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Orf virus (Parapoxvirus ovis, ORFV) is a dermatotropic virus causing pustular dermatitis in small ruminants and humans. We analysed isolated human primary keratinocytes (KC) and dermal fibroblasts (FB) for cell death and virus replication by infection with a patient-derived ORFV isolate. ORFV infection was associated with rapid induction of cell death in KC allowing for considerable virus removal. Upon infection with ORFV, KC and FB harboured intracytoplasmic ORFV and showed viral protein presence; however, missing virus spread indicated an abortive infection. Upon ORFV exposure, KC but not FB secreted the pro-inflammatory cytokine interleukin (IL)-6. ORFV infection enhanced the frequency of KC expressing intercellular adhesion molecule (ICAM)-1 which was independent of IL-6. Interestingly, ORFV inhibited ICAM-1 up-regulation on infected but not on non-infected KC. Even interferon-γ, a potent inducer of ICAM-1, up-regulated ICAM-1 only on non-infected KC. Transfer of ORFV-free supernatant from infected to non-infected KC induced ICAM-1 on non-infected KC pointing to the involvement of soluble mediator(s). Similarly as in KC, in FB interference with ICAM-1 up-regulation by ORFV infection was also observed. In conclusion, we shed light on epidermal and dermal defense mechanisms to ORFV infection and point to a novel ICAM-1-related immune evasion mechanism of ORFV in human skin.

Parapoxvirus (PPV) infections are of worldwide importance, particularly in sheep and goat herds. Owing to the zoonotic potential of all PPV species, they are a permanent threat to human health as well. The virus is also known to affect wildlife, as reported for pinnipeds, red deer and several other wild ruminants. PPVs found in red deer have been claimed as a unique species according to certain genomic features. So far infection of wildlife has been recognized because of clinical manifestation such as inflammation, stomatitis or typical pox-like lesions in the skin or mucous membranes. Here we report the use of targeted molecular diagnostics for the presence of PPV genomes in tonsil swabs of apparently healthy red deer in the Bavarian Alps. Out of 1764 swabs, 0.79 % tested positive for PPV genome presence. From one sample, PPV was successfully isolated in cell culture. This virus became the subject of complete genome characterization using next generation sequencing and various subsidiary PCR protocols. Strikingly, about a quarter of all ORFs were found to be larger than the corresponding ORFs in the reference PPV genome sequences used for comparison. To our knowledge this is the first genome-wide analysis that confirms red deer PPV as a unique species within the genus Parapoxvirus in Europe. Persistence of PPV in Alpine red deer indicates a source for virus transmission to susceptible livestock and hunters. The findings provide a further example of wildlife animals playing an important role as an inconspicuous reservoir of zoonotic diseases.

Bluetongue virus (BTV) antibodies were analysed in 27 Swiss calves born in 2016 at the age of 16-19 days using competitive enzyme-linked-immunosorbent-assay (cELISA) and virus neutralization test (VNT) (animal trial permission number: 75684). Obligatory documentation proved that 15 of 27 dams were BTV-8 vaccinated once or three times in 2008-2010. The offsprings of the non-vaccinated dams were seronegative. Two of three calves and 11 of 12 calves descending from dams who had been vaccinated one or three times, respectively, had BTV specific serum antibodies. As Switzerland is considered BTV-free from 2010 to 2016, it is likely that BTV-8 antibodies were transferred via colostrum. Furthermore, we confirmed neutralizing cross-reactivity of BTV-8 with BTV-4 antibodies as 5 samples positive for BTV-8 were also reactive with BTV-4 antibodies.

The avian-like swine influenza viruses emerged in 1979 in Belgium and Germany. Thereafter, they spread through many European swine-producing countries, replaced the circulating classical swine H1N1 influenza viruses, and became endemic. Serological and subsequent molecular data indicated an avian source, but details remained obscure due to a lack of relevant avian influenza virus sequence data. Here, the origin of the European avian-like swine influenza viruses was analysed using a collection of 16 European swine H1N1 influenza viruses sampled in 1979–1981 in Germany, the Netherlands, Belgium, Italy and France, as well as several contemporaneous avian influenza viruses of various serotypes. The phylogenetic trees suggested a triple reassortant with a unique genotype constellation. Time-resolved maximum clade credibility trees indicated times to the most recent common ancestors of 34–46 years (before 2008) depending on the RNA segment and the method of tree inference.

