Elsevier

Virus Research

Volume 181, 6 March 2014, Pages 11-21
Virus Research

Comparative and retrospective molecular analysis of Parapoxvirus (PPV) isolates

https://doi.org/10.1016/j.virusres.2013.12.015Get rights and content

Highlights

  • Comparative molecular analysis of 23 Parapoxvirus (PPV) isolates.

  • Two evaluated PCRs allowed PPV species discrimination and allocation of the isolates.

  • Zoonotic PPV infections are classified with respect to the source of infection.

  • Whole genome sequence data of an Orf virus and its human counterpart are provided.

  • PPV genomes showed conserved sequence features even after changing the host species.

Abstract

Species members of the genus Parapoxvirus (PPV) within the family Poxviridae cause contagious pustular dermatitis in small ruminants (Orf virus, ORFV) and mostly mild localized inflammation in cattle (bovine papular stomatitis virus, BPSV and pseudocowpox virus, PCPV). All PPVs are known to be zoonotic, leading to circumscribed skin lesions in humans, historically known as milker's nodules. Human PPV isolates are often ill defined concerning their allocation to an animal origin.

Here we present a comparative molecular analysis of a unique collection of 21 historic and recent human and animal PPV cell culture isolates (and two PPV DNA samples). Cell culture PPV propagation was restricted to primary ruminant fibroblasts and was strictly kept at low passages to avoid genomic changes by in vitro influences. For molecular arrangement of the isolate DNAs and their attribution to established PPV species DNA fragments of the PPVs were generated by two different discriminating PCR protocols, targeting the major part of the open reading frame (ORF) 011 (B2L gene) and the complete ORF 032. Multiple sequence alignments and phylogenetic analysis of both genes resulted in affiliation to the known PPV species. The sequences from the ORF 032 allowed discrimination of the isolate DNAs at a higher resolution. Human PPV isolates could be clearly assigned to the PPV species belonging to the reported or assumed animal host of transmission.

For the first time, a whole PPV genome sequence comparison of a human biopsy derived virus (B029) and its ovine counterpart (B015) originating from a defined Orf outbreak in Germany is provided, revealing their well conserved relationship. Thus human PPVs can be molecularly retraced to the PPV species indicating the animal of transmission. After transmission to the human host, molecular conservation of the animal's virus peculiarities indicative for a PPV species became evident.

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.

References (59)

  • A.A. Mercer et al.

    Comparative analysis of genome sequences of three isolates of Orf virus reveals unexpected sequence variation

    Virus Res.

    (2006)
  • G. Nagarajan et al.

    Sequence analysis of topoisomerase gene of pseudocowpox virus isolates from camels (Camelus dromedarius)

    Virus Res.

    (2011)
  • A.J. Robinson et al.

    Parapoxvirus of red deer: evidence for its inclusion as a new member in the genus parapoxvirus

    Virology

    (1995)
  • J.T. Sullivan et al.

    Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K

    Virology

    (1994)
  • T. Vikøren et al.

    A severe outbreak of contagious ecthyma (orf) in a free-ranging musk ox (Ovibos moschatus) population in Norway

    Vet. Microbiol.

    (2008)
  • F. Ballanger et al.

    Two giant orf lesions in a heart/lung transplant patient

    Eur. J. Dermatol.

    (2006)
  • A. Bankevich et al.

    SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing

    J. Comput. Biol.

    (2012)
  • Y. Bayindir et al.

    Investigation and analysis of a human orf outbreak among people living on the same farm

    New Microbiol.

    (2011)
  • P. Becher et al.

    Characterization of sealpox virus, a separate member of the parapoxviruses

    Arch. Virol.

    (2002)
  • J.J. Campanella et al.

    MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences

    BMC Bioinformatics

    (2003)
  • Centers for Disease Control and Prevention (CDC)

    Human Orf virus infection from household exposures – United States, 2009–2011

    MMWR Morb. Mortal. Wkly. Rep.

    (2012)
  • F. Dal Pozzo et al.

    Original findings associated with two cases of bovine papular stomatitis

    J. Clin. Microbiol.

    (2011)
  • T. Dashtseren et al.

    Camel contagious ecthyma (pustular dermatitis)

    Acta Virol.

    (1984)
  • D. Deane et al.

    Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins

    J. Gen. Virol.

    (2009)
  • G. Delhon et al.

    Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus

    J. Virol.

    (2004)
  • A.E. Friedmann-Kien et al.

    Milker's nodules: isolation of a poxvirus from a human case

    Science

    (1963)
  • U. Gassmann et al.

    Analysis of parapoxvirus genomes

    Arch. Virol.

    (1985)
  • K. Geerinck et al.

    A case of human orf in an immunocompromised patient treated successfully with cidofovir cream

    J. Med. Virol.

    (2001)
  • J. Goecks et al.

    Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences

    Genome Biol.

    (2010)
  • Cited by (0)

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