Comparative and retrospective molecular analysis of Parapoxvirus (PPV) isolates
Introduction
Parapoxviruses (PPVs) are commonly known as causative agent of infections and diseases worldwide, leading to a contagious pustular dermatitis (CPD), especially in the region of the lips, nostrils, oral mucosa and teats of domestic and wild ruminants. They are gaining importance primarily because of financial and economic losses in connection with disease outbreaks in livestock and because of their zoonotic potential.
In accordance with the current nomenclature there are four established species within the PPV genus, which belongs to the Chordopoxvirinae within the Poxviridae family:
The prototype member, Orf virus (ORFV) is endemic in most sheep and goat raising countries. Bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) mainly infect cattle but are differentiated by the manifestation locus on the muzzle and the teats, respectively. Parapoxvirus of red deer in New Zealand (PVNZ) is a newly defined species of the genus and, so far, solely relevant for infection of Cervids (Robinson and Mercer, 1995).
Additionally several new tentative species are a matter of debate, such as camel contagious ecthyma virus, reindeer parapoxvirus, muskox and seal parapoxvirus (Becher et al., 2002, Dashtseren et al., 1984, Tikkanen, 2004, Vikøren et al., 2008).
All PPVs are known to be zoonotic and infect humans after direct or indirect contact with infected animals, provoking local skin lesions historically known as milker's nodules (MN).
Albeit human PPV infections are considered to be rare and rather fortuitous events, infection rates of individuals at risk (e.g. slaughtermen, veterinarians, farmers and animal caretakers) are way higher. Also people not occupationally handling animals can get infected, particularly by visiting a petting zoo or by household exposure when handling contaminated furs, skin, or meat (Bayindir et al., 2011, CDC, 2012). Mass infection of people has been described linked to ritual sheep slaughter in celebration of Islamic holidays “Feast of Sacrifice” (Nougairede et al., 2013, Uzel et al., 2005).
The epitheliotropic PPVs enter via scarified or otherwise broken skin and subsequently can replicate locally in the human epidermis. Occasionally large and painful nodules evolve which are mostly localized on the hands; especially the index finger and less frequently at other skin sites, presumably by smear infection. Disease manifestation via localized lesions is benign and generally resolves within max. 1–2 month (Leavell et al., 1968), even without any medical treatment. However, there are individual cases described in the literature of severe complications; often linked to immunosuppression (Gurel et al., 2002, Larcher et al., 2009, Lederman et al., 2007). A common feature of PPV is the stimulation of vascular endothelial cells to proliferate leading to a tumor like clinical outcome described as “giant orf” in man (Ballanger et al., 2006, Geerinck et al., 2001). In sheep, severe lesion development is known as bloody lesion or cauliflower-like proliferation mostly complicated by secondary bacterial infections. Recently some exceptional co-infections with Vaccinia virus and ORFV or Vaccinia virus and PCPV in cattle were reported from Brazil (Abrahão et al., 2010, Sant’Ana et al., 2013).
PPVs possess a linear double-stranded DNA genome, which is approximately 130–150 kbp in size and exhibits an unusual high GC content (∼64%). While PPV isolates initially were defined by their host origin and the circumstances of infection and disease an increasing number of genomic data including whole genome sequences are used to define PPV species (Delhon et al., 2004, Hautaniemi et al., 2010, Mercer et al., 2006). The central region of the genome contains 88 genes that are present in the subfamily Chordopoxvirinae and mostly occur in a common order and orientation. The ends of the genomes are cross linked. The PPV unique and pathogenesis related genes are located in the terminal regions of the genomes (Mercer et al., 2006). Interestingly Gassmann et al. (1985) showed for the first time that the virus species ORFV, BPSV and PCPV can be differentiated by probes corresponding to terminal genomic regions.
A preferred target gene to generate PCR amplified DNA fragments for sequence analysis and comparison of PPV DNA is the open reading frame (ORF) 011 (B2L gene), the PPV orthologue of the vaccinia virus Copenhagen (VACV) gene F13L, which encodes the major envelope antigen p37K (Sullivan et al., 1994).
The PPV isolates of human origin mostly designated as PCPV are often ill defined concerning the source of animal transmission and they lack a clear position in nomenclature which should become more transparent by molecular data.
In this study PCR fragment sequences of historic and recent PPV isolates of national and international origin were compared. Two different genomic target sites were chosen for PCR based sequence generation. The major part of the coding region of the B2L gene was used for PCR fragment sequencing as well as the complete open reading frame 032 (ORF 032), another genomic region hypothesized to contain sufficient sequence heterogeneity for differentiation among the PPV species. A phylogenetic analysis should reveal PPV species peculiarities especially focused on PPV isolates collected from human cases in relation to the origin of animal transmission.
Section snippets
Virus isolates
Most of the PPV isolates used in this study (n = 23) were collected in Germany (n = 15) but in addition we could include DNA from two BPSV cases from Scotland (C. McInnes, Moredun Research Institute, Penicuik, Scotland), one BPSV isolate from Cameroon (K. A. Lay, Tübingen, Germany) and one from Iran (M. Hessami, Razi Institute, Karaj, Iran). Also ORFV cell culture isolates from Scotland (C. McInnes, Moredun Research Institute, Penicuik, Scotland), Sweden (Z. Dinter, Uppsala, Sweden) and Japan (D.
Cell culture isolation of PPV
All samples yielding a positive PPV particle diagnosis in electron microscopy were used for tissue culture propagation attempts. From the supernatants of 21 tissue samples from diseased men or animals (Table 1) PPV could be isolated in primary bovine cells. The occurrence of first signs of CPE lasted at least five days, in some cases up to ten days. After harvest of the first cell culture passage by repeated freeze thawing further cell passages were most efficient by simultaneous inoculation at
Discussion
As part of the PPV genomic core region both PCR amplified DNA fragments used in this study are well conserved. Above all the B2L gene is known to be conserved in PPV genomes. A PCR-protocol resulting in a 594 bp fragment of the B2L gene has been widely used for detection and diagnosis of PPVs and beyond that for identification of different PPV species (Hosamani et al., 2006, Inoshima et al., 2000, Inoshima et al., 2001, Nagarajan et al., 2011, Scagliarini et al., 2011). The PPV011-primer pair
Acknowledgments
We thank Anton Mayr a pioneer of German post war poxvirus research for his permanent enthusiasm and encouragement. We also thank A.A. Mercer and H.-J. Rzhia for fruitful discussions. Finally we thank S. Gellert and F. Sedlmaier for excellent technical support.
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