Five persons in France were infected with Orf virus after skin wounds were exposed to infected sheep tissues during Eid al-Adha, the Muslim Feast of Sacrifice. Infections were confirmed by electron microscopy, PCR, and sequence analysis. Prevention and control of this underdiagnosed disease can be achieved by educating physicians, slaughterhouse workers, and persons participating in Eid al-Adha.

In early 2009, four human cases of cowpox virus cutaneous infection in northern France, resulting from direct contact with infected pet rats (Rattus norvegicus), were studied. Pet rats, originating from the same pet store, were shown to be infected by a unique virus strain. Infection was then transmitted to humans who purchased or had contact with pet rats.

An increasing demand in livestock animal husbandry for intervention or emergency vaccination strategies requires a rapid onset of protection linked to prevention of infectious agent spread. Using the new recombinant parapoxvirus (PPV) Orf virus (ORFV) as a vaccine expressing the CSFV E2 glycoprotein we demonstrate that a single intra-muscular application confers solid protection. In the prime only concept, multi-site application of the vector vaccine turned out to be superior to single-site application as no pyrexia occurred after virulent CSFV challenge and CSFV neutralizing serum antibodies regularly were detectable before challenge. Vector virus vaccinated swine were able to cope with the lymphocyte and in particular B-cell depression in peripheral blood after challenge showing no clinical signs and no viremia. Early after challenge CSFV-specific IFN gamma production (IFN-gamma) and high neutralizing serum antibody titers clearly differentiated naïve from vaccinated and protected animals. After CSFV challenge neutralizing serum antibodies titers in vector vaccinated swine were significantly higher than those in sera from live attenuated vaccine primed animals. Horizontal challenge virus transmission was prevented under strict sentinel isolation before mingling but not in next-door stables separated by a wooden barrier at the day of challenge. The presence of CSFV-specific pre-challenge serum antibodies although in low titers is a good prognostic parameter for solid protection after ORFV vector vaccination even when a significant CSFV-specific IFN-gamma production was not detectable before challenge. A heterologous prime-boost regimen as a combination of prime with baculovirus-expressed glycoprotein E2 followed by boost with the parapoxvirus vector turned out to be a better immune stimulant than a homologous prime/boost with the modified live CSFV vaccine. A similar beneficial effect became evident when the challenge infection mimicked the booster vaccination after a single PPV vector prime.

In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of naive PBMC. Significant IFN-gamma production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1. The PRV-specific IFN-gamma secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-gamma production by PBMC clearly indicated that infection had occurred. Early significant IFN-gamma production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

Two lots of polyvalent live vaccines for human use against poliovirus were tested by reverse transcriptase (RT) and nested PCR for the presence of contaminating pestivirus RNA. By RT-PCR, samples from both lots showed a band of approximately 450 bp instead of 300 bp for the reference pestivirus strain used as positive control. After nested PCR, the template DNA (450 bp product) was not amplified, suggesting non-specificity of the previous amplification. Sequencing analysis confirmed the non-specificity of the 450 bp bands and revealed, respectively, 80 and 77% homology with a region in the VP1 gene of poliovirus type 1 in samples 1 and 2. This suggests that more caution should be taken in interpreting the results obtained by PCR, and that they should be confirmed by nested PCR or sequencing.

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and co-stimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.

Parapoxvirus (PPV) represents a genus of the poxviridae, and particularly PPV ovis (Orf virus, OV) seems to offer several potential advantages for the use of vector vaccine. Therefore, we started to investigate the genome of the highly attenuated OV strain D1701, which was only poorly characterised until now. Due to recombination of non-homologous sequences, part of the right hand end of the D1701 genome was duplicated and translocated to the opposite end of the genome. As a consequence gene deletion had occurred and the inverted terminal repeat region is increased. Results are described to identify viral genes, which are non-essential for virus replication and potentially influence viral pathogenesis, virulence, and host immunity. In more detail, we analysed the expression and functional activity of the OV-specific vascular endothelial growth factor (VEGF) gene homologue. Finally the construction and production of a D1701 mutant lacking the VEGF gene homologue is reported.

