Classical Swine Fever Virus N(pro) Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

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 N(pro). 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 N(pro), 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 N(pro) induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, N(pro) coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with N(pro). Finally, we show that N(pro) does not downregulate IRF7 expression

Classical swine fever virus replicon particles lacking the Erns^{\rm rns} gene: a potential marker vaccine for intradermal application

Classical swine fever virus replicon particles (CSF-VRP) deficient for E$^{\rm rns}$ were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E$^{\rm rns}$ (A187delE$^{\rm rns}$). For packaging of A187delE$^{\rm rns}$ into virus particles, porcine kidney cell lines constitutively expressing E$^{\rm rns}$ of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10$^7$ TCID$_{50}$ of VRP A187delE$^{\rm rns}$ elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-$\gamma$ producing cells. No anti-E$^{\rm rns}$ antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus

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

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

Recurrence of Hepatitis B Infection in Liver Transplant Patients Receiving Long-Term Hepatitis B Immunoglobulin Prophylaxis

BACKGROUND Long-term real-world data are relatively sparse regarding recurrence of chronic hepatitis B virus (HBV) infection after liver transplantation using hepatitis B immunoglobulin (HBIg) and nucleos(t)ide analogue (NUC) prophylaxis. MATERIAL AND METHODS Data from 371 adults transplanted for HBV-related disease at 20 European centers and given HBIg for ³12 months ± NUC therapy were analyzed retrospectively. RESULTS HBIg comprised Hepatect® (iv HBIgB; n=299), subcutaneous Zutectra® (sc HBIg, n=236), and other HBIg preparations (n=130); 93.5% received NUC therapy. Mean follow-up was 6.8±3.5 years. The primary efficacy variable, freedom from HBV recurrence, occurred in 95.7% of patients (95% CI [93.1%, 97.5%]). The observed incidence of recurrence was 16/371 (4.3%) (annual rate 0.65%); 5/16 patients with recurrence had discontinued HBIg and 7/16 had anti-HBs <100 IU/l. Excluding these 7 patients, the HBV recurrence rate was 2.4%. The recurrence rate while on HBIg therapy was 1 per 2069 months. In patients who discontinued HBIg, risk of HBV recurrence versus sc HBIg users was increased by 5.2-fold (1 per 1 603 versus 1 per 8379 treatment months). The annual rate of HBV-related hepatocellular carcinoma (HCC) recurrence was 1.7%. CONCLUSIONS These results support the long-term use of HBIg with NUC therapy as an effective management strategy to minimize risk of HBV recurrence and virus-related complications after liver transplantation