Reindeer Parapoxvirus : Molecular Biology and Detection

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

Reindeer Parapoxvirus

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

Genotyping and surveillance for scrapie in Finnish sheep

BACKGROUND: The progression of scrapie is known to be influenced by the amino acid polymorphisms of the host prion protein (PrP) gene. There is no breeding programme for TSE resistance in sheep in Finland, but a scrapie control programme has been in place since 1995. In this study we have analysed PrP genotypes of total of 928 purebred and crossbred sheep together with the data of scrapie survey carried out in Finland during 2002–2008 in order to gain knowledge of the genotype distribution and scrapie prevalence in Finnish sheep. RESULTS: The ARQ/ARQ genotype was the most common genotype in all breeds studied. ARR allele frequency was less than 12% in purebred Finnish sheep and in most genotypes heterozygous for ARR, the second allele was ARQ. The VRQ allele was not detected in the Grey race sheep of Kainuu or in the Aland sheep, and it was present in less than 6% of the Finnish Landrace sheep. Leucine was the most prominent amino acid found in codon 141. In addition, one novel prion dimorphisms of Q220L was detected. During the scrapie survey of over 15 000 sheep in 2002–2008, no classical scrapie cases and only five atypical scrapie cases were detected. CONCLUSIONS: The results indicate that the Finnish sheep populations have genetically little resistance to classical scrapie, but no classical scrapie was detected during an extensive survey in 2002–2008. However, five atypical scrapie cases emerged; thus, the disease is present in the Finnish sheep population at a low level

Integrated data management and validation platform for phosphorylated tandem mass spectrometry data

MS/MS is a widely used method for proteome-wide analysis of protein expression and PTMs. The thousands of MS/MS spectra produced from a single experiment pose a major challenge for downstream analysis. Standard programs, such as MASCOT, provide peptide assignments for many of the spectra, including identification of PTM sites, but these results are plagued by false-positive identifications. In phosphoproteomic experiments, only a single peptide assignment is typically available to support identification of each phosphorylation site, and hence minimizing false positives is critical. Thus, tedious manual validation is often required to increase confidence in the spectral assignments. We have developed phoMSVal, an open-source platform for managing MS/MS data and automatically validating identified phosphopeptides. We tested five classification algorithms with 17 extracted features to separate correct peptide assignments from incorrect ones using over 2600 manually curated spectra. The naïve Bayes algorithm was among the best classifiers with an AUC value of 97% and PPV of 97% for phosphotyrosine data. This classifier required only three features to achieve a 76% decrease in false positives as compared with MASCOT while retaining 97% of true positives. This algorithm was able to classify an independent phosphoserine/threonine data set with AUC value of 93% and PPV of 91%, demonstrating the applicability of this method for all types of phospho-MS/MS data. PhoMSVal is available at http://csbi.ltdk.helsinki.fi/phomsval.National Science Foundation (U.S.). Graduate Research Fellowship Progra

Viral haemorrhagic septicaemia virus (VHSV Id) infections are detected more consistently using syndromic vs. active surveillance

The eradication of viral haemorrhagic septicaemia virus (VHSV Id) from Finnish brackish-water rainbow trout Oncorhynchus mykiss farms located in the restriction zone in the Province of Åland, Baltic Sea, failed several times in the 2000s. The official surveillance programme was often unable to find VHSV-positive populations, leading to the misbelief in the fish farming industry that virus eradication could be achieved. The ability of 3 other surveillance programmes to detect infected fish populations was compared with the official programme. One programme involved syndromic surveillance based on the observation of clinical disease signs by fish farmers, while 2 programmes comprised active surveillance similar to the official programme, but included increased sampling frequencies and 2 additional tests. The syndromic surveillance concentrated on sending in samples for analysis when any sign of a possible infectious disease at water temperatures below 15°C was noticed. This programme clearly outperformed active surveillance. A realtime reverse transcriptase-polymerase chain reaction method proved to be at least as sensitive as virus isolation in cell culture in detecting acute VHSV infections. An ELISA method was used to test fish serum for antibodies against VHSV. The ELISA method may be a useful tool in VHSV eradication for screening populations during the follow-up period, before declaring an area free of infection

