Martin A. Hofmann

Genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland.

Nucleotide sequences analysis indicates that this virus is a new serotype of bluetongue virus.

Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

ABSTRACT Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.

Classical Swine Fever Virus Interferes with Cellular Antiviral Defense: Evidence for a Novel Function of Npro

ABSTRACT Classical swine fever virus (CSFV) replicates efficiently in cell lines and monocytic cells, including macrophages (MΦ), without causing a cytopathic effect or inducing interferon (IFN) secretion. In the present study, the capacity of CSFV to interfere with cellular antiviral activity was investigated. When the porcine kidney cell line SK-6 was infected with CSFV, there was a 100-fold increased capacity to resist to apoptosis induced by polyinosinic-polycytidylic acid [poly(IC)], a synthetic double-stranded RNA. In MΦ, the virus infection inhibited poly(IC)-induced alpha/beta IFN (type I IFN) synthesis. This interference with cellular antiviral defense correlated with the presence of the viral Npro gene. Mutants lacking the Npro gene (ΔNpro CSFV) did not protect SK-6 cells from poly(IC)-induced apoptosis, despite growth properties and protein expression levels similar to those of the wild-type virus. Furthermore, ΔNpro CSFV did not prevent poly(IC)-induced type I IFN production in MΦ but rather induced type I IFN in the absence of poly(IC) in both MΦ and the porcine kidney cell line PK-15, but not in SK-6 cells. With MΦ and PK-15, an impaired replication of the ΔNpro CSFV compared with wild-type virus was noted. In addition, ΔNpro CSFV, but not wild-type CSFV, could interfere with vesicular stomatitis virus replication in PK-15 cells. Taken together, these results provide evidence for a novel function associated with CSFV Npro with respect to the inhibition of the cellular innate immune system.

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains.

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.

Quantitative TaqMan® RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus

Since two different types of porcine reproductive and respiratory syndrome virus (PRRSV), the European (EU) and the North American (US) strain, occur or coexist in European swine herds, their rapid and reliable detection and differentiation is essential for disease surveillance. A quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) is described for PRRSV detection and strain differentiation. Sensitivity and specificity were compared with a conventional PRRSV RT-PCR and to the detection of both PRRSV types in cell cultures and both were found to be equal or superior to the reference methods. Reproducibility was tested and proved that the assay was very reliable. Standard dilutions included in each test allowed absolute quantitation of the amount of viral RNA. The TaqMan assay described below is time-saving, easy to handle, exhibits a decreased risk of cross-contamination and is highly sensitive and specific. It is, therefore, considered to be a powerful tool for the rapid detection and differentiation of PRRSV.

Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of Npro

ABSTRACT Pestiviruses prevent alpha/beta interferon (IFN-α/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral Npro nonstructural protein. Npro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire Npro gene resulted in attenuation in pigs. In order to elaborate on the role of the Npro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in Npro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of Npro. We describe two mutations in Npro that eliminate Npro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for Npro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-α/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-α/β induction is not a prerequisite for CSFV virulence.

An indirect ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen

An indirect enzyme-linked immunosorbent assay termed rnPRRS ELISA using baculovirus-expressed and affinity-purified viral nucleocapsid protein (rNC) antigen was developed for detecting antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) in swine sera. Sera (1395) originating from different European countries were used for the validation of this assay. The rnPRRS ELISA was capable of detecting antibodies in all sera known to contain anti-PRRSV antibodies, resulting in 100% sensitivity. The specificity was 95.8%. The rnPRRS ELISA was more sensitive compared to the most widely used tests for the diagnosis of porcine reproductive and respiratory syndrome (PRRS) (i) a commercially available ELISA; (ii) the indirect immunofluorescence assay (IIFA); and (iii) the immunoperoxidase monolayer assay (IPMA). The main advantage of the rnPRRS ELISA compared to an ELISA using whole virus antigen is the use of a single immunogenic protein instead of infectious PRRSV. The rnPRRS ELISA is suitable for routine diagnosis of PRRS and also for epidemiological surveys since it allows highly reliable testing of a large number of sera within a short period of time.

Low density blood granulocytic cells induced during classical swine fever are targets for virus infection

Classical swine fever virus infection of pigs causes a severe leukopenia and immunosuppression. In the present study, the kinetics of virus infection, and identification of target cells for the virus in peripheral blood were analysed. Virus infection was often not detectable before 5-7 days p.i. A minority of animals yielded detectable infected cells at 3 days p.i., but < 5% PBMC. It was not until 10 days p.i. that this figure increased-to 35-70% PBMC depending on the animal. Detailed analysis of Ficoll-Hypaque-purified PBMC identified the major population to be SWC3+SWC8+CD14+MHCII- granulocytic cells. Microscopic observations determined that these low density granulocytic cells in the PBMC from CSFV infected animals were indeed immature cells. Both the low density granulocytic cells and monocytes were major targets for CSFV infection in the peripheral blood. This is the first demonstration that low density granulocytic cells dominate the blood leukocyte population during CSF, and that such cells are targets for virus infection. The present work also demonstrates that the leukocyte population changes, such as B lymphocyte depletion and the relative dominance of myeloid cells in the blood during CSF, occur before virus infection of the affected cells. Thus, the pathological mechanism therein is not a direct consequence of virus infection.

Quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (PEDV)

Abstract The porcine epidemic coronavirus (PEDV), tentatively classified as a coronavirus, was adapted to Vero cells and a plaque test developed for infectivity titration, allowing us to test the biological and biophysical properties of the virus. Growth kinetics showed peak titers of 105.5 plaque-forming units ml−1 15 h after infection. Filtration experiments and electron microscopy revealed a particle diameter between 100 and 200 nm. The bouyant density of the virus was 1.18. The particle lost its infectivity on treatment with lipid solvents. Virus replication could not be inhibited by 5-iodo-2′-deoxyuridine. PEDV was moderately stable at 50°C, but heat sensitivity was not altered by divalent cations. At 4°C, the virus was stable between pH 5.0 and 9.0, but at 37°C stability was restricted to the pH range 6.5–7.5. Viral infectivity was not impaired by ultrasonication or by multiple freezing and thawing. PEDV was not neutralized by transmissible gastroenteritis virus antiserum. On the basis of the tests carried out, PEDV is a pleomorphic, enveloped RNA virus with a particle diameter of ∼ 150 nm and a bouyant density of 1.18. Infectivity depends on the presence of trypsin, and infected cells show a tendency to fuse and to form syncytia. All of these properties, as well as its physicochemical characteristics, allow PEDV to be classified as a coronavirus.

Detection of antibodies against classical swine fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2

Recombinant envelope protein E2 (gp55) of classical swine fever virus (CSFV) strain Alfort/187 was evaluated as an alternative to whole virus as ELISA antigen for the detection of antibodies against CSFV. A glycosylated and a non-glycosylated form of E2 was expressed in the baculovirus system. Six histidine residues added at the carboxy terminus of each of the recombinant proteins allowed purification by nickel-chelate affinity chromatography. Comparison of the antigenic properties of the two proteins in indirect and blocking ELISAs revealed that the glycosylated form resulted in both higher sensitivity and specificity. The indirect ELISA, using glycosylated E2, either derived from crude cell extract or affinity-purified, was validated by testing a total of 2719 porcine sera. Its final version proved to be as sensitive (98.3%) as the virus neutralization test when sera from infected pig herds were examined, and highly specific (99.6%) when applied to test negative sera. It is therefore suitable for large scale monitoring of classical swine fever.