Two new classical swine fever virus (CSFV) isolates obtained from naturally infected swine were found to exhibit a cytopathogenic (cp) phenotype. According to their reactivity with monoclonal antibodies (mabs) the isolates cpBW1 and cpMVP1 were classified as antigenic types "Lothringen'92" and "Flandern'90", respectively. In Northern blot analyses and PCR assays CSFV RNA of subgenomic length was detected in infected cells indicating the presence of defective interfering particles. Nucleotide sequencing of cDNA fragments obtained by reverse transcription (RT)-PCR allowed to localize in the genomes of the cpMVP1 and cpBW1 DIs deletions of 4,764 and 4,746, respectively. From the latter isolate a noncytopathogenic (noncp) biotype was obtained by cloning in cell culture. CSFV cpBW1 and a noncp isolate were used separately for infection of pigs. Viremia occurred much quicker after infection with cp virus and clinical signs were more prominent compared to animals infected with noncp virus.

The orf virus (OV) strain D1701 belongs to the genetically heterogenous parapoxvirus (PPV) genus of the family Poxviridae. The attenuated OV D1701 has been licensed as a live vaccine against contagious ecthyma in sheep. Detailed knowledge on the genetic structure and organization of this PPV vaccine strain is an important prerequisite to reveal possible genetic mechanisms of PPV attenuation. The present study demonstrates a genomic map of the approximately 158 kbp DNA of OV D1701 established by hybridization studies of cloned restriction fragments covering the complete viral genome. The results show an enlargement of the inverted terminal repeats (ITR) to up to 18 kbp due to recombination between nonhomologous sequences during cell culture adaptation. DNA sequencing of the region adjacent to the ITR junction revealed the absence of one open reading frame designated E2L. In contrast to a transposition-deletion variant of the New Zealand OV strain NZ2 (Fleming et al., 1995) the two genes E3L (a homologue of dUTPase) and G1L neighbouring E2L are retained in OV D1701. DNA and RNA analyses proved the presence of E2L gene in wild-type OV isolated directly from scab material. The data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and may represent one important viral gene in determining virulence and pathogenesis of OV.

a b s t r a c t 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. © 2013 Elsevier B.V. All rights reserved.

Prototypes of three poxvirus genera—orthopoxvirus (OPV), parapoxvirus (PPV), avipoxvirus (APV)—and Newcastle disease virus (NDV) as a control, as well as three recombinant OPV strains and one recombinant APV strain, were incubated in vitro with peripheral blood mononuclear leukocytes (PBML) of man, sheep and swine. Antiviral activity was determined in PBML culture supernatants at different time intervals after virus cell interaction using a cytopathic effect inhibition bioassay. Additionally, supernatants derived from human PBML were screened for interferons (IFN) alpha and gamma as well as for tumor necrosis factor by enzyme-linked immunosorbent assay. IFN titers reached a maximum 24 h after PBML stimulation at a multiplicity of infection (MOI) greater than 1. IFN alpha/beta was found to be responsible for the antiviral effect. Using a MOI ⩾ 1 the highly attenuated strain MVA was the only representant of vaccinia virus (VV) that induced significant amounts of IFN also as a lacZ recombinant. Replicable virus from five well-known VV strains as well as the Chinese VV strain Tien Tan (VV TT) as a recombinant vaccine failed to induce Ieukocyte IFN. Inactivated VV strain Elstree and the recombinant TT strain induced high titers of leukocyte IFN. Supernatants derived from human, porcine and ovine PBML stimulated with replicable PPV, native VV MVA and MVA lacZ recombinant or native APV and APV lacZ recombinant virus regularly contained IFN alpha. In contrast to NDV, neither specific antisera nor monoclonal antibodies were able to block the INF induction by VV and PPV.

