Nieuwe therapeutische strategieën voor de behandeling van infecties met Pestivirussen

The pestiviruses bovine viral diarrhoea viruses (BVDV), classical swine fever (CSFV) and border disease virus (BDV) are important pathogens for livestock. These viruses are worldwide responsible for significant economic losses. Infection of cattle with BVDV causes a spectrum of clinical signs, ranging from asymptomatic to severe disease potentially leading to death. The major losses due to BVDV infection include growth retardation and reduced milk production. BVDV crosses the placenta and infects the foetus, resulting, in persistently infected (PI) animals. PI animals continuously shed massive amount of virus during their life-time and serve as the reservoir of the virus in cattle herds. BVDV is endemic in bovine populations worldwide. In the last decades programs have been implemented to eliminate PI animals. The classic symptoms caused by infection with the CSFV in pigs are pyrexia, severe leucopoenia, haemorrhages and neurologic problems. This results in high morbidity and mortality. CSFV has been eradicated in parts of the world, i.e. the US, the EU and Australia, but it is still endemic in many other regions such as for example in Eastern European countries, Asia and South America. Vaccination and stamping out have been used for the control of CSFV. Since the control of CSFV in endemic regions remains problematic and since reintroduction of CSFV in virus-free regions results in a serious burden, new control measures for CSFV are needed. Moreover, given the enormous economic impact of BVDV, additional measures are needed for the control of this virus. Highly potent antiviral drugs may be an excellent tool to help control pestivirus infection. Efficient antiviral drugs have the benefit to result almost instantaneously in a protective effect (in contrast to vaccines where there is an immunity gap between vaccination and protection) and they may, in the case of CSFV control, be useful to close the immunity gap. Rapid control of outbreaks with CSFV will largely reduce the number of healthy animals that will need to be culled in the containment perimeter around the infected premises and help to minimize disruption of trade and thus to decrease the economic impact of the disease. Eliminating PI animals without prophylactic antivirals or vaccination of non-infected animals on a farm where a PI calf has been identified is currently regarded as an important control measure. In the last 10 to 15 years, our laboratory and others, identified different chemical classes of pestivirus inhibitors. The effect of two compounds (DB772 and BPIP) has been assessed in infected animals, DB772 in BVDV infected mini-zebu cows and the imidazopyridine BPIP (a compound discovered in our laboratory) in CSFV infected pigs. BPIP was shown to be effective in reducing viral load in infected pigs and to reduce transmission to sentinels. This provided the first proof-of-concept that a specific antiviral therapy has indeed great potential for the control of pestivirus infections in livestock. In a large scale screening effort aimed at identifying novel pestivirus inhibitors, we discovered several classes of potent and selective inhibitors of the in vitro replication of pestiviruses. To further optimize the antiviral activity hit-optimization programs were initiated for three different classes [2,6-bis(benzimidazol-2-yl)pyridine, BBP/CSFA-0; imidazolidine, BTB02541SC/CSFB-0; Quinolinecarboxamides, TO502-2403/CSFC-00 and TO505-6180/CSFC-0]. The structure-activity relationship (SAR) was carefully studied for these three chemical classes in an attempt to further improve the antiviral activity and selectivity. In parallel, the particular characteristics and mechanism of action of these (and yet some other) classes of compounds was studied. To this end, it was first attempted to select drug-resistant variants; which were next genotyped to identify in which gene (a) mutation(s) were accumulating. Surprisingly, for all classes of pestivirus inhibitors studied, the resistance-conferring mutations were located in the viral RNA-dependent RNA polymerase (RdRp). Moreover, the structurally very different classes of compounds proved all to be cross-resistant to each other as well as to earlier reported classes of pestivirus inhibitors (BPIP, AG110 and LZ37). All mutations identified were located in the fingertip of the RdRp. Remarkably, none of these compounds inhibited the activity of the purified BVDV RdRp but efficiently blocked the activity of viral replication complexes. Thus the different classes of pestivirus inhibitors all target a hotspot in the viral polymerase thereby inhibiting the functioning of the replication complex of which the RdRp is a component. Furthermore, our data demonstrate that this hotspot in the finger domain of the pestivirus RdRp is likely, and this in stark contrast to the situation for the related hepatitis virus, the only allosteric pocket in the polymerase. Inhibitors that bind to this pocket prevent the proper functioning of the RdRp (or interaction with other proteins or RNA) in the context of the viral replication complex. The findings reported in this thesis will contribute to the further development of additional control measures for pestiviral infections in livestock.status: publishe