The genus Pestivirus, family Flaviviridae, includes four economically important viruses of livestock, i.e., bovine viral diarrhea virus-1 (BVDV-1) and -2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). Erns and Npro, both expressed uniquely by pestiviruses, counteract the host’s innate immune defense by interfering with the induction of interferon (IFN) synthesis. The structural envelope protein Erns also exists in a soluble form and, by its endoribonuclease activity, degrades immunostimulatory RNA prior to their activation of pattern recognition receptors. Here, we show that at least three out of four positively-charged residues in the C-terminal glycosaminoglycan (GAG)-binding site of BVDV-Erns are required for efficient cell entry, and that a positively charged region more upstream is not involved in cell entry but rather in RNA-binding. Moreover, the C-terminal domain on its own determines intracellular targeting, as GFP fused to the C-terminal amino acids of Erns was found at the same compartments as wt Erns. In summary, RNase activity and uptake into cells are both required for Erns to act as an IFN antagonist, and the C-terminal amphipathic helix containing the GAG-binding site determines the efficiency of cell entry and its intracellular localization.

The genus Pestivirus, family Flaviviridae, includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, Npro and Erns, that pestiviruses evolved to regulate the host’s innate immune response. In addition to its function as a structural envelope glycoprotein, Erns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, Erns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host’s interferon (IFN) response. Here, we characterize the basic features of soluble Erns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various Erns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.

Bovine viral diarrhoea virus (BVDV) and Border disease virus (BDV) are closely related pestiviruses of cattle and sheep, respectively. Both viruses may be transmitted between either species, but control programs are restricted to BVDV in cattle. In 2008, a program to eradicate bovine viral diarrhoea (BVD) in cattle was started in Switzerland. As vaccination is prohibited, the cattle population is now widely naïve to pestivirus infections. In a recent study, we determined that nearly 10% of cattle are positive for antibodies to BDV. Here, we show that despite this regular transmission of BDV from small ruminants to cattle, we could only identify 25 cattle that were persistently infected with BDV during the last 12 years of the eradication program. In addition, by determining the BVDV and BDV seroprevalence in sheep in Central Switzerland before and after the start of the eradication, we provide evidence that BVDV is transmitted from cattle to sheep, and that the BVDV seroprevalence in sheep significantly decreased after its eradication in cattle. While BDV remains endemic in sheep, the population thus profited at least partially from BVD eradication in cattle. Importantly, on a national level, BVD eradication does not appear to be generally derailed by the presence of pestiviruses in sheep. However, with every single virus-positive cow, it is necessary to consider small ruminants as a potential source of infection, resulting in costly but essential investigations in the final stages of the eradication program.