The interferon (IFN)-inducing capacities of intact NDV virions, β-propiolactone-inactivated particles and several structural components were compared, using human PBML as the IFN producing cells. Intact and inactivated virions as well as the nucleocapsid fraction did not differ significantly in their IFN-inducing capacity. In contrast, genomic RNA as well as M protein fraction and envelopes induced IFN titres to a level of about 10% of those achieved with virions. NDV-induced IFN production could be blocked specifically by incubation with polychonal anti-NDV-monoclonal antibodies (mAbs) and with two of three anti-HN-mAbs, but not with anti-NDV-mAbs directed against the F, M or NP protein. In addition, IFN induction by fixed MDBK cells, expressing NDV surface proteins after infection with NDV Ulster, was inhibited by one of two anti-F-mAbs. The results suggest that the induction of IFN synthesis in human PBML is a complex process involving not only the HN protein but also the uncleaved F protein precursor, a component of the M protein fraction and — once having entered the cell — the genomic RNA.

The compulsory vaccination campaign against Bluetongue virus serotype eight (BTV-8) in Germany was exercised in the state of Bavaria using three commercial monovalent inactivated vaccines given provisional marketing authorisation for emergency use. In eleven Bavarian farms representing a cross sectional area of the state the immune reactions of sheep and cattle were followed over a two year period (2008–2009) using cELISA, a serum neutralisation test (SNT) and interferon gamma (IFN-γ) ELISPOT. For molecular diagnostics of BTV genome presence two recommended real time quantitative RT-PCR protocols were applied. The recommended vaccination scheme led to low or even undetectable antibody titers (ELISA) in serum samples of both cattle and sheep. A fourfold increase of the vaccine dose in cattle, however, induced higher ELISA titers and virus neutralising antibodies. Accordingly, repeated vaccination in sheep caused an increase in ELISA-antibody titers. BTV-8 neutralising antibodies occurred in most animals only after multiple vaccinations in the second year of the campaign. The secretion of interferon gamma (IFN-γ) in ELISPOT after in vitro re-stimulation of PBMC of BTV-8 vaccinated animals with BTV was evaluated in the field for the first time. Sera of BTV-8 infected or vaccinated animals neutralising BTV-8 could also neutralise an Italian BTV serotype 1 cell culture adapted strain and PBMC of such animals secreted IFN-γ when stimulated with BTV-1.

Bovine papular stomatitis virus was isolated from two calves in an animal house with biosafety level 3 confinement. The hypotheses on the origin of the infection, the interesting features of the partial amino acid sequences of the major envelope viral protein, and the importance of diagnostic tools available for animal diseases that are not listed by the World Organization for Animal Health (OIE) are discussed.

Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus Parapoxvirus . It displays only limited homology with orthopoxviruses and has some molecular features such as an unusual high GC content distinct from orthopoxviruses. Inactivated PPVO (iPPVO) displays strong immunostimulatory capacities mediating antiviral activity in vivo. The role of dendritic cells (DC) and the pattern recognition receptors and signaling requirements responsible for immunostimulation by iPPVO are unknown. We demonstrate here that bone marrow-derived plasmacytoid DC (BM-pDC) and bone marrow-derived conventional DC (BM-cDC) secrete alpha/beta interferon (IFN-α/β) in response to iPPVO. Furthermore, iPPVO induces tumor necrosis factor alpha (TNF-α) and interleukin-12/23p40 (IL-12/23p40) release and major histocompatibility complex class II (MHC-II), MHC-I, and CD86 upregulation by bone marrow-derived DC (BMDC). After engulfment, iPPVO is located in endosomal compartments and in the cytosol of BMDC. iPPVO elicits IFN-α/β by Toll-like receptor (TLR)-independent pathways in BM-cDC, since IFN-α/β release does not require myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-α release and enhanced expression of MHC-I and CD86 but not of MHC-II by BMDC chiefly requires MyD88 but not TLR2 or TLR4. Induction of IFN-α by iPPVO in BM-cDC occurred in the absence of IFN regulatory factor 3 (IRF3) but required the presence of IRF7, whereas iPPVO-triggered IFN-β production required the presence of either IRF7 or IRF3. These results provide the first evidence that iPPVO mediates its immunostimulatory properties by TLR-independent and TLR-dependent pathways and demonstrate an important role of cDC for IFN-α/β production.

