF, CB and EK participated in the planning of the project. EK was the leader of the project. ZL
and CM collected the samples. JA and RC, performed the
PCR and phylogenetic analysis. All authors read and
approved the final manuscript.
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Acknowledgements
We thank MSc. João R. dos Santos, Angela S. Lopes, Ilda M.V. Gamma, and
colleagues from Laboratório de Vírus (ICB-UFMG). Financial support was
provided by CNPq, CAPES and FAPEMIG. EGK, CAB and PCPF received
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