Parapoxviruses (PPVs) are zoonotic viruses which cause contagious pustular skin
infections of sheep, goats and cattle worldwide. In addition, they have more recently
been shown to infect other animals such as red deer, seals, camels and reindeer.
Cases of contagious pustular stomatitis in Finnish reindeer have been reported for
many years. This economically important disease occurs typically during winter
and is more common in the southern parts of the reindeer herding districts than in
the north. The first severe outbreak occurred in the winter 1992-1993, and during
the winter of 1999-2000 and in the late winter 2007 outbreaks of the disease were
again observed. Usual symptoms include diminished appetite, drooling, fever, and
later erosions and ulcerative lesions in the mouth. The aims of this study were to
establish specific and rapid detection methods for the causative agent of the disease
and characterize the viruses circulating in Finland.
The causative agent of reindeer pustular stomatitis was originally considered to
be Orf virus (ORFV) of the genus Parapoxvirus. PCR methods amplifying different
regions of the PPV genomes were developed to analyse clinical samples obtained
from outbreaks of the disease in reindeer and later from viruses isolated from the
disease of sheep and cattle in Finland. Subsequent phylogenetic analyses of the
Finnish PPVs, known members of the genus Parapoxvirus and selected members
of the subfamily Chordopoxvirinae were conducted to identify the virus species
isolated from reindeer. The results showed that the reindeer PPV from 1999-2000
is most closely related to the cattle PPV Pseudocowpox virus (PCPV) whereas the
PPV strains from the winter of 1992-1993 outbreak grouped with sheep ORFV
strains. Reindeer samples from the 2007 outbreak were identified as both PCPV
and ORFV. Analysis of the similarity between genes of reindeer PCPV and ORFV
isolates, Finnish sheep ORFV and cattle PCPV isolates indicated that these viruses
have been circulating among Finnish reindeer, cattle and sheep at least ten years.
Since the initial classification of the viruses causing pustular stomatitis in Finnish
reindeer relied solely on the partial sequence analysis of virion core- and EEV
envelope phospholipase protein sequences, the genome of PCPV-like reindeer
isolate (F00.120R) was sequenced by shotgun sequencing of plasmid sublibraries
of cosmids covering the central region of the genome, and by sequencing transposon
random insertion libraries of plasmids derived from each end of the genome. The
F00.120R and the genomic sequence of a reference strain of PCPV (VR634) were
annotated and analyzed in this study. This first characterization of PCPV genomes
revealed that F00.120R and VR634 are 135 and 145 kb in length and contain 131
and 134 putative genes, respectively. The organization of their genomes was found to be similar to that of other PPVs and both included 88 predicted genes that
are conserved across all sequenced poxviruses. F00.120R was found to have four,
possibly fragmented, genes at the left terminus and another near the central region
of the genome that are not present in ORFV or Bovine papular stomatitis virus
(BPSV; another PPV) genomes. In addition, the F00.120R genome was found to lack
six genes seen near the right genome terminus of other PPVs. Comparing the PPV
proteomes and whole genome phylogenetic analyses confirmed the classification of
PCPV as a separate species within the PPV genus and verified that the virus causing
pustular stomatitis in reindeer in 1999-2000 can be classified as PCPV.
The observed six gene deletion at the right terminus of the F00.120R genome
was further investigated in an attempt to use it in differentiating PCPV and ORFV
causing pustular stomatitis in reindeer. The preliminary PCR analyses of wild type
virus and early passages of F00.120R implied that the deletion of genes may have
arisen during cell culture of the virus. The sequence around the deleted region was
determined by sequencing two cloned overlapping PCR fragments from F00.120R
wt virus isolated from lesion material. The same region was sequenced from an
Italian PCPV field isolate (It1303). Further PCR analyses together with sequence
determination showed that a 5431 bp sequence containing genes 116-121 was likely
to have been deleted from the F00.120R genome prior to the 7th passage in cell
culture. In addition, genes 116-121 were present in It1303 and in other isolates of
reindeer and bovine PCPV isolated in Finland during the years 2005-2010. These
results indicate that the genome of reindeer PCPV is about 140 kbp in length and
has 137 genes instead of previously estimated length of 135 kbp and 131 genes; it
contains homologues of all known ORFV genes and this analysis further reinforces
the close genetic relationship between PCPV and ORFV