A H5N2 low pathogenic avian influenza virus (LPAIV) was isolated from a natural reservoir in Bavaria during a routine screen and was Used as a vaccine strain to Scrutinize the immune response involved in cross-protection after challenge infection with a H5N1highly pathogenic avian influenza Virus (HPAIV). The challenge virus was also isolated from a natural reservoir in Bavaria. Wild type, antibody deficient (mu MT), CD4(-/-) and CD8(-/-) mice were infected with the apathogenic H5N2 vaccine strain and challenge infection with a 100-fold MLD50 of the H5N1 strain was performed 80 days later. While 100% of the wild type and 100% of the CD8(-/-) mice stayed healthy, only 50% of the CD4(-/-) and none of the antibody deficient mice were protected. These results Support the view that the humoral immune response and to certain extends the CD4() T helper cells are a prerequisite for cross-protective immunity against H5 influenza virus. (C) 2008 Elsevier Ltd. All rights reserved

The almost simultaneous initial detections of avian influenza A H5N1 viruses in central Europe in February 2006, at a time devoid of migratory bird activity, raised the question of the origin of these viruses. This report presents molecular data from Europe providing evidence for multiple and spatially overlapping H5N1 introductions into Bavaria, Germany.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear.

Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.

International audience; The aim of this study was to determine the immunomodulatory effects of IL-12, IL-18 and CD154 (CD40 ligand, CD40L) in DNA-vaccination against the classical swine fever virus. Four recombinant plasmids were constructed including the CSFV coding region for the glycoprotein gp55/E2 alone or together with porcine IL-12, IL-18 or CD154 genes. Five groups of four pigs each were immunized intramuscularly (i.m.) three times with the respective constructs. The control group was inoculated with empty plasmid DNA. Eighteen days after the final immunization, the pigs were challenged with a lethal dose of CSFV strain Eystrup and monitored for a further 16 days. This study showed that co-delivery of IL-18 and CD154 induced an earlier appearance of serum antibodies, reduced B-cell deficiency after infection and protected pigs against a lethal CSFV infection. In contrast, co-delivery of IL-12 led to a reduced titer of neutralizing antibodies and protection against a lethal CSFV challenge in comparison to the other pigs and to pigs that were immunized with a gp55/E2 plasmid alone.

Cases of papular stomatitis in Finnish reindeer have been reported for many years. The causative agent was thought to beOrf virus(ORFV), one of theParapoxviridae, although this assumption was based mainly on clinical symptoms, pathology and electron microscopy. Here sequence analyses of the viral DNA isolated from a recent outbreak of disease in 1999–2000 are presented in comparison to that isolated from earlier outbreaks in 1992–1994. The results show that the virus isolated from the 1999–2000 outbreak is most closely related toPseudocowpox virus, whereas those from previous years grouped with ORFV. The present study describes a method for genetic characterization and classification of parapoxviruses (PPVs) and provides for the first time an extended phylogenetic analysis of PPVs isolated from Finland, established members of the genusParapoxvirusand selected members of the subfamilyChordopoxvirinae.

The present study provides for the first time an extended investigation of individual genes located at the near-terminal right end of the genome of parapoxvirus bovis 1, Bovine papular stomatitis virus (BPSV) strain B177 and Orf virus (ORFV). Comparison of the respective DNA sequences of ORFV strain D1701 (9.9 kbp) and BPSV B177 (7.7 kbp) revealed a very similar organization of closely related genes transcribed in a rightward orientation. The most salient findings of this study were: (i) the absence of the ORFV-specific vascular endothelial growth factor (VEGF-E) gene in the BPSV isolate; (ii) the presence of an interleukin-10 (IL-10) orthologue; and (iii) the detection of three new genes encoding ankyrin-repeat-containing polypeptides. These results not only contribute to potential improvements of future molecular differentiation between the parapoxvirus species, but also shed new light on different pathobiologies among parapoxviruses.