The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries

Background: During the last decade, active surveillance for transmissible spongiform encephalopathies in small ruminants has been intensive in Europe. In many countries this has led to the detection of cases of atypical scrapie which, unlike classical scrapie, might not be contagious. EU legislation requires, that following detection of a scrapie case, control measures including further testing take place in affected flocks, including the culling of genotype susceptible to classical scrapie. This might result in the detection of additional cases. The aim of this study was to investigate the occurrence of additional cases in flocks affected by atypical scrapie using surveillance data collected in Europe in order to ascertain whether atypical scrapie, is contagious. Results: Questionnaires were used to collect, at national level, the results of active surveillance and testing associated with flock outbreaks in 12 European countries. The mean prevalence of atypical scrapie was 5.5 (5.0-6.0) cases per ten thousand in abattoir surveillance and 8.1 (7.3-9.0) cases per ten thousand in fallen stock. By using meta-analysis, on 11 out of the 12 countries, we found that the probability of detecting additional cases of atypical scrapie in positive flocks was similar to the probability observed in animals slaughtered for human consumption (odds ratio, OR = 1.07, CI95%: 0.70-1.63) or among fallen stock (OR = 0.78, CI95%: 0.51-1.2). In contrast, when comparing the two scrapie types, the probability of detecting additional cases in classical scrapie positive flocks was significantly higher than the probability of detecting additional cases in atypical scrapie positive flocks (OR = 32.4, CI95%: 20.7-50.7). Conclusions: These results suggest that atypical scrapie is not contagious or has a very low transmissibility under natural conditions compared with classical scrapie. Furthermore this study stressed the importance of standardised data collection to make good use of the analyses undertaken by European countries in their efforts to control atypical and classical scrapie

Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer

Peer reviewe

Origins and Impacts of New Mammalian Exons

Mammalian genes are composed of exons, but the evolutionary origins and functions of new internal exons are poorly understood. Here, we analyzed patterns of exon gain using deep cDNA sequencing data from five mammals and one bird, identifying thousands of species-and lineage-specific exons. Most new exons derived from unique rather than repetitive intronic sequence. Unlike exons conserved across mammals, species-specific internal exons were mostly located in 5' UTRs and alternatively spliced. They were associated with upstream intronic deletions, increased nucleosome occupancy, and RNA polymerase II pausing. Genes containing new internal exons had increased gene expression, but only in tissues in which the exon was included. Increased expression correlated with the level of exon inclusion, promoter proximity, and signatures of cotranscriptional splicing. Altogether, these findings suggest that increased splicing at the 5' ends of genes enhances expression and that changes in 5' end splicing alter gene expression between tissues and between species.Peer reviewe

Mitochondrial toxicity of triclosan on mammalian cells

Effects of triclosan (5-chloro-2’-(2,4-dichlorophenoxy)phenol) on mammalian cells were investigated using human peripheral blood mono nuclear cells (PBMC), keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine pancreatic islets (MIN-6) and neuroblastoma cells (MNA) as targets. We show that triclosan (1 – 10 μg ml-1) depolarised the mitochondria, upshifted the rate of glucose consumption in PMBC, HaCaT, PK-15 and MNA, and subsequently induced metabolic acidosis. Triclosan induced a regression of insulin producing pancreatic islets into tiny pycnotic cells and necrotic death. Short exposure to low concentrations of triclosan (30 min, ≤ 1 μg / ml) paralysed the high amplitude tail beating and progressive motility of spermatozoa, within 30 min exposure, depolarized the spermatozoan mitochondria and hyperpolarised the acrosome region of the sperm head and the flagellar fibrous sheath (distal part of the flagellum). Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100 - 1000 fold lower that those permitted in consumer goods. The mitochondriotoxic mechanism of triclosan differs from that of valinomycin, cereulide and the enniatins by not involving potassium ionophoric activity.Peer reviewe

Infection with possible novel parapoxvirus in horse, Finland, 2013

A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus.</p