The first report of a vascular endothelial growth factor (VEGF)-like gene inOrf virusincluded the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41·1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates ofOrf virusderived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30·8% with an average amino acid identity between pairs of NZ2-like sequences of 86·1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

The present study is the first report on the functional activity of a parapoxvirus-encoded dUTPase. The dUTPase gene of the attenuated orf virus (ORFV), strain D1701, was expressed as a bacterial thioredoxin fusion protein.In vitroassays showed that ORFV dUTPase was highly specific for dUTP as substrate. The enzyme was active over a broad pH range (pH 6·0–9·0), with maximal enzymatic activity at pH 7·0 in the presence of Mg2+cations. Kinetic studies of the recombinant ORFV dUTPase revealed an apparentKmof 4·0 μM, which is more similar to that of the mammalian or African swine fever virus enzyme than to theKmof vaccinia virus dUTPase. Enzyme activity was also found with purified ORFV particles, indicating its virion association.

Viruses of the genus parapoxvirus from the family poxviridae cause widespread but localized diseases of small and large ruminants. The economically most important disease is contagious pustular dermatitis or contagious ecthyma among sheep and goats, often simply called orf. The parapoxviruses (PPV) can be transmitted to man leading to localized lesions that are named pseudocowpox or milkers' node as being mostly restricted to the hands and fingers. In cattle two forms of PPV manifestation are commonly observed, the bovine papular stomatitis in young calves and the occurrence of lesions at the udder of cows. We here report about the recent efforts in molecular characterization of orf viruses and the state of the art about the generation of orf virus recombinants. In addition the current knowledge on immune responses against orf viruses and some new data on the behaviour of orf virus recombinants under non-permissive conditions are reported.

To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82% of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFV-Glentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446–1460 of the CSFV non-structural protein NS2–3 could induce interferon-γ secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.

Live virus vaccines for human use, 29 monovalent vaccines against measles, mumps, rubella or polio, eight polyvalent vaccines against measles-mumps-rubella and one bacterial polyvalent vaccine against Streptococcus pneumoniae, were tested by reverse transcriptase-nested PCR for the presence of petivirus or pestivirus RNA. Twenty-four samples were selected from European manufacturers, ten were from U.S.A. and four from Japan. Five (13.1%) out of 38 tested samples were positive for pestivirus RNA. Three vaccines (rubella and two measles) were from Europe and two (mumps and rubella) from Japan. The 5'-untranslated genomic region of the contaminant pestivirus RNA were amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary structures, characteristic to genotypes, revealed that the cDNA sequences belonged to bovine viral diarrhea virus (BVDV). A cDNA sequence, detected from one measles sample, belonged to BVDV-1b genotype. Pestiviral cDNA detected from the Japanese mumps and rubella vaccine samples, belonged to the BVDV genotypes 1a and 1c, respectively. Analysis on two cDNA sequences detected from measles and rubella vaccine samples from Europe showed their appurtenance to a new genotype, BVDV-1d. These findings indicate that contamination by animal pestivirus may occur in biological products for human use.

Classical swine fever (CSF) is a severe hemorrhagic disease of swine caused by the pestivirus CSF virus (CSFV). Amino acid exchanges or deletions introduced by site-directed mutagenesis into the putative active site of the RNase residing in the glycoprotein E rns of CSFV abolished the enzymatic activity of this protein, as demonstrated with an RNase test suitable for detection of the enzymatic activity in crude cell extracts. Incorporation of the altered sequences into an infectious CSFV clone resulted in recovery of viable viruses upon RNA transfection, except for a variant displaying a deletion of the histidine codon at position 297 of the long open reading frame. These RNase-negative virus mutants displayed growth characteristics in tissue culture that were undistinguishable from wild-type virus and were stable for at least seven passages. In contrast to animals inoculated with an RNase-positive control virus, infection of piglets with an RNase-negative mutant containing a deletion of the histidine codon 346 of the open reading frame did not lead to CSF. Neither fever nor extended viremia could be detected. Animals infected with this mutant did not show decrease of peripheral B cells, a characteristic feature of CSF in swine. Animal experiments with four other mutants with either exchanges of codons 297 or 346 or double exchanges of both codons 297 and 346 showed that all these RNase-negative mutants were attenuated. All viruses with mutations affecting codon 346 were completely apathogenic, whereas those containing only changes of codon 297 consistently induced clinical symptoms for several days, followed by sudden recovery. Analyses of reisolated viruses gave no indication for the presence of revertants in the infected animals.

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.

There are two possibilities for virus contamination of foodstuff and bioproducts of animal origin: i) the presence of endogenous virus as a result of an acute or subclinical infection of animal raw material used for food processing or ii) contamination of food in the course of processing or thereafter. The latter must be considered as the highest risk for human consumers since the viral contamination mostly is caused by virus shedding people and the transmitted viruses are obligate human pathogens. Food from animals consumed as raw material (e.g. oysters) is listed in a high risk category concerning viral contamination (e.g. hepatovirus). Virus contamination of bioproducts such as vaccines, blood products or biological material used in surgery and for transplantations also is more hazardous because the application of contaminating virus usually occurs by circumvention of the natural barrier systems of the body. Moreover, in many cases immunosuppressed people are treated with bioproducts. Due to an enclosing shield of high protein and lipid content in food and bioproducts viruses are well protected against physical and chemical influences, however most preparation procedures for food are destructive for viruses. The detection of pseudorabies virus and pestivirus in biological fluids was tested using polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR and cell culture propagation. PCR is a powerful method to detect viral nucleic acid whereas the detection of infectious virus in cell cultures is more limited, e. g. due to protein and lipid destroying conditions. Virus contamination of bioproducts should be considered as a hazard no matter which method has been used for its detection. Examples are given about the contamination of cell lines and vaccines.

Despite many attempts to find reliable in vitro criteria for the efficacy of Biological Response Modifiers--BRMs (immunomodulators, paramunity inducers) animal challenge models are still the only way to demonstrate the totality of interlocking defense mechanisms. Challenge models with mouse pathogenic viruses provide an excellent possibility to study protective effects of BRMs against acute or chronic forms of viral diseases. For comparative studies two completely different virus challenge models--Pseudorabies PR and Stomatitis Vesicularis VSV--were developed with adult and baby NMRI mice respectively. The potency of BRMs in preventing lethal disease reveals significant differences depending upon the sort of BRM, the route of application and the time of pretreatment. Defense mechanisms important for the control of Pseudorabies virus infection in adult NMRI mice were tested in vitro (ex vivo) and correlated well with the degree of protection in vivo. Comparison of BRM efficacy in selective viral challenge models combined with screening of a variety of antiviral defense functions in infected animals in vitro provide reliable methods in demonstrating the potency of BRMs against viral infections.

Summary Experimental proof of paraspecific effects of purified and inactivated pox virus The paraspecific effect of pox viruses i.e. stimulation of the nonspecific part of the complex immune system, was demonstrated in vitro and in vivo for 3 different types of virus (vaccinia virus, avipox virus, parapox virus). By means of purification, concentration and inactivation, it was possible to prove that the paraspecific effect is brought about by the pox virus particle per se. The effect is induced by certain epitopes in the complex structure of the virus and is independent of virus multiplication. Both purified pox viruses and inactivated, purified pox viruses stimulate NK cell activity, the CSA and the formation of interferon, and reach higher efficacy indices in a challenge test with baby mice using stomatitis vesicularis virus than non-purified and non-inactivated cell culture material. The degree of purity of the pox virus preparations was established using the electron microscope and monoclonal antibodies. The purified and concentrated pox virus preparations proved to be pyrogen-free; they were not toxic to baby mice in vivo. An endotoxin-induced effect could be ruled out. The broad paraspecific effect of pox viruses and their structural components lends itself to use as paramunity inducers in prophylaxis and therapy. Zusammenfassung Die paraspezifische Wirkung von Pockenviren, d. h. die Stimulierung speziell des unspezifischen Teiles des komplexen Immunsystems, lies sich bei 3 verschiedenen Virusgenera (Vacciniavirus, Avipoxvirus, Parapoxvirus) in vitro und in vivo nachweisen. Mittels Reinigung, Konzentrierung und Inaktivierung wurde bewiesen, das die paraspezifische Wirkung durch das Pockenviruspartikelchen per se bedingt ist. Sie wird durch bestimmte Epitope in der komplexen Struktur des Virus induziert und ist unabhangig von der Vermehrung des Virus. Sowohl gereinigte als auch gereinigte und inaktivierte Pockenviren stimulieren die NK-Zell-Aktivitat, die CSA, die Interferonbildung und erreichen im Challenge-Test bei Babymausen mit dem Virus der Stomatitis Vesikularis hohere Wirkungsindices als nicht gereinigtes und nicht inaktiviertes Zellkulturmaterial. Der Reinheitsgrad der Pockenpraparate wurde im Elektronenmikroskop und mit Hilfe monoklonaler Antikorper uberpruft. Die gereinigten und konzentrierten Pockenviruspraparate erwiesen sich pyrogenfrei, sie waren in vivo fur Babymause nicht toxisch. Eine Endotoxin-induzierte Wirkung konnte ausgeschlossen werden. Die Nutzung der breiten Wirkung von Pockenviren bzw. ihrer Strukturkomponenten als Paramunitatsinducer bietet sich prophylaktisch wie therapeutisch an.

Summary Through in vitro stimulation of different cell types with various stimulating substances, production of mediators with antiviral activity in vivo was studied. In two lethal virus mouse challenge models — adult NMRI-mice challenged with pseudorabies virus (PRV) and baby mice challenged with stomatitis vesicularis virus (VSV) — antiviral efficacy of cell culture supernatants (CCSs) was demonstrated. In a plaque reduction assay, interferon activity in CCSs and in serum of CCS-treated mice was investigated. Prophylactic treatment with CCSs from murine hybridomas and human lymphomas — stimulated by inactivated parapoxvirus ovis — reduced the mortality rate of challenged mice significantly. None of the cell cultures secreted interferon into the medium, whereas intraperitoneal application of some CCSs induced interferon activity in serum of mice. A possibility for production of non-antigen-specific mediators was shown by appropriate stimulation of certain cell types in vitro. Prophylactic treatment with these mediators triggered non-specific defence mechanisms in mice against two lethal virus infections. Zusammenfassung Steigerung der unspezifischen Virusabwehr bei NMRI-Mausen durch Mediatoren aus Zellkulturen Mit verschiedenen Stimulationssubstanzen wurde versucht, in vitro unterschiedliche Zellarten zur Sekretion von Mediatoren mit antiviraler Wirksamkeit in vivo anzuregen. Die Prufung der antiviralen Wirksamkeit der Zellkulturuberstande (ZKU) erfolgte in zwei Virusbelastungsmodellen, wobei adulte NMRI-Mause mit Aujeszky-Virus und juvenile Mause mit Vesicularis Stomatitis-Virus belastet wurden. Mit einem Plaque-Reduktions-Test wurde die Interferon (IFN)-Aktivitat in einigen ZKU und im Serum ZKU-behandelter Tiere bestimmt. Vorbeugende Behandlung mit ZKU aus murinen Hybridomas bzw. humanen Lymphomas, die mit inaktiviertem Parapoxvirus ovis stimuliert waren, verringerte die Mortalitatsrate von virusbelasteten Mausen erheblich. Keine der Zellkulturen sezernierte IFN in den Uberstand, wahrend die intraperitoneale Verabreichung einiger ZKU IFN-Aktivitat im Serum der Mause induzierte. Die Versuche zeigen die Moglichkeit auf, bestimmte Zellarten mit geeigneten Stimulationssubstanzen zur Sekretion von Mediatoren anzuregen, mit denen sich bei Mausen in vivo die unspezifische Infektabwehr gegen bestimmte Virusinfektionen erhohen last.

The Chordopoxvirinae subfamily (Family Poxviridae) includes human and animal pathogens with zoonotic potential. Among them are viruses that are commonly used as viral and vaccination vectors, some ...